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DEPRESSION AND ANXIETY 26 : 965–975 (2009) Review GENETICS OF ANXIETY DISORDERS: THE COMPLEX ROAD FROM DSM TO DNA Jordan W. Smoller, M.D. Sc.D., Stefanie R. Block, B.A., and Mirella M. Young, M.A. Anxiety disorders are among the most common psychiatric disorders, affecting one in four individuals over a lifetime. Although our understanding of the etiology of these disorders is incomplete, familial and genetic factors are established risk factors. However, identifying the specific casual genes has been difficult. Within the past several years, advances in molecular and statistical genetic methods have made the genetic dissection of complex disorders a feasible project. Here we provide an overview of these developments, with a focus on their implications for genetic studies of anxiety disorders. Although the genetic and phenotypic complexity of the anxiety disorders present formidable challenges, advances in neuroimaging and experimental animal models of anxiety and fear offer important opportunities for discovery. Real progress in identifying the genetic basis of anxiety disorders will require integrative approaches that make use of these biologic tools as well as larger-scale genomic studies. If successful, such efforts may yield novel and more effective approaches for the prevention and treatment of these common and costly disorders. Depression and Anxiety r 2009 Wiley-Liss, Inc. 26:965–975, 2009. Key words: genetics; anxiety; genomewide association study; heritability A nxiety disorders are associated with an enormous burden of suffering as well as billions of dollars in direct and indirect economic costs. Although established treatments—including medications and cognitive-behavioral therapies—are effective for many patients, there is an ongoing need for improved treatment and prevention strategies. Progress in these areas will require a more complete understanding of the pathogenesis of these disorders. In light of the fact that family history is one of the best established risk factors for anxiety disorders, there has been great interest in efforts to identify the genetic basis of these conditions. Over the past two decades, molecular genetic approaches, including linkage and association analyses, have been applied to search for the relevant genes, but the genetic and phenotypic complexity of anxiety phenotypes has made progress difficult. In the past 5 years, the prospects for genetic research on complex disorders (including the anxiety disorders) have substantially improved with the advent of more powerful genomic approaches. Here, we will provide an overview of the current status of anxiety genetics with a focus on emerging issues in human and experimental animal research that will likely guide the research agenda in the coming years. Figure 1 outlines r 2009 Wiley-Liss, Inc. the successive research strategies used to explore the genetic of psychiatric illness. HOW DO WE KNOW THAT GENES CONTRIBUTE TO ANXIETY DISORDERS? The earliest evidence that anxiety disorders might have a genetic component came from family studies. Psychiatric Genetics Program in Mood and Anxiety Disorders, Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts Presented in part at the Scientific Research Symposium on the Genetics of Anxiety Disorders, 29th Annual Conference of the Anxiety Disordes Association of America, Albuquerque, NM. Correspondence to: Dr. Jordan W. Smoller, Department of Psychiatry and Center for Human Genetic Research, Massachusetts General Hospital, Simches Research Building, 185 Cambridge St., Boston, MA 02114. E-mail: [email protected] DOI 10.1002/da.20623 Published online in Wiley InterScience (www.interscience.wiley. com). 966 Smoller et al. Questions Study Methods Anxiety Disorders Is the disorder familial? Family study Major Depression Eating Disorders How much do genes contribute? Twin and adoption studies Alcohol Dependence What genes are involved? Linkage and association studies What do the genes do? Molecular biology and clinical studies Late Onset Alzheimer's disease Attention Deficit Disorder Bipolar Disorder Figure 1. Chain of psychiatric genetic research. Schizophrenia Since 1970s, numerous family studies have documented that the risk of specific anxiety disorders (including panic and phobic disorders, obsessive-compulsive disorder, and generalized anxiety disorder) is higher in first degree relatives of affected probands compared to relatives of unaffected controls.[1–16] Overall, firstdegree relatives have an approximately four- to six-fold increased risk of the proband’s disorder.