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

Rare coding variants that substantially affect function provide insights into the biology of a gene1-3. However, ascertaining the frequency of such variants 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. In total, 23% of the Regeneron Genetics Center Million Exome (RGC-ME) data come from individuals of African, East Asian, Indigenous American, Middle Eastern and South Asian ancestry. The catalogue includes more than 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 (LOF), we identify 3,988 LOF-intolerant genes, including 86 that were previously assessed as tolerant and 1,153 that lack established disease annotation. We also define regions of missense depletion at high resolution. Notably, 1,482 genes have regions that are depleted of missense variants despite being tolerant of 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 resource of coding variation from the RGC-ME dataset publicly accessible through a variant allele frequency browser.

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.

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.

Exome sequencing and characterization of 49,960 individuals in the UK Biobank.

The UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world1. Here we describe the release of exome-sequence data for the first 49,960 study participants, revealing approximately 4 million coding variants (of which around 98.6% have a frequency of less than 1%). The data include 198,269 autosomal predicted loss-of-function (LOF) variants, a more than 14-fold increase compared to the imputed sequence. Nearly all genes (more than 97%) had at least one carrier with a LOF variant, and most genes (more than 69%) had at least ten carriers with a LOF variant. We illustrate the power of characterizing LOF variants in this population through association analyses across 1,730 phenotypes. In addition to replicating established associations, we found novel LOF variants with large effects on disease traits, including PIEZO1 on varicose veins, COL6A1 on corneal resistance, MEPE on bone density, and IQGAP2 and GMPR on blood cell traits. We further demonstrate the value of exome sequencing by surveying the prevalence of pathogenic variants of clinical importance, and show that 2% of this population has a medically actionable variant. Furthermore, we characterize the penetrance of cancer in carriers of pathogenic BRCA1 and BRCA2 variants. Exome sequences from the first 49,960 participants highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.

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.).

Genome-wide association analysis of serum alanine and aspartate aminotransferase, and the modifying effects of BMI in 388k European individuals.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are biomarkers for liver health. Here we report the largest genome-wide association analysis to date of serum ALT and AST levels in over 388k people of European ancestry from UK biobank and DiscovEHR. Eleven million imputed markers with a minor allele frequency (MAF) ≥ 0.5% were analyzed. Overall, 300 ALT and 336 AST independent genome-wide significant associations were identified. Among them, 81 ALT and 61 AST associations are reported for the first time. Genome-wide interaction study identified 9 ALT and 12 AST independent associations significantly modified by body mass index (BMI), including several previously reported potential liver disease therapeutic targets, for example, PNPLA3, HSD17B13, and MARC1. While further work is necessary to understand the effect of ALT and AST-associated variants on liver disease, the weighted burden of significant BMI-modified signals is significantly associated with liver disease outcomes. In summary, this study identifies genetic associations which offer an important step forward in understanding the genetic architecture of serum ALT and AST levels. Significant interactions between BMI and genetic loci not only highlight the important role of adiposity in liver damage but also shed light on the genetic etiology of liver disease in obese individuals.

Patients With High Genome-Wide Polygenic Risk Scores for Coronary Artery Disease May Receive Greater Clinical Benefit From Alirocumab Treatment in the ODYSSEY OUTCOMES Trial.

