Study-wide Significance Research Articles

A Population-Specific Uncommon Variant in GRIN3A Associated with Schizophrenia

Genome-wide association studies have successfully identified several common variants showing robust association with schizophrenia. However, individually, these variants only produce a weak effect. To identify genetic variants with larger effect sizes, increasing attention is now being paid to uncommon and rare variants. From the 1000 Genomes Project data, we selected 47 candidate single nucleotide variants (SNVs), which were: 1) uncommon (minor allele frequency < 5%); 2) Asian-specific; 3) missense, nonsense, or splice site variants predicted to be damaging; and 4) located in candidate genes for schizophrenia and bipolar disorder. We examined their association with schizophrenia, using a Japanese case-control cohort (2012 cases and 2781 control subjects). Additional meta-analysis was performed using genotyping data from independent Han-Chinese case-control (333 cases and 369 control subjects) and family samples (9 trios and 284 quads). We identified disease association of a missense variant in GRIN3A (p.R480G, rs149729514, p = .00042, odds ratio [OR] = 1.58), encoding a subunit of the N-methyl-D-aspartate type glutamate receptor, with study-wide significance (threshold p = .0012). This association was supported by meta-analysis (combined p = 3.3 × 10(-5), OR = 1.61). Nominally significant association was observed in missense variants from FAAH, DNMT1, MYO18B, and CFB, with ORs of risk alleles ranging from 1.41 to 2.35. The identified SNVs, particularly the GRIN3A R480G variant, are good candidates for further replication studies and functional evaluation. The results of this study indicate that association analyses focusing on uncommon and rare SNVs are a promising way to discover risk variants with larger effects.

Association Between In Vivo Alcohol Metabolism and Genetic Variation in Pathways that Metabolize the Carbon Skeleton of Ethanol and NADH Reoxidation in the Alcohol Challenge Twin Study

Variation in alcohol metabolism affects the duration of intoxication and alcohol use. While the majority of genetic association studies investigating variation in alcohol metabolism have focused on polymorphisms in alcohol or aldehyde dehydrogenases, we have now tested for association with genes in alternative metabolic pathways that catalyze the carbon skeleton of ethanol (EtOH) and NADH reoxidation. Nine hundred fifty single nucleotide polymorphisms (SNPs) spanning 14 genes (ACN9, ACSS1, ACSS2, ALDH1A1, CAT, CYP2E1, GOT1, GOT2, MDH1, MDH2, SLC25A10, SLC25A11, SLC25A12, SLC25A13) were genotyped in 352 young adults who participated in an alcohol challenge study. Traits tested were blood alcohol concentration (BAC), breath alcohol concentration (BrAC), peak alcohol concentration, and rates of alcohol absorption and elimination. Allelic association was tested using quantitative univariate and multivariate methods. A CYP2E1 promoter SNP (rs4838767, minor allele frequency 0.008) exceeded the threshold for study-wide significance (4.01×10(-5) ) for 2 early BAC, 8 BrAC measures, and the peak BrAC. For each phenotype, the minor C allele was related to a lower alcohol concentration, most strongly for the fourth BrAC (p=2.07×10(-7) ) explaining ~8% of the phenotypic variance. We also observed suggestive patterns of association with variants in ALDH1A1 and on chromosome 17 near SLC25A11 for aspects of blood and breath alcohol metabolism. An SNP upstream of GOT1 (rs2490286) reached study-wide significance for multivariate BAC metabolism (p=0.000040). Overall, we did not find strong evidence that variation in genes coding for proteins that further metabolize the carbon backbone of acetaldehyde, or contribute to mechanisms for regenerating NAD from NADH, affects alcohol metabolism in our European-descent subjects. However, based on the breath alcohol data, variation in the promoter of CYP2E1 may play a role in preabsorptive or early hepatic alcohol metabolism, but more samples are required to validate this finding.

Genetic polymorphism and prostate cancer aggressiveness: a case-only study of 1,536 GWAS and candidate SNPs in African-Americans and European-Americans.