[17] Estimates of the familiality of posttraumatic stress disorder (PTSD) are more difficult to obtain because they would require matching family members on trauma exposure. Of course, the fact that anxiety disorders aggregate in families does not necessarily mean that genes contribute as family members share both genes and environmental exposures. One way to separately estimate the genetic and environmental components of disorder variance is to examine the phenotypic similarity of twins. Twin studies compare the similarity or concordance rate among identical (monozygotic) twins and nonidentical (dizygotic) twins and ask: given that one twin has the disorder, what’s the probability that the second twin does? If the concordance for a disorder is greater in the identical twins that suggests a genetic contribution—that is, their greater phenotypic similarity is attributable to their greater genetic similarity. One caveat is that if identical twins are treated more similarly by the environment that might make them appear more similar for nongenetic reasons. If the concordance rate for identical pairs, who are essentially clones, is not 100%, there must be something above and beyond the genetic variation involved. For the anxiety disorders, concordance rates have typically been in the range of 12–26% for monozygotic twins and 4–15% for dizygotic twins.[17] Twin studies allow us to estimate the ‘‘heritability’’ of a trait or disorder, an index of the proportion of phenotypic variance in a population that is attributable to genetic factors. That is, the heritability is an estimate of the proportion of disease risk that is due to variation in genes in a population. It is important to recognize that it is a population measure and does not, for example, measure what proportion of an individual’s anxiety disorder is due to genetic factors. Heritability can vary from 0 (no contribution of genetic variation to disease risk) to 100% (entirely due to genetic variation). For the anxiety disorders (including panic disorder, phobic disorders, GAD, OCD, and PTSD), heritability estimates from twin studies have consistently been in Depression and Anxiety 0 50 Approximate Heritability 100 Autism Figure 2. Approximate heritability of selected neuropsychiatric disorders. the range of 20–40%.[18–20] Thus, it is clear that variations in genes contribute to the risk of developing anxiety disorders. As shown in Figure 2, the heritability of anxiety disorders is similar to that for major depressive disorder, but less than a number of other psychiatric disorders. However, the magnitude of the heritability tells us little or nothing about the ‘‘genetic architecture’’ of a disorder—that is how many genes are involved or the magnitude of effects attributable to any contributing genes. For example, a heritability of 75% could represent the additive effects of three loci each accounting for 25% of the variance or 100 loci each accounting for 0.75%. As genetic architecture is the crucial determinant of how easy or difficult it will be to identify specific susceptibility genes, heritability offers little help beyond confirming that there are genes to be found. WHERE ARE THE GENES? As it has turned out, actually finding those genes proved more difficult than some initially expected. The root of this difficulty has to do with the fact that anxiety disorders, and other common medical disorders, are genetically ‘‘complex disorders’’ (Fig. 3). Many familiar medical genetic disorders are due to highly penetrant single gene mutations—for example, the autosomal dominant triplet repeat mutation underlying Huntington disease or the recessive point mutations that cause cystic fibrosis and sickle cell anemia. For such disorders, inheritance of the disease-causing variant produces illness with near certainty. In contrast, common diseases including the anxiety disorders are ‘‘complex’’ in that they reflect the influence of several or many genetic risk factors, each of which may have individually small effects. Moreover, the risk genes may require interactions with other genes (epistasis) or environmental factors (gene–environment interaction). In addition, there may be substantial genetic heterogeneity (different susceptibility genes segregating in different families). Detecting such small and diverse effects may be quite difficult. Review: Genetics of Anxiety Disorders Single Gene Disorder Complex Disorder Example: Huntington Disease (dominant inheritance) • • • Single gene causes disease • Disease requires one copy of mutation • • • 967 Not due to single gene Several or many genes may contribute Each may have small effect by itself Effects may depend on interaction with environment Figure 3. Single gene versus complex disorders. In the 1980s and early 1990s, after the mapping and later cloning of several important disease genes (including those causing Huntington disease and cystic fibrosis), there was great optimism that genes for psychiatric disorders would soon be found. With the recognition that the genetic architecture of psychiatric disorders was likely to be quite complex, this era of ‘‘irrational exuberance’’ gave way to a period of near hopelessness as researchers worried that mental illness genes might simply be out of reach. In general, efforts to localize and identify risk genes for anxiety disorders have relied on two well-established strategies. The first, linkage analysis, examines whether DNA markers spaced at intervals across the genome are co-inherited with the illness within families. Linkage analysis is well-suited to mapping genes of major effect (and, in fact, was the initial method that localized the genes for Huntington disease, cystic fibrosis, and many other single-gene medical genetic disorders). Essentially, this method asks the question ‘‘where in the genome are the disease genes located?’’ by determining whether markers in specific chromosomal regions are transmitted along with the phenotype more than expected by chance. These regions or ‘‘loci’’ are then considered likely to harbor susceptibility genes. Linkage studies of the anxiety disorders have implicated several chromosomal regions, although the results have been largely inconclusive (reviewed in[21]). Suggestive linkage for panic anxiety phenotypes has been reported for regions on chromosomes 1q,[22] 2q,[23] 7p,[24,25] 9q,[26] 12q,[27] 13q,[28,29] 15q,[23] and 22q.[28] Gelernter et al. conducted linkage analyses of phobic disorders in a set of pedigrees ascertained for panic disorder and reported evidence implicating chromosome 3q for agoraphobia,[22] 14q for specific phobia,[30] 16q for social phobia.[31] For OCD, suggestive evidence of linkage has been found for 3q27–q28,[32] 9p24,[33,34] 10p15,[35] and 14q.[36] In recent years, association analyses have become far more common than linkage for genetic studies of complex disorders. Association studies ask ‘‘which genes are involved?’’ by examining whether there is a correlation between specific alleles and the phenotype of interest. The most common design—the casecontrol study—is a standard epidemiologic design. For example, a case-control study of smoking and myocardial infarction (MI) would ascertain MI cases and unaffected controls and compare the prevalence of smoking between the two groups. If smokers are overrepresented among the cases (relative to controls), the inference would be that smoking is a cause of MI. The same approach is used in genetic association studies except that instead of smoking, the risk factor of interest is an allele or a genotype. Thus, we would ask, ‘‘are certain genotypes over-represented among cases?’’ To date, most association studies of anxiety disorders have focused on ‘‘candidate genes’’—that is, genes that are suspected to play a role in the disorder(s) based on earlier biological evidence (biological candidates) or, in some cases, because they are located within chromosomal regions previously implicated in linkage studies (positional candidates). Among the widely-studied biological candidate genes (sometimes referred to as ‘‘the usual suspects’’) are genes encoding receptors, transporters, and synthetic enzymes involved in neurotransmitter systems that are the target of therapeutic agents (e.g. serotonin, norepinephrine, glutamate, dopamine) and neuropeptides implicated in animal models of anxiety (e.g. the corticotropin releasing hormone system, neuropeptide Y, brain-derived neurotrophic factor). Two types of variations in these loci have been tested in most studies. The first are single nucleotide polymorphisms or SNPs—variations in single DNA bases. These are the most common form of variation in the genome, occurring on average at a frequency of 1 per 1000 bases of DNA sequence. The second comprise short repeated sequences of two to Depression and Anxiety 968 Smoller et al. four nucleotides that occur in stretches of variable length in and around genes (sometimes referred to as ‘‘microsatellites’’). In fact, the most extensively studied variant in genetic studies of anxiety and mood disorders is a di- (two-) nucleotide repeat that is present in the promoter region of the serotonin transporter gene (the so-called 5HTT promoter length polymorphism or 5HTTLPR). The serotonin transporter is the target of the most widely used pharmacotherapy for anxiety disorders—SSRI antidepressants. The ‘‘short‘‘ allele of the 5HTTLPR, lacking 44 base pairs of dinucleotide repeat sequence that are present in the ‘‘long’’ allele, confers reduced transcriptional activity of the serotonin transporter gene.[37] In addition, the polymorphism is common; the ‘‘short’’ allele has a frequency of approximately 45% among individuals of EuropeanAmerican ancestry.[38] These features have made the variation particularly appealing as a target of association studies, and the short allele has been associated with anxiety-related traits including neuroticism, harm avoidance, and, in some studies, anxiety disorders including social phobia, OCD, and PTSD.[39–44] However, the validity of these associations is difficult to establish. For example, there have been many negative studies and nonreplications with anxiety phenotypes. In addition, the polymorphism has been so widely studied that it has been associated with an implausibly large number of psychiatric and nonpsychiatric phenotypes, many of which are likely to be false positives. In a recent study of panic disorder, Strug et al.