Genome-wide Association Studies Signals Research Articles

Single Nucleotide Polymorphisms at a Distance from Aryl Hydrocarbon Receptor (AHR) Binding Sites Influence AHR Ligand-Dependent Gene Expression.

Greater than 90% of significant genome-wide association study (GWAS) single-nucleotide polymorphisms (SNPs) are in noncoding regions of the genome, but only 25.6% are known expression quantitative trait loci (eQTLs). Therefore, the function of many significant GWAS SNPs remains unclear. We have identified a novel type of eQTL for which SNPs distant from ligand-activated transcription factor (TF) binding sites can alter target gene expression in a SNP genotype-by-ligand–dependent fashion that we refer to as pharmacogenomic eQTLs (PGx-eQTLs)—loci that may have important pharmacotherapeutic implications. In the present study, we integrated chromatin immunoprecipitation-seq with RNA-seq and SNP genotype data for a panel of lymphoblastoid cell lines to identify 10 novel cis PGx-eQTLs dependent on the ligand-activated TF aryl hydrocarbon receptor (AHR)—a critical environmental sensor for xenobiotic (drug) and immune response. Those 10 cis PGx-eQTLs were eQTLs only after AHR ligand treatment, even though the SNPs did not create/destroy an AHR response element—the DNA sequence motif recognized and bound by AHR. Additional functional studies in multiple cell lines demonstrated that some cis PGx-eQTLs are functional in multiple cell types, whereas others displayed SNP-by-ligand–dependent effects in just one cell type. Furthermore, four of those cis PGx-eQTLs had previously been associated with clinical phenotypes, indicating that those loci might have the potential to inform clinical decisions. Therefore, SNPs across the genome that are distant from TF binding sites for ligand-activated TFs might function as PGx-eQTLs and, as a result, might have important clinical implications for interindividual variation in drug response.SIGNIFICANCE STATEMENTMore than 90% of single-nucleotide polymorphisms (SNPs) that are associated with clinical phenotypes are located in noncoding regions of the genome. However, the mechanisms of action of many of those SNPs have not been elucidated, and drugs may unmask functional expression quantitative trail loci (eQTLs). In the current study, we used drugs that bind to the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and identified SNPs that were associated with interindividual variation in gene expression following drug exposure—termed pharmacogenomic (PGx)-eQTLs. Possibly of greater significance, those PGx-eQTL SNPs were outside of AHR binding sites, indicating that they do not interrupt AHR DNA recognition. PGx-eQTLs such as those described in this work may have crucial implications for interindividual variation in drug.

GWAS contribution to atrial fibrillation and atrial fibrillation-related stroke: pathophysiological implications.

Atrial fibrillation (AF) and stroke are included in a group of complex traits that have been approached regarding of their study by susceptibility genetic determinants. Since 2007, several genome-wide association studies (GWAS) aiming to identify genetic variants modulating AF risk have been conducted. Thus, 11 GWAS have identified 26 SNPs (p <5× 10-2), of which 19 reached genome-wide significance (p <5×10-8). From those variants, seven were also associated with cardioembolic stroke and three reached genome-wide significance in stroke GWAS. These associations may shed a light on putative shared etiologic mechanisms between AF and cardioembolic stroke. Additionally, some of these identified variants have been incorporated in genetic risk scores in order to elucidate new approaches of stroke prediction, prevention and treatment.

