Genetic analysis for a shared biological basis between migraine and coronary artery disease

Objective: To apply genetic analysis of genome-wide association data to study the extent and nature of a shared biological basis between migraine and coronary artery disease (CAD). Methods: Four separate methods for cross-phenotype genetic analysis were applied on data from 2 large-scale genome-wide association studies of migraine (19,981 cases, 56,667 controls) and CAD (21,076 cases, 63,014 controls). The first 2 methods quantified the extent of overlapping risk variants and assessed the load of CAD risk loci in migraineurs. Genomic regions of shared risk were then identified by analysis of covariance patterns between the 2 phenotypes and by querying known genome-wide significant loci. Results: We found a significant overlap of genetic risk loci for migraine and CAD. When stratified by migraine subtype, this was limited to migraine without aura, and the overlap was protective in that patients with migraine had a lower load of CAD risk alleles than controls. Genes indicated by 16 shared risk loci point to mechanisms with potential roles in migraine pathogenesis and CAD, including endothelial dysfunction (PHACTR1) and insulin homeostasis (GIP). Conclusions: The results suggest that shared biological processes contribute to risk of migraine and CAD, but surprisingly this commonality is restricted to migraine without aura and the impact is in opposite directions. Understanding the mechanisms underlying these processes and their opposite relationship to migraine and CAD may improve our understanding of both disorders.

Functional evaluation of genetic variants associated with endometriosis near GREB1

STUDY QUESTION: Do DNA variants in the growth regulation by estrogen in breast cancer 1 (GREB1) region regulate endometrial GREB1 expression and increase the risk of developing endometriosis in women? SUMMARY ANSWER: We identified new single nucleotide polymorphisms (SNPs) with strong association with endometriosis at the GREB1 locus although we did not detect altered GREB1 expression in endometriosis patients with defined genotypes. WHAT IS ALREADY KNOWN: Genome-wide association studies have identified the GREB1 region on chromosome 2p25.1 for increasing endometriosis risk. The differential expression of GREB1 has also been reported by others in association with endometriosis disease phenotype. STUDY DESIGN, SIZE, DURATION: Fine mapping studies comprehensively evaluated SNPs within the GREB1 region in a large-scale data set (>2500 cases and >4000 controls). Publicly available bioinformatics tools were employed to functionally annotate SNPs showing the strongest association signal with endometriosis risk. Endometrial GREB1 mRNA and protein expression was studied with respect to phases of the menstrual cycle (n = 2-45 per cycle stage) and expression quantitative trait loci (eQTL) analysis for significant SNPs were undertaken for GREB1 [mRNA (n = 94) and protein (n = 44) in endometrium]. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants in this study are females who provided blood and/or endometrial tissue samples in a hospital setting. The key SNPs were genotyped using Sequenom MassARRAY. The functional roles and regulatory annotations for identified SNPs are predicted by various publicly available bioinformatics tools. Endometrial GREB1 expression work employed qRT-PCR, western blotting and immunohistochemistry studies. MAIN RESULTS AND THE ROLE OF CHANCE: Fine mapping results identified a number of SNPs showing stronger association (0.004 < P < 0.032) with endometriosis risk than the original GWAS SNP (rs13394619) (P = 0.034). Some of these SNPs were predicted to have functional roles, for example, interaction with transcription factor motifs. The haplotype (a combination of alleles) formed by the risk alleles from two common SNPs showed significant association (P = 0.026) with endometriosis and epistasis analysis showed no evidence for interaction between the two SNPs, suggesting an additive effect of SNPs on endometriosis risk. In normal human endometrium, GREB1 protein expression was altered depending on the cycle stage (significantly different in late proliferative versus late secretory, P < 0.05) and cell type (glandular epithelium, not stromal cells). However, GREB1 expression in endometriosis cases versus controls and eQTL analyses did not reveal any significant changes. LIMITATIONS, REASONS FOR CAUTION: In silico prediction tools are generally based on cell lines different to our tissue and disease of interest. Functional annotations drawn from these analyses should be considered with this limitation in mind. We identified cell-specific and hormone-specific changes in GREB1 protein expression. The lack of a significant difference observed following our GREB1 expression studies may be the result of moderate power on mixed cell populations in the endometrial tissue samples. WIDER IMPLICATIONS OF THE FINDINGS: This study further implicates the GREB1 region on chromosome 2p25.1 and the GREB1 gene with involvement in endometriosis risk. More detailed functional studies are required to determine the role of the novel GREB1 transcripts in endometriosis pathophysiology. STUDY FUNDING/COMPETING INTERESTS: Funding for this work was provided by NHMRC Project Grants APP1012245, APP1026033, APP1049472 and APP1046880. There are no competing interests.

