Determinants of mosaic chromosomal alteration fitness.

Clonal hematopoiesis (CH) is characterized by the acquisition of a somatic mutation in a hematopoietic stem cell that results in a clonal expansion. These driver mutations can be single nucleotide variants in cancer driver genes or larger structural rearrangements called mosaic chromosomal alterations (mCAs). The factors that influence the variations in mCA fitness and ultimately result in different clonal expansion rates are not well understood. We used the Passenger-Approximated Clonal Expansion Rate (PACER) method to estimate clonal expansion rate as PACER scores for 6,381 individuals in the NHLBI TOPMed cohort with gain, loss, and copy-neutral loss of heterozygosity mCAs. Our mCA fitness estimates, derived by aggregating per-individual PACER scores, were correlated (R2 = 0.49) with an alternative approach that estimated fitness of mCAs in the UK Biobank using population-level distributions of clonal fraction. Among individuals with JAK2 V617F clonal hematopoiesis of indeterminate potential or mCAs affecting the JAK2 gene on chromosome 9, PACER score was strongly correlated with erythrocyte count. In a cross-sectional analysis, genome-wide association study of estimates of mCA expansion rate identified a TCL1A locus variant associated with mCA clonal expansion rate, with suggestive variants in NRIP1 and TERT.

Determinants of mosaic chromosomal alteration fitness.

Clonal hematopoiesis (CH) is characterized by the acquisition of a somatic mutation in a hematopoietic stem cell that results in a clonal expansion. These driver mutations can be single nucleotide variants in cancer driver genes or larger structural rearrangements called mosaic chromosomal alterations (mCAs). The factors that influence the variations in mCA fitness and ultimately result in different clonal expansion rates are not well-understood. We used the Passenger-Approximated Clonal Expansion Rate (PACER) method to estimate clonal expansion rate for 6,381 individuals in the NHLBI TOPMed cohort with gain, loss, and copy-neutral loss of heterozygosity mCAs. Our estimates of mCA fitness were correlated (R 2 = 0.49) with an alternative approach that estimated fitness of mCAs in the UK Biobank using a theoretical probability distribution. Individuals with lymphoid-associated mCAs had a significantly higher white blood cell count and faster clonal expansion rate. In a cross-sectional analysis, genome-wide association study of estimates of mCA expansion rate identified TCL1A , NRIP1 , and TERT locus variants as modulators of mCA clonal expansion rate.

Age at Menopause, Leukocyte Telomere Length, and Coronary Artery Disease in Postmenopausal Women.

BACKGROUND: Premature menopause is a risk factor for accelerated cardiovascular aging, but underlying mechanisms remain incompletely understood. This study investigated the role of leukocyte telomere length (LTL), a marker of cellular aging and genomic instability, in the association of premature menopause with cardiovascular disease. METHODS: Participants from the UK Biobank and Women's Health Initiative with complete reproductive history and LTL measurements were included. Primary analyses tested the association between age at menopause and LTL using multivariable-adjusted linear regression. Secondary analyses stratified women by history of gynecologic surgery. Mendelian randomization was used to infer causal relationships between LTL and age at natural menopause. Multivariable-adjusted Cox regression and mediation analyses tested the joint associations of premature menopause and LTL with incident coronary artery disease. RESULTS: This study included 130 254 postmenopausal women (UK Biobank: n=122 224; Women's Health Initiative: n=8030), of whom 4809 (3.7%) had experienced menopause before age 40. Earlier menopause was associated with shorter LTL (meta-analyzed ß=-0.02 SD/5 years of earlier menopause [95% CI, -0.02 to -0.01]; P=7.2×10-12). This association was stronger and significant in both cohorts for women with natural/spontaneous menopause (meta-analyzed ß=-0.04 SD/5 years of earlier menopause [95% CI, -0.04 to -0.03]; P<2.2×10-16) and was independent of hormone therapy use. Mendelian randomization supported a causal association of shorter genetically predicted LTL with earlier age at natural menopause. LTL and age at menopause were independently associated with incident coronary artery disease, and mediation analyses indicated small but significant mediation effects of LTL in the association of menopausal age with coronary artery disease. CONCLUSIONS: Earlier age at menopause is associated with shorter LTL, especially among women with natural menopause. Accelerated telomere shortening may contribute to the heightened cardiovascular risk associated with premature menopause.

