Seon-Sook An

A whole genome association study of neuroticism using DNA pooling

We describe a multistage approach to identify single nucleotide polymorphisms (SNPs) associated with neuroticism, a personality trait that shares genetic determinants with major depression and anxiety disorders. Whole genome association with 452 574 SNPs was performed on DNA pools from ∼2000 individuals selected on extremes of neuroticism scores from a cohort of 88 142 people from southwest England. The most significant SNPs were then genotyped on independent samples to replicate findings. We were able to replicate association of one SNP within the PDE4D gene in a second sample collected by our laboratory and in a family-based test in an independent sample; however, the SNP was not significantly associated with neuroticism in two other independent samples. We also observed an enrichment of low P-values in known regions of copy number variations. Simulation indicates that our study had ∼80% power to identify neuroticism loci in the genome with odds ratio (OR)>2, and ∼50% power to identify small effects (OR=1.5). Since we failed to find any loci accounting for more than 1% of the variance, the heritability of neuroticism probably arises from many loci each explaining much less than 1%. Our findings argue the need for much larger samples than anticipated in genetic association studies and that the biological basis of emotional disorders is extremely complex.

Association between glutamic acid decarboxylase genes and anxiety disorders, major depression, and neuroticism

Abnormalities in the gamma-aminobutyric acid (GABA) neurotransmitter system have been noted in subjects with mood and anxiety disorders. Glutamic acid decarboxylase (GAD) enzymes synthesize GABA from glutamate, and, thus, are reasonable candidate susceptibility genes for these conditions. In this study, we examined the GAD1 and GAD2 genes for their association with genetic risk across a range of internalizing disorders. We used multivariate structural equation modeling to identify common genetic risk factors for major depression, generalized anxiety disorder, panic disorder, agoraphobia, social phobia and neuroticism (N) in a sample of 9270 adult subjects from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. One member from each twin pair for whom DNA was available was selected as a case or control based on scoring at the extremes of the genetic factor extracted from the analysis. The resulting sample of 589 cases and 539 controls was entered into a two-stage association study in which candidate loci were screened in stage 1, the positive results of which were tested for replication in stage 2. Several of the six single-nucleotide polymorphisms tested in the GAD1 region demonstrated significant association in both stages, and a combined analysis in all 1128 subjects indicated that they formed a common high-risk haplotype that was significantly over-represented in cases (P=0.003) with effect size OR=1.23. Out of 14 GAD2 markers screened in stage 1, only one met the threshold criteria for follow-up in stage 2. This marker, plus three others that formed significant haplotype combinations in stage 1, did not replicate their association with the phenotype in stage 2. Subject to confirmation in an independent sample, our study suggests that variations in the GAD1 gene may contribute to individual differences in N and impact susceptibility across a range of anxiety disorders and major depression.

Variants in nicotinic acetylcholine receptors α5 and α3 increase risks to nicotine dependence.

Nicotinic acetylcholine receptors bind to nicotine and initiate the physiological and pharmacological responses to tobacco smoking. In this report, we studied the association of α5 and α3 subunits with nicotine dependence and with the symptoms of alcohol and cannabis abuse and dependence in two independent epidemiological samples (n = 815 and 1,121, respectively). In this study, seven single nucleotide polymorphisms were genotyped in the CHRNA5 and CHRNA3 genes. In both samples, we found that the same alleles of rs16969968 (P = 0.0068 and 0.0028) and rs1051730 (P = 0.0237 and 0.0039) were significantly associated with the scores of Fagerström test for nicotine dependence (FTND). In the analyses of the symptoms of abuse/dependence of alcohol and cannabis, we found that rs16969968 and rs1051730 were significantly associated with the symptoms of alcohol abuse or dependence (P = 0.0072 and 0.0057) in the combined sample, but the associated alleles were the opposite of that of FTND. No association with cannabis abuse/dependence was found. These results suggested that the α5 and α3 subunits play a significant role in both nicotine dependence and alcohol abuse/dependence. However, the opposite effects with nicotine dependence and alcohol abuse/dependence were puzzling and future studies are necessary to resolve this issue.

Catechol-O-methyltransferase contributes to genetic susceptibility shared among anxiety spectrum phenotypes.

BACKGROUND Catechol-O-methyltransferase (COMT) has been investigated for its possible role in a wide range of psychiatric phenotypes. In particular, several studies support association of this gene with panic disorder and other anxiety-related traits. METHODS We examined the COMT gene for association with genetic risk across a range of anxiety spectrum phenotypes. We used multivariate structural equation modeling to select twin pairs scoring at the extremes of a latent genetic risk factor shared by neuroticism, several anxiety disorders, and major depression from a large population-based twin sample. With one member from each of these pairs, the resulting sample of 589 cases and 539 control subjects were entered into a two-stage association study in which genetic markers were screened in stage 1, the positive results of which were tested for replication in stage 2. RESULTS The functional val158met polymorphism (rs4680) plus nine other single nucleotide polymorphism markers selected to capture the major allelic variation across the COMT locus were analyzed for differences between cases and control subjects. Although the val (G) allele of rs4680 showed marginally significant association in our combined stage 1 plus stage 2 sample, a high-risk haplotype of this allele with the A allele of rs165599 was significantly over-represented in cases (p = 1.97e-5, odds ratio = 1.95). This haplotype also predicted individual differences in neuroticism and risk for several anxiety disorders and major depression. Consistent with prior studies, our findings are female-specific. CONCLUSIONS Variations in the COMT gene contribute to genetic risk shared across a range of anxiety-related phenotypes.

