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Rationale: Transposition of the great arteries (TGA) is one of the most severe types of congenital heart diseases. Understanding the clinical characteristics and pathogenesis of TGA is, therefore, urgently needed for patient management of this severe disease. However, the clinical characteristics and genetic cause underlying TGA remain largely unexplored. Objective: We sought to systematically examine the clinical characteristics and genetic cause for isolated nonsyndromic TGA. Methods and Results: We recruited 249 patients with TGA (66 family trios) and performed whole-exome sequencing. The incidence of patent ductus arteriosus in dextro-TGA (52.7%) and dextrocardia/mesocardia in congenitally corrected TGA (32.8%) were significantly higher than that in other subtypes. A high prevalence of bicuspid pulmonic valve (9.6%) was observed in patients with TGA. Similar results were observed in a replication group of TGA (n=132). Through a series of bioinformatics filtering steps, we obtained 82 candidate genes harboring potentially damaging de novo, loss of function, compound heterozygous, or X-linked recessive variants. Established congenital heart disease–causing genes, such as FOXH1 , were found among the list of candidate genes. A total of 19 ciliary genes harboring rare potentially damaging variants were also found; for example, DYNC2LI1 with a de novo putatively damaging variant. The enrichment of ciliary genes supports the roles of cilia in the pathogenesis of TGA. In total, 33% of the TGA probands had >1 candidate gene hit by putatively deleterious variants, suggesting that a portion of the TGA cases were probably affected by oligogenic or polygenic inheritance. Conclusions: The findings of clinical characteristic analyses have important implications for TGA patient stratification. The results of genetic analyses highlight the pathogenic role of ciliary genes and a complex genetic architecture underlying TGA.

BACKGROUND: Long QT syndrome (LQTS) is a hereditary disease that predisposes patients to life-threatening cardiac arrhythmias and sudden cardiac death. Our previous study of the human ether-à-go-go related gene (hERG)–encoded K(+) channel (K(v)11.1) supports an association between hERG and RING finger protein 207 (RNF207) variants in aggravating the onset and severity of LQTS, specifically T613M hERG (hERG(T613M)) and RNF207 frameshift (RNF207(G603fs)) mutations. However, the underlying mechanistic underpinning remains unknown. OBJECTIVE: The purpose of the present study was to test the role of RNF207 in the function of hERG-encoded K(+) channel subunits. METHODS: Whole-cell patch-clamp experiments were performed in human embryonic kidney (HEK 293) cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) together with immunofluorescent confocal and high resolution microscopy, auto-ubiquitinylation assays, and co-immunoprecipitation experiments to test the functional interactions between hERG and RNF207. RESULTS: Here, we demonstrated that RNF207 serves as an E3 ubiquitin ligase and targets misfolded hERG(T613M) proteins for degradation. RNF207(G603fs) exhibits decreased activity and hinders the normal degradation pathway; this increases the levels of hERG(T613M) subunits and their dominant-negative effect on the wild-type subunits, ultimately resulting in decreased current density. Similar findings are shown for hERG(A614V), a known dominant-negative mutant subunit. Finally, the presence of RNF207(G603fs) with hERG(T613M) results in significantly prolonged action potential durations and reduced hERG current in human-induced pluripotent stem cell–derived cardiomyocytes. CONCLUSION: Our study establishes RNF207 as an interacting protein serving as a ubiquitin ligase for hERG-encoded K(+) channel subunits. Normal function of RNF207 is critical for the quality control of hERG subunits and consequently cardiac repolarization. Moreover, our study provides evidence for protein quality control as a new paradigm in life-threatening cardiac arrhythmias in patients with LQTS.

The migratory cardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, developmental chronology, and transcriptomic states of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC‐associated congenital anomalies. Here, we perform single‐cell transcriptomic sequencing of 34,131 CNCC‐derived cells in mouse hearts covering eight developmental stages between E10.5 and P7. We report the presence of CNCC‐derived mural cells that comprise pericytes and microvascular smooth muscle cells (mVSMCs). Furthermore, we identify the transition from the CNCC‐derived pericytes to mVSMCs and the key regulators over the transition. In addition, our data support that many CNCC derivatives had already committed or differentiated to a specific lineage when migrating into the heart. We explore the spatial distribution of some critical CNCC‐derived subpopulations with single‐molecule fluorescence in situ hybridization. Finally, we computationally reconstruct the differentiation path and regulatory dynamics of CNCC derivatives. Our study provides novel insights into the cell lineages, developmental chronology, and regulatory dynamics of CNCC derivatives during development., A single‐cell transcriptomic atlas of mouse cardiac neural crest cell (CNCC) derivatives reveals that many had already committed or differentiated to a specific lineage when migrating into the heart.

