NHLBI Exome Sequencing Project Research Articles

An adaptive test for meta-analysis of rare variant association studies.

Single genome-wide studies may be underpowered to detect trait-associated rare variants with moderate or weak effect sizes. As a viable alternative, meta-analysis is widely used to increase power by combining different studies. The power of meta-analysis critically depends on the underlying association patterns and heterogeneity levels, which are unknown and vary from locus to locus. However, existing methods mainly focus on one or only a few combinations of the association pattern and heterogeneity level, thus may lose power in many situations. To address this issue, we propose a general and unified framework by combining a class of tests including and beyond some existing ones, leading to high power across a wide range of scenarios. We demonstrate that the proposed test is more powerful than some existing methods in simulation studies, then show their performance with the NHLBI Exome-Sequencing Project (ESP) data. One gene (B4GALNT2) was found by our proposed test, but not by others, to be statistically significantly associated with plasma triglyceride. The signal was driven by African-ancestry subjects but it was previously reported to be associated with coronary artery disease among European-ancestry subjects. We implemented our method in an R package aSPUmeta, publicly available at https://github.com/ytzhong/metaRV and will be on CRAN soon.

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

MON-376 A Novel Mutation in VHL Gene as Posible Cause of Metastasic Pheocromocytoma

A 62-year-old woman with no family history of hereditary diseases presented to the ER department in 2008 with the diagnosis of a subarachnoid hemorrhage that required urgent decompressive craniotomy. On February 2011, due to resistant hypertension, an abdominal CT scan was performed, revealing a left adrenal gland-dependent tumor of 70x86 mm, with additional increased normetanephrine levels in a 24-hour urine sample. Laparoscopic left adrenalectomy was performed on June 3, 2011, with a histological report of pheochromocytoma. It was also documented an elevation of iPTH (301 pg/ml) with corrected calcium of 11 mg/dL and phosphorus of 2.7 mg/dL. Parathyroidectomy was performed with the report of parathyroid adenoma. On January 2015, an abdominal CT scan was performed, revealing recurrence of the pheochromocytoma at the surgical site. A MIBG PET-CT confirmed the recurrence and detected a left retroperitoneal lesion close to the tail of the pancreas. Metastasectomy of the retroperitoneal lesions, distal pancreatectomy, and dissection of tumor adjacent to left kidney were performed with a histopathologic report of pheochromocytoma with nest pattern and extensive necrosis, negative margins, and three positive lymph nodes. During follow-up a significant increase of normetanephrine in 24-hour urine sample was documented (3 months after surgery). A Ga-DOTATOC PET-CT revealed the presence of hepatic metastases. In the 131-MIBG SPECT-CT performed one year later, a decreased height of C3 vertebral body with an abnormal concentration of the radiopharmaceutical in relation to secondary deposit was also found. On August 2017 the patient was treated with a therapeutic dose of 131-MIBG, achieving complete biochemical remission during 14 months of follow-up. On March 2018 an Invitae Multi-Cancer Panel reported the germline variant C. 416C > G (P. Ser139Cys) related to VHL without mutations in other pathogenic genes related to pheochromocytoma. VHL c.416C>G (at the cDNA level ) is a germline variant of Von Hipple Lindau (VHL) disease, also known as p.Ser139Cys (S139C) at the protein level. It results in the change of a Serine to a Cysteine (TCT>TGT), located in 3p25.3. This variant has been reported in population databases (rs587780732, ExAC 0.06%) and related to familiar erythrocytosis, monogenic diabetes (likely benign) and a type of autosomal recessive A1 deafness (uncertain significance); however, to our knowledge it has not been previously published in the literature as a pathogenic nor benign form of the VHL disease. It was also not observed in the NHLBI Exome Sequencing Project that included 6,500 individuals of European and African American ancestry, indicating it is not a common benign variant in these populations. Because of the lack of evidence to determine its role in disease, to date, it is considered a variant of uncertain significance.

Exome Sequencing Identifies TENM4 as a Novel Candidate Gene for Schizophrenia in the SCZD2 Locus at 11q14-21.

