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The V122I variant in transthyretin (TTR) is the most common amyloidogenic mutation worldwide. The aim of this study is to describe the cardiac phenotype and risk for adverse cardiovascular outcomes of young V122I TTR carriers in the general population.TTR genotypes were extracted from whole-exome sequence data in participants of the Dallas Heart Study. Participants with African ancestry, available V122I TTR genotypes (N = 1818) and either cardiac magnetic resonance imaging (n = 1364) or long-term follow-up (n = 1532) were included. The prevalence of V122I TTR carriers (45 ± 10 years) was 3.2% (n/N = 59/1818). The V122I TTR carriers had higher baseline left ventricular wall thickness (8.52 ± 1.82 vs 8.21 ± 1.62 mm, adjusted P = .038) than noncarriers, but no differences in other cardiac magnetic resonance imaging measures (P.05 for all). Although carrier status was not associated with amino terminal pro-B-type natriuretic peptide (NT-proBNP) at baseline (P = .79), V122I TTR carriers had a greater increase in NT-proBNP on follow-up than noncarriers (median 28.5 pg/mL, interquartile range 11.4-104.1 pg/mL vs median 15.9 pg/mL, interquartile range 0.0-43.0 pg/mL, adjusted P = .018). V122I TTR carriers were at a higher adjusted risk of heart failure (hazard ratio 3.82, 95% confidence interval 1.80-8.13, P.001), cardiovascular death (hazard ratio 2.65, 95% confidence interval 1.14-6.15, P = .023), and all-cause mortality (hazard ratio 1.95, 95% confidence interval 1.08-3.51, P = .026) in comparison with noncarriers.V122I TTR carrier status was associated with a greater increase in NT-proBNP, slightly greater left ventricular wall thickness, and a higher risk for heart failure, cardiovascular death, and all-cause mortality. These findings suggest the need to develop amyloidosis screening strategies for V122I TTR carriers.

Background Potassium levels regulate multiple physiologic processes. The heritability of serum potassium level is moderate, with published estimates varying from 17% to 60%, suggesting genetic influences. However, the genetic determinants of potassium levels are not generally known. Methods A whole-exome sequencing association study of serum potassium levels in 5812 subjects of the Old Order Amish was performed. A dietary salt intervention in 533 Amish subjects estimated interaction between p.R642G and sodium intake. Results A cluster of variants, spanning approximately 537 kb on chromosome 16q13, was significantly associated with serum potassium levels. Among the associated variants, a known pathogenic variant of autosomal recessive Gitelman syndrome (p.R642G SLC12A3) was most likely causal; there were no homozygotes in our sample. Heterozygosity for p.R642G was also associated with lower chloride levels, but not with sodium levels. Notably, p.R642G showed a novel association with lower serum BUN levels. Heterozygotes for p.R642G had a two-fold higher rate of self-reported bone fractures and had higher resting heart rates on a low-salt diet compared with noncarriers. Conclusions This study provides evidence that heterozygosity for a pathogenic variant in SLC12A3 causing Gitelman syndrome, a canonically recessive disorder, contributes to serum potassium concentration. The findings provide insights into SLC12A3 biology and the effects of heterozygosity on electrolyte homeostasis and related subclinical phenotypes that may have implications for personalized medicine and nutrition.

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., Exome sequences from the first 49,960 participants in the UK Biobank highlight the promise of genome sequencing in large population-based studies and are now accessible to the scientific community.

Introduction Obesity accounts for a substantial and growing burden of disease globally. Body adiposity is highly heritable, and human genetic studies can lead to biological and therapeutic insights. Rationale Whole-exome sequencing of hundreds of thousands of individuals is complementary to approaches used to date in obesity genetics and has the potential to identify rare protein-coding variants with large phenotypic impact. We sequenced the exomes of 645,626 individuals from the UK, the US, and Mexico and estimated associations of rare coding variants with body mass index (BMI), a measure of overall adiposity used to define obesity in clinical practice. We complemented exome sequencing with fine-mapping of common alleles, polygenic score analysis, and in vitro and in vivo modeling work. Results We identified 16 genes for which the burden of rare nonsynonymous variants was associated with BMI at exome-wide statistical significance (inverse-variance weighted meta-analysis P < 3.6 × 10−7), including associations at five brain-expressed G protein–coupled receptors (CALCR, MC4R, GIPR, GPR151, and GPR75). We observed an overrepresentation of genes highly expressed in the hypothalamus, a key center for the neuroendocrine regulation of energy balance. Protein-truncating variants in GPR75 were found in ~4/10,000 sequenced people and were associated with 1.8 kg/m2 lower BMI, 5.3 kg lower bodyweight, and 54% lower odds of obesity in heterozygous carriers. Knock out of Gpr75 in mice resulted in resistance to weight gain in a high-fat diet model, which was allele-dose dependent (25% and 44% lower weight gain, respectively, for heterozygous Gpr75−/+ mice and knockout Gpr75−/− mice compared with wild type) and accompanied by improved glycemic control and insulin sensitivity. Protein-truncating variants in CALCR were associated with higher BMI and obesity risk, whereas protein-truncating variants in GIPR and two missense alleles [Arg190→Gln (Arg190Gln), Glu288Gly], which we show result in loss of function in vitro, were associated with lower adiposity. Among monogenic obesity genes in the leptin-melanocortin pathway, heterozygous predicted loss-of-function variants in LEP, POMC, PCSK1, and MC4R (but not LEPR) were associated with higher BMI. Rare protein-truncating variants in UBR2, ANO4, and PCSK1 were associated with more than twofold higher odds of obesity in heterozygous carriers, similar to predicted-deleterious nonsynonymous variants in MC4R, which are considered the most common cause of monogenic obesity. Polygenic predisposition due to >2 million common genetic variants influenced the penetrance of obesity in rare variant carriers in an additive fashion. Conclusion These results suggest that inhibition of GPR75 may be a therapeutic strategy for obesity and illustrate the power of massive-scale exome sequencing for the identification of large-effect coding variant associations and drug targets for complex traits.

PurposeBirdshot Chorioretinopathy (BSCR) is strongly associated with HLA-A29. This study was designed to elucidate the genetic modifiers of BSCR in HLA-A29 carriers.MethodsWe sequenced the largest BSCR cohort to date, including 286 cases and 108 HLA-A29 positive controls to perform genome wide common and rare variant associations. We further typed the HLA alleles of cases and 45,386 HLA-A29 controls of European ancestry to identify HLA alleles that associate with BSCR risk.ResultsCarrying a second allele that belongs to the HLA-Aw19 broad antigen family (including HLA-A29, A30, A31, and A33) increases the risk for BSCR (OR=4.44, p=2.2e-03). This result was validated by comparing allele frequencies to large HLA-A29-controlled cohorts (n=45,386, OR>2.5, pConclusionsThe genetic factors increasing BSCR risk demonstrate a pattern of increased processing, as well as increased presentation of ERAP2 specific peptides. This suggests a mechanism in which exceeding a peptide presentation threshold activates the immune response in choroids of A29 carriers.