HIGH-RESOLUTION GENOMEWIDE PROMOTER-FOCUSED CONNECTOME IMPLICATES MICROGLIA CAUSAL GENES FOR ALZHEIMER’S DISEASE

Microglia are implicated as playing a central role in the pathogenesis of Alzheimer's Disease (AD). Indeed, genome-wide association studies (GWAS) have identified multiple genomic variants associated with inflammation and microglial immune response. That said, GWAS reports genomic signals associated with a given trait and not necessarily the precise localization of culprit effector genes. High resolution chromatin conformation capture-based techniques that detect contacts between distant regions of the genome offer a valuable tool to understand GWAS signals that reside almost exclusively in non-coding regions, principally through their presumed influence on regulatory elements. To improve on the low resolution of available Hi-C data, we expanded a high resolution Capture-C based method to simultaneously characterize the physical genome-wide interactions of all human promoters - which we applied to the human microglial cell line, HMC3. A custom Agilent SureSelect library was designed to target both ends of DpnII restriction fragments that overlap promoters of both protein-coding and noncoding transcripts, totaling 36,691 RNA baited fragments. Following sequencing, we investigated significant interactions at different resolutions. In parallel, we generated ATAC-seq open chromatin maps to filter for informative proxy (using a liberal r2>0.4) single nucleotide polymorphisms (SNPs) for each of 38 AD common independent sentinel SNPs reported to date beyond the APOE locus. ATAC-seq fine-mapping yielded 43 candidate SNPs in open chromatin for 14 of these loci. By further constraining on our promoter connectome data in HMC3 cells, at both one and four DpnII fragment resolution (median distance between interacting regions 24kb and 122kb, respectively; median region size = 265bp and 1,441bp, respectively), we observed contacts to “open” promoters for 12 putative target genes, relevant to four of the original GWAS loci. These included RTFDC1 at the ‘CASS4’ locus and MADD and PACSIN3 at the ‘CELF1’ locus. We observed informative contacts between proxy SNPs and putative effector genes for ∼11% of AD GWAS loci in the human microglia-specific context. Further efforts in other relevant cell types, where many of the other loci may in fact be principally operating, should shed light on additional signals. Follow-up functional studies are warranted to validate these findings.

Abstract 1594: Transcriptome-wide association study analysis of nasopharyngeal carcinoma

Abstract Arising from the epithelial cells in nasopharynx, nasopharyngeal carcinoma (NPC) is highly prevalent in Southeastern China, including Hong Kong and Taiwan. Genetic and environmental factors play important role in NPC susceptibility. Genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) located in major histocompatibility complex (MHC) region with strong evidence for association with NPC. Recently, combination of GWAS and expression quantitative trait loci (eQTL) datasets, transcriptome-wide association study (TWAS) was used to identify novel associations in transcriptome level. In this study, we conducted TWAS in NPC using two in house GWAS datasets and GTEx eQTL datasets from public domain. Most of the significant results were located in chromosome six MHC region. Our analysis identified significant TWAS genes that explain the most of the GWAS signals : HLA-A, GABBR1 and HLA-F genes with P values of 5.08E-13, 5.78E-09 and 2.30E-08, respectively. One of the novel genes, ZNRD1 (P = 8.22 E-08), was known to be involved in the tumorigenesis of esophageal cancer and liver cancer. Further analysis should be needed to understand the mechanisms between ZNRD1 association in NPC. Citation Format: Wen-Hui Su, Fu-Chun Chan. Transcriptome-wide association study analysis of nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1594.

Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes

Genetic studies promise to provide insight into the molecular mechanisms underlying type 2 diabetes (T2D). Variants associated with T2D are often located in tissue-specific enhancer clusters or super-enhancers. So far, such domains have been defined through clustering of enhancers in linear genome maps rather than in three-dimensional (3D) space. Furthermore, their target genes are often unknown. We have created promoter capture Hi-C maps in human pancreatic islets. This linked diabetes-associated enhancers to their target genes, often located hundreds of kilobases away. It also revealed >1,300 groups of islet enhancers, super-enhancers and active promoters that form 3D hubs, some of which show coordinated glucose-dependent activity. We demonstrate that genetic variation in hubs impacts insulin secretion heritability, and show that hub annotations can be used for polygenic scores that predict T2D risk driven by islet regulatory variants. Human islet 3D chromatin architecture, therefore, provides a framework for interpretation of T2D genome-wide association study (GWAS) signals.

GWAS of QRS duration identifies new loci specific to Hispanic/Latino populations.

