A deep catalogue of protein-coding variation in 983,578 individuals.

Rare coding variants that significantly impact function provide insights into the biology of a gene1-3. However, ascertaining their frequency requires large sample sizes4-8. Here, we present a catalogue of human protein-coding variation, derived from exome sequencing of 983,578 individuals across diverse populations. 23% of the Regeneron Genetics Center Million Exome data (RGC-ME) comes from non-European individuals of African, East Asian, Indigenous American, Middle Eastern, and South Asian ancestry. This catalogue includes over 10.4 million missense and 1.1 million predicted loss-of-function (pLOF) variants. We identify individuals with rare biallelic pLOF variants in 4,848 genes, 1,751 of which have not been previously reported. From precise quantitative estimates of selection against heterozygous loss-of-function, we identify 3,988 loss-of-function intolerant genes, including 86 that were previously assessed as tolerant and 1,153 lacking established disease annotation. We also define regions of missense depletion at high resolution. Notably, 1,482 genes have regions depleted of missense variants despite being tolerant to pLOF variants. Finally, we estimate that 3% of individuals have a clinically actionable genetic variant, and that 11,773 variants reported in ClinVar with unknown significance are likely to be deleterious cryptic splice sites. To facilitate variant interpretation and genetics-informed precision medicine, we make this important resource of coding variation from the RGC-ME accessible via a public variant allele frequency browser.

Variants in autophagy genes MTMR12 and FAM134A are putative modifiers of the hepatic phenotype in α1-antitrypsin deficiency.

BACKGROUND AND AIMS: In the classical form of α1-antitrypsin deficiency, a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here, we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were compared to sequences in well-characterized cohorts of homozygotes with or without liver disease, and then candidate sequence variants were tested for changes in the kinetics of α1-antitrypsin variant Z degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves. APPROACH AND RESULTS: Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of α1-antitrypsin variant Z in cells from the index patient, but both MTMR12 and FAM134A variants were needed to slow the degradation of α1-antitrypsin variant Z in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level. CONCLUSIONS: These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in α1-antitrypsin deficiency and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

Genome-wide association meta-analysis identifies risk loci for abdominal aortic aneurysm and highlights PCSK9 as a therapeutic target.

Abdominal aortic aneurysm (AAA) is a common disease with substantial heritability. In this study, we performed a genome-wide association meta-analysis from 14 discovery cohorts and uncovered 141 independent associations, including 97 previously unreported loci. A polygenic risk score derived from meta-analysis explained AAA risk beyond clinical risk factors. Genes at AAA risk loci indicate involvement of lipid metabolism, vascular development and remodeling, extracellular matrix dysregulation and inflammation as key mechanisms in AAA pathogenesis. These genes also indicate overlap between the development of AAA and other monogenic aortopathies, particularly via transforming growth factor ß signaling. Motivated by the strong evidence for the role of lipid metabolism in AAA, we used Mendelian randomization to establish the central role of nonhigh-density lipoprotein cholesterol in AAA and identified the opportunity for repurposing of proprotein convertase, subtilisin/kexin-type 9 (PCSK9) inhibitors. This was supported by a study demonstrating that PCSK9 loss of function prevented the development of AAA in a preclinical mouse model.

Implicating genes, pleiotropy, and sexual dimorphism at blood lipid loci through multi-ancestry meta-analysis.

BACKGROUND: Genetic variants within nearly 1000 loci are known to contribute to modulation of blood lipid levels. However, the biological pathways underlying these associations are frequently unknown, limiting understanding of these findings and hindering downstream translational efforts such as drug target discovery. RESULTS: To expand our understanding of the underlying biological pathways and mechanisms controlling blood lipid levels, we leverage a large multi-ancestry meta-analysis (N = 1,654,960) of blood lipids to prioritize putative causal genes for 2286 lipid associations using six gene prediction approaches. Using phenome-wide association (PheWAS) scans, we identify relationships of genetically predicted lipid levels to other diseases and conditions. We confirm known pleiotropic associations with cardiovascular phenotypes and determine novel associations, notably with cholelithiasis risk. We perform sex-stratified GWAS meta-analysis of lipid levels and show that 3-5% of autosomal lipid-associated loci demonstrate sex-biased effects. Finally, we report 21 novel lipid loci identified on the X chromosome. Many of the sex-biased autosomal and X chromosome lipid loci show pleiotropic associations with sex hormones, emphasizing the role of hormone regulation in lipid metabolism. CONCLUSIONS: Taken together, our findings provide insights into the biological mechanisms through which associated variants lead to altered lipid levels and potentially cardiovascular disease risk.

Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Integrating transcriptomics, metabolomics, and GWAS helps reveal molecular mechanisms for metabolite levels and disease risk.

Transcriptomics data have been integrated with genome-wide association studies (GWASs) to help understand disease/trait molecular mechanisms. The utility of metabolomics, integrated with transcriptomics and disease GWASs, to understand molecular mechanisms for metabolite levels or diseases has not been thoroughly evaluated. We performed probabilistic transcriptome-wide association and locus-level colocalization analyses to integrate transcriptomics results for 49 tissues in 706 individuals from the GTEx project, metabolomics results for 1,391 plasma metabolites in 6,136 Finnish men from the METSIM study, and GWAS results for 2,861 disease traits in 260,405 Finnish individuals from the FinnGen study. We found that genetic variants that regulate metabolite levels were more likely to influence gene expression and disease risk compared to the ones that do not. Integrating transcriptomics with metabolomics results prioritized 397 genes for 521 metabolites, including 496 previously identified gene-metabolite pairs with strong functional connections and suggested 33.3% of such gene-metabolite pairs shared the same causal variants with genetic associations of gene expression. Integrating transcriptomics and metabolomics individually with FinnGen GWAS results identified 1,597 genes for 790 disease traits. Integrating transcriptomics and metabolomics jointly with FinnGen GWAS results helped pinpoint metabolic pathways from genes to diseases. We identified putative causal effects of UGT1A1/UGT1A4 expression on gallbladder disorders through regulating plasma (E,E)-bilirubin levels, of SLC22A5 expression on nasal polyps and plasma carnitine levels through distinct pathways, and of LIPC expression on age-related macular degeneration through glycerophospholipid metabolic pathways. Our study highlights the power of integrating multiple sets of molecular traits and GWAS results to deepen understanding of disease pathophysiology.

A multi-layer functional genomic analysis to understand noncoding genetic variation in lipids.

A major challenge of genome-wide association studies (GWASs) is to translate phenotypic associations into biological insights. Here, we integrate a large GWAS on blood lipids involving 1.6 million individuals from five ancestries with a wide array of functional genomic datasets to discover regulatory mechanisms underlying lipid associations. We first prioritize lipid-associated genes with expression quantitative trait locus (eQTL) colocalizations and then add chromatin interaction data to narrow the search for functional genes. Polygenic enrichment analysis across 697 annotations from a host of tissues and cell types confirms the central role of the liver in lipid levels and highlights the selective enrichment of adipose-specific chromatin marks in high-density lipoprotein cholesterol and triglycerides. Overlapping transcription factor (TF) binding sites with lipid-associated loci identifies TFs relevant in lipid biology. In addition, we present an integrative framework to prioritize causal variants at GWAS loci, producing a comprehensive list of candidate causal genes and variants with multiple layers of functional evidence. We highlight two of the prioritized genes, CREBRF and RRBP1, which show convergent evidence across functional datasets supporting their roles in lipid biology.

Germline Mutations in CIDEB and Protection against Liver Disease.