Copy-number variations (CNVs) represent a significant proportion of the genetic differences between individuals and many CNVs associate causally with syndromic disease and clinical outcomes. Here, we characterize the landscape of copy-number variation and their phenome-wide effects in a sample of 472,228 array-genotyped individuals from the UK Biobank. In addition to population-level selection effects against genic loci conferring high mortality, we describe genetic burden from potentially pathogenic and previously uncharacterized CNV loci across more than 3,000 quantitative and dichotomous traits, with separate analyses for common and rare classes of variation. Specifically, we highlight the effects of CNVs at two well-known syndromic loci 16p11.2 and 22q11.2, previously uncharacterized variation at 9p23, and several genic associations in the context of acute coronary artery disease and high body mass index. Our data constitute a deeply contextualized portrait of population-wide burden of copy-number variation, as well as a series of dosage-mediated genic associations across the medical phenome.

Loss of function variants in NOTCH1 cause left ventricular outflow tract obstructive defects (LVOTO). However, the risk conferred by rare and non-coding variants in NOTCH1 for LVOTO remains largely uncharacterized. In a cohort of 49 families affected by hypoplastic left heart syndrome, a severe form of LVOTO, we discovered predicted loss of function NOTCH1 variants in 6% of individuals. Rare or low-frequency missense variants were found in 16% of families. To make a quantitative estimate of the genetic risk posed by variants in NOTCH1 for LVOTO, we studied associations of 400 coding and non-coding variants in NOTCH1 in 1,085 cases and 332,788 controls from the UK Biobank. Two rare intronic variants in strong linkage disequilibrium displayed significant association with risk for LVOTO amongst European-ancestry individuals. This result was replicated in an independent analysis of 210 cases and 68,762 controls of non-European and mixed ancestry. In conclusion, carrying rare predicted loss of function variants in NOTCH1 confer significant risk for LVOTO. In addition, the two intronic variants seem to be associated with an increased risk for these defects. Our approach demonstrates the utility of population-based datasets in quantifying the specific risk of individual variants for disease related phenotypes.

Genetic factors undoubtedly affect the development of congenital heart disease (CHD) but still remain ill defined. We sought to identify genetic risk factors associated with CHD and to accomplish a functional analysis of SNP-carrying genes. We performed a genome-wide association study (GWAS) of 4034 White patients with CHD and 8486 healthy controls. One SNP on chromosome 5q22.2 reached genome-wide significance across all CHD phenotypes and was also indicative for septal defects. One region on chromosome 20p12.1 pointing to the MACROD2 locus identified 4 highly significant SNPs in patients with transposition of the great arteries (TGA). Three highly significant risk variants on chromosome 17q21.32 within the GOSR2 locus were detected in patients with anomalies of thoracic arteries and veins (ATAV). Genetic variants associated with ATAV are suggested to influence the expression of WNT3, and the variant rs870142 related to septal defects is proposed to influence the expression of MSX1. We analyzed the expression of all 4 genes during cardiac differentiation of human and murine induced pluripotent stem cells in vitro and by single-cell RNA-Seq analyses of developing murine and human hearts. Our data show that MACROD2, GOSR2, WNT3, and MSX1 play an essential functional role in heart development at the embryonic and newborn stages.