BackgroundThe electrocardiographically quantified QRS duration measures ventricular depolarization and conduction. QRS prolongation has been associated with poor heart failure prognosis and cardiovascular mortality, including sudden death. While previous genome-wide association studies (GWAS) have identified 32 QRS SNPs across 26 loci among European, African, and Asian-descent populations, the genetics of QRS among Hispanics/Latinos has not been previously explored.MethodsWe performed a GWAS of QRS duration among Hispanic/Latino ancestry populations (n = 15,124) from four studies using 1000 Genomes imputed genotype data (adjusted for age, sex, global ancestry, clinical and study-specific covariates). Study-specific results were combined using fixed-effects, inverse variance-weighted meta-analysis.ResultsWe identified six loci associated with QRS (P<5x10-8), including two novel loci: MYOCD, a nuclear protein expressed in the heart, and SYT1, an integral membrane protein. The top SNP in the MYOCD locus, intronic SNP rs16946539, was found in Hispanics/Latinos with a minor allele frequency (MAF) of 0.04, but is monomorphic in European and African descent populations. The most significant QRS duration association was with intronic SNP rs3922344 (P = 1.19x10-24) in SCN5A/SCN10A. Three other previously identified loci, CDKN1A, VTI1A, and HAND1, also exceeded the GWAS significance threshold among Hispanics/Latinos. A total of 27 of 32 previously identified QRS duration SNPs were shown to generalize in Hispanics/Latinos.ConclusionsOur QRS duration GWAS, the first in Hispanic/Latino populations, identified two new loci, underscoring the utility of extending large scale genomic studies to currently under-examined populations.

Genetic associations of breast and prostate cancer are enriched for regulatory elements identified in disease-related tissues.

Although genome-wide association studies (GWAS) have identified hundreds of risk loci for breast and prostate cancer, only a few studies have characterized the GWAS association signals across functional genomic annotations with a particular focus on single nucleotide polymorphisms (SNPs) located in DNA regulatory elements. In this study, we investigated the enrichment pattern of GWAS signals for breast and prostate cancer in genomic functional regions located in normal tissue and cancer cell lines. We quantified the overall enrichment of SNPs with breast and prostate cancer association p values < 1 × 10-8 across regulatory categories. We then obtained annotations for DNaseI hypersensitive sites (DHS), typical enhancers, and super enhancers across multiple tissue types, to assess if significant GWAS signals were selectively enriched in annotations found in disease-related tissue. Finally, we quantified the enrichment of breast and prostate cancer SNP heritability in regulatory regions, and compared the enrichment pattern of SNP heritability with GWAS signals. DHS, typical enhancers, and super enhancers identified in the breast cancer cell line MCF-7 were observed with the highest enrichment of genome-wide significant variants for breast cancer. For prostate cancer, GWAS signals were mostly enriched in DHS and typical enhancers identified in the prostate cancer cell line LNCaP. With progressively stringent GWAS p value thresholds, an increasing trend of enrichment was observed for both diseases in DHS, typical enhancers, and super enhancers located in disease-related tissue. Results from heritability enrichment analysis supported the selective enrichment pattern of functional genomic regions in disease-related cell lines for both breast and prostate cancer. Our results suggest the importance of studying functional annotations identified in disease-related tissues when characterizing GWAS results, and further demonstrate the role of germline DNA regulatory elements from disease-related tissue in breast and prostate carcinogenesis.

Genetic studies of abdominal MRI data identify genes regulating hepcidin as major determinants of liver iron concentration

Excess liver iron content is common and is linked to the risk of hepatic and extrahepatic diseases. We aimed to identify genetic variants influencing liver iron content and use genetics to understand its link to other traits and diseases. First, we performed a genome-wide association study (GWAS) in 8,289 individuals from UK Biobank, whose liver iron level had been quantified by magnetic resonance imaging, before validating our findings in an independent cohort (n = 1,513 from IMI DIRECT). Second, we used Mendelian randomisation to test the causal effects of 25 predominantly metabolic traits on liver iron content. Third, we tested phenome-wide associations between liver iron variants and 770 traits and disease outcomes. We identified 3 independent genetic variants (rs1800562 [C282Y] and rs1799945 [H63D] in HFE and rs855791 [V736A] in TMPRSS6) associated with liver iron content that reached the GWAS significance threshold (p <5 × 10-8). The 2 HFE variants account for ∼85% of all cases of hereditary haemochromatosis. Mendelian randomisation analysis provided evidence that higher central obesity plays a causal role in increased liver iron content. Phenome-wide association analysis demonstrated shared aetiopathogenic mechanisms for elevated liver iron, high blood pressure, cirrhosis, malignancies, neuropsychiatric and rheumatological conditions, while also highlighting inverse associations with anaemias, lipidaemias and ischaemic heart disease. Our study provides genetic evidence that mechanisms underlying higher liver iron content are likely systemic rather than organ specific, that higher central obesity is causally associated with higher liver iron, and that liver iron shares common aetiology with multiple metabolic and non-metabolic diseases. Excess liver iron content is common and is associated with liver diseases and metabolic diseases including diabetes, high blood pressure, and heart disease. We identified 3 genetic variants that are linked to an increased risk of developing higher liver iron content. We show that the same genetic variants are linked to higher risk of many diseases, but they may also be associated with some health advantages. Finally, we use genetic variants associated with waist-to-hip ratio as a tool to show that central obesity is causally associated with increased liver iron content.