BACKGROUND: Exome sequencing in hundreds of thousands of persons may enable the identification of rare protein-coding genetic variants associated with protection from human diseases like liver cirrhosis, providing a strategy for the discovery of new therapeutic targets. METHODS: We performed a multistage exome sequencing and genetic association analysis to identify genes in which rare protein-coding variants were associated with liver phenotypes. We conducted in vitro experiments to further characterize associations. RESULTS: The multistage analysis involved 542,904 persons with available data on liver aminotransferase levels, 24,944 patients with various types of liver disease, and 490,636 controls without liver disease. We found that rare coding variants in APOB, ABCB4, SLC30A10, and TM6SF2 were associated with increased aminotransferase levels and an increased risk of liver disease. We also found that variants in CIDEB, which encodes a structural protein found in hepatic lipid droplets, had a protective effect. The burden of rare predicted loss-of-function variants plus missense variants in CIDEB (combined carrier frequency, 0.7%) was associated with decreased alanine aminotransferase levels (beta per allele, -1.24 U per liter; 95% confidence interval [CI], -1.66 to -0.83; P = 4.8×10-9) and with 33% lower odds of liver disease of any cause (odds ratio per allele, 0.67; 95% CI, 0.57 to 0.79; P = 9.9×10-7). Rare coding variants in CIDEB were associated with a decreased risk of liver disease across different underlying causes and different degrees of severity, including cirrhosis of any cause (odds ratio per allele, 0.50; 95% CI, 0.36 to 0.70). Among 3599 patients who had undergone bariatric surgery, rare coding variants in CIDEB were associated with a decreased nonalcoholic fatty liver disease activity score (beta per allele in score units, -0.98; 95% CI, -1.54 to -0.41 [scores range from 0 to 8, with higher scores indicating more severe disease]). In human hepatoma cell lines challenged with oleate, CIDEB small interfering RNA knockdown prevented the buildup of large lipid droplets. CONCLUSIONS: Rare germline mutations in CIDEB conferred substantial protection from liver disease. (Funded by Regeneron Pharmaceuticals.).

Whole Exome Sequencing Enhanced Imputation Identifies 85 Metabolite Associations in the Alpine CHRIS Cohort.

Metabolites are intermediates or end products of biochemical processes involved in both health and disease. Here, we take advantage of the well-characterized Cooperative Health Research in South Tyrol (CHRIS) study to perform an exome-wide association study (ExWAS) on absolute concentrations of 175 metabolites in 3294 individuals. To increase power, we imputed the identified variants into an additional 2211 genotyped individuals of CHRIS. In the resulting dataset of 5505 individuals, we identified 85 single-variant genetic associations, of which 39 have not been reported previously. Fifteen associations emerged at ten variants with >5-fold enrichment in CHRIS compared to non-Finnish Europeans reported in the gnomAD database. For example, the CHRIS-enriched ETFDH stop gain variant p.Trp286Ter (rs1235904433-hexanoylcarnitine) and the MCCC2 stop lost variant p.Ter564GlnextTer3 (rs751970792-carnitine) have been found in patients with glutaric acidemia type II and 3-methylcrotonylglycinuria, respectively, but the loci have not been associated with the respective metabolites in a genome-wide association study (GWAS) previously. We further identified three gene-trait associations, where multiple rare variants contribute to the signal. These results not only provide further evidence for previously described associations, but also describe novel genes and mechanisms for diseases and disease-related traits.

A multiancestry genome-wide association study of unexplained chronic ALT elevation as a proxy for nonalcoholic fatty liver disease with histological and radiological validation.

Nonalcoholic fatty liver disease (NAFLD) is a growing cause of chronic liver disease. Using a proxy NAFLD definition of chronic elevation of alanine aminotransferase (cALT) levels without other liver diseases, we performed a multiancestry genome-wide association study (GWAS) in the Million Veteran Program (MVP) including 90,408 cALT cases and 128,187 controls. Seventy-seven loci exceeded genome-wide significance, including 25 without prior NAFLD or alanine aminotransferase associations, with one additional locus identified in European American-only and two in African American-only analyses (P < 5 × 10-8). External replication in histology-defined NAFLD cohorts (7,397 cases and 56,785 controls) or radiologic imaging cohorts (n = 44,289) replicated 17 single-nucleotide polymorphisms (SNPs) (P < 6.5 × 10-4), of which 9 were new (TRIB1, PPARG, MTTP, SERPINA1, FTO, IL1RN, COBLL1, APOH and IFI30). Pleiotropy analysis showed that 61 of 77 multiancestry and all 17 replicated SNPs were jointly associated with metabolic and/or inflammatory traits, revealing a complex model of genetic architecture. Our approach integrating cALT, histology and imaging reveals new insights into genetic liability to NAFLD.

