The genomic history of the Middle East.

The Middle East region is important to understand human evolution and migrations but is underrepresented in genomic studies. Here, we generated 137 high-coverage physically phased genome sequences from eight Middle Eastern populations using linked-read sequencing. We found no genetic traces of early expansions out-of-Africa in present-day populations but found Arabians have elevated Basal Eurasian ancestry that dilutes their Neanderthal ancestry. Population sizes within the region started diverging 15-20 kya, when Levantines expanded while Arabians maintained smaller populations that derived ancestry from local hunter-gatherers. Arabians suffered a population bottleneck around the aridification of Arabia 6 kya, while Levantines had a distinct bottleneck overlapping the 4.2 kya aridification event. We found an association between movement and admixture of populations in the region and the spread of Semitic languages. Finally, we identify variants that show evidence of selection, including polygenic selection. Our results provide detailed insights into the genomic and selective histories of the Middle East.

Pro106Leu MPL mutation is associated with thrombocytosis and a low risk of thrombosis, splenomegaly and marrow fibrosis.

The P106L mutation in the human myeloproliferative leukemia virus oncogene (MPL) was shown to be associated with hereditary thrombocythemia in Arabs. The clinical and bone marrow (BM) features of P106L mutation are unknown. Genetic databases at two tertiary hospitals in Saudi Arabia were searched to identify patients with the MPL P106L mutation. Clinical data were collected retrospectively and the BM aspirates and biopsies were independently reviewed by two hematopathologists. In total, 115 patients were included. Median age was 33 years of which 31 patients were pediatric and 65 were female. The mutation was homozygous in 87 patients. Thrombocytosis was documented in 107 patients, with a median platelet count of 667 × 109/L. The homozygous genotype was associated with a higher platelet count. Thirty-three patients had an evaluable BM and clustering of megakaryocytes was observed in 30/33 patients. At the time of last follow-up, 114 patients were alive. The median follow-up was 7.8 years from the time of thrombocytosis. No patients developed disease progression to myelofibrosis. The P106L mutation was associated with marked thrombocytosis at a younger age and with a low risk of thrombosis, splenomegaly, and marrow fibrosis. The BM demonstrated normal or hypocellular marrow with megakaryocyte clusters.

Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.

Rare variants in NR2F2 cause congenital heart defects in humans.

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters.

Using large sequencing data sets to refine intragenic disease regions and prioritize clinical variant interpretation.

PURPOSE: Classification of novel variants is a major challenge facing the widespread adoption of comprehensive clinical genomic sequencing and the field of personalized medicine in general. This is largely because most novel variants do not have functional, genetic, or population data to support their clinical classification. METHODS: To improve variant interpretation, we leveraged the Exome Aggregation Consortium (ExAC) data set (N = ~60,000) as well as 7,000 clinically curated variants in 132 genes identified in more than 11,000 probands clinically tested for cardiomyopathies, rasopathies, hearing loss, or connective tissue disorders to perform a systematic evaluation of domain level disease associations. RESULTS: We statistically identify regions that are most sensitive to functional variation in the general population and also most commonly impacted in symptomatic individuals. Our data show that a significant number of exons and domains in genes strongly associated with disease can be defined as disease-sensitive or disease-tolerant, leading to potential reclassification of at least 26% (450 out of 1,742) of variants of uncertain clinical significance in the 132 genes. CONCLUSION: This approach leverages domain functional annotation and associated disease in each gene to prioritize candidate disease variants, increasing the sensitivity and specificity of novel variant assessment within these genes.Genet Med advance online publication 22 September 2016.

Using population data for assessing next-generation sequencing performance.

MOTIVATION: During the past 4 years, whole-exome sequencing has become a standard tool for finding rare variants causing Mendelian disorders. In that time, there has also been a proliferation of both sequencing platforms and approaches to analyse their output. This requires approaches to assess the performance of different methods. Traditionally, criteria such as comparison with microarray data or a number of known polymorphic sites have been used. Here we expand such approaches, developing a maximum likelihood framework and using it to estimate the sensitivity and specificity of whole-exome sequencing data. RESULTS: Using whole-exome sequencing data for a panel of 19 individuals, we show that estimated sensitivity and specificity are similar to those calculated using microarray data as a reference. We explore the effect of frequency misspecification arising from using an inappropriately selected population and find that, although the estimates are affected, the rankings across procedures remain the same. AVAILABILITY AND IMPLEMENTATION: An implementation using Perl and R can be found at busso.ncl.ac.uk (Username: igm101; Password: Z1z1nts).

Improving hearing loss gene testing: a systematic review of gene evidence toward more efficient next-generation sequencing-based diagnostic testing and interpretation.