1724-P: Interaction between Genetic Variants and Hemoglobin A1c Level on the Risk of Sight-Threatening Diabetic Retinopathy in Patients with Type 2 Diabetes

Objectives: While hemoglobin A1c (HbA1c) is a well-established risk factor of diabetic retinopathy (DR), it only accounts for limited variation in DR risk. Recent genome-wide association studies (GWAS) have identified some susceptibility single nucleotide polymorphisms (SNPs) for DR. This study aimed to evaluate the possible SNPxHbA1c interaction for the risk of sight-threatening DR (STDR) among patients with type 2 diabetes (T2DM). Methods: Sixty-nine SNPs that showed top association signals (P&amp;lt;5x10-4; r2&amp;lt;0.9) in previous GWAS of DR were genotyped in 1106 STDR cases and 2069 non-DR controls. The associations between the SNPs and STDR were examined by multiple logistic regression analyses with adjustment for the traditional risk factors. HbA1c level was dichotomized according to a cut-off of 7% as suggested for an optimal glycemic control of diabetes. Interaction with HbA1c was examined by implementing an interaction term of SNP x dichotomized HbA1c in the logistic regression model. Results: COL5A1 rs59126004 (P=0.016;OR=0.82), IGSF21-KLHDC7A rs3007729 (P=0.022;OR=0.86), LOC728275-LOC728316 rs227455 (P=0.031;OR=0.88), C6orf170 rs17083119 (P=0.035;OR=0.82) and SLC25A32 rs3098241 (P=0.046;OR=0.88) showed significant associations with STDR. Among these, COL5A1 rs59126004 also showed a significant interaction with HbA1c for the risk of STDR (Pinteraction=0.002). Stratified analysis showed that the minor allele of this SNP was associated with a reduced risk of STDR only in the patients with HbA1c &amp;lt;7% (P=1.79x10-4; OR=0.58), but not in patients with HbA1c ≥7%. Conclusions: We have demonstrated a potential interaction between the COL5A1 rs59126004 and HbA1c for the susceptibility to STDR. Further studies to validate our findings are warranted. Disclosure K. Ng: None. C. Lee: None. Y. Woo: None. H. Fong: None. R. Wong: None. K.S. Lam: None. C.Y. Cheung: None. Funding Hong Kong Health and Medical Research Fund; Hong Kong Special Administrative Region

321-OR: A Genetic Locus on Chromosome 1p36 Associated with Cardiovascular Autonomic Neuropathy in Type 2 Diabetes