Alcohol consumption level and alcohol use disorder (AUD) diagnosis are moderately heritable traits. We conduct genome-wide association studies of these traits using longitudinal Alcohol Use Disorder Identification Test-Consumption (AUDIT-C) scores and AUD diagnoses in a multi-ancestry Million Veteran Program sample (N = 274,424). We identify 18 genome-wide significant loci: 5 associated with both traits, 8 associated with AUDIT-C only, and 5 associated with AUD diagnosis only. Polygenic Risk Scores (PRS) for both traits are associated with alcohol-related disorders in two independent samples. Although a significant genetic correlation reflects the overlap between the traits, genetic correlations for 188 non-alcohol-related traits differ significantly for the two traits, as do the phenotypes associated with the traits’ PRS. Cell type group partitioning heritability enrichment analyses also differentiate the two traits. We conclude that, although heavy drinking is a key risk factor for AUD, it is not a sufficient cause of the disorder., The genetic underpinnings of alcohol use disorder and consumption are incompletely understood. Here, the authors perform GWAS for Alcohol Use Disorder (AUD) Identification Test-Consumption scores and AUD diagnosis from electronic health records of 274,424 individuals and identify a total of 18 associated loci.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a respiratory illness that can result in hospitalization or death. We used exome sequence data to investigate associations between rare genetic variants and seven COVID-19 outcomes in 586,157 individuals, including 20,952 with COVID-19. After accounting for multiple testing, we did not identify any clear associations with rare variants either exome wide or when specifically focusing on (1) 13 interferon pathway genes in which rare deleterious variants have been reported in individuals with severe COVID-19, (2) 281 genes located in susceptibility loci identified by the COVID-19 Host Genetics Initiative, or (3) 32 additional genes of immunologic relevance and/or therapeutic potential. Our analyses indicate there are no significant associations with rare protein-coding variants with detectable effect sizes at our current sample sizes. Analyses will be updated as additional data become available, and results are publicly available through the Regeneron Genetics Center COVID-19 Results Browser.

Estimates from genome-wide association studies (GWAS) represent a combination of the effect of inherited genetic variation (direct effects), demography (population stratification, assortative mating) and genetic nurture from relatives (indirect genetic effects). GWAS using family-based designs can control for demography and indirect genetic effects, but large-scale family datasets have been lacking. We combined data on 159,701 siblings from 17 cohorts to generate population (between-family) and within-sibship (within-family) estimates of genome-wide genetic associations for 25 phenotypes. We demonstrate that existing GWAS associations for height, educational attainment, smoking, depressive symptoms, age at first birth and cognitive ability overestimate direct effects. We show that estimates of SNP-heritability, genetic correlations and Mendelian randomization involving these phenotypes substantially differ when calculated using within-sibship estimates. For example, genetic correlations between educational attainment and height largely disappear. In contrast, analyses of most clinical phenotypes (e.g. LDL-cholesterol) were generally consistent between population and within-sibship models. We also report compelling evidence of polygenic adaptation on taller human height using within-sibship data. Large-scale family datasets provide new opportunities to quantify direct effects of genetic variation on human traits and diseases.

More than 800 million people in the world suffer from chronic kidney disease (CKD). Genome-wide association studies (GWAS) have identified hundreds of loci where genetic variants are associated with kidney function; however, causal genes and pathways for CKD remain unknown. Here, we performed integration of kidney function GWAS and human kidney–specific expression quantitative trait analysis and identified that the expression of beta-mannosidase (MANBA) was lower in kidneys of subjects with CKD risk genotype. We also show an increased incidence of renal failure in subjects with rare heterozygous loss-of-function coding variants in MANBA using phenome-wide association analysis of 40,963 subjects with exome sequencing data. MANBA is a lysosomal gene highly expressed in kidney tubule cells. Deep phenotyping revealed structural and functional lysosomal alterations in human kidneys from subjects with CKD risk alleles and mice with genetic deletion of Manba. Manba heterozygous and knockout mice developed more severe kidney fibrosis when subjected to toxic injury induced by cisplatin or folic acid. Manba loss altered multiple pathways, including endocytosis and autophagy. In the absence of Manba, toxic acute tubule injury induced inflammasome activation and fibrosis. Together, these results illustrate the convergence of common noncoding and rare coding variants in MANBA in kidney disease development and demonstrate the role of the endolysosomal system in kidney disease development.

The clinical impact of rare loss-of-function variants has yet to be determined for most genes. Integration of DNA sequencing data with electronic health records (EHRs) could enhance our understanding of the contribution of rare genetic variation to human disease1. By leveraging 10,900 whole-exome sequences linked to EHR data in the Penn Medicine Biobank, we addressed the association of the cumulative effects of rare predicted loss-of-function variants for each individual gene on human disease on an exome-wide scale, as assessed using a set of diverse EHR phenotypes. After discovering 97 genes with exome-by-phenome-wide significant phenotype associations (P

Rapid decline of glomerular filtration rate estimated from creatinine (eGFRcrea) is associated with severe clinical endpoints. In contrast to cross-sectionally assessed eGFRcrea, the genetic basis for rapid eGFRcrea decline is largely unknown. To help define this, we meta-analyzed 42 genome-wide association studies from the Chronic Kidney Diseases Genetics Consortium and United Kingdom Biobank to identify genetic loci for rapid eGFRcrea decline. Two definitions of eGFRcrea decline were used: 3 mL/min/1.73m(2)/year or more ("Rapid3"; encompassing 34,874 cases, 107,090 controls) and eGFRcrea decline 25% or more and eGFRcrea under 60 mL/min/1.73m(2) at follow-up among those with eGFRcrea 60 mL/min/1.73m(2) or more at baseline ("CKDi25"; encompassing 19,901 cases, 175,244 controls). Seven independent variants were identified across six loci for Rapid3 and/or CKDi25: consisting of five variants at four loci with genome-wide significance (near UMOD-PDILT (2), PRKAG2, WDR72, OR2S2) and two variants among 265 known eGFRcrea variants (near GATM, LARP4B). All these loci were novel for Rapid3 and/or CKDi25 and our bioinformatic follow-up prioritized variants and genes underneath these loci. The OR2S2 locus is novel for any eGFRcrea trait including interesting candidates. For the five genome-wide significant lead variants, we found supporting effects for annual change in blood urea nitrogen or cystatin-based eGFR, but not for GATM or (LARP4B). Individuals at high compared to those at low genetic risk (8-14 vs. 0-5 adverse alleles) had a 1.20-fold increased risk of acute kidney injury (95% confidence interval 1.08-1.33). Thus, our identified loci for rapid kidney function decline may help prioritize therapeutic targets and identify mechanisms and individuals at risk for sustained deterioration of kidney function., Zweitveröffentlichungen der Universität Potsdam : Reihe der Digital Engineering Fakultät; 19

Summary Osteoarthritis affects over 300 million people worldwide. Here, we conduct a genome-wide association study meta-analysis across 826,690 individuals (177,517 with osteoarthritis) and identify 100 independently associated risk variants across 11 osteoarthritis phenotypes, 52 of which have not been associated with the disease before. We report thumb and spine osteoarthritis risk variants and identify differences in genetic effects between weight-bearing and non-weight-bearing joints. We identify sex-specific and early age-at-onset osteoarthritis risk loci. We integrate functional genomics data from primary patient tissues (including articular cartilage, subchondral bone, and osteophytic cartilage) and identify high-confidence effector genes. We provide evidence for genetic correlation with phenotypes related to pain, the main disease symptom, and identify likely causal genes linked to neuronal processes. Our results provide insights into key molecular players in disease processes and highlight attractive drug targets to accelerate translation., Graphical abstract, Highlights • A multicohort study identifies 52 previously unknown osteoarthritis genetic risk variants • Similarities and differences in osteoarthritis genetic risk depend on joint sites • Osteoarthritis genetic components are associated with pain-related phenotypes • High-confidence effector genes highlight potential targets for drug intervention, A multicohort genome-wide association meta-analysis of osteoarthritis highlights the impact of joint site types on the features of genetic risk variants and the link between osteoarthritis genetics and pain-related phenotypes, pointing toward potential targets for therapeutic intervention.