Inference of the genetic architecture underlying BMI and height with the use of 20,240 sibling pairs

Evidence that complex traits are highly polygenic has been presented by population-based genome-wide association studies (GWASs) through the identification of many significant variants, as well as by family-based de novo sequencing studies indicating that several traits have a large mutational target size. Here, using a third study design, we show results consistent with extreme polygenicity for body mass index (BMI) and height. On a sample of 20,240 siblings (from 9,570 nuclear families), we used a within-family method to obtain narrow-sense heritability estimates of 0.42 (SE = 0.17, p = 0.01) and 0.69 (SE = 0.14, p = 6 x 10(-)(7)) for BMI and height, respectively, after adjusting for covariates. The genomic inflation factors from locus-specific linkage analysis were 1.69 (SE = 0.21, p = 0.04) for BMI and 2.18 (SE = 0.21, p = 2 x 10(-10)) for height. This inflation is free of confounding and congruent with polygenicity, consistent with observations of ever-increasing genomic-inflation factors from GWASs with large sample sizes, implying that those signals are due to true genetic signals across the genome rather than population stratification. We also demonstrate that the distribution of the observed test statistics is consistent with both rare and common variants underlying a polygenic architecture and that previous reports of linkage signals in complex traits are probably a consequence of polygenic architecture rather than the segregation of variants with large effects. The convergent empirical evidence from GWASs, de novo studies, and within-family segregation implies that family-based sequencing studies for complex traits require very large sample sizes because the effects of causal variants are small on average.

No genetic support for a contribution of prostaglandins to the aetiology of androgenetic alopecia

MADAM, Androgenetic alopecia (AGA) is a common age-dependent trait, characterized by a progressive loss of hair from the scalp. The hair loss may commence during puberty and up to 80% of white men experience some degree of AGA during their lifetime.1 Research has established that two essential aetiological factors for AGA are a genetic predisposition and the presence of androgens (male sex hormones).1,2 A recent meta-analysis of genome-wide association studies (GWAS) has increased the number of identified loci associated with this trait at the molecular level to a total of eight.3 However, despite these successes, a large fraction of the genetic contribution remains to be identified. One way to identify further genetic loci is to combine the resource of GWAS datasets with knowledge about specific biological factors likely to be involved in the development of disease. The focused evaluation of a limited number of candidate genes in GWAS datasets avoids the necessity for extensive correction for multiple testing, which typically limits the power for detecting genetic loci at a genome-wide level.4 Because the presence of genetic association suggests that candidate genes are likely to operate early in the causative chain of events leading to the phenotype, this approach may also function to favour biological pathways for their importance in the development of AGA.

Genome-wide association study of major depressive disorder: New results, meta-analysis, and lessons learned

Major depressive disorder (MDD) is a common complex disorder with a partly genetic etiology. We conducted a genome-wide association study of the MDD2000+ sample (2431 cases, 3673 screened controls and >1 M imputed single-nucleotide polymorphisms (SNPs)). No SNPs achieved genome-wide significance either in the MDD2000+ study, or in meta-analysis with two other studies totaling 5763 cases and 6901 controls. These results imply that common variants of intermediate or large effect do not have main effects in the genetic architecture of MDD. Suggestive but notable results were: (a) gene-based tests suggesting roles for adenylate cyclase 3 (ADCY3, 2p23.3) and galanin (GAL, 11q13.3); published functional evidence relates both of these to MDD and serotonergic signaling; (b) support for the bipolar disorder risk variant SNP rs1006737 in CACNA1C (P=0.020, odds ratio=1.10), and; (c) lack of support for rs2251219, a SNP identified in a meta-analysis of affective disorder studies (P=0.51). We estimate that sample sizes 1.8- to 2.4-fold greater are needed for association studies of MDD compared with those for schizophrenia to detect variants that explain the same proportion of total variance in liability. Larger study cohorts characterized for genetic and environmental risk factors accumulated prospectively are likely to be needed to dissect more fully the etiology of MDD.