Cardiovascular autonomic neuropathy (CAN) is an independent predictor of cardiovascular disease (CVD) morbidity and all-cause and CVD mortality in diabetes. In light of its significant heritability, we aimed to identify genetic predictors of CAN through an unbiased genome-wide association study (GWAS) among whites in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) cohort, comprising individuals with type 2 diabetes (T2D) and high CVD risk. CAN was defined based on indices of heart rate variability derived from 10-s resting electrocardiograms, namely, as the lowest quartile of standard deviation of normally conducted R-R intervals (SDNN &amp;lt;7.813 ms) and highest quartile of QT index (QTI &amp;gt;104.32%). Applying logistic regression models, we conducted a GWAS testing 6.8 million common single nucleotide polymorphisms (SNPs), with cases (N=807) having CAN at baseline and/or during follow-up and controls (N=3,144) being without CAN at end of follow-up. The top signal, rs779142, on chromosome 1p36, was associated with 35% increased odds of CAN (OR=1.35 [1.20-1.51], P=1.9 ×10-7). Among incident cases (N=499), this SNP reached GWAS significance (OR=1.48 [1.29-1.69], P=1.7 ×10-8). In a time-to-event analysis, rs779142 was associated with increased risk of progression to CAN (HR=1.45 [1.28-1.64], P=6.9 ×10-9) - an effect that was independent of the glycemic control arm assignments within the trial (P for interaction = 0.40). Genes lying within 1Mb of the lead SNP included KCNAB2 - a potassium voltage-gated channel associated with fatal cardiac arrhythmia, and PER3 - period circadian clock 3, which is involved in the regulation of the cardiac sympathovagal balance. To conclude, we have identified a locus on 1p36 having a genome-wide significant effect on the development of incident CAN in T2D. Pending further studies, these findings may provide better mechanistic insights into CAN pathophysiology as well as potential new therapeutic targets against this complication. Disclosure Y. Tang: None. H. Shah: None. X. Sun: None. B.A. Perkins: Advisory Panel; Self; Abbott, Boehringer Ingelheim International GmbH, Boehringer Ingelheim International GmbH, Insulet Corporation. Research Support; Self; Boehringer Ingelheim International GmbH. Other Relationship; Self; Abbott, Boehringer Ingelheim International GmbH, Lilly Diabetes, Medtronic, Novo Nordisk Inc., Sanofi. M.J. Wagner: None. A. Motsinger-Reif: None. J.B. Buse: Consultant; Self; Neurimmune AG. Research Support; Self; AstraZeneca, National Center for Advancing Translational Sciences, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk A/S, Sanofi, vTv Therapeutics. Stock/Shareholder; Self; Mellitus Health, PhaseBio Pharmaceuticals, Inc., Stability Health. Other Relationship; Self; ADOCIA, AstraZeneca, Dance Biopharm Holdings Inc., Eli Lilly and Company, MannKind Corporation, NovaTarg, Novo Nordisk A/S, Senseonics, vTv Therapeutics, Zafgen, Inc. J. Mychaleckyj: None. R. Pop-Busui: Consultant; Self; Novo Nordisk A/S. Research Support; Self; AstraZeneca. Other Relationship; Self; American Diabetes Association. A. Doria: Research Support; Self; Sanofi-Aventis. Funding National Institutes of Health

A Genetic Locus on Chromosome 2q24 Predicting Peripheral Neuropathy Risk in Type 2 Diabetes: Results From the ACCORD and BARI 2D Studies.

Genetic factors have been postulated to be involved in the etiology of diabetic peripheral neuropathy (DPN), but their identity remains mostly unknown. The aim of this study was to conduct a systematic search for genetic variants influencing DPN risk using two well-characterized cohorts. A genome-wide association study (GWAS) testing 6.8 million single nucleotide polymorphisms was conducted among participants of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) clinical trial. Included were 4,384 white case patients with type 2 diabetes (T2D) and prevalent or incident DPN (defined as a Michigan Neuropathy Screening Instrument clinical examination score >2.0) and 784 white control subjects with T2D and no evidence of DPN at baseline or during follow-up. Replication of significant loci was sought among white subjects with T2D (791 DPN-positive case subjects and 158 DPN-negative control subjects) from the Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes (BARI 2D) trial. Association between significant variants and gene expression in peripheral nerves was evaluated in the Genotype-Tissue Expression (GTEx) database. A cluster of 28 SNPs on chromosome 2q24 reached GWAS significance (P < 5 × 10−8) in ACCORD. The minor allele of the lead SNP (rs13417783, minor allele frequency = 0.14) decreased DPN odds by 36% (odds ratio [OR] 0.64, 95% CI 0.55–0.74, P = 1.9 × 10−9). This effect was not influenced by ACCORD treatment assignments (P for interaction = 0.6) or mediated by an association with known DPN risk factors. This locus was successfully validated in BARI 2D (OR 0.57, 95% CI 0.42–0.80, P = 9 × 10−4; summary P = 7.9 × 10−12). In GTEx, the minor, protective allele at this locus was associated with higher tibial nerve expression of an adjacent gene (SCN2A) coding for human voltage-gated sodium channel NaV1.2 (P = 9 × 10−4). To conclude, we have identified and successfully validated a previously unknown locus with a powerful protective effect on the development of DPN in T2D. These results may provide novel insights into DPN pathogenesis and point to a potential target for novel interventions.

