Interview with Dr. Francis Collins on Genetic Information Nondiscrimination Act of 2008

DIFERENCIAS SEXOGENERICAS EN DEPRESION

Feature Review
Molecular Psychiatry (2010) 15, 23–28;; published online 22 September 2009
Sex, trauma, stress hormones and depression
E Young Dr. Elizabeth Young died on September 1, 2009 and that the article on pages 23-28 is the last paper that she wrote?1,✠ and A Korszun2
1Molecular and Behavioral Neurosciences Institute, University of Michigan, Ann Arbor, MI, USA
2Barts and The London School of Medicine, Queen Mary University of London, London, UK
Correspondence: Professor A Korszun, Centre for Psychiatry, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. E-mail: a.korszun@qmul.ac.uk
✠Deceased.
Abstract
Although few studies dispute that there are gender differences in depression, the etiology is still unknown. In this review, we cover a number of proposed factors and the evidences for and against these factors that may account for gender differences in depression. These include the possible role of estrogens at puberty, differences in exposure to childhood trauma, differences in stress perception between men and women and the biological differences in stress response. None of these factors seem to explain gender differences in depression. Finally, we do know that when depressed, women show greater hypothalamic–pituitary–adrenal (HPA) axis activation than men and that menopause with loss of estrogens show the greatest HPA axis dysregulation. It may be the constantly changing steroid milieu that contributes to these phenomena and vulnerability to depression.
Keywords: depression, sex differences, estrogen, progesterone, HPA axis, stress

Introduction
Depression is a multifactorial disorder in which adaptation to stressors undoubtedly has a crucial role. Although genetic vulnerability is critical to the development of depression, the incidence of depression is very low in the absence of environmental stressors;1 and in approximately 75% of cases of depression, there is a precipitating life event.2, 3 An understanding of the pathoetiology of depression must provide an explanation for the high predominance of women with depressive disorders. Thus, the relationship between gender, trauma, gonadal steroids stress and depression, and in sex differences in the stress hormone response, comprises both an intriguing and fruitful area of research. Furthermore, there may be gender differences in stress hormones found only in the depressed patients.
The occurrence of gender differences in depression has been replicated in numerous studies across cultures.4 This difference arises at puberty and persists throughout the reproductive years.5 Does estrogen cause depression? Studies by Angold6 have found a relationship between the rise in estrogen and testosterone levels and the rising incidence of depression in girls during adolescence. In women with an earlier episode of depression, the periods of rapidly changing gonadal steroid concentrations, such as those occurring premenstrually or postpartum, mark particularly vulnerable times for the occurrence of depressive symptoms. Studies by O'Hara et al.7, 8 confirm that a history of depressive episodes increases the risk of both postpartum ‘blues’ and postpartum major depression. Recent studies on two epidemiological cohorts9, 10 have found an increased incidence of depressive symptoms and major depression during the menopause transition. The findings of Freeman et al.10, 11 with regard to estrogen showed that both high and low estrogen were associated with depression. More recently, their data suggest that variability in estrogen levels may drive depression, that is, those women who show rapid changes from high to low estrogen and vice versa are those who develop depressive symptoms during the perimenopause transition. Finally, estrogen levels are lower in women with major depression, possibly predisposing women to depression.12
Do sex differences in stress and trauma account for greater incidence of depression?
Given the link between stress and depression, perhaps women are more likely to experience major life stressors. Research does not suggest that this is the case, and the overall incidence of major life stressors is the same between sexes. However, the depressogenic effects of stressors are influenced by gender, and hence women are more susceptible to stressors that affect more distant relationships.13 Kendler et al. found that women were more sensitive to the depressogenic effects of problems in getting along with others in their interpersonal network but men were more sensitive to the depressogenic effects of work problems and separation problems.14 Neither study found that the rate of stressful events could account for excess rates of depression in women.
Perhaps men and women perceive stress differently? In an analysis of a stratified representative sample of 2387 adults, Cohen and Williamson15 found no meaningful sex differences in the perceived stress scale. A large-scale multicountry European study by de Smet et al.,16 consisting of 34 972 subjects, found little support for differences in job strain between men and women. There were sex differences in the sense of control but not in the ratings of strain. These studies do not support that differences in stress perception would account for sex differences in depression.
Greater exposure to childhood abuse, including sexual abuse, is another leading explanation for gender differences in depression. Early-life stress and trauma have been found to sensitize animals to later stressors17 and some have suggested that the higher rates of trauma in women might explain the gender differences in depression. However, epidemiological data do not support that women necessarily have more childhood trauma. Studies by Kessler and Magee,18 examining the American Changing Lives longitudinal survey, found greater rates of childhood sexual abuse in women. Furthermore, in general, men are more likely to experience trauma than women. Studies examining population-based samples (generally from school-based samples) of childhood abuse in either young adults18, 19, 20, 21 or late adolescence conclude either no gender difference in exposure to trauma or greater exposure in boys than in girls.19, 20, 21, 22, 23 A recent meta-analysis by Tolin and Foa24 reached similar conclusions that (1) existing studies do not support greater exposure to trauma in women and (2) that overall studies do not support sex differences in childhood trauma. Two studies conducted by a health maintenance organization in San Diego, involving 9460 individuals, analyzed both childhood sexual abuse and other forms of trauma.25, 26 Although the first study, focusing on childhood abuse in women, found a strong association, the second study26 evaluated both men and women with childhood abuse and concluded, ‘For both men and the women, the risk of each outcome was increased at a similar magnitude. For example, compared with no sexual abuse, there was a twofold increased risk for suicide attempts for both men and women (P=0.05) Similarly, there was a 40% increased risk of marrying an alcoholic for both men and women who reported CSA compared with those not reporting CSA (P=0.05)’. It should be further noted that the participants in these studies were all in their mid 50s. It is quite possible that there was some recall bias and those with long histories of depression attributed their depression to events such as childhood abuse when there may well have been other causes. Finally, the study by Breslau et al.,27 analyzing data on adult and childhood trauma from the Detroit Area Study, concluded that trauma does not increase the risk of depression except through post-traumatic stress disorder (PTSD) and that the rates of PTSD are not high enough to account for the excess of depression in women.
It should be noted that the hypothalamic–pituitary–adrenal (HPA) picture shown by women with childhood abuse is not the classic HPA axis picture of major depression. These women show enhanced sensitivity to dexamethasone28 and lower cortisol than their matched control women.29 Although this study by Heim et al.29 did show an exaggerated response to a stressor, the Trier Social Stress Test (TSST), we observed the same exaggerated response to the same stressor in patients with depression plus a comorbid anxiety disorder,30 raising the possibility that it was comorbid anxiety, rather than childhood abuse, that accounted for the differences between groups. Although the study by Breslau et al.,27 analyzing data on adult and childhood trauma from the Detroit Area Study, concluded that trauma does not increase the risk of depression except through PTSD, all studies conclude that women are more sensitive to the development of PTSD after trauma. Furthermore, women are more likely to develop depression in the face of significant life events. This raises the question of sex differences in the biological response to stress.
Sex differences in stress systems
Living organisms survive by maintaining a complex dynamic equilibrium or homeostasis that is constantly challenged by intrinsic or extrinsic stressors. These stressors set into motion responses aimed at preserving homeostasis, including activation of the HPA axis. A hormonal cascade is initiated with the release of corticotropin-releasing hormone (CRH) that triggers the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary corticotrope, and that, in turn, triggers the release of adrenal glucocorticoids. The stress response is turned off by glucocorticoid feedback at the brain and pituitary sites. It has been shown in both rats and humans that the stress response is sexually dimorphic; and studies in rats and humans have suggested that gonadal steroids have an important role in modulating the HPA axis, acting particularly on sensitivity to glucocorticoid negative feedback.31 The effects may be on glucocorticoid receptors, on brain CRH systems or on the responsiveness to CRH.
Studies by Kirschbaum et al.32 have observed sex differences in response to one stress, the TSST. However, the difference is not what would be predicted: men show a greater ACTH response to the stressor. However, the plasma cortisol response is the same in both men and women. Our studies with this same stressor on 96 subjects reached exactly the same conclusions that men show a greater ACTH response to the stressor than women but the cortisol response to the stressor is the same.30 The majority of cortisol in plasma is bound to cortisol steroid-binding globulin. Examining saliva cortisol, the free or active cortisol leads to the conclusions that women show smaller saliva cortisol responses to stress, but it is dependent upon the menstrual cycle phase. Thus, during the follicular phase, women show much smaller responses to the TSST, whereas during the luteal phase their saliva cortisol response to the TSST is the same as men.32 Furthermore, oral contraceptives profoundly decrease the free cortisol response to this stressor.33 We have similar data confirming large menstrual cycle difference in free (saliva) cortisol response to the TSST in normal women, as well as a markedly blunted response in oral contraceptive users (Young, unpublished data).
We have also examined sex differences in response to cortisol infusion. Infusion of cortisol ‘turns off’ corticotroph secretion within 15 min of the onset of a rise in cortisol in both premenopausal female and age-matched male control subjects.34 After the termination of infusion, men showed a continued inhibition of corticotroph secretion for 60 min, whereas women were resistant to cortisol infusion and begin to secrete again in 1 h. This difference seems to be dependent on the menstrual cycle phase. Women with follicular phase plasma progesterone concentrations showed patterns of suppression of corticotroph secretion similar to the men. However, women with progesterone concentrations typical of the luteal phase showed rebound corticotroph secretion after termination of cortisol infusion.34
Similar studies by Altemus et al.35 have found decreased cortisol suppression to dexamethasone during the luteal phase of the menstrual cycle, as compared with the follicular phase in the same women. Furthermore, menstrual cycle hormonal changes influenced the expression of glucocorticoid receptor mRNA in lymphocytes, resulting in a decrease in receptors in the luteal phase of the menstrual cycle35 and suggesting that decreases in glucocorticoid receptors may explain the decreased response to dexamethasone. In our studies of basal ACTH and cortisol secretion for 24 h, we did not observe any menstrual cycle differences in either ACTH or cortisol.36 Nor have we found menstrual cycle differences in response to metyrapone blockade (Altemus and Young, unpublished data). Taken together, these data suggest that the increased stress responsiveness observed during the luteal phase may well be a function of decreased sensitivity to the glucocorticoid negative feedback.
Which ovarian steroids influence the HPA axis?
Having shown that there are both sex differences and changes across the menstrual cycle in normal subjects, we next turn to an analysis regarding which specific ovarian hormone is the critical hormone. We used rodent models and examined the effects of estrogen antagonists and ovariectomy, with and without estrogen replacement, and found that estradiol decreased stress responsiveness.37 Similar findings have been shown in sheep and humans.38, 39 Treatment of normal men with estradiol for 48 h results in increased stress responsiveness.40 However, this could be a function of decreases in testosterone that inhibits stress responsiveness.41 Estradiol administered to postmenopausal women had no effect on the response to the stressor, TSST, but it did decrease the response to the dex-CRH challenge.33
In a clever study design in normally menstruating women, Roca et al.42, 43 analyzed women treated with Lupron, which leads to a loss of all gonadal steroids, followed by estradiol or progesterone add-back phases. They examined the response to exercise stress and found that the exercise stress response was increased during the progesterone ‘add-back’ phase but not during the estrogen ‘add-back’ phase. This supports a role for progesterone in functioning as an antagonist of glucocorticoids. Thus, so far, the data from human studies suggest that estradiol acts as an additional ‘brake’ on the HPA axis but progesterone may impair glucocorticoid negative feedback.
Sex differences in HPA axis regulation in depression
Abnormalities of the HPA axis, as manifested by hypercortisolemia, and disruption of the circadian rhythm of cortisol secretion are well-established phenomena in depression.44, 45 In addition, the failure of cortisol and ACTH suppression with dexamethasone has also been described.44 As HPA dysregulation is the most consistent neuroendocrine abnormality in depression, and depressive disorders occur twice as commonly in women than in men, we asked whether there are sex differences in HPA axis function in depressed patients. We studied baseline cortisol secretion in the morning in 16 depressed patients and 16 age- and sex-matched control patients and found predictably increased cortisol secretion in the group as a whole.34 However, there were also clear sex differences: male patients and their matched controls showed the same plasma cortisol concentration, whereas female depressed patients showed significantly higher mean plasma cortisol concentration than their matched controls. Removal of glucocorticoid negative feedback using metyrapone, a glucocorticoid synthesis inhibitor, showed increased central drive in depressed patients in the evening.46 The response to metyrapone also showed sex differences; only the female depressed patients manifested rebound corticotroph secretion in comparison with their matched controls and the males did not. We conducted 24-h studies with ACTH clamped under metyrapone, and found that depressed women showed significantly increased central drive (presumably because of CRH) only over the 1600–2200 hours time period, replicating our earlier finding. Depressed men showed a significantly decreased response to metyrapone in this same time period compared with the matched control men. The maximum ACTH response using metyrapone was identical between depressed women and their matched controls. In contrast, depressed men showed a significantly decreased maximal ACTH response compared with either their matched controls or with the depressed women. These data argue for a specific circadian time period (late evening) rather than increased CRH–ACTH across the 24-h day as the critical time period in women.47
Dexamethasone non-suppression and menopause We also examined the effect of loss of gonadal steroids at menopause on HPA axis regulation in depressed women,31 using corticotroph secretion as our end point. We conducted these studies on 51 depressed women; 36 of whom were premenopausal and 15 postmenopausal. The premenopausal women showed a significantly lower incidence of corticotroph non-suppressors (44%) than the postmenopausal women (non-suppressor=81%). Using a step-wise regression with a number of independent variables and corticotroph non-suppression to dexamethasone as the dependent variable, we found that only age and baseline cortisol had a significant effect on corticotroph non-suppression; but when age and menopausal status were compared, menopausal status showed a stronger correlation and, combined with cortisol, yielded a correlation coefficient of 0.817. This suggests that menopausal status, in conjunction with cortisol hypersecretion, is a critical variable in the development of HPA dysregulation in women, as manifested by resistance to dexamethasone, and accounts for 65% of the variance. Furthermore, the lower rate of cortisol non-suppression in premenopausal women suggests that gonadal steroids may modulate the HPA axis and exert some protective effect against the high levels of endogenous glucocorticoids (cortisol).
In his formulation of the glucocorticoid cascade hypothesis, Sapolsky48 has suggested that stress and repeated bouts of hypercortisolemia lead to downregulation of glucocorticoid receptors, which in turn results in further glucocorticoid hypersecretion, eventually leading to loss of hippocampal neurons, that is, a ‘glucocorticoid feedforward cascade’.48 His studies in rats have suggested that aging is a critical variable, and that aging rats showed downregulation of glucocorticoid receptors, failure to shut off stress-induced glucocorticoid secretion and hippocampal neuronal loss. Aging is also associated with HPA axis dysregulation in depression.49, 50, 51 We tested the hypothesis that the recurrent episodes of hypercortisolemia, occurring in recurrent depression, lead to progressive HPA axis dysregulation. We divided patients into first episode versus recurrent unipolar depression and examined differences in the rates of pituitary non-suppression. We found no effect of recurrent episodes but observed that, within both recurrent and first-episode subgroups, aging was associated with a higher incidence of HPA axis dysregulation. As 16 of the 20 subjects over 50 years were women, we cannot determine if aging is also a factor in men. However, in women, aging seemed to be a more important factor than the absolute number of episodes, and the previous analysis suggested that menopausal status was the critical variable in aging.
In summary, menopause itself is not associated with increases in plasma cortisol concentrations in depressed women, but it is associated with an increase in dexamethasone resistance. Resistance to dexamethasone suppression is strongly associated with an increased baseline cortisol secretion, in combination with age or menopause, and thus seems to reflect the development of glucocorticoid receptor resistance in the context of low estrogen.
Conclusions
Women are more likely to develop depression than men with the onset of depression beginning during puberty. They are also more likely than men to develop PTSD after exposure to trauma,52 despite similar rates of trauma exposure. Gender differences in exposure to trauma, in the number of significant life stressors experienced or in the perception of stress, do not explain gender differences in depression. Gender differences between the magnitude of the biological stress response also do not explain the increased sensitivity of women to the psychiatric sequelae of stress. However, it is clear that ovarian hormones do regulate the magnitude of the ACTH and cortisol responses to stressors. Furthermore, they strongly influence the HPA axis picture in depressed women. The interactions of organizational differences in female brains with cyclical gonadal steroid hormone changes after puberty, followed by menopause and the loss of these same steroids, suggest that stress responsiveness and susceptibility to stress-related disorders could vary substantially over the lifetime of women. Among patients with major depression, depressed women show greater HPA axis dysregulation than depressed men. There is certainly evidence that the increased vulnerability to depression in women arises at puberty, when gonadal steroids first begin to regularly influence the HPA axis of girls. Although this review focused on the HPA axis, ovarian steroids influence many other brain systems and all of these systems are exposed to cyclic changes in ovarian steroids on a monthly basis; the major increases in these steroids are followed by steep decreases with pregnancy and childbirth, and then finally loss of the steroid effects at menopause. It may be that the continually changing steroid milieu is the major factor sensitizing women to stress.
References
1.Kendler KS, Kessler RC, Walters EE, MacLean C, Neale MC, Heath AC et al. Stressful life events, genetic liability and onset of an episode of major depression in women. Am J Psychiatry 1995; 152: 833–842. PubMed ISI ChemPort
2.Brown GW, Harris T. Social Origins of Depression: A Study of Psychiatric Disorder in Women. Free Press: New York, 1978.
3.Frank E, Anderson B, Reynolds C, Ritenour A, Kupfer DJ. Life events and the research diagnostic criteria endogenous subtype: a confirmation of the distinction using the Bedford College methods. Arch Gen Psychiatry 1994; 51: 519–524. PubMed ChemPort
4.Nolen-Hoeksema S. Sex differences in unipolar depression: evidence and theory. Psychol Bull 1987; 101: 259–282. Article PubMed ChemPort
5.Kessler RC, McGonagle KA, Swartz M, Blazer DG, Nelson CB. Sex and depression in the National Comorbidity Survey. I: Lifetime prevalence, chronicity and recurrence. J Affect Disord 1993; 29: 85–96. Article PubMed ISI ChemPort
6.Angold A, Costello EJ, Erkanli A, Worthman CM. Pubertal changes in hormone levels and depression in girls. Psychol Med 1999; 29: 1043–1053. Article PubMed ChemPort
7.O'Hara MW. Social support, life events and depression during pregnancy and the puerperium. Arch Gen Psychiatry 1986; 43: 569–573. PubMed ChemPort

PREVENCION DEL SUICIDIO

Perspective
Molecular Psychiatry (2010) 15, 12–17;
The 2009 Nobel conference on the role of genetics in promoting suicide prevention and the mental health of the population
D Wasserman1, L Terenius2, J Wasserman1 and M Sokolowski1
1Department of Public Health Sciences, The National Prevention of Suicide and Mental Ill-Health (NASP), Karolinska Institute (KI), Stockholm, Sweden
2Department of Clinical Neuroscience, Center for Molecular Medicine (CMM), Karolinska Institute, Stockholm, Sweden
Correspondence: Professor D Wasserman, Department of Public Health Sciences, The National Prevention of Suicide and Mental Ill-Health (NASP), Karolinska Institute, Box 230, S-171 77 Stockholm, Sweden. E-mail: Danuta.Wasserman@ki.se

A 3-day Nobel Conference entitled ‘The role of genetics in promoting suicide prevention and the mental health of the population’ was held at the Nobel Forum, Karolinska Institute (KI) in Stockholm, Sweden, during 8–10 June 2009. The conference was sponsored by the Nobel Assembly for Physiology or Medicine and organized by the National Prevention for Suicide and Mental Ill-Health and the Center for Molecular Medicine at KI. The program consisted of 19 invited presentations, covering the genetic basis of mood/psychotic disorders and substance abuse in relation to suicide, with topics ranging from cellular-molecular mechanisms to (endo)phenotypes of mental disorders at the level of the individual and populations. Here, we provide an overview based on the highlights of what was presented.
Keywords: Nobel conference, genetics, suicide, mental health
Day 1
As introduced by Hans Jörnvall from Karolinska Institute (KI), former Secretary of the Nobel Assembly awarding the Nobel Prize in Medicine or Physiology, the avenue provided a needed opportunity to present both the current progress and future challenges and directions in the field. This statement turned out to be fulfilled during the coming 3 days. The first day provided a comprehensive overview of this field as a complex scientific endeavor, from the population and society perspectives, to the molecular genetic levels.

Sessions 1 ‘Prevention of mental ill-health and suicide, clinical and public health perspective’ and 2 ‘The family genetics of suicide’ (chaired by José Bertolote; Botucatu Medical School, Sao Paulo State University, Brazil)
These sessions dealt with the epidemiologic (Christina Hoven; Columbia University and NY State Psychiatric Institute, NY, USA) and clinical (Alan Apter; Sackler School of Medicine, Tel-Aviv, Israel) complexity of suicidal behavior (SB) and with family (David Brent; University of Pittsburgh, School of Medicine, USA) and twin (Nancy Pedersen; KI, Sweden) studies. The complexity creates difficulties in the clinic in determining which of the suicide attempters (SA) are at risk for death by completed suicide (SC). Together, the most consistent messages were that SB is a major and rising public health problem, seriously affecting the individual, family, and society (for example with the increasing SC rates in adolescents in Europe, and in adult SC related to, for example, financial stress),1 that suicide attempts (SA)/SC are distinct (as behaviors with intent to die) from both suicide ideation, which is widely spread both in the general population and in psychiatric patients, but not clearly associated with suicide mortality,2 as well as distinct from deliberate self-harm, in familial transmission.3 Furthermore, the psychiatric (endo)phenotypes of both impulsive aggression (externalizing) and mood disorders (MD; internalizing) are of importance in SA/SC, childhood abuse (CA; sexual or physical) appear to have essential roles on SB rates at the level of both populations and single families (indicating gene–environment interactions (GxEs) influencing heritability, being in the range of 20–50% for SA and SC, compared with ~70% for major depression) and important gender differences exist in relation to suicide (with SC mainly among the males), as is also found with these psychiatric endophenotypes.

Session 3 ‘The genetics of neurosystems in mental ill-health and suicidality’ (chaired by Vsevolod Rozanov; Odessa National Mechnikov University, Ukraine)
The beginning of the session (J John Mann; Columbia University and NY State Psychiatric Institute, NY, USA) focused on the genetics of the abnormal/‘stress-sensitized’ brain (in response to CA) in development of SB and depression, involving reciprocal links between the hypothalamic–pituitary–adrenal (HPA) and serotonergic (5HT) systems. The latter is the most extensively studied neurobiological aspect of suicide to date and results concerning 5HT transporter (SLC6A4) and TPH2 were shown. The progress of our understanding of genetics in SB and of the related psychiatric endophenotypes of, for example, MD, impulsivity and aggression, was shown to involve the combination of various biological endophenotypes; for SB being, for example, a dysregulated HPA-system (assayed by dexamethasone suppression test/cortisol levels), lowered 5-HIAA cerebrospinal fluid levels and lowered 5HTT binding in orbital (ventromedial) prefrontal cortex (OFC) in post-mortem brains of suicides, and lowered brain activity in the dorsolateral PFC (DLPFC)/anterior cingulate cortex (ACC), correlating with suicide intent, impulsivity and verbal fluency in SA.4 These results were related to the genetics of SB by association and linkage, for example SNPs in CRHR1 associating with the levels of salivary cortisol, and identification of a SNP causing production of a truncated TPH2-isoform, as identified by re-sequencing.5 On-going epigenetics studies used correlation between the exposure to CA, with methylation status and binding of H3K5Me3 to promotors by ChIP sequencing, in the Rhesus model.

The second speaker (Dan Rujescu; Ludwig Maximilians University, Germany) showed that while several other 5HT genes have also been shown to relate with aggression and suicide (for example TPH1, ABCG1, and COMT), non-5HT genes influencing the aggression-phenotype in, for example, knock-out mice (for example NOS1, TACR1, and DDC) have subsequently shown association to SB as well, motivating further gene search. Using a hypothesis-free approach, a GWAS in healthy subjects associated neuroticism with a neural cell adhesion gene involved in forebrain development, MDGA2.6 Furthermore, RNA expression arrays on post-mortem suicidal brains, revealed 124 genes being differentially expressed in suicide, in genomic locations that did not match with those of previously studied candidate genes.7 Gene ontology analyses showed that the genes were mainly categorized in roles of CNS development, homophilic cell adhesion, regulation of cell proliferation, and transmission of nerve impulse.7
Day 2
The substantial genetic overlap between different diagnoses is being increasingly demonstrated, and the conference would onward be dealing in various ways with the (heterogeneity of the) phenotype at different levels of observations or measurement, neurobiological functional complexity, and deterministic vs probabilistic, as well as hypothesis-free vs hypothesis-driven, approaches to studies of genes and genetic variants.

Session 1 ‘Influences of Genes in mental ill-health and suicide’ (chaired by Julio Licinio; University of Miami Health System, USA)
The day started by showing the results of a major GWAS of bipolar disorder (Peter McGuffin; King's College London, UK), implicating CACNA1C and ANK3 genes (involved in voltage-gated transmission), as well as the ‘usual suspects’ (DISC1, DTNBP1, NRG1, BDNF, and COMT),8 whereby the CACNA1C was recently showed having signals in also schizophrenia and bipolar disorder.9 Suicide severity was under investigation among these cases. The next talk (Danuta Wasserman; KI, Sweden) presented the results on a main regulator of the stress-responsive HPA-system, the CRHR1 candidate gene, of likely main importance in the impaired stress resilience of many psychiatric diseases. Although CRH circuits interact extensively with the function of all monoaminergic systems and CRHR1 antagonists are being evaluated for use as antidepressants, there are surprisingly few, but very consistent results, on the importance of CRHR1 variants in depression, treatment response, and SB. Association and linkage of several CRHR1 SNPs in the 5′-region was found, with depression intensity among males who had performed SA (used as main phenotype).10 One SNP was exonic and affected putative binding of SR proteins, indicating it could act by controlling alternative splicing, or by (alternative) transcriptional (start site) regulation of (unknown) CRHR1 transcripts. Third talk (Lars Terenius; KI, Sweden) showed results of the experimental search for molecular understanding of mu-opioid-type receptors (MORs), in reward and euphoria of alcohol use, the latter having substantial influence in, for example, aggression and SB. A novel explanatory hypothesis could be formulated by use of advanced fluorescence imaging of MOR plasma membrane lipid dynamics in live cells, showing that 20 mM of ethanol altered MOR mobility and surface density in a complex biphasic pattern, which could be counteracted by pre-treatment with antagonist Naltrexone, otherwise used for preventing relapse in alcoholism.11 He also showed statistics on mortality within 1 year of diagnosis, with equal frequency of SC in alcoholics and patients with endogenous depression. The final talk of the session (Gil Zalsman; Sackler School of Medicine, Tel-Aviv, Israel) reminded about the importance of events during the late development of children and adolescents.12 A GWAS on brains of suicides showed evidence for ROR1, CD44, FOXN3, DHX15, genes matching with certain gene ontology categories implicated by the group of D Rujescu et al. in gene expression arrays.7 However, such studies should also take into account the effects of exposure to CA and the gender differences in behavior, arising largely during puberty.
Session 2 ‘Genes and drug interplay in mental ill-health: treatments and pharmacogenetics’ (chaired by Lil Träskman-Bendz; University of Lund, Sweden)
Pharmacogenetics involves identifying and characterizing present and novel drug targets, and the session was marked by the search for larger and more specific genetic effects, either by studying rarer genetic variants, subgroups having a broader diagnose/condition, or by study in context of molecular and cellular functions as well as of intermediate brain function phenotypes, thus taking a step closer to deterministic, rather than probabilistic studies of psychiatric genetics.
The first talk (Gustavo Turecki; McGill University, Canada) showed the utility of genome-wide expression studies,13 here presenting a novel strategy for dealing with genetic heterogeneity by using an Extreme Value Analysis (EVA), examining genes in top 5% and with >3-fold change in gene expression over 17 different brain regions in subgroups of suicides. Results showed reduction in mainly astrocytic gap junction genes GJA1 and GJB6, and transcription factor SOX9, among ~20% of the suicides. Experiments showed the regulation by SOX9 by the promotors of GJA1 and GJB6, and also of several other genes from the EVA (NTRK2, SLC1A3 and –A2, and GLUL). Combined with the results of others,14 a model was proposed that deficient calcium currents might result in less astroglial glutamate release and stimulation, resulting in depressed mood.
The second talk (Daniel Weinberger; NIMH, USA) demonstrated how a translational approach, including the use of intermediate brain dysfunction phenotypes and functional molecular-cellular studies, can facilitate the understanding of the genetic effects in psychiatric diseases.15 One such example was that of frontal lobe cortical processing efficiency (relevant in schizophrenia), having an inverted U-shape relation with DA signaling, a target for antipsychotic treatment. The alterations of the DA synapse by, for example, functional alleles in genes COMT and DTNBP1, having large degrees of epistasis, are consistently explained in relation to disruptions of the optimal DA-signaling ratio. Other genes explored similarly in relation to this intermediate phenotype were GRM3 and a novel, brain- and primate-specific isoform of KCNH2.16 It was suggested that future studies must focus on detailed understanding of such molecular-genetic networks in relation to specific and measured brain system functions, typical of the diagnosis.17
The third and final presentation of the day (Hans-Jürgen Möller; talk given by D Rujescu; both from Ludwig Maximilians University, Germany) showed results from a study of the dynamic aspect of pharmacogenetics, in the attempt to explore why, for example, 25–40% of schizophrenic or depressed do not respond to the initial treatment. Candidate gene studies of antidepressant treatment response shown were those of ABRB1, COMT, MAOA, and MAOB, as well as the results of a recent meta-analysis.18 Single-gene associations have also been shown for DRD2, 3, and 4, GRM3, HRH1, HTR2A, −2C and −6, SLC6A4, COMT, MDR1, MTHFR, NEF3, NRG1 in treatment response of antipsychotics. A test of a larger array of SNPs in multiple (72) candidate genes found an oligogenic configuration of 23 genes that related to the time of recovery during haloperidol treatment among 70–85% of early responder (but not among non-responders). The talk ended by an overview of several recent large-scale association studies of schizophrenia, using either common SNPs or rarer CNVs (showing genetic overlap also with autism and mental retardation), for example the accumulation of multiple exon-deleting CNVs in NRX1 gene among schizophrenics (OR ~9).19 The presentation ended by a statement that both the study of common and rare variants show genetic overlap between different diagnoses, for example as recently demonstrated in GWAS-data for schizophrenia and bipolar disorder,20, 21 with an ~60% genetic overlap as shown in family studies.22
Day 3
The last day recapitulated several important aspects mentioned during the conference, with focus on cognitive endophenotypes, gene-environment interplay during childhood/adolescence and methodological approaches, which may guide current and future investigations.

Session 1 ‘Cognition and mental ill-health’ (chaired by Marco Sarchiapone; University of Molise, Italy)
The first speaker (Philippe Courtet; CHU Montpellier, France) showed alterations in activity of the lateral OFC (BA47, as well as BA6 and ACC, BA32) in relation to deficiencies in decision making and (socially) emotional stimuli (by combining Iowa Gambling Task, response to angry/neutral/happy faces and fMRI) among male SA, overlapping with regions previously shown to have 5HT dysfunction in suicides. Polymorphisms in 5HT genes (5HTT, TPH1 and -2, MAOA) correlated with the improvement in the latter phase of the test, that is with a deficiency in switching to more advantageous choices.23 Preliminary results also suggested that stress-response genes, mainly CRHR1, modulated the decision-making process. It was concluded that lateral OFC activity is a marker for impaired decision making in SB (maybe relating to valuation of future consequences), and that the further reciprocal involvements of ACC (for example in psychological pain processing to specific social stimuli), DLPFC (affecting, for example, impulsivity, intent, and verbal fluency) and amygdala was hypothesized accumulate in SB, particularly in combination with depressed mood (maladaptative response).
The second presentation (Jouko Lönnqvist; University of Helsinki, Finland) generally overviewed the use of psychological cognitive measures to refine the clinical diagnoses. Individuals may have impairments but not be diagnosed with disorder. Cognitive impairments can be confounded by medication and drug abuse. A continuum of decreasing cognitive functions is observed in a spectrum ranging from BPD to schizophrenia.24 The importance of optimal stress levels for cognitive functions can be described with an inverted U-shape curve. Memory is enhanced in relation to the stressful or emotionally arousing events, but if being very intense is causative of aversive memory traces, as observed in PTSD and anxiety disorders. Chronic/repeated stress can likewise have enduring effects on the brain structures involved in cognition, partly through the activation of HPA axis, and depend on the timing during life.25 Exposure in prenatal periods can cause negative programming effects in amygdala, hippocampus, and PFC. During childhood, the hippocampus is the most sensitive to CA, and can cause decreased or increased cortisol levels. In adolescence, the PFC is most vulnerable to extended cortisol response, whereas in adult, the effects are negative in relation to neurogenesis. Thus, interventions should start as early as possible to minimize such effects by environment.