Genome-wide association studies have established a number of replicated single nucleotide polymorphisms (SNPs) for susceptibility to prostate cancer (CaP), but it is unclear whether these susceptibility SNPs are also associated with disease aggressiveness. This study evaluates whether such replication SNPs or other candidate SNPs are associated with CaP aggressiveness in African-American (AA) and European-American (EA) men. A 1,536 SNP panel which included 34 genome-wide association study (GWAS) replication SNPs, 38 flanking SNPs, a set of ancestry informative markers, and SNPs in candidate genes and other areas was genotyped in 1,060 AA and 1,087 EA men with incident CaP from the North Carolina-Louisiana Prostate Cancer Project (PCaP). Tests for association were conducted using ordinal logistic regression with a log-additive genotype model and a 3-category CaP aggressiveness variable. Four GWAS replication SNPs (rs2660753, rs13254738, rs10090154, rs2735839) and seven flanking SNPs were associated with CaP aggressiveness (P < 0.05) in three genomic regions: One at 3p12 (EA), seven at 8q24 (5 AA, 2 EA), and three at 19q13 at the kallilkrein-related peptidase 3 (KLK3) locus (two AA, one AA and EA). The KLK3 SNPs also were associated with serum prostate-specific antigen (PSA) levels in AA (P < 0.001) but not in EA. A number of the other SNPs showed some evidence of association but none met study-wide significance levels after adjusting for multiple comparisons. Some replicated GWAS susceptibility SNPs may play a role in CaP aggressiveness. However, like susceptibility, these associations are not consistent between racial groups.

Circadian gene variants and susceptibility to type 2 diabetes: a pilot study.

BackgroundDisruption of endogenous circadian rhythms has been shown to increase the risk of developing type 2 diabetes, suggesting that circadian genes might play a role in determining disease susceptibility. We present the results of a pilot study investigating the association between type 2 diabetes and selected single nucleotide polymorphisms (SNPs) in/near nine circadian genes. The variants were chosen based on their previously reported association with prostate cancer, a disease that has been suggested to have a genetic link with type 2 diabetes through a number of shared inherited risk determinants.Methodology/Principal FindingsThe pilot study was performed using two genetically homogeneous Punjabi cohorts, one resident in the United Kingdom and one indigenous to Pakistan. Subjects with (N = 1732) and without (N = 1780) type 2 diabetes were genotyped for thirteen circadian variants using a competitive allele-specific polymerase chain reaction method. Associations between the SNPs and type 2 diabetes were investigated using logistic regression. The results were also combined with in silico data from other South Asian datasets (SAT2D consortium) and white European cohorts (DIAGRAM+) using meta-analysis. The rs7602358G allele near PER2 was negatively associated with type 2 diabetes in our Punjabi cohorts (combined odds ratio [OR] = 0.75 [0.66–0.86], p = 3.18×10−5), while the BMAL1 rs11022775T allele was associated with an increased risk of the disease (combined OR = 1.22 [1.07–1.39], p = 0.003). Neither of these associations was replicated in the SAT2D or DIAGRAM+ datasets, however. Meta-analysis of all the cohorts identified disease associations with two variants, rs2292912 in CRY2 and rs12315175 near CRY1, although statistical significance was nominal (combined OR = 1.05 [1.01–1.08], p = 0.008 and OR = 0.95 [0.91–0.99], p = 0.015 respectively).Conclusions/significanceNone of the selected circadian gene variants was associated with type 2 diabetes with study-wide significance after meta-analysis. The nominal association observed with the CRY2 SNP, however, complements previous findings and confirms a role for this locus in disease susceptibility.

Large-Scale Gene-Centric Meta-Analysis across 39 Studies Identifies Type 2 Diabetes Loci

To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10(-9)) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10(-6)). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10(-7)) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10(-15)). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10(-8)). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups.

Genetic determinants for promoter hypermethylation in the lungs of smokers: a candidate gene-based study.

The detection of tumor suppressor gene promoter methylation in sputum-derived exfoliated cells predicts early lung cancer. Here, we identified genetic determinants for this epigenetic process and examined their biologic effects on gene regulation. A two-stage approach involving discovery and replication was used to assess the association between promoter hypermethylation of a 12-gene panel and common variation in 40 genes involved in carcinogen metabolism, regulation of methylation, and DNA damage response in members of the Lovelace Smokers Cohort (N = 1,434). Molecular validation of three identified variants was conducted using primary bronchial epithelial cells. Association of study-wide significance (P < 8.2 × 10(-5)) was identified for rs1641511, rs3730859, and rs1883264 in TP53, LIG1, and BIK, respectively. These single-nucleotide polymorphisms (SNP) were significantly associated with altered expression of the corresponding genes in primary bronchial epithelial cells. In addition, rs3730859 in LIG1 was also moderately associated with increased risk for lung cancer among Caucasian smokers. Together, our findings suggest that genetic variation in DNA replication and apoptosis pathways impacts the propensity for gene promoter hypermethylation in the aerodigestive tract of smokers. The incorporation of genetic biomarkers for gene promoter hypermethylation with clinical and somatic markers may improve risk assessment models for lung cancer.