BACKGROUND: Alirocumab, an antibody that blocks PCSK9 (proprotein convertase subtilisin/kexin type 9), was associated with reduced major adverse cardiovascular events (MACE) and death in the ODYSSEY OUTCOMES trial (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab). In this study, higher baseline levels of low-density lipoprotein cholesterol (LDL-C) predicted greater benefit from alirocumab treatment. Recent studies indicate high polygenic risk scores (PRS) for coronary artery disease (CAD) identify individuals at higher risk who derive increased benefit from statins. We performed post hoc analyses to determine whether high PRS for CAD identifies higher-risk individuals, independent of baseline LDL-C and other known risk factors, who might derive greater benefit from alirocumab treatment. METHODS: ODYSSEY OUTCOMES was a randomized, double-blind, placebo-controlled trial comparing alirocumab or placebo in 18 924 patients with acute coronary syndrome and elevated atherogenic lipoproteins despite optimized statin treatment. The primary endpoint (MACE) comprised death of CAD, nonfatal myocardial infarction, ischemic stroke, or unstable angina requiring hospitalization. A genome-wide PRS for CAD comprising 6 579 025 genetic variants was evaluated in 11 953 patients with available DNA samples. Analysis of MACE risk was performed in placebo-treated patients, whereas treatment benefit analysis was performed in all patients. RESULTS: The incidence of MACE in the placebo group was related to PRS for CAD: 17.0% for high PRS patients (>90th percentile) and 11.4% for lower PRS patients (≤90th percentile; P<0.001); this PRS relationship was not explained by baseline LDL-C or other established risk factors. Both the absolute and relative reduction of MACE by alirocumab compared with placebo was greater in high versus low PRS patients. There was an absolute reduction by alirocumab in high versus low PRS groups of 6.0% and 1.5%, respectively, and a relative risk reduction by alirocumab of 37% in the high PRS group (hazard ratio, 0.63 [95% CI, 0.46-0.86]; P=0.004) versus a 13% reduction in the low PRS group (hazard ratio, 0.87 [95% CI, 0.78-0.98]; P=0.022; interaction P=0.04). CONCLUSIONS: A high PRS for CAD is associated with elevated risk for recurrent MACE after acute coronary syndrome and a larger absolute and relative risk reduction with alirocumab treatment, providing an independent tool for risk stratification and precision medicine.

Homozygosity for a mutation affecting the catalytic domain of tyrosyl-tRNA synthetase (YARS) causes multisystem disease.

Aminoacyl-tRNA synthetases (ARSs) are critical for protein translation. Pathogenic variants of ARSs have been previously associated with peripheral neuropathy and multisystem disease in heterozygotes and homozygotes, respectively. We report seven related children homozygous for a novel mutation in tyrosyl-tRNA synthetase (YARS, c.499C > A, p.Pro167Thr) identified by whole exome sequencing. This variant lies within a highly conserved interface required for protein homodimerization, an essential step in YARS catalytic function. Affected children expressed a more severe phenotype than previously reported, including poor growth, developmental delay, brain dysmyelination, sensorineural hearing loss, nystagmus, progressive cholestatic liver disease, pancreatic insufficiency, hypoglycemia, anemia, intermittent proteinuria, recurrent bloodstream infections and chronic pulmonary disease. Related adults heterozygous for YARS p.Pro167Thr showed no evidence of peripheral neuropathy on electromyography, in contrast to previous reports for other YARS variants. Analysis of YARS p.Pro167Thr in yeast complementation assays revealed a loss-of-function, hypomorphic allele that significantly impaired growth. Recombinant YARS p.Pro167Thr demonstrated normal subcellular localization, but greatly diminished ability to homodimerize in human embryonic kidney cells. This work adds to a rapidly growing body of research emphasizing the importance of ARSs in multisystem disease and significantly expands the allelic and clinical heterogeneity of YARS-associated human disease. A deeper understanding of the role of YARS in human disease may inspire innovative therapies and improve care of affected patients.

Bi-allelic CCDC47 Variants Cause a Disorder Characterized by Woolly Hair, Liver Dysfunction, Dysmorphic Features, and Global Developmental Delay.