The UK Biobank Exome Sequencing Consortium (UKB-ESC) is a unique private/public partnership between the UK Biobank and eight biopharma companies that will sequence the exomes of all ∼500,000 UK Biobank participants. Here we describe early results from the exome sequence data generated by this consortium for the first ∼200,000 UKB subjects and the key features of this project that enabled the UKB-ESC to come together and generate this data.Exome sequencing data from the first 200,643 UKB enrollees are now accessible to the research community. Approximately 10M variants were observed within the targeted regions, including: 8,086,176 SNPs, 370,958 indels and 1,596,984 multi-allelic variants. Of the ∼8M variants observed, 84.5% are coding variants and include 2,139,318 (25.3%) synonymous, 4,549,694 (53.8%) missense, 453,733 (5.4%) predicted loss-of-function (LOF) variants (initiation codon loss, premature stop codons, stop codon loss, splicing and frameshift variants) affecting at least one coding transcript. This open access data provides a rich resource of coding variants for rare variant genetic studies, and is particularly valuable for drug discovery efforts that utilize rare, functionally consequential variants.Over the past decade, the biopharma industry has increasingly leveraged human genetics as part of their drug discovery and development strategies. This shift was motivated by technical advances that enabled cost-effective human genetics research at scale, the emergence of electronic health records and biobanks, and a maturing understanding of how human genetics can increase the probability of successful drug development. Recognizing the need for large-scale human genetics data to drive drug discovery, and the unique value of the open data access policies and contribution terms of the UK Biobank, the UKB-ESC was formed. This precompetitive collaboration has further strengthened the ties between academia and industry and provided teams an unprecedented opportunity to interact with and learn from the wider research community.

SUMMARYAging is characterized by degeneration in cellular and organismal functions leading to increased disease susceptibility and death. Although our understanding of aging biology in model systems has increased dramatically, large-scale sequencing studies to understand human aging are now just beginning. We applied exome sequencing and association analyses (ExWAS) to identify age-related variants on 58,470 participants of the DiscovEHR cohort. Linear Mixed Model regression analyses of age at last encounter revealed variants in genes known to be linked with clonal hematopoiesis of indeterminate potential, which are associated with myelodysplastic syndromes, as top signals in our analysis, suggestive of age-related somatic mutation accumulation in hematopoietic cells despite patients lacking clinical diagnoses. In addition to APOE, we identified rare DISP2 rs183775254 (p = 7.40×10−10) and ZYG11A rs74227999 (p = 2.50×10−08) variants that were negatively associated with age in either both sexes combined and females, respectively, which were replicated with directional consistency in two independent cohorts. Epigenetic mapping showed these variants are located within cell-type-specific enhancers, suggestive of important transcriptional regulatory functions. To discover variants associated with extreme age, we performed exome-sequencing on persons of Ashkenazi Jewish descent ascertained for extensive lifespans. Case-Control analyses in 525 Ashkenazi Jews cases (Males ≥ 92 years, Females ≥ 95years) were compared to 482 controls. Our results showed variants in APOE (rs429358, rs6857), and TMTC2 (rs7976168) passed Bonferroni-adjusted p-value, as well as several nominally-associated population-specific variants. Collectively, our Age-ExWAS, the largest performed to date, confirmed and identified previously unreported candidate variants associated with human age.

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.

BackgroundIntestinal 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.ObjectiveTo genetically diagnose two first-degree cousins once removed, who presented with PLE at ages 22 and 2.5 years.MethodsFamily-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.ResultsWe 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.ConclusionsBiallelic 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.

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, we identified ∼66,000 close (first- and second-degree) relationships within DiscovEHR involving 55.6% of study participants. Our simulation results project that >70% of the cohort will be involved in these close relationships as DiscovEHR scales to 250,000 recruited individuals. We reconstructed 12,574 pedigrees 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 through reconstructed pedigrees, including a tandem duplication in LDLR causing familial hypercholesterolemia. 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.

BackgroundLoss-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. MethodsWe 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. ResultsIn th...

Alleles associated with lower levels of LDL cholesterol (LDL-C) have recently been associated with an increased risk of type 2 diabetes (T2D), highlighting the complex relationship between LDL-C and diabetes. This observation begs the question of whether LDL-C–raising alleles are associated with a decreased risk of T2D. This issue was recently addressed in a large familial hypercholesterolemia (FH) screening study, which reported a lower prevalence of self-reported diabetes in FH subjects than in age-matched relatives without FH. To extend this observation, we tested the association of FH with diabetes status and glycemia in a large Amish population enriched for the FH-associated APOB R3527Q variant that included 640 APOB R3527Q carriers and 4,683 noncarriers. Each copy of the R3527Q T allele was associated with a 74.9 mg/dL increase in LDL-C. There was little difference in T2D prevalence between subjects with (5.2%) and without (4.5%) the R3527Q allele (P = 0.23), and there was no association between R3527Q variant and impaired fasting glucose, fasting glucose or insulin, or oral glucose tolerance test–derived measures. Our data provide no evidence supporting an association between the APOB R3527Q variant and T2D or glycemia and highlight the asymmetry of the LDL-C–T2D relationship and/or the gene/variant-dependent specificity of the LDL-C–T2D association.

SUMMARYThe UK Biobank is a prospective study of 502,543 individuals, combining extensive phenotypic and genotypic data with streamlined access for researchers around the world. Here we describe the first tranche of large-scale exome sequence data for 49,960 study participants, revealing approximately 4 million coding variants (of which ~98.4% have frequency < 1%). The data includes 231,631 predicted loss of function variants, a >10-fold increase compared to imputed sequence for the same participants. Nearly all genes (>97%) had ≥1 predicted loss of function carrier, and most genes (>69%) had ≥10 loss of function carriers. We illustrate the power of characterizing loss of function variation in this large population through association analyses across 1,741 phenotypes. In addition to replicating a range of established associations, we discover novel loss of function 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 significance in this population, finding that 2% of the population has a medically actionable variant. Additionally, we leverage the phenotypic data to characterize the relationship between rare BRCA1 and BRCA2 pathogenic variants and cancer risk. Exomes from the first 49,960 participants are now made accessible to the scientific community and highlight the promise offered by genomic sequencing in large-scale population-based studies.

Ca(2+) 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 Ca(2+) store, and dysregulation of ER Ca(2+) 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 Ca(2+)-binding ER transmembrane protein CCDC47. CCDC47, also known as calumin, has been shown to bind Ca(2+) 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 Ca(2+) storage, impaired Ca(2+) signaling mediated by the IP(3)R Ca(2+) release channel, and reduced ER Ca(2+) refilling via store-operated Ca(2+) entry. These results, together with the previously described role of CCDC47 in Ca(2+) signaling and development, suggest that bi-allelic loss-of-function variants in CCDC47 underlie the pathogenesis of this multisystem disorder.