The clinical evaluation of a genetic syndrome relies upon recognition of a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. However, individuals displaying a single phenotype of a complex syndrome may not meet criteria for clinical diagnosis or genetic testing. Here, we present a phenome-wide association study (PheWAS) approach to systematically explore the phenotypic expressivity of common and rare alleles in genes associated with four well-described syndromic diseases (Alagille (AS), Marfan (MS), DiGeorge (DS), and Noonan (NS) syndromes) in the general population. Using human phenotype ontology (HPO) terms, we systematically mapped 60 phenotypes related to AS, MS, DS and NS in 337,198 unrelated white British from the UK Biobank (UKBB) based on their hospital admission records, self-administrated questionnaires, and physiological measurements. We performed logistic regression adjusting for age, sex, and the first 5 genetic principal components, for each phenotype and each variant in the target genes (JAG1, NOTCH2 FBN1, PTPN1 and RAS-opathy genes, and genes in the 22q11.2 locus) and performed a gene burden test. Overall, we observed multiple phenotype-genotype correlations, such as the association between variation in JAG1, FBN1, PTPN11 and SOS2 with diastolic and systolic blood pressure; and pleiotropy among multiple variants in syndromic genes. For example, rs11066309 in PTPN11 was significantly associated with a lower body mass index, an increased risk of hypothyroidism and a smaller size for gestational age, all in concordance with NS-related phenotypes. Similarly, rs589668 in FBN1 was associated with an increase in body height and blood pressure, and a reduced body fat percentage as observed in Marfan syndrome. Our findings suggest that the spectrum of associations of common and rare variants in genes involved in syndromic diseases can be extended to individual phenotypes within the general population., Author summary Standard medical evaluation of genetic syndromes relies upon recognizing a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. This may lead to missing diagnoses in patients with silent or a low expressed form of the syndrome. Here we take advantage of a rich electronic health record, various phenotypic measurements, and genetic information in 337,198 unrelated white British from the UKBB, to study the relation between single syndromic disease phenotypes and genes related to syndromic disease. We show multiple phenotype-genotype associations in concordance with phenotypes variations found in syndromic diseases. For example, we show that a commonly found variant in FBN1 was associated with high standing/sitting height ratio and reduced body fat percentage as observed in individuals with Marfan syndrome. Our findings suggest that common and rare alleles in syndromic disease genes are causative of individual component phenotypes present in a general population; further research is needed to characterize the pleiotropic effect of alleles in syndromic genes in persons without the syndromic disease.

The clinical evaluation of a genetic syndrome relies upon recognition of a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. However, individuals displaying a few phenotypes of a complex syndrome may not meet criteria for clinical diagnosis or genetic testing. Here, we present a phenome-wide association study (PheWAS) approach to systematically explore pleiotropy of common and rare alleles in genes associated with four well-described syndromic diseases (Alagille (AS), Marfan (MS), DiGeorge (DS), and Noonan (NS) syndromes) in the general population.Using human phenotype ontology (HPO) terms, we systematically mapped 60 phenotypes related to AS, MS, DS and NS in 337,198 unrelated white British from the UK Biobank (UKBB) based on their hospital admission records, self-administrated questionnaires, and physiological measurements. We performed logistic regression adjusting for age, sex, and the first 5 genetic principal components, for each phenotype and each variant in the target genes (JAG1, TBX1, FBN1, PTPN11, NOTCH2, and MAP2K1) and performed a gene burden testing.Overall, we observed multiple phenotype-genotype correlations, such as the association between variation in JAG1, FBN1, PTPN11 and SOS2 with diastolic and systolic blood pressure; and pleiotropy among multiple variants in syndromic genes. For example, rs11066309 in PTPN11 was significantly associated with a lower body mass index, an increased risk of hypothyroidism and a smaller size for gestational age, all in concordance with NS-related phenotypes. Similarly, rs589668 in FBN1 was associated with an increase in body height and blood pressure, and a reduced body fat percentage as observed in Marfan syndrome.Our findings suggest that the spectrum of associations of common and rare variants in genes involved in syndromic diseases can be extended to individual phenotypes within the general population.Author SummaryStandard medical evaluation of genetic syndromes relies upon recognizing a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. This may lead to missing diagnoses in patients with silent or a low expressed form of the syndrome. Here we take advantage of a rich electronic health record, various phenotypic measurements, and genetic information in 337,198 unrelated white British from the UKBB, to study the relation between single syndromic disease phenotypes and genes related to syndromic disease. We show multiple phenotype-genotypes associations in concordance with phenotypes variations found in syndromic diseases. For example, we show that mutation in FBN1 was associated with high standing/sitting height ratio and reduced body fat percentage as observed in individuals with Marfan syndrome. Our findings suggest that common and rare alleles in SD genes are causative of individual component phenotypes present in a general population; further research is needed to characterize the pleiotropic effect of alleles in syndromic genes in persons without the syndromic disease.

