Xiangning Chen

Multiple Independent Loci at Chromosome 15q25.1 Affect Smoking Quantity: a Meta-Analysis and Comparison with Lung Cancer and COPD

Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls (all smokers), and 2,614 COPD cases and 3,568 COPD-free controls (all smokers). We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking (mutually adjusted p-values<10−35 and <10−8 respectively). Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10−6). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10−20) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.

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 the catechol- o -methyltransferase (COMT) gene are associated with schizophrenia in Irish high-density families

The enzyme catechol-o-methyltransferase (COMT) transfers a methyl group from adenosylmethionine to catecholamines including the neurotransmitters dopamine, epinephrine and norepinephrine. This methylation results in the degradation of catecholamines. The involvement of the COMT gene in the metabolic pathway of these neurotransmitters has made it an attractive candidate gene for many psychiatric disorders. In this article, we reported our study of association of COMT with schizophrenia in Irish families with a high density of schizophrenia. Three single nucleotide polymorphisms (SNPs) were genotyped for the 274 such families and within-family transmission disequilibrium tests were performed. SNP rs4680, which is the functional Val/Met polymorphism, showed modest association with the disease by the TRANSMIT, FBAT and PDT programs, while the other two SNPs were negative. These SNPs showed lower level of LDs with each other in the Irish subjects than in Ashkenazi Jews. Haplotype analysis indicated that a haplotype, haplotype A-G-A for SNPs rs737865-rs4680-rs165599, was preferentially transmitted to the affected subjects. This was different from the reported G-G-G haplotype found in Ashkenazi Jews, but both haplotypes shared the Val allele. We concluded that COMT gene is associated with schizophrenia and carries a small but significant risk to the susceptibility in the Irish subjects.

Schizophrenia Gene Networks and Pathways and Their Applications for Novel Candidate Gene Selection

Background Schizophrenia (SZ) is a heritable, complex mental disorder. We have seen limited success in finding causal genes for schizophrenia from numerous conventional studies. Protein interaction network and pathway-based analysis may provide us an alternative and effective approach to investigating the molecular mechanisms of schizophrenia. Methodology/Principal Findings We selected a list of schizophrenia candidate genes (SZGenes) using a multi-dimensional evidence-based approach. The global network properties of proteins encoded by these SZGenes were explored in the context of the human protein interactome while local network properties were investigated by comparing SZ-specific and cancer-specific networks that were extracted from the human interactome. Relative to cancer genes, we observed that SZGenes tend to have an intermediate degree and an intermediate efficiency on a perturbation spreading throughout the human interactome. This suggested that schizophrenia might have different pathological mechanisms from cancer even though both are complex diseases. We conducted pathway analysis using Ingenuity System and constructed the first schizophrenia molecular network (SMN) based on protein interaction networks, pathways and literature survey. We identified 24 pathways overrepresented in SZGenes and examined their interactions and crosstalk. We observed that these pathways were related to neurodevelopment, immune system, and retinoic X receptor (RXR). Our examination of SMN revealed that schizophrenia is a dynamic process caused by dysregulation of the multiple pathways. Finally, we applied the network/pathway approach to identify novel candidate genes, some of which could be verified by experiments. Conclusions/Significance This study provides the first comprehensive review of the network and pathway characteristics of schizophrenia candidate genes. Our preliminary results suggest that this systems biology approach might prove promising for selection of candidate genes for complex diseases. Our findings have important implications for the molecular mechanisms for schizophrenia and, potentially, other psychiatric disorders.

Regulator of G-protein signaling 4 (RGS4) gene is associated with schizophrenia in Irish High Density Families

The regulator of the G‐protein signaling 4 (RGS4) gene was shown to have a different expression pattern in schizophrenia patients in a microarray study. A family‐based study subsequently implicated the association of this gene with schizophrenia. We replicated the study with our sample from the Irish Study of High Density Schizophrenia Families (ISHDSF). Single marker transmission disequilibrium tests (TDT) for the four core SNPs showed modest association for SNP 18 (using a narrow diagnostic approach with FBAT P = 0.044; with PDT P = 0.0073) and a trend for SNP 4 (with FBAT P = 0.1098; with PDT P = 0.0249). For SNP 1 and 7, alleles overtransmitted to affected subjects were the same as previously reported. Haplotype analyses suggested that haplotype G‐G‐G for SNP1–4–18, which is the most abundant haplotype (42.3%) in the Irish families, was associated with the disease (narrow diagnosis, FBAT P = 0.0061, PDT P = 0.0498). This was the same haplotype implicated in the original study. While P values were not corrected for multiple testing because of the clear prior hypothesis, these results could be interpreted as supporting evidence for the association between RGS4 and schizophrenia.