Exome sequencing in bipolar disorder identifies AKAP11 as a risk gene shared with schizophrenia.

We report results from the Bipolar Exome (BipEx) collaboration analysis of whole-exome sequencing of 13,933 patients with bipolar disorder (BD) matched with 14,422 controls. We find an excess of ultra-rare protein-truncating variants (PTVs) in patients with BD among genes under strong evolutionary constraint in both major BD subtypes. We find enrichment of ultra-rare PTVs within genes implicated from a recent schizophrenia exome meta-analysis (SCHEMA; 24,248 cases and 97,322 controls) and among binding targets of CHD8. Genes implicated from genome-wide association studies (GWASs) of BD, however, are not significantly enriched for ultra-rare PTVs. Combining gene-level results with SCHEMA, AKAP11 emerges as a definitive risk gene (odds ratio (OR) = 7.06, P = 2.83 × 10-9). At the protein level, AKAP-11 interacts with GSK3B, the hypothesized target of lithium, a primary treatment for BD. Our results lend support to BD's polygenicity, demonstrating a role for rare coding variation as a significant risk factor in BD etiology.

Genome-wide association studies of metabolites in Finnish men identify disease-relevant loci.

Few studies have explored the impact of rare variants (minor allele frequency < 1%) on highly heritable plasma metabolites identified in metabolomic screens. The Finnish population provides an ideal opportunity for such explorations, given the multiple bottlenecks and expansions that have shaped its history, and the enrichment for many otherwise rare alleles that has resulted. Here, we report genetic associations for 1391 plasma metabolites in 6136 men from the late-settlement region of Finland. We identify 303 novel association signals, more than one third at variants rare or enriched in Finns. Many of these signals identify genes not previously implicated in metabolite genome-wide association studies and suggest mechanisms for diseases and disease-related traits.

Genome-wide analysis provides genetic evidence that ACE2 influences COVID-19 risk and yields risk scores associated with severe disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters human host cells via angiotensin-converting enzyme 2 (ACE2) and causes coronavirus disease 2019 (COVID-19). Here, through a genome-wide association study, we identify a variant (rs190509934, minor allele frequency 0.2-2%) that downregulates ACE2 expression by 37% (P = 2.7 × 10-8) and reduces the risk of SARS-CoV-2 infection by 40% (odds ratio = 0.60, P = 4.5 × 10-13), providing human genetic evidence that ACE2 expression levels influence COVID-19 risk. We also replicate the associations of six previously reported risk variants, of which four were further associated with worse outcomes in individuals infected with the virus (in/near LZTFL1, MHC, DPP9 and IFNAR2). Lastly, we show that common variants define a risk score that is strongly associated with severe disease among cases and modestly improves the prediction of disease severity relative to demographic and clinical factors alone.

Heterozygous variants of CLPB are a cause of severe congenital neutropenia.

Severe congenital neutropenia is an inborn disorder of granulopoiesis. Approximately one third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 severe congenital neutropenia cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase that is implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the adenosine triphosphate-binding pocket. Both genetic loss of CLPB and expression of CLPB variants result in impaired granulocytic differentiation of human hematopoietic progenitor cells and increased apoptosis. These CLPB variants associate with wild-type CLPB and inhibit its adenosine triphosphatase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.

The power of genetic diversity in genome-wide association studies of lipids.