Background: In pulmonary arterial hypertension (PAH), pathological changes in pulmonary arterioles progressively raise pulmonary artery pressure and increase pulmonary vascular resistance, leading to right heart failure and high mortality rates. Recently, the first potassium channelopathy in PAH, because of mutations in KCNK3 , was identified as a genetic cause and pharmacological target. Methods: Exome sequencing was performed to identify novel genes in a cohort of 99 pediatric and 134 adult-onset group I PAH patients. Novel rare variants in the gene identified were independently identified in a cohort of 680 adult-onset patients. Variants were expressed in COS cells and function assessed by patch-clamp and rubidium flux analysis. Results: We identified a de novo novel heterozygous predicted deleterious missense variant c.G2873A (p.R958H) in ABCC8 in a child with idiopathic PAH. We then evaluated all individuals in the original and a second cohort for rare or novel variants in ABCC8 and identified 11 additional heterozygous predicted damaging ABCC8 variants. ABCC8 encodes SUR1 (sulfonylurea receptor 1)—a regulatory subunit of the ATP-sensitive potassium channel. We observed loss of ATP-sensitive potassium channel function for all ABCC8 variants evaluated and pharmacological rescue of all channel currents in vitro by the SUR1 activator, diazoxide. Conclusions: Novel and rare missense variants in ABCC8 are associated with PAH. Identified ABCC8 mutations decreased ATP-sensitive potassium channel function, which was pharmacologically recovered.

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 found to be associated with reduced levels of ALT (P=4.20×10(−12)) and AST (P=6.2×10(−10)). Among DiscovEHR study participants, this variant was found to be 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 is associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.)

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We performed exome and whole-genome DNA sequencing and RNA sequencing on purified CTCL and matched normal cells. The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NF-κB signaling, chromatin remodeling and DNA damage response. CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs versus 1.0 somatic single-nucleotide variant per CTCL). These findings have implications for new therapeutics.

MotivationDetection of abnormal karyotypes from whole-exome sequencing has significant clinical potential, enabling a primary screen for chromosomal anomalies among samples undergoing short-read sequencing for nucleotide resolution genomic characterization.ResultsWe present KaryoScan, a high-throughput method for detecting chromosomal anomalies within large cohort exome sequencing studies. We detect and validate autosomal and sex chromosomal aneuploidies in a large exome sequencing cohort, and demonstrate detection of smaller and complex events (partial chromosome, mosaic, copy neutral, and complex rearrangements), representing the range of anomalies that can be uncovered from the exome.Availabilityhttps://github.com/rgcgithub/karyoscan

Bone morphogenetic protein 2 (BMP2) in chromosomal region 20p12 belongs to a gene superfamily encoding TGF-β-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.

Copy number variants (CNVs) are a substantial source of genomic variation and contribute to a wide range of human disorders. Gene-disrupting exonic CNVs have important clinical implications as they can underlie variability in disease presentation and susceptibility. The relationship between exonic CNVs and clinical traits has not been broadly explored at the population level, primarily due to technical challenges. We surveyed common and rare CNVs in the exome sequences of 50,726 adult DiscovEHR study participants with linked electronic health records (EHRs). We evaluated the diagnostic yield and clinical expressivity of known pathogenic CNVs, and performed tests of association with EHR-derived serum lipids, thereby evaluating the relationship between CNVs and complex traits and phenotypes in an unbiased, real-world clinical context. We identified CNVs from megabase to exon-level resolution, demonstrating reliable, high-throughput detection of clinically relevant exonic CNVs. In doing so, we created a catalog of high-confidence common and rare CNVs and refined population frequency estimates of known and novel gene-disrupting CNVs. Our survey among an unselected clinical population provides further evidence that neuropathy-associated duplications and deletions in 17p12 have similar population prevalence but are clinically under-diagnosed. Similarly, adults who harbor 22q11.2 deletions frequently had EHR documentation of neurodevelopmental/neuropsychiatric disorders and congenital anomalies, but not a formal genetic diagnosis (i.e., deletion). In an exome-wide association study of lipid levels, we identified a novel five-exon duplication within LDLR segregating in a large kindred with features of familial hypercholesterolemia. Exonic CNVs provide new opportunities to understand and diagnose human disease.

Summary: Whole-exome sequencing (WES) studies have demonstrated the contribution of de novo loss-of-function single-nucleotide variants (SNVs) to autism spectrum disorder (ASD). However, challenges in the reliable detection of de novo insertions and deletions (indels) have limited inclusion of these variants in prior analyses. By applying a robust indel detection method to WES data from 787 ASD families (2,963 individuals), we demonstrate that de novo frameshift indels contribute to ASD risk (OR = 1.6; 95% CI = 1.0–2.7; p = 0.03), are more common in female probands (p = 0.02), are enriched among genes encoding FMRP targets (p = 6 × 10−9), and arise predominantly on the paternal chromosome (p < 0.001). On the basis of mutation rates in probands versus unaffected siblings, we conclude that de novo frameshift indels contribute to risk in approximately 3% of individuals with ASD. Finally, by observing clustering of mutations in unrelated probands, we uncover two ASD-associated genes: KMT2E (MLL5), a chromatin regulator, and RIMS1, a regulator of synaptic vesicle release. : Insertions and deletions (indels) have proven especially difficult to detect in exome sequencing data. Dong et al. now identify indels in exome data for 787 autism spectrum disorder (ASD) families. They demonstrate association between de novo indels that alter the reading frame and ASD. Furthermore, by observing clustering of indels in unrelated probands, they uncover two additional ASD-associated genes: KMT2E (MLL5), a chromatin regulator, and RIMS1, a regulator of synaptic vesicle release.

Upon detection of pathogen-associated molecular patterns, innate immune receptors initiate inflammatory responses. These receptors include cytoplasmic NOD-like receptors (NLRs) whose stimulation recruits and proteolytically activates caspase-1 within the inflammasome, a multiprotein complex. Caspase-1 mediates the production of interleukin-1 family cytokines (IL1FCs), leading to fever and inflammatory cell death (pyroptosis). Mutations that constitutively activate these pathways underlie several autoinflammatory diseases with diverse clinical features. We describe a family with a previously unreported syndrome featuring neonatal-onset enterocolitis, periodic fever, and fatal or near-fatal episodes of autoinflammation. We show that the disease is caused by a de novo gain-of-function mutation in NLRC4 encoding a p.Val341Ala substitution in the HD1 domain of the protein that cosegregates with disease. Mutant NLRC4 causes constitutive IL1FC production and macrophage cell death. Infected macrophages from affected individuals are polarized toward pyroptosis and exhibit abnormal staining for inflammasome components. These findings identify and describe the cause of a life-threatening but treatable autoinflammatory disease that underscores the divergent roles of the NLRC4 inflammasome.