Schizophrenia is a complex psychiatric disorder with high genetic heterogeneity, however, the contribution of rare mutations to the disease etiology remains to be further elucidated. We herein performed exome sequencing in a Han Chinese schizophrenia family and identified a missense mutation (c.6724C>T, p.R2242C) in the teneurin transmembrane protein 4 (TENM4) gene in the SCZD2 locus, a region previously linked to schizophrenia at 11q14-21. The mutation was confirmed to co-segregate with the schizophrenia phenotype in the family. Subsequent investigation of TENM4 exons 31, 32, and 33 adjacent to the p.R2242C mutation revealed two additional missense mutations in 120 sporadic schizophrenic patients. Residues mutated in these mutations, which are predicted to be deleterious to protein function, were highly conserved among vertebrates. These rare mutations were not detected in 1000 Genomes, NHLBI Exome Sequencing Project databases, or our in-house 1136 non-schizophrenic control exomes. Analysis of RNA-Seq data showed that TENM4 is expressed in the brain with high abundance and specificity. In line with the important role of TENM4 in central nervous system development, our findings suggested that increased rare variants in TENM4 could be associated with schizophrenia, and thus TENM4 could be a novel candidate gene for schizophrenia in the SCZD2 locus.

Siblings with Rare Hemophilia a Genetic Mutation and Normal Factor VIII Levels

BACKGROUNDHemophilia A is an X-linked inherited disorder which affects 1 in 5,000 males and is caused by mutations in the factor VIII (FVIII) gene. Hemophilia is typically diagnosed by measurement of FVIII procoagulant (FVIII:C) activity. Molecular genetic testing of the FVIII gene identifies pathogenic variants in as many as 98% of individuals with hemophilia A. The specific genetic test performed varies and must take into account the severity of hemophilia and the gene affected. Hemophilia A families with severe disease may have genetic analysis for intron 22 and intron 1 mutation performed followed by DNA sequencing if intron analysis is uninformative. Families with mild to moderate disease often need upfront gene sequencing for diagnosis.OBJECTIVETo describe a case series of siblings with FVIII gene mutation having normal Factor VIII activity levels, with grandfather found to have similar gene mutationDESIGN/METHOD: Case SeriesRESULTSCase 1An 8 year old boy with presumed Von Willebrand disease (VWD) was seen in our clinic for evaluation for bleeding disorders. Testing prior to being seen in clinic showed Factor VIII level of 37%, Von Willebrand factor activity of 53% and Von Willebrand factor antigen of 103%. He had post-operative hemorrhage after tonsillectomy and adenoidectomy at 3 years of age, as well as frequent epistaxis. Family history was significant for sibling with epistaxis, a maternal grandfather with VWD and sister with menorrhagia. Subsequently, his maternal grandfather was found to have mild hemophilia A based on genetic testing which brought into question the child's diagnosis of mild Type 1 VWD. Repeat testing showed Factor VIII 64%, Von Willebrand factor antigen 126% and Ristocetin cofactor activity 93%. Genetic testing revealed he was hemizygous for the pathogenic variant, c.1621A>T (p.Thr541Ser), in the FVIII gene. He is doing well with normal Factor VIII levels, without significant bleeding episodes and remains on DDAVP as needed.Case 2A 7 year old boy with presumed history of type 1 VWD was seen in our clinic for evaluation for a bleeding disorder. He had a history of cephalohematoma after skull fracture, as well as frequent epistaxis. Testing prior to being seen in our clinic showed Von Willebrand factor antigen of 87%, Von Willebrand factor activity of 69%, and Factor VIII activity of 32%. Family history was as reported for his sibling (Case 1). In our clinic, he had genetic testing performed that was not consistent with VWD Type 2N (Normandy phenotype). Repeat labs in our clinic showed Von Willebrand factor antigen of 100%, Von Willebrand factor activity of 92%, and Factor VIII activity of 50%. He was treated with Von Willebrand factor replacement prior to planned foot surgery as well as DDAVP. Subsequent to identification of a FVIII gene mutation in his maternal grandfather and brother, testing revealed he was hemizygous for the same mutation detailed in Case 1. He is doing well with normal Factor VIII levels, no significant bleeding and remains on DDAVP as needed for bleeding.CONCLUSIONThe c.1621A>T (p.Thr541Ser) variant which occurs in exon 11 of the FVIII gene, has been is known to cause mild hemophilia A. The Thr541Ser substitution is rare and is present in ~0.009% of the NHLBI exome sequencing project. This is a unique case series of siblings with a rare mutation having normal Factor VIII levels and grandfather with similar mutation who was eventually also diagnosed with mild hemophilia A. The underlying gene mutation is an important but not sole determinant of residual FVIII:C in hemophilia A patients. As our understanding of pathophysiologic process of causative genetic event and baseline FVIII:C in patients is limited, this case series highlights the current diagnostic challenges in the mild Hemophilia A population and the importance of appropriate diagnostic techniques to avoid delayed diagnosis. Correct identification of mutations also provides the opportunity to define molecular consequences and identify residues important for factor VIII activity. DisclosuresCarpenter:Bayer: Honoraria; Kedrion Biopharmaceuticals: Consultancy; Nationwide Children's Hospital: Speakers Bureau; Kane County State's Attorney: Consultancy; 4th Judicial District Attorney's Office- Colorado: Consultancy; Kedrion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; HEMA Biologics: Consultancy; CSL Behring: Speakers Bureau; National Hemophilia Foundation (Impact Education): Speakers Bureau; Genentech Incorporated: Membership on an entity's Board of Directors or advisory committees; American Academy of Pediatrics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk Pharmaceuticals, Inc: Consultancy; Novo Nordisk: Consultancy.