PURPOSE: With next generation sequencing technology improvement and cost reductions, it has become technically feasible to sequence a large number of genes in one diagnostic test. This is especially relevant for diseases with large genetic and/or phenotypic heterogeneity, such as hearing loss. However, variant interpretation remains the major bottleneck. This is further exacerbated by the lack in the clinical genetics community of consensus criteria for defining the evidence necessary to include genes on targeted disease panels or in genomic reports, and the consequent risk of reporting variants in genes with no relevance to disease. METHODS: We describe a systematic evidence-based approach for assessing gene-disease associations and for curating relevant genes for different disease aspects, including mode of inheritance, phenotypic severity, and mutation spectrum. RESULTS: By applying this approach to clinically available hearing loss gene panels with a total of 163 genes, we show that a significant number (45%) of genes lack sufficient evidence of association with disease and thus are expected to increase uncertainty and patient anxiety, in addition to intensifying the interpretation burden. Information about all curated genes is summarized. Our retrospective analysis of 539 hearing loss cases tested by our previous OtoGenomeV2 panel demonstrates the impact of including genes with weak disease association in laboratory wet-bench and interpretation processes. CONCLUSION: Our study is, to our knowledge, the first to highlight the urgent need for defining the clinical validity of gene-disease relationships for more efficient and accurate clinical testing and reporting.Genet Med 18 6, 545-553.

Exome sequencing identifies rare variants in multiple genes in atrioventricular septal defect.

PURPOSE: The genetic etiology of atrioventricular septal defect (AVSD) is unknown in 40% cases. Conventional sequencing and arrays have identified the etiology in only a minority of nonsyndromic individuals with AVSD. METHODS: Whole-exome sequencing was performed in 81 unrelated probands with AVSD to identify potentially causal variants in a comprehensive set of 112 genes with strong biological relevance to AVSD. RESULTS: A significant enrichment of rare and rare damaging variants was identified in the gene set, compared with controls (odds ratio (OR): 1.52; 95% confidence interval (CI): 1.35-1.71; P = 4.8 × 10(-11)). The enrichment was specific to AVSD probands, compared with a cohort without AVSD with tetralogy of Fallot (OR: 2.25; 95% CI: 1.84-2.76; P = 2.2 × 10(-16)). Six genes (NIPBL, CHD7, CEP152, BMPR1a, ZFPM2, and MDM4) were enriched for rare variants in AVSD compared with controls, including three syndrome-associated genes (NIPBL, CHD7, and CEP152). The findings were confirmed in a replication cohort of 81 AVSD probands. CONCLUSION: Mutations in genes with strong biological relevance to AVSD, including syndrome-associated genes, can contribute to AVSD, even in those with isolated heart disease. The identification of a gene set associated with AVSD will facilitate targeted genetic screening in this cohort.

Recessive nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum is caused by homozygous protein-truncating mutations of WDR73.

We describe a novel nephrocerebellar syndrome on the Galloway-Mowat syndrome spectrum among 30 children (ages 1.0 to 28 years) from diverse Amish demes. Children with nephrocerebellar syndrome had progressive microcephaly, visual impairment, stagnant psychomotor development, abnormal extrapyramidal movements and nephrosis. Fourteen died between ages 2.7 and 28 years, typically from renal failure. Post-mortem studies revealed (i) micrencephaly without polymicrogyria or heterotopia; (ii) atrophic cerebellar hemispheres with stunted folia, profound granule cell depletion, Bergmann gliosis, and signs of Purkinje cell deafferentation; (iii) selective striatal cholinergic interneuron loss; and (iv) optic atrophy with delamination of the lateral geniculate nuclei. Renal tissue showed focal and segmental glomerulosclerosis and extensive effacement and microvillus transformation of podocyte foot processes. Nephrocerebellar syndrome mapped to 700 kb on chromosome 15, which contained a single novel homozygous frameshift variant (WDR73 c.888delT; p.Phe296Leufs*26). WDR73 protein is expressed in human cerebral cortex, hippocampus, and cultured embryonic kidney cells. It is concentrated at mitotic microtubules and interacts with α-, ß-, and γ-tubulin, heat shock proteins 70 and 90 (HSP-70; HSP-90), and the carbamoyl phosphate synthetase 2/aspartate transcarbamylase/dihydroorotase multi-enzyme complex. Recombinant WDR73 p.Phe296Leufs*26 and p.Arg256Profs*18 proteins are truncated, unstable, and show increased interaction with α- and ß-tubulin and HSP-70/HSP-90. Fibroblasts from patients homozygous for WDR73 p.Phe296Leufs*26 proliferate poorly in primary culture and senesce early. Our data suggest that in humans, WDR73 interacts with mitotic microtubules to regulate cell cycle progression, proliferation and survival in brain and kidney. We extend the Galloway-Mowat syndrome spectrum with the first description of diencephalic and striatal neuropathology.

Defective presynaptic choline transport underlies hereditary motor neuropathy.

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies.

Recessive HYDIN mutations cause primary ciliary dyskinesia without randomization of left-right body asymmetry.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307(∗)), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus.

Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

Combined NGS approaches identify mutations in the intraflagellar transport gene IFT140 in skeletal ciliopathies with early progressive kidney Disease.

Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.

Case report: Birk-Landau-Perez syndrome linked to the SLC30A9 gene-identification of additional cases and expansion of the phenotypic spectrum.

Birk-Landau-Perez syndrome (BILAPES) is an autosomal recessive cerebro-renal syndrome associated with genetic defects in the SLC30A9 gene, initially reported in 2017 in six individuals belonging to a large Bedouin kindred. The SLC30A9 gene encodes a putative mitochondrial zinc transporter with ubiquitous expression, the highest found in the brain, kidney, and skeletal muscle. Since the first report, only one additional affected patient has been described, but there were some inconsistencies, such as hearing loss, failure to thrive, and neuroimaging findings between the clinical presentation of the disease in the Bedouin family and the second patient. Here, we present two more patients from a consanguineous Middle Eastern family with features of chronic kidney disease, neurodevelopmental regression, ataxia, hearing loss, and eye abnormalities, which were largely consistent with BILAPES. Whole-exome sequencing detected a homozygous in-frame deletion c.1049_1051delCAG (p.Ala350del) in the SLC30A9 gene, which was the same variant detected in the patients from the primary literature report and the variant segregated with disease in the family. However, in the patients described here, brain MRI showed cerebellar atrophy, which was not a cardinal feature of the syndrome from the primary report. Our findings provide further evidence for SLC30A9-associated BILAPES and contribute to defining the clinical spectrum.

Consensus Recommendations for the Diagnosis, Biomarker Testing, and Clinical Management of Advanced or Metastatic Non-small Cell Lung Cancer With Mesenchymal-Epithelial Transition Exon 14 Skipping Mutations in the Middle East, Africa, and Russia.

Mesenchymal-epithelial transition exon 14 (METex14) skipping mutations occur in about 3%-4% of patients with non-small cell lung cancer (NSCLC). This is an aggressive subtype associated with poor prognosis. METex14 skipping is a potentially targetable mutation. Targeted therapy is a promising treatment modality for patients with advanced/metastatic METex14-mutant NSCLC. Performing systematic molecular testing to detect the driver mutation is essential for initiating targeted therapy. However, there is a lack of guidelines on molecular testing for assessing the eligibility of patients for targeted therapy. Therefore, a multidisciplinary panel consisting of experts from the Middle East, Africa, and Russia convened via a virtual advisory board meeting to provide their insights on various molecular testing techniques for the diagnosis of METex14 skipping mutation, management of patients with targeted therapies, and developing consensus recommendations for improving the processes. The expert panel emphasized performing molecular testing and liquid biopsy before treatment initiation and tissue re-biopsy for patients with failed molecular testing. Liquid biopsy was recommended as complementary to tissue biopsy for disease monitoring and prognosis. Selective MET inhibitors were recommended as the first and subsequent lines of therapy. These consensus recommendations will facilitate the management of METex14 skipping NSCLC in routine practice and warrant optimum outcomes for these patients.

Isolated Bone Marrow Non-Langerhans Cell Histiocytosis Preceding RUNX1-Mutated Acute Myeloid Leukemia: Case Report and Literature Review.

OBJECTIVES: The prevalence of concomitant myeloid neoplasms was recently reported to be unexpectedly high among adults without non-Langerhans cell histiocytosis (non-LCH); however, the coexistence of non-LCH with RUNX1 genetic aberration has not been reported previously. METHODS: Herein, we report a 23-year-old woman with severe pancytopenia diagnosed with non-LCH following presentation with pancytopenia and marrow examination showing histiocytosis positive for CD45, CD68, CD136, and lysozyme but negative for CD1a, langerin, and S100. RESULTS: Whole-exome sequencing showed RUNX1 mutation and NF1 mutation. In the ensuing 6 months, she developed hepatosplenomegaly, and repeat bone marrow evaluation was diagnostic of acute myeloid leukemia (AML). Repeat mutational analysis showed again presence of RUNX1 mutation. She underwent induction therapy but died of septic shock. CONCLUSIONS: The demonstration of RUNX1 mutation in both non-LCH and AML bone marrow specimens at differing time points is suggestive of a biologic association of both distinct disease entities.

Artificial Intelligence Approach for Variant Reporting.

Purpose: Next-generation sequencing technologies are actively applied in clinical oncology. Bioinformatics pipeline analysis is an integral part of this process; however, humans cannot yet realize the full potential of the highly complex pipeline output. As a result, the decision to include a variant in the final report during routine clinical sign-out remains challenging. Methods: We used an artificial intelligence approach to capture the collective clinical sign-out experience of six board-certified molecular pathologists to build and validate a decision support tool for variant reporting. We extracted all reviewed and reported variants from our clinical database and tested several machine learning models. We used 10-fold cross-validation for our variant call prediction model, which derives a contiguous prediction score from 0 to 1 (no to yes) for clinical reporting. Results: For each of the 19,594 initial training variants, our pipeline generates approximately 500 features, which results in a matrix of > 9 million data points. From a comparison of naive Bayes, decision trees, random forests, and logistic regression models, we selected models that allow human interpretability of the prediction score. The logistic regression model demonstrated 1% false negativity and 2% false positivity. The final models' Youden indices were 0.87 and 0.77 for screening and confirmatory cutoffs, respectively. Retraining on a new assay and performance assessment in 16,123 independent variants validated our approach (Youden index, 0.93). We also derived individual pathologist-centric models (virtual consensus conference function), and a visual drill-down functionality allows assessment of how underlying features contributed to a particular score or decision branch for clinical implementation. Conclusion: Our decision support tool for variant reporting is a practically relevant artificial intelligence approach to harness the next-generation sequencing bioinformatics pipeline output when the complexity of data interpretation exceeds human capabilities.

Loss of PCLO function underlies pontocerebellar hypoplasia type III.

OBJECTIVE: To identify the genetic cause of pontocerebellar hypoplasia type III (PCH3). METHODS: We studied the original reported pedigree of PCH3 and performed genetic analysis including genome-wide single nucleotide polymorphism genotyping, linkage analysis, whole-exome sequencing, and Sanger sequencing. Human fetal brain RNA sequencing data were then analyzed for the identified candidate gene. RESULTS: The affected individuals presented with severe global developmental delay and seizures starting in the first year of life. Brain MRI of an affected individual showed diffuse atrophy of the cerebrum, cerebellum, and brainstem. Genome-wide single nucleotide polymorphism analysis confirmed the linkage to chromosome 7q we previously reported, and showed no other genomic areas of linkage. Whole-exome sequencing of 2 affected individuals identified a shared homozygous, nonsense variant in the PCLO (piccolo) gene. This variant segregated with the disease phenotype in the pedigree was rare in the population and was predicted to eliminate the PDZ and C2 domains in the C-terminus of the protein. RNA sequencing data of human fetal brain showed that PCLO was moderately expressed in the developing cerebral cortex. CONCLUSIONS: Here, we show that a homozygous, nonsense PCLO mutation underlies the autosomal recessive neurodegenerative disorder, PCH3. PCLO is a component of the presynaptic cytoskeletal matrix, and is thought to be involved in regulation of presynaptic proteins and synaptic vesicles. Our findings suggest that PCLO is crucial for the development and survival of a wide range of neuronal types in the human brain.

High throughput exome coverage of clinically relevant cardiac genes.

BACKGROUND: Given the growing use of whole-exome sequencing (WES) for clinical diagnostics of complex human disorders, we evaluated coverage of clinically relevant cardiac genes on WES and factors influencing uniformity and depth of coverage of exonic regions. METHODS: Two hundred and thirteen human DNA samples were exome sequenced via Illumina HiSeq using different versions of the Agilent SureSelect capture kit. 50 cardiac genes were further analyzed including 31 genes from the American College of Medical Genetics (ACMG) list for reporting of incidental findings and 19 genes associated with congenital heart disease for which clinical testing is available. Gene coordinates were obtained from two databases, CCDS and Known Gene and compared. Read depth for each region was extracted from the exomes and used to assess capture variability between kits for individual genes, and for overall coverage. GC content, gene size, and inter-sample variability were also tested as potential contributors to variability in gene coverage. RESULTS: All versions of capture kits (designed based on Consensus coding sequence) included only 55% of known genomic regions for the cardiac genes. Although newer versions of each Agilent kit showed improvement in capture of CCDS regions to 99%, only 64% of Known Gene regions were captured even with newer capture kits. There was considerable variability in coverage of the cardiac genes. 10 of the 50 genes including 6 on the ACMG list had less than the optimal coverage of 30X. Within each gene, only 32 of the 50 genes had the majority of their bases covered at an interquartile range ≥30X. Heterogeneity in gene coverage was modestly associated with gene size and significantly associated with GC content. CONCLUSIONS: Despite improvement in overall coverage across the exome with newer capture kit versions and higher sequencing depths, only 50% of known genomic regions of clinical cardiac genes are targeted and individual gene coverage is non-uniform. This may contribute to a bias with greater attribution of disease causation to mutations in well-represented and well-covered genes. Improvements in WES technology are needed before widespread clinical application.