[45] reported association with an intronic SNP in the serotonin transporter gene (SLC6A4) but no association with the 5HTTLPR polymorphism. A number of other genes have been reported to be associated with anxiety disorders in multiple studies, though in virtually all cases, nonreplications have also been reported. Meta-analyses can be useful in providing a summary estimate of the evidence in favor a putative susceptibility gene or variant and allow the pooling of data to achieve the power needed to support or reject a candidate gene. As shown in Table 1, a small handful of genes have been associated with anxiety disorders in multiple studies. Table 1 shows the only specific variants for which the same allele has been associated with a given anxiety disorders in more than one study at a statistical threshold of Pr.01. Candidate gene studies of gene–environment interaction have begun to appear, but this remains an under-utilized strategy for genetic studies of anxiety. Such analyses are particularly relevant for understanding the pathogenesis of PTSD that, by definition, requires exposure to an environmental trauma. Despite the fact that trauma exposure is common, only a subset of victims develop PTSD and it seems likely that genetic variation contributes to this vulnerability. For example, Binder et al.[46] recently reported that SNPs in the FKBP5, which regulates glucocorticoid receptor sensitivity, were associated with PTSD severity among victims of child abuse (but not other trauma). Candidate gene studies have been plagued by an ‘‘inconvenient truth’’: despite the claims made for the importance of specific genes in anxiety or other psychiatric disorders, it has been very difficult to establish a role for them based on association studies. There are likely several reasons for this. First, the likelihood that a reported candidate gene association is a true positive is related to the gene’s earlier probability of association. There are more than 20,000 genes in the human genome, and a substantial fraction are brainexpressed. Any of these could be considered a candidate gene for neuropsychiatric phenotypes like the anxiety disorders. However, the earlier probability is low that the specific candidate gene selected for a given study is in fact causally related to the disorder (as our understanding of the pathogenesis of anxiety disorders remains limited). In this circumstance, an association with a candidate gene in a single study is likely to be a false positive. Other factors contributing to falsepositive reports of association include inadequate correction for multiple testing, inadequate genotyping quality control, confounding due to population stratification (differences in genetic background between cases and controls) and the ‘‘winner’s curse’’ (an ascertainment bias in which the initial report of an association effect size is likely to be inflated).[47] Given TABLE 1. Candidate gene studies associated (at Pr.01) with specific anxiety disorders in at least two independent studies Disorder PD OCD PTSD Gene Variant Risk allele ] Positive studies/total studies 5HT2AR COMT COMT COMT DRD2 FKBP5 rs6313 (T102C) rs4680 (val158met) rs4680 (val158met) rs4680 (val158met) Taq1 (rs1800497) rs3800373 C G (Met) A (Val) G (Met) A1 C 2/7 2/13 2/13 4/16 2/5 2/2 PD, Panic disorder; OCD, Obsessive-compulsive disorder; PTSD, Posttraumatic stress disorder. Pr.01. Meta-analysis has not supported an association with PD.[113] Supported by recent meta-analysis.[114] Based on studies in English-language publications. References available upon request. Depression and Anxiety Review: Genetics of Anxiety Disorders the explosive proliferation of genetic association studies and the likelihood that a large proportion of reported findings are false positives, replication in adequately-powered independent samples has become the sine qua non for establishing valid genotype–phenotype associations.[48] At the same time, many association studies may be at risk for Type II error—that is, missing true associations. Most complex disorders are believed to be highly polygenic and common variants underlying these disorders are expected to have very modest effect sizes (e.g. odds ratios for susceptibility alleles on the order of 1.1–1.4).[49] Reliably detecting such effects requires extremely large sample sizes—on the order of thousands of cases and controls—much larger than the samples that have typically been examined in candidate gene studies. In addition, studies may examine only a fraction of the variants in a given candidate gene, further increasing the risk of false-negative conclusions about such genes. WHY IT’S HARD TO GO FROM DSM TO DNA These methodological considerations have contributed to the difficulty in establishing susceptibility variants for anxiety disorders and other psychiatric disorders. But there are additional difficulties to overcome (Fig. 4). The genetic complexity of these disorders presents additional challenges, including the existence of genetic heterogeneity, epistasis, and gene–environment interactions (see earlier), all of which increase the sample size requirements for successful association studies. But there is another important reason challenge that has made it difficult to establish the specific genes that account for the heritability of psychiatric disorders—that is, to go ‘‘from DSM to DNA.’’ The problem of ‘‘phenotypic complexity’’ is a challenge that may be more daunting for psychiatric genetics than for other fields of medical genetics. As reviewed earlier,[21] the current definitions of anxiety disorders are relatively recent, dating to DSM-III in 1980, and the boundaries among them are far from clear. Family studies have demonstrated Challenge • Genetic Complexity p y – Multiple genes of small effect – Gene–gene & gene–environment interaction • Phenotypic Complexity – Limitations of diagnostic categories Strategy ⇑Sample Size and Genomewide studies ⇑ Effect Size: Identity more powerful phenotypes Figure 4. Complex disorders: Why it’s hard to go from DSM to DNA. 969 significant familial co-aggregation of DSM-IV anxiety disorders (for review, see [21]). For example, relatives of probands with social phobia have an increased risk of agoraphobia and panic disorder in addition to social phobia itself. Relatives of probands with generalized anxiety disorder are at increased risk of both GAD and panic disorder; and relatives of probands with OCD are at increased risk for panic and phobic disorders as well as OCD. Twin studies have confirmed that genes contribute to the familial clustering of anxiety disorders. In a large population-based twin sample, Hettema et al.[18] identified two genetic factors underlying the major anxiety disorders with one factor primarily predisposing to PD, agoraphobia, and GAD and the other primarily influencing specific phobias. Social phobia showed overlapping influences of both factors. Anxiety-related personality traits may underlie these cross-disorder effects. For example, neuroticism and introversion are heritable traits that are associated with a range of anxiety disorders. Twin studies have shown that, taken together, genetic influences on neuroticism and introversion entirely account for genetic variation in risk of social phobia and agoraphobia.[50] GO BIG OR GO DEEP Broadly speaking, investigators have pursued two strategies to address the complexity of psychiatric disorders and enhance the power to find susceptibility genes. The first has been to vastly increase the scope of genetic studies—both in terms of genes and sample size. The second has been to try to maximize detectable effect sizes by examining phenotypes that may be more direct expressions of anxiety disorder genes than are the disorders themselves. The first strategy, that of ‘‘going big’’, has been enabled by dramatic recent advances in our understanding of the genome (Fig. 5). Following the sequencing of the human genome in 2001,[51] several large-scale projects have provided key tools for • Up to 1 million genetic markers can be examined i lt ly simultaneous • The “whole haystack” contained on a single gene “chip” • Before 2006, only a handful of genes had been found for any common medical disorders di d Disease Genes/Loci Discovered 2006-2008 Diabetes >25 Cardiovascular >20 Inflammatory Bowel Disease >30 Autoimmune Disease >20 Obesity Cancer >20 >30 Total >400 Figure 5. Genomewide association studies: a major advance for complex trait genetics. Depression and Anxiety 970 Smoller et al. genomic analyses. The International HapMap project,[52] which cataloged genomic variation across different populations, has provided a database of SNPs that have made genomewide association analysis (GWAS) feasible. Instead of limiting the search to pre-specified candidate genes, GWAS analysis provides a hypothesis-free (also called, unbiased) survey of the entire genome in a single experiment. These studies make use of the fact that the alleles of many SNPs are strongly correlated—or, more technically, there is extensive ‘‘linkage disequilibrium’’ across the genome. Thus, a given SNP may provide information about other (correlated) SNPs, so that common genetic variation throughout the genome can be assayed without having to genotype all of the millions of individual SNPs that actually exist. By selecting a reduced set of SNPs that efficiently ‘‘tag’’ nearby genetic variants, the entire genome can be interrogated using DNA chips that simultaneously assay up to one million or more SNPs.[53] Because of the large number of statistical tests involved, very large sample sizes and stringent statistical measures to control false-positive rates are needed. A consensus has emerged that a P value threshold of at least 10 7 (or, more stringently, 5 10 8) is the appropriate genomewide correction to account for the approximately 1 million independent tests involved in surveying the entire genome.[54,55] Nevertheless, the GWAS approach has already unequivocally identified several genes and variants that influence complex disorders including diabetes, autoimmune disease, cardiovascular disorders, cancer, and others.[56] Before 2006, only a handful of susceptibility variants had been found for common medical disorders. In the last 3 years, largely through the application of GWAS analysis, this number has increased to several hundred.