Abstract B13: BLM, FOXO3, FOXK2, FOXM1, FOXR1 genes as therapeutic targets to neuroblastoma

Abstract Due to heterogeneity of the neuroblastoma (NB) tumor, next-generation personalized medicine may identify therapeutic targets. Gene expression alterations of BLM (Bloom Syndrome RecQ Like Helicase) and FOX (Forkhead-box) family have been associated with an increased risk for cancer and resistance to chemotherapy drugs. Considering the potential implication of these genes on the clinical management of NB, we correlated clinical parameters, such as survival and treatments with genetic profiles of 11 NB patients stage 3 and 4 with MYCN-amplified (4 patients) or MYCN-non-amplified (7 patients) and one patient stage 2B and MYCN-non-amplified. Patients were treated with doxorubicin, etoposide, topotecan, cyclophosphamide, vincristine, and carboplatin. Targeted DNA sequencing, methylation array, and mRNA expression were performed on tumor and blood peripheral of these patients. Multiplex ligation-dependent probe amplification (MLPA). All variants considered important were validated by Sanger sequencing. Polymorphisms were identified using dbSNP138, 1000 genomes Project Consortium and NHLBI Exome Sequencing Project (ESP) databases with minor allele frequencies recorded from each database. In this study, we observed in a patient with 17 months, diagnosed with MYCN-non amplified (11q-,+17p,+9p) stage 3 NB, a variant in FOXO3 gene (rs201947198) and BLM gene (rs28384991) with low frequency in the population (0.001% and 1%, respectively). FOXO3 and BLM gene expressions were decreased while FOXM1 gene expression was increased. This patient received chemotherapy and 2 years after the end of therapy, NB recurred locally. Six years after the diagnosis, the patient is still alive and receives chemotherapy. In other case, we found a patient of 13 months, diagnosed with MYCN amplified (+2p,+3p,+17q) stage 3 NB, three synonymous variants in BLM gene (rs2227933,rs2227934,rs1063147). These variants have been shown to affect protein levels and function. We also identified an important variant in FOXO3 gene (rs 111556510) with low frequency in the population. FOXM1 mutation was not found in this patient and FOXM1 RNAm was increased. This patient received chemotherapy and despite the treatment, died 6 months after the diagnosis. As relapse with a second event, we found two variants in BLM gene (rs2227935 and rs7167216), in a patient diagnosed at 6 months, stage 2B, MYCN-non-amplified (+2p;+3p;-11q;+17) NB, who received chemotherapy but has recurred three times at the same local site, despite the treatment. These variants are polymorphism low frequency and databases of the prediction algorithms, considered the SNP p.P868L damage. The variant identified in FOXR1 gene (c.C517G:p.R173G). This variant has not been described to NB. Further, hypermethylation in the promoter region of FOXK2 was correlated with decreased FOXK2 RNAm. Many studies have shown that epigenetic silencing of FOXK2 gene increases resistance to chemotherapy drugs. Other patients (3 MYCN-amplified and 6 MYCN-non-amplified) were found only polymorphisms synonymous in the BLM gene (rs2227933, rs2227934, and rs1063147). Moreover, variants or alterations in FOXO3, FOXR1, FOXK2 expression were not found. However, FOXM1 expression was increased in these patients. Conclusion: Many studies have shown that personalized oncology plays an important role in treatment of cancer patients. In this study, we demonstrated that variants in FOXO3, FOXR1 and BLM genes, FOXM1 expression and silencing epigenetic of FOXK2 found in 3/12 patients with refractory relapse disease may be candidate targets for personalized therapy in neuroblastoma. Citation Format: Thamiris Magalhaes Gimenez, Nathalia Halley Neves, Andreia Rangel Santos, Fabio A. Marchi, Leslie Kulikowski, Lilian M. Cristofani, Estela M. Novak, Vicente Odone Filho. BLM, FOXO3, FOXK2, FOXM1, FOXR1 genes as therapeutic targets to neuroblastoma [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B13.