Session 2 ‘Gene-environment interplay and mental ill-health’ (chaired by Avi Weizman; Sackler School of Medicine, Israel)
The roles of stress and environment was the next main focus, and the first speaker (Kenneth S Kendler; Virginia Commonwealth University, USA) provided an overview of epidemiological modeling of possible pathways from genes and environment, to the outcome of SB. Starting with models of direct (main) effects, this included the effects of axis I internalizing and externalizing psychiatric disorders and axis II personality disorders (characterized by general emotionality, impulsivity, and avoidant traits), as well as of childhood (sexual abuse, poor parenting, harsch discipline, and parental loss) and current (stressful life events) adversity, respectively. Further models were about the indirect effect of genes into selecting exposure to adverse environments (gene–environment correlations),26 as well as GxEs with current adversity (stressful life events), interactions between childhood and current adversity (environment–environment interactions), interaction between genes and culture (social acceptability affecting heritability), dynamic pathways from genes to environment and back again (bidirectional relationships between social selection and social influence), as well as the age dependency of GxEs (for example with GxEs causing alcohol use occurring mainly in ages 8–11 years, whereas GxEs were not detected in adults, showing stronger main effects of environment).
Variations in genes of the 5HT system are associated with depression and are subject to GxEs (Lars Oreland; Uppsala University, Sweden), as carriers of the S-allele of 5HTTLPR (and low-activity allele of MAO-A) have decreased CSF 5-HIAA levels among peer-reared Rhesus monkey's (but not mother-reared). The GxE of the 5HTTLPR is specific for females only, as males having the S-allele 5HTTLPR are stress-sensitized (high ACTH response) irrespective of the rearing conditions. Gender differences in the 5HT system have also been demonstrated in humans by various approaches, for example with gender-specific behavioral responses to acute tryptophan depletion, with opposite alleles of MAO-A showing GxEs with CA on antisocial behavior/criminality,27 as well as with changes in the structure/activity of the OFC shown by fMRI, specifically among male MAOA L-allele carriers, changes related to increased risk for impulsive violence.28 Gender differences in the 5HT system functioning may in part be caused by, for example, epigenetic modulation of MAOA, or by the regulation by estrogen. The influence of estrogen may relate to its crucial roles in the glucocorticoid receptor (GR)-dependent negative feedback of the HPA axis (HPA dysregulation being an endophenotype of, for example, SB), which may further involve life-long epigenetic reprogramming of GR (NR3C1) after exposure to CA, as found among male suicide victims.29
Session 3 ‘Methodology in Genetic Studies’ (chaired by Alec Roy; New Jersey Medical School, USA)
The last session suggested solutions to current challenges in the field. The road ahead (David Goldman; NIAAA/NIH, USA) must deal with exploring the remaining unknown ~90% of the genetic variation, the ‘dark matter,’ attributing to the variance of complex diseases. Strategies suggested were to refine the phenotypes (to study measurable intermediate brain phenotypes in more homogenous samples), to study genetics in better contexts (in relation to the common denominator of stress in all psychiatric diseases, for example CA-exposure, or by detailed study the effects of functional alleles in explaining identified brain intermediate phenotype, for example the inverted U-shape function of cognitive performance and DA signaling),15 in relation to epigenetics (investigating histone-binding combined with methylation-status and allelic variants), and to find the rare variants that co-segregate by using (massive parallel) re-sequencing. Examples of these approaches were the identification of a functional SNP in Neuropeptide Y (NPY), which showed reducing penetrance on the continuum from molecular (gene expression), intermediate brain function (for example emotion-induced activation in fMRI and activation of the μ-opioid system in brain areas processing pain), to behavioral phenotypes (for example trait anxiety).30 Another study used re-sequencing of genes in Finns, found a rare SNP introducing a stop codon in HTR2B (Q20*), which co-segregated in the families with extreme dyscontrol behaviors, for example extremely impulsive/violent criminal offences and SA.
The final talk (Jessica Lasky-Su; Harvard School of Public Health, USA) presented the set of tools to be used for association studies using families (parent-offspring units, that is trios).31 The tools provided in these Family Based Association Tests (FBATs) for the ‘GWAS-era,’ can be used to, for example, increase the signal-to-noise and to control for multiple testing by an estimated power-ranking screening procedure, as well as increasing the heritability of the phenotype with FBAT-PC (principal components) by combining multiple measures of a phenotype. It was suggested that the results from GWAS should primarily be judged in terms of how robust the signals are, for example signals from multiple SNPs from the same gene (as well as the convergence across multiple related measures or intermediate phenotypes). Furthermore, genetic effect associations can be dependent on the age (of onset).
Summary
In general, although much work remains to be done before it will be possible to implement the present genetic findings into clinical benefits for patients and the society at large, the basis of exploration and future potential have been firmly established. The complex genetic causality appears to involve a combination of (often within the same gene) both common variants of usually smaller effects, of which many are by now known and included in, for example, GWAS, as well as many rare variants with often larger phenotypic effects, being specific for a smaller subset of cases. As rarer variants become more easily identified and screened for, by the further development of sequencing technologies, it will likely shift the main focus of field from probabilistic investigations in relation to psychiatric phenomena (that is symptoms and diagnoses), into a mechanistic understanding of underlying brain system dysfunction, understood as propagating events acting from molecular, cellular, and neurosystems, to behavioral levels. Such understanding will also help to resolve how, for example, gene expression changes, by a combination of alleles and epialleles, resulting in profound phenotypic effects, by occurring in a certain genetic background and in localized areas of specific cell types. Detailed investigations of the structure and function of complex biological mechanisms, which often also involve epistasis, can nevertheless be understood in context of networks of genes acting at specific neurobiological pathways, sometimes leading to observable symptoms, thus presenting novel opportunities for truly personalized medicine, such as treatments by targeting of genetic propensities, directly or indirectly. The path taken can well result in the removal of many nosological boundaries of current diagnoses based on symptoms, to be replaced by a classification based on constellations of genetic markers, brain function intermediate phenotypes (at both molecular, cellular, and neurosystems levels), assays of past stressful exposures to epigenetic imprints, and outcomes of appropriate psychometric tests. The Conference was characterized by vivid and extremely stimulating discussions, a generous exchange of information and reflections, and the initiation of novel, future collaborations.
References
1.Hoven C, Wasserman D, Wasserman C, Mandell D. Awareness in nine countries: a public health approach to suicide prevention. Leg Med 2009; 11: 13–17. Article
2.Brent DA, Mann JJ. Family genetic studies, suicide, and suicidal behavior. Am J Med Genet C Semin Med Genet 2005; 133C: 13–24.
3.Bertolote JM, Fleischmann A, De Leo D, Wasserman D. Suicidal thoughts, suicide plans, and attempts in the general population on different continents. In: Wasserman D, Wasserman C (eds). Suicidology and Suicide Prevention: A Global Perspective. Oxford University Press: Oxford, UK, 2009, pp 99–104.
4.Mann JJ, Arango VA, Avenevoli S, Brent DA, Champagne FA, Clayton P et al. Candidate endophenotypes for genetic studies of suicidal behavior. Biol Psychiatry 2009; 65: 556–563. Article PubMed ChemPort
5.Haghighi F, Bach-Mizrachi H, Huang YY, Arango V, Shi S, Dwork AJ et al. Genetic architecture of the human tryptophan hydroxylase 2 Gene: existence of neural isoforms and relevance for major depression. Mol Psychiatry 2008; 13: 813–820. Article PubMed ChemPort
6.van den Oord EJ, Kuo PH, Hartmann AM, Webb BT, Moller HJ, Hettema JM et al. Genomewide association analysis followed by a replication study implicates a novel candidate gene for neuroticism. Arch Gen Psychiatry 2008; 65: 1062–1071. Article PubMed
7.Thalmeier A, Dickmann M, Giegling I, Schneider B, Hartmann MA, Maurer K et al. Gene expression profiling of post-mortem orbitofrontal cortex in violent suicide victims. Int J Neuropsychopharmacol 2008; 11: 217–228. Article PubMed ChemPort

EDUCACION SEXUAL O EDUCACION DE LA SEXUALIDAD

Educación sexual o educación de la sexualidad
Por: Francisco Delfín Lara *,

Miércoles, 25 de Noviembre de 2009
Esta es una problemática que involucra a varios actores y que afecta a la sociedad entera
GUANAJUATO
Antes de entrar de lleno al tema vale la pena revisar algunos conceptos que nos permitirán una mejor comprensión.
EN EL ARTE
En el arte Les amants René Magritte 1898-1967 Bruselas colección privada

Sexo: Se trata de un concepto biológico, se refiere a la serie de características físicas que permiten establecer las diferencias entre machos y hembras de la misma especie.
Ha animales en los cuales es fácil distinguir al macho de la hembra por ejemplo: león y leona; el gallo y la gallina. Existen otros en los cuales resulta difícil distinguir, a simple vista al macho de la hembra como en los pericos africanos.
Los seres humanos nos encontramos entre ambos grupos.
Género: Este concepto es nuevo y de orden sociocultural. Según la Organización Panamericana de la Salud y la Organización Mundial de la Salud (OPS/OMS, 2000) significa: Suma de valores y actitudes, prácticas o características culturales basadas en el sexo . Es decir, el comportamiento de los seres humanos no está biológicamente determinado son las sociedades las que lo moldean de acuerdo a sus gustos, necesidades o caprichos.
Papeles genéricos: comportamientos específicos que las sociedades esperan adopte el individuo por el hecho de ser hombre o mujer; su no cumplimiento se sanciona de diversas formas . Se nos ha educado para que ellas barran, cocinen y cuiden a los niños; en cambio ellos deben salir a trabajar y si no colaboran en los quehaceres domésticos ni modo, son hombres.
Querámoslo o no el trato desigual entre varones y féminas sigue vigente.
Erotismo: Capacidad humana de experimentar la excitación sexual e incluso llegar al orgasmo a través de los recuerdos, imaginación y fantasía, por supuesto los contactos físicos también caben en esta definición .
Orientación sexual : organización específica del erotismo o vínculo emocional en relación al sexo de la persona involucrada o por quien se siente atracción (OPS/OMS, 2000).
Cuando
Es hora de tomar ‘el toro por los cuernos’ e involucrarnos todos, Estado y padres de familia en una educación sexual completael individuo es atraído por personas del mismo sexo se dice que es Homosexual , si le atraen personas del otro sexo es Heterosexual y si le atraen personas de uno u otro sexo es Bisexual . Desde hace más de 30 años, la Asociación Psiquiátrica Americana y junto con ella las asociaciones serias y de reconocido prestigio internacional señalaron que ninguna de las tres orientaciones antes mencionadas son patológicas, es decir, no son enfermedades, ni perversiones ni aberraciones.
Cuando alguien, frunciendo el ceño y cerrando los puños se refiere a "los perversos homosexuales" podemos estar seguros que habla en base a sus prejuicios y no desde una perspectiva científica.
Salud sexual : es la experiencia del proceso permanente de consecución de bienestar físico, psicológico y sociocultural relacionado con la sexualidad (OPS/OMS, 2000).
Lo interesante de esta definición es que no se restringe a la ausencia de enfermedades y se desliga de la reproducción porque la gente responde que tiene relaciones sexuales por: deseo, amor, compromiso, negocio, obligación, por cumplir, aburrimiento pero casi nadie las tiene porque desea reproducirse.
¿ Recuerda usted cuándo fue la vez más reciente que hizo el amor con el específico y exclusivo deseo de reproducirse ?
Masturbación: Una gran cantidad de investigadores asegura que el presente término proviene del latín: Manus strupare , lo cual se traduce como: la mano que daña, profana, ensucia o mancha .
En concreto tiene una connotación negativa. Sin embargo y de acuerdo con la mayoría de las investigaciones sobre sexualidad realizadas en varios países, se trata de una actividad practicada por la mayoría de los varones y más de la mitad de las mujeres; a lo largo de la vida; con independencia del estado civil; que no daña y que en la actualidad es una opción en el ámbito de la terapia sexual.
Sabías que...
En Canadá, en donde la educación sexual forma parte de una campaña sistemática del Estado, el índice de embarazos en adolescentes es la mitad del que se registra en México.
Educación de la sexualidad : Desde hace años, en todo el mundo, se ha expresado una honda preocupación por el tema de la sexualidad, sobre todo por lo que toca a la infancia y la juventud.
Los primeros programas formales surgieron en Escandinavia y de ahí, de manera gradual llegaron a diferentes naciones.
En Estados Unidos de Norteamérica se han usado varios modelos entre los que destacan:
Educación sexual basada en la abstinencia : considera que hablar de sexualidad hará que los jóvenes se lancen a una vida sexual desenfrenada.
Durante los gobiernos de George W. Bush recibió enormes financiamientos. Dicen que tener relaciones prematrimoniales ocasiona daños físicos y psicológicos, lo cual es falso.
Insisten en tener relaciones sexuales hasta después de casarse pero no se hablaba de métodos anticonceptivos o si los nombraban es para decir que, su uso resulta peligroso.
Al comprobar que los embarazos en adolescentes seguían aumentando obligaron a las y los jóvenes a firmar un documento donde se comprometían a tener relaciones sexuales hasta después del matrimonio; pese a ello chicas y chicos siguieron teniendo relaciones sexuales a edades tempranas pero, sin protegerse porque desconocían el uso de anticonceptivos y el condón.
Educación sexual integral : en este modelo se sugiere dar información sobre la sexualidad a los jóvenes; se les anima a tomar decisiones de forma responsable.
Se enfatiza que la abstinencia es una estrategia muy segura pero se les enseña cómo prevenir: embarazos no deseados e infecciones de transmisión sexual.
A últimas fechas se ha comprobado que esta estrategia es la más adecuada porque la juventud estadounidense tiene tasas de embarazo más alto que otros países de Europa y Asia.
Es hora de tomar el toro por los cuernos , los padres debemos educar desde la más tierna infancia pero no olvidar que lo hacemos con el ejemplo.
Progenitores autoritarios y que todo prohíben sobran pero, hacen falta madres y padres que brinden confianza y escuchen a sus hijos, les apoyen, respeten sus derechos y les animen a responsabilizarse de sus actos.
La escuela y el profesorado requiere preparación actualizada pero por encima de todo comprometerse a respetar al alumnado para que de ese modo coadyuven a formar mejores seres humanos.
* Médico cirujano UNAM. Sexólogo educador: Grupo Interdisciplinario de Sexología e Instituto Mexicano de Sexología. Miembro fundador de Profesionistas en Psicoterapia Sexual Integral A. C. Autor del libro: Sex populi , Editorial Alfil.

BASES MOLECULARES DEL CANCER COLORECTAL

NEW ENG J MED,Volume 361:2449-2460 December 17, 2009 Number 25
Molecular Basis of Colorectal Cancer
Sanford D. Markowitz, M.D., Ph.D., and Monica M. Bertagnolli, M.D.

Every year in the United States, 160,000 cases of colorectal cancer are diagnosed, and 57,000 patients die of the disease, making it the second leading cause of death from cancer among adults.1 The disease begins as a benign adenomatous polyp, which develops into an advanced adenoma with high-grade dysplasia and then progresses to an invasive cancer.2 Invasive cancers that are confined within the wall of the colon (tumor–node–metastasis stages I and II) are curable, but if untreated, they spread to regional lymph nodes (stage III) and then metastasize to distant sites (stage IV).3,4,5 Stage I and II tumors are curable by surgical excision, and up to 73% of cases of stage III disease are curable by surgery combined with adjuvant chemotherapy.3,4,6 Recent advances in chemotherapy have improved survival, but stage IV disease is usually incurable.3,4
The clinical behavior of a colorectal cancer results from interactions at many levels (Figure 1). The challenges are to understand the molecular basis of individual susceptibility to colorectal cancer and to determine factors that initiate the development of the tumor, drive its progression, and determine its responsiveness or resistance to antitumor agents. This review summarizes areas of current knowledge, recognizing that the topics presented are only fragments of the total picture.
Figure 1. Multifactorial Colorectal Carcinogenesis.
The molecular events that drive the initiation, promotion, and progression of colorectal cancer occur on many interrelated levels. This dynamic process involves interactions among environmental influences, germ-line factors dictating individual cancer susceptibility, and accumulated somatic changes in the colorectal epithelium.
Genomic Instability
The loss of genomic stability can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations. In this disease, genomic instability takes several forms, each with a different cause (Table 1).7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26
Chromosomal Instability
The most common type of genomic instability in colorectal cancer is chromosomal instability, which causes numerous changes in chromosomal copy number and structure.7 Chromosomal instability is an efficient mechanism for causing the physical loss of a wild-type copy of a tumor-suppressor gene, such as APC, P53, and SMAD family member 4 (SMAD4), whose normal activities oppose the malignant phenotype.2,27,28 In colorectal cancer, there are numerous rare inactivating mutations of genes whose normal function is to maintain chromosomal stability during replication, and in the aggregate, these mutations account for most of the chromosomal instability in such tumors.8 In contrast to some other cancers, colorectal cancer does not commonly involve amplification of gene copy number29 or gene rearrangement.
DNA-Repair Defects
In a subgroup of patients with colorectal cancer, there is inactivation of genes required for repair of base–base mismatches in DNA, collectively referred to as mismatch-repair genes (Figure 2 and Figure 3). The inactivation can be inherited, as in hereditary nonpolyposis colon cancer (HNPCC), also known as the Lynch syndrome, or acquired, as in tumors with methylation-associated silencing of a gene that encodes a DNA mismatch-repair protein.
Figure 2. Genes and Growth Factor Pathways That Drive the Progression of Colorectal Cancer.
In the progression of colon cancer, genetic alterations target the genes that are identified at the top of the diagram. The microsatellite instability (MSI) pathway is initiated by mismatch-repair (MMR) gene mutation or by aberrant MLH1 methylation and is further associated with downstream mutations in TGFBR2 and BAX. Aberrant MLH1 methylation and BRAF mutation are each associated with the serrated-adenoma pathway. The question mark indicates that genetic or epigenetic changes specific to metastatic progression have not been identified. Key growth factor pathways that are altered during colon neoplasia are shown at the bottom of the diagram. CIN denotes chromosomal instability, EGFR epidermal growth factor receptor, 15-PGDH 15-prostaglandin dehydrogenase, and TGF-β transforming growth factor β.
Figure 3. Genetic Instability Pathways That Drive Colon Neoplasias.
Shown are the overlapping relationships that define the major pathways of genomic instability in colon cancers: chromosomal instability, microsatellite instability caused by defects in DNA mismatch-repair genes that are either inherited as germ-line defects (e.g., in hereditary nonpolyposis colon cancer) or somatically acquired (e.g., by aberrant methylation and epigenetic silencing of MLH1), and the CpG island methylator phenotype.
In patients with HNPCC, germ-line defects in mismatch-repair genes (primarily MLH1 and MSH2) confer a lifetime risk of colorectal cancer of about 80%, with cancers evident by the age of 45 years, on average.10,11,12,13,30,31 The loss of mismatch-repair function in patients with HNPCC is due not only to the mutant germ-line mismatch-repair gene but also to somatic inactivation of the wild-type parental allele.31 Genomic instability arising from mismatch-repair deficiency dramatically accelerates the development of cancer in patients with HNPCC — some cancers arise within 36 months after normal results on colonoscopy.32 For this reason, yearly colonoscopy is recommended for carriers of an HNPCC mutation,30,32 and prophylactic colectomy should be considered for patients with high-grade lesions. Germ-line mutations of another mismatch-repair gene, MSH6, attenuates the predisposition to familial cancer.9,33,34 Somatic inactivation of mismatch-repair genes occurs in approximately 15% of patients with nonfamilial colorectal cancer. In these patients, biallelic silencing of the promoter region of the MLH1 gene by promoter methylation inactivates mismatch repair15,16,17 (Figure 2 and Figure 3).
The loss of mismatch-repair function is easy to recognize by the associated epiphenomenon of microsatellite instability, in which the inability to repair strand slippage within repetitive DNA sequence elements changes the size of the mononucleotide or dinucleotide repeats (microsatellites) that are scattered throughout the genome. Mismatch-repair deficiency can also be detected by immunohistochemical analysis, which can identify the loss of one of the mismatch-repair proteins.14,35,36,37 Cancers characterized by mismatch-repair deficiency arise primarily in the proximal colon, and in sporadic cases, they are associated with older age and female sex.30 In mismatch-repair deficiency, tumor-suppressor genes, such as those encoding transforming growth factor β (TGF-β) receptor type II (TGFBR2) and BCL2-associated X protein (BAX), which have functional regions that contain mononucleotide or dinucleotide repeat sequences, can be inactivated.2,27,28
An alternative route to colorectal cancer involves germ-line inactivation of a base excision repair gene, mutY homologue (MUTYH, also called MYH).25,33 The MYH protein excises from DNA the 8-oxoguanine product of oxidative damage to guanine.24,25,33 In persons who carry two inactive germ-line MYH alleles, a polyposis phenotype develops, with a risk of colorectal cancer of nearly 100% by the age of 60 years.33 MYH-associated polyposis is increasingly recognized: one third of all persons in whom 15 or more colorectal adenomas develop have MYH-associated polyposis.33 The diagnosis requires genetic testing, which is facilitated by two mutations, Y165C and G382D, that together account for 85% of cases.33 Thus far, somatic inactivation of MYH has not been detected in colorectal cancer.
Aberrant DNA Methylation
Epigenetic silencing of genes, mostly mediated by aberrant DNA methylation, is another mechanism of gene inactivation in patients with colorectal cancer.18,20 A methylated form of cytosine in which a methyl group is attached to carbon 5 (5-methylcytosine) defines a fifth DNA base, introduced by DNA methylases that modify cytosines within CpG dinucleotides.18 In the normal genome, cytosine methylation occurs in areas of repetitive DNA sequences outside of exons; it is largely excluded from the CpG-rich "CpG islands" in the promoter regions of approximately half of all genes.18 By comparison, in the colorectal-cancer genome, there is a modest global depletion of cytosine methylation but considerable acquisition of aberrant methylation within certain promoter-associated CpG islands.18 This aberrant promoter-associated methylation can induce epigenetic silencing of gene expression.18 In sporadic colorectal cancer with microsatellite instability, somatic epigenetic silencing blocks the expression of MLH1.18
Among the loci that can undergo aberrant methylation in colorectal cancer, a subgroup seems to become aberrantly methylated as a group, a phenomenon called the CpG island methylator phenotype (CIMP, or CIMP-high).18,19 The molecular mechanism for CIMP remains unknown, but the phenomenon is reproducibly observed in about 15% of colorectal cancers and is present in nearly all such tumors with aberrant methylation of MLH118,19,21,38 (Figure 2 and Figure 3). The pathogenetic consequence of MLH1 silencing is well established, but the contribution of other epigenetic silencing events to colorectal carcinogenesis remains an area of ongoing study. An intermediate level of aberrant methylation in CIMP may define a subtype (i.e., CIMP2 and CIMP-low) that is thought to account for 30% of CIMP cases.22,23 A third pattern of aberrant methylation is exemplified by exon 1 of the gene encoding vimentin. Although this locus is not expressed by normal colon mucosa or colorectal cancer, it is aberrantly methylated in 53 to 83% of patients with colorectal cancer in a pattern that is independent of CIMP.39,40
Mutational Inactivation of Tumor-Suppressor Genes
APC
Colorectal cancers acquire many genetic changes, but certain signaling pathways are clearly singled out as key factors in tumor formation (Figure 2 and Table 2).41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62 One of these changes, the activation of the Wnt signaling pathway, is regarded as the initiating event in colorectal cancer.2,28,43 Wnt signaling occurs when the oncoprotein β-catenin binds to nuclear partners (members of the T-cell factor–lymphocyte enhancer factor family) to create a transcription factor that regulates genes involved in cellular activation.2,28,43 The β-catenin degradation complex controls levels of the β-catenin protein by proteolysis. A component of this complex, APC, not only degrades β-catenin but also inhibits its nuclear localization.
Table 2. Tumor-Suppressor Genes and Oncogenes Commonly Associated with Colorectal Cancer.
The most common mutation in colorectal cancer inactivates the gene that encodes the APC protein. In the absence of functional APC — the brake on β-catenin — Wnt signaling is inappropriately and constitutively activated. Germ-line APC mutations give rise to familial adenomatous polyposis, an inherited cancer-predisposition syndrome in which more than 100 adenomatous polyps can develop; in carriers of the mutant gene, the risk of colorectal cancer by the age of 40 years is almost 100%.2,30,43 Somatic mutations and deletions that inactivate both copies of APC are present in most sporadic colorectal adenomas and cancers.2,43 In a small subgroup of tumors with wild-type APC, mutations of β-catenin that render the protein resistant to the β-catenin degradation complex activate Wnt signaling.2,41,42,43
TP53
The inactivation of the p53 pathway by mutation of TP53 is the second key genetic step in colorectal cancer. In most tumors, the two TP53 alleles are inactivated, usually by a combination of a missense mutation that inactivates the transcriptional activity of p53 and a 17p chromosomal deletion that eliminates the second TP53 allele.2,27,28,44,45 Wild-type p53 mediates cell-cycle arrest and a cell-death checkpoint, which can be activated by multiple cellular stresses.63 The inactivation of TP53 often coincides with the transition of large adenomas into invasive carcinomas.64 In many colorectal cancers with mismatch-repair defects, TP53 remains wild-type, though in these cancers the activity of the p53 pathway is probably attenuated by mutations in the BAX inducer of apoptosis.2,28
TGF-β Tumor-Suppressor Pathway
The mutational inactivation of TGF-β signaling is a third step in the progression to colorectal cancer.50 In about one third of colorectal cancers, somatic mutations inactivate TGFBR2.47,49,50,65,66 In tumors with mismatch-repair defects, TGFBR2 is inactivated by distinctive frameshift mutations in a polyadenine repeat within the TGFBR2 coding sequence.47 In at least half of all colorectal cancers with wild-type mismatch repair, TGF-β signaling is abolished by inactivating missense mutations that affect the TGFBR2 kinase domain or, more commonly, mutations and deletions that inactivate the downstream TGF-β pathway component SMAD4 or its partner transcription factors, SMAD2 and SMAD3.29,47,49,50,51,65,66,67,68 Mutations that inactivate the TGF-β pathway coincide with the transition from adenoma to high-grade dysplasia or carcinoma.69
Activation of Oncogene Pathways
RAS and BRAF
Several oncogenes play key roles in promoting colorectal cancer (Figure 2 and Table 2). Oncogenic mutations of RAS and BRAF, which activate the mitogen-activated protein kinase (MAPK) signaling pathway, occur in 37% and 13% of colorectal cancers, respectively.21,55,57,70,71 RAS mutations, principally in KRAS, activate the GTPase activity that signals directly to RAF. BRAF mutations signal BRAF serine–threonine kinase activity, which further drives the MAPK signaling cascade.70,71 BRAF mutations are detectable even in small polyps,21 and as compared with RAS mutations, they are more common in hyperplastic polyps, serrated adenomas, and proximal colon cancers, particularly in those with the CIMP phenotype (Figure 3). Patients with numerous and large hyperplastic lesions, a condition termed the hyperplastic polyposis syndrome, have an increased risk of colorectal cancer, with disease progression occurring through an intermediate lesion with a serrated luminal border on histologic analysis.18,22,38,58,59
Phosphatidylinositol 3-Kinase
One third of colorectal cancers bear activating somatic mutations in PI3KCA, which encodes the catalytic subunit of phosphatidylinositol 3-kinase (PI3K).72 Less common genetic alterations that may substitute for PI3KCA mutations include loss of PTEN, an inhibitor of PI3K signaling, as well as amplification of insulin receptor substrate 2 (IRS2), an upstream activator of PI3K signaling, and coamplification of AKT and PAK4, which are downstream mediators of PI3K signaling.73
Sequencing the Colorectal-Cancer Genome
Advances in DNA sequencing technology have made it possible to sequence the entire coding genome of a human cancer. Colorectal cancer provided the first example of the power of this technology, with high-throughput sequencing of 18,000 members of the Reference Sequence (RefSeq) database of the National Center for Biotechnology Information.65,66 Cancer-associated somatic mutations were identified in 848 genes. Of these, 140 were identified as candidate cancer genes that probably contributed to the cancer phenotype because they were mutated in at least two colorectal cancers and when corrected for gene size showed more mutations than expected by chance.
The average stage IV colorectal-cancer genome bears 15 mutated candidate cancer genes and 61 mutated passenger genes (very-low-frequency mutational events). The predominance of low-frequency mutations in candidate cancer genes implies enormous genetic heterogeneity among colorectal cancers, which mirrors the heterogeneity of the clinical behavior of colorectal cancers. The high degree of genetic heterogeneity makes it difficult to determine the clinical effect of individual mutational events. Moreover, these initial results are probably conservative, because some mutations, which were initially labeled as rare "passengers" in colorectal cancer, have subsequently emerged as common and are probably pathogenetic in other cancer types (e.g., an IDH1 mutation noted initially in one colorectal cancer but subsequently in many gliomas).65,66,74
High-throughput sequencing of the colorectal-cancer genome has identified new common mutational targets. These include the ephrin receptors EPHA3 and EPHB6 (receptor tyrosine kinases), which together are mutated in 20% of colorectal cancers, and FBXW7, which functions in a protein degradation pathway that regulates levels of cyclin E and is mutated in 14% of colorectal cancers.65,66,75 An important challenge is to reduce the complexity of the 140 candidate cancer genes by identifying the biologic pathways and processes that are common downstream targets of multiple mutational events.
Genomic Changes and Tumor Progression
The sequence of transformation from adenoma to carcinoma, as initially formulated,2,28,43 was a model of the development of colorectal cancer in which specific tumor-promoting mutations are progressively acquired. This model predicts the presence of mutations that dictate specific tumor characteristics, such as the presence of regional or distant metastases (Figure 2). Unexpectedly, the examination of results of full-genome sequencing from primary colorectal cancers and distant metastases in the same patient showed no new mutations in the metastases,76 implying that new mutations are not required to enable a tumor cell to leave the primary tumor and seed a distant site. Because the ongoing generation of mutations serves as a molecular clock, the finding that all the mutations in a metastasis are also present in the primary tumor implies that metastatic seeding is rapid, requiring less than 24 months from the appearance of the final mutation in the primary tumor.76
Growth Factor Pathways
Aberrant Regulation of Prostaglandin Signaling
The activation of growth factor pathways is common in colorectal cancer (Figure 2). An early and critical step in the development of an adenoma is the activation of prostaglandin signaling.77,78 This abnormal response can be induced by inflammation or mitogen-associated up-regulation of COX-2, an inducible enzyme that mediates the synthesis of prostaglandin E2, an agent strongly associated with colorectal cancer.78 Prostaglandin E2 activity can also be increased by the loss of 15-prostaglandin dehydrogenase (15-PGDH), the rate-limiting enzyme in catalyzing degradation of prostaglandin.79,80,81 Increased levels of COX-2 are found in approximately two thirds of colorectal cancers,78,82 and there is loss of 15-PGDH in 80% of colorectal adenomas and cancers.79 Clinical trials have shown that the inhibition of COX-2 by nonsteroidal antiinflammatory drugs prevents the development of new adenomas83,84,85,86 and mediates regression of established adenomas.87
Epidermal Growth Factor Receptor
Epidermal growth factor (EGF) is a soluble protein that has trophic effects on intestinal cells. Clinical studies have supported an important role of signaling through the EGF receptor (EGFR) in a subgroup of colorectal cancers.88,89,90,91 EGFR mediates signaling by activating the MAPK and PI3K signaling cascades. Recent clinical data have shown that advanced colorectal cancer with tumor-promoting mutations of these pathways — including activating mutations in KRAS,92,93,94 BRAF,95,96 and the p110 subunit of PI3K97 — do not respond to anti-EGFR therapy.
Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) that is produced in states of injury or during the growth of normal tissue drives the production of new stromal blood vessels (angiogenesis). Clinical studies have suggested a role for angiogenic pathways in the growth and lethal potential of colorectal cancer. Treatment with the anti-VEGF antibody bevacizumab added an average of 4.7 months to the overall survival of patients with advanced colorectal cancer (15.6 months with standard therapy).98 The identification of molecular distinctions between cancers that benefit from this treatment and those that do not remains a challenge.
Stem-Cell Pathways
Stem cells in colorectal cancers are believed to be uniquely endowed with the capacity to renew themselves.99,100,101,102 Single colorectal-cancer stem cells, by definition, can lodge in a permissive site, such as the liver, and produce a metastasis. Currently, it is not possible to isolate individual colorectal-cancer stem cells, although certain cell-surface proteins (e.g., CD133, CD44, CD166, and aldehyde dehydrogenase 1) are promising markers. Normal stem cells that reside in the colonic crypt rely on adhesive and soluble stromal–epithelial interactions to maintain division and differentiation. The extent of alterations in these regulatory mechanisms in colorectal-cancer stem cells is a promising area of investigation, since agents that control the growth of colorectal-cancer stem cells could theoretically be used for cancer prevention and treatment.
Predictive and Prognostic Markers
One ongoing challenge is to translate the wealth of knowledge regarding colorectal-cancer genomics into clinically applicable predictive or prognostic tests (Table 3). The relation between mutations in EGFR signaling components RAS and BRAF and anti-EGFR therapy is currently the only application of colorectal-cancer genomics to treatment.92,93,94,95,96 A few genomic markers are useful for prognosis. For example, germ-line mutations in tumor-suppressor genes, such as APC, MLH1, and MSH2, indicate a very high risk of colorectal cancer and guide the frequency of colorectal-cancer surveillance and recommendations for prophylactic surgery. Other somatic markers have modest or unconfirmed prognostic value and are not currently used to direct care. Sporadic colorectal cancers with a mismatch-repair deficiency generally have a favorable prognosis35,103,105,108; poor survival in stage II and III colon cancers is associated with the loss of p27 (a proapoptotic regulator of the cell cycle109) or the loss of heterozygosity at chromosomal location 18q.105
Table 3. Prognostic and Predictive DNA Markers in Colorectal Cancer.
Noninvasive Molecular Detection
The development of molecular diagnostics for the early detection of colorectal cancer is an important translation of colon-cancer genetics into clinical practice. One example is the development of assays to detect mutations that are specific to colorectal cancer and cancer-associated aberrant DNA methylation in fecal DNA from patients with colorectal cancer or advanced adenomas. These assays have a sensitivity of 46 to 77% for detecting early-stage colorectal cancer, which is superior to the sensitivity of testing for fecal occult blood although their superiority in preventing death from cancer has not been shown.39,110,111,112,113 Stool DNA testing for colorectal cancer has been added to the cancer-screening guidelines of the American Cancer Society114 and appears to be equally sensitive for detecting advanced adenomas.115 Although still in the developmental stage, assays for detecting plasma cell-free DNA may also be clinically useful,115 and assays for tumor-specific plasma protein or RNA profiles also remain targets of research. Questions that remain to be resolved are the optimal interval between serial tests and the performance and cost-effectiveness of stool DNA testing as compared with those of newer immunochemical fecal occult-blood tests.116
Genetic Influences in Population Susceptibility
Genetic epidemiology and twin studies indicate that 35 to 100% of colorectal cancers and adenomas develop in persons with an inherited susceptibility to the disease.117,118,119 In addition, an HNPCC-like syndrome occurs in some families without any evidence of defects in mismatch repair.120 Several genomic loci that could harbor highly penetrant susceptibility genes have been identified with the use of linkage approaches,121,122,123 but the underlying mutations are unknown. Genomewide association studies have also identified germ-line DNA variants that are strongly associated with susceptibility, but the clinical use of these results is probably limited, since the relative risk associated with these variants is low.124,125,126,127,128,129
Conclusions
Studies that aid in the understanding of colorectal cancer on a molecular level have provided important tools for genetic testing for high-risk familial forms of the disease, predictive markers for selecting patients for certain classes of drug therapies, and molecular diagnostics for the noninvasive detection of early cancers. In addition, biologic pathways that could form the basis of new therapeutic agents have been identified. Although some high-frequency mutations are attractive targets for drug development, common signaling pathways downstream from these mutations may also be tractable as therapeutic targets. Recent progress in molecular assays for the early detection of colorectal cancer indicates that understanding the genes and pathways that control the earliest steps of the disease and individual susceptibility can contribute to clinical management in the near term.
An understanding of the signals that dictate the metastatic phenotype will provide the information necessary to develop drugs to control or prevent advanced disease. The considerable recent advances encourage us to believe that improvements in our knowledge of the molecular basis of colorectal cancer will continue to reduce the burden of this disease.
References
Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2008. CA Cancer J Clin 2008;58:71-96. [Free Full Text]
Kinzler KW, Vogelstein B. Colorectal tumors. In: Vogelstein B, Kinzler KW, eds. The genetic basis of human cancer. 2nd ed. New York: McGraw-Hill, 2002:583-612.
Libutti SK, Saltz LB, Tepper JE. Colon cancer. In: DeVita VT Jr, Lawrence TS, Rosenberg SA, eds. DeVita, Hellman, and Rosenberg's cancer: principles and practice of oncology. Vol. 1. Philadelphia: Lippincott Williams & Wilkins, 2008:1232-84.
Compton C, Hawk ET, Grochow L, Lee F, Ritter M, Niederhuber JE. Colon cancer. In: Abeloff MD, Armitage J, Niederhuber JE, Kastan MB, McKenna GW, eds. Abeloff's clinical oncology. Philadelphia: Churchill Livingstone, 2008:1477-534.
Markowitz SD, Dawson DM, Willis J, Willson JK. Focus on colon cancer. Cancer Cell 2002;1:233-236. [CrossRef][Web of Science][Medline]
André T, Boni C, Mounedji-Boudiaf L, et al. Oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment for colon cancer. N Engl J Med 2004;350:2343-2351. [Free Full Text]

PENSAMIENTOS SOBRE LA MUERTE

Ending End-of-Life Phobia — A Prescription for Enlightened Health Care Reform
Posted by NEJM • December 16th, 2009 •
Benjamin W. Corn, M.D.
Reform is coming to U.S. health care. A sense of urgency regarding the redesign of policies prevailed well before Barack Obama was elected president under the banner of change. A debate is now raging over a wide span of topics, including prescription plans, physician reimbursement, and solutions for the medical liability problem. But thought leaders have been remarkably reticent with respect to one aspect of the health care system: end-of-life care. Given that patients with terminal illness require a disproportionate concentration of expenditures, the silence is deafening. Sure, the summer’s controversy over “death panels” provided fodder for late-night comedians, but just under the surface of the joking there was evidence of America’s uneasiness with the hard conversations that lay ahead. Why has it been so difficult to initiate a dialogue about matters pertaining to a subject that defines the human experience?
As a cancer specialist, I am actually not surprised by this state of affairs. Patients, family members, and (yes) even many of my colleagues have great difficulty in coping with thoughts of death.1 Sophisticated mechanisms, ranging from the modern approach to dying (i.e., doing so outside the home, supported by high-tech equipment) to the use of euphemisms and gallows humor, have been developed to help us deny and sanitize death. People in general are most comfortable deferring questions that relate to their finitude.
But try as we may to avoid it, death has a way of intruding on our lives. Beyond my encounters inside the hospital, I am flooded by constant reminders of this point. Driving home from work last month, as a traffic jam began to clear, I noted that the remains from a fatal motor vehicle accident were being removed from the shoulder of the highway. The sight was horrific. As I write these words, a friend “tweets” that his uncle has lapsed into an irreversible coma. And several years ago, I was stunned to hear that a violent murder had been committed in the quiet suburban New York neighborhood where I grew up. These are dreadful developments that force me to confront my mortality.
Every human being fears death in his or her unique way. This anxiety often shunts us onto a path of least resistance, veering away from the processing of basic existential issues. But this diversion may represent a lost opportunity, since an awareness of death can catalyze positive transformations.2
Recently, a 52-year-old businesswoman who was treated for colorectal cancer in 2006 came into my office for her follow-up visit. We were just shy of the magical 3-year milestone, which connotes cure if there is no evidence of disease recurrence. Unfortunately, a routine scan disclosed widespread metastases in the liver, lungs, and bone. The woman was understandably distraught and overwhelmed by the need to choose a chemotherapy regimen. However, when she decided to tackle what she termed “ultimate issues,” such as loneliness and suffering, she was able to marshal the courage to ease up on career commitments and focus on resolving long-standing conflicts, especially her estrangement from an older brother. She concluded that they had stopped speaking because of petty concerns, and she persevered until forgiveness was found.
Many of the patients I work with uncover a profound appreciation of life after contemplating the implications of the end of life. Most people can actually be taught to turn an outright crisis into an occasion for growth. So my modest suggestion is that a willingness to ponder death can bring about renewed clarity in our thinking and our priorities.3 Once we are ready to process the feelings that death evokes, we will be better equipped to take on the challenging ramifications of terminal illness.
The high-voltage nature of the topic was clear when the “death panel” controversy first surfaced. The term — introduced on Sarah Palin’s Facebook page and subsequently ranked as a finalist on many word-of-the-year lists — has come to connote a theoretical body that determines which patients deserve to live when health care is rationed. The trigger for the concept was a small provision embedded in the lengthy House bill that would permit Medicare coverage for an end-of-life consultation once every 5 years. In an op-ed piece in the New York Times, Representative Earl Blumenauer (D-OR) explained that his motivation in composing the clause was to create a mechanism to “reimburse doctors for having a thoughtful conversation to prepare patients and families for the delicate, complex and emotionally demanding decisions surrounding end-of-life.”4 These intentions seem noble, yet even this seemingly cautious and reasonable language apparently scared people.
What animates these fears? I believe that patients perceive their vulnerability when such discussions are initiated. Moreover, patients (as well as the caregivers and potential surrogates who support them) may fear that the physicians who broach the topic are striving to cut costs and compromise services. This worry is recognizable to any oncologist who has ever peered into the eyes of a patient with refractory cancer who yearns to try third-line chemotherapy. At the same time, however, policymakers are sincerely worried about a disconnect between society’s expectations from its medical system and what might realistically be offered as we go forward. My concern is for the vulnerable patients, but my goal is to reach a level of maturity that enables these conversations to transpire in a manner that is indeed thoughtful.
Some people may never be comfortable with this subject, but others might welcome a dialogue, provided that three principles are affirmed: personal autonomy, the sanctity of life, and a climate of balance. The first two terms are admittedly quite subjective, but most people easily sense when these values are being respected or violated. To achieve a climate of balance, however, I believe that we will need to add some safeguards into the equation.
During my residency training in the 1980s, the concept of conservative therapy for early-stage breast cancer was gaining interest. New ground was being broken, and it had to be done gently. In many hospitals, surgeons who were enamored of mastectomy and radiation oncologists who saw a rationale for the combination of lumpectomy and radiotherapy agreed to be simultaneously present in the room as patients tried to navigate the clinical controversy and determine what was best for them. In most circumstances, tactful exchanges were carried out without rancor and without questioning of the experts’ hidden agenda. In that environment, patients felt safe. If similar conversations were led by a team consisting of patient advocates (e.g., chaplains) and medical experts espousing countervailing views regarding the use of resources at life’s end, perhaps we could restore the requisite trust for this essential dialogue.
As the U.S. health care system braces for reform, end-of-life concerns must be confronted squarely. Will hospice services be expanded? Should expensive experimental therapies that only slightly prolong life be deemed reimbursable? Can our profession evolve nuanced strategies for resolving questions of medical futility?5 Can we find creative ways for restoring dignity to the dying process? Should national guidelines be created for physician-assisted suicide? There is no shortage of topics that require attention.
Concerns over the end of life will never die. But denial of our mortality is no longer an option. If we muster the courage to address the last collective phobia of the Western world, we may generate ideas for truly comprehensive health care reform and better living.
References
1.Shanafelt T, Adjei A, Meyskens FL. When your favorite patient relapses: physician grief and well-being in the practice of oncology. J Clin Oncol 2003;21:2616-2619. [Free Full Text]

2.Yalom ID. Staring at the sun: overcoming the terror of death. San Francisco: Jossey-Bass, 2008.

3.Rosenbaum ME, Loas J, Ferguson K. Using reflection activities to enhance teaching about end-of-life care. J Palliat Med 2005;8:1186-1195. [CrossRef][Web of Science][Medline]

4.Blumenauer E. My near death panel experience. New York Times. November 15, 2009.

5.Burns JP, Truog RD. Futility: a concept in evolution. Chest 2007;132:1987-1993. [Free Full Text]

DISTROFIA MUSCULAR DE DUCHENNE

J Neurol Neurosurg Psychiatry 2009;80:320-325
Research paper
Disability and survival in Duchenne muscular dystrophy
M Kohler1, C F Clarenbach1, C Bahler2, T Brack1, E W Russi1,3, K E Bloch1,3
+ Author Affiliations
1Pulmonary Division, University Hospital of Zurich, Zurich, Switzerland
2Mathilde Escher Heim, Zurich, Switzerland
3Zurich Centre for Integrative Human Physiology, University of Zurich, Switzerland
Dr K E Bloch, Pulmonary Division, University Hospital of Zurich, Raemistrasse 100, 8091 Zurich, Switzerland; konrad.bloch@usz.ch
Abstract
Background: Duchenne muscular dystrophy (DMD) leads to progressive impairment of muscle function, respiratory failure and premature death. Longitudinal data on the course of physical disability and respiratory function are sparse.
Objectives: To assess prospectively physical impairment and disability, respiratory function and survival in patients with DMD over several years to describe the course of the disease with current care.
Methods: In 43 patients with DMD, aged 5–35 years, yearly assessments of physical disability by the Duchenne muscular dystrophy physical Impairment and Dependence on care (DID) score, ranging from 9 (no disability) to 80 (complete dependence), and forced vital capacity (FVC), were obtained over a mean time interval of 5.4 (SD 2.1) years.
Results: DID scores were correlated with age according to a hyperbolic function (f = 85.3×age/(10.05+age), R = 0.62, p<0.0001). FVC declined exponentially with age (f = 139.1×exp(−0.08×age), R = 0.52, p<0.0001). Mean age at which patients lost their ambulation was 9.4 (SD 2.4) years and they became dependent on an electric wheelchair at 14.6 (4.0) years. Age at the beginning of assisted ventilation was 19.8 (3.9) years, Three patients died during the observation period. The estimated probability of survival to age 30 years was 85% (median survival was 35 years).
Conclusions: Our detailed observations of the progression of physical disability, dependence on care and respiratory impairment in patients with DMD from childhood to adult life is valuable for predicting the clinical course with current medical care. Compared with historical data, survival has improved considerably.
Duchenne muscular dystrophy (DMD) is the most common form of the inherited muscular dystrophies affecting approximately 1 in 3300 male births. The disorder is caused by mutations in the gene located at Xp21 which codes for the dystrophin protein. DMD leads to progressive muscular weakness, severe physical disability and ultimately death.1 2 Most patients with DMD become wheelchair users in childhood, and they depend largely on their parents for their daily activities and care.3 In more advanced stages of the disease, the progressive spinal and chest wall deformity and impairment of respiratory muscle function leads to hypercapnic respiratory failure, and cardiac muscle involvement may entail congestive heart failure.4 Recent studies suggest that non-invasive positive pressure ventilation and other supportive measures prolong survival of patients with DMD well into adulthood.5 6
With prolongation of life, physical impairment and dependence on care increases because of the progressive nature of the disorder but data on this topic from adult patients with DMD are limited or outdated.1 3 7 8 We are aware of only one cohort for whom longitudinal data on physical disability in adult patients with DMD were reported.7 9 Prediction of profiles was limited by the small number of patients included and a relatively short follow-up time.7 Knowledge of the course of physical impairment in DMD is of great importance to patients, parents and health care providers because it allows for a realistic outlook on the progression of the disease, facilitates planning of long term care, and defines the specific problems and needs of patients with DMD.
Previously, conventional evaluation of physical impairment has mainly focused on motor deficits based on measurement of muscle strength3 10 but such measures might not appropriately capture the clinically relevant functional disabilities of the patients. To overcome this limitation, several scores have been proposed to assess physical impairment in patients with neuromuscular diseases but they were not specifically designed for patients with DMD of various age ranges.11–15 Therefore, we recently introduced a simple DMD specific score that assesses the various aspects of physical impairment and dependency on help by others and technical aids5 that we termed the DID score (Duchenne muscular dystrophy physical Impairment and Dependency score).
In this study, we applied the DID score to prospectively investigate the long term course of physical impairment and dependence on care, along with lung function and survival in a cohort of 43 patients with DMD. The aim was to provide current profiles of the clinical presentation and natural history of DMD from childhood into adult life in order to predict the individual clinical course of the disease with current medical care.
METHODS
Patients
Patients with DMD living or attending school in a facility specialised in the care of patients with muscular dystrophy, the Mathilde-Escher-Heim, Zurich, Switzerland, were prospectively enrolled during the period from 1999 to 2006. The diagnosis of DMD was based on standard criteria: progressive symmetrical muscle weakness and other signs and symptoms starting before the age of 5 years; elevated serum creatinine kinase activity; muscle biopsy and/or genetic analysis; and, in some, a family history consistent with X chromosome linked recessive inheritance.16 Information on genetic analysis was available from 10 patients (nine patients had a deletion in at least one of the exons 48–52 of the DMD gene locus whereas a duplication mutation was found in one patient). The study protocol was approved by the local ethics committee and patients gave informed consent to participate.
Measurements
A physical examination, including measurement of body weight and height, was performed and body mass index (BMI) was calculated. Height was used for calculation of reference values of pulmonary function. It was determined by a flexible ruler fitted along the contours of the body, from the head, along the vertebral spine and the back of the legs, to the heels to account for kyphoscoliosis and leg contractures.
Spirometry was performed in the sitting position with a flowmeter attached to a flanged rubber mouthpiece with the nose occluded.17 Reference values for ages up to 17 years18 and above19 were computed.
Physical impairment and the inability to perform activities of daily living and dependence on others and on technical aids was evaluated with the DID score specifically developed at our centre for patients with DMD, as described previously (see appendix online).5 The DID score consists of the following eight aspects of daily life: mobility without technical aids, mobility with technical aids, transfers (eg, from bed to wheelchair), changes in body position, dressing, static body control, feeding and breathing. Each aspect is rated with up to 10 points, with higher scores reflecting greater impairment and disability. The sum score of all eight domains is calculated as a measure of overall impairment, disability and dependency, with a minimal value of 9 (no impairment in any of the domains) and a maximum value of 80 points (completely impaired and dependent on help by others in all domains). The DID score was prospectively applied on a yearly basis.
For evaluation of interobserver agreement, two experienced physical therapists independently applied the DID score to all patients with DMD alive in 2006.
Data analysis
Data are expressed as means (SD). Regression analysis was used to determine the relationships among the DID score, forced vital capacity (FVC) and age. Survival probability was calculated by the Kaplan–Meier method. Interobserver agreement between DID scores obtained by two observers was evaluated by Pearson’s correlation and by calculating the mean difference (bias) and limits of agreement (±2 SD).20 A probability value of <0.05 was considered statistically significant.
RESULTS
Demographics
Forty-three patients with DMD with a mean age of 15.3 (SD 5.2) years and a BMI of 20.0 (7.3) kg/m2 at enrolment were followed over a time period of 5.4 (2.1) years (range 1–8). The course of weight, height and BMI is shown for selected ages in table 1.
Milestones in the clinical course of Duchenne muscular dystrophyPhysical impairment and dependency
In total, 227 yearly assessments were performed in 43 patients. Milestones of the clinical course in patients with DMD are described in table 1. Individual trends in DID scores over time are shown in fig 1. Progression of physical impairment and disability, as reflected by the DID score at any particular age, was strongly correlated with age (in years) according to the hyperbolic function: DID score = 85.33×age/(10.05+age), R = 0.62, p<0.0001. The DID scores of 19 out of 20 patients (95%) whose initial score was above the fitted hyperbolic line remained above this line. Thus an advanced disability at the first observation was associated with a subsequent further continuous progression in disability. The DID score of only nine patients crossed the fitted line on a total of 11 occasions, eight in an upward direction (worse) but only three in a downward direction (improved) (χ2 test, p = 0.03).
Duchenne muscular dystrophy physical Impairment and Dependence on care (DID) scores obtained at yearly intervals in 43 patients (n = 227 assessments). The age related progressive limitation in activities of daily living and dependency on care is described by the function f = 85.33×age/(10.05+age) and represented by the bold line. Thin lines connect data of individual patients.Follow-up assessments of the eight domains of the DID score revealed that the impairment in the domains “mobility without technical aid”, “mobility with technical aid”, “transfer”, “changes in body position” and “getting dressed” started at around the age of 5 years and increased rapidly until the age of 10–15 years with a subsequent plateau at the maximum level of disability. In contrast, progression of impairment in the domains “static body control”, “eating and drinking” and “breathing” was moderate in early life but accelerated in adulthood (fig 2).
Trends for the eight components of the yearly Duchenne muscular dystrophy physical Impairment and Dependence on care (DID) scores shown in fig 1 (n = 227 assessments, 43 patients). Lines connect data for individual patients. A rapid progression in impairment with asymptotically approached maximal values in early life was noted in scores reflecting mobility, transfer, changing position and dressing. In contrast, the need for assistance in eating and drinking, breathing and static body control was modest in childhood but showed an accelerated increase in adulthood.For some domains of the DID score specific milestones in the clinical course of the disease are listed in table 1. Mean age at which patients lost their ambulation was 9.4 (2.4) years (range 6–15). They became dependent on an electric wheelchair at 14.6 (4.0) years (range 11–28). Thirty-three patients (77%) had undergone spinal fusion surgery at a mean age of 14.2 (2.6) years (range 8–21). Age when food and drinks had to be given to a patient by a caregiver was 18.2 (4.2) years (range 12–23), and the age at which 22 of the patients required assisted mechanical ventilation was 19.8 (3.9) years (range 14–31). In eight patients (19%) a gastrostomy had been performed at a mean age of 24.7 (4.9) years (range 20–34) because of feeding problems.
DID scores obtained independently by two observers in 40 patients were closely correlated (r = 0.96, p<0.0001) with a mean difference of 2.1 points and limits of agreement (±2 SD of bias) of 4.6 points.
Lung function
FVC revealed an exponential decline with age according to the function: FVC = 139.1×exp(−0.08*age), R = 0.52, p<0.0001 (fig 3). Mean age at which FVC fell below 1 litre was 18.1 (5.0) years (range 13–31).
Forced vital capacity (FVC) values progressively decreased with advancing age. Thin lines connect data of individual patients (n = 170 assessments in 43 patients; the most severely disabled patients were unable to perform spirometry). The bold line represents an exponential decay function (f = 139.1×exp(-0.08×age)).Twenty-two of the 43 patients (51%) received long term assisted mechanical ventilation for chronic respiratory failure. Seventeen of these patients were ventilated non-invasively with a nasal or oral–nasal mask, and five patients via tracheotomy. Mean age at the beginning of mechanical ventilation was 19.8 (3.9) years (range 14–31) and mean FVC was 0.73 (0.34) litre (20 (10)% of predicted).
Survival
Only three of the 43 patients with DMD died during the follow-up period. One patient died because of heart failure due to cardiomyopathy at the age of 35 years, one patient died suddenly 8 days after tracheotomy at the age of 22 years and one patient died of respiratory failure at the age of 24 years. Kaplan–Meier analysis revealed a median survival of 35 years. The probability of surviving 10 years after initiation of assisted mechanical ventilation was 68% (fig 4).
(A) Kaplan–Meier curve showing the cumulative survival in 43 patients with Duchenne muscular dystrophy. (B) Cumulative survival in 22 of the 43 patients following initiation of assisted positive pressure ventilation.
DISCUSSION
We prospectively investigated the clinical course in patients with DMD from childhood to adult life and identified milestones of disease progression. The strength of our study is the detailed observation of the course of physical impairment and dependence on care over several years by using the DID score along with spirometry and survival in a large patient cohort. The current update on the clinical course of DMD is valuable for patients, families and health professionals as an adjunct in planning medical care and future life. The median survival of 35 years compares favourably with historical data and presumably reflects advances in assisted mechanical ventilation and other supportive care.
We recorded the clinical course of patients with DMD using a recently introduced instrument, the DID score, that assesses eight different aspects of impairment and disability in daily life.5 The DID score quantifies the physical impairment in patients with DMD with high interobserver agreement and identified clinical milestones such as loss of ambulation at a mean age of 9.4 years, dependence on an electric wheelchair at 14.6 years, dependence on being dressed and fed by caregivers at 18.2 years and requirement for assisted mechanical ventilation at 19.8 years. In contrast with other (generic) scores such as the index of activities of daily living which was designed for the elderly,11 the DID score incorporates aspects of disability typical for patients with DMD and relevant to their entire life span. Similar to the EK score, which focuses on disability in non-ambulatory patients with DMD,15 the DID score assesses wheelchair mobility, transfer, static body control and dependence on help with eating and drinking. But unlike the EK score, the DID score also incorporates observations on mobility without technical aids (see appendix, domain 1 online), and specifically addresses the need for assisted mechanical ventilation (see appendix, domain 8 online) which has become an essential life saving component of care.
The DID scores progressed rapidly in childhood and subsequently approached maximal values corresponding to nearly total dependence on caregivers and technical aids such as a wheelchair and mechanical ventilation. Individual grades of disability at a certain age varied, possibly related to differences in treatment and lifestyle,21–23 although all patients with DMD in our study received medical care by the same institution. We observed an increase in DID scores over time according to a hyperbolic function (fig 2). Almost all patients (95%) who initially scored above the regression line did so in every subsequent follow-up (fig 1). Scores for the eight domains incorporated into the DID score followed two different patterns (fig 2): the progression in the domains ”mobility without technical aid”, “mobility with technical aid”, “transfer”, “changes of position” and “getting dressed” followed a hyperbolic function characterised by a steep initial increase to an asymptotically approached maximum value, while the scores for the domains ”static body control”, “eating and drinking” and “breathing” progressed exponentially indicating that these functions were severely affected late in the course. This may be related to the different types of muscles involved in these functions, as proximal skeletal muscles, which contain large muscle fibres, are affected early in the course of DMD, whereas muscles containing small calibre fibres are relatively spared initially, so that breathing and eating are more gradually impaired, a finding supported by animal models.24 25
FVC progressed with advancing age in accordance with our previous report and that of others.5 6 Lung function rapidly deteriorated in young patients but declined less steeply thereafter (fig 3). Correspondingly, a more rapid yearly FVC decline of 8.5% was reported in patients with DMD at 10–20 years of age, whereas the decline was reduced to 6.2% per year above the age of 20 years.26 It has been previously reported that patients with DMD are more likely to develop chronic respiratory failure if their vital capacity falls below 1 litre and the 5 year survival rate was only 8% if assisted ventilation was not provided.27 In an early study, Brooke and colleagues3 found that patients with DMD passed this milestone at a median age of 13.5 years whereas in the current study FVC was reduced to 1 litre at a considerably older age of 18.1 years. A possible explanation is the more general application of glucocorticosteroid therapy and other changes in treatment within the past two decades.23 Unfortunately, there was no reliable information on previous glucocorticosteroid therapy available from our study cohort and thus we have not been able to explore the potential impact of this treatment on lung function. The less rapid reduction of FVC compared with earlier studies is unlikely related to the use of cough assist devices as our patients used such devices only during periods of increased mucus production (eg, during chest infections). Half of our patients had less than 20% of predicted FVC by the age of 20 years which corresponds approximately to the age at which assisted mechanical ventilation was initiated (on average 19.8 years). Similar to our findings, Toussaint and colleagues6 reported a mean age of 19.4 years and FVC of 21% predicted at the time of initiation of positive pressure ventilation.
Median survival of 35 years which we observed (fig 4) is considerably higher than the median survival of 14.4–20.5 years reported in patients not treated with assisted ventilation,27–29 and 26–33 years in patients receiving long term mechanical ventilation.6 30 31 The probability of surviving 5 years after initiation of assisted ventilation was 70% in two studies analysing this outcome.6 32 In our cohort, survival 5 and 10 years after beginning assisted mechanical ventilation was 82% and 68%, respectively (fig 4). The recent improvement in survival of patients with DMD may not only reflect advances in treatment but also the changing attitudes of caregivers regarding various therapeutic options (eg, therapy of cardiomyopathy, and spinal fusion surgery).30
In conclusion, our prospective longitudinal study provides novel data on the clinical course of DMD from childhood to adult life which updates and extends earlier cross sectional observations. The DID score is a valuable instrument to describe the distinct patterns of disease progression in several domains of physical impairment and dependence on care. Our observations on clinical milestones and on the improved survival represent essential information for patients with DMD, their families and caregivers as a basis for planning and evaluating care and therapeutic interventions.
REFERENCES
1.↵Bushby KM. Genetic and clinical correlations of Xp21 muscular dystrophy. J Inher Metab Dis 1992;15:551–4.[CrossRef][Medline][Web of Science]2.↵Hoffman EP, Brown RH, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919–28.[CrossRef][Medline][Web of Science]3.↵Brooke MH, Fenichel GM, Griggs RC, et al. Duchenne muscular dystrophy: patterns of clinical progression and effects of supportive therapy. Neurology 1989;39:475–81.[Abstract/FREE Full text]4.↵Smith PE, Calverley PM, Edwards RH, et al. Practical problems in the respiratory care of patients with muscular dystrophy. N Engl J Med 1987;316:1197–205.[Medline][Web of Science]5.↵Kohler M, Clarenbach CF, Boni L, et al. Quality of life, physical disability, and respiratory impairment in Duchenne muscular dystrophy. Am J Respir Crit Care Med 2005;172:1032–6.[Abstract/FREE Full text]6.↵Toussaint M, Steens M, Wasteels G, et al. Diurnal ventilation via mouthpiece: survival in end-stage Duchenne patients. Eur Respir J 2006;28:549–55.[Abstract/FREE Full text]

ASIMETRIA SEXUAL DEL CEREBRO LIMBICO

Hot Topics
Neuropsychopharmacology (2010) 35, 340–341;
Sex-related functional asymmetry in the limbic brain
Timothy Koscik1,3, Antoine Bechara2 and Daniel Tranel1,3
1Division of Behavioral Neurology and Cognitive Neuroscience, Department of Neurology, University of Iowa College of Medicine, Iowa City, IA, USA
2Department of Psychology, Brain and Creativity Institute, University of Southern California, Los Angels, CA, USA
3Neuroscience Graduate Program, University of Iowa, Iowa City, IA, USA
Correspondence: Daniel Tranel, E-mail: daniel-tranel@uiowa.edu
At a macroscopic level, the brains of both men and women are very similar. However, closer inspection shows striking differences between the sexes in the structure–function relationships in the brain. Interpersonal, emotional, and occupational success depends critically on effective navigation of the social world. At the same time, individuals of each sex have different goals—only women bear children, and this basic function likely sets the agenda for the individual and societal goals of many women. Men do not bear children and may be driven by an agenda emphasizing acquiring and maintaining resources and power. Thus, sex differences in social-emotional regions of the brain might be adaptive. The limbic regions of the brain and closely connected regions, especially the ventromedial prefrontal cortex (VMPC) and amygdala, are important for social-emotional processing. Furthermore, as nearly all brain regions have homologous versions in each hemisphere, this may be a substrate on which divergent selection resulted in sex-related functional asymmetry.

Studies of neurological patients have shown that the right VMPC and amygdala appear to be critical for social-emotional functioning and decision making in men, whereas the left VMPC and amygdala appear to be more important for these functions in women (Tranel et al, 2005; Tranel and Bechara, 2009). For example, a man with a unilateral right VMPC lesion, who was well educated and had worked successfully as a minister, was entirely unable to return to any form of gainful employment after his brain damage. He requires supervision for daily tasks and demonstrates severe disturbances in behavior and emotional regulation, including impulsivity and poor judgment. By contrast, a man with a unilateral left VMPC lesion was able to return to his job at a grain elevator and remains successfully employed there. He is remarkably free of disturbances to his social life and emotional functioning (Tranel et al, 2005). Moreover, preliminary evidence from the Trust Game, a multiplayer neuroeconomics task, suggests that women with left VMPC lesions and men with right VMPC lesions trust others less (ie, they invest less in others) and display more frequent acts of negative interpersonal reciprocity (ie, they return less than the amount invested). Similarly, women with lesions to the left amygdala and men with lesions to the right amygdala appear less risk averse than do men and women with lesions to the opposite amygdala. This evidence converges with other research, including (1) fMRI studies showing similar patterns of lateralized activations in these regions in response to social and emotional paradigms (eg, Killgore and Yurgelun-Todd, 2001; Cahill et al, 2004); (2) studies showing sex differences in orbitofrontal-dependent behavioral paradigms, such as the Iowa Gambling Task (eg, Overman, 2004); and (3) studies showing increased functional connectivity with the right amygdala of men and with the left amygdala of women (eg, Kilpatrick et al, 2006). Altogether, such evidence suggests that the right VMPC and amygdala in men as well as the left VMPC and amygdala in women are important for social-emotional functions. Potentially, the left limbic dominance observed in women reflects a need for expertise in interpersonal relationships (eg, the need to bear and rear children, maintain in-group cohesion, etc.), whereas the right limbic dominance observed in men could reflect a need for expertise in inter-group relations (eg, warfare, out-group relations, leverage of critical resources, etc.).
Sex-related functional asymmetry in the VMPC and amygdala may prove to be at the heart of the complementary social roles that both men and women have in human society. It is incontrovertible that men and women deserve equal opportunities to participate in society; it is also clear that men and women are neither biologically nor behaviorally identical. Sex-related functional asymmetry may be one way that evolution has capitalized on the capacity of homologous brain regions to process information differently and shaped our brains to meet the demands of both the sexes with unique reproductive and social roles.
References
Cahill L, Uncapher M, Kilpatrick L, Alkire MT, Turner J (2004). Sex-related hemispheric lateralization of amygdala function in emotionally influenced memory: an fMRI investigation. Learn Mem 11: 261–266. Article PubMed ISI
Killgore WDS, Yurgelun-Todd DA (2001). Sex differences in amygdala activation during the perception of facial affect. Neuroreport 12: 2543–2547. Article PubMed ChemPort
Kilpatrick LA, Zald DH, Pardo JV, Cahil LF (2006). Sex-related differences in amygdala functional connectivity during resting conditions. Neuroimage 30: 452–461. Article PubMed ChemPort
Overman WH (2004). Sex differences in early childhood, adolescence, and adulthood on cognitive tasks that rely on orbital prefrontal cortex. Brain Cogn 55: 134–147. Article PubMed
Tranel D, Bechara A (2009). Sex-related functional asymmetry of the amygdala: preliminary evidence using a case-matched lesion approach. Neurocase 15: 217–234. Article
Tranel D, Damasio H, Denburg NL, Bechara A (2005). Does gender play a role in functional asymmetry of ventromedial prefrontal cortex? Brain 128: 2872–2881. Article PubMed

NEUROCIRCUITOS Y MECANISMOS GENETICOS EN ESQUIZOFRENIA

Neuropsychopharmacology Reviews
Neuropsychopharmacology (2010) 35, 258–277;
Executive Function, Neural Circuitry, and Genetic Mechanisms in Schizophrenia
Daniel Paul Eisenberg1 and Karen Faith Berman1
1Section on Integrative Neuroimaging and Clinical Brain Disorders Branch, Genes, Cognition, and Psychosis Program, National Institute of Mental Health, NIH, DHHS, Bethesda, MD, USA
Abstract
After decades of research aimed at elucidating the pathophysiology and etiology of schizophrenia, it has become increasingly apparent that it is an illness knowing few boundaries. Psychopathological manifestations extend across several domains, impacting multiple facets of real-world functioning for the affected individual. Even within one such domain, arguably the most enduring, difficult to treat, and devastating to long-term functioning—executive impairment—there are not only a host of disrupted component processes, but also a complex underlying dysfunctional neural architecture. Further, just as implicated brain structures (eg, dorsolateral prefrontal cortex) through postmortem and neuroimaging techniques continue to show alterations in multiple, interacting signaling pathways, so too does evolving understanding of genetic risk factors suggest multiple molecular entry points to illness liability. With this expansive network of interactions in mind, the present chapter takes a systems-level approach to executive dysfunction in schizophrenia, by identifying key regions both within and outside of the frontal lobes that show changes in schizophrenia and are important in cognitive control neural circuitry, summarizing current knowledge of their relevant functional interactions, and reviewing emerging links between schizophrenia risk genetics and characteristic executive circuit aberrancies observed with neuroimaging methods.
Keywords: schizophrenia, cognition, executive function, working memory, neuroimaging, genetics
INTRODUCTION
Psychiatry has long appreciated deficits in higher-order thought processes in schizophrenia, with relative sparing of many basic cognitive abilities (Kraepelin, 1909–1913), and indeed, the modern era of neuropsychological research has accumulated data from schizophrenic patients showing significant impairments in complex tasks requiring a range of advanced cognitive processes, collectively described as executive functions (Bozikas et al, 2006; Carter et al, 2001; Tan et al, 2006; Weinberger et al, 1986). Executive functions rely heavily on frontal lobe structures and include: directed attention and inhibition, task management, planning, monitoring, and coding of representations in working memory (Smith and Jonides, 1999). Subsets of these functions have shown a close relationship to both negative symptoms (O'Leary et al, 2000; Pantelis et al, 2001), thought disorder (Perlstein et al, 2001; Stirling et al, 2006), and functional outcomes in schizophrenia (Kurtz et al, 2005; Liddle, 2000), in line with the suggestion that frontal lobe dysfunction is crucially important in schizophrenic psychopathology (Elvevag and Goldberg, 2000; Weinberger et al, 1994). Accumulated evidence from over two decades of neuroimaging experiments has confirmed executive-task-related functional abnormalities of the prefrontal cortex in schizophrenia; however, despite the numerous replications of this finding, the precise nature of illness-related frontal local circuit aberrancies contributing to executive dysfunction remains incompletely defined and remains the focus of ongoing investigation. Furthermore, because (1) functional abnormalities in schizophrenia are not exclusive to the frontal cortex, and (2) executive processes, though heavily reliant on the frontal cortex, also require cooperation from structures outside of the frontal lobes, schizophrenia research has increasingly turned its eye toward discerning how extended neural circuit dynamics contribute to illness-related cognitive phenotypes. Ultimately, if both these local prefrontal and extended distributed network characteristics in schizophrenia are relevant to the neurobiology of this disorder, then it may be possible to examine these systems through the lens of genetics. As schizophrenia likely involves multiple molecular pathways, this approach is invaluable in clarifying the mechanistic steps between risk genes, neuronal cellular function, neural circuits, and clinical morbidity. This chapter reviews the contributions of regions implicated in both schizophrenia and executive processing to local and extended neural circuits as well as describes recent advances in understanding relationships between these circuits and schizophrenia risk genes. Emphasis is placed on seven interconnected brain regions that have each prominently shown: (1) neuropathological and/or neurophysiological abnormalities in schizophrenia, (2) relevance to executive functioning and aberrant activity during executive processing in schizophrenia, and (3) abnormal functional relationships with other included regions during executive processing in schizophrenia. Additionally, nine genes are highlighted, each with variation showing both (1) evidence for contribution to risk of schizophrenia and (2) association with schizophrenia executive neuroimaging phenotypes that include circuits involving the emphasized regions.
EXECUTIVE CIRCUITS WITHIN THE FRONTAL LOBES
Ever since seminal regional blood flow studies showing specific and replicable frontal lobe dysfunction during executive task challenge in schizophrenia (Berman et al, 1986; Weinberger et al, 1986), better characterization of executive control circuits within the frontal lobes has remained at the forefront of schizophrenia research efforts. Investigations of abstract rule inference (Berman et al, 1995; Buchsbaum et al, 2005; Monchi et al, 2001), conflict management and monitoring (Macdonald et al, 2000; Pardo et al, 1990), verbal fluency (Frith et al, 1991; Gourovitch et al, 2000), and working memory (Cohen et al, 1997; Tsuchida and Fellows, 2009) in healthy individuals consistently show reliance on key frontal regions, most notably, the dorsolateral, ventrolateral, and anterior cingulate cortices. Abnormal functional measures in each of these regions have been shown in schizophrenia during these same paradigms (Becker et al, 2008; Berman et al, 1986; Callicott et al, 2003b; Kerns et al, 2005; Spence et al, 2000; Weinberger et al, 1986), bolstering the hypothesis of frontal primacy in schizophrenic pathophysiology (Elvevag and Goldberg, 2000; Weinberger et al, 1994) but increasing the imperative to understand how these disparate frontal nodes interact in concert during illness.
Dorsolateral Prefrontal Cortex
Numerous lines of evidence converge to implicate abnormalities of the dorsolateral prefrontal cortex (DLPFC)—the prototypical center of higher-order cognitive processing—in schizophrenia pathophysiology. Though they have neither shown gross evidence of degeneration (eg, gliosis) nor a diagnostic lesion, postmortem studies of schizophrenia patient DLPFC tissue have nonetheless shown support for perturbations of excitatory cells: increased pyramidal cell density (Selemon et al, 1995, 1998), reduced pyramidal neuron dendritic spine density (Glantz and Lewis, 2000), altered NMDA receptor subunit expression (Akbarian et al, 1996); inhibitory cells: reduced GAD67 and GAT1 mRNA expression (Akbarian et al, 1995; Volk et al, 2000); and dopaminergic afferents: reduced tyrosine hydroxylase expressing afferent axons (Akil et al, 1999), reduced DARPP-32 concentrations (Albert et al, 2002; Ishikawa et al, 2007). To what degree each of these and other cellular pathological findings are primary effects or secondary to other local disturbances (eg, other cellular pathological changes) or more distant alterations (eg, abnormal afferents from other frontal lobe structures or extrafrontal structures, Wang and Deutch, 2007) requires dedicated future study.
In vivo patient studies have further substantiated DLPFC pathological changes. Complimentary data from both region of interest (ROI) studies (Andreasen et al, 1994b; Nopoulos et al, 1995) and voxel-based morphometric studies (Cannon et al, 2002; Fornito et al, 2009; Giuliani et al, 2005) have reported statistically reduced DLPFC volumes in schizophrenia. Though not as consistently reported as reduced medial temporal lobe volumes (Honea et al, 2005), this finding, in concert with increased neuronal density, has been interpreted as a result of decreased DLPFC neuropil (Selemon et al, 1998). Importantly, reduced DLPFC gray matter volume is significantly more pronounced in patients with greater executive dysfunction, as measured by the Wisconsin Card Sorting Test (Rüsch et al, 2007). Neuronal measures of the DLPFC in schizophrenia have also shown abnormalities, particularly reduced N-acetylapartate (NAA), a measure of neuronal integrity, which has been repeatedly found in magnetic resonance spectroscopy studies (Abbott and Bustillo, 2006) and has shown relevance to cognitive function in schizophrenia: NAA levels in patients show a positive correlation with the degree of DLPFC working memory activation as measured by [15O]H2O PET (Bertolino et al, 2000b). Notably, DLPFC D1 receptor binding, measured by [11C]NNC112 PET in medication-free schizophrenic patients, has been shown to be both increased and correlated with working memory impairment in schizophrenia (Abi-Dargham et al, 2002). When measured with [11C]SCH23390, however, D1 binding in the prefrontal cortex appears reduced (Okubo et al, 1997). Interestingly, these conflicting data actually correspond well with rodent models of subchronic dopaminergic depletion, which increases [11C]NNC112 binding but paradoxically decreases [11C]SCH23390 binding (Guo et al, 2003) and are hypothesized to reflect compensatory responses to reduced prefrontal dopaminergic input from the midbrain. White matter abnormalities of the DLPFC have also been described (Schlösser et al, 2007). Taken together, these cytopathological, structural, and neuroreceptor mapping findings predict both a prominent role for disrupted dorsolateral prefrontal cortical function and related aberrant interactions between this region and other brain structures contributing to executive dysfunction in schizophrenia.
In line with the former assertion, a plethora of functional imaging studies of schizophrenia have shown alterations in DLPFC physiology in response to executive cognitive demands. Replication of reduced relative frontal activity (Ingvar and Franzén, 1974), in the DLPFC during executive tasks, has been frequent in the past two decades, and has been observed in medicated, medication-free, and medication-naïve patients (Barch et al, 2001, 2003; Berman et al, 1986, 1992; Callicott et al, 1998; Camchong et al, 2006; Cannon et al, 2005; Cantor-Graae et al, 1991; Carter et al, 1998; Catafau et al, 1994; Curtis et al, 1998; Driesen et al, 2008; Fletcher et al, 1998; Glahn et al, 2005; Goldberg et al, 1990; Liu et al, 2002; Mcdowell et al, 2002; Meyer-Lindenberg et al, 2001, 2002; Parellada et al, 1994, 1998; Perlstein et al, 2001; 2003; Ragland et al, 1998; Rubia et al, 1994; 2001; Schlösser et al, 2007; Steinberg et al, 1996; Volz et al, 1997; Weinberger et al, 1986; Yurgelun-Todd et al, 1996). Furthermore, there is evidence that the DLPFC dysfunction as described in schizophrenia is not solely explained by attentional or global cognitive impairment (Berman et al, 1988), nor is it a result of neuropsychiatric illness, generally, as patients with major depression (Barch et al, 2003; Berman et al, 1993) and Huntington's (Goldberg et al, 1990) do not exhibit this finding. However, abnormally increased DLPFC activation has also been reported (Callicott et al, 2003b; Manoach et al, 1999; 2000; Potkin et al, 2009; Thermenos et al, 2005), often in fMRI studies of higher performing patient cohorts, and has been consequently labeled as inefficient prefrontal processing (Callicott et al, 2003b; Manoach et al, 1999; Potkin et al, 2009) because greater activation is required to achieve a given performance level. Notably, some studies have found that better performing patients show more hyperactivation in the DLPFC whereas poorer performing patients hypoactivate the DLPFC (Callicott et al, 2003b; Karlsgodt et al, 2007, 2009; Manoach et al, 2000); however, even this behavioral–physiological relationship shows variability, being susceptible to dopaminergic manipulation (Daniel et al, 1991) and may differ from healthy volunteers (Karlsgodt et al, 2009). Reconciliation of these findings remains a matter of debate, but several authors have proposed that they rest in part on variation in task demands and individual performance capacities. This hypothesis features an inverted U-shaped load–response curve, such that as task demands increase, activation initially rises until physiological capacity is reached, after which, activation falls (Fletcher et al, 1998). In schizophrenia, this curve may be shifted to the left (Jansma et al, 2004; Perlstein et al, 2003), resulting in hyperactivation (ie, inefficient signal) at lower relative task loads (where performance matching with healthy volunteers is more attainable) and hypoactivation (ie, inadequate signal) at higher relative task loads (where performance is likely to be significantly worse in schizophrenia) (Callicott et al, 2003b; Manoach 2003). This curve may also be flattened in patients, resulting in less neural response to varying load levels (Johnson et al, 2006). Thus, more activation is not always better; rather, its significance depends on individual capacity and task load. Other investigations have emphasized the impact of greater morphological variability in schizophrenia. Such variability can affect the topographical distribution of activation patterns, which, in turn, can weaken group-averaged data for a given region, despite potentially equivalent or stronger activations at the individual level (Manoach, 2003; Park et al, 2004). Additional factors, such as specific task paradigm characteristics (Barbalat et al, 2009; Curtis et al, 1999; Holmes et al, 2005; Macdonald et al, 2005; Quintana et al, 2003), clinical heterogeneity, and medication status (see Weiss et al, 2003 versus Weiss et al, 2007, showing greater activation during a modified Stroop paradigm when medicated patients were studied, but the opposite finding when a separate cohort of unmedicated patients was studied), may also have a role. Regardless of the cause of directional discrepancies, because most studies of DLPFC connectivity (covariance with activity in other brain regions) show abnormal disconnection with other neocortical structures important for executive function (Bassett et al, 2008; Kim et al, 2003; Schlösser et al, 2003; Spence et al, 2000; Tan et al, 2006; Whitfield-Gabrieli et al, 2009; Wolf et al, 2007; Woodward et al, 2009; Yasuno et al, 2005) and because even patients who overactivate the DLPFC still often do not achieve a higher performance on executive tasks than their healthy control comparators, it is clear that DLPFC is dysfunctional in schizophrenia. In the context of the cellular pathological, structural, and neuroreceptor imaging DLPFC findings, such altered DLPFC physiology seems to be an expected and robust illness-related phenotype reflecting reduced neurophysiological resources in which microcircuits are either overtaxed or overwhelmed.
Ventrolateral Prefrontal Cortex
Though less well studied in schizophrenia than the DLPFC, the ventrolateral prefrontal cortex (VLPFC), judged to be preferentially involved in working memory storage and rehearsal processes rather than information manipulation (Wager and Smith, 2003), may show less cellular abnormalities. For instance, the increased density of pyramidal neurons in DLPFC does not seem to exist in the VLPFC (Selemon et al, 2003). Nonetheless, activation differences have been reported in schizophrenia during executive tasks including working memory (Callicott et al, 2003b; Scheuerecker et al, 2008; Schneider et al, 2007; Stevens et al, 1998; Tan et al, 2005), motor response inhibition (Kaladjian et al, 2007), and attentional tasks (Schneider et al, 2007), raising the question of exactly how this region contributes to executive processing networks in psychotic illness. In line with the theory that VLPFC is recruited in a compensatory fashion during DLPFC-taxing tasks in schizophrenia, patients have shown increased VLPFC activation in conjunction with reduced DLPFC activation during manipulation in a verbal working memory task (Tan et al, 2005). More recent data examining functional connectivity suggest that this potential compensatory mechanism cannot simply be described as increased relative activation, but rather, increased dominance and assumption of DLPFC's nodal role in extended executive circuitry. Tan et al (2006), for instance, used the n-back working memory fMRI paradigm to show that high-performing healthy control subjects evidenced greater DLPFC relative to VLPFC activation with greater working memory load, whereas volunteers with schizophrenia showed the opposite pattern. Remarkably, in control subjects, DLPFC showed more robust functional connectivity with a posterior parietal region, whereas in patients, the VLPFC showed greater parietal functional connectivity (Tan et al, 2006). Of note, this echoes report of abnormally increased 'structural connectivity' (the correlation between gray matter volumes of two or more brain structures across individuals) between ventral prefrontal cortex and inferior parietal lobule (IPL) in schizophrenia (Buchanan et al, 2004). Likewise, these results are similar to findings from a word-encoding fMRI paradigm, in which schizophrenic patients showed reduced DLPFC-temporal and increased VLPFC-temporal functional connectivity (Wolf et al, 2007). Thus, increased VLPFC relative to DLPFC prominence in executive neural networks may characterize altered and often inadequate (by behavioral performance measures) circuit-level strategies in schizophrenia during DLPFC-activating executive tasks.
Anterior Cingulate
As in DLPFC, postmortem experiments in schizophrenia have identified alterations in the neurons of the anterior cingulate cortex (ACC), a paralimbic structure also within the frontal lobes. These include abnormalities in a wide range of proteins (Clark et al, 2006), increased glutamatergic vertical fibers—presumably associative afferents—in layers II and IIIa (Benes et al, 1992b), reduced layer IV pyramidal cell density (Benes et al, 2001), and a number of findings related to GABAergic neurons (Torrey et al, 2005), such as reduced concentration of neurons expressing GAD67 mRNA (Woo et al, 2004) but increased superficial layer GABAA receptor binding (Benes et al, 1992a), though this last finding was not seen in receptor imaging studies in vivo (Verhoeff et al, 1999). Corroborating experiments using structural and spectroscopic MRI have further documented reductions in anterior cingulate volume (Baiano et al, 2007; Goldstein et al, 1999), gray matter concentration (Kubicki et al, 2002; Meda et al, 2008; Rüsch et al, 2007), and NAA levels (Wood et al, 2007), as well as increased glutamine (Theberge et al, 2002). Additionally, PET studies have shown reduced D2/3 binding in this region (Buchsbaum et al, 2006; Suhara et al, 2002; Yasuno et al, 2005). Many of these volumetric (Szeszko et al, 2000), morphometric (Eack et al, 2008), spectroscopic (Ohrmann et al, 2008), and neuroreceptor (Ko et al, 2009; Lumme et al, 2007) indices have shown robust relationships with executive functioning in healthy control subjects.
In light of these findings, it is perhaps not surprising that executive functions reliant on anterior cingulate activity elicit abnormal ACC responses in schizophrenia. For example, during tasks that include conflict and error monitoring, subjects with schizophrenia show both worse performance (in select, but not all, studies: less error-related reaction time slowing, posterror behavioral adjustments and more errors) and less anterior cingulate activation (Andreasen et al, 1992; Carter et al, 1997, 2001; Dolan et al, 1995; Ford et al, 2004; Kerns et al, 2005; Krabbendam et al, 2009; Laurens et al, 2003; Polli et al, 2008; Rubia et al, 2001; Salgado-Pineda et al, 2004; Volz et al, 1999; Weiss et al, 2007; Yucel et al, 2002; but see Weiss et al, 2003), suggesting a deficit in self-monitoring processes required to signal conflicts between response and maintained rule representations (Macdonald et al, 2000). Similar reductions in anterior cingulate activation during verbal fluency tasks have also been reported in several (Boksman et al, 2005; Broome et al, 2009; Fletcher et al, 1996; Fu et al, 2005), but not all (Ragland et al, 2008), investigations. It is notable that dopamine agonist administration results in marked augmentation of ACC activation during verbal fluency in schizophrenia, but not healthy volunteers (Dolan et al, 1995), implicating either aberrant modulatory mesencephalic input to this region and/or postsynaptic dopaminergic signaling dysregulation in this region. Robust evidence for augmented basal ganglia sensitivity to dopamine agonists in schizophrenia (Abi-Dargham et al, 1998; Laruelle et al, 1996) offers circumstantial support for the former hypothesis. As the anterior cingulate shows heterogeneous functional topography, it is important to note that the majority of the above-cited findings localize to the dorsal anterior cingulate, consistent with a more cognitive specialization of this region (Drevets and Raichle, 1998), though a few reports also feature rostral anterior cingulate findings (Laurens et al, 2003; Polli et al, 2008), perhaps reflecting motivational components of task performance and monitoring (Polli et al, 2008). The absence of subgenual anterior cingulate cortical findings suggest that this region likely does not have an important function in executive task performance, in line with its predominantly affective role (Drevets and Raichle, 1998).
Given the above-cited cellular, structural, and functional abnormalities in ACC and DLPFC, effective neural cooperation between these structures in the service of executive processing in schizophrenia is critical but unlikely. Indeed, preclinical and clinical studies are both suggestive of disrupted DLPFC–ACC communication. Efferent projections from the anterior cingulate (BA32) synapse both on excitatory and inhibitory target cells in the supragranular layers of DLPFC (BA9), the latter being predominantly calbindin-positive GABAergic neurons (Medalla and Barbas, 2009) that inhibit distal pyramidal spines in the theorized service of dampening distracting stimuli (Wang et al, 2004). This is in contrast to projections within DLPFC regions (BA46 BA9), in which inhibitory targets are less robust and more frequently calretinin-positive cells that synapse on inhibitory interneurons, thereby promoting disinhibitory effects on DLPFC pyramidal cells (Medalla and Barbas, 2009). Notably, the density of calbindin-positive, but not calretinin-positive, GABAergic neurons may be reduced in the superficial layers of the DLPFC in schizophrenia (Beasley et al, 2002; Sakai et al, 2008) but see (Daviss and Lewis, 1995; Tooney and Chahl, 2004), suggesting one potential basis for disrupted ACC–DLPFC neural transmission, resulting in increased noise at the level of higher-order cognitive representations (Winterer et al, 2004). Likewise, reciprocal connections from DLPFC and other cortical regions to the ACC may also be affected as suggested indirectly by superficial cortical layer abnormalities within the ACC (Benes et al, 1992a, 1992b). Reduced white matter integrity (fractional anisotropy measured by DTI) in the cingulum bundle, which shows a relationship with impaired Wisconsin Card Sorting Task performance (Kubicki et al, 2003), offers another reason to predict altered communication between the anterior cingulate and prefrontal regions. In any case, frontocingulate functional dysconnectivity has been explicitly described during verbal fluency (Spence et al, 2000) and modified continuous performance tasks (Honey et al, 2005) in schizophrenia. Further, structural equation modeling of regional D2/3 receptor binding has shown altered connectivity from other frontal cortical regions (as well as thalamus and parietal cortex) to the anterior cingulate (Yasuno et al, 2005).
EXTENDED EXECUTIVE CIRCUITS
Despite historical emphasis on frontal circuits in investigations aimed at understanding cognitive pathophysiology in schizophrenia, recent studies have amassed considerable evidence that a systems-level disruption, including but not limited to frontal cortical dysfunction, is at play. During executive tasks, functional neuroimaging of patients shows abnormal activation not only in the frontal lobes, but also similarly in other distributed brain regions typically recruited by executive task demands (Jansma et al, 2004). Several of these regions have also shown cellular, structural, or neurochemical abnormalities in schizophrenia and include (1) the IPL, which has consistently shown significant contributions to a range of executive functions in neurophysiological experiments and may be a particularly important support to frontal executive circuits as a working memory storage buffer (Jonides et al, 1998); (2) the medial temporal cortex/hippocampus, which may provide specific contextual/stimulus–stimulus association consolidation for abstract rule establishment during select executive tasks, such as the Wisconsin Card Sort (Graham et al, 2009), but is normally suppressed during other executive functions (eg, working memory); (3) the basal ganglia/caudate, which is important for cognitive flexibility (Eslinger and Grattan, 1993), and along with the thalamus, may provide a gating function for prefrontal-bound information during working memory (Frank et al, 2001; Landau et al, 2009); and (4) the thalamus, which is an essential pathway within cortico-striatal-thalamic-cortical loops and shows prefrontal-like participation in working-memory-related neural transmission (Tanibuchi and Goldman-Rakic, 2003). Furthermore, particular disturbances of communication among these and frontal regions, often measured through fMRI or PET functional connectivity methodologies, suggest inefficient circuit dynamics that may underlie executive dysfunction. Thus, studies in recent years have increasingly attended to extrafrontal regions both to show novel cellular and molecular biological markers of disease, and to understand the critical contributions of extrafrontal regions to these circuits.
Inferior Parietal Lobule
Though preclinical data implicating the IPL in schizophrenia are scarce, structural imaging findings in this region are not. Reductions in parietal gray matter volume in schizophrenia relative to healthy individuals have been reported in a handful of studies (Buchanan et al, 2004; Frederikse et al, 2000; Goldstein et al, 1999; Hulshoff Pol et al, 2001; Kubicki et al, 2002; Nierenberg et al, 2005; Schlaepfer et al, 1994; Wolf et al, 2008; Zhou et al, 2007) and are more pronounced in patients with passivity delusions (Maruff et al, 2005) and greater cognitive impairment (Wolf et al, 2008). Schizophrenia patients also show significantly greater structural variability (Yoon et al, 2006) and reversed or absent hemispheric asymmetry (Buchanan et al, 2004; Niznikiewicz et al, 2000; Zhou et al, 2007) in this region. Reductions in parietal white matter have also been found in patients with prominent negative symptoms (Zetzsche et al, 2008). It is notable that child onset schizophrenia patients show early and accelerated parietal volume loss over time (Thompson et al, 2001).
Regions in the IPL (BA 40), in addition to lateral prefrontal cortices and anterior cingulate, show reliable activation during prototypical executive function tasks, such as the Wisconsin Card Sorting Test, as well as during component executive processes, such as response inhibition and set shifting (Buchsbaum et al, 2005). In conjunction with structural imaging evidence for abnormalities in this area and executive dysfunction in schizophrenia, this would predict parietal functional deficits detectible during executive task performance. Indeed, akin to findings in the prefrontal cortex, reductions in parietal activation during working memory (Barch and Csernansky, 2007; Broome et al, 2009; Jansma et al, 2004; Kindermann et al, 2004; Schlagenhauf et al, 2008; Schlösser et al, 2007; Schneider et al, 2007), semantic integration (Kuperberg et al, 2008), and selective attention (modified Stroop) (Weiss et al, 2007) have been commonly observed in schizophrenia subjects (but see Lee et al, 2008; Ragland et al, 2008; Thermenos et al, 2005, showing increases). Recent data also suggest the possibility that hallucinating patients may have less working-memory-associated parietal activation than nonhallucinating patients (Wible et al, 2009).
The inferior parietal and prefrontal cortices share key involvement in executive processing and important anatomical connections. In view of both of these regions' structural and functional abnormalities in schizophrenia, it is likely that communication between these structures, particularly during executive tasks, is abnormal as well in patients. The superior longitudinal fasciculus, which links parietal and prefrontal cortical areas, shows reduced fractional anisotropy, a measure of white matter integrity, in schizophrenia (Shergill et al, 2007) suggestive of impaired prefrontal–parietal interactions. This notion has been advanced by several functional connectivity studies as well: for instance, DLPFC–IPL connectivity during the n-back working memory task is reduced in schizophrenia (Kim et al, 2003; Tan et al, 2006), though the results of two other studies have been mixed (Barch and Csernansky, 2007; Schlosser et al, 2003). Similarly, during a choice reaction-time test (Woodward et al, 2009) and the AX version of the continuous performance task (Yoon et al, 2008), both of which require less executive resources than the n-back working memory test, prefrontal–IPL connectivity is also reduced in schizophrenia. Even resting state regional glucose metabolism shows this pattern (Mallet et al, 1998), substantiating the pervasive nature of this functional disconnection.
Temporal Cortex/Hippocampus
The medial temporal cortex has been the focus of a large number of investigations and findings of regional pathological changes in schizophrenia. Postmortem examination of the hippocampal formation has shown a number of abnormalities in schizophrenia, including reduced pyramidal cell size (Arnold et al, 1995; Benes et al, 1991; Zaidel et al, 1997) (but see Highley et al, 2003), reduced dendritic spine density (Rosoklija et al, 2000), reduced spinophilin mRNA expression (Law et al, 2004), reduced microtubule-associated proteins (Arnold et al, 1991), reduced BDNF (Durany et al, 2001), reduced mossy fiber terminal density (Kolomeets et al, 2007), reduced synaptic protein levels (Browning et al, 1993; Sawada et al, 2005; Young et al, 1998), alterations in NMDA receptor subtypes (reduced NR1 and increased NR2B) (Gao et al, 2000) and reduced non-NMDA ionotropic glutamate receptors (Harrison et al, 1991), as well as reduced mRNA expression of DISC1 binding partners (FEZ1, NUDEL, and LIS1) (Lipska et al, 2006b), among others.
Hippocampal volume reductions in schizophrenia have been shown by both voxel-based and ROI methodologies (Honea et al, 2005; Nelson et al, 1998; Weiss et al, 2005; Wright et al, 2000) are seen even when compared with patients' unaffected monozygotic twins, implicating nongenetic contributions to this finding (Suddath et al, 1990), and are present at the onset of psychosis (Bogerts et al, 1990). Furthermore, in patients, but not healthy individuals, hippocampal volume predicts the degree of prefrontal hypoactivation during the Wisconsin Card Sorting Test (Weinberger et al, 1992), leading to the hypothesis that fronto-limbic circuits may be particularly central to schizophrenia pathophysiology linked to cognitive dysfunction. Compelling rodent models have elaborated on this interaction: neonatal ventral hippocampal lesions in rodents disrupt medial temporal–prefrontal afferentation and produce numerous schizophrenia-like phenotypes after adolescence (Lipska and Weinberger, 2000) including working memory deficits (Lipska et al, 2002) and reduced prefrontal NAA (Bertolino et al, 2002), suggesting that, in fact, medial temporal lobe afferentation is critical to prefrontal cortical development and subsequent executive processing. Additionally, reductions in fractional anisotropy of temporal white matter, including the fornix (Fitzsimmons et al, 2009) and inferior longitudinal fasciculus (Ashtari et al, 2007), suggest compromised integrity of key bidirectional white matter tracts of the hippocampus, including those that communicate with the prefrontal cortex.
On the framework of these observations, recent functional imaging experiments have uncovered abnormalities of hippocampal–prefrontal interactions during executive tasks, particularly working memory, in schizophrenia. During the n-back working memory task, which is not thought to rely substantially on hippocampal processing, the hippocampus is deactivated and disengaged from prefrontal and inferior parietal regions (Meyer-Lindenberg et al, 2005b). However, patients with schizophrenia show impaired suppression of this region in the contexts of hypoactivated DLPFC (Meyer-Lindenberg et al, 2001), hyperactivated VLPFC (Thermenos et al, 2005), or hyperactivated basal ganglia (Kawasaki et al, 1992). Precise examination of hippocampal–DLPFC interactions during the 0-back sensorimotor task in health and in schizophrenia shows an inverse correlation between these regions; however, in patients, but not healthy volunteers, this relationship remains inappropriately robust and regionally specific during the 2-back working memory condition (Meyer-Lindenberg et al, 2005b). As noted by Meyer-Lindenberg et al, impairment in modulating fronto-limbic circuitry in response to executive challenge could be predicted by an etiological model (Lipska et al, 2002) centered on early abnormal hippocampal physiology and connectivity resulting in subsequent retarded maturation of DLPFC and aberrant reciprocal innervation back to the hippocampus. Continued investigation of this hypothesis will require more direct studies of frontohippocampal circuitry during executive task challenge and will need to address other aspects of executive circuit abnormalities, including the role of medial temporal and prefrontal dopaminergic signaling (Aalto et al, 2005) in relation to basal ganglia function (Saunders et al, 1998).
Basal Ganglia
Postmortem examinations of the neostriatum implicating its involvement in schizophrenia have reported several findings, including: increased corticostriatal dendritic spine density (Kung et al, 1998; Roberts et al, 2005, 2008), reduced axonic mitochondria (Kung and Roberts, 1999), reduced GABA and glutamate uptake sites (Simpson et al, 1992), reduced cholinergic interneurons (Holt et al, 1999), increased dopamine concentrations (Mackay et al, 1982), and increased D2/3 and more robustly D4 receptors (Mackay et al, 1982; Murray et al, 1995; Seeman et al, 1993).
In vivo PET imaging studies have found upregulation of striatal D2 receptors as well, even in medication-nave patients (Wong et al, 1986). Though there have been several negative studies, the weight of the literature supports an effect (Kestler et al, 2001; Laruelle, 1998). Striatal presynaptic dopamine synthesis and storage, measured by PET L-DOPA radiotracers, is increased in the schizophrenia prodrome (Howes et al, 2009) and in patients who fulfill full diagnostic criteria regardless of medication status (medicated, medication free, and neuroleptic naïve) (Hietala et al, 1995, 1999; Lindström et al, 1999; Mcgowan et al, 2004; Meyer-Lindenberg et al, 2002; Nozaki et al, 2009; Reith et al, 1994) (but see Dao-Castellana et al, 1997, showing only greater variability in patients and Elkashef et al, 2000 showing decreases in ventral striatum). Greater amphetamine-induced striatal dopamine release (D2 receptor radioligand displacement) in schizophrenia, measured by PET and SPECT, has also been well documented (Abi-Dargham et al, 1998; Breier et al, 1997; Laruelle et al, 1996). Taken together, these results establish abnormally heightened dopaminergic signaling in the striatum in schizophrenia.
As functional imaging studies have outlined a role for the striatum, and the caudate in particular, in spatial working memory (Postle and D'Esposito, 1999), planning (Owen et al, 1996), interference management (Vernaleken et al, 2007), and verbal working memory (Chang et al, 2007; Koch et al, 2008; Landau et al, 2009; Lewis et al, 2004; Rypma et al, 1999), striatal disinhibition may contribute to executive dysfunction in schizophrenia. This is in accord with the anatomy of basal ganglia-thalamo-cortical tracts, which features significant innervation of the above-discussed prefrontal regions (Middleton and Strick, 2002), and with the working memory deficits that arise from anterior neostriatal lesions in nonhuman primates, which can be remarkably similar to deficits seen with prefrontal lesions (Goldman and Rosvold, 1972). Conversely, striatal disinhibition in schizophrenia may be compensatory for or directly result from prefrontal dysfunction, as reciprocal corticostriatal modulation of the basal ganglia is also robust in the healthy individual. Bolstering prefrontal dopaminergic signaling with locally administered dopamine agonists results in reduced striatal dopamine release (Jaskiw et al, 1991; Kolachana et al, 1995). Likewise, frontal lesions result in exaggerated striatal dopamine release (Flores et al, 1996; Jaskiw et al, 1990a, 1990b; Pycock et al, 1980), and surgical disconnection of frontostriatal circuitry impairs delayed alternation task performance in rats (Dunnett et al, 2005). Thus, to what degree abnormal neural activity in the striatum during executive functioning in schizophrenia is a primary or secondary phenomenon remains an unresolved question.
Nonetheless, hypothesizing that frontostriatal circuits are dominant (Pantelis et al, 1997) and specific (Badcock et al, 2005) contributors to schizophrenic cognitive impairments, a number of investigations have elucidated frontostriatal circuit abnormalities relevant to executive dysfunction in schizophrenia. Indirect evidence from functional imaging studies has been suggestive of striatal dysfunction (hyperactivation) in schizophrenia during inhibition tasks (Rubia et al, 2001), verbal fluency (Ragland et al, 2008), numeric working memory (Manoach et al, 2000), and the Wisconsin Card Sort Task (Kawasaki et al, 1992; Rubin et al, 1991, 1994). Perhaps the strongest evidence for the frontostriatal hypothesis comes from multimodal imaging approaches. Reduced DLPFC NAA shows an inverse relationship with amphetamine-induced striatal dopamine release, measured with [11C]raclopride PET, in patients with schizophrenia but not control subjects (Bertolino et al, 2000a). Though DLPFC NAA has been related to executive functioning in schizophrenia (Bertolino et al, 2000b), better characterization of striatal dysregulation and disturbed prefrontal physiology requires in vivo examination of both of these factors. This was recently achieved by Meyer-Lindenberg et al who studied schizophrenia patients and healthy volunteers with both [15O]H2O PET during the Wisconsin Card Sorting Task and [18F]DOPA PET. Patients showed greater striatal presynaptic dopamine synthesis and storage and reduced prefrontal activation during the Card Sort compared with healthy individuals, and moreover, in patients these two abnormalities were highly correlated (Meyer-Lindenberg et al, 2002). These remarkable and predicted associations invite speculation that breakdowns in prefrontal neuronal integrity and function result in impaired restraint on striatal circuits, yielding an inflexible, dysregulated circuit. However, given the correlative nature of these findings, further testing is needed to establish causality.
Thalamus
As the thalamus is a central entry point for frontal cortex-bound projections, including those from the striatum, there has been great interest in investigating this region for pathology in schizophrenia. Reductions in mediodorsal nucleus volume (Byne et al, 2002), neuronal number (Pakkenberg, 1990; Popken et al, 2000), and multiple ionotropic glutamatergic receptor types (NMDA, AMPA, Kainate) exist in thalamic nuclei (most prominently, mediodorsal and centromedial) of schizophrenia postmortem tissue (Ibrahim et al, 2000). Upregulation of exitatory amino-acid transporter (types 1 and 2) (Smith et al, 2001a) and vesicular glutamate transporter (Smith et al, 2001b) has also been reported in the same regions.
In vivo data showing illness-associated thalamic volume reductions (Andreasen et al, 1994a; Gur et al, 1998; Hulshoff Pol et al, 2001; Konick and Friedman, 2001) and reduced NAA measured by magnetic resonance techniques (Auer et al, 2001; Deicken et al, 2000; Ende et al, 2001) align well with reports of cellular neuropathological findings in the mediodorsal nuclei of schizophrenic patients. Reductions in D2/3 receptor binding in the mediodorsal and pulvinar thalamic nuclei have also recently been documented (Buchsbaum et al, 2006; Talvik et al, 2003).
As thalamic activity is associated with the working memory (Callicott et al, 1999; Rypma et al, 1999), the Wisconsin Card Sorting Task (Goldberg et al, 1998), and the verbal fluency (Basho et al, 2007), pathological changes in this region in schizophrenia predict abnormal physiological responses to executive challenge. Indeed, hypoactivation of this region during working memory tasks (Andrews et al, 2006; Camchong et al, 2006; Mendrek et al, 2004; Schlösser et al, 2008) has been well replicated (though, see Manoach et al, 2000 showing hyperactivation). The mediodorsal nucleus of the thalamus shows reduced glucose metabolism in patients performing a modified California Verbal Learning Test (Hazlett et al, 2004) and manifests reduced connectivity with both regions in the DLPFC and medial temporal lobe (Mitelman et al, 2005). This agrees well with the possibility of structural derangement of prefrontal- and anterior cingulate-thalamic connections, as suggested by recent DTI studies using tractography (Kunimatsu et al, 2008) and fractional anisotropy measurements (Zou et al, 2008). In contrast, schizophrenia patients have shown increased thalamo-ventrolateral prefrontal and thalamo-dorsolateral prefrontal connectivity by structural equation modeling during an fMRI n-back paradigm (Schlösser et al, 2003). Though Schlosser and Mitelman used very different methodologies, it remains unclear how to reconcile their opposing results without additional experimentation.
THE INFLUENCE OF SCHIZOPHRENIA RISK GENES ON EXECUTIVE CIRCUITRY
As schizophrenia is highly heritable (Cardno and Gottesman, 2000), and healthy relatives of patients show executive task impairments and associated neuroimaging phenotypes, which are qualitatively similar to their affected family member but attenuated (Callicott et al, 2003a; Macdonald et al, 2008), and given a core role for executive dysfunction in schizophrenia, it is likely that functional variation in specific schizophrenia risk genes will impact aspects of the above-reviewed neurocircuit dynamics in predictable ways. Building on the endophenotype approach originally proposed by Gottesman and Sheilds (1972), recent advances in imaging genetics have begun to provide remarkably convergent evidence supporting this hypothesis, as delineated below (see Table 1). Such advances are crucial, in part, because among the multitude of molecular pathways impacting the interacting neural systems relevant to schizophrenia, any one candidate risk gene variant is likely to contribute only a nominal effect to the complex behavioral phenotype that establishes the clinical diagnosis, and the gene variants discussed here are no exception. However, the experiments reviewed below have nonetheless been able to detect robust genetic effects by using neuroimaging techniques to assay 'intermediate' phenotypes at the neural systems level—a level of organization that is closer to the actual impact of a single gene variation—rather than measuring diagnosis itself (Mier et al, 2009). One particular strength of this approach is the ability to examine risk gene effects in healthy individuals that do not possess many of the confounds inherent in studying patients, such as medication exposure and psychotic symptoms, which has resulted in the majority of studies employing healthy populations; but by the same token, much work is still needed to better understand the effects of these genetic variants in the complex clinical and genetic context of schizophrenia.
COMT
Variation in the gene coding for catechol-O-methyltransferase (COMT), an enzyme central to cortical synaptic dopamine catabolism modestly influences risk of illness and has garnered significant attention for providing insight into the biological underpinnings of the imaging phenotype of schizophrenia. The rs4680 single nucleotide polymorphism (SNP) has been best studied, and the valine risk allele confers thermostability, permitting greater enzymatic activity and thereby reduced dopaminergic tone in cortical synapses. In a seminal paper by Egan et al and subsequent replications, the valine risk allele reliably predicts worse performance but increased dorsolateral prefrontal and anterior cingulate physiological response to the n-back task in both schizophrenic individuals and their unaffected siblings (Egan et al, 2001). This work has been extended to show that predicted prefrontal dopaminergic tone by combined genotype and pharmacological condition follow an inverted U-shaped response during working memory, such that risk allele homozygotes have improved and protective allele homozygotes have worse prefrontal efficiency in response to amphetamine (Mattay et al, 2003). Notably, functional variation in the COMT gene is not limited to the rs4680 SNP, but rather includes other polymorphisms, including a P2 promoter region SNP and a 3' region SNP. These three SNPs show nonlinear interacting effects on prefrontal efficiency during working memory task performance, in agreement with predictions of resultant cortical dopaminergic catabolic rates, and highlight the complexity of genetic contributions to functional neuroimaging phenotypes, even within a single gene (Meyer-Lindenberg et al, 2006). To add to this complexity, the ability of COMT to regulate cortical dopamine relies on other genetically determined cellular resources, as suggested by studies of MTHFR by Roffman et al (2008). Variation in MTHFR (rs1801133), which also shows association with schizophrenia risk (Gilbody et al, 2007), regulates the availability of methyl groups for use by COMT and, in combination with rs4680, predicts DLPFC activation during working memory in a manner consistent with the above-mentioned inverted U-shaped curve. Taken together, these data provide further support for the proposition that suboptimal prefrontal dopamine signaling contributes to the prefrontal imaging phenotypes of executive dysfunction in schizophrenia.
Importantly, recent investigations have expanded this line of inquiry to assess the impact of genetically defined cortical dopamine tone on distributed circuitry relevant to executive function, and a number of results have emerged that are consistent with the data and putative mechanisms regarding schizophrenia itself, reviewed above. For instance, during the n-back working memory task, valine carriers show increased ventrolateral relative to dorsolateral prefrontal engagement and increased ventrolateral relative to dorsolateral connectivity with parietal regions, as had been seen in schizophrenia earlier (Tan et al, 2007). Additionally, just as inappropriate prefrontal–hippocampal coupling persists during working memory in schizophrenia patients (Meyer-Lindenberg et al, 2005b), during a recognition memory task that activates the hippocampus, carriers of COMT rs4680 valine alleles show disadvantageous increased prefrontal–hippocampal connectivity (Bertolino et al, 2006). Finally, in agreement with the above-highlighted frontostriatal circuit abnormalities in schizophrenia, particularly disinhibited presynaptic striatal dopaminergic signaling in association with DLPFC hypofunction, postmortem data show that COMT valine alleles predict increased tyrosine hydroxylase mRNA expression in the midbrain (Akil et al, 2003), origin of dopaminergic projections to the striatum. Corroborating this effect are in vivo data describing COMT genotype effects on the relationship between midbrain dopamine storage and prefrontal activation during the n-back task: in met homozygotes, this relationship was negative, but in val carriers, it was positive (Meyer-Lindenberg et al, 2005a). This has been interpreted as a downstream effect of genetically conferred variation of prefrontal dopaminergic neurotransmission, as midbrain relative to cortical COMT expression is weak (Kastner et al, 1994), such that suboptimal prefrontal output to mesencephalic inhibitory cells results in exaggerated activity of dopamine neurons projecting to the striatum.
RGS4
RGS4 is an important modulator of central dopamine, glutamate, and neuregulin G-protein receptor systems, and transcript expression in the DLPFC of schizophrenia patients has been shown to be reduced (Mirnics et al, 2001). An SNP (rs951436 C A) in the gene coding for this protein is associated with both schizophrenia (Chowdari et al, 2002) and reductions in DLPFC volumes (Prasad et al, 2005). Buckholtz et al (2007a, 2007b) studied this risk SNP in a large group of healthy individuals undergoing functional MRI scans during the n-back task and found that individuals carrying more risk alleles evidenced greater activation in the left ventrolateral PFC, but less activation in the right lateral PFC, temporal cortex, and caudate (Buckholtz et al, 2007a). Similar to investigations in COMT (Tan et al, 2007) and schizophrenia itself (Tan et al, 2006), examination of functional connectivity between these differentially activated nodes showed that risk alleles impaired cooperativity between right hemispheric nodes activated by the task (eg, DLPFC, PPC) but exaggerated cooperativity between VLPFC and nodes deactivated by task (eg, mPFC, superior temporal cortex, posterior cingulate, and parahippocampal gyrus) (Buckholtz et al, 2007a). Notably, when regional brain activations during the n-back task are examined with consideration of both COMT and RGS4 genotypes, there exists an epistatic interaction, such that RGS4 risk allele-associated greater DLPFC and midbrain activation occurs only in the context of COMT risk allele carriers (Buckholtz et al, 2007b). Regardless of whether this interaction occurs biologically at the molecular (eg, COMT regulating RGS4 gene expression, Lipska et al, 2006a) or systems level (eg, inefficient executive circuits being more susceptible to RGS4 effects) (Buckholtz et al, 2007b), these data highlight the complex contribution of schizophrenia risk gene networks to executive processing.
GRM3
An SNP in the gene coding for the metabotropic type II glutamate receptor mGluR3, GRM3 (rs6465084), results in weakly increased risk for schizophrenia, reduced prefrontal excitatory amino-acid transporter 2 mRNA expression (EEAT2), worse verbal fluency performance, and reduced DLPFC neuronal integrity as measured by magnetic resonance spectroscopy (Egan et al, 2004; Marenco et al, 2006). As in COMT, during the n-back working memory task, greater DLPFC BOLD signal activation for the same performance level ('prefrontal inefficiency') is seen in carriers of the risk SNP (Egan et al, 2004). However, this finding of GRM3 risk allele-associated prefrontal inefficiency during working memory, as in RGS4, has been replicated in COMT rs4680 risk allele carriers but not in methionine homozygotes, suggesting an epistatic interaction between these two risk genes. Furthermore, carriers of both COMT and GRM3 risk alleles show disproportionately greater VLPFC over DLPFC connectivity with parietal regions activated by this task (Tan et al, 2007), similar to the schizophrenia phenotype (Tan et al, 2006).
PPP1R1B
Dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32) is abundant in the striatum and has a key function in modulating dopaminergic postsynaptic intracellular signaling through multifaceted effects on protein kinases (Svenningsson et al, 2004). One common haplotype in the PPP1R1B gene coding for DARPP-32 shows an association with schizophrenia, with worse IQ, verbal fluency, working memory, and Wisconsin Card Sorting performance, with reduced striatal volumes, with reduced striatal BOLD activation during the n-back, and with increased frontostriatal connectivity. Notably, both the activation and connectivity findings were replicated in a separate cohort during performance of an emotional face-matching task (Meyer-Lindenberg et al, 2007).
PRODH
A functional haplotype (rs4819756 and rs2870983 and rs450046 minor alleles) in the proline oxydase gene, PRODH, shows increased enzymatic activity, risk for schizophrenia, diminished striatal volumes, reduced striatal BOLD activation, and increased frontostriatal connectivity during the n-back task (Kempf et al, 2008). Despite significant differences between the functions of proline oxydase and DARPP-32, these results are remarkably similar to those of PPP1R1B and converge on circuitry (prefrontal-neostriatal) that is dysregulated in schizophrenia (Meyer-Lindenberg et al, 2002).
AKT-1
AKT-1 is an intracellular signaling protein that has an important function in dopamine-mediated neurotransmission (Beaulieu et al, 2005; Wei et al, 2007) and has shown reduced expression in schizophrenic brains (Emamian et al, 2004) and lymphocytes (Tan et al, 2008). Further, several reports have found an association between a functional AKT-1 genetic variations and schizophrenia (Emamian et al, 2004; Tan et al, 2008). One such variation, an SNP, rs1130233, additionally shows a relationship with neuropsychological assessments of executive function as well as n-back-related prefrontal activation. The risk allele also imparts reduced prefrontal and caudate volumes, in agreement with its hypothesized impact on frontostriatal circuitry, though formal testing of functional connectivity has not been performed at this date (Tan et al, 2008).
DISC-1
The disrupted in schizophrenia (DISC-1) gene codes for a protein abundant in the hippocampus, which partners with Nudel and other dynein complex proteins to impact centrosomal function, neurite outgrowth, and neuronal migration (Kamiya et al, 2005). Variations in DISC-1 are associated with schizophrenia (Callicott et al, 2005; Ekelund et al, 2004; Hennah et al, 2003; Hodgkinson et al, 2004), and recent multimodal imaging data have evidenced an effect of the DISC-1 Ser704Cys polymorphism on hippocampal structure and function in healthy adults (Callicott et al, 2005). Specifically, serine homozygotes showed reduced hippocampal gray matter volume, lower hippocampal N-acetyl aspartate, and during the n-back working memory task, abnormally greater hippocampal activation (Callicott et al, 2005). These results align well with the impaired suppression of medial temporal lobe activity during executive processing seen in schizophrenia (Meyer-Lindenberg et al, 2001). Furthermore, during verbal fluency task performance, serine homozygotes show increased prefrontal activation (Prata et al, 2008), though to what degree frontotemporal connectivity is directly influenced by this polymorphism remains to be tested.
ZNF804A
In a recent genome-wide association study, an SNP (rs1344706) in ZNF804A, a gene coding for a protein of unclear function but potential gene regulatory ability, showed independent, significant association with schizophrenia (O'Donovan et al, 2008). Comparing healthy individuals with either no, one, or two risk alleles, Esslinger et al (2009) have found that the number of risk alleles predicted greater prefrontal–hippocampal functional connectivity during the n-back working memory task, just as had been described earlier in patients (Meyer-Lindenberg et al, 2005b), reinforcing the fact that greater functional connectivity (especially with a dysfunctional prefrontal cortex, as in schizophrenia), not only less, can be the risk phenotype. Better understanding of the biology of ZNF804A is needed to clarify the nature of this observation, but it is nonetheless remarkable that a risk gene without a priori evidence for either prefrontal or hippocampal involvement can so clearly show a predicted illness circuit phenotype in this way.
NRG-1
Neuregulin1 (NRG-1) isoforms and its receptor ErbB4 have important functions in potentially illness-relevant neural processes, including neuronal migration, axonal guidance and myelination, synaptic plasticity, and glutamatergic dendritic spine maturation (Barros et al, 2009; Mei and Xiong, 2008). Variation in the NRG-1 gene has shown association with schizophrenia diagnosis, behavioral abnormalities in mouse models responsive to antipsychotic medication (Li et al, 2006; Stefansson et al, 2002), and altered neuregulin isoform expression (Law et al, 2006).
In a group of individuals at high risk of developing schizophrenia by virtue of strong family history, carrier status of an NRG1 risk allele (SNP8NRG243177 polymorphism, which influences neuregulin transcript expression, Law et al, 2006) predicted development of psychotic symptoms as well as reduced activation in medial prefrontal and temporo-occipital regions during a sentence completion task (Hall et al, 2006).
The number of NRG-1 risk alleles carried in healthy adults correlates with reduced semantic verbal fluency performance and reduced anterior cingulate, inferior frontal, and middle temporal activation measured by fMRI BOLD signal (Kircher et al, 2009). Disrupted microstructural connectivity in association with the risk allele of this same polymorphism is evidence by reduced white matter density and fractional anisotropy in the anterior limb of the internal capsule (Mcintosh et al, 2007), which contains important axonal fibers linking the prefrontal cortex with other nodes in the extended executive network. Future work is needed to confirm these findings and determine to what degree these abnormalities explain functional differences in individuals with different allelic risk loads.
SUMMARY AND FUTURE DIRECTIONS
Key brain regions that show postmortem and in vivo evidence for disarray in schizophrenia are important in executive functioning, and are physiologically abnormal during executive challenge in patients, evidence characteristically aberrant interactions and remarkable susceptibility to variation in putative schizophrenia risk genes. DLPFC dysfunction and aberrant functional connectivity, relatively increased VLPFC involvement in executive circuitry, ACC, and IPL dysfunction and reduced coupling with DLPFC, impairment in suppression of medial temporal activity during certain executive challenges, prefrontal disinhibition of mesostriatal dopaminergic signaling, and reduced thalamofrontal cooperativity not only form a complex landscape of circuit changes in schizophrenia, but also, in selected subsets of these, create quantifiable links to emerging molecular footprints of genetic predisposition to psychosis. Systematic work is needed to better characterize the dynamics of these systems-level abnormalities in response to particular executive task demands, pharmacological interventions, and genetic environments.
Specifically, several avenues of research promise to provide invaluable insights into pathophysiology and ultimately targeted treatment of this devastating illness.
To address accumulating evidence of genetic heterogeneity underlying the disorder and concomitant variability in psychopathological and neuropsychological profiles, all of which may have contributed to apparent inconsistencies in the literature, more extensive genetically, clinically, and cognitively stratified studies are necessary. Likewise, longitudinal studies directed at understanding both naturalistic and pharmacologically induced fluctuations in executive network function are essential to assess the stability of circuit perturbations in schizophrenia over the course of illness and treatment. Additionally, developing advanced methodologies to bridge molecular and physiological data and fuel both candidate risk gene discovery and biological validation has become increasingly important. One such approach is to use neurocircuit risk phenotypes as quantitative trait variables to identify genetic factors contributing to executive dysfunction in psychotic disorders. Potkin et al (2008) have begun to implement this strategy with DLPFC activation alone as the quantitative trait variable, yielding novel results. As efforts to characterize and quantify the above-outlined systems-level circuitry disruptions in schizophrenia advance, bringing greater predictive power for diagnosis and treatment response to nuanced functional imaging phenotypes, this reverse mapping—from imaging to genes—may become increasingly valuable for understanding illness pathophysiology and for developing pharmacogenetic models. Similarly, development of robust data-driven analytical techniques, such as parallel independent components analysis (Liu et al, 2009) to meaningfully combine highly dimensional genetic and imaging datasets in a coordinated and comprehensive fashion may eventually help shed light on the underlying structures of each. Finally, because inherited variation in DNA sequences, though incredibly useful for identifying key molecular pathways to schizophrenia as illustrated above, is likely only a partial contributor to illness brain phenotypes, it will be progressively more important to explore connections between executive circuit dynamics and de novo mutations (Stefansson et al, 2008), epigenetics (Huang and Akbarian, 2007), and gene–environment interactions (Caspi et al, 2005; Nicodemus et al, 2008) associated with schizophrenia.
In summary, schizophrenia patients show a remarkable number of characteristic abnormalities of executive circuitry, evident in vivo with functional neuroimaging techniques, the topography of which corresponds well to other pathological findings in postmortem tissue and in vivo neurochemical (magnetic resonance spectroscopy, neuroreceptor mapping) assays. A growing list of candidate schizophrenia risk genes show variation in executive circuit dynamics, akin to that in illness, suggesting that increasing attention to genetic and genetic–environmental interactions yields promise for better understanding the biology of executive dysfunction in schizophrenia.
References
Aalto S, Bruck A, Laine M, Nagren K, Rinne JO (2005). Frontal and temporal dopamine release during working memory and attention tasks in healthy humans: a positron emission tomography study using the high-affinity dopamine d2 receptor ligand [11c]flb 457. J Neurosci 25: 2471–2477. Article PubMed ISI ChemPort
Abbott C, Bustillo J (2006). What have we learned from proton magnetic resonance spectroscopy about schizophrenia? A critical update. Curr Opin Psychiatry 19: 135–139. Article PubMed
Abi-Dargham A, Gil R, Krystal J, Baldwin RM, Seibyl JP, Bowers M et al (1998). Increased striatal dopamine transmission in schizophrenia: confirmation in a second cohort. Am J Psychiatry 155: 761–767. PubMed ISI ChemPort
Abi-Dargham A, Mawlawi O, Lombardo I, Gil R, Martinez D, Huang Y et al (2002). Prefrontal dopamine d1 receptors and working memory in schizophrenia. J Neurosci 22: 3708–3719. Evidence of upregulated D1 receptors in the DLPFC in schizophrenia, potentially in response to reduced dopaminergic input previously shown. PubMed ISI ChemPort
Akbarian S, Kim JJ, Potkin SG, Hagman JO, Tafazzoli A, Bunney Jr WE et al (1995). Gene expression for glutamic acid decarboxylase is reduced without loss of neurons in prefrontal cortex of schizophrenics. Arch Gen Psychiatry 52: 258–266. PubMed ISI ChemPort