National Institutes of Health (NIH), Monarch Initiative [OD #5R24OD011883]; Forums for Integrative Phenomics [U13 CA221044-01]; NCATS Data Translator [1OT3TR002019]; NCATS National Center for Digital Health Informatics Innovation [U24 TR002306];NIH Data Commons [1 OT3 OD02464-01 UNCCH]; Cost Action CA 16118 Neuro-MIG; British Heart Foundation Programme Grant [RG/13/5/30112]; Division of Intramural Research; NIAID; NIH; E-RARE project Hipbi-RD [01GM1608]; European Union's Horizon 2020 Research and Innovation Programme [779257]. Funding for open access charge: NIH; Donald A. Roux Family Fund (to P.N.R.). The Human Phenotype Ontology (HPO) - a standardized vocabulary of phenotypic abnormalities associated with 7000+ diseases - is used by thousands of researchers, clinicians, informaticians and electronic health record systems around the world. Its detailed descriptions of clinical abnormalities and computable disease definitions have made HPO the de facto standard for deep phenotyping in the field of rare disease. The HPO's interoperability with other ontologies has enabled it to be used to improve diagnostic accuracy by incorporating model organism data. It also plays a key role in the popular Exomiser tool, which identifies potential disease-causing variants from whole-exome or whole-genome sequencing data. Since the HPO was first introduced in 2008, its users have become both more numerous and more diverse. To meet these emerging needs, the project has added new content, language translations, mappings and computational tooling, as well as integrations with external community data. The HPO continues to collaborate with clinical adopters to improve specific areas of the ontology and extend standardized disease descriptions. The newly redesigned HPO website (www.human-phenotype-ontology.org) simplifies browsing terms and exploring clinical features, diseases, and human genes.

Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

Loss of function variants in NOTCH1 cause left ventricular outflow tract obstructive defects (LVOTO) in a small percentage of families. Clinical surveys report an increased prevalence of missense variants in NOTCH1 in family members of individuals with LVOTO and other types of congenital heart disease (CHD). However, the risk conferred by rare variants in NOTCH1 for LVOTO remains largely uncharacterized. In a cohort of 49 families affected by hypoplastic left heart syndrome, a severe form of LVOTO, we discovered predicted loss of function NOTCH1 variants in 6% of individuals. Rare missense variants were found in an additional 16% of families. To make a quantitative estimate of the genetic risk posed by variants in NOTCH1 for LVOTO, we studied associations of 400 coding and non-coding variants in NOTCH1 in 271 adult cases and 333,571 controls from the UK Biobank. Two rare intronic variants in strong linkage disequilibrium displayed significant association with risk for LVOTO (g.chr9:139427582C>T, Odds Ratio 16.9, p=3.12e-6; g.chr9:139435649C>T, Odds Ratio 19.6, p = 2.44e-6) amongst European-ancestry British individuals. This result was replicated in an independent analysis of 51 cases and 68,901 controls of non-European and mixed ancestry. We conclude that carrying rare predicted loss of function variants or either of two intronic variants in NOTCH1 confer significant risk for LVOTO. Our approach demonstrates the utility of population-based datasets in quantifying the specific risk of individual variants for disease related phenotypes.Author summaryCongenital heart defects are the most common class of birth defect and are present in 1% of live births. Although CHD cases are often clustered in families, and thus the causal variant(s) are seemingly inherited, finding genetic variants causing these defects has been challenging. With the knowledge that variation in the NOTCH1 gene previously has been associated with CHDs affecting the left side of the heart, our aim was to further investigate the role of different types of NOTCH1 variants in left sided CHDs in two cohorts – a cohort of Finnish families with severe lesions affecting the left side of the heart, and the UK Biobank population including individuals with less severe left-sided lesions such as bicuspid aortic valve, congenital aortic stenosis, and coarctation of the aorta. We found a causal loss-of-function NOTCH1 variant in 6% of the families in the Finnish cohort and in the UK Biobank cohort, we identified two rare variants in the non-coding region of NOTCH1, associated with severe left-sided CHDs. These findings support screening of NOTCH1 loss-of-function variants in patients with severe left sided congenital heart defects and suggests that non-coding region variants in NOTCH1 play a role in CHDs.

