A quantitative-trait genome-wide association study of alcoholism risk in the community: findings and implications

BACKGROUND Given moderately strong genetic contributions to variation in alcoholism and heaviness of drinking (50% to 60% heritability) with high correlation of genetic influences, we have conducted a quantitative trait genome-wide association study (GWAS) for phenotypes related to alcohol use and dependence. METHODS Diagnostic interview and blood/buccal samples were obtained from sibships ascertained through the Australian Twin Registry. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed with 8754 individuals (2062 alcohol-dependent cases) selected for informativeness for alcohol use disorder and associated quantitative traits. Family-based association tests were performed for alcohol dependence, dependence factor score, and heaviness of drinking factor score, with confirmatory case-population control comparisons using an unassessed population control series of 3393 Australians with genome-wide SNP data. RESULTS No findings reached genome-wide significance (p = 8.4 x 10(-8) for this study), with lowest p value for primary phenotypes of 1.2 x 10(-7). Convergent findings for quantitative consumption and diagnostic and quantitative dependence measures suggest possible roles for a transmembrane protein gene (TMEM108) and for ANKS1A. The major finding, however, was small effect sizes estimated for individual SNPs, suggesting that hundreds of genetic variants make modest contributions (1/4% of variance or less) to alcohol dependence risk. CONCLUSIONS We conclude that: - 1) meta-analyses of consumption data may contribute usefully to gene discovery; - 2) translation of human alcoholism GWAS results to drug discovery or clinically useful prediction of risk will be challenging, and; - 3) through accumulation across studies, GWAS data may become valuable for improved genetic risk differentiation in research in biological psychiatry (e.g., prospective high-risk or resilience studies).

A genome-wide association study of Cloninger's temperament scales: implications for the evolutionary genetics of personality

Variation in personality traits is 30-60% attributed to genetic influences. Attempts to unravel these genetic influences at the molecular level have, so far, been inconclusive. We performed the first genome-wide association study of Cloninger's temperament scales in a sample of 5117 individuals, in order to identify common genetic variants underlying variation in personality. Participants' scores on Harm Avoidance, Novelty Seeking, Reward Dependence, and Persistence were tested for association with 1,252,387 genetic markers. We also performed gene-based association tests and biological pathway analyses. No genetic variants that significantly contribute to personality variation were identified, while our sample provides over 90% power to detect variants that explain only 1% of the trait variance. This indicates that individual common genetic variants of this size or greater do not contribute to personality trait variation, which has important implications regarding the genetic architecture of personality and the evolutionary mechanisms by which heritable variation is maintained.

A genome wide linkage scan for dizygotic twinning in 525 families of mothers of dizygotic twins

BACKGROUND: The tendency to conceive dizygotic (DZ) twins is a complex trait influenced by genetic and environmental factors. To search for new candidate loci for twinning, we conducted a genome-wide linkage scan in 525 families using microsatellite and single nucleotide polymorphism marker panels. METHODS AND RESULTS: Non-parametric linkage analyses, including 523 families containing a total of 1115 mothers of DZ twins (MODZT) from Australia and New Zealand (ANZ) and The Netherlands (NL), produced four linkage peaks above the threshold for suggestive linkage, including a highly suggestive peak at the extreme telomeric end of chromosome 6 with an exponential logarithm of odds \[(exp)LOD] score of 2.813 (P = 0.0002). Since the DZ twinning rate increases steeply with maternal age independent of genetic effects, we also investigated linkage including only families where at least one MODZT gave birth to her first set of twins before the age of 30. These analyses produced a maximum expLOD score of 2.718 (P = 0.0002), largely due to linkage signal from the ANZ cohort, however, ordered subset analyses indicated this result is most likely a chance finding in the combined dataset. Linkage analyses were also performed for two large DZ twinning families from the USA, one of which produced a peak on chromosome 2 in the region of two potential candidate genes. Sequencing of FSHR and FIGLA, along with INHBB in MODZTs from two large NL families with family specific linkage peaks directly over this gene, revealed a potentially functional variant in the 5' untranslated region of FSHR that segregated with the DZ twinning phenotype in the Utah family. CONCLUSION: Our data provide further evidence for complex inheritance of familial DZ twinning.