Ca2+ signaling is vital for various cellular processes including synaptic vesicle exocytosis, muscle contraction, regulation of secretion, gene transcription, and cellular proliferation. The endoplasmic reticulum (ER) is the largest intracellular Ca2+ store, and dysregulation of ER Ca2+ signaling and homeostasis contributes to the pathogenesis of various complex disorders and Mendelian disease traits. We describe four unrelated individuals with a complex multisystem disorder characterized by woolly hair, liver dysfunction, pruritus, dysmorphic features, hypotonia, and global developmental delay. Through whole-exome sequencing and family-based genomics, we identified bi-allelic variants in CCDC47 that encodes the Ca2+-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca2+ with low affinity and high capacity. In mice, loss of Ccdc47 leads to embryonic lethality, suggesting that Ccdc47 is essential for early development. Characterization of cells from individuals with predicted likely damaging alleles showed decreased CCDC47 mRNA expression and protein levels. In vitro cellular experiments showed decreased total ER Ca2+ storage, impaired Ca2+ signaling mediated by the IP3R Ca2+ release channel, and reduced ER Ca2+ refilling via store-operated Ca2+ entry. These results, together with the previously described role of CCDC47 in Ca2+ signaling and development, suggest that bi-allelic loss-of-function variants in CCDC47 underlie the pathogenesis of this multisystem disorder.

Profiling and Leveraging Relatedness in a Precision Medicine Cohort of 92,455 Exomes.

Large-scale human genetics studies are ascertaining increasing proportions of populations as they continue growing in both number and scale. As a result, the amount of cryptic relatedness within these study cohorts is growing rapidly and has significant implications on downstream analyses. We demonstrate this growth empirically among the first 92,455 exomes from the DiscovEHR cohort and, via a custom simulation framework we developed called SimProgeny, show that these measures are in line with expectations given the underlying population and ascertainment approach. For example, within DiscovEHR we identified ∼66,000 close (first- and second-degree) relationships, involving 55.6% of study participants. Our simulation results project that >70% of the cohort will be involved in these close relationships, given that DiscovEHR scales to 250,000 recruited individuals. We reconstructed 12,574 pedigrees by using these relationships (including 2,192 nuclear families) and leveraged them for multiple applications. The pedigrees substantially improved the phasing accuracy of 20,947 rare, deleterious compound heterozygous mutations. Reconstructed nuclear families were critical for identifying 3,415 de novo mutations in ∼1,783 genes. Finally, we demonstrate the segregation of known and suspected disease-causing mutations, including a tandem duplication that occurs in LDLR and causes familial hypercholesterolemia, through reconstructed pedigrees. In summary, this work highlights the prevalence of cryptic relatedness expected among large healthcare population-genomic studies and demonstrates several analyses that are uniquely enabled by large amounts of cryptic relatedness.

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease.

BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10-12) and AST (P=6.2×10-10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).

Large-scale exome datasets reveal a new class of adaptor-related protein complex 2 sigma subunit (AP2σ) mutations, located at the interface with the AP2 alpha subunit, that impair calcium-sensing receptor signalling.

Mutations of the sigma subunit of the heterotetrameric adaptor-related protein complex 2 (AP2σ) impair signalling of the calcium-sensing receptor (CaSR), and cause familial hypocalciuric hypercalcaemia type 3 (FHH3). To date, FHH3-associated AP2σ mutations have only been identified at one residue, Arg15. We hypothesized that additional rare AP2σ variants may also be associated with altered CaSR function and hypercalcaemia, and sought for these by analysing >111 995 exomes (>60 706 from ExAc and dbSNP, and 51 289 from the Geisinger Health System-Regeneron DiscovEHR dataset, which also contains clinical data). This identified 11 individuals to have 9 non-synonymous AP2σ variants (Arg3His, Arg15His (x3), Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) with 3 of the 4 individuals who had Arg15His and Met117Ile AP2σ variants having mild hypercalcaemia, thereby indicating a prevalence of FHH3-associated AP2σ mutations of ∼7.8 per 100 000 individuals. Structural modelling of the novel eight AP2σ variants (Arg3His, Ala44Thr, Phe52Tyr, Arg61His, Thr112Met, Met117Ile, Glu122Gly and Glu142Lys) predicted that the Arg3His, Thr112Met, Glu122Gly and Glu142Lys AP2σ variants would disrupt polar contacts within the AP2σ subunit or affect the interface between the AP2σ and AP2α subunits. Functional analyses of all eight AP2σ variants in CaSR-expressing cells demonstrated that the Thr112Met, Met117Ile and Glu142Lys variants, located in the AP2σ α4-α5 helical region that forms an interface with AP2α, impaired CaSR-mediated intracellular calcium (Cai2+) signalling, consistent with a loss of function, and this was rectified by treatment with the CaSR positive allosteric modulator cinacalcet. Thus, our studies demonstrate another potential class of FHH3-causing AP2σ mutations located at the AP2σ-AP2α interface.