The study of orphan diseases, defined by the US Food and Drug Administration as any condition that affects

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk., Author Summary Congenital heart disease (CHD) is a leading cause of childhood morbidity in the developed world. There are few prevalent clinical risk factors and though it is possible that up to 90% of risk for CHD may be genetic, the number of genes clinically associated with disease is small. Rather than grouping disparate CHD phenotypes as other studies have done, we studied a single specific malformation- the atrioventricular septal defect (AVSD). Instead of recurrent variation in a handful of genes, we observed de novo and inherited variation in 19 genes associated with human disease, syndromic loci, and genes implicated in cardiac development by mouse knockout. The number of loci identified support the longstanding hypothesis of a complex oligogenic inheritance for a single malformation and suggest that analyses of CHD data to include inherited variation may uncover additional loci contributing risk for cardiac malformations.

Atrioventricular septal defects (AVSDs) are a frequent but not universal component of Down syndrome (DS), while AVSDs in otherwise normal individuals have no well-defined genetic basis. The contribution of copy number variation (CNV) to specific congenital heart disease (CHD) phenotypes including AVSD is unknown. We hypothesized that de novo CNVs on chromosome 21 might cause isolated sporadic AVSDs, and separately that CNVs throughout the genome might constitute an additional genetic risk factor for AVSD in patients with DS. We utilized a custom oligonucleotide arrays targeted to CNV hotspots that are flanked by large duplicated segments of high sequence identity. We assayed 29 euploid and 50 DS individuals with AVSD, and compared to general population controls. In patients with isolated-sporadic AVSD we identified two large unique deletions outside of chromosome 21 not seen in the expanded set of 8,635 controls, each overlapping with larger deletions associated with similar CHD reported in the DECIPHER database. There was a small duplication in one patient with DS and AVSD. We conclude that isolated sporadic AVSDs may be occasionally associated with large de novo genomic structural variation outside of chromosome 21. The absence of CNVs on chromosome 21 in patients with isolated sporadic AVSD suggests that sub-chromosomal duplications or deletions of greater than 150 kbp on chromosome 21 do not cause sporadic AVSDs. Large CNVs do not appear to be an additive risk factor for AVSD in the DS population. © 2012 Wiley Periodicals, Inc.

Objective Murine genetic models suggest that function of the 12/15-LOX enzyme promotes atherosclerosis. We tested the hypothesis that exonic and/or promoter single nucleotide polymorphisms (SNPs) in the human 12/15-LOX gene ( ALOX15 ) alter the risk of symptomatic coronary artery disease (CAD). Methods and results We resequenced ALOX15 and then genotyped a common promoter and a less common novel coding SNP (T560M) in 1809 subjects with CAD and 1734 controls from Kaiser Permanente including a subset of participants of the Coronary Artery Risk Development in Young Adults study. We found no association between the promoter SNP and the risk of CAD. However, heterozygote carriers of the 560M allele had an increased risk of CAD (adjusted OR, 1.62; P =0.02) compared to non-carriers. In vitro studies demonstrated a 20-fold reduction in the catalytic activity of 560M when compared to 560T. We then genotyped T560M in 12,974 participants of the Atherosclerosis Risk in Communities study and similarly found that heterozygote carriers had an increased risk of CAD compared to non-carriers (adjusted HR, 1.31; P =0.06). In both population studies, homozygote carriers were rare and associated with a non-significant decreased risk of CAD compared to non-carriers (adjusted OR, 0.55; P =0.63 and HR, 0.93; P =0.9). Conclusions A coding SNP in ALOX15 (T560M) results in a near null variant of human 12/15-LOX. Assuming a co-dominant mode of inheritance, this variant does not protect against CAD. Assuming a recessive mode of inheritance, the effect of this mutation remains unclear, but is unlikely to provide a protective effect to the degree suggested by mouse knockout studies.

Recent human genetic studies suggest that allelic variants of leukotriene pathway genes influence the risk of clinical and subclinical atherosclerosis. We sequenced the promoter, exonic, and splice site regions of ALOX5 and ALOX5AP and then genotyped 7 SNPs in ALOX5 and 6 SNPs in ALOX5AP in 1,552 cases with clinically significant coronary artery disease (CAD) and 1,583 controls from Kaiser Permanente including a subset of participants of the coronary artery risk development in young adults study. A nominally significant association was detected between a promoter SNP in ALOX5 (rs12762303) and CAD in our subset of white/European subjects (adjusted odds ratio per minor allele, log-additive model, 1.32; P = 0.002). In this race/ethnic group, rs12762303 has a minor allele frequency of 15% and is tightly linked to variation at the SP1 variable tandem repeat promoter polymorphism. However, the association between CAD and rs12762303 could not be reproduced in the atherosclerosis risk in communities study (hazard rate ratio per minor allele; 1.08, P = 0.1). Assuming a recessive mode of inheritance, the association was not significant in either population study but our power to detect modest effects was limited. No significant associations were observed between all other SNPs and the risk of CAD. Overall, our findings do not support a link between common allelic variation in or near ALOX5 or ALOX5AP and the risk of CAD. However, additional studies are needed to exclude modest effects of promoter variation in ALOX5 on the risk of CAD assuming a recessive mode of inheritance.