African-specific alleles modify risk for asthma at the 17q12-q21 locus in African Americans.

BACKGROUND: Asthma is the most common chronic disease in children, occurring at higher frequencies and with more severe disease in children with African ancestry. METHODS: We tested for association with haplotypes at the most replicated and significant childhood-onset asthma locus at 17q12-q21 and asthma in European American and African American children. Following this, we used whole-genome sequencing data from 1060 African American and 100 European American individuals to identify novel variants on a high-risk African American-specific haplotype. We characterized these variants in silico using gene expression and ATAC-seq data from airway epithelial cells, functional annotations from ENCODE, and promoter capture (pc)Hi-C maps in airway epithelial cells. Candidate causal variants were then assessed for correlation with asthma-associated phenotypes in African American children and adults. RESULTS: Our studies revealed nine novel African-specific common variants, enriched on a high-risk asthma haplotype, which regulated the expression of GSDMA in airway epithelial cells and were associated with features of severe asthma. Using ENCODE annotations, ATAC-seq, and pcHi-C, we narrowed the associations to two candidate causal variants that are associated with features of T2 low severe asthma. CONCLUSIONS: Previously unknown genetic variation at the 17q12-21 childhood-onset asthma locus contributes to asthma severity in individuals with African ancestries. We suggest that many other population-specific variants that have not been discovered in GWAS contribute to the genetic risk for asthma and other common diseases.

Genetic determinants of telomere length from 109,122 ancestrally diverse whole-genome sequences in TOPMed.

Genetic studies on telomere length are important for understanding age-related diseases. Prior GWAS for leukocyte TL have been limited to European and Asian populations. Here, we report the first sequencing-based association study for TL across ancestrally-diverse individuals (European, African, Asian and Hispanic/Latino) from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program. We used whole genome sequencing (WGS) of whole blood for variant genotype calling and the bioinformatic estimation of telomere length in n=109,122 individuals. We identified 59 sentinel variants (p-value <5×10-9) in 36 loci associated with telomere length, including 20 newly associated loci (13 were replicated in external datasets). There was little evidence of effect size heterogeneity across populations. Fine-mapping at OBFC1 indicated the independent signals colocalized with cell-type specific eQTLs for OBFC1 (STN1). Using a multi-variant gene-based approach, we identified two genes newly implicated in telomere length, DCLRE1B (SNM1B) and PARN. In PheWAS, we demonstrated our TL polygenic trait scores (PTS) were associated with increased risk of cancer-related phenotypes.

Multi-Site Observational Study to Assess Biomarkers for Susceptibility or Resilience to Chronic Pain: The Acute to Chronic Pain Signatures (A2CPS) Study Protocol.

Chronic pain has become a global health problem contributing to years lived with disability and reduced quality of life. Advances in the clinical management of chronic pain have been limited due to incomplete understanding of the multiple risk factors and molecular mechanisms that contribute to the development of chronic pain. The Acute to Chronic Pain Signatures (A2CPS) Program aims to characterize the predictive nature of biomarkers (brain imaging, high-throughput molecular screening techniques, or "omics," quantitative sensory testing, patient-reported outcome assessments and functional assessments) to identify individuals who will develop chronic pain following surgical intervention. The A2CPS is a multisite observational study investigating biomarkers and collective biosignatures (a combination of several individual biomarkers) that predict susceptibility or resilience to the development of chronic pain following knee arthroplasty and thoracic surgery. This manuscript provides an overview of data collection methods and procedures designed to standardize data collection across multiple clinical sites and institutions. Pain-related biomarkers are evaluated before surgery and up to 3 months after surgery for use as predictors of patient reported outcomes 6 months after surgery. The dataset from this prospective observational study will be available for researchers internal and external to the A2CPS Consortium to advance understanding of the transition from acute to chronic postsurgical pain.

Mendelian randomization supports bidirectional causality between telomere length and clonal hematopoiesis of indeterminate potential.