Genome-wide and gene-based association implicates FRMD6 in alzheimer disease

Genome-wide association studies (GWAS) that allow for allelic heterogeneity may facilitate the discovery of novel genes not detectable by models that require replication of a single variant site. One strategy to accomplish this is to focus on genes rather than markers as units of association, and so potentially capture a spectrum of causal alleles that differ across populations. Here, we conducted a GWAS of Alzheimer disease (AD) in 2,586 Swedes and performed gene-based meta-analysis with three additional studies from France, Canada, and the United States, in total encompassing 4,259 cases and 8,284 controls. Implementing a newly designed gene-based algorithm, we identified two loci apart from the region around APOE that achieved study-wide significance in combined samples, the strongest finding being for FRMD6 on chromosome 14q (P = 2.6 × 10(-14)) and a weaker signal for NARS2 that is immediately adjacent to GAB2 on chromosome 11q (P = 7.8 × 10(-9)). Ontology-based pathway analyses revealed significant enrichment of genes involved in glycosylation. Results suggest that gene-based approaches that accommodate allelic heterogeneity in GWAS can provide a complementary avenue for gene discovery and may help to explain a portion of the missing heritability not detectable with single nucleotide polymorphisms (SNPs) derived from marker-specific meta-analysis.

Combined analysis of three genome-wide association studies on vWF and FVIII plasma levels

BackgroundElevated levels of factor VIII (FVIII) and von Willebrand Factor (vWF) are well-established risk factors for cardiovascular diseases, in particular venous thrombosis. Although high, the heritability of these traits is poorly explained by the genetic factors known so far. The aim of this work was to identify novel single nucleotide polymorphisms (SNPs) that could influence the variability of these traits.MethodsThree independent genome-wide association studies for vWF plasma levels and FVIII activity were conducted and their results were combined into a meta-analysis totalling 1,624 subjects.ResultsNo single nucleotide polymorphism (SNP) reached the study-wide significance level of 1.12 × 10-7 that corresponds to the Bonferroni correction for the number of tested SNPs. Nevertheless, the recently discovered association of STXBP5, STX2, TC2N and CLEC4M genes with vWF levels and that of SCARA5 and STAB2 genes with FVIII levels were confirmed in this meta-analysis. Besides, among the fifteen novel SNPs showing promising association at p < 10-5 with either vWF or FVIII levels in the meta-analysis, one located in ACCN1 gene also showed weak association (P = 0.0056) with venous thrombosis in a sample of 1,946 cases and 1,228 controls.ConclusionsThis study has generated new knowledge on genomic regions deserving further investigations in the search for genetic factors influencing vWF and FVIII plasma levels, some potentially implicated in VT, as well as providing some supporting evidence of previously identified genes.

GWAS SNP Replication among African American and European American men in the North Carolina–Louisiana prostate cancer project (PCaP)

Genome-wide association studies (GWAS) have identified numerous common SNPs associated with prostate cancer (CaP) risk in men of European descent. This study evaluates GWAS SNPs associated with CaP in African Americans (AAs) and European Americans (EA). Eight hundred SNPs were genotyped, including 32 from European-based GWAS and 35 flanking SNPs, in 417 AA and 455 EA cases from the NC-LA Prostate Cancer Project (PCaP) and compared to 925 AA and 1,687 EA controls from Illumina's iControlDB. The 32 GWAS SNPs were evaluated for their predictive power to discriminate between cases and controls using ROC curves. Of the 32 GWAS SNPs, 13 were significant at P < 0.05 in EA and 4 in AA (rs6983267, rs7017300, rs1859962, rs6501455). Three of 35 flanking SNPs, all from chromosome 8q, reached study-wide significance (P < 3.5 × 10(-5)); 2 in AA (rs10505476 rs6985504) and 1 in EA (rs16901970). Among the remaining 656 SNPs, 2 were associated with CaP (P < 3.5 × 10(-5)): rs1472606 (OR: 1.43 in EA) and rs9351265 (OR: 1.48 in AA) both in intergenic regions. For the 32 GWAS SNPs, ROC plots yielded AUC estimates too low for clinical use (EA AUC = 0.60 and AA AUC = 0.56). This study confirms a large proportion of CaP associated regions implicated by European-based GWAS and provides evidence that some regions may be important in AA CaP risk. Despite the identification of a large panel of GWAS replicated SNPs for CaP, this panel is not appropriate for clinical screening.