Six novel susceptibility Loci for early-onset androgenetic alopecia and their unexpected association with common diseases

Androgenetic alopecia (AGA) is a highly heritable condition and the most common form of hair loss in humans. Susceptibility loci have been described on the X chromosome and chromosome 20, but these loci explain a minority of its heritable variance. We conducted a large-scale meta-analysis of seven genome-wide association studies for early-onset AGA in 12,806 individuals of European ancestry. While replicating the two AGA loci on the X chromosome and chromosome 20, six novel susceptibility loci reached genome-wide significance (p = 2.62x10(-)(9)-1.01x10(-)(1)(2)). Unexpectedly, we identified a risk allele at 17q21.31 that was recently associated with Parkinson's disease (PD) at a genome-wide significant level. We then tested the association between early-onset AGA and the risk of PD in a cross-sectional analysis of 568 PD cases and 7,664 controls. Early-onset AGA cases had significantly increased odds of subsequent PD (OR = 1.28, 95% confidence interval: 1.06-1.55, p = 8.9x10(-)(3)). Further, the AGA susceptibility alleles at the 17q21.31 locus are on the H1 haplotype, which is under negative selection in Europeans and has been linked to decreased fertility. Combining the risk alleles of six novel and two established susceptibility loci, we created a genotype risk score and tested its association with AGA in an additional sample. Individuals in the highest risk quartile of a genotype score had an approximately six-fold increased risk of early-onset AGA [odds ratio (OR) = 5.78, p = 1.4x10(-)(8)(8)]. Our results highlight unexpected associations between early-onset AGA, Parkinson's disease, and decreased fertility, providing important insights into the pathophysiology of these conditions.

Association between ORMDL3, IL1RL1 and a deletion on chromosome 17q21 with asthma risk in Australia

Genome-wide association studies followed by replication provide a powerful approach to map genetic risk factors for asthma. We sought to search for new variants associated with asthma and attempt to replicate the association with four loci reported previously (ORMDL3, PDE4D, DENND1B and IL1RL1). Genome-wide association analyses of individual single nucleotide polymorphisms (SNPs), rare copy number variants (CNVs) and overall CNV burden were carried out in 986 asthma cases and 1846 asthma-free controls from Australia. The most-associated locus in the SNP analysis was ORMDL3 (rs6503525, P = 4.8 x 10(-)(7)). Five other loci were associated with P < 10(-)(5), most notably the chemokine CXC motif ligand 14 (CXCL14) gene (rs31263, P = 7.8 x 10(-)(6)). We found no evidence for association with the specific risk variants reported recently for PDE4D, DENND1B and ILR1L1. However, a variant in IL1RL1 that is in low linkage disequilibrium with that reported previously was associated with asthma risk after accounting for all variants tested (rs10197862, gene wide P = 0.01). This association replicated convincingly in an independent cohort (P = 2.4 x 10(-)(4)). A 300-kb deletion on chromosome 17q21 was associated with asthma risk, but this did not reach experiment-wide significance. Asthma cases and controls had comparable CNV rates, length and number of genes affected by deletions or duplications. In conclusion, we confirm the association between asthma risk and variants in ORMDL3 and identify a novel risk variant in IL1RL1. Follow-up of the 17q21 deletion in larger cohorts is warranted.

Replication of the association of common rs9939609 variant of FTO with increased BMI in an Australian adult twin population but no evidence for gene by environment (G x E) interaction

OBJECTIVE: To further investigate a common variant (rs9939609) in the fat mass- and obesity-associated gene (FTO), which recent genome-wide association studies have shown to be associated with body mass index (BMI) and obesity. DESIGN: We examined the effect of this FTO variant on BMI in 3353 Australian adult male and female twins. RESULTS: The minor A allele of rs9939609 was associated with an increased BMI (P=0.0007). Each additional copy of the A allele was associated with a mean BMI increase of approximately 1.04 kg/m(2) (approximately 3.71 kg). Using variance components decomposition, we estimate that this single-nucleotide polymorphism accounts for approximately 3% of the genetic variance in BMI in our sample (approximately 2% of the total variance). By comparing intrapair variances of monozygotic twins of different genotypes we were able to perform a direct test of gene by environment (G x E) interaction in both sexes and gene by parity (G x P) interaction in women, but no evidence was found for either. CONCLUSIONS: In addition to supporting earlier findings that the rs9939609 variant in the FTO gene is associated with an increased BMI, our results indicate that the associated genetic effect does not interact with environment or parity.

Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution

Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10−9 to P = 1.8 × 10−40) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10−3 to P = 1.2 × 10−13). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.

Trait components provide tools to dissect the genetic susceptibility of migraine

The commonly used "end diagnosis" phenotype that is adopted in linkage and association studies of complex traits is likely to represent an oversimplified model of the genetic background of a disease. This is also likely to be the case for common types of migraine, for which no convincingly associated genetic variants have been reported. In headache disorders, most genetic studies have used end diagnoses of the International Headache Society (IHS) classification as phenotypes. Here, we introduce an alternative strategy; we use trait components--individual clinical symptoms of migraine--to determine affection status in genomewide linkage analyses of migraine-affected families. We identified linkage between several traits and markers on chromosome 4q24 (highest LOD score under locus heterogeneity [HLOD] 4.52), a locus we previously reported to be linked to the end diagnosis migraine with aura. The pulsation trait identified a novel locus on 17p13 (HLOD 4.65). Additionally, a trait combination phenotype (IHS full criteria) revealed a locus on 18q12 (HLOD 3.29), and the age at onset trait revealed a locus on 4q28 (HLOD 2.99). Furthermore, suggestive or nearly suggestive evidence of linkage to four additional loci was observed with the traits phonophobia (10q22) and aggravation by physical exercise (12q21, 15q14, and Xp21), and, interestingly, these loci have been linked to migraine in previous studies. Our findings suggest that the use of symptom components of migraine instead of the end diagnosis provides a useful tool in stratifying the sample for genetic studies.

Genetic variation in the LDL receptor-related protein 5 (LRP5) gene : Association with bone health and metabolic parameters

Bone mass accrual and maintenance are regulated by a complex interplay between genetic and environmental factors. Recent studies have revealed an important role for the low-density lipoprotein receptor-related protein 5 (LRP5) in this process. The aim of this thesis study was to identify novel variants in the LRP5 gene and to further elucidate the association of LRP5 and its variants with various bone health related clinical characteristics. The results of our studies show that loss-of-function mutations in LRP5 cause severe osteoporosis not only in homozygous subjects but also in the carriers of these mutations, who have significantly reduced bone mineral density (BMD) and increased susceptibility to fractures. In addition, we demonstrated for the first time that a common polymorphic LRP5 variant (p.A1330V) was associated with reduced peak bone mass, an important determinant of BMD and osteoporosis in later life. The results from these two studies are concordant with results seen in other studies on LRP5 mutations and in association studies linking genetic variation in LRP5 with BMD and osteoporosis. Several rare LRP5 variants were identified in children with recurrent fractures. Sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses revealed no disease-causing mutations or whole-exon deletions. Our findings from clinical assessments and family-based genotype-phenotype studies suggested that the rare LRP5 variants identified are not the definite cause of fractures in these children. Clinical assessments of our study subjects with LPR5 mutations revealed an unexpectedly high prevalence of impaired glucose tolerance and dyslipidaemia. Moreover, in subsequent studies we discovered that common polymorphic LRP5 variants are associated with unfavorable metabolic characteristics. Changes in lipid profile were already apparent in pre-pubertal children. These results, together with the findings from other studies, suggest an important role for LRP5 also in glucose and lipid metabolism. Our results underscore the important role of LRP5 not only in bone mass accrual and maintenance of skeletal health but also in glucose and lipid metabolism. The role of LRP5 in bone metabolism has long been studied, but further studies with larger study cohorts are still needed to evaluate the specific role of LRP5 variants as metabolic risk factors.