Human genetic studies support an inverse causal relationship between leukocyte telomere length (LTL) and coronary artery disease (CAD), but directionally mixed effects for LTL and diverse malignancies. Clonal hematopoiesis of indeterminate potential (CHIP), characterized by expansion of hematopoietic cells bearing leukemogenic mutations, predisposes both hematologic malignancy and CAD. TERT (which encodes telomerase reverse transcriptase) is the most significantly associated germline locus for CHIP in genome-wide association studies. Here, we investigated the relationship between CHIP, LTL, and CAD in the Trans-Omics for Precision Medicine (TOPMed) program (n = 63,302) and UK Biobank (n = 47,080). Bidirectional Mendelian randomization studies were consistent with longer genetically imputed LTL increasing propensity to develop CHIP, but CHIP then, in turn, hastens to shorten measured LTL (mLTL). We also demonstrated evidence of modest mediation between CHIP and CAD by mLTL. Our data promote an understanding of potential causal relationships across CHIP and LTL toward prevention of CAD.

Detecting Gene-Environment Interaction for Maternal Exposures Using Case-Parent Trios Ascertained Through a Case With Non-Syndromic Orofacial Cleft.

Two large studies of case-parent trios ascertained through a proband with a non-syndromic orofacial cleft (OFC, which includes cleft lip and palate, cleft lip alone, or cleft palate alone) were used to test for possible gene-environment (G × E) interaction between genome-wide markers (both observed and imputed) and self-reported maternal exposure to smoking, alcohol consumption, and multivitamin supplementation during pregnancy. The parent studies were as follows: GENEVA, which included 1,939 case-parent trios recruited largely through treatment centers in Europe, the United States, and Asia, and 1,443 case-parent trios from the Pittsburgh Orofacial Cleft Study (POFC) also ascertained through a proband with an OFC including three major racial/ethnic groups (European, Asian, and Latin American). Exposure rates to these environmental risk factors (maternal smoking, alcohol consumption, and multivitamin supplementation) varied across studies and among racial/ethnic groups, creating substantial differences in power to detect G × E interaction, but the trio design should minimize spurious results due to population stratification. The GENEVA and POFC studies were analyzed separately, and a meta-analysis was conducted across both studies to test for G × E interaction using the 2 df test of gene and G × E interaction and the 1 df test for G × E interaction alone. The 2 df test confirmed effects for several recognized risk genes, suggesting modest G × E effects. This analysis did reveal suggestive evidence for G × Vitamin interaction for CASP9 on 1p36 located about 3 Mb from PAX7, a recognized risk gene. Several regions gave suggestive evidence of G × E interaction in the 1 df test. For example, for G × Smoking interaction, the 1 df test suggested markers in MUSK on 9q31.3 from meta-analysis. Markers near SLCO3A1 also showed suggestive evidence in the 1 df test for G × Alcohol interaction, and rs41117 near RETREG1 (a.k.a. FAM134B) also gave suggestive significance in the meta-analysis of the 1 df test for G × Vitamin interaction. While it remains quite difficult to obtain definitive evidence for G × E interaction in genome-wide studies, perhaps due to small effect sizes of individual genes combined with low exposure rates, this analysis of two large case-parent trio studies argues for considering possible G × E interaction in any comprehensive study of complex and heterogeneous disorders such as OFC.

A Multiancestry Sex-Stratified Genome-Wide Association Study of Spontaneous Clearance of Hepatitis C Virus.

BACKGROUND: Spontaneous clearance of acute hepatitis C virus (HCV) infection is more common in women than in men, independent of known risk factors. METHODS: To identify sex-specific genetic loci, we studied 4423 HCV-infected individuals (2903 male, 1520 female) of European, African, and Hispanic ancestry. We performed autosomal, and X chromosome sex-stratified and combined association analyses in each ancestry group. RESULTS: A male-specific region near the adenosine diphosphate-ribosylation factor-like 5B (ARL5B) gene was identified. Individuals with the C allele of rs76398191 were about 30% more likely to have chronic HCV infection than individuals with the T allele (OR, 0.69; P = 1.98 × 10-07), and this was not seen in females. The ARL5B gene encodes an interferon-stimulated gene that inhibits immune response to double-stranded RNA viruses. We also identified suggestive associations near septin 6 and ribosomal protein L39 genes on the X chromosome. In box sexes, allele G of rs12852885 was associated with a 40% increase in HCV clearance compared with the A allele (OR, 1.4; P = 2.46 × 10-06). Septin 6 facilitates HCV replication via interaction with the HCV NS5b protein, and ribosomal protein L39 acts as an HCV core interactor. CONCLUSIONS: These novel gene associations support differential mechanisms of HCV clearance between the sexes and provide biological targets for treatment or vaccine development.