[57] In the past 2 years, genomewide analysis of common SNPs and rare copy number variations (deletions or duplications of typically 10 kilobases to a few megabases of DNA sequence) have been strongly associated with psychiatric disorders including autism,[58,59] to bipolar disorder (BPD),[60] and SCZ.[61–65] To date, few applications of GWAS methods to anxiety phenotypes have been reported. The first published GWAS of panic disorder examined 200 cases and 200 controls of Japanese origin.[66] Seven SNPs in or near genes met a threshold of Po10 6 (for a false discovery rate of o.05). The strongest signals were observed for SNPs in TMEM16B (top SNP 5 rs12579350, P 5 3.7 10 9), a gene on chromsome 12p13 whose function remains poorly understood, and PKP1 (top SNP 5 rs860554, P 5 4.6 10 8), a gene on 1q32 involved in cytoskeleton/cell membrane interactions. None of the ‘‘usual suspect’’ candidate genes showed evidence of association. These results await replication. GWAS analyses of the anxiety-related trait neuroticism have also appeared[67–69] though no loci explaining more than 1% of the variance in the trait have emerged. In one GWAS study, Van den Oord Depression and Anxiety et al.[69] reported moderate evidence of association for SNPs in MAMDC1, a gene thought to be involved in neuronal migration, with neuroticism in a US sample that approached genomewide significance when combined with GWAS data from an independent German sample. Overall, the results to date suggest that neuroticism is likely to be highly polygenic and identifying the genes that contribute to its overall heritability may require very large samples. The other major strategy for enhancing the power of genetic studies has been to define phenotypes that may be more proximal expressions of genes that confer susceptibility to the target disorder (‘‘going deep’’). These putative endophenotypes or intermediate phenotypes are familial or heritable traits that are thought to underlie the clinically diagnosed disorders. The hope is that genes that may have only modest effects on the disorder would have larger effects on these more elemental traits. It is here that genetic studies of anxiety disorders have advantages over studies of many other psychiatric disorders. In particular, research in neuroimaging and experimental animal models has identified key elements of the neural circuitry and biological pathways that mediate anxiety and fear behavior. Functional MRI (fMRI) studies have shown that anxiety disorders are associated with altered limbic reactivity during emotion processing tasks. For example, a meta-analysis of fMRI studies[70] found increased activity of amygdala and insula among individuals with social and specific phobias and PTSD compared with healthy controls. PTSD was further characterized by reduced activity of the anterior cingulate and ventromedial prefrontal cortices. Such phenotypes provide attractive targets for genetic studies because they reflect the underlying functional biology that lies between genetic variation and disorder risk. A number of specific candidate polymorphisms have shown evidence of association with brain measures of emotional reactivity related to anxiety. Of these, the most widely studied has been the 5HTTLPR polymorphism that was first associated with amygdala reactivity by Harriri et al.[71] Since then several studies have replicated an association between the short allele of this variation and increased amygdala reactivity to emotional stimuli. Further analysis has suggested that short allele carriers exhibit reduced connectivity of the anterior cingulated and amygdala, suggesting that this variant may enhance fear reactivity by reducing cortical inhibition of amygdala responses to threat.[72] A recent meta-analysis of 14 studies (total N 5 339) supported the association of the short allele with increased amygdala reactivity (overall P 5.002).[73] Animal models of anxiety-related traits have also been used to characterize the neurobiologic basis of fear and anxiety phenotypes. Several fear and anxietyrelated traits appear to be evolutionarily conserved phenotypes that resemble primate and human anxiety. Rodent assays of fear behavior are arguably, along with substance abuse models, the best validated animal Review: Genetics of Anxiety Disorders models of human psychopathology. They also provide powerful tools for dissecting the role of specific genes in anxiety-related traits. For example, genetic mapping in large-scale mouse crosses led to the localization and ultimate identification of a quantitative trait gene, rgs2, that influences murine anxious temperament.[74] Gene targeting approaches, in which specific genes can be inserted (transgenic mice) or deleted (knockout mice) from the genome, have allowed researchers to examine the effect of specific genes on anxiety-related behaviors.[75] As an example, mice in which the serotonin 1A receptor was deleted were found to exhibit increased anxiety behaviors.[76] Using a tissue-specific conditional rescue strategy, Gross et al.