NEUROCIRCUITOS DEL MIEDO,STRESS Y ANSIEDAD

Neuropsychopharmacology Reviews
Neuropsychopharmacology (2010) 35, 169–191; ; published online 22 July 2009
The Neurocircuitry of Fear, Stress, and Anxiety Disorders
Lisa M Shin1,2 and Israel Liberzon3,4
1Department of Psychology, Tufts University, Medford, MA, USA
2Department of Psychiatry, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA
3Psychiatry Service, Ann Arbor Veterans Affairs Medical Center, Ann Arbor, MI, USA
4Department of Psychiatry, University of Michigan, Ann Arbor, MI, USA
Anxiety disorders are a significant problem in the community, and recent neuroimaging research has focused on determining the brain circuits that underlie them. Research on the neurocircuitry of anxiety disorders has its roots in the study of fear circuits in animal models and the study of brain responses to emotional stimuli in healthy humans. We review this research, as well as neuroimaging studies of anxiety disorders. In general, these studies have reported relatively heightened amygdala activation in response to disorder-relevant stimuli in post-traumatic stress disorder, social phobia, and specific phobia. Activation in the insular cortex appears to be heightened in many of the anxiety disorders. Unlike other anxiety disorders, post-traumatic stress disorder is associated with diminished responsivity in the rostral anterior cingulate cortex and adjacent ventral medial prefrontal cortex. Additional research will be needed to (1) clarify the exact role of each component of the fear circuitry in the anxiety disorders, (2) determine whether functional abnormalities identified in the anxiety disorders represent acquired signs of the disorders or vulnerability factors that increase the risk of developing them, (3) link the findings of functional neuroimaging studies with those of neurochemistry studies, and (4) use functional neuroimaging to predict treatment response and assess treatment-related changes in brain function.
Keywords: amygdala, fMRI, PET, anterior cingulate, insula, hippocampus
INTRODUCTION
Anxiety disorders are marked by excessive fear (and avoidance), often in response to specific objects or situations and in the absence of true danger, and they are extremely common in the general population. According to a recent epidemiological study, the lifetime prevalence of any anxiety disorder is 28.8% (Kessler et al, 2005). Anxiety disorders are associated with impaired workplace performance and hefty economic costs (Greenberg et al, 1999), as well as an increased risk of cardiovascular morbidity and mortality (Albert et al, 2005; Kawachi et al, 1994; Smoller et al, 2007). Given that anxiety disorders are a significant problem in the community, recent neuroimaging research has focused on determining the brain circuits that underlie them to inform the use of existing treatments and guide the possible development of new treatments. In the future, neuroimaging studies of anxiety disorders may also prove to be clinically helpful in the prediction of treatment response.
Given that excessive fear is a key component of anxiety disorders, it is not surprising that the search for the neurocircuitry of anxiety disorders has its roots in and has been closely intertwined with studies of fear circuits in animal models. A large volume of experimental work has examined the neurocircuitry associated with fear responses, mainly in rodents, using primarily fear conditioning, inhibitory avoidance, and fear-potentiated startle models. Key components of fear circuitry including the amygdala (and its subnuclei), nucleus accumbens (including bed nucleus of stria terminalis BNST), hippocampus, ventromedial hypothalamus, periaqueductal gray, a number of brain stem nuclei, thalamic nuclei, insular cortex, and some prefrontal regions (mainly infralimbic cortex) have been identified in these studies (for recent reviews see Davis, 2006; Maren, 2008; Quirk and Mueller, 2008). These regions have their respective roles in the various components of fear processing such as the perception of threat or of unconditioned stimuli, the pairing of an unconditioned stimulus and conditioned response (learning/conditioning), the execution of efferent components of fear response, and the modulation of fear responses through potentiation, contextual modulation, or extinction. Some key findings from animal literature, such as the central role of amygdaloid nuclei in the acquisition of fear conditioning and expression of fear responses, the involvement of the hippocampus in contextual processing, and the importance of the infralimbic cortex in extinction recall, have been replicated across different studies and laboratories. These basic components of fear circuitry are well preserved across species and likely support similar functions in humans. Animal work using in vivo electrophysiological recording, tracing and lesions/reversible inactivation techniques was indispensable in acquiring this knowledge. Some recent work had even suggested that there might be separate fear and anxiety systems orchestrated through the central nucleus of the amygdala and the BNST, respectively (Davis, 2006). These types of findings are particularly exciting as they might allow for a better focus on the neurocircuits involved in pathological anxiety.
On the other hand, other important issues, such as the exact neuroanatomical region that stores fear memory traces, or the exact role of a particular process (eg, the role of reconsolidation in fear memory, Nader and Hardt, 2009), or of a particular region (eg, the insular cortex) are intensely debated and actively studied. Nevertheless, the basic fear-related neurocircuitry identified in rodents is a useful place to start examining anxiety-related neurocircuitry in humans. It is important to note that the exact roles of many brain regions are yet to be firmly established and could differ across species. Even regions such as the amygdala, hippocampus, and nucleus accumbens might be involved in different, additional, or even unique tasks in humans (eg, the role of the hippocampus in explicit verbal memory in humans). Finally, there are major differences between human anxiety/anxiety disorders and fear conditioning models in animals. These differences include the frequent absence of clear unconditioned stimuli (US) in human anxiety disorders, and the central roles of avoidance and cognitive components (eg, anticipatory anxiety) in humans. These unique characteristics of anxiety disorders suggest potential involvement of other brain regions in addition to those identified in rodents, such as areas of prefrontal cortex that are more unique to humans. Thus, although animal studies are indispensable in understanding basic fear neurocircuitry, in vivo human studies are critical for understanding the neurocircuitry of anxiety disorders.
In this review, we will discuss three main topics: (1) fear neurocircuitry in healthy humans; (2) stress as a normal response to internal and external stimuli, and (3) anxiety disorders as defined in human psychopathology. The first of these topics will include a discussion of Pavlovian fear conditioning and extinction, pharmacologically induced fear and anxiety states, and the assessment of emotional stimuli in humans. In the third topic, we review the role of brain regions such as the amygdala, medial prefrontal cortex (including the rostral anterior cingulate cortex (rACC) and dorsal anterior cingulate cortex (dACC)), hippocampus, and insular cortex in anxiety disorders (Figure 1). Finally, we discuss some of the limitations of neuroimaging studies of anxiety disorders as well as the directions that we expect the field to take in the near future. (For an outline of this review see the Appendix.)
FEAR NEUROCIRCUITRY IN HUMANS
Pavlovian Fear Conditioning and Extinction
Fear conditioning In its most basic form, Pavlovian fear conditioning involves repeatedly presenting a previously neutral conditioned stimulus (CS; eg, a tone) with an aversive unconditioned stimulus (US; eg, a shock). After repeated pairings, the CS alone comes to elicit a conditioned fear response (eg, increased freezing, fear-potentiated startle, or skin conductance responses). Pavlovian fear conditioning has been used as a testable and translational, though admittedly simplistic, model of the acquisition of fears that might be relevant to some anxiety disorders like phobias and possibly to some aspects of post-traumatic stress disorder (PTSD).
Studies of Pavlovian fear conditioning in non-humans have highlighted the importance of the amygdala in the acquisition of fear conditioning (LeDoux, 2000; LeDoux et al, 1990; Pare et al, 2004; Sananes and Davis, 1992). Similarly, functional neuroimaging studies in humans have reported amygdala activation during fear conditioning (Alvarez et al, 2008; Barrett and Armony, 2009; Buchel et al, 1998, 1999; Cheng et al, 2003, 2006; Gottfried and Dolan, 2004; Knight et al, 2004, 2005; LaBar et al, 1998; Milad et al, 2007b; Morris and Dolan, 2004; Pine et al, 2001; Tabbert et al, 2006), even when the CS is presented below perceptual thresholds (Critchley et al, 2002; Knight et al, 2009; Morris et al, 2001) and even when more complex USs are used (Doronbekov et al, 2005; Klucken et al, 2009). In addition, amygdala activity has been associated with skin conductance changes during fear conditioning (Cheng et al, 2006; Furmark et al, 1997; LaBar et al, 1998; Phelps et al, 2001). Interestingly, amygdala activation in humans also has been observed in response to cues following (1) verbal instructions that discriminate between cues that predict shock vs safety (even though no shock was actually administered) (Phelps et al, 2001), and (2) observational fear learning, whereby participants watch a video of another person experiencing a Pavlovian fear-conditioning paradigm (Olsson et al, 2007). What exactly amygdaloid activation represents in these latter paradigms is not entirely clear. It could suggest for example that: (1) higher order centers that decipher the anticipated predictive value of the cue, or that learn from observation using empathy, convey information to the amygdala, or (2) alternatively, that the human amygdala is less specific in its responses and is more sensitive to contextual modulation in the absence of a US. These interpretations could have potentially different implications for the understanding of the role of the amygdala in anxiety disorders.
Fear conditioning is also associated with increased activation in the dACC and rACC (Alvarez et al, 2008; Buchel et al, 1998, 1999; Dunsmoor et al, 2007; Klucken et al, 2009; LaBar et al, 1998; Marschner et al, 2008; Milad et al, 2007a, 2007b; Morris and Dolan, 2004; Phelps et al, 2004). Activation in the dACC and rACC also occurs during observational fear learning (Olsson et al, 2007). In addition, dACC activation is positively correlated with differential skin conductance responses (Milad et al, 2007a). Fear conditioning studies (involving both specific CSs and contexts) also commonly report insular cortex activation (Alvarez et al, 2008; Buchel et al, 1999; Buchel et al, 1998; Critchley et al, 2002; Dunsmoor et al, 2007; Gottfried and Dolan, 2004; Klucken et al, 2009; Knight et al, 2009; Marschner et al, 2008; Morris and Dolan, 2004; Phelps et al, 2001, 2004) and hippocampal activation (Alvarez et al, 2008; Buchel et al, 1999; Knight et al, 2004, 2009; Lang et al, 2009; Marschner et al, 2008).
Extinction Extinction learning occurs when a CS that previously predicted a US no longer does so, and over time, the conditioned response (eg, freezing or elevated skin conductance responses) decreases. Extinction learning or, more likely, the later recall of this learning involves the ventromedial prefrontal cortex (vmPFC) (Milad and Quirk, 2002; Morgan et al, 1993; Quirk et al, 2000, 2003, 2006) in rodents. Functional neuroimaging studies of healthy humans have reported vmPFC activation during extinction (Barrett and Armony, 2009; Gottfried and Dolan, 2004; Kalisch et al, 2006; Milad et al, 2007b) and the later recall of extinction (Milad et al, 2007b; Phelps et al, 2004). Skin conductance measures of extinction memory are positively correlated with vmPFC activation (Milad et al, 2007b; Phelps et al, 2004) and vmPFC cortical thickness (Milad et al, 2005). Activation of the amygdala and insular cortex also may occur during extinction learning or recall (Gottfried and Dolan, 2004; LaBar et al, 1998; Milad et al, 2007b; Phelps et al, 2004), and greater amygdala responses during extinction have been associated with higher trait anxiety (Barrett and Armony, 2009). Finally, extinction can be modulated by context (ie, the surroundings in which extinction takes place), and the hippocampus has a role in this process. In rodents, dorsal hippocampal lesions reduce the context-dependence of extinction (Bouton et al, 2006). In a recent fMRI study, hippocampal activation to the CS+ occurred in the extinction context but not in the conditioning context (Kalisch et al, 2006). Hippocampal activation was also positively correlated with vmPFC activation in this study (Kalisch et al, 2006), suggesting that hippocampal–vmPFC interactions may be important for the contextual modulation of extinction.
Fear States and Responses to Emotional Stimuli
Pharmacological challenge Another way to examine the mediating functional neuroanatomy of fear or anxiety is to use specific pharmacological agents to provoke such states in healthy individuals during PET or fMRI scanning. For example, cholecystokinin-4 (CCK-4) is associated with increases in subjective states of fear and anxiety, as well as increased activation in the amygdala, insular cortex, claustrum, cerebellum, brain stem, and the ACC (Benkelfat et al, 1995; Eser et al, 2009; Javanmard et al, 1999; Schunck et al, 2006). In addition, two studies reported dACC increases during anticipatory anxiety preceding the CCK administration (Eser et al, 2009; Javanmard et al, 1999). It is important to keep in mind, however, that CCK-B receptor agonists like pentagastrin also have direct effects on stress axis stimulation independent of their effects on subjective experience of distress/fear (Abelson et al, 2005, 2008). Procaine administration has been associated with elevated subjective ratings of fear/anxiety, activation of the amygdala, ACC, and insular cortex (Ketter et al, 1996; Servan-Schreiber et al, 1998), and deactivation of neocortical structures (Servan-Schreiber et al, 1998). Furthermore, amygdala activity was positively correlated with subjective reports of anxiety (Ketter et al, 1996; Servan-Schreiber et al, 1998). Interestingly, those subjects who did not have a panic attack in response to procaine had greater activation in the rACC compared with those who did have a panic attack (Servan-Schreiber et al, 1998), consistent with the idea that the rACC may perform a regulatory or inhibitory function (Mayberg, 1997). The alpha-2 adrenergic antagonist yohimbine has likewise been associated with increased normalized blood flow in medial prefrontal cortex, insular cortex, and cerebellum in healthy individuals (Cameron et al, 2000). A major caveat in the interpretation of pharmacological challenge studies, however, is the difficulty in disentangling the effects that are specific to fear induction from the direct effect of a pharmacological agent on regional brain activity and from the non-specific effects of the pharmacological agent.
Emotional stimuli Over the past two decades, functional neuroimaging studies have shown that a core set of brain regions mediate responses to emotional stimuli in healthy humans. (For reviews, see Phan et al, 2002; Phan et al, 2004b). The relevance of these studies to fear/anxiety circuitry is two-fold: (1) A significant number of these emotional activation paradigms utilize stimuli that depict and/or elicit fear, and (2) these studies shed light on more general emotion-generating neurocircuitry. PET and fMRI studies have reported amygdala activation in response to emotionally negative photographs (Britton et al, 2006; Hariri et al, 2002; Irwin et al, 1996; Lane et al, 1997a; Paradiso et al, 1999; Phan et al, 2003b; Reiman et al, 1997; Taylor et al, 1998), odors (Zald and Pardo, 1997) and tastes (Zald et al, 1998). Several studies have reported amygdala activation to positive stimuli as well (Garavan et al, 2001; Hamann and Mao, 2002; Hamann et al, 1999, 2002; Liberzon et al, 2003), which suggests that the amygdala responds more broadly to emotionally arousing and/or salient stimuli (Phan et al, 2004b). Reappraisal of emotionally negative photographs is associated with reduced amygdala activation (Ochsner et al, 2002) and increased ventromedial prefrontal cortex activation (Urry et al, 2006). Finally, amygdala activation during encoding of emotionally arousing stimuli is correlated with the subsequent recollection of those stimuli (Cahill et al, 1996; Dolcos et al, 2004, 2005; Hamann et al, 1999).
Medial prefrontal cortex, including the rACC, also activates in response to emotional pictures (Lane et al, 1997a, 1997b; Phan et al, 2003a, 2003b, 2004a; Reiman et al, 1997) and may mediate self-referential processing (Kelley et al, 2002; Lane et al, 1997a; Zysset et al, 2002). Although the medial prefrontal cortex may activate regardless of task or valence, the rACC may be more likely to activate when a cognitive task is performed during scanning (Phan et al, 2002). Ventromedial PFC responses to fear-related images have been negatively associated with cortisol reactivity (Root et al, 2009). The dACC also activates in response to emotional photographs (Britton et al, 2006; Teasdale et al, 1999) and aversive tastes (Zald et al, 1998). Finally, the insular cortex is responsive to aversive stimuli (Phan et al, 2004a), internally generated sadness (Lane et al, 1997b; Reiman et al, 1997) and disgust-related stimuli (Britton et al, 2006).
Emotional facial expressions Interestingly, the same neurocircuitry that has been implicated in fear/anxiety responses in humans is readily activated by stimuli that are not intrinsically threatening, but may convey information regarding the presence of threat in the environment or about the fearful emotional state of others. Responses in the amygdala are readily elicited by photographs of facial expressions, especially those of fear (Breiter et al, 1996a; Davis and Whalen, 2001; Fitzgerald et al, 2006; Morris et al, 1996; Vuilleumier and Pourtois, 2007; Whalen et al, 2001), even when presented below conscious awareness (Morris et al, 1998; Whalen et al, 1998, 2004). Emotional facial expressions have also been associated with activation in the dACC, rACC, medial frontal gyrus, and insular cortex (Fitzgerald et al, 2006; Gorno-Tempini et al, 2001; Morris et al, 1996; Phillips et al, 1997, 2004; Sabatini et al, 2009; Sprengelmeyer et al, 1998).
Brain responses to the relatively ambiguous facial expression of surprise have been shown, in some studies, to depend on the extent to which individual subjects interpreted these expressions as positive or negative; more negative interpretations were associated with greater amygdala and lower ventral medial prefrontal cortex activation (Kim et al, 2003). These findings are consistent with the notion that the amygdala and medial prefrontal cortex are reciprocally modulated (eg, Garcia et al, 1999). Furthermore, the experimental manipulation of the context in which surprise facial expressions are presented alters brain activation patterns in a similar way: surprise expressions associated with a negative context elicited more amygdala activation than those associated with a positive context (Kim et al, 2004). These amygdala activations were positively correlated with activation in the dACC (Kim et al, 2004).
Of relevance to our later discussion of anxiety disorders are findings that suggest that healthy individuals with high scores on anxiety measures have greater amygdala and insular cortex responses to emotional (angry, fearful, and happy) faces and less rACC activation than participants with normative scores on these measures (Bishop et al, 2004a, 2004b; Stein et al, 2007). Similarly, trait anxiety has been positively correlated with amygdala responses to neutral faces (Somerville et al, 2004).
Summary
Studies of fear conditioning, pharmacologically induced fear, and responses to emotional stimuli and facial expressions have provided evidence that the human amygdala, although responsive to multiple salient stimuli, responds reliably and potentially preferably to stimuli that predict threat and can be involved in mediating fear/anxiety states. Given that patients with anxiety disorders experience fear and distress in response to possible predictors of threat, the amygdala has been hypothesized to be hyperresponsive in some anxiety disorders. In the next section, we will review the evidence related to this hypothesis.
Studies of extinction have highlighted the potential involvement of the vmPFC and hippocampus in the process of learning and remembering that stimuli that used to predict threat no longer do. One possible reason for exaggerated fear, anxiety, and distress in patients with anxiety disorders is that these emotional responses fail to extinguish or that extinction learning is not recalled even when specific cues no longer predict threat. Indeed, some studies have reported impaired extinction in several anxiety disorders, such as PTSD (Blechert et al, 2007; Milad et al, 2008; Orr et al, 2000; Peri et al, 2000; Wessa and Flor, 2007). Thus, the vmPFC and hippocampus are clear regions of interest in functional neuroimaging studies of anxiety disorders.
Finally, both the animal literature and studies reviewed above suggest that the dACC (and its likely homolog prelimbic cortex) and insular cortex respond to emotional stimuli or those that predict threat. As with the amygdala, hippocampus, and vmPFC, these regions are involved in multiple other functions; however, they might also have important roles in specific aspects of anxiety. For example, the insular cortex is thought to mediate the monitoring of internal body states, and has been found to be hyperresponsive in anxiety-prone individuals (Paulus and Stein, 2006). In summary, research on healthy individuals has suggested that all of these brain regions are prime candidates to examine in patients with anxiety disorders.
STRESS
An important and often overlooked aspect of the fear/anxiety neurocircuitry is its overlap and interaction with the neurocircuitry that orchestrates the stress response. It is important to note that the concept of 'stress' used here is relatively specific. It does not encompass general concepts of 'subjective distress' or 'performance load.' Although these are useful concepts, they are heterogeneous by nature and are not likely to be associated with a specific neurocircuitry. On the other hand, the concept of a stress system that leads to activation of limbic–hypothalamo–pituitary–adrenal axis (LHPA) and secretion of stress hormones like corticotropin-releasing hormone (CRH), adrenocorticotropic hormone, and cortisol is quite specific and is likely to be highly relevant to the neurocircuitry of fear and anxiety. The neurocircuitry governing LHPA activation has been the focus of intense studies in rodents, primates, and humans because it has been repeatedly linked to the neurobiology of mood disorders (which is addressed in detail elsewhere in this volume), but the evidence linking LHPA axis abnormalities to anxiety disorders has been less consistent, sometimes confusing, and often oversimplified. At the same time, some of the same brain regions are implicated in both anxiety and stress responses, suggesting that these responses are interrelated and can influence each other. Furthermore, anxiety and mood disorders are highly comorbid, suggesting that some common abnormalities in neurocircuitry might be present in both disorders. In the following few paragraphs, we briefly address only the structural overlap in neurocircuits and the effects of stress system activation (or stress hormones) on anxiety/fear neurocircuitry.
Epidemiologically, major depression is highly comorbid with anxiety disorders like PTSD, panic disorder, and social phobia (Reiger et al, 1990), and anxiety symptoms are highly prevalent in depression (Frances et al, 1990). Furthermore, major subcortical components of the LHPA axis (eg, hypothalamus, hippocampus, amygdala, and BNST) have also been identified as key components of anxiety/fear neurocircuitry (albeit sometimes involving different subnuclei, for example paraventricular nuclei vs ventromedial hypothalamus for LHPA and fear neurocircuitry, respectively). More recently with the introduction of in vivo imaging methodologies in LHPA/stress research, the role of cortical structures like the insula and dorsal mPFC in the activation and inhibition of stress response, respectively, has been reported (Liberzon and Martis, 2006) as well as the role of subgenual ACC in self-induced sadness and depression (Mayberg et al, 1999). Together, these findings suggest a significant overlap in structures involved in the stress response and those involved in fear/anxiety responses (eg medial prefrontal cortex, insula, amygdala, hippocampus, and BNST). Finally, with respect to neurotransmitters involved, CRH is likely involved in the orchestration of both LHPA axis activity and many anxiety/fear responses. (For a review see Heim and Nemeroff, 2001.)
The activation of these overlapping regions in functional neuroimaging studies does not necessarily signify, however, activation of both the fear/anxiety response and the LHPA axis. As a matter of fact, activation of fear/anxiety does not necessarily activate an LHPA stress response, even in highly fearful (phobic) individuals (Curtis et al, 1976). In turn, activation of LHPA axis is not necessarily experienced subjectively as fear or anxiety. For example, morning awakening, food intake, and nausea all lead to LHPA axis activation without notable increases in subjective sense of fear. It is becoming increasingly clear that specific characteristics of experience (novelty, control, social support, etc.) are more salient for LHPA axis activation than degree of subjective distress or fear (Abelson et al, 2007). These facts help to better understand the findings of non-specific, or even sometimes counterintuitive, findings regarding the LHPA and stress responses in anxiety disorders such as panic disorder (Abelson et al, 2007) and PTSD (Yehuda, 2006; Yehuda et al, 1991). This also suggests that activation in specific cortical regions like mPFC or insula cannot be readily interpreted as a component of the fear response, and has to be considered within a context of a specific experiment, subjective report, symptoms, neuroendocrine profile, etc.
With these caveats in mind, important findings about stress exposure and LHPA axis activation affecting fear/anxiety responses have been accumulating. These can be seen in two general categories: (1) the immediate effects of stress or of stress hormones on fear/anxiety responses (eg, stress or stress hormone exposure immediately precedes, or is present during the fear/anxiety responses), and (2) delayed or developmental effects, (eg, stress exposure during developmentally sensitive periods, like early childhood, modulates fear/anxiety responses later in life). In the former category, it has been reported that exposure to acute stress in healthy individuals potentiates the anxiety response (Grillon et al, 2007). In addition, stress exposure (Trier Social Stress Test) led to enhanced galvanic skin responses to conditioned stimuli (CS+) during fear conditioning (Jackson et al, 2006). Interestingly, stress modulates fear responses differentially in men and women. Differential effects of stress on fear/anxiety in females vs males also have been demonstrated in animal studies. Chronic stress exposure led to impaired extinction recall of fear conditioning in male but not female rats (Baran et al, 2009). Stress exposure in animal studies also led to enhancement in contextual fear conditioning (Cordero et al, 2003). The effects of stress hormone exposure are somewhat more difficult to interpret because higher endogenous cortisol levels were associated with higher skin conductance responses (SCR) (Jackson et al, 2006), whereas administration of exogenous cortisol led to decreased SCR (Stark et al, 2006).
With respect to delayed effects of stress during the vulnerable developmental period, the findings are somewhat complex. Studies of early maternal separation in rodents (Plotsky and Meaney, 1993) and variable foraging in primates (Coplan et al, 1996) have revealed long-term alteration in stress axis responses and key neurotransmitter systems (for review see Heim and Nemeroff, 2001). In addition, recent findings of gene-by-environment interactions in PTSD (Binder et al, 2008) also point toward the possibility that early childhood experience might modify fear/anxiety neurocircuitry and contribute to the development of anxiety disorders. Direct evidence of these effects on fear/anxiety behavior is less convincing, however, as maternal separation in rat pups, which did alter relevant neurotransmitter systems, did not result in significantly enhanced startle response or decreased open field exploration as compared with non-separated animals (Caldji et al, 2000). Similarly, mixed results have been obtained in other studies where rats exposed to severe sporadic stress spent more time in open arms of an elevated plus maze but displayed increases in defensive probe burying behavior. Furthermore, animals exposed to milder chronic stress showed opposite changes (Pohl et al, 2007).
The character of the stress exposure (mild vs severe, prolonged vs short, predictable vs non-predictable) and the sex of the individual emerge as important variables that can define the long-term effects of stress exposure, but more experimental data are clearly needed. Similarly, little is known about the specific mechanisms by which stress exposure modulates fear/anxiety circuitry. It has been suggested, as stated above, that early developmental stress exposure alters fear/anxiety circuitry via altered sensitivity and responsivity of the CRH and adrenergic systems, and recent advances in morphological work had suggested a potential mechanism for the effects of stress on fear conditioning and extinction. Chronic stress decreases dendritic branching in the hippocampus (McEwen, 2001) and mPFC (Liston et al, 2006; Radley et al, 2004), but increases dendritic branching in the amygdala (Mitra et al, 2005; Vyas et al, 2006). This pattern could lead to increased conditioning and impaired extinction, and both of these processes could contribute changes in anxiety/fear-related behaviors. Future research addressing these important questions will be needed to fully understand the impact of stress/LHPA axis activation on fear/anxiety and the underlying neurocircuitry.
ANXIETY DISORDERS
Posttraumatic Stress Disorder
PTSD can develop in individuals who (1) were exposed to an event or events that involved the threat of death or serious injury and (2) reacted with intense fear, helplessness or horror (APA, 2000). Individuals with PTSD reexperience the traumatic event in the form of nightmares, intrusive recollections, flashbacks, and physiological arousal and distress in response to reminders of trauma. These patients may attempt to avoid reminders of the trauma and may experience a restricted range of effect, especially positive effect. Finally, patients with PTSD report hyperarousal symptoms, such as hypervigilance, exaggerated startle, and difficulty sleeping or concentrating (APA, 2000).
Neurocircuitry models of PTSD implicate the amygdala, mPFC, and hippocampus (Rauch et al, 1998b, 2006). According to some models, the amygdala is hyperresponsive in PTSD, which may account for exaggerated fear responses and the persistence of traumatic memories. In addition, portions of the vmPFC (including the rACC) are hyporesponsive and fail to inhibit the amygdala. It is not clear which of the two regions 'drives' the overall outcome, but a hyperresponsive amygdala and hyporesponsive mPFC may potentially lead to deficits in extinction, emotion regulation, attention, and contextual processing (Liberzon and Sripada, 2008). Abnormal hippocampal function may contribute to deficits in contextual processing, as well as impairments in memory and neuroendocrine dysregulation. Although not originally included in early neurocircuitry models, the dACC and insular cortex may have a role in PTSD as well. Recent studies have suggested that the dACC is hyperresponsive in PTSD, perhaps underlying exaggerated fear learning. Finally, the insular cortex appears to be hyperresponsive in PTSD and other anxiety disorders, consistent with the notion that the insula may mediate anxiety proneness (Paulus and Stein, 2006; Simmons et al, 2006). (For other models, see Elzinga and Bremner, 2002; Hamner et al, 1999; Layton and Krikorian, 2002.)
Amygdala

Several studies have reported increased amygdala activation in PTSD relative to comparison groups in response to trauma-related imagery (Shin et al, 1997; Shin et al, 2004a), combat-related sounds or smells (Liberzon et al, 1999; Pissiota et al, 2002; Vermetten et al, 2007), trauma-related photographs or words (Driessen et al, 2004; Hendler et al, 2003; Morey et al, 2009; Protopopescu et al, 2005), fear conditioning (Bremner et al, 2005), and fearful facial expressions (Bryant et al, 2008b; Rauch et al, 2000; Shin et al, 2005; Williams et al, 2006). Exaggerated amygdala activation in PTSD has also been found at rest (Chung et al, 2006; Semple et al, 2000) and during the completion of neutral attention and memory tasks (Bryant et al, 2005; Shin et al, 2004b). Several studies, however, have found no differential response in the amygdala in PTSD (eg, Bremner et al, 1999a; Lanius et al, 2001) or even decreased responsivity to negative stimuli (Phan et al, 2006a). Interestingly, resilience to PTSD may be associated with relatively decreased amygdala activation (Britton et al, 2005; Osuch et al, 2008), and amygdala lesions may reduce the occurrence of PTSD (Koenigs et al, 2008). In support of the potential role of amygdala in PTSD, some studies have reported that amygdala activation is positively correlated with PTSD symptom severity (Armony et al, 2005; Dickie et al, 2008; Pissiota et al, 2002; Protopopescu et al, 2005; Rauch et al, 2000; Shin et al, 2004a). Similarly, response to cognitive-behavioral treatment is associated with a decrease in amygdala activation (Felmingham et al, 2007; Peres et al, 2007), and relatively higher pre-treatment amygdala activation is predictive of a less favorable response to cognitive-behavioral therapy (Bryant et al, 2008a).
Relatively few studies have examined amygdala structure and neurochemistry in PTSD. Two studies have reported trends for smaller amygdala volumes in PTSD (Bremner et al, 1997; Wignall et al, 2004), but several others have not (Bonne et al, 2001; De Bellis et al, 2001a; Fennema-Notestine et al, 2002; Gilbertson et al, 2002; Gurvits et al, 1996; Lindauer et al, 2004b). One recent study using PET and 11C-carfentanil has reported diminished mu-opioid receptor binding in the extended amygdala in trauma-exposed individuals with vs without PTSD (Liberzon et al, 2007b). Another study has found decreased [11C]flumazenil binding in the left amygdala in PTSD subjects compared with trauma-exposed control participants, consistent with altered GABAergic function in this disorder (Geuze et al, 2008a), although two other studies have not reported this finding (Bremner et al, 2000a; Fujita et al, 2004).
Medial prefrontal cortex Functional neuroimaging studies of PTSD have reported decreased activation or failure to activate the mPFC (including the rACC, medial frontal gyrus, and subcallosal cortex) during traumatic script-driven imagery (Bremner et al, 1999a; Britton et al, 2005; Lanius et al, 2001; Lindauer et al, 2004a; Shin et al, 1999, 2004a), the presentation of trauma-related stimuli (Bremner et al, 1999b; Hou et al, 2007; Yang et al, 2004), and negative, non-traumatic stimuli (Kim et al, 2007; Lanius et al, 2003; Phan et al, 2006a; Shin et al, 2005; Williams et al, 2006). Relatively diminished activation of the mPFC in PTSD also has been shown during extinction (Bremner et al, 2005), emotional Stroop interference (Bremner et al, 2004; Shin et al, 2001), emotional word retrieval (Bremner et al, 2003b), non-emotional cognitive tasks (Bryant et al, 2005; Semple et al, 2000) and at rest (Semple et al, 2000). Furthermore, mPFC activation appears to be inversely correlated with PTSD symptom severity (Britton et al, 2005; Dickie et al, 2008; Hopper et al, 2007; Kim et al, 2007; Shin et al, 2004a, 2005; Williams et al, 2006) and positively correlated with pre-scan cortisol levels (Liberzon et al, 2007a). Finally, increased mPFC activation following treatment has been positively associated with symptomatic improvement (Felmingham et al, 2007; Lansing et al, 2005; Peres et al, 2007; Seedat et al, 2004), although not all studies have shown this, perhaps due to paradigm-related methodological differences (Bryant et al, 2008a).
Most of the findings summarized above reflect activation peaks in rostral ACC and ventral portions of the mPFC. In contrast, more dorsal regions of the ACC (ie, the dACC) appear to have either normal or exaggerated responsivity in PTSD during fear conditioning, interference tasks, an auditory oddball task, and at rest (Bremner et al, 2005; Bryant et al, 2005; Felmingham et al, 2009; Pannu Hayes et al, 2009; Shin et al, 2001, 2007, in press).
The findings of several studies suggest diminished volumes or gray matter densities in the ACC in PTSD (Corbo et al, 2005; Kasai et al, 2008; Rauch et al, 2003; Woodward et al, 2006; Yamasue et al, 2003), and smaller ACC volumes have been associated with greater PTSD symptom severity (Woodward et al, 2006; Yamasue et al, 2003). In a study of monozygotic twins discordant for trauma exposure, diminished gray matter densities in pregenual ACC were not found in the identical twins of the PTSD participants, suggesting that this gray matter density decrease is likely an acquired sign of the disorder rather than a familial risk factor (Kasai et al, 2008).
Magnetic resonance spectroscopy (MRS) studies have revealed diminished N-acetyl aspartate (NAA) levels in the ACC in PTSD (De Bellis et al, 2001b, 2001b; Ham et al, 2007a; Mahmutyazicioglu et al, 2005; Schuff et al, 2008). Furthermore, NAA levels in the pregenual ACC were negatively correlated with the severity of reexperiencing symptoms (Ham et al, 2007a). Two recent studies have reported decreased benzodiazepine receptor binding in the mPFC in PTSD (Bremner et al, 2000a; Geuze et al, 2008a), although one other study failed to find this effect (Fujita et al, 2004).
Hippocampus