Cannabinoid Receptor 1 Gene Association With Nicotine Dependence

CONTEXT The endogenous cannabinoid system has been implicated in drug addiction in animal models. The cannabinoid receptor 1 (CNR1) gene is 1 of the 2 receptors expressed in the brain. It has been reported to be associated with alcoholism and multiple drug abuse and dependence. OBJECTIVE To test the hypothesis that the CNR1 gene is associated with nicotine dependence. DESIGN Genotype-phenotype association study. Ten single-nucleotide polymorphisms were genotyped in the CNR1 gene in 2 independent samples. For the first sample (n = 688), a 3-group case-control design was used to test allele association with smoking initiation and nicotine dependence. For the second sample (n = 961), association was assessed with scores from the Fagerström Test for Nicotine Dependence (FTND). Settings Population samples selected from the Mid-Atlantic Twin Registry. PARTICIPANTS White patients aged 18 to 65 years who met the criteria of inclusion. MAIN OUTCOME MEASURES Fagerström Tolerance Questionnaire and FTND scores. RESULTS Significant single-marker and haplotype associations were found in both samples, and the associations were female specific. Haplotype 1-1-2 of markers rs2023239-rs12720071-rs806368 was associated with nicotine dependence and FTND score in the 2 samples (P < .001 and P = .009, respectively). CONCLUSION Variants and haplotypes in the CNR1 gene may alter the risk for nicotine dependence, and the associations are likely sex specific.

Association study between the serotonin 1A receptor (HTR1A) gene and neuroticism, major depression, and anxiety disorders.

The serotonin neurotransmitter system in general, and the serotonin 1A receptor in particular, has been broadly implicated in the pathophysiology of mood and anxiety disorders, although the results of genetic association studies have been mixed. In this study, we examined the serotonin 1A receptor gene, HTR1A, for its association with shared genetic risk across a range of anxiety and depression‐related phenotypes. Using multivariate structural equation modeling, we selected twin pairs from the population‐based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, and several anxiety disorders. One member from each selected pair was entered into a 2‐stage, case‐control association study for the HTR1A gene. In the resulting sample of 589 cases and 539 controls, four SNPs spanning the HTR1A locus, including the C(−1019)G functional promoter polymorphism (rs6295), were screened in stage 1, the positive results of which were tested for replication in stage 2. While one marker met threshold significance criteria in stage 1, this association was not replicated in stage 2. Post‐hoc analyses did not reveal association to any of the specific psychiatric phenotypes. Our data suggests that the HTR1A gene may not play a major role in the genetic susceptibility underlying depressive and anxiety‐related phenotypes.

Lack of association between the amiloride-sensitive cation channel 2 (ACCN2) gene and anxiety spectrum disorders.

Objective Ion channels are involved in a wide range of central nervous system functions and have been implicated in several neuropsychiatric disorders. Rodent studies suggest that the acid-sensing ion channel, ASIC1, may play a role in fear conditioning, a model for human anxiety disorders. In this study, we examined, for the first time, the human analog of ASIC1, the amiloride-sensitive cation channel 2 (ACCN2) gene, for its association with genetic risk across a range of anxiety spectrum phenotypes. Methods Using multivariate structural equation modeling, we selected twin pairs scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, generalized anxiety disorder, panic disorder, agoraphobia, and social phobia, from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. One member from each selected pair for whom DNA was available was entered into a 2-stage, case–control association study for the ACCN2 gene. In the resulting sample of 589 cases and 539 controls, a total of seven single nucleotide polymorphisms that represented the major allelic variation across the ACCN2 locus were screened in stage 1, the positive results of which were tested for replication in stage 2. Results Although several markers or haplotypic combinations met threshold significance criteria in stage 1, their association was not replicated in stage 2. Post hoc analyses did not reveal significant association to the specific psychiatric phenotypes. Conclusions Although the ACCN2 gene may play a role in rodent fear conditioning, we could not detect association with genetic risk shared among human anxiety spectrum disorders.