[77] found that expression of the gene in the hippocampus and cortex (but not in the raphe nuclei) is sufficient to rescue the anxiety phenotype of the knockout mice. They further showed that the timing of gene expression is crucial: using a conditional knockout that inactivated the gene in either early development or adulthood they demonstrated that function of the gene in the early postnatal period determines anxiety-like behavior in the adult. Such studies can demonstrate the neuroanatomic and developmental importance of candidate genes. Of note, human studies have implicated the 5HT1A receptor gene and its expression in panic and phobic anxiety disorders.[78–80] Expression profiling in mouse brain tissue provides another powerful tool for identifying genes relevant to anxiety phenotypes. For example, Hovatta et al.[81] compared mRNA expression levels of approximately 10,000 genes in limbic brain regions from mouse strains that differed in anxiety-proneness. They identified 17 genes whose expression levels correlated with anxiety behavior and went on to use gene targeting methods to implicate two of these genes (glyoxylase 1 and glutathione reductase 1) as causally related to the anxiety phenotypes. In a subsequent study, Donner et al.[82] examined variants in the human orthologues of these 17 genes in a sample of 321 cases and 653 controls form a population-based Finnish sample. They observed nominally significant association (Po.01) for social phobia (with variants in ALAD and CDH2), panic disorder (EPB41L4Aa and PSAP), and generalized anxiety disorder (PTGDS, DYNLL2, and EPB41L4A). Gene targeting methods in rodents also allow us to go in the opposite direction—from human studies to experimental animal models—to characterize the functional significance of risk alleles. Several studies have suggested that the Val66Met polymorphism in the brain derived neurotrophic factor (BDNF) gene is associated with a variety of neuropsychiatric phenotypes relevant to mood and anxiety disorders. Chen et al.[83] demonstrated that mice engineered to express the human Met allele exhibit increased anxiety-related behaviors and are resistant to SSRI treatment. Primate models of anxious temperament have also provided opportunities to examine genetic and 971 gene–environment effects on anxiety. Anxious temperament (behavioral inhibition) is heritable in rhesus macaques and is associated with hyper-reactivity of limbic brain circuits that have also been implicated in human anxious temperament.[84,85] Furthermore, monkeys carrying the short allele of the rhesus 5HTTLPR exhibit anxious temperament and altered stress hormone responses, particularly after early adversity.[86–88] In an example of combining insights from animal models and intermediate phenotypes, we have been studying the effects of mouse anxiety genes on behavioral and biological traits underlying social anxiety disorder[89] with a focus on the temperamental profile known as behavioral inhibition to the unfamiliar (BI).[90] Several features of the BI phenotype make it particularly attractive for studying the genetic basis of anxiety. BI is observable in laboratory protocols as early as 14 months of age and consists of a stable tendency to be cautious, quiet, and behaviorally restrained in situations of novelty. Longitudinal research has also demonstrated that BI is a stable profile in childhood[91] and is a familial and developmental risk factor for anxiety disorders, particularly social anxiety disorder.[92–94] Estimates of the heritability of BI (in the range of 40–75%) have exceeded those for the anxiety disorders themselves,[95,96] and biologic features of BI have been documented including evidence of sympathetic hyperreactivity,[97] activation of the HPA axis, and asymmetric (right4left) frontal EEG activity.[97–100] Functional MRI studies have shown that adults classified as inhibited in early childhood exhibit increased amygdala reactivity to novel and emotional stimuli.[101,102] In addition, behavioral inhibition has also been described and well-studied in animal models including rodents and monkeys. Like children with BI, fearful mice exhibit inhibition of behavior and autonomic arousal in response to novel, unfamiliar, or threatening environments.[103] As in humans, anxious temperament appears to be highly heritable in rodent and primate models.[85,104,105] As noted earlier, Rgs2 was identified by positional mapping as a contributor to mouse anxious temperament phenotypes resembling BI.[74] The gene encodes a regulator of G-protein signaling that accelerates the deactivation of G-proteins that are second messengers for neurotransmitters including norepinephrine and serotonin.[106] We observed association between variants in the human orthologue (RGS2) with BI in a family-based sample in which children had undergone laboratorybased temperament assessments.