Some functional neuroimaging studies have reported decreased hippocampal activation during symptomatic states (Bremner et al, 1999a) and during memory tasks that involve neutral or emotional stimuli (Astur et al, 2006; Bremner et al, 2003a, 2003b; Moores et al, 2008; Shin et al, 2004b). One study found reduced glucose metabolic rate in the hippocampus at rest (Molina et al, 2007), and another reported that successful treatment was related to increased hippocampal activation (Peres et al, 2007). Other studies, however, have reported increased activation in the hippocampus in PTSD (Geuze et al, 2007, 2008b; Sachinvala et al, 2000; Semple et al, 2000; Thomaes et al, 2009; Werner et al, 2009) or a positive correlation between hippocampal activation and PTSD symptom severity (Osuch et al, 2001; Shin et al, 2004b). The direction of hippocampal functional abnormalities appears to depend in part on the type of task and the type of statistical analysis used.
Hippocampal volumes appear to be diminished in PTSD in some (Bossini et al, 2008; Bremner et al, 1995, 1997, 2003a; Gilbertson et al, 2002; Gurvits et al, 1996; Karl et al, 2006; Kitayama et al, 2005; Smith, 2005; Stein et al, 1997; Villarreal et al, 2002a; Wignall et al, 2004; Winter and Irle, 2004; Woon and Hedges, 2008), but not all studies (Bonne et al, 2001; Carrion et al, 2001; De Bellis et al, 1999, 2002; Fennema-Notestine et al, 2002; Golier et al, 2005; Pederson et al, 2004). Hippocampal volumes have been inversely associated with verbal memory deficits (Bremner et al, 1995), combat exposure severity (Gurvits et al, 1996), dissociative symptom severity (Bremner et al, 2003a; Stein et al, 1997), depression severity (Villarreal et al, 2002a), and PTSD symptom severity (Bremner et al, 2003a; Gilbertson et al, 2002; Villarreal et al, 2002a). Spectroscopy studies of hippocampus have reported decreased NAA in the hippocampus, often interpreted as consistent with decreased neuronal integrity (Brown et al, 2003; Freeman et al, 1998; Ham et al, 2007a; Mohanakrishnan Menon et al, 2003; Schuff et al, 2001; Villarreal et al, 2002b). The results of two studies suggest that hippocampal volumes may increase following treatment with serotonin reuptake inhibitors (Bossini et al, 2007; Vermetten et al, 2003).
Whether decreased volumes can explain abnormal hippocampal activation in PTSD is not entirely clear, although the findings of at least two studies suggest that functional abnormalities might be still present even if the volumetric differences are controlled statistically (Bremner et al, 2003a; Shin et al, 2004b). The origin of decreased hippocampal volumes is not known, although the results of one twin study suggest that diminished hippocampal volumes may be a familial risk factor for developing PTSD following psychological trauma (Gilbertson et al, 2002).
With regard to neurochemistry, one recent PET study found decreased [11C]flumazenil binding in the hippocampus (as well as thalamus and cortical areas) suggesting diminished benzodiazepine–GABAA function in the hippocampus in PTSD (Geuze et al, 2008a).
Insular cortex Relative to comparison groups, increased activation in the insular cortex has been found in PTSD during script-driven imagery (Lanius et al, 2007; Lindauer et al, 2008), fear conditioning and extinction (Bremner et al, 2005), the anticipation of negative images (Simmons et al, 2008), the retrieval of emotional or neutral stimuli (Bremner et al, 2003b; Werner et al, 2009; Whalley et al, 2009), aversive smells and painful stimuli (Geuze et al, 2007; Vermetten et al, 2007), and the performance of an emotional Stroop task (Shin et al, 2001). Insular cortex activation has been found to be positively correlated with measures of symptom severity (Carrion et al, 2008; Hopper et al, 2007; Osuch et al, 2001) and post-scan plasma adrenocorticotropic hormone levels (Liberzon et al, 2007a). Although greater insular activation in PTSD has been confirmed by a recent voxel-wise meta-analysis (Etkin and Wager, 2007), a few studies have reported either no group differences in insular activation or relatively decreased activation in PTSD (Bremner et al, 1999a, 2004; Molina et al, 2007; Moores et al, 2008; Phan et al, 2006a; Shin et al, 1999).
Voxel-based morphometry studies of PTSD have reported reduced gray matter density in the insular cortex (Chen et al, 2006; Corbo et al, 2005; Kasai et al, 2008). In one study, gray matter density in the insular cortex was negatively correlated with reexperiencing; that is, lower gray matter density was associated with greater reexperiencing (Kasai et al, 2008).
One recent PET-[11C]flumazenil study has reported decreased benzodiazepine–GABAA receptor binding in bilateral insular cortex in PTSD (Geuze et al, 2008a).
Summary

In general, the functional neuroimaging findings in PTSD support the hypothesis that the amygdala is hyperresponsive and ventral portions of medial prefrontal cortex are hyporesponsive, at least in some groups of PTSD patients. Indeed, the latter finding appears to be one of the most robust in the literature (Etkin and Wager, 2007) (Table 1). In addition, albeit in a small number of studies, reduced volumes and gray matter densities in the ACC have been fairly consistently reported. Furthermore, emerging evidence suggests that the dACC and insular cortex may be hyperresponsive in PTSD, although insular cortex hyperresponsivity does not appear to be specific to PTSD (Etkin and Wager, 2007). Finally, the majority of studies have found diminished hippocampal volumes in PTSD patients. Hippocampal function appears to be abnormal as well, although the direction of the abnormality seems to depend on the type of task completed during neuroimaging.
Panic Disorder
Panic disorder patients experience recurrent, unexpected panic attacks, along with a persistent concern about having future attacks, or worry about the implications of the attacks, or a significant change in behavior related to the attacks (APA, 2000). A panic attack is a discrete episode of intense fear, discomfort, and sympathetic nervous system arousal that occurs in the absence of true danger (APA, 2000). According to one of the neurocircuitry models of panic disorder, the 'fear network,' which includes the amygdala, hippocampus, thalamus, and brain stem structures, is hypersensitive. Furthermore, frontal cortex fails to provide top-down inhibitory input to the amygdala, leading to exaggerated amygdala activation and unnecessary activation of the entire fear network, resulting in a panic attack (Coplan and Lydiard, 1998; Gorman et al, 2000). This type of model is similar to the models proposed for other anxiety disorders such as PTSD. Indeed, PTSD patients often suffer from comorbid panic attacks (Falsetti and Resnick, 1997). Although it is quite possible that PTSD and panic disorder indeed share similar pathophysiological components, it will be important in the future to identify abnormalities that are disorder specific and are responsible for disorder-specific symptomatology.
Amygdala

Hyperactivation of the amygdala in panic disorder has been reported in response to panic-related words (van den Heuvel et al, 2005b) and neutral faces (Pillay et al, 2007). One recent PET study found greater resting glucose metabolism in the amygdala (Sakai et al, 2005), although amygdala glucose metabolism did not change after effective treatment with cognitive-behavioral therapy (Sakai et al, 2006). The possibility that amygdala hyperactivation is present in a subgroup of panic disorder patients has been supported by recent studies examining the effect of genotypes within patients with panic disorder. These have revealed greater amygdala activation in carriers of the COMT 158val allele (Domschke et al, 2008), the 5-HT1A x1019 GG allele, and the short allele of the serotonin transporter polymorphism (Domschke et al, 2006). In contrast, two studies found relatively decreased amygdala activation during anticipatory anxiety (Boshuisen et al, 2002) and in response to fearful facial expressions (Pillay et al, 2006), although the latter finding may be attributed to the fact that panic disorder patients were taking antidepressants, which have been found to decrease amygdala activation (Harmer et al, 2006).
The volumetric findings in the amygdala of panic disorder patients are very sparse. One study reported smaller bilateral amygdala volumes in panic disorder compared with healthy participants (Massana et al, 2003). The same group reported reduced levels of creatine and phosphocreatine in the right medial temporal lobe (including the amygdala and part of the hippocampus) in panic disorder. This was interpreted as potentially representing a hypermetabolic state in the right medial temporal region (Massana et al, 2002), which would be consistent with the findings of Sakai et al (2005). Finally, with respect to relevant neurotransmission findings, SPECT and PET studies have reported decreased GABA–benzodiazepine receptor binding in the medial temporal lobes (Kaschka et al, 1995; Malizia et al, 1998) and decreased 5-HT1A receptor binding in the amygdala in panic disorder (Nash et al, 2008).
Medial prefrontal cortex Consistent with the literature on pharmacologically induced fear and panic in healthy volunteers, studies of panic disorder have revealed increased rACC activation during imagery of high vs low anxiety situations (Bystritsky et al, 2001), anticipatory anxiety (Boshuisen et al, 2002), and in response to happy faces in panic disorder (Pillay et al, 2007), although medication was a potential confound in the latter study. Dorsal ACC hyperresponsivity has also been reported in panic disorder in one study (Pillay et al, 2007). Panic disorder patients who are carriers of the COMT 158val allele appear to have less medial prefrontal deactivation and greater orbitofrontal activation in response to emotional facial expressions (Domschke et al, 2008). In contrast, panic disorder subjects with the 5-HT1A x1019 GG allele had less ACC, medial prefrontal cortex, and orbitofrontal activation in response to fearful facial expressions (Domschke et al, 2006).
Volumetric evidence is again sparse, but it appears that both dACC and rACC might be exhibiting similar types of change. Gray matter volumes appear to be reduced in the rACC (Asami et al, 2008; Uchida et al, 2008) and dACC (Asami et al, 2008), and white matter integrity, as measured by DTI, appears to be enhanced in the rACC in panic disorder (Han et al, 2008).
Two studies have reported decreased GABAA–benzodiazepine receptor binding in the ACC and medial prefrontal cortex, including in the dACC (Hasler et al, 2008; Malizia et al, 1998), and one study found a negative correlation between panic attack symptom severity and benzodiazepine receptor binding in dorsal medial frontal gyrus (Bremner et al, 2000b). An MRS study reported increased lactate and choline levels in the rACC (Ham et al, 2007b), and two PET studies have reported decreased 5-HT1A receptor binding in the anterior cingulate in panic disorder (Nash et al, 2008; Neumeister et al, 2004).
Hippocampus The evidence for hippocampal involvement in panic disorder comes mainly from studies of metabolism and perfusion. Two PET studies of panic disorder have reported abnormalities in the laterality of hippocampal resting glucose metabolic rates (Nordahl et al, 1990, 1998), and two PET studies have found greater resting glucose metabolism in the hippocampus in patients with panic disorder (Bisaga et al, 1998; Sakai et al, 2005). In contrast, a SPECT study has reported reduced perfusion in the hippocampus in panic disorder (De Cristofaro et al, 1993). Two studies have reported decreased GABAA–benzodiazepine receptor binding in the hippocampus (Bremner et al, 2000b; Malizia et al, 1998), and one study has reported the opposite finding (Hasler et al, 2008).
Insular cortex With regard to the insular cortex in panic disorder, studies have reported decreased activity during anticipatory anxiety (Boshuisen et al, 2002), increased gray matter volume (Protopopescu et al, 2006; Uchida et al, 2008), decreased 5-HT1A receptor binding (Nash et al, 2008), and decreased GABAA–benzodiazepine receptor binding (Malizia et al, 1998).
Brain stem Functional neuroimaging studies have reported increased glucose metabolic rates (Sakai et al, 2005) and increased activity during anticipatory anxiety (Boshuisen et al, 2002) in the brain stem in panic disorder. Gray matter volume appears to be increased in the midbrain and pons (Protopopescu et al, 2006; Uchida et al, 2008). Decreased GABAA–benzodiazepine receptor binding was reported in the pons (Malizia et al, 1998), and two studies have found decreased 5-HT1A receptor binding in the raphe nucleus in panic disorder (Nash et al, 2008; Neumeister et al, 2004).
Summary

Several studies have provided evidence consistent with amygdala and brain stem hyperresponsivity in panic disorder. Activation in rACC and dACC appears to be increased, and gray matter volumes in these regions appear to be decreased. Several studies have reported decreased GABAA–benzodiazepine and 5-HT1A receptor binding in the amygdala, medial prefrontal cortex, insular cortex, and brain stem in panic disorder. A common limitation seen in several neuroimaging studies of this disorder is the inclusion of participants taking psychiatric medications (Domschke et al, 2006, 2008; Han et al, 2008; Pillay et al, 2006, 2007; Uchida et al, 2008). Thus, the findings of such studies should be interpreted cautiously pending replication in medication-free participants.
Social Phobia
Social phobia (or social anxiety disorder) is characterized by a marked and persistent fear of social or performance situations involving possible scrutiny by others (APA, 2000). The fear of embarrassment and distress can lead to avoidance of social situations and impairment in social, occupational, and academic functioning. The amygdala and medial prefrontal cortex have been considered important regions of interest in this disorder (Amaral, 2002; Liebowitz et al, 2005; Mathew et al, 2001; Stein et al, 2002a; Stein, 1998).
Amygdala

Exaggerated amygdala responses in social phobia have been observed during public speaking (Tillfors et al, 2001), the anticipation of public speaking (Lorberbaum et al, 2004; Tillfors et al, 2002), negative comments (Blair et al, 2008a), and in response to neutral, angry, contemptuous, happy, and schematic angry facial expressions (Birbaumer et al, 1998; Blair et al, 2008b; Cooney et al, 2006; Evans et al, 2008; Gentili et al, 2008; Guyer et al, 2008; Phan et al, 2006b; Schneider et al, 1999; Stein et al, 2002b; Straube et al, 2004a, 2005; Veit et al, 2002; Yoon et al, 2007). One study found temporally delayed amygdala responses to faces in a social phobia group compared with a healthy control group (Campbell et al, 2007). In addition, amygdala responses in social phobia have been positively correlated with self-reported fear increases (Tillfors et al, 2001), severity of social anxiety symptoms (Blair et al, 2008b; Evans et al, 2008; Guyer et al, 2008; Phan et al, 2006b), and state-trait anxiety scores (Cooney et al, 2006). The genetic predisposition for social phobia has been supported by the finding that social phobia patients with a short allele of the serotonin transporter polymorphism had greater amygdala responses during public speaking compared to those with long alleles (Furmark et al, 2004). Finally, amygdala activation during public speaking in social phobia appears to decrease with successful treatment (Furmark et al, 2002, (2005). In contrast, one recent study found decreased amygdala activation during script-driven imagery of anxiety-provoking social situations and a mental arithmetic task in social phobia (Kilts et al, 2006). One study has reported reduced 5-HT1A receptor binding in the amygdala in this disorder (Lanzenberger et al, 2007).
Medial prefrontal cotex Exaggerated rACC activation in social phobia has been found in response to facial expressions of fear (Blair et al, 2008b) and disgust (Amir et al, 2005), and in response to pictures of peers with whom patients did not want to interact (Guyer et al, 2008). One study reported greater orbitofrontal cortex activation to angry vs neutral prosody (Quadflieg et al, 2008). In contrast, other studies have found decreased activation (Van Ameringen et al, 2004) and decreased glucose metabolic rates in the ventromedial prefrontal cortex (Evans et al, 2009), which increased following treatment with tiagabine (Evans et al, 2009). Glutamate/creatine and NAA/creatine ratios appear to be elevated in the rACC and are correlated with symptom severity (Phan et al, 2005).
Studies of dACC function in social phobia also have been somewhat mixed. Greater dACC activation has been reported in response to negative comments (Blair et al, 2008a) and harsh or disgusted facial expressions (Amir et al, 2005; Phan et al, 2006b). Another study reported greater dorsal medial frontal activation in response to harsh faces (Stein et al, 2002b). Treatment with nefazodone-decreased activation in dACC (Kilts et al, 2006). In contrast, other studies have found decreased dACC activation to schematic angry faces (Evans et al, 2008) and in anticipation of public speaking (Lorberbaum et al, 2004), as well as decreased glucose metabolism at rest (Evans et al, 2009). One study found temporally delayed medial prefrontal cortex/dACC responses to faces in social phobia, which may help to account for the heterogeneity of findings in the literature regarding this structure (Campbell et al, 2007). Finally, one study reported reduced 5-HT1A receptor binding in the anterior cingulate in social phobia, although whether this finding occurred in the dorsal or rostral ACC was not specified (Lanzenberger et al, 2007).
Insular cortex Insular cortex activation appears to be elevated in social phobia during the anticipation of public speaking (Lorberbaum et al, 2004) and in response to emotional facial expressions (Amir et al, 2005; Gentili et al, 2008; Straube et al, 2004a, 2005; Yoon et al, 2007), including schematic facial expressions (Evans et al, 2008; Straube et al, 2004a). Two studies, however, found decreased insular cortex activation during public speaking (Tillfors et al, 2001) and during an implicit sequence-learning task in social phobia (Sareen et al, 2007). One study has found reduced 5-HT1A receptor binding in the insular cortex in this disorder (Lanzenberger et al, 2007).
Striatum

One recent study has found reduced caudate activation during an implicit sequence-learning task in generalized social phobia (Sareen et al, 2007). Other studies have found reduced D2 receptor binding and dopamine transporter densities in the striatum in social phobia (Schneier et al, 2000; Tiihonen et al, 1997), although a recent study failed to replicate these findings (Schneier et al, 2009).
Summary

Exaggerated amygdala activation has been the most consistent functional neuroimaging finding in the social phobia literature. Although several studies have reported exaggerated rACC and insular cortex activation as well, a number of other studies have reported contradictory findings. Future research will have to: (1) address questions regarding ACC/mPFC and insular involvement, potentially utilizing cognitive activation tasks to probe ACC function, and (2) attempt to identify the neurocircuitry specific to social phobia (eg, the regions that contribute to the perception/interpretation of social stimuli as particularly anxiety/fear inducing).
Specific Phobia
Specific phobias aremarked by excessive, unreasonable and persistent fear of specific objects or situations such as small animals, flying, enclosed places, heights, and blood/injury (APA, 2000). The fear and avoidance causes significant distress and/or impairment in occupational, academic, or social functioning. Specific phobia is a relatively common disorder, with a lifetime prevalence of 7–11% (APA, 2000). Early models of the etiology of phobias centered on fear conditioning and extinction, and therefore implicated the amygdala and medial prefrontal cortex. Admittedly, such fear conditioning models are likely to be incomplete given that (1) many individuals with phobias cannot recall a conditioning event, and (2) only a small number of common stimuli or situations are the objects of phobias (Fyer, 1998). Nevertheless, fear conditioning and extinction models have been useful in guiding neuroimaging researchers toward examining amygdala, medial prefrontal cortex, and insular cortex function in this disorder.
Amygdala

Exaggerated amygdala activation in individuals with specific phobia has been observed in response to phobia-related pictures (Dilger et al, 2003; Goossens et al, 2007a, 2007b; Schienle et al, 2005, 2007; Straube et al, 2006b; Veltman et al, 2004; Wendt et al, 2008). In addition, treatment has been associated with decreased amygdala activation (Goossens et al, 2007b; Schienle et al, 2007). However, numerous studies have not reported exaggerated amygdala activation in specific phobia (Hermann et al, 2007; Larson et al, 2006; Paquette et al, 2003; Straube et al, 2004b, 2007; Wik et al, 1996, 1997; Wright et al, 2003), perhaps in part due to methodological differences, such as the use of different imaging modalities, type of stimuli (verbal vs pictorial), mode of presentation (video clips vs stills), or phobia-unrelated facial expressions. Preliminary findings assessing the NK1 receptor have suggested enhanced levels of endogenous neuropeptide substance P (commonly associated with stress and negative affect) in the amygdala in phobics when presented with phobia-relevant pictures (Michelgard et al, 2007).
Medial prefrontal cortex The findings with regard to the rACC in specific phobia are mixed, with two studies showing less rACC activation in phobia groups in response to phobia-related vs neutral pictures (Hermann et al, 2007; Schienle et al, 2007), and two studies reporting enhanced rACC activation to phobia-related stimuli (Britton et al, 2009; Pissiota et al, 2003). Activation in the rACC appears to be positively correlated with anticipatory anxiety in phobia patients (Straube et al, 2007). The rACC has been shown to be thicker in participants with specific phobias relative to control participants without phobias (Rauch et al, 2004).
In contrast, the results of functional neuroimaging studies are much more consistent with regard to the dACC, which has been found to be hyperresponsive to phobia-related stimuli (Goossens et al, 2007a, 2007b; Straube et al, 2006a, 2008b) or the anticipation of such stimuli (Straube et al, 2007). In addition, dACC activation in specific phobia decreases after habituation (Veltman et al, 2004) and cognitive-behavioral treatment (Goossens et al, 2007b; Straube et al, 2006a).
Insular cortex Recent research has reported exaggerated insular cortex activation in specific phobia vs control cohorts in response to phobia- or fear-related pictures, videos, and words (Dilger et al, 2003; Goossens et al, 2007a, 2007b; Schienle et al, 2005; Straube et al, 2004b, 2006b, 2007; Wendt et al, 2008), as well as fearful facial expressions (Wright et al, 2003). In addition, treatment studies have reported decreased insula activation following cognitive-behavioral treatment (Goossens et al, 2007b; Schienle et al, 2007; Straube et al, 2006a). Insular cortex also appears to be thicker (bilaterally) in participants with specific phobia as compared with healthy control participants (Rauch et al, 2004).
Summary

The amygdala, dACC and insular cortex all appear to be hyperresponsive to phobia-related stimuli in specific phobia. These abnormalities tend to normalize with successful treatment. The findings are few and mixed with regard to the rACC.
Obsessive–Compulsive Disorder
Patients with obsessive–compulsive disorder (OCD) experience recurrent, unwanted thoughts or images (obsessions) that cause distress, and engage in excessive ritualistic behaviors or mental acts (compulsions) that are typically carried out in response to the obsessions (APA, 2000). Because OCD is covered more extensively in another chapter, we only very briefly discuss it here (see also Friedlander and Desrocher, 2006; Menzies et al, 2008). In general, abnormalities in thalamo-cortico–striatal loops have been posited to account for the repetitive quality and the cognitive and motor content of the obsessions and compulsions in OCD. One neurocircuitry model of OCD posits that the striatum (caudate nucleus) functions abnormally, leading to inefficient gating in the thalamus (Graybiel and Rauch, 2000; Rauch et al, 1998a). This may lead to hyperactivity in the orbitofrontal cortex and the anterior cingulate cortex, which may mediate intrusive thoughts and anxiety, respectively. Compulsions may serve to recruit the striatum and to achieve thalamic gating, thereby neutralizing the obsessions and reducing anxiety. Thus, the fear/anxiety-related brain regions that we have focused on so far (eg, amygdala, mPFC, insula, and hippocampus) do not appear to mediate the core OCD symptomatology. It is interesting to note here that the neurocircuitry model of OCD is much further developed (eg, nodes, links, and directionality are better specified) than the models of other anxiety disorders. There could be a number of factors contributing to this: (1) OCD has been studied longer than other anxiety disorders; (2) The nature of OCD symptoms (for example the predominant cognitive component) implicated neurocircuitry that is better understood or is organized in a way that renders itself easier for the development of circuitry-based models. Whatever the reason might be, one of the goals for future research in other anxiety disorders should be to strive to develop neurocircuitry hypotheses that are comparable in detail and specifications to the models that are already available for OCD.
Although some functional neuroimaging studies have found elevated amygdala activation in OCD (Breiter et al, 1996b; van den Heuvel et al, 2004; Van Laere et al, 2006), the majority have not, even with facial expression paradigms that are known to activate the amygdala in healthy subjects and patients with other anxiety disorders (Cannistraro et al, 2004). The preponderance of neuroimaging studies have identified functional and/or structural abnormalities in the components of thalamo–cortico–striatal loops: striatum (Bartha et al, 1998; Baxter et al, 1987, 1988; Chen et al, 2004; Rauch et al, 1994, 1997; Remijnse et al, 2006; Robinson et al, 1995; Rosenberg et al, 1997; van den Heuvel et al, 2005a), thalamus (Atmaca et al, 2006; Chen et al, 2004; Fitzgerald et al, 2000; Gilbert et al, 2000), orbitofrontal cortex (Baxter et al, 1987, 1988; Chamberlain et al, 2008; Chen et al, 2004; Kang et al, 2004; Rauch et al, 1994; Remijnse et al, 2006; Valente et al, 2005), and the ACC (Ebert et al, 1997; Fitzgerald et al, 2005; Rauch et al, 1994; Valente et al, 2005). In addition, recent receptor imaging studies in OCD have revealed reduced serotonin transporter availability in the thalamus and midbrain (Reimold et al, 2007), as well as reduced 5-HT2A receptor availability in the ACC and other frontal cortical areas (Perani et al, 2008). One interesting and unanswered question remains: If OCD is indeed associated with very high anxiety states, especially linked to specific worries or compulsions, why doesn't the circuitry associated with other anxiety disorders or state anxiety in healthy controls have a more prominent role in the expression of this anxiety?
Generalized Anxiety Disorder
Generalized anxiety disorder (GAD) is characterized by excessive diffuse anxiety and worry that is difficult to control. Patients with GAD may experience restlessness, fatigue, irritability, muscle tension, and sleep and concentration difficulties (APA, 2000). Relatively few neuroimaging studies of GAD exist in the literature and some of the findings are conflicting; however, some studies have implicated the amygdala and medial prefrontal cortex in this disorder.
Amygdala

Exaggerated amygdala activation in response to fearful (McClure et al, 2007b) and masked angry facial expressions (Monk et al, 2008) and during the anticipation of aversive photographs (Nitschke et al, 2009) has been reported in patients with GAD. In a mixed cohort of subjects with GAD and social phobia, subjects who had a low tolerance for uncertainty had elevated amygdala activation during a decision-making task (Krain et al, 2008). In a study of adolescents with GAD, amygdala responses were positively correlated with GAD symptom severity (Monk et al, 2008). However, other studies have not found exaggerated amygdala activation in GAD (Blair et al, 2008b; Whalen et al, 2008). In a recent treatment study, greater pre-treatment left amygdala activation to fearful faces was associated with a less favorable response to venlafaxine (Whalen et al, 2008); in contrast, a different study on a pediatric GAD sample found that greater pre-treatment left amygdala activation to fearful faces was associated with a more favorable response to cognitive-behavioral treatment (McClure et al, 2007a). One study has reported larger amygdala volumes in pediatric GAD (De Bellis et al, 2000a).
Medial prefrontal cortex Although the literature is currently very small, it appears that medial prefrontal cortex activation may be elevated in GAD. Activation in dACC and rACC appears to be elevated in response to fearful facial expressions in adolescents with GAD (McClure et al, 2007b). In a mixed cohort of subjects with GAD and social phobia, those with higher intolerance for uncertainty had elevated rACC and subgenual ACC activation during a decision-making task (Krain et al, 2008). In addition, dorsal ACC activation in response to worry vs neutral statements declined significantly after treatment with citalopram (Hoehn-Saric et al, 2004). Finally, greater pre-treatment rACC activation in response to fearful facial expressions (Whalen et al, 2008) and the anticipation of emotional stimuli (Nitschke et al, 2009) was associated with a more favorable response to venlafaxine. Regions in lateral prefrontal cortex also appear to show exaggerated activation (Monk et al, 2006) and elevated NAA/creatine ratios in GAD (Mathew et al, 2004).
Summary

Although there is some evidence for exaggerated amygdala and medial prefrontal cortex responses in GAD, there are too few studies to form conclusions about the role of these structures in the pathophysiology of GAD. Future functional neuroimaging studies using fearful facial expressions and tasks that probe medial prefrontal cortex function (eg, extinction, emotion regulation, or interference tasks) might be able to contribute the much needed additional information regarding amygdala and medial prefrontal cortex function in GAD.
Summary
Overall, the findings of functional neuroimaging studies are consistent with the notion of exaggerated amygdala activation to specific stimuli in a number of anxiety disorders, especially social phobia, specific phobia, and PTSD. Facial expressions have been especially effective probes of amygdala responses in social phobia and PTSD. Interestingly, the data regarding amygdala function in panic disorder are still inconclusive, and given that relatively few studies have examined amygdala function in GAD, additional research is needed to make meaningful comparisons of amygdala responses between various anxiety disorders. Although increased amygdala activation has been observed in a few studies of OCD, the overall pathophysiology of OCD appears to be localized to a different brain circuit. One potential conceptualization of these findings is that amygdala hyperactivation is a common pathway for exaggerated anxiety/fear that is triggered by specific stimuli. Thus anxiety disorders that manifest increased fear/anxiety that is associated with specific identifiable stimuli (eg, PTSD, social phobia, and specific phobia) will have evidence of exaggerated amygdala reactivity. Panic attacks, on the other hand, can occur in the absence of such stimuli and might thus involve activation of other structures within fear/anxiety neurocircuitry (such as brain stem nuclei, periaqueductal gray, and mPFC). If this is the case, it can explain why the degree of amygdala activation in panic disorder may vary depending on the presence of identified panic-related stimuli and/or other brain regions activated. Conceptually, the hyperresponsivity of amygdala and brain stem is consistent with cognitive and somatic manifestation of panic attacks; however, hyperactivation of the anterior cingulate is probably more consistent with compensatory/regulatory roles rather than reflecting panic-specific pathophysiology.
Consistent with the findings of the meta-analysis of Etkin and Wager (2007), relatively diminished rACC activation has been reported fairly consistently in PTSD, but not in other anxiety disorders. Thus, relatively diminished rACC function may be specific to PTSD and could reflect abnormalities in recall/contextualization of fear memories. Activation in other regions like the dACC and insular cortex appears to be elevated in PTSD and in several of the other anxiety disorders. Exaggerated activation in these regions may reflect various aspects of anxiety/fear response, such as anticipatory anxiety, interoceptive components, autobiographic memory, or anxiety proneness (Paulus and Stein, 2006; Shin et al, in press; Simmons et al, 2006). Together these findings can be conceptualized as an evidence of hyperresponsive threat detection, autobiographic memory, and somatic/physiological reactivity systems in PTSD, accompanied by the failure in regulatory regions responsible for safety signaling, fear extinction, and stimulus appraisal, together leading to aberrant contextual processing of the threat-related stimuli.
The hippocampus was studied most frequently in PTSD and panic disorder, but rarely in the other disorders. Thus, whether similar structural and functional abnormalities in the hippocampus occur in other anxiety disorders is uncertain. Although the evidence for hippocampal involvement in memory and contextual processing is strongly supported by animal studies, and deficits in hippocampal function (eg, contextual processing) are consistent with the PTSD model outlined above, more information is needed to understand the role of the hippocampus in anxiety disorders.
However, neuroimaging studies of anxiety disorders are not without limitations. On the technical side, the spatial resolution of functional neuroimaging techniques is limited, and even those techniques with the best spatial resolution (fMRI) cannot accurately differentiate between very small adjacent structures (such as subnuclei of the amygdala). This, in addition to the presence of susceptibility artifacts (in the case of fMRI studies), makes resolution of potentially important structures, such as the hypothalamus and brain stem nuclei, particularly problematic for studies of fear/anxiety neurocircuitry. In addition, the temporal resolution of some of the imaging techniques used (ie, PET and SPECT) makes it difficult to detect quick and transient responses to stimuli. On the clinical side, the use of medications (ie, antidepressants or benzodiazepines) in patient groups can affect brain activation and represent a confounding factor. However, for regions such as the amygdala, medications tend to normalize amygdala responses rather than exaggerate them (Harmer et al, 2006; Paulus et al, 2005). Comorbidity represents a challenge as well. The majority of patients with anxiety disorders have comorbid conditions, such as depression. Although it is tempting to exclude from neuroimaging studies those anxiety disorder patients with comorbid conditions, this procedure raises concerns about the ability to generalize findings to the larger population of individuals with anxiety disorders. Recent studies have attempted to deal with this issue by separating anxiety disorder patients into groups with and without comorbidity (Kemp et al, 2007; Lanius et al, 2007).
FUTURE RESEARCH DIRECTIONS
Although much information that is relevant to the pathophysiology of anxiety disorders has been gained over the last two decades, many research questions remain to be answered. As we have noted previously, questions remain regarding the specific roles of the amygdala, medial prefrontal cortex, insula, and hippocampus in the anxiety disorders. In addition, one of the more general and basic questions is whether the functional abnormalities identified in anxiety disorders represent acquired signs of the disorders or vulnerability factors that increase the risk of developing the disorders. For example, does amygdala hyperresponsivity occur after the symptoms of social phobia, specific phobia, or PTSD appear? Or does the amygdala hyperresponsivity precede the development of symptoms and increase the risk for developing them? Two types of studies could be used to try to address these questions. First, longitudinal studies that include functional neuroimaging could assess functional activation before the onset of anxiety symptoms (or before trauma exposure in the case of PTSD) to determine whether baseline amygdala activation predicts (or increases the risk for) subsequent anxiety disorder diagnoses. One recent study using a variant of this longitudinal design has yielded findings suggesting that amygdala activation may represent a vulnerability factor. In this study, amygdala activation in response to novel faces was studied in adults who were categorized in childhood as either behaviorally inhibited or uninhibited (Schwartz et al, 2003). Behavioral inhibition in childhood is a known risk factor for the development of social anxiety later in life (Biederman et al, 2001; Schwartz et al, 1999). Amygdala activation was greater in the inhibited group as compared with the uninhibited group, and this finding remained even when inhibited subjects with social phobia were removed from the analyses. Although this study does not provide definitive proof that exaggerated amygdala activation is a vulnerability factor for the development of social phobia, it does lend some support to the idea.
The second type of study that can help determine whether functional abnormalities are acquired characteristics or vulnerability factors involves studying the identical twins of probands with vs without the disorder in question. With regard to PTSD, Roger Pitman and colleagues have studied combat veterans with PTSD and their combat-unexposed identical co-twins without PTSD, as well as combat veterans who never had PTSD and their identical combat-unexposed co-twins without PTSD. Structural and functional abnormalities that are observed in both the individuals with PTSD and their identical trauma-unexposed co-twins likely represent familial vulnerability factors, whereas abnormalities that are observed only in the individuals with PTSD would be consistent with acquired characteristics. Using this type of design, diminished hippocampal volumes (Gilbertson et al, 2002) and dACC hypermetabolism (Shin et al, in press) appear to be familial vulnerability factors, whereas diminished gray matter volumes in the rACC appear to be acquired characteristics (Kasai et al, 2008).
This twin design, however, is unable to determine whether familial vulnerability is attributable to genetic vs environmental factors. Future studies will be needed to determine whether the functional abnormalities that appear to be familial vulnerability factors are associated with specific genotypes. For example, dACC hypermetabolism has been reported in healthy carriers of the short allele of the serotonin transporter polymorphism (Graff-Guerrero et al, 2005). The prevalence of the short allele appears to be elevated in PTSD (Lee et al, 2005), and the finding of dACC hypermetabolism in individuals with PTSD and their identical co-twins could be at least in part attributable to the presence of this short allele. Future studies will evaluate this possibility. Studies that examine links between specific genotypes and endophenotypes (eg, neuroimaging or neuroendocrine measures) that may predispose individuals to various anxiety disorders following environmental modulation (during development or in adulthood) are likely to prove very valuable in future research.
Much more progress is urgently needed in examining in vivo neurochemistry in anxiety disorders. SPECT, PET, and MRS studies are needed to link functional neuroimaging data with cellular and molecular changes that might be driving these abnormalities. Finally, another major future direction would entail using functional neuroimaging to predict treatment response in patients with anxiety disorders. This type of research has recently begun, and some studies have reported that greater amygdala activation at a pre-treatment baseline predicts a less favorable response to (1) cognitive-behavioral therapy in PTSD (Bryant et al, 2008a), and (2) venlafaxine in GAD (Whalen et al, 2008). Future studies will be needed to determine whether specific pre-treatment functional neuroimaging profiles can distinguish between those who will respond to medication vs cognitive-behavioral treatments. One ultimate goal of this research would be to determine whether functional neuroimaging measures can be used to guide treatment choice for individual patients.
References
Abelson JL, Khan S, Liberzon I, Erickson TM, Young EA (2008). Effects of perceived control and cognitive coping on endocrine stress responses to pharmacological activation. Biol Psychiatry 64: 701–707. Article PubMed ChemPort
Abelson JL, Khan S, Liberzon I, Young EA (2007). HPA axis activity in patients with panic disorder: review and synthesis of four studies. Depress Anxiety 24: 66–76. Article PubMed
Abelson JL, Liberzon I, Young EA, Khan S (2005). Cognitive modulation of the endocrine stress response to a pharmacological challenge in normal and panic disorder subjects. Arch Gen Psychiatry 62: 668–675. Article PubMed ISI
Albert CM, Chae CU, Rexrode KM, Manson JE, Kawachi I (2005). Phobic anxiety and risk of coronary heart disease and sudden cardiac death among women. Circulation 111: 480–487. Article PubMed
Alvarez RP, Biggs A, Chen G, Pine DS, Grillon C (2008). Contextual fear conditioning in humans: cortical-hippocampal and amygdala contributions. J Neurosci 28: 6211–6219. Article
Amaral DG (2002). The primate amygdala and the neurobiology of social behavior: implications for understanding social anxiety. Biol Psychiatry 51: 11–17. Article PubMed ISI

ENFERMEDADES TELOMERICAS

NEW ENG J MED,Volume 361:2353-2365 December 10, 2009 Number 24
Telomere Diseases
Rodrigo T. Calado, M.D., Ph.D., and Neal S. Young, M.D.