SIRT1 activates MAO-A in the brain to mediate anxiety and exploratory drive.

SIRT1 is a NAD(+)-dependent deacetylase that governs a number of genetic programs to cope with changes in the nutritional status of cells and organisms. Behavioral responses to food abundance are important for the survival of higher animals. Here we used mice with increased or decreased brain SIRT1 to show that this sirtuin regulates anxiety and exploratory drive by activating transcription of the gene encoding the monoamine oxidase A (MAO-A) to reduce serotonin levels in the brain. Indeed, treating animals with MAO-A inhibitors or selective serotonin reuptake inhibitors (SSRIs) normalized anxiety differences between wild-type and mutant animals. SIRT1 deacetylates the brain-specific helix-loop-helix transcription factor NHLH2 on lysine 49 to increase its activation of the MAO-A promoter. Both common and rare variations in the SIRT1 gene were shown to be associated with risk of anxiety in human population samples. Together these data indicate that SIRT1 mediates levels of anxiety, and this regulation may be adaptive in a changing environment of food availability.

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.

Evaluation of a Susceptibility Gene for Schizophrenia: Genotype Based Meta-Analysis of RGS4 Polymorphisms from Thirteen Independent Samples

BACKGROUND Associations between schizophrenia (SCZ) and polymorphisms at the regulator of G-protein signaling 4 (RGS4) gene have been reported (single nucleotide polymorphisms [SNPs] 1, 4, 7, and 18). Yet, similar to other SCZ candidate genes, studies have been inconsistent with respect to the associated alleles. METHODS In an effort to resolve the role for RGS4 in SCZ susceptibility, we undertook a genotype-based meta-analysis using both published and unpublished family-based and case-control samples (total n = 13,807). RESULTS The family-based dataset consisted of 10 samples (2160 families). Significant associations with individual SNPs/haplotypes were not observed. In contrast, global analysis revealed significant transmission distortion (p = .0009). Specifically, analyses suggested overtransmission of two common haplotypes that account for the vast majority of all haplotypes. Separate analyses of 3486 cases and 3755 control samples (eight samples) detected a significant association with SNP 4 (p = .01). Individual haplotype analyses were not significant, but evaluation of test statistics from individual samples suggested significant associations. CONCLUSIONS Our collaborative meta-analysis represents one of the largest SCZ association studies to date. No individual risk factor arose from our analyses, but interpretation of these results is not straightforward. Our analyses suggest risk due to at least two common haplotypes in the presence of heterogeneity. Similar analysis for other putative susceptibility genes is warranted.

The μ-opioid receptor gene and smoking initiation and nicotine dependence

The gene encoding the mu-opioid receptor (OPRM1) is reported to be associated with a range of substance dependence. Experiments in knockout mice indicate that the mu-opioid receptor may mediate reinforcing effects of nicotine. In humans, opioid antagonist naltrexone may reduce the reinforcing effects of tobacco smoking. Additionally, the OPRM1 gene is located in a region showing linkage to nicotine dependence. The OPRM1 is thus a plausible candidate gene for smoking behavior. To investigate whether OPRM1 contributes to the susceptibility of smoking initiation and nicotine dependence, we genotyped 11 SNPs in the gene for 688 Caucasian subjects of lifetime smokers and nonsmokers. Three SNPs showed nominal significance for smoking initiation and one reached significance for nicotine dependence. The global test for three-marker (rs9479757-rs2075572-rs10485057) haplotypes was significant for smoking initiation (p = 0.0022). The same three-marker haplotype test was marginal (p = 0.0514) for nicotine dependence. These results suggest that OPRM1 may be involved in smoking initiation and nicotine dependence.