[89] In particular, a haplotype of SNPs spanning the gene conferred a three-fold increased likelihood of inhibited temperament in these children. The same alleles were associated with introversion, a trait for which BI is a developmental precursor, in a sample of 744 adults. Finally, we examined whether the gene is associated with brain endophenotypes common to anxious temperament and anxiety disorders. The same allele of a SNP (rs4606, previously associated with reduced RGS2 Depression and Anxiety 972 Smoller et al. expression) that was associated with BI and introversion, also predicted increased amygdala and insula response to emotional stimuli in a sample (N 5 55) of adults who underwent fMRI. Taken together, these results suggest that genetic variation associated with reduced expression of RGS2 contributes to increased reactivity of limbic brain structures modulating anxious temperament and social anxiety. Although these findings require replication, they highlight the potential utility of combining clues from behavioral and developmental research, neuroimaging, and animal models for dissecting the genetic basis of anxiety disorders. However, the role of RGS2 in the etiology of anxiety disorders remains unclear as (nominal) association has been reported for the rs4606 SNP and panic disorder,[107] PTSD,[108] and GAD,[109] but with the allele opposite to that associated with BI. CONCLUSIONS AND FUTURE DIRECTIONS Several conclusions have emerged from the past two decades of research on the familial and genetic basis of anxiety disorders. First, it is clear that all of the major DSM-IV anxiety disorders run in families and are heritable. In addition to motivating molecular genetic studies, this provides evidence that the clinical diagnostic categories capture phenotypes that are under genetic influence. However, the second conclusion that can be drawn from family and twin studies is that the familial and genetic boundaries among these disorders are not sharply defined. They co-aggregate in families and are genetically correlated, suggesting that the genetic and environmental risk factors among subsets of these disorders are shared. This creates a challenge for efforts to define phenotypes for gene mapping studies. We can also be confident that the anxiety disorders, like other common psychiatric and medical syndromes are genetically complex. It is likely that many genes of modest effect contribute and that the expression of anxiety disorder phenotypes involves interactions among genes and between genes and environmental influences. To date, molecular genetic studies (linkage and association studies) have implicated several specific genes but few findings have been robustly replicated. This is at least partly attributable to the limited set of genes that have been examined and the need for much larger studies to achieve sufficient power to detect susceptibility genes. How can we move the ball forward? Lessons from other successful studies of other complex diseases suggest a few directions. First, genetic association studies will require much larger samples than have been examined to date. While the underlying genetic architecture of the anxiety disorders remains obscure, GWAS studies that have established risk genes for diseases like diabetes, autoimmune disease, and cardiovascular disease[110] suggest that common risk alleles are likely to have very Depression and Anxiety small effect sizes. Indeed, identifying genes influencing height, a highly heritable phenotype, required GWAS studies of tens of thousands of cases and controls,[111] with rigorous replication in independent samples. Success in these other areas of medicine has required extensive collaboration and the formation of research consortia to achieve the kinds of power that are simply impossible for individual research groups. Investigators interested in the genetics of anxiety have recently formed a collaborative network (the ANxiety Genetics Study Team, or ANGST) that may provide such a resource for identifying anxiety disorder genes. Recent evidence from genomewide studies of schizophrenia and autism have highlighted the possibility that the genetic architecture of complex psychiatric phenotypes involves a combination of common variants of small effect and rarer variants of larger effect (e.g. copy number variations). Genomewide analyses of copy number variation have not yet been undertaken for the anxiety disorders. In the coming years, advances in highthroughput whole genome DNA sequencing will facilitate genomewide studies of rare variants, but again, large sample sizes will be needed for such analyses. Progress in genomic biology is creating other exciting opportunities for molecular studies of complex disease. For example, epigenetic modification of DNA and associated proteins appears to have important effects on gene expression that may underlie environmental influences on disease risk. Preclinical studies in rodents have suggested that early adversity can exert lasting effects on stress responsivity through such mechanisms.[112] The role of epigenetic effects on human anxiety is a fertile area for research. In any case, the strong likelihood that environmental factors interact with genetic risk to confer vulnerability to anxiety disorders underscores the need for further research on gene–environment interaction. 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