A versatile gene-based test for genome-wide association studies

We have derived a versatile gene-based test for genome-wide association studies (GWAS). Our approach, called VEGAS (versatile gene-based association study), is applicable to all GWAS designs, including family-based GWAS, meta-analyses of GWAS on the basis of summary data, and DNA-pooling-based GWAS, where existing approaches based on permutation are not possible, as well as singleton data, where they are. The test incorporates information from a full set of markers (or a defined subset) within a gene and accounts for linkage disequilibrium between markers by using simulations from the multivariate normal distribution. We show that for an association study using singletons, our approach produces results equivalent to those obtained via permutation in a fraction of the computation time. We demonstrate proof-of-principle by using the gene-based test to replicate several genes known to be associated on the basis of results from a family-based GWAS for height in 11,536 individuals and a DNA-pooling-based GWAS for melanoma in approximately 1300 cases and controls. Our method has the potential to identify novel associated genes; provide a basis for selecting SNPs for replication; and be directly used in network (pathway) approaches that require per-gene association test statistics. We have implemented the approach in both an easy-to-use web interface, which only requires the uploading of markers with their association p-values, and a separate downloadable application.

A simple and fast two-locus quality control test to detect false positives due to batch effects in genome-wide association studies

The impact of erroneous genotypes having passed standard quality control (QC) can be severe in genome-wide association studies, genotype imputation, and estimation of heritability and prediction of genetic risk based on single nucleotide polymorphisms (SNP). To detect such genotyping errors, a simple two-locus QC method, based on the difference in test statistic of association between single SNPs and pairs of SNPs, was developed and applied. The proposed approach could detect many problematic SNPs with statistical significance even when standard single SNP QC analyses fail to detect them in real data. Depending on the data set used, the number of erroneous SNPs that were not filtered out by standard single SNP QC but detected by the proposed approach varied from a few hundred to thousands. Using simulated data, it was shown that the proposed method was powerful and performed better than other tested existing methods. The power of the proposed approach to detect erroneous genotypes was approximately 80% for a 3% error rate per SNP. This novel QC approach is easy to implement and computationally efficient, and can lead to a better quality of genotypes for subsequent genotype-phenotype investigations.

Genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1

Migraine is a common episodic neurological disorder, typically presenting with recurrent attacks of severe headache and autonomic dysfunction. Apart from rare monogenic subtypes, no genetic or molecular markers for migraine have been convincingly established. We identified the minor allele of rs1835740 on chromosome 8q22.1 to be associated with migraine (P = 5.38 x 10(-)(9), odds ratio = 1.23, 95% CI 1.150-1.324) in a genome-wide association study of 2,731 migraine cases ascertained from three European headache clinics and 10,747 population-matched controls. The association was replicated in 3,202 cases and 40,062 controls for an overall meta-analysis P value of 1.69 x 10(-)(1)(1) (odds ratio = 1.18, 95% CI 1.127-1.244). rs1835740 is located between MTDH (astrocyte elevated gene 1, also known as AEG-1) and PGCP (encoding plasma glutamate carboxypeptidase). In an expression quantitative trait study in lymphoblastoid cell lines, transcript levels of the MTDH were found to have a significant correlation to rs1835740 (P = 3.96 x 10(-)(5), permuted threshold for genome-wide significance 7.7 x 10(-)(5). To our knowledge, our data establish rs1835740 as the first genetic risk factor for migraine.

Genome-wide linkage analysis of multiple measures of neuroticism of 2 large cohorts from Australia and the Netherlands

CONTEXT People meeting diagnostic criteria for anxiety or depressive disorders tend to score high on the personality scale of neuroticism. Studying this personality dimension can give insights into the etiology of these important psychiatric disorders. OBJECTIVES To undertake a comprehensive genome-wide linkage study of neuroticism using large study samples that have been measured multiple times and to compare the results between countries for replication and across time within countries for consistency. DESIGN Genome-wide linkage scan. SETTING Twin individuals and their family members from Australia and the Netherlands. PARTICIPANTS Nineteen thousand six hundred thirty-five sibling pairs completed self-report questionnaires for neuroticism up to 5 times over a period of up to 22 years. Five thousand sixty-nine sibling pairs were genotyped with microsatellite markers. METHODS Nonparametric linkage analyses were conducted in MERLIN-REGRESS for the mean neuroticism scores averaged across time. Additional analyses were conducted for the time-specific measures of neuroticism from each country to investigate consistency of linkage results. RESULTS Three chromosomal regions exceeded empirically derived thresholds for suggestive linkage using mean neuroticism scores: 10p 5 Kosambi cM (cM) (Dutch study sample), 14q 103 cM (Dutch study sample), and 18q 117 cM (combined Australian and Dutch study sample), but only 14q retained significance after correction for multiple testing. These regions all showed evidence for linkage in individual time-specific measures of neuroticism and 1 (18q) showed some evidence for replication between countries. Linkage intervals for these regions all overlap with regions identified in other studies of neuroticism or related traits and/or in studies of anxiety in mice. CONCLUSIONS Our results demonstrate the value of the availability of multiple measures over time and add to the optimism reported in recent reviews for replication of linkage regions for neuroticism. These regions are likely to harbor causal variants for neuroticism and its related psychiatric disorders and can inform prioritization of results from genome-wide association studies.