Mutant human Cu/Zn superoxide dismutase 1 (SOD1) is associated with motor neuron toxicity and death in an inherited form of amyotrophic lateral sclerosis (ALS; Lou Gehrig disease). One aspect of toxicity in motor neurons involves diminished fast axonal transport, observed both in transgenic mice and, more recently, in axoplasm isolated from squid giant axons. The latter effect appears to be directly mediated by misfolded SOD1, whose addition activates phosphorylation of p38 MAPK and phosphorylation of kinesin. Here, we observe that several different oligomeric states of a fusion protein, comprising ALS-associated human G85R SOD1 joined with yellow fluorescent protein (G85R SOD1YFP), which produces ALS in transgenic mice, inhibited anterograde transport when added to squid axoplasm. Inhibition was blocked both by an apoptosis signal-regulating kinase 1 (ASK1; MAPKKK) inhibitor and by a p38 inhibitor, indicating the transport defect is mediated through the MAPK cascade. In further incubations, we observed that addition of the mammalian molecular chaperone Hsc70, abundantly associated with G85R SOD1YFP in spinal cord of transgenic mice, exerted partial correction of the transport defect, associated with diminished phosphorylation of p38. Most striking, the addition of the molecular chaperone Hsp110, in a concentration substoichiometric to the mutant SOD1 protein, completely rescued both the transport defect and the phosphorylation of p38. Hsp110 has been demonstrated to act as a nucleotide exchange factor for Hsc70 and, more recently, to be able to cooperate with it to mediate protein disaggregation. We speculate that it can cooperate with endogenous squid Hsp(c)70 to mediate binding and/or disaggregation of mutant SOD1 protein, abrogating toxicity.

The recent discovery that nevus sebaceus is a mosaic RASopathy represents a major breakthrough in research on epidermal nevi. In this issue, both Levinsohn et al. and Sun et al. confirm this advancement with results obtained through whole-exome sequencing. Further molecular studies will almost certainly show that sebaceous and keratinocytic nevi are different disorders, although there is some clinical overlap.

The Dark Side of Langerhans Cells Several immune cell populations reside in the skin and are thought to provide a protective barrier against infections and to act as sentinels against malignant transformation. However, studies in mice that lack Langerhans cells, a subset of dendritic cells, have suggested that these cells may actually promote tumorigenesis. Using a mouse model of squamous cell carcinoma, Modi et al. (p. 104 ) now reveal how Langerhans cells may promote the transformation of skin epithelial cells. In response to the carcinogen 7,12-dimethylbenz[α]anthracene (DMBA), Langerhans cells increased their expression of the cytochrome P-450 enzyme CYP1B1, which can metabolize DMBA to the mutagenic DMBA- trans -3,4-diol. Thus, besides their functions in regulating the adaptive immune response, Langerhans cells may participate in the metabolism of environmental carcinogens.

The melanocortin 4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) and a key molecule in the regulation of energy homeostasis. At least 159 substitutions in the coding region of human MC4R (hMC4R) have been described experimentally; over 80 of those occur naturally, and many have been implicated in obesity. However, assessment of the presumably functionally essential residues remains incomplete. Here we have performed a complete in silico mutagenesis analysis to assess the functional essentiality of all possible nonnative point mutants in the entire hMC4R protein (332 residues). We applied SNAP, which is a method for quantifying functional consequences of single amino acid (AA) substitutions, to calculate the effects of all possible substitutions at each position in the hMC4R AA sequence. We compiled a mutability score that reflects the degree to which a particular residue is likely to be functionally important. We performed the same experiment for a paralogue human melanocortin receptor (hMC1R) and a mouse orthologue (mMC4R) in order to compare computational evaluations of highly related sequences. Three results are most salient: 1) our predictions largely agree with the available experimental annotations; 2) this analysis identified several AAs that are likely to be functionally critical, but have not yet been studied experimentally; and 3) the differential analysis of the receptors implicates a number of residues as specifically important to MC4Rs vs. other GPCRs, such as hMC1R.—Bromberg, Y., Overton, J., Vaisse, C., Leibel, R. L., Rost, B. In silico mutagenesis: a case study of the melanocortin 4 receptor.

Summary Transcript regulation is essential for cell function, and misregulation can lead to disease. Despite technologies to survey the transcriptome, we lack a comprehensive understanding of transcript kinetics, which limits quantitative biology. This is an acute challenge in embryonic development, where rapid changes in gene expression dictate cell fate decisions. By ultra-high-frequency sampling of Xenopus embryos and absolute normalization of sequence reads, we present smooth gene expression trajectories in absolute transcript numbers. During a developmental period approximating the first 8 weeks of human gestation, transcript kinetics vary by eight orders of magnitude. Ordering genes by expression dynamics, we find that "temporal synexpression" predicts common gene function. Remarkably, a single parameter, the characteristic timescale, can classify transcript kinetics globally and distinguish genes regulating development from those involved in cellular metabolism. Overall, our analysis provides unprecedented insight into the reorganization of maternal and embryonic transcripts and redefines our ability to perform quantitative biology.

Adrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood.To utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations present in ACC.Screening for somatic mutation events and copy number alterations (CNAs) was performed by comparative analysis of tumors and matched normal samples from 41 patients with ACC.In total, 966 nonsynonymous somatic mutations were detected, including 40 tumors with a mean of 16 mutations per sample and one tumor with 314 mutations. Somatic mutations in ACC-associated genes included TP53 (8/41 tumors, 19.5%) and CTNNB1 (4/41, 9.8%). Genes with potential disease-causing mutations included GNAS, NF2, and RB1, and recurrently mutated genes with unknown roles in tumorigenesis comprised CDC27, SCN7A, and SDK1. Recurrent CNAs included amplification at 5p15.33 including TERT (6/41, 14.6%) and homozygous deletion at 22q12.1 including the Wnt repressors ZNRF3 and KREMEN1 (4/41 9.8% and 3/41, 7.3%, respectively). Somatic mutations in ACC-established genes and recurrent ZNRF3 and TERT loci CNAs were mutually exclusive in the majority of cases. Moreover, gene ontology identified Wnt signaling as the most frequently mutated pathway in ACCs.These findings highlight the importance of Wnt pathway dysregulation in ACC and corroborate the finding of homozygous deletion of Wnt repressors ZNRF3 and KREMEN1. Overall, mutations in either TP53 or CTNNB1 as well as focal CNAs at the ZNRF3 or TERT loci denote mutually exclusive events, suggesting separate mechanisms underlying the development of these tumors.

Background Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels. Results A panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross. Conclusions This technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-979) contains supplementary material, which is available to authorized users.

Pathologically elevated serum levels of fibroblast growth factor-23 (FGF23), a bone-derived hormone that regulates phosphorus homeostasis, result in renal phosphate wasting and lead to rickets or osteomalacia. Rarely, elevated serum FGF23 levels are found in association with mosaic cutaneous disorders that affect large proportions of the skin and appear in patterns corresponding to the migration of ectodermal progenitors. The cause and source of elevated serum FGF23 is unknown. In those conditions, such as epidermal and large congenital melanocytic nevi, skin lesions are variably associated with other abnormalities in the eye, brain and vasculature. The wide distribution of involved tissues and the appearance of multiple segmental skin and bone lesions suggest that these conditions result from early embryonic somatic mutations. We report five such cases with elevated serum FGF23 and bone lesions, four with large epidermal nevi and one with a giant congenital melanocytic nevus. Exome sequencing of blood and affected skin tissue identified somatic activating mutations of HRAS or NRAS in each case without recurrent secondary mutation, and we further found that the same mutation is present in dysplastic bone. Our finding of somatic activating RAS mutation in bone, the endogenous source of FGF23, provides the first evidence that elevated serum FGF23 levels, hypophosphatemia and osteomalacia are associated with pathologic Ras activation and may provide insight in the heretofore limited understanding of the regulation of FGF23.