Common variants in the TPH1 and TPH2 regions are not associated with persistent ADHD in a combined sample of 1,636 adult cases and 1,923 controls from four European populations

The tryptophan hydroxylase 1 and 2 (TPH1 and TPH2) genes encode the rate-limiting enzymes in the serotonin biosynthesis. Genetic variants in both genes have been implicated in several psychiatric disorders. For attention-deficit/hyperactivity disorder (ADHD) in children, the results are conflicting, and little is known about their role in adult ADHD patients. We therefore first genotype-tagged all common variants within both genes in a Norwegian sample of 451 patients with a diagnosis of adult ADHD and 584 controls. Six of the single nucleotide polymorphisms (SNPs) were subsequently genotyped in three additional independent European Caucasian samples of adult ADHD cases and controls from the International Multicenter persistent ADHD Collaboration (IMpACT). None of the SNPs reached formal study-wide significance in the total meta-analysis sample of 1,636 cases and 1,923 controls, despite having a power of >80% to detect a variant conferring an OR = 1.25 at P = 0.001 level. Only the TPH1 SNP rs17794760 showed nominal significance [OR = 0.84 (0.71-1.00), P = 0.05]. In conclusion, in the single largest ADHD genetic study of TPH1 and TPH2 variants presented to date (n = 3,559 individuals), we did not find consistent evidence for a substantial effect of common genetic variants on persistent ADHD.

A systematic gene-based screen of chr4q22–q32 identifies association of a novel susceptibility gene, DKK2 , with the quantitative trait of alcohol dependence symptom counts

Studies of alcohol dependence (AD) have consistently found evidence of linkage on chromosome 4q21-q32. A genome-wide linkage scan in the Irish Affected Sib Pair Study of Alcohol Dependence (IASPSAD) sample also provided its strongest evidence of linkage on chromosome 4q22-q32 using an index of AD severity based on the count of DSM-IV AD symptoms (ADSX; LOD = 4.59). We conducted a systematic, gene-centric association study using 518 LD-tagging single nucleotide polymorphisms (SNPs) in the 65 known and predicted genes within the 1-LOD interval surrounding the linkage peak. Case-only regression analysis with the quantitative variable of ADSX was performed in the 562 genetically independent cases; nominal support for association was demonstrated by 32 tagging SNPs in 14 genes. We did not observe study-wide significance, but gene-wise correction for multiple testing with the Nyholt procedure yielded empirical evidence of association with two genes, DKK2 (dickkopf homolog 2) (P = 0.007) and EGF (epidermal growth factor) (P = 0.025) in the IASPSAD sample. Three SNPs in DKK2 (rs427983; rs419558; rs399087) demonstrated empirical significance. Assessment of possible replication in 847 cases of European descent from a large independent sample, the Collaborative Study of the Genetics of Alcoholism, yielded replication for DKK2 but not EGF. We observed genotypic and phenotypic replication for DKK2 with the three SNPs yielding significant association with ADSX in the IASPSAD sample. Haplotype-specific expression measurements in post-mortem tissue samples suggested a functional role for DKK2. This evidence notwithstanding, replication is needed before confidence can be placed in these findings.

Fine Mapping on Chromosome 10q22-q23 Implicates Neuregulin 3 in Schizophrenia

Linkage studies have implicated 10q22-q23 as a schizophrenia (SZ) susceptibility locus in Ashkenazi Jewish (AJ) and Han Chinese from Taiwan populations. To further explore our previous linkage signal in the AJ population (NPL score: 4.27, empirical p = 2 x 10(-5)), we performed a peakwide association fine mapping study by using 1414 SNPs across approximately 12.5 Mb in 10q22-q23. We genotyped 1515 AJ individuals, including 285 parent-child trios, 173 unrelated cases, and 487 unrelated controls. We analyzed the binary diagnostic phenotype of SZ and 9 heritable quantitative traits derived from a principal components factor analysis of 73 items from our consensus diagnostic ratings and direct assessment interviews. Although no marker withstood multiple test correction for association with the binary SZ phenotype, we found strong evidence of association by using the "delusion" factor as the quantitative trait at three SNPs (rs10883866, rs10748842, and rs6584400) located in a 13 kb interval in intron 1 of Neuregulin 3 (NRG3). Our best p value from family-based association analysis was 7.26 x 10(-7). We replicated this association in the collection of 173 unrelated AJ cases (p = 1.55 x 10(-2)), with a combined p value of 2.30 x 10(-7). After performing 10,000 permutations of each of the phenotypes, we estimated the empirical study-wide significance across all 9 factors (90,000 permutations) to be p = 2.7 x 10(-3). NRG3 is primarily expressed in the central nervous system and is one of three paralogs of NRG1, a gene strongly implicated in SZ. These biological properties together with our linkage and association results strongly support NRG3 as a gene involved in SZ.

Family-based SNP association study on 8q24 in bipolar disorder.