CDK10 Mutations in Humans and Mice Cause Severe Growth Retardation, Spine Malformations, and Developmental Delays

In five separate families, we identified nine individuals affected by a previously unidentified syndrome characterized by growth retardation, spine malformation, facial dysmorphisms, and developmental delays. Using homozygosity mapping, array CGH, and exome sequencing, we uncovered bi-allelic loss-of-function CDK10 mutations segregating with this disease. CDK10 is a protein kinase that partners with cyclin M to phosphorylate substrates such as ETS2 and PKN2 in order to modulate cellular growth. To validate and model the pathogenicity of these CDK10 germline mutations, we generated conditional-knockout mice. Homozygous Cdk10-knockout mice died postnatally with severe growth retardation, skeletal defects, and kidney and lung abnormalities, symptoms that partly resemble the disease's effect in humans. Fibroblasts derived from affected individuals and Cdk10-knockout mouse embryonic fibroblasts (MEFs) proliferated normally; however, Cdk10-knockout MEFs developed longer cilia. Comparative transcriptomic analysis of mutant and wild-type mouse organs revealed lipid metabolic changes consistent with growth impairment and altered ciliogenesis in the absence of CDK10. Our results document the CDK10 loss-of-function phenotype and point to a function for CDK10 in transducing signals received at the primary cilia to sustain embryonic and postnatal development.

HIPred: an integrative approach to predicting haploinsufficient genes.

MotivationA major cause of autosomal dominant disease is haploinsufficiency, whereby a single copy of a gene is not sufficient to maintain the normal function of the gene. A large proportion of existing methods for predicting haploinsufficiency incorporate biological networks, e.g. protein-protein interaction networks that have recently been shown to introduce study bias. As a result, these methods tend to perform best on well-studied genes, but underperform on less studied genes. The advent of large genome sequencing consortia, such as the 1000 genomes project, NHLBI Exome Sequencing Project and the Exome Aggregation Consortium creates an urgent need for unbiased haploinsufficiency prediction methods.ResultsHere, we describe a machine learning approach, called HIPred, that integrates genomic and evolutionary information from ENSEMBL, with functional annotations from the Encyclopaedia of DNA Elements consortium and the NIH Roadmap Epigenomics Project to predict haploinsufficiency, without the study bias described earlier. We benchmark HIPred using several datasets and show that our unbiased method performs as well as, and in most cases, outperforms existing biased algorithms.Availability and ImplementationHIPred scores for all gene identifiers are available at: https://github.com/HAShihab/HIPred.Supplementary information Supplementary data are available at Bioinformatics online.

Guidelines for Large-Scale Sequence-Based Complex Trait Association Studies: Lessons Learned from the NHLBI Exome Sequencing Project

Massively parallel whole-genome sequencing (WGS) data have ushered in a new era in human genetics. These data are now being used to understand the role of rare variants in complex traits and to advance the goals of precision medicine. The technological and computing advances that have enabled us to generate WGS data on thousands of individuals have also outpaced our ability to perform analyses in scientifically and statistically rigorous and thoughtful ways. The past several years have witnessed the application of whole-exome sequencing (WES) to complex traits and diseases. From our analysis of NHLBI Exome Sequencing Project (ESP) data, not only have a number of important disease and complex trait association findings emerged, but our collective experience offers some valuable lessons for WGS initiatives. These include caveats associated with generating automated pipelines for quality control and analysis of rare variants; the importance of studying minority populations; sample size requirements and efficient study designs for identifying rare-variant associations; and the significance of incidental findings in population-based genetic research. With the ESP as an example, we offer guidance and a framework on how to conduct a large-scale association study in the era of WGS.

Association Between Absolute Neutrophil Count and Variation at TCIRG1: The NHLBI Exome Sequencing Project.