Detection of circular RNA expression and related quantitative trait loci in the human dorsolateral prefrontal cortex

BackgroundCircular RNAs (circRNAs) are implicated in various biological processes. As a layer of the gene regulatory network, circRNA expression is also an intermediate phenotype bridging genetic variation and phenotypic changes. Thus, analyzing circRNA expression variation will shed light on molecular fundamentals of complex traits and diseases.ResultsWe systematically characterize 10,559 high-quality circRNAs in 589 human dorsolateral prefrontal cortex samples. We identify biological and technical factors contributing to expression heterogeneity associated with the expression levels of many circRNAs, including the well-known circRNA CDR1as. Combining the expression levels of circRNAs with genetic cis-acting SNPs, we detect 196,255 circRNA quantitative trait loci (circQTLs). By characterizing circQTL SNPs, we find that partial circQTL SNPs might influence circRNA formation by altering the canonical splicing site or the reverse complementary sequence match. Additionally, we find that a subset of these circQTL SNPs is highly linked to genome-wide association study signals of complex diseases, especially schizophrenia, inflammatory bowel disease, and type II diabetes mellitus.ConclusionsOur results reveal technical, biological, and genetic factors affecting circRNA expression variation among individuals, which lead to further understanding of circRNA regulation and thus of the genetic architecture of complex traits or diseases.

Integrating Signals from Sperm Methylome Analysis and Genome-Wide Association Study for a Better Understanding of Male Fertility in Cattle.

Decreased male fertility is a big concern in both human society and the livestock industry. Sperm DNA methylation is commonly believed to be associated with male fertility. However, due to the lack of accurate male fertility records (i.e., limited mating times), few studies have investigated the comprehensive impacts of sperm DNA methylation on male fertility in mammals. In this study, we generated 10 sperm DNA methylomes and performed a preliminary correlation analysis between signals from sperm DNA methylation and signals from large-scale (n = 27,214) genome-wide association studies (GWAS) of 35 complex traits (including 12 male fertility-related traits). We detected genomic regions, which experienced DNA methylation alterations in sperm and were associated with aging and extreme fertility phenotypes (e.g., sire-conception rate or SCR). In dynamic hypomethylated regions (HMRs) and partially methylated domains (PMDs), we found genes (e.g., HOX gene clusters and microRNAs) that were involved in the embryonic development. We demonstrated that genomic regions, which gained rather than lost methylations during aging, and in animals with low SCR were significantly and selectively enriched for GWAS signals of male fertility traits. Our study discovered 16 genes as the potential candidate markers for male fertility, including SAMD5 and PDE5A. Collectively, this initial effort supported a hypothesis that sperm DNA methylation may contribute to male fertility in cattle and revealed the usefulness of functional annotations in enhancing biological interpretation and genomic prediction for complex traits and diseases.

Multiple SCN5A variant enhancers modulate its cardiac gene expression and the QT interval

The rationale for genome-wide association study (GWAS) results is sequence variation in cis-regulatory elements (CREs) modulating a target gene's expression as the major cause of trait variation. To understand the complete molecular landscape of one of these GWAS loci, we performed in vitro reporter screens in cardiomyocyte cell lines for CREs overlapping nearly all common variants associated with any of five independent QT interval (QTi)-associated GWAS hits at the SCN5A-SCN10A locus. We identified 13 causal CRE variants using allelic reporter activity, cardiomyocyte nuclear extract-based binding assays, overlap with human cardiac tissue DNaseI hypersensitive regions, and predicted impact of sequence variants on DNaseI sensitivity. Our analyses identified at least one high-confidence causal CRE variant for each of the five sentinel hits that could collectively predict SCN5A cardiac gene expression and QTi association. Although all 13 variants could explain SCN5A gene expression, the highest statistical significance was obtained with seven variants (inclusive of the five above). Thus, multiple, causal, mutually associated CRE variants can underlie GWAS signals.

Allele-specific enhancers mediate associations between LCAT and ABCA1 polymorphisms and HDL metabolism.