Previous linkage studies have identified chromosome 8q24 as a promising positional candidate region to search for bipolar disorder (BP) susceptibility genes. We, therefore, sought to identify BP susceptibility genes on chromosome 8q24 using a family-based association study of a dense panel of SNPs selected to tag the known common variation across the region of interest. A total of 1,458 SNPs across 16 Mb of 8q24 were examined in 3,512 subjects, 1,954 of whom were affected with BP, from 737 multiplex families. Single-locus tests were carried out with FBAT and Geno-PDT, and multi-locus test were carried out with HBAT and multi-locus Geno-PDT. None of the SNPs were associated with BP in the single-locus tests at a level that exceeded our threshold for study-wide significance (P < 3.00 x 10(-5)). However, there was consistent evidence at our threshold for the suggestive level (P < 7.00 x 10(-4)) from both the single locus and multi-locus tests of associations with SNPs in the genes ADCY8, ST3GAL1, and NSE2. Multi-locus analyses suggested joint effects between ADCY8 and ST3GAL1 (P = 3.00 x 10(-4)), with at least one copy of the "high risk" allele required at both genes for association with BP, consistent with a jointly dominant-dominant model of action. These findings with ADCY8 and ST3GAL1 warrant further investigation in order to confirm the observed associations and their functional significance for BP susceptibility.

Genomewide linkage survey of nicotine dependence phenotypes

Background A comprehensive understanding of the etiology and neurobiology of nicotine dependence is not available. We sought to identify genomic regions that might contain etiologically-relevant loci using genomewide univariate and bivariate linkage analyses. Methods We conducted secondary data analyses of 626 all possible sibling pairs ascertained in Ireland and Northern Ireland on the basis of alcohol dependence. A set of 1020 short tandem repeat genetic markers were genotyped in all subjects. The phenotypes analyzed were the Fagerström Test for Nicotine Dependence (FTND), a history of nicotine dependence, the number of symptoms of alcohol dependence (AlcSx), and a history of alcohol dependence. Genomewide linkage analyses were conducted with non-parametric and variance components methods. Findings For the bivariate variance component analysis of the continuous FTND and AlcSx scores, multipoint LOD scores were >4 in two genomic regions—an 11 cM region on chr7 (D7S2252–D7S691, empirical p = 0.0006) and an 8 cM region on chr18 flanking D18S63 (empirical p = 0.0007). These findings did not exceed a conservative estimate of study-wide significance. The remaining sets of findings had considerably smaller or less consistent peak signals. Notably, strong linkage signal at D4S1611 for AlcSx from a prior report (PMID 16534506) was not found when jointly analyzed with FTND. Interpretation Replication is required. However, chromosomes 7 and 18 may contain genetic loci relevant to the etiology of nicotine-related phenotypes.

Size Matters: The Unexpected Challenge of Detecting Linkage in Large Cohorts

Size Matters: The Unexpected Challenge of Detecting Linkage in Large Cohorts

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<h3>Background</h3> Studies have identified many common genetic associations that influence renal function and all-cause CKD, but these explain only a small fraction of variance in these traits. The contribution of rare variants has not been systematically examined. <h3>Methods</h3> We performed exome sequencing of 3150 individuals, who collectively encompassed diverse CKD subtypes, and 9563 controls. To detect causal genes and evaluate the contribution of rare variants we used collapsing analysis, in which we compared the proportion of cases and controls carrying rare variants per gene. <h3>Results</h3> The analyses captured five established monogenic causes of CKD: variants in <i>PKD1</i>, <i>PKD2</i>, and <i>COL4A5</i> achieved study-wide significance, and we observed suggestive case enrichment for <i>COL4A4</i> and <i>COL4A3</i>. Beyond known disease-associated genes, collapsing analyses incorporating regional variant intolerance identified suggestive dominant signals in <i>CPT2</i> and several other candidate genes. Biallelic mutations in <i>CPT2</i> cause carnitine palmitoyltransferase II deficiency, sometimes associated with rhabdomyolysis and acute renal injury. Genetic modifier analysis among cases with <i>APOL1</i> risk genotypes identified a suggestive signal in <i>AHDC1</i>, implicated in Xia–Gibbs syndrome, which involves intellectual disability and other features. On the basis of the observed distribution of rare variants, we estimate that a two- to three-fold larger cohort would provide 80% power to implicate new genes for all-cause CKD. <h3>Conclusions</h3> This study demonstrates that rare-variant collapsing analyses can validate known genes and identify candidate genes and modifiers for kidney disease. In so doing, these findings provide a motivation for larger-scale investigation of rare-variant risk contributions across major clinical CKD categories.