Monoallelic BMP2 Variants Predicted to Result in Haploinsufficiency Cause Craniofacial, Skeletal, and Cardiac Features Overlapping Those of 20p12 Deletions.

Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-ß-signaling proteins involved in bone and cartilage biology. Monoallelic deletions of 20p12 are variably associated with cleft palate, short stature, and developmental delay. Here, we report a cranioskeletal phenotype due to monoallelic truncating and frameshift BMP2 variants and deletions in 12 individuals from eight unrelated families that share features of short stature, a recognizable craniofacial gestalt, skeletal anomalies, and congenital heart disease. De novo occurrence and autosomal-dominant inheritance of variants, including paternal mosaicism in two affected sisters who inherited a BMP2 splice-altering variant, were observed across all reported families. Additionally, we observed similarity to the human phenotype of short stature and skeletal anomalies in a heterozygous Bmp2-knockout mouse model, suggesting that haploinsufficiency of BMP2 could be the primary phenotypic determinant in individuals with predicted truncating variants and deletions encompassing BMP2. These findings demonstrate the important role of BMP2 in human craniofacial, skeletal, and cardiac development and confirm that individuals heterozygous for BMP2 truncating sequence variants or deletions display a consistent distinct phenotype characterized by short stature and skeletal and cardiac anomalies without neurological deficits.

Genetic and Pharmacologic Inactivation of ANGPTL3 and Cardiovascular Disease.

BACKGROUND: Loss-of-function variants in the angiopoietin-like 3 gene (ANGPTL3) have been associated with decreased plasma levels of triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol. It is not known whether such variants or therapeutic antagonism of ANGPTL3 are associated with a reduced risk of atherosclerotic cardiovascular disease. METHODS: We sequenced the exons of ANGPTL3 in 58,335 participants in the DiscovEHR human genetics study. We performed tests of association for loss-of-function variants in ANGPTL3 with lipid levels and with coronary artery disease in 13,102 case patients and 40,430 controls from the DiscovEHR study, with follow-up studies involving 23,317 case patients and 107,166 controls from four population studies. We also tested the effects of a human monoclonal antibody, evinacumab, against Angptl3 in dyslipidemic mice and against ANGPTL3 in healthy human volunteers with elevated levels of triglycerides or LDL cholesterol. RESULTS: In the DiscovEHR study, participants with heterozygous loss-of-function variants in ANGPTL3 had significantly lower serum levels of triglycerides, HDL cholesterol, and LDL cholesterol than participants without these variants. Loss-of-function variants were found in 0.33% of case patients with coronary artery disease and in 0.45% of controls (adjusted odds ratio, 0.59; 95% confidence interval, 0.41 to 0.85; P=0.004). These results were confirmed in the follow-up studies. In dyslipidemic mice, inhibition of Angptl3 with evinacumab resulted in a greater decrease in atherosclerotic lesion area and necrotic content than a control antibody. In humans, evinacumab caused a dose-dependent placebo-adjusted reduction in fasting triglyceride levels of up to 76% and LDL cholesterol levels of up to 23%. CONCLUSIONS: Genetic and therapeutic antagonism of ANGPTL3 in humans and of Angptl3 in mice was associated with decreased levels of all three major lipid fractions and decreased odds of atherosclerotic cardiovascular disease. (Funded by Regeneron Pharmaceuticals and others; ClinicalTrials.gov number, NCT01749878 .).