Elizabeth Blackburn, Jack Szostak, and Carol Greider were recently awarded the Nobel Prize in Physiology or Medicine for their elucidation of the structure and maintenance of telomeres (the tips of chromosomes). These investigators discovered that telomeres are DNA sequences with a structure that protects chromosomes from erosion and that a specific enzyme, telomerase, is involved in their repair after mitosis.1,2 In this review, we discuss the medical implications of these discoveries.
Telomeres were causally connected to human disease when mutations in the DKC1 gene were detected in a rare inherited form of bone marrow failure.3 DKC1 encodes a protein of the telomerase complex.4 Telomeres are short in many patients with inherited or apparently acquired aplastic anemia, and mutations affecting telomerase have been identified in these forms of aplastic anemia; telomerase mutations also have been associated with fibrosis of the lungs and the liver. Moreover, the telomerase gene is a susceptibility locus for cancer, and short telomeres may be risk factors for cardiovascular disease. Thus, a common molecular mechanism appears to underlie a range of clinical entities. An understanding of the role of telomeres in disease has important implications for diagnosis, genetic counseling, clinical management, and therapy.
Telomeres and Telomerase
Telomeres and telomerase provide protection against threats to the genome that arise from a difficulty inherent in the asymmetric replication of DNA. Without telomeres, genetic material would be lost every time a cell divides. DNA polymerase requires an RNA primer with a 3' hydroxyl donor group to initiate DNA replication, during which the "end-replication problem" arises.5 The primer dissociates as the DNA polymerase moves along the template strand, leaving behind a gap at the ends of chromosomes. As a result, the newly synthesized DNA strand is shorter than the original template. Telomeres and telomerase ameliorate this problem by providing a repetitive template for enzymatic repair of the ends of chromosomes, thereby avoiding the loss of genetically encoded information during mitosis.
Telomeres consist of repetitive DNA sequences coated by capping proteins (shelterin) at the ends of linear chromosomes (Figure 1). In human cells, telomeres consist of hundreds to thousands of TTAGGG tandem repeats in the leading strand.7 A single-stranded 3'-hydroxyl overhang is generated by the catalytic addition of telomeric repeats to the 3' end and by postreplicative processing of the lagging strand. Shelterin proteins, which coat the telomeric DNA sequence,8 serve as a molecular signal to prevent the cellular DNA repair machinery from mistaking telomeres for double-stranded DNA breaks.
Figure 1. Telomere Structure.
As shown in Panel A, telomeres are located at the ends of linear chromosomes; they are composed of hundreds to thousands of tandem DNA repeat sequences: hexameric TTAGGG in the leading strand and CCCTAA in the lagging strand in humans. Protective proteins associated with telomere DNA are collectively termed shelterin (TRF1, TRF2, TIN2, POT1, TPP1, and RAP1). The 3' end of the telomeric leading strand terminates as a single-stranded overhang, which folds back and invades the double-stranded telomeric helix, forming the T loop. As shown in Panel B, telomeres can be directly visualized under the microscope at the ends of metaphase chromosomes (four telomere signals per chromosome) by fluorescence in situ hybridization (FISH). (Image provided by Peter Lansdorp, M.D., Ph.D.) Average telomere length can be measured by several methods: a technique that combines flow cytometry and FISH (flow-FISH), Southern blotting, and a quantitative polymerase-chain-reaction (qPCR) assay. Flow-FISH can measure the telomere length in different cell subgroups, such as granulocytes or CD4+ T lymphocytes; Southern blotting reveals length and length heterogeneity; and qPCR is a rapid assay that requires very small amounts of DNA. As shown in Panel C, the average length of telomeres in human leukocytes varies, ranging from approximately 11 kb at birth (in umbilical-cord blood) to 6 kb at 90 years of age. Telomere loss is most rapid early in life, and over a life span it is not linear but follows a third-order polynomial. Data are from Yamaguchi et al.6
When they are too short, telomeres signal the arrest of cell proliferation, senescence, and apoptosis. This process explains the interruption of proliferation in cultured human cells — the "Hayflick limit" (Figure 2).9 If protective mechanisms, such as the TP53 tumor-suppressor gene, are inactive, thus allowing continued proliferation, telomeres become extremely short and dysfunctional; end-to-end fusions ultimately cause chromosomal instability. Conversely, cells transfected with the telomerase gene can proliferate indefinitely.10
Figure 2. Consequences of Telomere Erosion in the Cell.
Telomeres inexorably shorten with every cell division, and telomere attrition is an inevitable physiological consequence of aging. Telomere shortening also may be iatrogenic; for example, telomere shortening occurs after bone marrow transplantation, in which highly proliferative hematopoietic stem cells and progenitor cells reconstitute hematopoiesis. Environmental factors also may accelerate telomere loss. In addition, telomere attrition may be genetic; there may be an inherited inability to elongate telomeres as a result of mutations in components of the telomerase complex. When telomeres become critically short, inappropriately capped chromosomes or telomere-free ends emerge, which lead to cell senescence or apoptosis. If the cell overrides senescence and continues to proliferate (e.g., because of inactive p53), uncapped telomeres may cause end-to-end fusion of chromosomes, breakage-fusion-bridge cycles, aneuploidy, and chromosomal translocations.
To avoid the attrition of telomeres, germ-line cells and some somatic cells produce telomerase, an enzyme that catalyzes DNA synthesis to maintain telomere length. Telomerase reverse transcriptase (TERT) uses the telomerase RNA component (TERC) as a template to synthesize telomere DNA (Figure 3). The catalytic unit of telomerase contains two copies each of TERT, TERC, and dyskerin (encoded by the DKC1 gene), and proteins that stabilize the complex.
Figure 3. The Telomerase Complex and Its Components.
The enzyme telomerase reverse transcriptase (TERT), its RNA component (TERC), the protein dyskerin, and other associated proteins (NHP2, NOP10, and GAR1) are shown. Telomerase catalytically adds TTAGGG hexameric nucleotide repeats to the 3'-hydroxyl end of the telomeric leading strand, using a specific sequence in the RNA component as the template. TERT contains three major domains: the N-terminal region, the reverse-transcriptase motifs, and the C-terminal region, all containing evolutionarily conserved motifs. TERC contains 451 nucleotides in seven conserved regions (CR1 through CR7), including the template (CR1), and an H/ACA box, a hairpin nucleotide sequence characteristic of a class of small nucleolar RNAs involved in RNA processing.
Telomerase has functions other than elongating telomeres.11 For example, telomerase overexpression in adult mice mobilizes stem cells and induces stem-cell proliferation in the absence of telomere elongation by modulation of the wingless in drosophila (Wnt)–β-catenin signaling pathway.12
Mice in which telomerase genes have been knocked out have been used to model the role of these genes in higher organisms. However, differences in telomere biology between mice and humans preclude ideal modeling of human biology in the mouse system. Moreover, in contrast to mice in the wild, laboratory strains have very long telomeres, and the first generation of telomerase-knockout mice does not show critical telomere shortening or a phenotype. Tissue abnormalities usually appear after the fifth generation; by the sixth generation, mice are infertile and hematopoietic progenitor function is defective.
Diseases of Telomeres
Bone Marrow Failure
Hematopoietic dysfunction caused by defective telomere structure and repair has a broad clinical spectrum. The manifestations may occur from birth to late adulthood, they may range in severity from no abnormalities or mild hematologic abnormalities to extreme pancytopenia, and they may be associated with anomalies, as in dyskeratosis congenita. Telomere mutations are inherited, but penetrance can vary, even within pedigrees.
Dyskeratosis Congenita
A form of ectodermal dysplasia, dyskeratosis congenita, is characterized by a triad of signs: dystrophic nails, patchy skin hyperpigmentation, and oral leukoplakia (Figure 4). Mucocutaneous findings are present in infancy, and bone marrow failure follows in the first or second decade; aplastic anemia is usually fatal. There is variation in the clinical presentation, other organ systems may be involved, and pulmonary disease can be lethal.14 A particularly severe variant of dyskeratosis congenita is the Hoyeraal–Hreidarsson syndrome of progressive pancytopenia, neurologic manifestations of microcephaly, ataxia, and growth retardation in young children. Dyskeratosis congenita may occasionally be first diagnosed in midlife, with only minimally abnormal blood counts.15
Figure 4. Pathologic Consequences of Telomere Erosion in Organs and Tissues.
The childhood syndrome dyskeratosis congenita is characterized clinically by the mucocutaneous triad of nail dystrophy (Panel A), reticular skin hyperpigmentation or hypopigmentation (Panel B), and leukoplakia (Panel C) (reprinted from Alter,13 with the permission of the publisher). In the bone marrow, telomere shortening confers a predisposition to aplastic anemia (Panel D, Giemsa stain) and progression to myelodysplasia and acute myeloid leukemia (Panel E, Giemsa stain). Leukemic bone marrow is characterized by an increased number of myeloid blasts (arrowheads) and dysplasia or dyserythropoiesis (asterisk). In the lungs, telomere attrition can be clinically manifested as pulmonary fibrosis and radiologically characterized by diffuse fibrosis predominantly in the subpleural region (Panel F). Histologically, fibrotic zones alternate with less affected parenchyma (Panel G, hematoxylin and eosin). Telomere shortening in the liver has diverse histologic appearances, including cirrhosis with inflammation (Panel H, hematoxylin and eosin) and nodular regenerative hyperplasia (Panel I, reticulin stain).
Family studies led to the recognition of mutations affecting telomere maintenance. Most cases of dyskeratosis occur in boys. Genetic analysis of multiplex pedigrees linked the phenotype to the Xq28 region, and the gene was named DKC1.3 The encoded protein, dyskerin, is a small nucleolar protein that binds to RNA (including ribosomal RNA and TERC) and affects many cell functions.
After the discovery of DKC1 mutations, very short telomeres were detected in all patients with dyskeratosis congenita.16 Mutations were then sought in the RNA template gene in autosomal dominant pedigrees.4,17 Genetic screening revealed heterozygous mutations in TERC, homozygous mutations in NOP1018 and NHP219 (genes encoding proteins that, like dyskerin, associate with the complex), and in TERT. Recently, mutated TINF2 was detected in autosomal dominant dyskeratosis congenita20; loss of this protein in the shelterin complex causes extremely short telomeres.8 There is no clear relation between specific mutations and phenotype, but patients with the shortest telomeres (as in the Hoyeraal–Hreidarsson syndrome) tend to have the most severe disease. These data indicate that dyskeratosis congenita is the result of defective repair or protection of telomeres. An indication of the complexity of the pathways involved in the disease is the fact that a genetic defect has not yet been identified in most patients with this syndrome.
There is no agreed-on case definition of dyskeratosis congenita. As a result, there may be uncertainty regarding the characteristics of a patient with a mutation. We recommend reserving the diagnosis of dyskeratosis congenita for well-defined kindreds with abnormalities of the integument and an early onset of visceral-organ involvement. Laboratory testing to detect dyskeratosis congenita is also problematic. The presence of very short telomeres in lymphocytes, detected by means of flow cytometry and fluorescence in situ hybridization in commercial laboratories, appears to distinguish dyskeratosis congenita from other constitutional syndromes involving bone marrow failure.21
Telomere dysfunction has several peculiar and important clinical consequences. Some families show "anticipation," or worsening manifestations of disease in succeeding generations as a consequence of inadequate repair of telomeres in germ-line cells.22 With the exception of DKC1 deficiency, which is complete because of X-linkage of the gene, dominance of the telomere repair defect as a result of heterozygous TERC and TERT mutations is due to haploinsufficiency.23 The remaining normal gene might be induced to compensate (e.g., by means of sex hormone therapy). Both male and female sex hormones up-regulate TERT expression and telomerase function in cultured hematopoietic cells.24 Within families, genetically normal members may inherit short telomeres from one parent with the mutation, but it is unclear whether, with their normal telomere repair capacity, they are at risk for disease. Pedigree interpretation occasionally can be complicated by mosaicism, and girls and women may have a mild phenotype with a single X-linked DKC1 mutation.14
The treatment of dyskeratosis congenita has not been studied systematically. Androgens improve blood counts in about 60% of patients.15 Experience with bone marrow transplantation in dyskeratosis congenita is limited: children have been cured, but multiorgan complications often occur.25,26 Regardless of the severity of bone marrow manifestations or therapy, lifelong monitoring for cancer is imperative.27,28
Acquired Aplastic Anemia
Measurements of telomere length in hematopoietic cells preceded the discovery of the mutations in dyskeratosis. Studies of clinical specimens were stimulated by the potential role of telomeres as "mitotic clocks," or surrogates of the cell's proliferative history.29 For example, telomeres of leukocytes are shorter in transplant recipients than in their donors, at least transiently.30,31 In studies that showed that telomere length was short in some patients with acquired aplastic anemia, it was assumed to be due to proliferative stress on limited numbers of hematopoietic stem cells.32,33 After the cause of dyskeratosis congenita was revealed, this explanation became suspect.
Systematic screening of patients with apparently acquired bone marrow failure showed a few patients with TERC mutations34; they were adults with a diagnosis of acquired disease and without the physical signs of dyskeratosis congenita.35 Hematologic abnormalities, if any, in other family members with TERC mutations were often mild and not progressive, but the bone marrow was very hypocellular, hematopoietic progenitors were diminished, and there were elevated levels of circulating hematopoietic growth factors. Bone marrow transplantation from a histocompatible sibling with an unrecognized TERC mutation culminated in early death from graft failure in one patient; this led to the selection of an unrelated donor rather than a sibling donor in a second patient.
Mutations in TERT were first discovered in patients with aplastic anemia.6,36 As with TERC, most patients were adults with a recent onset of bone marrow failure; in some patients, there was progression from moderate to severe pancytopenia, and in others, blood counts remained stable. Family histories were not obvious for blood disease, but phenotyping of pedigrees showed a range of blood counts in TERT mutation carriers. The failure of organs other than the bone marrow, including the liver and the lung, was also associated with TERT mutations.
Overall, mutations in telomerase genes (but not in DKC1) appear to explain the short telomeres detected in about 10% of patients with aplastic anemia. Mutations are associated with short telomere length (adjusted for the patient's age) of blood leukocytes. The enzymatic activity of mutant telomerase is decreased. As in dyskeratosis congenita, heterozygosity causes disease by means of a dominant mechanism of haploinsufficiency.
Pulmonary Fibrosis
The characteristics of idiopathic pulmonary fibrosis are cough, dyspnea, impaired gas exchange, and reduced lung volume. Pathologically, there is patchy fibrosis of the lungs and interstitial inflammation, normal lung alternating with fibrosis, inflammation, and collagen deposition (Figure 4F and 4G). In about 20% of patients with dyskeratosis congenita, lung complications ultimately diagnosed as pulmonary fibrosis develop15; there is often a family history of interstitial pneumonia.17,37,38 Respiratory failure is also a common fatal complication after hematopoietic stem-cell transplantation in patients with dyskeratosis.26,39 These associations led to the discovery of telomerase mutations in about 15% of patients with familial idiopathic pulmonary fibrosis.40,41,42 In these kindreds, there was also bone marrow failure or liver cirrhosis.41,43 Smoking is common among affected patients, suggesting a role of environmental factors in the development of the disease. Telomerase mutations are also sometimes present in patients with sporadic idiopathic pulmonary fibrosis.41 Many more patients with idiopathic pulmonary fibrosis have short telomeres than identified mutations, suggesting as-yet-unidentified genetic abnormalities in a higher proportion of patients.43
Liver Disease
Some patients with dyskeratosis congenita have liver abnormalities or fatal liver complications after bone marrow transplantation.15,39 Patients with pulmonary fibrosis and short telomeres can also have cryptogenic hepatic cirrhosis; this implicates telomere loss in both fibrotic processes. We have observed that many relatives of patients with aplastic anemia and a telomerase mutation have liver disease.44 Liver diseases associated with TERT and TERC mutations are mainly fibrosis with inflammation and nodular regenerative hyperplasia, a leading cause of noncirrhotic portal hypertension (Figure 4H and 4I). The genetic basis of the peculiar familial association of bone marrow, liver, and lung disease38,45,46 is telomere erosion and telomerase mutations.
In summary, telomere diseases can be viewed as a spectrum, from genes acting as determinants of the stereotypical dyskeratosis congenita syndrome to genetic risk factors in specific types of organ failure and fibrosis (Figure 5A).
Figure 5. Telomere Erosion and Human Diseases.
Panel A shows a Venn diagram of mutations of the telomerase complex and human telomere diseases. Dyskeratosis congenita is the most evident and severe manifestation of genetic lesions causing telomere diseases, with high genetic penetrance and congenital clinical manifestations. However, telomerase mutations may be less penetrant and induce single-organ damage in adults without suggestive family histories and the classic physical signs of dyskeratosis congenita. Thus, telomerase mutations represent risk factors rather than genetic determinants in aplastic anemia, pulmonary fibrosis, and liver cirrhosis. Environmental, epigenetic, and other genetic factors probably contribute to disease development in these patients. Panel B shows the relationship between telomere shortening and the risk of cancer. In dyskeratosis congenita, in which genetic penetrance is high, the risk of the development of cancer — particularly head and neck squamous-cell carcinoma and acute myeloid leukemia — also is elevated. In addition, patients with aplastic anemia are at risk for the development of clonal malignant disorders, but the risk is lower than that among patients with dyskeratosis congenita. Similarly, short telomeres appear to predict the progression of chronic inflammatory gastrointestinal states to adenocarcinoma. In multiple genomewide association studies, the TERT locus has appeared as a significant susceptibility locus for a variety of cancers, but at relatively low odds ratios. Shaded areas representing diseases and disease states are not drawn to scale.
Telomere Attrition and Cancer
Animal Models of Telomere Maintenance and Chromosomal Instability
Chromosomal instability was originally postulated by Boveri in 1914 as a fundamental event in the origin of tumors.47 This inference from studies of sea urchins is applicable to the gross derangement in the number and structure of chromosomes in most cancers. Cell experiments and animal models led to the proposal that telomere attrition is a mechanism for the loss or gain of chromosomes.48,49,50,51,52 When telomere maintenance is disrupted in yeast, the few cells that escape senescence show chromosomal abnormalities; in the absence of telomerase, mutation rates increase as a result of terminal chromosome deletions and repeated cycles of break-fusion-bridge rearrangements.53 In late-generation Terc-knockout mice, short telomeres cause chromosomal instability through end-to-end fusions. Apoptosis removes most of these cells, but they can be rescued if DNA damage is not adequately monitored. Thus, in Terc–/– mice that are also deficient in p53, a variety of cancers develop in association with nonreciprocal translocations, mimicking human malignant conditions.54
Accelerated Telomere Attrition, Inflammation, and Malignant Transformation
In humans, studies are often limited by the necessity to measure telomeres in leukocytes, the uncertain significance of telomere length in tumor cells (in which up-regulated telomerase or the alternative pathway of telomere repair may allow evasion of cell senescence), and the difficulty of performing longitudinal assessments. Nevertheless, telomere length has been linked to several types of cancer. The finding of short telomeres in colorectal cancer suggested that telomere loss contributes to tumorigenesis and genetic instability of the malignant cell.55 Telomerase deficiency has been detected in the histologically normal mucosa of patients with inflammatory bowel disease (cancer cells expressed high telomerase activity).56 Losses of chromosomes in nondysplastic tissue in patients with ulcerative colitis were associated with telomere shortening and the appearance of anaphase bridges, especially in patients in whom cancer developed.57
The major risk factor for esophageal cancer is the chronic inflammation of Barrett's esophagus. In one study, the telomere length in leukocytes at first presentation was inversely proportional to the risk of later esophageal cancer; possible explanations were a genetic predisposition to defective repair of DNA in mucosa or long-term exposure to oxidative stress that provokes cell proliferation.58 Short telomeres, visualized directly by means of fluorescence staining of biopsy specimens of Barrett's esophagus, and chromosomal instability associated with dysplastic changes suggest that both are early events in the development of esophageal cancer.59 Telomeres of leukocytes that are short relative to the patient's age have been implicated as a risk factor60,61 or biomarker for many solid tumors, but not for all of them; breast cancer is one exception.60,61,62,63
Hematologic and other cancers develop in patients with bone marrow failure. In a National Cancer Institute review of 50 cases of classic dyskeratosis congenita, the authors found markedly elevated risks of tumors (overall, about 11 times as high as in the general population), especially head and neck squamous-cell carcinomas, skin and anorectal cancers, and acute myeloid leukemia.64 In our study of acquired aplastic anemia, the telomere length of leukocytes was the major predictor of clonal evolution: almost all patients in whom monosomy 7 myelodysplastic syndrome and acute myeloid leukemia developed were in the lowest quartile of telomere length when they first presented with bone marrow failure.65 Because leukemia is linked to TERT and TERC mutations in some pedigrees, we screened multiple cohorts of patients with acute myeloid leukemia.66 Constitutional TERT mutations were detected in about 9% of these patients and were strongly associated with the risk of cytogenetic abnormalities. In small studies, telomere length has been associated with the risk of leukemic transformation from myelodysplasia after chemotherapy and autologous hematopoietic-cell transplantation,67,68,69 and short telomeres of blast cells have been correlated with chromosomal abnormalities.70,71
TERT and the Risk of Cancer
Genomewide association studies have shown polymorphisms in the TERT gene at a higher frequency than normal in patients with cancer. However, the level of risk is much lower than in individual diseases or in patient populations that are assessed serially, for malignant conditions that develop in the setting of inflammation. For example, a massive genomewide scan of more than 30,000 European patients with cancer and 45,000 control DNA samples showed an association between the TERT locus and 5 of 16 cancers, including basal-cell cancer of the skin and cancers of the lung, bladder, prostate, and cervix (all tumors that are caused in part by environmental factors); the overall risks were relatively small (relative risk, 1.12 to 1.21) but consistent across diverse ethnic populations.72 Similar statistical associations have been reproducible for lung cancer in genomewide association studies involving large European populations73 and in Chinese patients, in whom risk was also linked to short telomeres.74 TERT-locus polymorphisms have been associated with susceptibility to gliomas73,75 and renal-cell carcinoma76; they have also been associated with relative resistance to melanoma77 and breast cancer.78
In summary, elomere shortening can be related to the risk of cancer, ranging from high rates of specific cancers in dyskeratosis congenita to modest contributions to oncogenesis in general. In some specific inflammatory and immune diseases, telomere attrition may be the critical factor in promoting the development of cancer (Figure 5B).
Telomeres, Degenerative Diseases, and Aging
Telomeres and Heart Disease
Telomere length has been connected with cardiovascular complications, but the associations have varied across studies, exploratory epidemiologic surveys often do not correct for multiple variables, and there is no accepted pathophysiological link.79,80,81,82,83,84 In one study, endothelial progenitor-cell telomeres were shorter in patients with coronary artery disease than in healthy persons, and intensive lipid-lowering therapy both reduced oxidative DNA damage and prevented further telomere attrition.85 In one of many epidemiologic surveys, people with short leukocyte telomeres were at risk for coronary disease, which appeared to be attenuated by statin therapy.86 In the Cardiovascular Health Study, shortened telomeres corresponded with a risk of myocardial infarction among younger patients that was three times as high as the risk among older patients,87 and in the Heart and Soul Study, shorter-than-normal telomeres were a biomarker for the risk of death in patients with stable coronary artery disease.88 Telomere length was short in a study involving British patients with premature myocardial infarction.89 In the Framingham Heart Study, shortened telomere length correlated with carotid-artery intimal thickening.90
Telomeres and Aging
Does telomere biology explain physiologic aging in humans?52,91 Telomere attrition leads to cell senescence and the Hayflick phenomenon. Telomerase defects affect yeast viability and replication and account for the shortened life span of knockout mice. In addition, under specific conditions, telomere length is a "mitotic clock" for a cell's proliferative history, and telomere loss is linked to DNA damage by reactive oxygen species, which accumulates over time. Proteins that are released from dysfunctional cells because of telomere shortening have been proposed as biomarkers of aging and age-dependent degenerative diseases.92
Dyskeratosis congenita is sometimes misclassified with aging syndromes such as Hutchinson–Gilford progeria and Werner's syndrome, but typical patients with this condition do not appear old, nor do they prematurely have atherosclerosis, Alzheimer's disease, or other classic characteristics of aging such as osteopenia or type 2 diabetes. Many people with TERT and TERC mutations have normal life expectancies. Telomere shortening is not uniform among tissues (e.g., the brain and heart show little shortening) or among the various cells within a tissue. It is not known whether the decline with age in bone marrow cellularity and lung compliance, as well as nodular regeneration in autopsies of the elderly, is due to physiologic telomere attrition. Inbred mice, despite their long telomeres, do age. Decreases in telomere length over a human life span93 do not establish telomere shortening as the cause of aging.
Modulation of Telomerase Activity
Because telomerase is activated in leukemia and solid tumors, the repair complex has been targeted in drug therapy for malignant conditions.79 Since telomere shortening is a risk factor for cancer in patients with dyskeratosis congenita and those with immune-mediated or inflammatory diseases such as aplastic anemia, ulcerative colitis, and Barrett's esophagus, and since telomere attrition may underlie many cancers and degenerative diseases of aging, strategies to maintain or delay telomere attrition may be useful. Prevention of accelerated telomere attrition also may be necessary for effective stem-cell therapies.80
Telomere maintenance and telomerase activation are highly regulated. Although twin studies show telomere length to be largely genetically determined,81,82 some modulation is environmental.83 In tissue culture, exposure to reactive oxygen species appears to accelerate telomere shortening.84 In vitro, hormones and growth factors affect telomerase activity,94 including hematopoietic growth factors.95,96 TERT can be directly activated by the tumor-suppressor protein c-Myc,97 and other components of the repair complex are influenced by specific ubiquitin ligases and protein kinases. Smoking status, diet, socioeconomic status, stress level, and lifestyle might influence telomere dynamics.62,98,99,100 The sex hormones directly increase TERT transcription and telomerase activity in human cells.24,101 Natural and synthetic androgens can restore telomerase activity to normal levels in cells in patients with TERT and TERC mutations24; this probably explains the benefit of these agents in syndromes involving hematopoietic failure. Sex hormones might be used in the treatment of other telomere diseases, such as pulmonary fibrosis and hepatic cirrhosis, for which we now have no effective therapies. Sex-hormone replacement and the use of sex hormones in pharmacologic doses could also be therapeutic in patients with accelerated telomere attrition and a known risk of secondary malignant conditions (e.g., after intensive chemotherapy or hematopoietic stem-cell transplantation). The theoretical desirability of hormone replacement in older healthy persons to "stabilize" telomere loss would need to be balanced against the risks of undesirable effects on secondary sex organs and known associated malignant conditions.
References
Szostak JW, Blackburn EH. Cloning yeast telomeres on linear plasmid vectors. Cell 1982;29:245-255. [CrossRef][Web of Science][Medline]
Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405-413. [CrossRef][Web of Science][Medline]
Heiss NS, Knight SW, Vulliamy TJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet 1998;19:32-38. [Web of Science][Medline]
Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 1999;402:551-555. [CrossRef][Medline]
Olovnikov AM. Principle of marginotomy in template synthesis of polynucleotides. Dokl Akad Nauk SSSR 1971;201:1496-1499. [Medline]
Yamaguchi H, Calado RT, Ly H, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med 2005;352:1413-1424. [Free Full Text]
Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci U S A 1988;85:6622-6626. [Free Full Text]
de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 2005;19:2100-2110. [Free Full Text]
Blasco MA. Telomere length, stem cells and aging. Nat Chem Biol 2007;3:640-649. [CrossRef][Web of Science][Medline]
Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA. Creation of human tumour cells with defined genetic elements. Nature 1999;400:464-468. [CrossRef][Medline]
Sarin KY, Cheung P, Gilisonee E, et al. Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature 2005;436:1048-1052. [CrossRef][Medline]
Choi J, Southworth LK, Sarin KY, et al. TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program. PLoS Genet 2008;4:e10-e10. [CrossRef][Medline]
Alter BP. Inherited bone marrow failure syndromes. In: Nathan DG, Orkin SH, Look AT, Ginsburg D, eds. Nathan and Oski's hematology of infancy and childhood. 6th ed. Philadelphia: W.B. Saunders, 2003:280-365.
Knight S, Vulliamy T, Copplestone A, Gluckman E, Mason P, Dokal I. Dyskeratosis Congenita (DC) Registry: identification of new features of DC. Br J Haematol 1998;103:990-996. [CrossRef][Web of Science][Medline]
Dokal I. Dyskeratosis congenita in all its forms. Br J Haematol 2000;110:768-779. [CrossRef][Web of Science][Medline]