Inherited causes of clonal haematopoiesis in 97,691 whole genomes.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown1. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer2-4 and coronary heart disease5-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP)6. Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.

Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program.

Human induced pluripotent stem cell (hiPSC) lines have previously been generated through the NHLBI sponsored NextGen program at nine individual study sites. Here, we examined the structural integrity of 506 hiPSC lines as determined by copy number variations (CNVs). We observed that 149 hiPSC lines acquired 258 CNVs relative to donor DNA. We identified six recurrent regions of CNVs on chromosomes 1, 2, 3, 16 and 20 that overlapped with cancer associated genes. Furthermore, the genes mapping to regions of acquired CNVs show an enrichment in cancer related biological processes (IL6 production) and signaling cascades (JNK cascade & NFκB cascade). The genomic region of instability on chr20 (chr20q11.2) includes transcriptomic signatures for cancer associated genes such as ID1, BCL2L1, TPX2, PDRG1 and HCK. Of these HCK shows statistically significant differential expression between carrier and non-carrier hiPSC lines. Overall, while a low level of genomic instability was observed in the NextGen generated hiPSC lines, the observation of structural instability in regions with known cancer associated genes substantiates the importance of systematic evaluation of genetic variations in hiPSCs before using them as disease/research models.

Whole genome sequencing of orofacial cleft trios from the Gabriella Miller Kids First Pediatric Research Consortium identifies a new locus on chromosome 21.

Orofacial clefts (OFCs) are among the most prevalent craniofacial birth defects worldwide and create a significant public health burden. The majority of OFCs are non-syndromic, and the genetic etiology of non-syndromic OFCs is only partially determined. Here, we analyze whole genome sequence (WGS) data for association with risk of OFCs in European and Colombian families selected from a multicenter family-based OFC study. This is the first large-scale WGS study of OFC in parent-offspring trios, and a part of the Gabriella Miller Kids First Pediatric Research Program created for the study of childhood cancers and structural birth defects. WGS provides deeper and more specific genetic data than using imputation on present-day single nucleotide polymorphic (SNP) marker panels. Genotypes of case-parent trios at single nucleotide variants (SNV) and short insertions and deletions (indels) spanning the entire genome were called from their sequences using human GRCh38 genome assembly, and analyzed for association using the transmission disequilibrium test. Among genome-wide significant associations, we identified a new locus on chromosome 21 in Colombian families, not previously observed in other larger OFC samples of Latin American ancestry. This locus is situated within a region known to be expressed during craniofacial development. Based on deeper investigation of this locus, we concluded that it contributed risk for OFCs exclusively in the Colombians. This study reinforces the ancestry differences seen in the genetic etiology of OFCs, and underscores the need for larger samples when studying for OFCs and other birth defects in populations with diverse ancestry.

Association of DNA Methylation Differences With Schizophrenia in an Epigenome-Wide Association Study.