Neutrophils are a key component of innate immunity. Individuals with low neutrophil count are susceptible to frequent infections. Linkage and association between congenital neutropenia and a single rare missense variant in TCIRG1 have been reported in a single family. Here, we report on nine rare missense variants at evolutionarily conserved sites in TCIRG1 that are associated with lower absolute neutrophil count (ANC; p = 0.005) in 1,058 participants from three cohorts: Atherosclerosis Risk in Communities (ARIC), Coronary Artery Risk Development in Young Adults (CARDIA), and Jackson Heart Study (JHS) of the NHLBI Grand Opportunity Exome Sequencing Project (GO ESP). These results validate the effects of TCIRG1 coding variation on ANC and suggest that this gene may be associated with a spectrum of mild to severe effects on ANC.

Mutation screening of SCN2A in schizophrenia and identification of a novel loss-of-function mutation.

There is a growing body of evidence suggesting a shared genetic susceptibility between many neuropsychiatric disorders, including schizophrenia, autism, intellectual disability (ID) and epilepsy. The sodium channel, voltage-gated type II α subunit gene SCN2A has been shown to exhibit loss-of-function (LoF) mutations in individuals with seizure disorders, ID, autism and schizophrenia. The role of LoF mutations in schizophrenia is still uncertain with only one such mutation identified to date. To seek additional evidence for a role for LoF mutations at SCN2A in schizophrenia we performed mutation screening of the entire coding sequence in 980 schizophrenia cases. Given an absence of LoF mutations in a public exome cohort (ESP6500, N=6503), we did not additionally sequence controls. We identified a novel, nonsense (i.e. stop codon) mutation in one case (E169X) that is absent in 4300 European-American and 2203 African-American individuals from the NHLBI Exome Sequencing Project. This is the second LoF allele identified in a schizophrenia case to date. We also show a novel, missense variant, V1282F, that occurs in two cases and is absent in the control dataset. We argue that very rare, LoF mutations at SCN2A act in a moderately penetrant manner to increase the risk of developing several neuropsychiatric disorders including seizure disorders, ID, autism and schizophrenia.

In Silico Analysis of FMR1 Gene Missense SNPs.

The FMR1 gene, a member of the fragile X-related gene family, is responsible for fragile X syndrome (FXS). Missense single-nucleotide polymorphisms (SNPs) are responsible for many complex diseases. The effect of FMR1 gene missense SNPs is unknown. The aim of this study, using in silico techniques, was to analyze all known missense mutations that can affect the functionality of the FMR1 gene, leading to mental retardation (MR) and FXS. Data on the human FMR1 gene were collected from the Ensembl database (release 81), National Centre for Biological Information dbSNP Short Genetic Variations database, 1000 Genomes Browser, and NHLBI Exome Sequencing Project Exome Variant Server. In silico analysis was then performed. One hundred-twenty different missense SNPs of the FMR1 gene were determined. Of these, 11.66% of the FMR1 gene missense SNPs were in highly conserved domains, and 83.33% were in domains with high variety. The results of the in silico prediction analysis showed that 31.66% of the FMR1 gene SNPs were disease related and that 50% of SNPs had a pathogenic effect. The results of the structural and functional analysis revealed that although the R138Q mutation did not seem to have a damaging effect on the protein, the G266E and I304N SNPs appeared to disturb the interaction between the domains and affect the function of the protein. This is the first study to analyze all missense SNPs of the FMR1 gene. The results indicate the applicability of a bioinformatics approach to FXS and other FMR1-related diseases. I think that the analysis of FMR1 gene missense SNPs using bioinformatics methods would help diagnosis of FXS and other FMR1-related diseases.

Rare variant associations with waist-to-hip ratio in European-American and African-American women from the NHLBI-Exome Sequencing Project.

Waist-to-hip ratio (WHR), a relative comparison of waist and hip circumferences, is an easily accessible measurement of body fat distribution, in particular central abdominal fat. A high WHR indicates more intra-abdominal fat deposition and is an established risk factor for cardiovascular disease and type 2 diabetes. Recent genome-wide association studies have identified numerous common genetic loci influencing WHR, but the contributions of rare variants have not been previously reported. We investigated rare variant associations with WHR in 1510 European-American and 1186 African-American women from the National Heart, Lung, and Blood Institute-Exome Sequencing Project. Association analysis was performed on the gene level using several rare variant association methods. The strongest association was observed for rare variants in IKBKB (P=4.0 × 10(-8)) in European-Americans, where rare variants in this gene are predicted to decrease WHRs. The activation of the IKBKB gene is involved in inflammatory processes and insulin resistance, which may affect normal food intake and body weight and shape. Meanwhile, aggregation of rare variants in COBLL1, previously found to harbor common variants associated with WHR and fasting insulin, were nominally associated (P=2.23 × 10(-4)) with higher WHR in European-Americans. However, these significant results are not shared between African-Americans and European-Americans that may be due to differences in the allelic architecture of the two populations and the small sample sizes. Our study indicates that the combined effect of rare variants contribute to the inter-individual variation in fat distribution through the regulation of insulin response.