A Unique Presentation of Infantile-Onset Colitis and Eosinophilic Disease without Recurrent Infections Resulting from a Novel Homozygous CARMIL2 Variant.

PURPOSE: This study aimed to characterize the clinical phenotype, genetic basis, and consequent immunological phenotype of a boy with severe infantile-onset colitis and eosinophilic gastrointestinal disease, and no evidence of recurrent or severe infections. METHODS: Trio whole-exome sequencing (WES) was utilized for pathogenic variant discovery. Western blot (WB) and immunohistochemical (IHC) staining were used for protein expression analyses. Immunological workup included in vitro T cell studies, flow cytometry, and CyTOF analysis. RESULTS: WES revealed a homozygous variant in the capping protein regulator and myosin 1 linker 2 (CARMIL2) gene: c.1590C>A; p.Asn530Lys which co-segregated with the disease in the nuclear family. WB and IHC analyses demonstrated reduced protein levels in patient's cells compared with controls. Moreover, comprehensive immunological workup revealed severely diminished blood-borne regulatory T cell (Treg) frequency and impaired in vitro CD4+ T cell proliferation and Treg generation. CyTOF analysis showed significant shifts in the patient's innate and adaptive immune cells compared with healthy controls and ulcerative colitis patients. CONCLUSIONS: Pathogenic variants in CARMIL2 have been implicated in an immunodeficiency syndrome characterized by recurrent infections, occasionally with concurrent chronic diarrhea. We show that CARMIL2-immunodeficiency is associated with significant alterations in the landscape of immune populations in a patient with prominent gastrointestinal disease. This case provides evidence that CARMIL2 should be a candidate gene when diagnosing children with very early onset inflammatory and eosinophilic gastrointestinal disorders, even when signs of immunodeficiency are not observed.

Inactivating Variants in ANGPTL4 and Risk of Coronary Artery Disease.

BACKGROUND: Higher-than-normal levels of circulating triglycerides are a risk factor for ischemic cardiovascular disease. Activation of lipoprotein lipase, an enzyme that is inhibited by angiopoietin-like 4 (ANGPTL4), has been shown to reduce levels of circulating triglycerides. METHODS: We sequenced the exons of ANGPTL4 in samples obtain from 42,930 participants of predominantly European ancestry in the DiscovEHR human genetics study. We performed tests of association between lipid levels and the missense E40K variant (which has been associated with reduced plasma triglyceride levels) and other inactivating mutations. We then tested for associations between coronary artery disease and the E40K variant and other inactivating mutations in 10,552 participants with coronary artery disease and 29,223 controls. We also tested the effect of a human monoclonal antibody against ANGPTL4 on lipid levels in mice and monkeys. RESULTS: We identified 1661 heterozygotes and 17 homozygotes for the E40K variant and 75 participants who had 13 other monoallelic inactivating mutations in ANGPTL4. The levels of triglycerides were 13% lower and the levels of high-density lipoprotein (HDL) cholesterol were 7% higher among carriers of the E40K variant than among noncarriers. Carriers of the E40K variant were also significantly less likely than noncarriers to have coronary artery disease (odds ratio, 0.81; 95% confidence interval, 0.70 to 0.92; P=0.002). K40 homozygotes had markedly lower levels of triglycerides and higher levels of HDL cholesterol than did heterozygotes. Carriers of other inactivating mutations also had lower triglyceride levels and higher HDL cholesterol levels and were less likely to have coronary artery disease than were noncarriers. Monoclonal antibody inhibition of Angptl4 in mice and monkeys reduced triglyceride levels. CONCLUSIONS: Carriers of E40K and other inactivating mutations in ANGPTL4 had lower levels of triglycerides and a lower risk of coronary artery disease than did noncarriers. The inhibition of Angptl4 in mice and monkeys also resulted in corresponding reductions in these values. (Funded by Regeneron Pharmaceuticals.).