For most complex traits, the majority of SNPs identified through genome-wide association studies (GWAS) reside within noncoding regions that have no known function. However, these regions are enriched for the regulatory enhancers specific to the cells relevant to the specific trait. Indeed, many of the GWAS loci that have been functionally characterized lie within enhancers that regulate expression levels of key genes. In order to identify polymorphisms with potential allele-specific regulatory effects, we developed a bioinformatics pipeline that harnesses epigenetic signatures as well as transcription factor (TF) binding motifs to identify putative enhancers containing a SNP with potential allele-specific TF binding in linkage disequilibrium (LD) with a GWAS-identified SNP. We applied the approach to GWAS findings for blood lipids, revealing 7 putative enhancers harboring associated SNPs, 3 of which lie within the introns of LCAT and ABCA1, genes that play crucial roles in cholesterol biogenesis and lipoprotein metabolism. All 3 enhancers demonstrated allele-specific in vitro regulatory activity in liver-derived cell lines. We demonstrated that these putative enhancers are in close physical proximity to the promoters of their respective genes, in situ, likely through chromatin looping. In addition, the associated alleles altered the likelihood of transcription activator STAT3 binding. Our results demonstrate that through our approach, the LD blocks that contain GWAS signals, often hundreds of kilobases in size with multiple SNPs serving as statistical proxies to the true functional site, can provide an experimentally testable hypothesis for the underlying regulatory mechanism linking genetic variants to complex traits.

Evidence for Recent Polygenic Selection on Educational Attainment and Intelligence Inferred from Gwas Hits: A Replication of Previous Findings Using Recent Data

Genetic variants identified by three large genome-wide association studies (GWAS) of educational attainment (EA) were used to test a polygenic selection model. Weighted and unweighted polygenic scores (PGS) were calculated and compared across populations using data from the 1000 Genomes (n = 26), HGDP-CEPH (n = 52) and gnomAD (n = 8) datasets. The PGS from the largest EA GWAS was highly correlated to two previously published PGSs (r = 0.96–0.97, N = 26). These factors are both highly predictive of average population IQ (r = 0.9, N = 23) and Learning index (r = 0.8, N = 22) and are robust to tests of spatial autocorrelation. Monte Carlo simulations yielded highly significant p values. In the gnomAD samples, the correlation between PGS and IQ was almost perfect (r = 0.98, N = 8), and ANOVA showed significant population differences in allele frequencies with positive effect. Socioeconomic variables slightly improved the prediction accuracy of the model (from 78–80% to 85–89%), but the PGS explained twice as much of the variance in IQ compared to socioeconomic variables. In both 1000 Genomes and gnomAD, there was a weak trend for lower GWAS significance SNPs to be less predictive of population IQ. Additionally, a subset of SNPs were found in the HGDP-CEPH sample (N = 127). The analysis of this sample yielded a positive correlation with latitude and a low negative correlation with distance from East Africa. This study provides robust results after accounting for spatial autocorrelation with Fst distances and random noise via an empirical Monte Carlo simulation using null SNPs.

Evidence for Recent Polygenic Selection on Educational Attainment and Intelligence Inferred from Gwas Hits: A Replication of Previous Findings Using Recent Data

Genetic variants identified by three large genome-wide association studies (GWAS) of educational attainment (EA) were used to test a polygenic selection model. Weighted and unweighted polygenic scores (PGS) were calculated and compared across populations using data from the 1000 Genomes (n = 26), HGDP-CEPH (n = 52) and gnomAD (n = 8) datasets. The PGS from the largest EA GWAS was highly correlated to two previously published PGSs (r = 0.96–0.97, N = 26). These factors are both highly predictive of average population IQ (r = 0.9, N = 23) and Learning index (r = 0.8, N = 22) and are robust to tests of spatial autocorrelation. Monte Carlo simulations yielded highly significant p values. In the gnomAD samples, the correlation between PGS and IQ was almost perfect (r = 0.98, N = 8), and ANOVA showed significant population differences in allele frequencies with positive effect. Socioeconomic variables slightly improved the prediction accuracy of the model (from 78–80% to 85–89%), but the PGS explained twice as much of the variance in IQ compared to socioeconomic variables. In both 1000 Genomes and gnomAD, there was a weak trend for lower GWAS significance SNPs to be less predictive of population IQ. Additionally, a subset of SNPs were found in the HGDP-CEPH sample (N = 127). The analysis of this sample yielded a positive correlation with latitude and a low negative correlation with distance from East Africa. This study provides robust results after accounting for spatial autocorrelation with Fst distances and random noise via an empirical Monte Carlo simulation using null SNPs.