As whole exome sequencing (WES) and whole genome sequencing (WGS) transition from research tools to clinical diagnostic tests, it is increasingly critical for sequencing methods and analysis pipelines to be technically accurate. The Genome in a Bottle Consortium has recently published a set of benchmark SNV, indel, and homozygous reference genotypes for the pilot whole genome NIST Reference Material based on the NA12878 genome. We examine the relationship between human genome complexity and genes/variants reported to be associated with human disease. Specifically, we map regions of medical relevance to benchmark regions of high or low confidence. We use benchmark data to assess the sensitivity and positive predictive value of two representative sequencing pipelines for specific classes of variation. We observe that the accuracy of a variant call depends on the genomic region, variant type, and read depth, and varies by analytical pipeline. We find that most false negative WGS calls result from filtering while most false negative WES variants relate to poor coverage. We find that only 74.6 % of the exonic bases in ClinVar and OMIM genes and 82.1 % of the exonic bases in ACMG-reportable genes are found in high-confidence regions. Only 990 genes in the genome are found entirely within high-confidence regions while 593 of 3,300 ClinVar/OMIM genes have less than 50 % of their total exonic base pairs in high-confidence regions. We find greater than 77 % of the pathogenic or likely pathogenic SNVs currently in ClinVar fall within high-confidence regions. We identify sites that are prone to sequencing errors, including thousands present in publicly available variant databases. Finally, we examine the clinical impact of mandatory reporting of secondary findings, highlighting a false positive variant found in BRCA2. Together, these data illustrate the importance of appropriate use and continued improvement of technical benchmarks to ensure accurate and judicious interpretation of next-generation DNA sequencing results in the clinical setting.

International audience; The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain

Whole genome comparisons of distantly related species effectively predict biologically important sequences--core genes and cis-acting regulatory elements (REs)--but require experimentation to verify biological activity. To examine the efficacy of comparative genomics in identification of active REs from anonymous, non-coding (NC) sequences, we generated a novel alignment of the human and draft zebrafish genomes, and contrasted this set to existing human and fugu datasets. We tested the transcriptional regulatory potential of candidate sequences using two in vivo assays. Strict selection of non-genic elements which are deeply conserved in vertebrate evolution identifies 1744 core vertebrate REs in human and two fish genomes. We tested 16 elements in vivo for cis-acting gene regulatory properties using zebrafish transient transgenesis and found that 10 (63%) strongly modulate tissue-specific expression of a green fluorescent protein reporter vector. We also report a novel quantitative enhancer assay with potential for increased throughput based on normalized luciferase activity in vivo. This complementary system identified 11 (69%; including 9 of 10 GFP-confirmed elements) with cis-acting function. Together, these data support the utility of comparative genomics of distantly related vertebrate species to identify REs and provide a scaleable, in vivo quantitative assay to define functional activity of candidate REs.

Introduction: Perinatal LQTS represents a severe form of long-QT syndrome with poor outcomes and early genotype-specific treatment is limited by the 2 month turnaround time of standard panel genetic testing. Hypothesis: We aimed to provide a molecular diagnosis within a clinically actionable timeframe. Methods: We performed rapid CLIA-certified whole genome sequencing on two infants with perinatal long-QT syndrome delivering a molecular diagnosis at 10-days of life. Whole cell patch clamping and single cell genotyping were also performed. Results: In Case #1 we discovered a previously characterized variant in KCNH2 which was paternally inherited, however whole genome sequencing provided an unbiased assessment of the entire catalog of human genes revealing a second maternally inherited modifier variant in RNF207. In Case #2 we discovered a novel mutation leucine replacing valine (V1762L) at residue 1762 in the SNC5A sodium channel. Whole-cell patch clamping experiments show the V1762L mutation causes a profound defect in late sodium current ~4.5 fold greater than the wild-type channel. A single cell analysis demonstrated that the mutation was present in the genome of only 3 of 36 individually isolated and genotyped patient cells, suggesting mosaicism. Conspicuously, standard panel genetic testing was negative. Conclusions: We report here the earliest molecular diagnoses of LQTS, and demonstrate that rapid whole genome sequencing may be fruitfully applied to perinatal LQTS. In case #1 we hypothesize that a polygenic inheritance may explain the early and severe perinatal presentation, and have identified a putative modifier gene for LQTS in RNF207. The observation of mosaicism in case #2 suggests that in studies of inherited disease, mosaicism represents a common mechanism by which causal variation may be missed.