Genetic Clues to Meningioma Meningiomas are the most common primary brain tumors in adults. Located within the layer of tissue covering the brain, these tumors are usually slow-growing and benign but can cause serious neurological complications. About half of these tumors have mutations in the neurofibromin 2 gene ( NF2 ). To identify other genes that contribute to meningioma pathogenesis, Clark et al. (p. 1077 , published online 24 January) performed genome sequence analysis on 300 tumors. Meningiomas fell into two general classes: benign tumors located at the skull base—which tend to harbor mutations in the TRAF7, KLF4, AKT1 , and SMO genes—and higher-grade tumors located in the cerebral and cerebellar hemispheres harbor mutations in NF2.

Adrenal aldosterone-producing adenomas (APAs) constitutively produce the salt-retaining hormone aldosterone and are a common cause of severe hypertension. Recurrent mutations in the potassium channel KCNJ5 that result in cell depolarization and Ca2+ influx cause ~40% of these tumors1. We found five somatic mutations (four altering glycine 403, one altering isoleucine 770) in CACNA1D, encoding a voltage-gated calcium channel, among 43 non-KCNJ5-mutant APAs. These mutations lie in S6 segments that line the channel pore. Both result in channel activation at less depolarized potentials, and glycine 403 mutations also impair channel inactivation. These effects are inferred to cause increased Ca2+ influx, the sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa2. Remarkably, we identified de novo mutations at the identical positions in two children with a previously undescribed syndrome featuring primary aldosteronism and neuromuscular abnormalities. These findings implicate gain of function Ca2+ channel mutations in aldosterone-producing adenomas and primary aldosteronism.

Exome sequencing of patients with congenital heart disease (CHD) and their unaffected parents reveals an excess of strong-effect, protein-altering de novo mutations in genes expressed in the developing heart, many of which regulate chromatin modification in key developmental genes; collectively, these mutations are predicted to account for approximately 10% of severe CHD cases. This paper demonstrates that de novo mutations with large effect have a role in the pathogenesis of at least 10% of cases of congenital heart disease (CHD). Using exome sequence analysis in parent–offspring trios Richard Lifton and colleagues compared the frequency of de novo mutations, identified by exome sequencing, in 362 CHD parent–offspring trios and 264 control trios. Gene ontology analysis demonstrated significant enrichment of de novo protein-altering mutation of genes involved in chromatin modification, notably a marked enrichment of genes involved in the production, removal and reading of methylation of histone H3K4 and H3K27. Congenital heart disease (CHD) is the most frequent birth defect, affecting 0.8% of live births1. 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 methylation2,3,4. There are also two de novo mutations in SMAD2, which regulates H3K27 methylation in the embryonic left–right organizer5. The combination of both activating (H3K4 methylation) and inactivating (H3K27 methylation) chromatin marks characterizes ‘poised’ promoters and enhancers, which regulate expression of key developmental genes6. These findings implicate de novo point mutations in several hundreds of genes that collectively contribute to approximately 10% of severe CHD.

Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer. We analyzed the mutational landscape of USC by whole-exome sequencing of 57 cancers, most of which were matched to normal DNA from the same patients. The distribution of the number of protein-altering somatic mutations revealed that 52 USC tumors had fewer than 100 (median 36), whereas 5 had more than 3,000 somatic mutations. The mutations in these latter tumors showed hallmarks of defects in DNA mismatch repair. Among the remainder, we found a significantly increased burden of mutation in 14 genes. In addition to well-known cancer genes (i.e., TP53 , PIK3CA , PPP2R1A , KRAS , FBXW7 ), there were frequent mutations in CHD4/Mi2b , a member of the NuRD–chromatin-remodeling complex, and TAF1 , an element of the core TFIID transcriptional machinery. Additionally, somatic copy-number variation was found to play an important role in USC, with 13 copy-number gains and 12 copy-number losses that occurred more often than expected by chance. In addition to loss of TP53 , we found frequent deletion of a small segment of chromosome 19 containing MBD3 , also a member of the NuRD–chromatin-modification complex, and frequent amplification of chromosome segments containing PIK3CA , ERBB2 (an upstream activator of PIK3CA), and CCNE1 (a target of FBXW7-mediated ubiquitination). These findings identify frequent mutation of DNA damage, chromatin remodeling, cell cycle, and cell proliferation pathways in USC and suggest potential targets for treatment of this lethal variant of endometrial cancer.

Background: Hereditary hemochromatosis (HH), one of the most common single gene disorders that alter the body's ability to regulate iron, causes excess iron deposition in liver, heart, pancreas, joints and pituitary gland, with resultant cirrhosis, cancer, diabetes, heart and joint diseases over time. HH can be effectively treated, but if untreated, the increased iron storage can lead to severe organ damage, and death. Therefore, early identification and treatment is imperative. Most severe cases of HH result from a common mutation in the HFE gene (C282Y), but other HFE mutations have also been identified (H63D and S65C), associated with milder disease. Caucasians of northern European descent are at highest risk, and there is ∼1 million people in the US with these mutations, and 10% of Caucasians are carriers. Some individuals are asymptomatic due to the low penetrance, and variable genotype-phenotype correlations. Aims: To determine the prevalence of known HFE mutations, identify potential novel variants, and compare the clinical & laboratory findings with HH genotypes for genotype-phenotype correlations. Materials and Methods: Through the collaboration of Geisinger Health System with the Regeneron, 31,058 MyCode biobank participants were evaluated for HFE gene mutations by analyzing exome sequence data from peripheral blood. Results: Compared to controls, significant differences were noted in males with C282Y/C282Y, C282Y/H63D, and H63D/H63D genotypes for ferritin, serum iron, iron binding capacity (IBC), and transferrin saturation (TS), while C282Y and H63D carriers did not show a significant difference for ferritin, but did for other parameters when compared to non-carriers. Slight differences were noted in females when compared to males, with TS also being significant in C282Y/S65C, H63D/S65C, and S65C/WT females, but not ferritin in C282Y/C282Y or IBC in H63D/H63D individuals. Cirrhosis, hepatocellular carcinoma (HCC), and oral cancers were more prevalent in the C282Y/C282Y genotype. HCC, esophagus and urinary cancers were higher in those with H63D/H63D, while, non-melanoma skin cancers were more prevalent in H63D/WT, and uterine and urinary cancers were more prevalent in the S65C/WT genotype. The patients diagnosed with ICD-9 code of HH were significantly lower than the prevalence of these genotypes, indicating a potential issue of underdiagnosis, or misdiagnosis. One short coming of our evaluation is that the results have not been confirmed by assessment of the medical charts. Conclusion: This large scale genomic study allowed us to identify carriers of HH-associated genetic variants, and uncover potential deficiencies in the current screening algorithm. This will enable the implementation of processes to promote precision medicine, minimize misdiagnosis/underdiagnosis, and maximize outcomes in the management of HH patients. Citation Format: Mehmet Tahir Aslan, Anita Mathew, Ferit Akova, Raghu Metpally, David J. Carey, Heinric Williams, Marc S. Williams, John Overton, Aris Baras, Adam M. Cook, Ryan D. Colonie, Nefize Sertac Kip. Decoding >30 thousand individuals to analyze the most common genetic disorder: Hereditary hemochromatosis (HH). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5222.