A novel TUFM homozygous variant in a child with mitochondrial cardiomyopathy expands the phenotype of combined oxidative phosphorylation deficiency 4.

Translation of mitochondrial-specific DNA is required for proper mitochondrial function and energy production. For this purpose, an elaborate network of dedicated molecular machinery including initiation, elongation and termination factors exists. We describe a patient with an unusual phenotype and a novel homozygous missense variant in TUFM (c.344A>C; p.His115Pro), encoding mtDNA translation elongating factor Tu (EFTu). To date, only four patients have been reported with bi-allelic mutations in TUFM, leading to combined oxidative phosphorylation deficiency 4 (COXPD4) characterized by severe early-onset lactic acidosis and progressive fatal infantile encephalopathy. The patient presented here expands the phenotypic features of TUFM-related disease, exhibiting lactic acidosis and dilated cardiomyopathy without progressive encephalopathy. This warrants the inclusion of TUFM in differential diagnosis of metabolic cardiomyopathy. Cases that further refine genotype-phenotype associations and characterize the molecular basis of mitochondrial disorders allow clinicians to predict disease prognosis, greatly impacting patient care, as well as provide families with reproductive planning options.

De novo mutations in histone-modifying genes in congenital heart disease.

Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births. Many cases occur sporadically and impair reproductive fitness, suggesting a role for de novo mutations. Here we compare the incidence of de novo mutations in 362 severe CHD cases and 264 controls by analysing exome sequencing of parent-offspring trios. CHD cases show a significant excess of protein-altering de novo mutations in genes expressed in the developing heart, with an odds ratio of 7.5 for damaging (premature termination, frameshift, splice site) mutations. Similar odds ratios are seen across the main classes of severe CHD. We find a marked excess of de novo mutations in genes involved in the production, removal or reading of histone 3 lysine 4 (H3K4) methylation, or ubiquitination of H2BK120, which is required for H3K4 methylation. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left-right organizer. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes 'poised' promoters and enhancers, which regulate expression of key developmental genes. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Establishing the role of PLVAP in protein-losing enteropathy: a homozygous missense variant leads to an attenuated phenotype.

BACKGROUND: Intestinal integrity is essential for proper nutrient absorption and tissue homeostasis, with damage leading to enteric protein loss, that is, protein-losing enteropathy (PLE). Recently, homozygous nonsense variants in the plasmalemma vesicle-associated protein gene (PLVAP) were reported in two patients with severe congenital PLE. PLVAP is the building block of endothelial cell (EC) fenestral diaphragms; its importance in barrier function is supported by mouse models of Plvap deficiency. OBJECTIVE: To genetically diagnose two first-degree cousins once removed, who presented with PLE at ages 22 and 2.5 years. METHODS: Family-based whole exome sequencing was performed based on an autosomal recessive inheritance model. In silico analyses were used to predict variant impact on protein structure and function. RESULTS: We identified a rare homozygous variant (NM_031310.2:c.101T>C;p.Leu34Pro) in PLVAP, which co-segregated with the disease. Leu34 is predicted to be located in a highly conserved, hydrophobic, α-helical region within the protein's transmembrane domain, suggesting Leu34Pro is likely to disrupt protein function and/or structure. Electron microscopy and PLVAP immunohistochemistry demonstrated apparently normal diaphragm morphology, predicted to be functionally affected. CONCLUSIONS: Biallelic missense variants in PLVAP can cause an attenuated form of the PLE and hypertriglyceridaemia syndrome. Our findings support the role of PLVAP in the pathophysiology of PLE, expand the phenotypic and mutation spectrums and underscore PLVAP's importance in EC barrier function in the gut.