IMPORTANCE: DNA methylation may play an important role in schizophrenia (SZ), either directly as a mechanism of pathogenesis or as a biomarker of risk. OBJECTIVE: To scan genome-wide DNA methylation data to identify differentially methylated CpGs between SZ cases and controls. DESIGN, SETTING, AND PARTICIPANTS: Epigenome-wide association study begun in 2008 using DNA methylation levels of 456 513 CpG loci measured on the Infinium HumanMethylation450 array (Illumina) in a consortium of case-control studies for initial discovery and in an independent replication set. Primary analyses used general linear regression, adjusting for age, sex, race/ethnicity, smoking, batch, and cell type heterogeneity. The discovery set contained 689 SZ cases and 645 controls (n = 1334), from 3 multisite consortia: the Consortium on the Genetics of Endophenotypes in Schizophrenia, the Project among African-Americans To Explore Risks for Schizophrenia, and the Multiplex Multigenerational Family Study of Schizophrenia. The replication set contained 247 SZ cases and 250 controls (n = 497) from the Genomic Psychiatry Cohort. MAIN OUTCOMES AND MEASURES: Identification of differentially methylated positions across the genome in SZ cases compared with controls. RESULTS: Of the 689 case participants in the discovery set, 477 (69%) were men and 258 (37%) were non-African American; of the 645 controls, 273 (42%) were men and 419 (65%) were non-African American. In our replication set, cases/controls were 76% male and 100% non-African American. We identified SZ-associated methylation differences at 923 CpGs in the discovery set (false discovery rate, <0.2). Of these, 625 showed changes in the same direction including 172 with P < .05 in the replication set. Some replicated differentially methylated positions are located in a top-ranked SZ region from genome-wide association study analyses. CONCLUSIONS AND RELEVANCE: This analysis identified 172 replicated new associations with SZ after careful correction for cell type heterogeneity and other potential confounders. The overlap with previous genome-wide association study data can provide potential insights into the functional relevance of genetic signals for SZ.

Identification of Rare Variants in ATP8B4 as a Risk Factor for Systemic Sclerosis by Whole-Exome Sequencing.

OBJECTIVE: To determine the contribution of rare variants as genetic modifiers of the expressivity, penetrance, and severity of systemic sclerosis (SSc). METHODS: We performed whole-exome sequencing of 78 European American patients with SSc, including 35 patients without pulmonary arterial hypertension (PAH) and 43 patients with PAH. Association testing of case-control probability for rare variants was performed using the unified sequence kernel association test with optimal kernel weighting and small sample adjustment by comparing all SSc patients with a reference population of 3,179 controls from the Exome Sequencing Project 5,500 exome data set. Replication genotyping was performed in an independent sample of 3,263 patients (415 patients with SSc and 2,848 controls). We conducted expression profiling of messenger RNA from 61 SSc patients (19 without PAH and 42 with PAH) and 41 corresponding controls. RESULTS: The ATP8B4 gene was associated with a significant increase in the risk of SSc (P = 2.77 × 10(-7)). Among the 64 ATP8B4 variants tested, a single missense variant, c.1308C>G (F436L, rs55687265), provided the most compelling evidence of association (P = 9.35 × 10(-10), odds ratio [OR] 6.11), which was confirmed in the replication cohort (P = 0.012, OR 1.86) and meta-analysis (P = 1.92 × 10(-7), OR 2.5). Genes involved in E3 ubiquitin-protein ligase complex (ASB10) and cyclic nucleotide gated channelopathies (CNGB3) as well as HLA-DRB5 and HSPB2 (heat-shock protein 27) provided additional evidence of association (P < 10(-5)). Differential ATP8B4 expression was observed among the SSc patients compared to the controls (P = 0.0005). CONCLUSION: ATP8B4 may represent a putative genetic risk factor for SSc and pulmonary vascular complications.

Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci.

Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.

Evidence of gene-environment interaction for two genes on chromosome 4 and environmental tobacco smoke in controlling the risk of nonsyndromic cleft palate.

Nonsyndromic cleft palate (CP) is one of the most common human birth defects and both genetic and environmental risk factors contribute to its etiology. We conducted a genome-wide association study (GWAS) using 550 CP case-parent trios ascertained in an international consortium. Stratified analysis among trios with different ancestries was performed to test for GxE interactions with common maternal exposures using conditional logistic regression models. While no single nucleotide polymorphism (SNP) achieved genome-wide significance when considered alone, markers in SLC2A9 and the neighboring WDR1 on chromosome 4p16.1 gave suggestive evidence of gene-environment interaction with environmental tobacco smoke (ETS) among 259 Asian trios when the models included a term for GxE interaction. Multiple SNPs in these two genes were associated with increased risk of nonsyndromic CP if the mother was exposed to ETS during the peri-conceptual period (3 months prior to conception through the first trimester). When maternal ETS was considered, fifteen of 135 SNPs mapping to SLC2A9 and 9 of 59 SNPs in WDR1 gave P values approaching genome-wide significance (10(-6)