Multiple studies have confirmed the contribution of rare de novo copy number variations (CNVs) to the risk for Autism Spectrum Disorders (ASD).1-3 While de novo single nucleotide variants (SNVs) have been identified in affected individuals,4 their contribution to risk has yet to be clarified. Specifically, the frequency and distribution of these mutations has not been well characterized in matched unaffected controls, data that are vital to the interpretation of de novo coding mutations observed in probands. Here we show, via whole-exome sequencing of 928 individuals, including 200 phenotypically discordant sibling pairs, that highly disruptive (nonsense and splice-site) de novo mutations in brain-expressed genes are associated with ASD and carry large effects (OR=5.65; CI: 1.44-22.2; p=0.01 asymptotic test). Based on mutation rates in unaffected individuals, we demonstrate that multiple independent de novo SNVs in the same gene among unrelated probands reliably identifies risk alleles, providing a clear path forward for gene discovery. Among a total of 279 identified de novo coding mutations, there is a single instance in probands, and none in siblings, in which two independent nonsense variants disrupt the same gene, SCN2A (Sodium Channel, Voltage-Gated, Type II, Alpha Subunit), a result that is highly unlikely by chance (p=0.005).

The ubiquitous overexpression of agouti-signaling protein (ASP), a paracrine-signaling molecule that regulates pigment-type switching in the hair follicle of the mouse, is responsible for the obesity and yellow pelage of the Yellow mouse (A(y)). Mahogany (Attractin, Atrn/mg) and mahoganoid (Mahogunin Ring Finger-1, Mgrn1/md) are mutations epistatic to A(y). These mutations have been described as suppressors of ASP action, blocking its antagonizing effects on the melanocortin 1 and 4 receptors (MC1R and MC4R) in the skin and the brain, respectively, via unknown mechanisms. Here, we describe the molecular bases for the md- and mg-dependent rescue of the A(y) phenotype at the MC4R. We show that overexpression of ASP inhibits the rise in cAMP levels in response to α-melanocyte-stimulating hormone, an MC4R agonist, by blocking ligand binding and by directing MC4R trafficking to the lysosome. Loss-of-function of either attractin or MGRN1 blocks ASP-dependent MC4R degradation and promotes increased trafficking of internalized MC4R to the cell surface, but it does not restore α-melanocyte-stimulating hormone-dependent cAMP signaling. We propose that MGRN1 and attractin are components of an evolutionarily conserved receptor trafficking pathway and that the md and mg mutations rescue the A(y) phenotypes by a primarily cAMP-independent mechanism promoting trafficking of MC4R and likely MC1R away from the lysosome toward the cell surface.

In 404 Lepob/ob F2 progeny of a C57BL/6J (B6) x DBA/2J (DBA) intercross, we mapped a DBA-related quantitative trait locus (QTL) to distal Chr1 at 169.6 Mb, centered about D1Mit110, for diabetes-related phenotypes that included blood glucose, HbA1c, and pancreatic islet histology. The interval was refined to 1.8 Mb in a series of B6.DBA congenic/subcongenic lines also segregating for Lepob. The phenotypes of B6.DBA congenic mice include reduced β-cell replication rates accompanied by reduced β-cell mass, reduced insulin/glucose ratio in blood, reduced glucose tolerance, and persistent mild hypoinsulinemic hyperglycemia. Nucleotide sequence and expression analysis of 14 genes in this interval identified a predicted gene that we have designated “Lisch-like” (Ll) as the most likely candidate. The gene spans 62.7 kb on Chr1qH2.3, encoding a 10-exon, 646–amino acid polypeptide, homologous to Lsr on Chr7qB1 and to Ildr1 on Chr16qB3. The largest isoform of Ll is predicted to be a transmembrane molecule with an immunoglobulin-like extracellular domain and a serine/threonine-rich intracellular domain that contains a 14-3-3 binding domain. Morpholino knockdown of the zebrafish paralog of Ll resulted in a generalized delay in endodermal development in the gut region and dispersion of insulin-positive cells. Mice segregating for an ENU-induced null allele of Ll have phenotypes comparable to the B.D congenic lines. The human ortholog, C1orf32, is in the middle of a 30-Mb region of Chr1q23-25 that has been repeatedly associated with type 2 diabetes., Author Summary Type 2 diabetes (T2D) accounts for over 90% of instances of diabetes and is a leading cause of medical morbidity and mortality. Twin studies indicate a strong polygenic contribution to susceptibility within the context of obesity. Although approximately ten genes making important contributions to individual risk have been identified, it is clear that others remain to be identified. In this study, we intercrossed obese, diabetes-resistant and diabetes-prone mouse strains to implicate a genetic interval on mouse Chr1 associated with reduced β-cell numbers and elevated blood glucose. We narrowed the region using molecular genetics and computational approaches to identify a novel gene we designated “Lisch-like” (Ll). The orthologous human genetic interval has been repeatedly implicated in T2D. Mice with an induced mutation that reduces Ll expression are impaired in both β-cell development and glucose metabolism, and reduced expression of the homologous gene in zebrafish disrupts islet development. Ll is expressed in organs implicated in the pathophysiology of T2D (hypothalamus, islets, liver, and skeletal muscle) and is predicted to encode a transmembrane protein that could mediate cholesterol transport and/or convey signals related to cell division. Either mechanism could mediate effects on β-cell mass that would predispose to T2D.

WOS: 000365765400011, PubMed ID: 26539891, Development of the human nervous system involves complex interactions among fundamental cellular processes and requires a multitude of genes, many of which remain to be associated with human disease. We applied whole exome sequencing to 128 mostly consanguineous families with neurogenetic disorders that often included brain malformations. Rare variant analyses for both single nucleotide variant (SNV) and copy number variant (CNV) alleles allowed for identification of 45 novel variants in 43 known disease genes, 41 candidate genes, and CNVs in 10 families, with an overall potential molecular cause identified in >85% of families studied. Among the candidate genes identified, we found PRUNE, VARS, and DHX37 in multiple families and homozygous loss-of-function variants in AGBL2, SLC18A2, SMARCA1, UBQLN1, and CPLX1. Neuroimaging and in silico analysis of functional and expression proximity between candidate and known disease genes allowed for further understanding of genetic networks underlying specific types of brain malformations., U.S. National Human Genome Research Institute (NHGRI) NHLBI grant [U54HG006542]; NINDSUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Neurological Disorders & Stroke (NINDS) [RO1 NS058529, K23NS078056]; NHGRIUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Human Genome Research Institute (NHGRI) [5U54HG003273]; Medical Genetics Research Fellowship Program [T32 GM07526]; Regeneron, We thank all the family members and collaborators who participated in this study. This work was supported by U.S. National Human Genome Research Institute (NHGRI) NHLBI grant U54HG006542 to the Baylor-Hopkins Center for Mendelian Genomics, NINDS grant RO1 NS058529 to J. R. L., and NHGRI 5U54HG003273 to R. A. G. T. H. is supported by the Medical Genetics Research Fellowship Program (T32 GM07526). W. W. is supported by Career Development Award K23NS078056 from NINDS. The authors would like to thank the ExAC and the groups that provided exome variant data for comparison. A full list of contributing groups can be found at http://exac.broadinstitute.org/about. J.R.L. has stock ownership in 23andMe and Lasergen and is a paid consultant for Regeneron. J. R. L. is also a coinventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine derives revenue from the chromosomal microarray analysis and clinical exome sequencing offered in the Medical Genetics Laboratory (https://www.bcm.edu/geneticlabs/). W. K. C. is a paid consultant for Regeneron and BioReference Laboratories. C.G.-J. and J. D. O. are employees of the RGC.