Association study of CREB1 with Major Depressive Disorder and related phenotypes

Cyclic AMP response element binding protein (CREB) has been implicated in behavioral models of anxiety and depression, antidepressant response in humans, and suicide. One group reported a female‐specific association of the CREB1 gene in early‐onset Major Depressive Disorder (MDD), while another found no evidence of association with this phenotype. In this study, we sought to examine the evidence for association of the CREB1 gene to MDD and related phenotypes. We used multivariate structural equation modeling to identify and select twin pairs that scored at the extremes of a latent genetic risk factor shared by MDD, neuroticism, and several anxiety disorders from the Virginia Twin Registry. Using one member from each of these pairs, the resulting sample of 589 cases (including 473 subjects with lifetime MDD) and 539 controls were entered into a 2‐stage association study in which genetic markers were screened in stage 1, the positive results of which were tested for replication in stage 2. Eight SNP markers selected to capture the major allelic variation across the haplotype block containing CREB1 were analyzed for differences between cases and controls. Several markers showed criterion differences between cases and controls in the stage 1 sample with some evidence of sex specific effects. However, none of these markers were significant in stage 2 in either sex individually or combined. Our data suggests that common variations in the CREB1 gene do not appear to increase susceptibility for MDD or related phenotypes.

Follow-up association study of novel neuroticism gene MAMDC1.

Neuroticism is a personality trait reflecting a tendency towards negative affective states. Extant research supports its role as a risk marker for, and its potential shared genetic basis with, mood and anxiety disorders (Hettema et al., 2006). Our group recently performed a genomewide association study (GWAS) of neuroticism in 1227 US Caucasion participants in which the most promising markers were subsequently tested for replication in a German sample (N = 1880) (van den Oord et al., 2008). The strongest associations derived from four correlated single-nucleotide polymorphisms (SNPs) in the gene MAMDC1 (also known as MDGA2), with P values of 10−5 to 10−6 in the original sample and 0.006–0.025 in the replication sample and with the same direction of effects. Despite these encouraging results, conventional wisdom emphasizes the need for replication of such GWAS findings in multiple independent samples (McCarthy et al., 2008). In this study, we attempted a second replication of the MAMDC1 gene for association with neuroticism in 2722 US Caucasion participants from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders [see (Kendler and Prescott, 1999) for sample details]. Neuroticism was assessed with the Eysenck Personality Questionnaire, the same instrument used in the original GWAS analysis. The four SNPs in MAMDC1 that showed association in the original study (rs7151262, rs1288334, rs1959813, and rs3007105) were in very high linkage disequilibrium, each tagging the same two haplotypes accounting for 94% of the haplotypic variation. Using the Tagger module of HAPLOVIEW 4.0 (Barrett et al., 2005) with HapMap Phase II data, we confirmed their high correlation (r2 = 1) in a 12kb block within MAMDC1. We genotyped one marker (rs7151262) that captured 11 of 13 HapMap SNPs with minor allele frequency >0.05 at r2 > 0.8 in this block. This SNP was tested for association through linear regression assuming an additive genetic effect, as in the original study. The genotypes for rs7151262 were available for 2686 participants in our sample. They were in Hardy—Weinberg equilibrium and the minor allele frequency (0.40) was the same as that found in the prior US and German samples. No association of rs7151262 with neuroticism was found (P > 0.6), although there was a nonsignificant trend for mean neuroticism scores to increase with the number of copies of the minor allele, consistent with the direction of effects in the original study. In a large, independent sample, we attempted to replicate an association identified in a prior GWAS study between neuroticism and marker rs7151262 within the MAMDC1 gene. We followed ‘best practice’ recommendations of testing the same marker with the same phenotype in a sample of similar ethnicity as in the original study (McCarthy et al., 2008). Our large sample possessed sufficient power (about 80%) to detect the predicted effect size attributable to this marker (explaining 0.3% of individual variation in neuroticism estimated from the original German replication sample). Allele frequencies obtained in the current sample were similar to those in the original samples, suggesting comparability of genetic make-up of the samples and genotyping accuracy. Despite satisfying all of these conditions, the current analysis failed to detect a significant association signal, underscoring the importance of multiple replication attempts in independent samples for GWAS findings.

Genetic association between RGS1 and internalizing disorders

Objective Quantitative trait loci identified in animal models provide potential candidate susceptibility loci for human disorders. In this study, we investigated whether internalizing disorders (anxiety disorders, major depression, and neuroticism) were associated with a region on human chromosome 1 syntenic with a quantitative trait locus for rodent emotionality. Methods We genotyped 31 single-nucleotide polymorphisms in genes located on chromosome 1q31.2 in a two-stage association study of 1128 individuals chosen for a high or a low genetic risk for internalizing disorders from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders. Results None of the individual single-nucleotide polymorphisms showed consistent association across stages. A four-marker haplotype in the regulator of G-protein signaling 1 gene (RGS1) was significantly associated with decreased internalizing risk in both stages, whereas another showed a nominal association with a higher risk. Conclusion Our data suggest that markers in the RGS1 gene might be in linkage disequilibrium with a protective allele that reduces the risk of anxiety and depressive disorders.