Increased blood lipid levels are heritable risk factors of cardiovascular disease with varied prevalence worldwide owing to different dietary patterns and medication use1. Despite advances in prevention and treatment, in particular through reducing low-density lipoprotein cholesterol levels2, heart disease remains the leading cause of death worldwide3. Genome-wideassociation studies (GWAS) of blood lipid levels have led to important biological and clinical insights, as well as new drug targets, for cardiovascular disease. However, most previous GWAS4-23 have been conducted in European ancestry populations and may have missed genetic variants that contribute to lipid-level variation in other ancestry groups. These include differences in allele frequencies, effect sizes and linkage-disequilibrium patterns24. Here we conduct a multi-ancestry, genome-wide genetic discovery meta-analysis of lipid levels in approximately 1.65 million individuals, including 350,000 of non-European ancestries. We quantify the gain in studying non-European ancestries and provide evidence to support the expansion of recruitment of additional ancestries, even with relatively small sample sizes. We find that increasing diversity rather than studying additional individuals of European ancestry results in substantial improvements in fine-mapping functional variants and portability of polygenic prediction (evaluated in approximately 295,000 individuals from 7 ancestry groupings). Modest gains in the number of discovered loci and ancestry-specific variants were also achieved. As GWAS expand emphasis beyond the identification of genes and fundamental biology towards the use of genetic variants for preventive and precision medicine25, we anticipate that increased diversity of participants will lead to more accurate and equitable26 application of polygenic scores in clinical practice.

Genetic and functional evidence links a missense variant in B4GALT1 to lower LDL and fibrinogen.

Increased blood levels of low-density lipoprotein cholesterol (LDL-C) and fibrinogen are independent risk factors for cardiovascular disease. We identified associations between an Amish-enriched missense variant (p.Asn352Ser) in a functional domain of beta-1,4-galactosyltransferase 1 (B4GALT1) and 13.9 milligrams per deciliter lower LDL-C (P = 4.1 × 10­19) and 29 milligrams per deciliter lower plasma fibrinogen (P = 1.3 × 10­5). B4GALT1 gene­based analysis in 544,955 subjects showed an association with decreased coronary artery disease (odds ratio = 0.64, P = 0.006). The mutant protein had 50% lower galactosyltransferase activity compared with the wild-type protein. N-linked glycan profiling of human serum found serine 352 allele to be associated with decreased galactosylation and sialylation of apolipoprotein B100, fibrinogen, immunoglobulin G, and transferrin. B4galt1 353Ser knock-in mice showed decreases in LDL-C and fibrinogen. Our findings suggest that targeted modulation of protein galactosylation may represent a therapeutic approach to decreasing cardiovascular disease.

Exome sequencing and analysis of 454,787 UK Biobank participants.

A major goal in human genetics is to use natural variation to understand the phenotypic consequences of altering each protein-coding gene in the genome. Here we used exome sequencing1 to explore protein-altering variants and their consequences in 454,787 participants in the UK Biobank study2. We identified 12 million coding variants, including around 1 million loss-of-function and around 1.8 million deleterious missense variants. When these were tested for association with 3,994 health-related traits, we found 564 genes with trait associations at P ≤ 2.18 × 10-11. Rare variant associations were enriched in loci from genome-wide association studies (GWAS), but most (91%) were independent of common variant signals. We discovered several risk-increasing associations with traits related to liver disease, eye disease and cancer, among others, as well as risk-lowering associations for hypertension (SLC9A3R2), diabetes (MAP3K15, FAM234A) and asthma (SLC27A3). Six genes were associated with brain imaging phenotypes, including two involved in neural development (GBE1, PLD1). Of the signals available and powered for replication in an independent cohort, 81% were confirmed; furthermore, association signals were generally consistent across individuals of European, Asian and African ancestry. We illustrate the ability of exome sequencing to identify gene-trait associations, elucidate gene function and pinpoint effector genes that underlie GWAS signals at scale.

Sequencing of 640,000 exomes identifies GPR75 variants associated with protection from obesity.

Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein-coupled receptors (CALCR, MC4R, GIPR, GPR151, and GPR75). Protein-truncating variants in GPR75 were observed in ~4/10,000 sequenced individuals and were associated with 1.8 kilograms per square meter lower BMI and 54% lower odds of obesity in the heterozygous state. Knock out of Gpr75 in mice resulted in resistance to weight gain and improved glycemic control in a high-fat diet model. Inhibition of GPR75 may provide a therapeutic strategy for obesity.