TO THE EDITOR Woolly hair nevus (WHN) is a mosaic disorder characterized by distinct patterns of tightly curled scalp hair which can appear concurrently with epidermal nevi (EN) at other sites (Peteiro et al., 1989; Venugopal et al., 2012). Woolly hair is also found in congenital disorders resulting from mutations affecting diverse cellular components including intermediate filament, adherens junction, and signal transduction proteins (Harel and Christiano, 2012). Embryonic somatic mutation causes mosaic disorders which appear in patterns of ectodermal progenitor dorsovental migration. Somatic mutations causing mosaic disorders including Proteus syndrome (Lindhurst et al., 2011), port-wine stains (Shirley et al., 2013), and EN (Levinsohn et al., 2013; Sun et al., 2013) have been found using exome sequencing. Recognizing that exome sequencing would permit identification of mutations causing WHN, we ascertained two cases. Our first (WHN100, Figure 1a-d) was a 10 year-old girl without history of developmental delay who had regions of slightly curly hair over her occipital scalp from infancy which progressively curled with no scalp surface change and lie alongside areas of straight hair. She has hyperpigmented patches on her neck, trunk, and arms, with more keratotic lesions on her distal extremities, and acanthosis nigricans in both axillae. There was linear palmar keratoderma (PPK) and hyperkeratosis over most metacarpophalangeal and some proximal interphalangeal joints. Given concurrent PPK and woolly hair, clinical concern for Naxos or Carvajal syndromes led to regular cardiology evaluations that found no abnormalities. Figure 1 Clinical features of index cases with woolly hair nevi. On the scalp, woolly hair nevus presents with a portion of the scalp exhibiting patches of curly, thin, hair intermixed with regions of normal, straight hair, as observed in WHN100 and WHN101. On ... Our second case (WHN101, Figure 1e-h) was a 6 year-old girl whose hair developed at age one and consisted of a mixture of poker-straight hair and curly, thin hair. In infancy, she developed linear dyspigmentation on the right arm and trunk, which became more raised and scaly on the distal extremities over time. She had normal development, with no cardiac or ophthalmic abnormalities found on routine physical examination, cardiac MRI and serial electrocardiograms. Clinical suspicion of mosaic Naxos or Caravajal syndrome motivated clinical sequencing of DSP, DSC1, DSG1, JUP, PKP2, and TMEM43; no mutations were found. To determine the genetic basis of WHN, we performed paired whole exome sequencing of DNA isolated from affected tissue and blood in both cases (Supplementary Figure 2). Data was analyzed to identify somatic single nucleotide variants (SNVs), deletions and insertions (Supplementary Methods). A somatic heterozygous HRAS c.34G>A, p.G12S substitution was found in each (Figure 2a). There was no evidence of loss of heterozygosity (LOH) (Supplementary Figure 3) or secondary mutation somatic mutation, suggesting that HRAS mutation alone is sufficient to cause WHN. Sanger sequencing confirmed mutation presence in affected tissue (Figure 2b, c). To determine if this mutation causes woolly hair, we prepared DNA from hair bulbs of straight and curly hair obtained from affected individual WHN101, finding the HRAS p.G12S mutation in curly hair only (Figure 2d, Supplementary Figure 1). Figure 2 Somatic HRAS p.G12S mutation causes WHN. (a) In WHN100 and WHN101, exome sequencing of affected tissue and blood was performed. Tissue-specific SNVs are annotated bychromosome, position, base change, protein consequence, and numbers of reference and non-reference ... Consistent with somatic mosaicism in an epidermal progenitor, prior cases of WHN have been reported with concurrent keratinocytic epidermal nevi (KEN). KEN result from somatic mutations in HRAS, KRAS, PIK3CA, FGFR3, and NRAS (Hafner et al., 2012) including the HRAS p.G12S mutation found in WHN (Hafner et al., 2011). Furthermore, Costello syndrome (CS), in which patients present with developmental delay, high birth weight, feeding difficulties, failure to thrive, cardiac anomalies, and curly hair, results from germline heterozygous HRAS mutations, including p.G12S (Gripp and Lin, 2012; Siegel et al., 2012). The timing of somatic mutation during embryonic development determines extent of cutaneous involvement and presence of other systemic abnormalities (Moss et al., 1993). Notably, somatic activating HRAS mutations are found in most cases of nevus sebaceus (NS), a mosaic lesion which typically appears on the scalp and features alopecia, papillomatosis, and marked sebaceus hyperplasia (Groesser et al., 2012; Levinsohn et al., 2013; Sun et al., 2013). These features contrast with those of WHN in which hair is present but curly, and sebaceous hyperplasia is absent. Given that WHN and NS are both caused by somatic HRAS mutations, we hypothesize that their phenotypic divergence may derive from relative potency of the mutant allele with respect to MAP kinase activation. HRAS mutations in WHN and NS fall within the finger loop of HRAS, replacing glycine residues with larger amino acids which prevent GTP hydrolysis (Malumbres and Barbacid, 2003). Though comparison of the WHN p.G12S mutation and the common NS p.G13R mutation has not been performed, HRAS codon 12 serine substitutions have been shown to be less activating than arginine, aspartic acid or valine substitutions (Fasano et al., 1984). To evaluate the frequency of HRAS mutation in NS, we screened 116 archival scalp NS lesions for HRAS and KRAS mutation. We found 88 HRAS and 9 KRAS mutations. HRAS p.G13R was present in 85 NS and p.G12S was not found (Supplementary Table 2). In prior reports, 64 additional samples were screened, and HRAS p.G12S mutations were not found (Levinsohn et al., 2013; Sun et al., 2013). In one report, 3 specimens with HRAS p.G12S mutations were identified; in 2 there was a concurrent HRAS p.G13R mutation, and in one, the lesion was on the ear, a site at which it could be difficult to distinguish EN and NS (Groesser et al., 2012). These data combined with evidence from CS suggest that more strongly activating RAS mutations may cause the alopecia and sebaceous hyperplasia found in NS, and the more mildly activating p.G12S mutation causes woolly hair phenotypes. In summary, we find somatic HRAS c.34G>A, p.G12S mutation in affected tissue from two cases with mosaic woolly hair and EN. Consistent with reports of WHN and in KEN, the identified p.G12S mutation causes an EN phenotype on the body, but the finding of curly hair on the scalp suggests that WHN represents a mosaic RASopathy with phenotype determined by location, either due to distinct epidermal progenitor types or site-specific mesenchymal interactions. We hypothesize that in contrast to strongly activating RAS mutations found in NS which drive hair follicle progenitors toward sebocyte differentiation, the more weakly activating mutation found in WHN permits an intermediate phenotype with abnormal curly hair growth but without sebaceous hyperplasia.