Background The advent of clinical next generation sequencing is rapidly changing the landscape of rare disease medicine. Molecular diagnosis of long QT syndrome (LQTS) can affect clinical management, including risk stratification and selection of pharmacotherapy on the basis of the type of ion channel affected, but results from the current gene panel testing requires 4–16 weeks before return to clinicians. Objective A term female infant presented with 2:1 atrioventricular block and ventricular arrhythmias consistent with perinatal LQTS, requiring aggressive treatment including epicardial pacemaker and cardioverter-defibrillator implantation and sympathectomy on day of life 2. We sought to provide a rapid molecular diagnosis for the optimization of treatment strategies. Methods We performed Clinical Laboratory Improvement Amendments–certified rapid whole genome sequencing (WGS) with a speed-optimized bioinformatics platform to achieve molecular diagnosis at 10 days of life. Results We detected a known pathogenic variant in KCNH2 that was demonstrated to be paternally inherited by follow-up genotyping. The unbiased assessment of the entire catalog of human genes provided by WGS revealed a maternally inherited variant of unknown significance in a novel gene. Conclusion Rapid clinical WGS provides faster and more comprehensive diagnostic information at 10 days of life than does standard gene panel testing. In selected clinical scenarios such as perinatal LQTS, rapid WGS can provide more timely and clinically actionable information than can a standard commercial test.

Somatic mosaicism is a condition that arises when DNA mutations lead to genetic variations within an individual. Recently, an infant with LQTS characterized by episodes of 2:1 block (i.e., 2 atrial/His-bundle activations for every one ventricular activation) was found to have a novel SCN5a mutation (V1762L) in ∼20% of cardiomyocytes, causing a 10-fold increase in late sodium current within affected cells. However, the mechanism by which V1762L mosaicism could give rise to this clinical phenotype remained unknown. We hypothesized that 2:1 block could arise from 20% V1762L mosaicism in the PS, regardless of its spatial distribution.To test this hypothesis, we used a biophysically-detailed 3D computational model of the ventricles and PS calibrated to approximate the size/geometry of the human neonatal heart. Versions of the model incorporating 20% V1762L mosaicism with diffuse or clustered distributions in the ventricles and PS were generated using a stochastic approach. To examine the arrhythmogenic propensity of the heart models, simulated normal sinus rhythm (coupling interval, CI=400ms) was interrupted by two consecutive abnormally timed stimuli (variable CIs: 300-700ms in 50ms intervals).Ventricular mosaicism alone did not lead to 2:1 block. However, once mosaicism was incorporated into the PS, a majority of all pacing sequences in each model (diffuse: 79%, clustered: 80%) resulted in 2:1 block. Even pacing sequences with CIs within the physiological range for infant heart rates (CI=400-600ms) resulted in 2:1 block (diffuse: 48%, clustered: 52%). Left bundle branch block, a highly arrhythmogenic activation pattern, was also observed (diffuse: 4%, clustered: 2%).We demonstrated a possible link between cell-scale phenotypic variability and organ-scale arrhythmogenesis by showing that 20% V1762L mosaicism in the PS, regardless of spatial distribution, can result in 2:1 block.

High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework., Author Summary Technological advances have dramatically reduced the cost of sequencing the human genome. Tools for analyzing such data across families including annotation of clinically important variants and aggregation of variants for personalizing drug prescriptions have been developed but few are publically available. Here we describe such tools then demonstrate their application in several distinct data sets. In particular, we use the tools to define the genetic basis of a new congenital arrhythmia syndrome.

Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of ~;1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome, the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.