Genome-wide association study for subclinical atherosclerosis in major arterial territories in the NHLBI's Framingham Heart Study

INTRODUCTION:Subclinical atherosclerosis (SCA) measures in multiple arterial beds are heritable phenotypes that are associated with increased incidence of cardiovascular disease. We conducted a genome-wide association study (GWAS) for SCA measurements in the community-based Framingham Heart Study.METHODS:Over 100,000 single nucleotide polymorphisms (SNPs) were genotyped (Human 100K GeneChip, Affymetrix) in 1345 subjects from 310 families. We calculated sex-specific age-adjusted and multivariable-adjusted residuals in subjects tested for quantitative SCA phenotypes, including ankle-brachial index, coronary artery calcification and abdominal aortic calcification using multi-detector computed tomography, and carotid intimal medial thickness (IMT) using carotid ultrasonography. We evaluated associations of these phenotypes with 70,987 autosomal SNPs with minor allele frequency [greater than or equal to] 0.10, call rate [greater than or equal to] 80%, and Hardy-Weinberg p-value [greater than or equal to] 0.001 in samples ranging from 673 to 984 subjects, using linear regression with generalized estimating equations (GEE) methodology and family-based association testing (FBAT). Variance components LOD scores were also calculated.RESULTS:There was no association result meeting criteria for genome-wide significance, but our methods identified 11 SNPs with p < 10-5 by GEE and five SNPs with p < 10-5 by FBAT for multivariable-adjusted phenotypes. Among the associated variants were SNPs in or near genes that may be considered candidates for further study, such as rs1376877 (GEE p < 0.000001, located in ABI2) for maximum internal carotid artery IMT and rs4814615 (FBAT p = 0.000003, located in PCSK2) for maximum common carotid artery IMT. Modest significant associations were noted with various SCA phenotypes for variants in previously reported atherosclerosis candidate genes, including NOS3 and ESR1. Associations were also noted of a region on chromosome 9p21 with CAC phenotypes that confirm associations with coronary heart disease and CAC in two recently reported genome-wide association studies. In linkage analyses, several regions of genome-wide linkage were noted, confirming previously reported linkage of internal carotid artery IMT on chromosome 12. All GEE, FBAT and linkage results are provided as an open-access results resource at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.CONCLUSION:The results from this GWAS generate hypotheses regarding several SNPs that may be associated with SCA phenotypes in multiple arterial beds. Given the number of tests conducted, subsequent independent replication in a staged approach is essential to identify genetic variants that may be implicated in atherosclerosis.

Genetic analysis of coronary artery disease single nucleotide polymorphisms in diabetic nephropathy

Background. Diabetic nephropathy is a leading cause of end-stage renal disease. Premature mortality is common in patients with nephropathy, largely due to cardiovascular disease. Genetic variants implicated in macrovascular disease are therefore excellent candidates to assess for association with diabetic nephropathy. Recent genome-wide association studies have identified a total of 15 single-nucleotide polymorphisms (SNPs) that are reproducibly associated with cardiovascular disease.

Methods. We initially assessed these SNPs for association in UK type 1 diabetic patients with (cases; n = 597) and without (controls; n = 502) nephropathy using iPLEXTM and TaqMan® assays. Replication studies were performed with DNA genotyped in a total of 2668 individuals from the British Isles.

Results. One SNP (rs4420638) on chromosome 19q13 was found to be significantly associated with diabetic nephropathy before (P = 0.0002) and after correction for multiple testing (Pcorrected = 0.002). We replicated this finding in a phenotypically similar case–control collection comprising 709 individuals with type 1 diabetes (P = 0.002; combined P < 0.00001; OR = 1.54, 95% CI: 1.29–1.84).

Conclusions. Our case–control data suggest that rs4420638, or a functional SNP in linkage disequilibrium with this SNP, may be associated with diabetic nephropathy.

Genetic Variation in the alpha 7 Nicotinic Acetylcholine Receptor is Associated with Delusional Symptoms in Alzheimer's Disease

Psychotic symptoms are common in Alzheimer's disease (AD) and have a negative impact oil quality of life. It is suggested that psychotic symptoms may be attributed to genetic risk factors which are revealed during neurodegeneration. CHRNA7, the gene for the alpha 7 nicotinic acetylcholine receptor, has been associated with schizophrenia in linkage and association Studies. Hence we investigated single SNPs and haplotypes in CHRNA7 in relation to AD with psychosis in a large, well-characterised and previously described cohort within the Northern Ireland population. A significant association between delusions and the T allele of rs6494223 (P = 0.014, OR = 1.63, Cl 1.22-2.17) was found. This suggests that the alpha 7 receptor may be a suitable target for the treatment of AD with psychosis.

Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease

Background: Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes. Methodology: We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset.