Exome Sequencing Project Research Articles

Abstract 3886: Comparing the mutational landscape of African American and Caucasian lung cancers

Abstract Background: The overall death rate from lung cancer is higher in African-Americans (AA) compared to Caucasians (CAU). Understanding differences in the prevalence and type of somatic alterations between races may illuminate differences in prognosis and lead to the reduction of outcome disparities by more precisely targeting patients’ treatment. Methods: Formalin-fixed paraffin embedded tumor samples were collected from Baptist Cancer Center, Memphis, TN. DNA was extracted and sonicated to 250 bp following Covaris FFPE DNA Extraction & Purification protocol and further purified using Agencourt AMPure XP beads. The OncoPanel_v2 target enrichment panel (Agilent SureSelect) was used for hybrid capture of 502 cancer-related genes. Samples were pooled and sequenced on an Illumina HiSeq2500 to a mean depth of coverage of 210x. Tumors with >30x sequencing depth over >80% of targeted bases were considered for analysis. MuTect and SomaticIndelDetector were used to identify somatic single nucleotide variants (SNVs) and short insertions or deletions (indels), respectively. As matched normal DNA was not available for these samples, analysis was limited to variants previously observed in tumors as described in the Catalogue of Somatic Mutations in Cancer (COSMIC) database, and variants were excluded if found in the germline dataset of the Exome Sequencing Project (ESP). Results: 510 tumor specimens from 242 Black and 268 White patients (with self-reported race) were analyzed including 320 adenocarcinomas and 142 squamous cell carcinomas. Pathological classification was independently reviewed and confirmed for 374 of 472 cases for an overall concordance rate of 79%. Using principle component analysis (PCA) on germline SNVs, we observed that the biological ancestry was different than the self-reported race for 1.5% of patients. Mutational frequencies for genes with known roles in adenocarcinoma such as KRAS and EGFR were not significantly different between tumors from Black and White patients (Fisher's exact p-value > 0.05). Amplification rates for NKX2-1, MET, MDM2, and MYC and homozygous deletion rates for CDKN2A were also not significantly different between populations. Translocations involving ALK and ROS1 were detected in tumors from Black patients demonstrating that these events are present in both populations. Similarly, mutational frequencies for genes such as PIK3CA and PTEN were not significantly different across populations in squamous cell carcinomas. Conclusions: These results demonstrate that lung cancers from Black patients are more similar to Whites than East Asians with respect to genes such as EGFR, and suggest that clinical trials of targeted therapies could significantly benefit patients in both populations. Citation Format: Joshua Campbell, Christopher Lathan, Lynette Sholl, Matthew Ducar, Mikenah Vega, Ling Lin, Aaron Thorner, Nick Faris, Paul van Hummelen, Raymond Osarogiagbon, Matthew Meyerson, Laura MacConaill. Comparing the mutational landscape of African American and Caucasian lung cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2015-3886

Abstract 4796: A founder mutation in the BAP1 gene among four caucasian families with high incidences of malignant peritoneal mesothelioma and uveal melanoma: a molecular and genealogical study in a 10-generation BAP1 cancer syndrome kindred

Abstract The BAP1 cancer syndrome is characterized by a high incidence of malignant mesothelioma (MM), uveal melanoma (UM), cutaneous melanoma (CM), clear cell renal cell carcinoma (ccRCC), and it is expected that the clinical phenotype will continue to broaden in scope. Molecular screening for BAP1 gene mutations among 29 patients selected for clinically apparent familial MM led to the identification of a heterozygous C base deletion mutation (c.1832delC, p.Leu573fs*3) shared by four of those patients. The frame shift deletion is predicted to truncate the BAP1 protein, and immunohistochemistry analysis of tumor specimens revealed predominant cytoplasmic staining. We genotyped 650K SNPs of the four MM samples and four controls and carried out principal component analysis and whole-genome identity-by-descent analysis using publicly available genotype data from the 1000 Genomes Project, UK10K Project, and NHIBL Exome Sequencing Project. These analyses showed that the four MM patients are of Central Europe ancestry and that some are related by a kinship coefficient of 0.0186. Haplotype analysis showed the presence of significantly shared segments around the BAP1 gene (LOD>37.1). The pairwise extent of shared segments between any of the four MM patients ranged in length from 9.1 to 34.2 Mbp and indicates the c.1832delC variant originated in a recent common ancestor within five to ten generations. Through a combined molecular genomic and genealogical approach, we ascertained, to our knowledge, the largest known genealogically connected BAP1 cancer syndrome kindred (K4), whose members can trace descent from a common ancestor in the 17th century. This pedigree provided a unique opportunity to examine effects of BAP1 alteration on tumor expression on various body sites and would facilitate study of gene-gene and gene-environment interaction involving the BAP1 gene. It also suggests that molecular screening of the BAP1 gene, coupled with genealogical research, would be an effective strategy for the early detection and early intervention for BAP1-associated malignancies. Citation Format: Erin Flores, Mitsuru Emi, Todd Johnson, Tatsuhiko Tsunoda, Dusty Behner, Harriet Hoffman, Mary Hesdorffer, Masaki Nasu, Andrea Napolitano, Francine Baumann, Haining Yang, Michele Carbone. A founder mutation in the BAP1 gene among four caucasian families with high incidences of malignant peritoneal mesothelioma and uveal melanoma: a molecular and genealogical study in a 10-generation BAP1 cancer syndrome kindred. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4796. doi:10.1158/1538-7445.AM2015-4796

Genome-wide gene-gene interaction analysis for next-generation sequencing.

The critical barrier in interaction analysis for next-generation sequencing (NGS) data is that the traditional pairwise interaction analysis that is suitable for common variants is difficult to apply to rare variants because of their prohibitive computational time, large number of tests and low power. The great challenges for successful detection of interactions with NGS data are (1) the demands in the paradigm of changes in interaction analysis; (2) severe multiple testing; and (3) heavy computations. To meet these challenges, we shift the paradigm of interaction analysis between two SNPs to interaction analysis between two genomic regions. In other words, we take a gene as a unit of analysis and use functional data analysis techniques as dimensional reduction tools to develop a novel statistic to collectively test interaction between all possible pairs of SNPs within two genome regions. By intensive simulations, we demonstrate that the functional logistic regression for interaction analysis has the correct type 1 error rates and higher power to detect interaction than the currently used methods. The proposed method was applied to a coronary artery disease dataset from the Wellcome Trust Case Control Consortium (WTCCC) study and the Framingham Heart Study (FHS) dataset, and the early-onset myocardial infarction (EOMI) exome sequence datasets with European origin from the NHLBI's Exome Sequencing Project. We discovered that 6 of 27 pairs of significantly interacted genes in the FHS were replicated in the independent WTCCC study and 24 pairs of significantly interacted genes after applying Bonferroni correction in the EOMI study.

Mucolipidosis types II and III and non-syndromic stuttering are associated with different variants in the same genes.

Homozygous mutations in GNPTAB and GNPTG are classically associated with mucolipidosis II (ML II) alpha/beta and mucolipidosis III (ML III) alpha/beta/gamma, which are rare lysosomal storage disorders characterized by multiple pathologies. Recently, variants in GNPTAB, GNPTG, and the functionally related NAGPA gene have been associated with non-syndromic persistent stuttering. In a worldwide sample of 1013 unrelated individuals with non-syndromic persistent stuttering we found 164 individuals who carried a rare non-synonymous coding variant in one of these three genes. We compared the frequency of these variants with those in population-matched controls and genomic databases, and their location with those reported in mucolipidosis. Stuttering subjects displayed an excess of non-synonymous coding variants compared to controls and individuals in the 1000 Genomes and Exome Sequencing Project databases. We identified a total of 81 different variants in our stuttering cases. Virtually all of these were missense substitutions, only one of which has been previously reported in mucolipidosis, a disease frequently associated with complete loss-of-function mutations. We hypothesize that rare non-synonymous coding variants in GNPTAB, GNPTG, and NAGPA may account for as much as 16% of persistent stuttering cases, and that variants in GNPTAB and GNPTG are at different sites and may in general, cause less severe effects on protein function than those in ML II alpha/beta and ML III alpha/beta/gamma.

Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

SummaryPlatelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) < 0.05%. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21%) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF<1% and 22 with MAF ≥ 1%). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Comparison of GENCODE and RefSeq gene annotation and the impact of reference geneset on variant effect prediction.

BackgroundA vast amount of DNA variation is being identified by increasingly large-scale exome and genome sequencing projects. To be useful, variants require accurate functional annotation and a wide range of tools are available to this end. McCarthy et al recently demonstrated the large differences in prediction of loss-of-function (LoF) variation when RefSeq and Ensembl transcripts are used for annotation, highlighting the importance of the reference transcripts on which variant functional annotation is based.ResultsWe describe a detailed analysis of the similarities and differences between the gene and transcript annotation in the GENCODE and RefSeq genesets. We demonstrate that the GENCODE Comprehensive set is richer in alternative splicing, novel CDSs, novel exons and has higher genomic coverage than RefSeq, while the GENCODE Basic set is very similar to RefSeq. Using RNAseq data we show that exons and introns unique to one geneset are expressed at a similar level to those common to both. We present evidence that the differences in gene annotation lead to large differences in variant annotation where GENCODE and RefSeq are used as reference transcripts, although this is predominantly confined to non-coding transcripts and UTR sequence, with at most ~30% of LoF variants annotated discordantly. We also describe an investigation of dominant transcript expression, showing that it both supports the utility of the GENCODE Basic set in providing a smaller set of more highly expressed transcripts and provides a useful, biologically-relevant filter for further reducing the complexity of the transcriptome.ConclusionsThe reference transcripts selected for variant functional annotation do have a large effect on the outcome. The GENCODE Comprehensive transcripts contain more exons, have greater genomic coverage and capture many more variants than RefSeq in both genome and exome datasets, while the GENCODE Basic set shows a higher degree of concordance with RefSeq and has fewer unique features. We propose that the GENCODE Comprehensive set has great utility for the discovery of new variants with functional potential, while the GENCODE Basic set is more suitable for applications demanding less complex interpretation of functional variants.

Are all the previously reported genetic variants in limb girdle muscular dystrophy genes pathogenic?

Hundreds of variants in autosomal genes associated with the limb girdle muscular dystrophies (LGMDs) have been reported as being causative. However, in most cases the proof of pathogenicity derives from their non-occurrence in hundreds of healthy controls and/or from segregation studies in small families. The limited statistics of the genetic variations in the general population may hamper a correct interpretation of the effect of variants on the protein. To clarify the meaning of low-frequency variants in LGMD genes, we have selected all variants described as causative in the Leiden Open Variation Database and the Human Gene Mutation Database. We have systematically searched for their frequency in the NHLBI GO Exome Sequencing Project (ESP) and in our internal database. Surprisingly, the ESP contains about 4% of the variants previously associated with a dominant inheritance and about 9% of those associated with a recessive inheritance. The putative disease alleles are much more frequent than those estimated considering the disease prevalence. In conclusion, we hypothesize that a number of disease-associated variants are non-pathogenic and that other variations are not fully penetrant, even if they affect the protein function, suggesting a more complex genetic mechanisms for such heterogeneous disorders.

Prevalence estimation for monogenic autosomal recessive diseases using population-based genetic data.

Genetic methods can complement epidemiological surveys and clinical registries in determining prevalence of monogenic autosomal recessive diseases. Several large population-based genetic databases, such as the NHLBI GO Exome Sequencing Project, are now publically available. By assuming Hardy-Weinberg equilibrium, the frequency of individuals homozygous in the general population for a particular pathogenic allele can be directly calculated from a sample of chromosomes where some harbor the pathogenic allele. Further assuming that the penetrance of the pathogenic allele(s) is known, the prevalence of recessive phenotypes can be determined. Such work can inform public health efforts for rare recessive diseases. A Bayesian estimation procedure has yet to be applied to the problem of estimating disease prevalence from large population-based genetic data. A Bayesian framework is developed to derive the posterior probability density of monogenic, autosomal recessive phenotypes. Explicit equations are presented for the credible intervals of these disease prevalence estimates. A primary impediment to performing accurate disease prevalence calculations is the determination of truly pathogenic alleles. This issue is discussed, but in many instances remains a significant barrier to investigations solely reliant on statistical interrogation--functional studies can provide important information for solidifying evidence of variant pathogenicity. We also discuss several challenges to these efforts, including the population structure in the sample of chromosomes, the treatment of allelic heterogeneity, and reduced penetrance of pathogenic variants. To illustrate the application of these methods, we utilized recently published genetic data collected on a large sample from the Schmiedeleut Hutterites. We estimate prevalence and calculate 95% credible intervals for 13 autosomal recessive diseases using these data. In addition, the Bayesian estimation procedure is applied to data from a central European study of hereditary fructose intolerance. The methods described herein show a viable path to robustly estimating both the expected prevalence of autosomal recessive phenotypes and corresponding credible intervals using population-based genetic databases that have recently become available. As these genetic databases increase in number and size with the advent of cost-effective next-generation sequencing, we anticipate that these methods and approaches may be helpful in recessive disease prevalence calculations, potentially impacting public health management, health economic analyses, and treatment of rare diseases.

An efficient and scalable analysis framework for variant extraction and refinement from population-scale DNA sequence data.

The analysis of next-generation sequencing data is computationally and statistically challenging because of the massive volume of data and imperfect data quality. We present GotCloud, a pipeline for efficiently detecting and genotyping high-quality variants from large-scale sequencing data. GotCloud automates sequence alignment, sample-level quality control, variant calling, filtering of likely artifacts using machine-learning techniques, and genotype refinement using haplotype information. The pipeline can process thousands of samples in parallel and requires less computational resources than current alternatives. Experiments with whole-genome and exome-targeted sequence data generated by the 1000 Genomes Project show that the pipeline provides effective filtering against false positive variants and high power to detect true variants. Our pipeline has already contributed to variant detection and genotyping in several large-scale sequencing projects, including the 1000 Genomes Project and the NHLBI Exome Sequencing Project. We hope it will now prove useful to many medical sequencing studies.

Analysis of Sequence Data Under Multivariate Trait-Dependent Sampling

High-throughput DNA sequencing allows for the genotyping of common and rare variants for genetic association studies. At the present time and for the foreseeable future, it is not economically feasible to sequence all individuals in a large cohort. A cost-effective strategy is to sequence those individuals with extreme values of a quantitative trait. We consider the design under which the sampling depends on multiple quantitative traits. Under such trait-dependent sampling, standard linear regression analysis can result in bias of parameter estimation, inflation of Type I error, and loss of power. We construct a likelihood function that properly reflects the sampling mechanism and uses all available data. We implement a computationally efficient EM algorithm and establish the theoretical properties of the resulting maximum likelihood estimators. Our methods can be used to perform separate inference on each trait or simultaneous inference on multiple traits. We pay special attention to gene-level association tests for rare variants. We demonstrate the superiority of the proposed methods over standard linear regression through extensive simulation studies. We provide applications to the Cohorts for Heart and Aging Research in Genomic Epidemiology Targeted Sequencing Study and the National Heart, Lung, and Blood Institute Exome Sequencing Project. Supplementary materials for this article are available online.

Accumulation of rare variants in the arylsulfatase G (ARSG) gene in task-specific dystonia.

Musician's dystonia and writer's cramp are examples of task-specific dystonia. Recently, the arylsulfatase G (ARSG) locus was suggested to be associated with musician's dystonia and writer's cramp by a genome-wide association study. To test for the presence of causal variants, the entire coding region and exon-intron boundaries of ARSG were sequenced in DNA samples from 158 musician's dystonia patients which were collected at the University of Music, Drama, and Media (Hanover, Germany), and 72 patients with writer's cramp which were recruited at the Academic Medical Centers in Amsterdam and Groningen, the Netherlands. The frequency of variants within ARSG was compared to publically available data at the exome variant server (EVS) from the NHLBI GO Exome Sequencing Project. We identified 11 single nucleotide variants (SNVs) in the patients including eight non-synonymous substitutions. All variants have previously been reported at EVS including two SNVs with a reported minor allele frequency <1%. One rare missense variant, rs61999318 (p.I493T), was significantly enriched in the group of writer's cramp patients compared to European Americans in EVS database (p = 0.0013). In patients with writer's cramp, there was an overall enrichment for rare, protein-changing variants compared to controls (p < 0.01). In conclusion, we did not detect any conclusive mutation in ARSG. However, we showed an association with rs61999318 in patients with writer's cramp that contributed to an overall enrichment for rare, protein-changing variants in these patients. Thus, our data provide further support for a role of ARSG variants in task-specific dystonia, especially writer's cramp.

Role of common and rare variants in SCN10A: results from the Brugada syndrome QRS locus gene discovery collaborative study.

Brugada syndrome (BrS) remains genetically heterogeneous and is associated with slowed cardiac conduction. We aimed to identify genetic variation in BrS cases at loci associated with QRS duration. A multi-centre study sequenced seven candidate genes (SCN10A, HAND1, PLN, CASQ2, TKT, TBX3, and TBX5) in 156 Caucasian SCN5A mutation-negative BrS patients (80% male; mean age 48) with symptoms (64%) and/or a family history of sudden death (47%) or BrS (18%). Forty-nine variants were identified: 18 were rare (MAF <1%) and non-synonymous; and 11/18 (61.1%), mostly in SCN10A, were predicted as pathogenic using multiple bioinformatics tools. Allele frequencies were compared with the Exome Sequencing and UK10K Projects. SKAT methods tested rare variation in SCN10A finding no statistically significant difference between cases and controls. Co-segregation analysis was possible for four of seven probands carrying a novel pathogenic variant. Only one pedigree (I671V/G1299A in SCN10A) showed co-segregation. The SCN10A SNP V1073 was, however, associated strongly with BrS [66.9 vs. 40.1% (UK10K) OR (95% CI) = 3.02 (2.35-3.87), P = 8.07 × 10-19]. Voltage-clamp experiments for NaV1.8 were performed for SCN10A common variants V1073, A1073, and rare variants of interest: A200V and I671V. V1073, A200V and I671V, demonstrated significant reductions in peak INa compared with ancestral allele A1073 (rs6795970). Rare variants in the screened QRS-associated genes (including SCN10A) are not responsible for a significant proportion of SCN5A mutation negative BrS. The common SNP SCN10A V1073 was strongly associated with BrS and demonstrated loss of NaV1.8 function, as did rare variants in isolated patients.

Actionable exomic incidental findings in 6503 participants: challenges of variant classification.

Recommendations for laboratories to report incidental findings from genomic tests have stimulated interest in such results. In order to investigate the criteria and processes for assigning the pathogenicity of specific variants and to estimate the frequency of such incidental findings in patients of European and African ancestry, we classified potentially actionable pathogenic single-nucleotide variants (SNVs) in all 4300 European- and 2203 African-ancestry participants sequenced by the NHLBI Exome Sequencing Project (ESP). We considered 112 gene-disease pairs selected by an expert panel as associated with medically actionable genetic disorders that may be undiagnosed in adults. The resulting classifications were compared to classifications from other clinical and research genetic testing laboratories, as well as with in silico pathogenicity scores. Among European-ancestry participants, 30 of 4300 (0.7%) had a pathogenic SNV and six (0.1%) had a disruptive variant that was expected to be pathogenic, whereas 52 (1.2%) had likely pathogenic SNVs. For African-ancestry participants, six of 2203 (0.3%) had a pathogenic SNV and six (0.3%) had an expected pathogenic disruptive variant, whereas 13 (0.6%) had likely pathogenic SNVs. Genomic Evolutionary Rate Profiling mammalian conservation score and the Combined Annotation Dependent Depletion summary score of conservation, substitution, regulation, and other evidence were compared across pathogenicity assignments and appear to have utility in variant classification. This work provides a refined estimate of the burden of adult onset, medically actionable incidental findings expected from exome sequencing, highlights challenges in variant classification, and demonstrates the need for a better curated variant interpretation knowledge base.

Triglyceride-rich lipoproteins and coronary artery disease risk: new insights from human genetics.

Despite ample success in reducing coronary artery disease (CAD) risk through reduction of low-density lipoprotein cholesterol (LDL-C), there remains substantial residual risk.1–4 Recent prospective studies have demonstrated that elevated triglycerides (TGs) are independent predictors of CAD risk.5–9 Furthermore, TGs are strongly associated with incident CAD events in patients with low LDL-C levels treated with statin.10 Thus, triglyceride-rich lipoproteins (TRLs) offer a potentially orthogonal risk factor to LDL-C for lowering CAD risk, but only if TRLs are causally associated with atherosclerotic disease.11 Human genetics has the potential to reveal the causal relationships of biomarkers found to be associated with disease outcomes.12–15 For example, genetic variants associated with plasma LDL-C levels are consistently associated with CAD risk in the right direction,15–18 consistent with a causal relationship. Importantly, similar studies have causally implicated the key TG-regulating enzyme lipoprotein lipase (LPL) in CAD risk. A common gain-of-function LPL variant, S447X, confers an antiatherogenic lipid profile characterized by low levels of TGs, and in several studies, it has been associated with lower incidence of vascular disease or myocardial infarction (MI).19–25 Conversely, several loss-of-function (LOF) LPL variants associated with elevated TG levels have been reported to be associated with increased CAD risk.21,26 Furthermore, multiple genome-wide association studies in the last 5 years have identified common noncoding variants at the LPL gene locus associated with both TG and CAD risk in the same direction.27–29 Beyond LPL itself, common variants that influence TG levels are significantly associated with CAD risk even after adjusting for their effects on other lipid traits.30 Do et al30 surveyed 185 single-nucleotide polymorphisms (SNPs) that were genome-wide significantly associated with ≥1 plasma lipid trait and identified a subset of …

American Heart Association Cardiovascular Genome-Phenome Study: foundational basis and program.

The American Heart Association has established the Cardiovascular Genome-Phenome Study (CV-GPS) as a means by which to achieve the goal of using modern genomics and phenotyping optimally to combat cardiovascular disease (CVD). CVD remains the leading cause of death in the United States and has become a major cause of morbidity and mortality worldwide. The course of CVD begins at an early age and evolves throughout life. Typically, risk factor precursors (eg, prehypertension, borderline abnormal lipids) are first observed in adolescence or young adulthood. By middle age, they develop into full-fledged cardiometabolic risk factors and subclinical disease, which usually become clinically apparent in older age. Importantly, risk factors for CVD reflect the complex interplay between genetic and environmental factors and demonstrate the complexity of the multiple determinants of disease. Editorial see p 17 Many of the genetic and environmental determinants of CVD have been identified over the past 50 years, and successful preventive and therapeutic strategies have been developed as a result. However, many potential genetic or acquired disease drivers remain unaccounted for, as illustrated by the observation that the major CVD risk factors combined account for only a fraction of the population-attributable risk.1 In addition, variations in CVD risk exist among individuals with similar risk profiles. Part of these differences may reflect differences in profiles of known risk factors that have not yet been fully refined but also individual differences in underlying genetic modifiers of such risk factors. Modern genomics, with its ability to provide ideally unbiased analysis of the entire genome, offers an approach to the ascertainment of all genetic determinants of CVD. Furthermore, the increasingly broad range of “omics” methods, including RNA-Seq, modern proteomics, metabolomics, and metabonomics, provide deeper and more refined molecular detail by which to define an individual’s genome and its relationship to (patho)phenotype. …

Clinical characterization and genotype analysis of idiopathic mental retardation in male patients with epilepsy

Objective To detect genetic causes of idiopathic mental retardation /developmental delay in 20 male patients with epilepsy and to analyze their clinical characteristics of positive mutation carriers. Methods The families, consisted of the patient and his parents were recruited.Genomic DNA was extracted from peripheral blood, and candidate gene mutation screening was carried out by next-generation sequencing technology.Mutations in positive gene were verified by polymerase chain reaction(PCR) and direct sequencing. Results Three missense mutations were identified among 3 patients out of 20 cases, with a detection rate of 15%.They were: OPHN1 gene c. 1996 C>G, RAB39B gene c. 542 C>T and AFF2 gene c. 427 A>T, none of which had been reported before.All of these mutations were likely to be pathogenic based on gene function, evolutionary conservation, variant frequency in normal population(NHLBI Exome Sequencing Project and 1 000 Genomes), bioinformatics prediction and inheritance patterns.In addition, all 3 genes disrupted were residing on the X chromosome previously demonstrated to be associated with X-linked mental retardation(XLMR), indicating that they were probably pathogenic or might serve as one of the risk factors. Conclusions Abnormal function of genes on the X chromosomal is one of the most impotent causes of XLMR.X chromosomal gene mutation screening would be recommended for male children suffering from idiopathic mental retardation with epilepsy. Key words: Mental retardation; Epilepsy; Male; Gene mutation

Exome sequencing as a tool for short stature gene discovery: analysis of a Korean family with pseudohypoparathyroidism

A 4 bp deletion in GNAS has known to be the causal mutation for pseudohypoparathyroidism. In this study, we performed sanger sequencing on exons of GNAS and identified the 4 bp causal deletion in Korean family with pseudohypoparathyroidism. Despite the finding of the deletion, underlying genetics of short stature in the family is unclear since paternal lineage showed history of short stature and mother didn’t show short stature. Therefore, we performed exome sequencing to explore putative causal mutations responsible for short stature phenotype in the Korean family with pseudohypoparathyroidism. Initially, we discovered 58,829 variants from four members of the family by exome sequencing. Subsequent variant filtering removed possible non-causal variants based on public databases including 1,000 genomes project and Exome Sequencing Project. After selecting dominant functional variants inherited from the father, we identified 40 putative candidates. Among them, the strongest possible candidate would be ALPL that has known to be related with hypophosphatasia. In conclusion, our results could provide additional insight into the understanding of genetics related with short stature phenotype.

A massively parallel pipeline to clone DNA variants and examine molecular phenotypes of human disease mutations.

Understanding the functional relevance of DNA variants is essential for all exome and genome sequencing projects. However, current mutagenesis cloning protocols require Sanger sequencing, and thus are prohibitively costly and labor-intensive. We describe a massively-parallel site-directed mutagenesis approach, “Clone-seq”, leveraging next-generation sequencing to rapidly and cost-effectively generate a large number of mutant alleles. Using Clone-seq, we further develop a comparative interactome-scanning pipeline integrating high-throughput GFP, yeast two-hybrid (Y2H), and mass spectrometry assays to systematically evaluate the functional impact of mutations on protein stability and interactions. We use this pipeline to show that disease mutations on protein-protein interaction interfaces are significantly more likely than those away from interfaces to disrupt corresponding interactions. We also find that mutation pairs with similar molecular phenotypes in terms of both protein stability and interactions are significantly more likely to cause the same disease than those with different molecular phenotypes, validating the in vivo biological relevance of our high-throughput GFP and Y2H assays, and indicating that both assays can be used to determine candidate disease mutations in the future. The general scheme of our experimental pipeline can be readily expanded to other types of interactome-mapping methods to comprehensively evaluate the functional relevance of all DNA variants, including those in non-coding regions.

αIIbβ3 Variants Defined By Next Generation Sequencing: Implications for Predicting Variants Likely to Cause Glanzmann Thrombasthenia and Alloimmune Disorders

# Authors contributed equally to this work.~ Currently at Genomics England Ltd, London, United KingdomNext generation sequencing is transforming our understanding of human genetic variation and is becoming a routine part of human genetic analysis. The identification of millions of new variants, which are mainly rare and assessing their implications for human health presents new challenges to researchers and clinicians. We have analyzed missense variants in the ITGB2A and ITGB3 genes obtained from whole exome and whole genome sequencing (WES & WGS) data from 5 databases: The Human Genome Mutation Database, the 1000 Genomes project, the UK10K Whole Exome Sequencing project, the UK10K Whole Genome Sequencing project, and The National Heart, Lung and Blood Institute Exome Sequencing Project. Together, these encompass variants of the platelet αIIbβ3 integrin receptor from ~32,000 alleles derived from 16,108 individuals. We identified 111 missense variants that have previously been associated with Glanzmann thrombasthenia (GT), 20 variants associated with alloimmune thrombocytopenia, and 5 variants associated with aniso/macrothrombocytopenia. None of the GT variants were found in the last four databases, indicating that they have minor allele frequencies (MAF) less than ~0.01%, attesting to both their rarity and the likelihood that they entered the population within the last ~2,500 years.We also identified 114 novel missense variants in ITGB2A affecting ~11% of the amino acids and 68 novel missense variants in ITGB3 affecting ~9% of the amino acids. 96% of the novel variants had MAF <0.1%, indicating their rarity. Based on sequence conservation, MAF, and/or location of the substituted residue on a complete model of αIIbβ3 that suggested a possible effect on protein folding, we selected three novel variants (αIIb P943A and P176H, and β3 C547G) that affect amino acids previously associated with GT for expression in HEK 293 cells. Both αIIb P176H and β3 C547G severely affected αIIbβ3 expression, whereas αIIb P943A had only a partial effect on expression and no effect on DTT-induced fibrinogen binding. We were not surprised that the latter variant did not have a severe effect on expression or function because it has an MAF (0.46%) that is much higher than the MAFs of the other GT-causing variants.To estimate the percentage of the 114 novel identified variants that are likely to be deleterious we used 3 different algorithms, CADD, Polyphen 2-HDVI, and SIFT. The algorithms showed moderate concordance in their rankings of the likelihood that a variant is deleterious. To compare their predictive powers, we performed receiver operating characteristic (ROC) analysis based on their ability to discriminate confirmed GT missense variants (positive controls) from alloantigens (negative controls); the area under the curve (AUC) values were 0.91, 0.88, and 0.90, respectively. At cutoff values that achieved greater than 95% sensitivity for each algorithm: 1) the specificity values were 75%, 65%, and 60%, and 2) the percentages of novel αIIb+β3 missense variants predicted to be deleterious were 43%, 56%, and 58%. Polyphen 2-HDVI and SIFT identified αIIb P176H and β3 C547G as highly likely to be deleterious and αIIb P943A as much less likely to be deleterious, whereas CADD did not differentiate them in the same way. We conclude that ~1.1% of individuals in the populations studied carry at least one missense variant in αIIb or β3 and that 0.6% carry a variant that might be deleterious and therefore may result in a hemorrhagic GT-like phenotype. The rarity of almost all of the novel missense variants identified indicates that they entered the population recently. Despite having detailed knowledge of the structure and function of αIIbβ3, it is difficult to predict with certainty the impact of any single missense variant. This will pose serious challenges as more individuals undergo WES and WGS; we anticipate that linkage to health record data, as will happen for the UK 100,000 Genomes project, will aid clinical interpretation. Finally, “hypomorphic” gene variants that produce only a partial decrease in expression, such as αIIb P943A, may contribute to the wide variation in αIIbβ3 surface expression observed in the healthy population. DisclosuresNo relevant conflicts of interest to declare.

Two Novel, Co-Inherited, Telomerase Reverse Transcriptase (TERT) Mutations Resulting in Inherited Aplastic Anemia and Telomere Shortening

Inherited aplastic anemia syndromes generally present early in the first decade of life with tri-lineage bone marrow failure. Patients with a more gradual presentation of marrow failure may be missed in later childhood or misdiagnosed as a primary thrombocytopenia or neutropenia syndromes. This family cohort demonstrates a very slow evolution of inherited aplastic anemia thus suggesting that marrow failure with shortened telomere length be considered in the initial work up of unexplained mild to moderate thrombocytopenia and megaloblastic anemia.Patients: A family is described where 3 of 4 children developed aplastic anemia, of varying intensity, over a period of 5-6 years starting at age 10 years. The onset was slow initially with the two oldest children presenting with only mild thrombocytopenia initially (30-40,000 ul/ml) picked up on routine screening at ages 10 yr, female, and 12y, male siblings . They remained stable for a period of two–three years, however with monitoring it became evident that they were developing a mild megaloblastic anemia after 5 years The whole family was screened and an additional child was found to have mild megaloblastic changes and a platelet count of 100,000 ul/ml (9 yr Male). All three affected children demonstrated tri-lineage failure on bone marrow biopsy, but cytogenetic studies and breakage studies were all normal. The parents and a younger sister had normal complete blood counts. There was no history of consanguinity, but detailed family history revealed premature greying of the hair, and pulmonary fibrosis on the paternal side in three generations, although the father is currently unaffected. On the maternal side of the family there is a history of bleeding in maternal aunts, uncles and grandfather, but the mother only has a history of anemia with pregnancy or dieting. On physical exam all patients with aplastic anemia have premature greying of the hair in their twenties but are otherwise normal in appearance without dysmorphic features or skeletal abnormalities.Genetic and Telomere Analysis: Initial workup included analysis for inherited aplastic anemia including Fanconi, Shwachmann Diamond variants, Dyskeratosis Congenita, and Congenital Amegakaryocytosis all of which were negative. With Telomere length (TL) analysis, all three affected patients were found to have Telomere Lengths far less than the first percentile, while the parents and the unaffected sibling were normal for age (Repeat Diagnostics, Vancouver). Additional gene sequencing of WRAP 53, DKC1, NHP2, NOP10, TERC, and TERT was performed on the index case (Ambrey Genetics, Aliso Viejo, CA), and all genes were normal except for TERT, where he was found to be compound heterozygote for two novel TERT mutations in exon 2 at codons F101L and R312W. Family studies revealed that the mother carried the codon R312W mutation, and the father carried the F101L variant. All affected aplastic anemia siblings were also compound heterozygotes and the unaffected sibling had neither mutation. Familial testing confirms that these alterations are on opposite chromosomes (in trans). The codon F101L variant located in exon 2 of the TERT gene, results in phenylalanine being replaced by leucine, an amino acid with highly similar properties. The codon R312W variant is also located in exon 2 of the TERT gene. In this mutation, the arginine at codon 312 is replaced by tryptophan, an amino acid with very dissimilar properties. Neither variant has been previously reported in population based cohorts including: Database of Single Nucleotide Polymorphisms (dbSNP), NHLBI Exome Sequencing Project (ESP), and 1000 Genomes Project. Both mutations are predicted to be deleterious by SIFT in silico analyses. Additional analysis using our qPCR based Telomere Length assay and Telomerase activity confirm the diagnosis and are used to screen for similar TL abnormalities in patients with marrow failure.Conclusion: Although neither variant alone results in anemia in the carrier parent, in combination, these mutations appear to result in aplastic anemia in offspring who are compound heterozygotes. Previous reports of other mutations in exon 2 of the TERT gene can also result in marrow failure. Whole genome analysis and telomerase expression studies currently underway will help to elucidate the presence of modifying genes which may impact the variable phenotype between affected siblings in this family. DisclosuresNo relevant conflicts of interest to declare.