Genome-wide association studies of quantitative traits with related individuals: Little (power) lost but much to be gained

For complex disease genetics research in human populations, remarkable progress has been made in recent times with the publication of a number of genome-wide association scans (GWAS) and subsequent statistical replications. These studies have identified new genes and pathways implicated in disease, many of which were not known before. Given these early successes, more GWAS are being conducted and planned, both for disease and quantitative phenotypes. Many researchers and clinicians have DNA samples available on collections of families, including both cases and controls. Twin registries around the world have facilitated the collection of large numbers of families, with DNA and multiple quantitative phenotypes collected on twin pairs and their relatives. In the design of a new GWAS with a fixed budget for the number of chips, the question arises whether to include or exclude related individuals. It is commonly believed to be preferable to use unrelated individuals in the first stage of a GWAS because relatives are 'over-matched' for genotypes. In this study, we quantify that for GWAS of a quantitative phenotype, relative to a sample of unrelated individuals surprisingly little power is lost when using relatives. The advantages of using relatives are manifold, including the ability to perform more quality control, the choice to perform within-family tests of association that are robust to population stratification, and the ability to perform joint linkage and association analysis. Therefore, the advantages of using relatives in GWAS for quantitative traits may well outweigh the small disadvantage in terms of statistical power.

A genome-wide linkage scan provides evidence for both new and previously reported loci influencing common migraine

Latent class analysis was performed on migraine symptom data collected in a Dutch population sample (N = 12,210, 59% female) in order to obtain empirical groupings of individuals suffering from symptoms of migraine headache. Based on these heritable groupings (h(2) = 0.49, 95% CI: 0.41-0.57) individuals were classified as affected (migrainous headache) or unaffected. Genome-wide linkage analysis was performed using genotype data from 105 families with at least 2 affected siblings. In addition to this primary phenotype, linkage analyses were performed for the individual migraine symptoms. Significance levels, corrected for the analysis of multiple traits, were determined empirically via a novel simulation approach. Suggestive linkage for migrainous headache was found on chromosomes 1 (LOD = 1.63; pointwise P = 0.0031), 13 (LOD = 1.63; P = 0.0031), and 20 (LOD = 1.85; P = 0.0018). Interestingly, the chromosome 1 peak was located close to the ATP1A2 gene, associated with familial hemiplegic migraine type 2 (FHM2). Individual symptom analysis produced a LOD score of 1.97 (P = 0.0013) on chromosome 5 (photo/phonophobia), a LOD score of 2.13 (P = 0.0009) on chromosome 10 (moderate/severe pain intensity) and a near significant LOD score of 3.31 (P = 0.00005) on chromosome 13 (pulsating headache). These peaks were all located near regions previously reported in migraine linkage studies. Our results provide important replication and support for the presence of migraine susceptibility genes within these regions, and further support the utility of an LCA-based phenotyping approach and analysis of individual symptoms in migraine genetic research. Additionally, our novel "2-step" analysis and simulation approach provides a powerful means to investigate linkage to individual trait components.

A comparison of DNA pools constructed following whole genome amplification for two-stage SNP genotyping designs

Genotyping in DNA pools reduces the cost and the time required to complete large genotyping projects. The aim of the present study was to evaluate pooling as part of a strategy for fine mapping in regions of significant linkage. Thirty-nine single nucleotide polymorphisms (SNPs) were analyzed in two genomic DNA pools of 384 individuals each and results compared with data after typing all individuals used in the pools. There were no significant differences using data from either 2 or 8 heterozygous individuals to correct frequency estimates for unequal allelic amplification. After correction, the mean difference between estimates from the genomic pool and individual allele frequencies was .033. A major limitation of the use of DNA pools is the time and effort required to carefully adjust the concentration of each individual DNA sample before mixing aliquots. Pools were also constructed by combining DNA after Multiple Displacement Amplification (MDA). The MDA pools gave similar results to pools constructed after careful DNA quantitation (mean difference from individual genotyping .040) and MDA provides a rapid method to generate pools suitable for some applications. Pools provide a rapid and cost-effective screen to eliminate SNPs that are not polymorphic in a test population and can detect minor allele frequencies as low as 1% in the pooled samples. With current levels of accuracy, pooling is best suited to an initial screen in the SNP validation process that can provide high-throughput comparisons between cases and controls to prioritize SNPs for subsequent individual genotyping.