Haploinsufficiency of ARHGAP42 is associated with hypertension.

Family studies have established that the heritability of blood pressure is significant and genome-wide association studies (GWAS) have identified numerous susceptibility loci, including one within the non-coding part of Rho GTPase-activating protein 42 gene (ARHGAP42) on chromosome 11q22.1. Arhgap42-deficient mice have significantly elevated blood pressure, but the phenotypic effects of human variants in the coding part of the gene are unknown. In a Danish cohort of carriers with apparently balanced chromosomal rearrangements, we identified a family where a reciprocal translocation t(11;18)(q22.1;q12.2) segregated with hypertension and obesity. Clinical re-examination revealed that four carriers (age 50-77 years) have had hypertension for several years along with an increased body mass index (34-43 kg/m2). A younger carrier (age 23 years) had normal blood pressure and body mass index. Mapping of the chromosomal breakpoints with mate-pair and Sanger sequencing revealed truncation of ARHGAP42. A decreased expression level of ARHGAP42 mRNA in the blood was found in the translocation carriers relative to controls and allele-specific expression analysis showed monoallelic expression in the translocation carriers, confirming that the truncated allele of ARHGAP42 was not expressed. These findings support that haploinsufficiency of ARHGAP42 leads to an age-dependent hypertension. The other breakpoint truncated a regulatory domain of the CUGBP Elav-like family member 4 (CELF4) gene on chromosome 18q12.2 that harbours several GWAS signals for obesity. We thereby provide additional support for an obesity locus in the CELF4 domain.

Genetic and epigenetic architecture of paternal origin contribute to gestation length in cattle

The length of gestation can affect offspring health and performance. Both maternal and fetal effects contribute to gestation length; however, paternal contributions to gestation length remain elusive. Using genome-wide association study (GWAS) in 27,214 Holstein bulls with millions of gestation records, here we identify nine paternal genomic loci associated with cattle gestation length. We demonstrate that these GWAS signals are enriched in pathways relevant to embryonic development, and in differentially methylated regions between sperm samples with long and short gestation length. We reveal that gestation length shares genetic and epigenetic architecture in sperm with calving ability, body depth, and conception rate. While several candidate genes are detected in our fine-mapping analysis, we provide evidence indicating ZNF613 as a promising candidate for cattle gestation length. Collectively, our findings support that the paternal genome and epigenome can impact gestation length potentially through regulation of the embryonic development.

Functional characterization of the C7ORF76 genomic region, a prominent GWAS signal for osteoporosis in 7q21.3.

Genome-wide association studies (GWAS) have repeatedly identified genetic variants associated with bone mineral density (BMD) and osteoporotic fracture in non-coding regions of C7ORF76, a poorly studied gene of unknown function. The aim of the present study was to elucidate the causality and molecular mechanisms underlying the association. We re-sequenced the genomic region in two extreme BMD groups from the BARCOS cohort of postmenopausal women to search for functionally relevant variants. Eight selected variants were tested for association in the complete cohort and 2 of them (rs4342521 and rs10085588) were found significantly associated with lumbar spine BMD and nominally associated with osteoporotic fracture. cis-eQTL analyses of these 2 SNPs, together with SNP rs4727338 (GWAS lead SNP in Estrada et al., Nat Genet. 44:491-501, 2012), performed in human primary osteoblasts, disclosed a statistically significant influence on the expression of the proximal neighbouring gene SLC25A13 and a tendency on the distal SHFM1. We then studied the functionality of a putative upstream regulatory element (UPE), containing rs10085588. Luciferase reporter assays showed transactivation capability with a strong allele-dependent effect. Finally, 4C-seq experiments in osteoblastic cell lines showed that the UPE interacted with different tissue-specific enhancers and a lncRNA (LOC100506136) in the region. In summary, this study is the first one to analyse in depth the functionality of C7ORF76 genomic region. We provide functional regulatory evidence for the rs10085588, which may be a causal SNP within the 7q21.3 GWAS signal for osteoporosis.