Identification of IDUA and WNT16 phosphorylation-related non-synonymous polymorphisms for bone mineral density in meta-analyses of genome-wide association studies

Protein phosphorylation regulates a wide variety of cellular processes. Thus, we hypothesize that single-nucleotide polymorphisms (SNPs) that may modulate protein phosphorylation could affect osteoporosis risk. Based on a previous conventional genome-wide association (GWA) study, we conducted a three-stage meta-analysis targeting phosphorylation-related SNPs (phosSNPs) for femoral neck (FN)-bone mineral density (BMD), total hip (HIP)-BMD, and lumbar spine (LS)-BMD phenotypes. In stage 1, 9593 phosSNPs were meta-analyzed in 11,140 individuals of various ancestries. Genome-wide significance (GWS) and suggestive significance were defined by α = 5.21 × 10–6 (0.05/9593) and 1.00 × 10–4, respectively. In stage 2, nine stage 1–discovered phosSNPs (based on α = 1.00 × 10–4) were in silico meta-analyzed in Dutch, Korean, and Australian cohorts. In stage 3, four phosSNPs that replicated in stage 2 (based on α = 5.56 × 10–3, 0.05/9) were de novo genotyped in two independent cohorts. IDUA rs3755955 and rs6831280, and WNT16 rs2707466 were associated with BMD phenotypes in each respective stage, and in three stages combined, achieving GWS for both FN-BMD (p = 8.36 × 10–10, p = 5.26 × 10–10, and p = 3.01 × 10–10, respectively) and HIP-BMD (p = 3.26 × 10–6, p = 1.97 × 10–6, and p = 1.63 × 10–12, respectively). Although in vitro studies demonstrated no differences in expressions of wild-type and mutant forms of IDUA and WNT16B proteins, in silico analyses predicts that WNT16 rs2707466 directly abolishes a phosphorylation site, which could cause a deleterious effect on WNT16 protein, and that IDUA phosSNPs rs3755955 and rs6831280 could exert indirect effects on nearby phosphorylation sites. Further studies will be required to determine the detailed and specific molecular effects of these BMD-associated non-synonymous variants. © 2015 American Society for Bone and Mineral Research.

Draft genome sequence of caloramator mitchellensis, a thermoanaerobe isolated from the waters of the Great Artesian Basin

The genome sequence of Caloramator mitchellensis strain VF08, a rod-shaped, heterotrophic, strictly anaerobic bacterium iso-lated from the free-flowing waters of a Great Artesian Basin (GAB) bore well located in Mitchell, an outback Queensland town in Australia, is reported here. The analysis of the 2.42-Mb genome sequence indicates that the attributes of the genome are consistent with its physiological and phenotypic traits.

Identification of a novel FGFRL1 MicroRNA target site polymorphism for bone mineral density in meta-analyses of genome-wide association studies

MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNAs' Target Sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)-, and femoral neck (FN)-bone mineral density (BMD). In stage I, 41,102 poly-miRTSs were meta-analyzed in 7 cohorts with a genome-wide significance (GWS) α=0.05/41,102=1.22×10-6. By applying α=5×10-5 (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P-value=7.67×10-6 and 1.58×10-5) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P-value=5.08×10-3) at α=0.10/11=9.09×10-3. PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P-value=7.55×10-6) at α=0.05/2=0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P-value=8.87×10-12). Dual-luciferase reporter assays demonstrated that FGFRL1 3' untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation. © The Author 2015. Published by Oxford University Press. All rights reserved.

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10-8) level: 14q24.2 (rs227425, P-value 3.98 × 10-13, SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10-9, CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n 5 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis. © The Author 2013. Published by Oxford University Press.

Whole-genome sequencing identifies EN1 as a determinant of bone density and fracture

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 x 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 x 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 x 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.