Rare variant associations with waist-to-hip ratio in European-American and African-American women from the NHLBI-Exome Sequencing Project

Rare variant associations with waist-to-hip ratio in European-American and African-American women from the NHLBI-Exome Sequencing Project

Diagnostic yield of molecular autopsy in patients with sudden arrhythmic death syndrome using targeted exome sequencing.

The targeted genetic screening of Sudden Arrhythmic Death Syndrome (SADS) probands in a molecular autopsy has a diagnostic yield of up to 35%. Exome sequencing has the potential to improve this yield. The primary aim of this study is to examine the feasibility and diagnostic utility of targeted exome screening in SADS victims, utilizing familial clinical screening whenever possible. To determine the feasibility and diagnostic yield of targeted exome sequencing deoxyribonucleic acid (DNA) was isolated from 59 SADS victims (mean age 25 years, range 1-51 years). Targeted exome sequencing of 135 genes associated with cardiomyopathies and ion channelopathies was performed on the Illumina HiSeq2000 platform. Non-synonymous, loss-of-function, and splice-site variants with a minor allele frequency <0.02% in the NHLBI exome sequencing project and an internal set of control exomes were prioritized for analysis followed by <0.5% frequency threshold secondary analysis. First-degree relatives were offered clinical screening for inherited cardiac conditions. Seven probands (12%) carried very rare (<0.02%) or novel non-sense candidate mutations and 10 probands (17%) had previously published rare (0.02-0.5%) candidate mutations-a total yield of 29%. Co-segregation fully confirmed two private SCN5A Na channel mutations. Variants of unknown significance were detected in a further 34% of probands. Molecular autopsy using targeted exome sequencing has a relatively low diagnostic yield of very rare potentially disease causing mutations. Candidate pathogenic variants with a higher frequency in control populations are relatively common and should be interpreted with caution.

Evaluating pathogenic dementia variants in posterior cortical atrophy

Posterior cortical atrophy (PCA) is an understudied visual impairment syndrome most often due to “posterior Alzheimer's disease (AD)” pathology. Case studies detected mutations in PSEN1, PSEN2, GRN, MAPT, and PRNP in subjects with clinical PCA. To detect the frequency and spectrum of mutations in known dementia genes in PCA, we screened 124 European-American subjects with clinical PCA (n = 67) or posterior AD neuropathology (n = 57) for variants in genes implicated in AD, frontotemporal dementia, and prion disease using NeuroX, a customized exome array. Frequencies in PCA of the variants annotated as pathogenic or potentially pathogenic were compared against ∼4300 European-American population controls from the NHLBI Exome Sequencing Project. We identified 2 rare variants not previously reported in PCA, TREM2 Arg47His, and PSEN2 Ser130Leu. No other pathogenic or potentially pathogenic variants were detected in the screened dementia genes. In this first systematic variant screen of a PCA cohort, we report 2 rare mutations in TREM2 and PSEN2, validate our previously reported APOE ε4 association, and demonstrate the utility of NeuroX.

Inference of Super-exponential Human Population Growth via Efficient Computation of the Site Frequency Spectrum for Generalized Models

The site frequency spectrum (SFS) and other genetic summary statistics are at the heart of many population genetic studies. Previous studies have shown that human populations have undergone a recent epoch of fast growth in effective population size. These studies assumed that growth is exponential, and the ensuing models leave an excess amount of extremely rare variants. This suggests that human populations might have experienced a recent growth with speed faster than exponential. Recent studies have introduced a generalized growth model where the growth speed can be faster or slower than exponential. However, only simulation approaches were available for obtaining summary statistics under such generalized models. In this study, we provide expressions to accurately and efficiently evaluate the SFS and other summary statistics under generalized models, which we further implement in a publicly available software. Investigating the power to infer deviation of growth from being exponential, we observed that adequate sample sizes facilitate accurate inference; e.g., a sample of 3000 individuals with the amount of data expected from exome sequencing allows observing and accurately estimating growth with speed deviating by ≥10% from that of exponential. Applying our inference framework to data from the NHLBI Exome Sequencing Project, we found that a model with a generalized growth epoch fits the observed SFS significantly better than the equivalent model with exponential growth (P-value ). The estimated growth speed significantly deviates from exponential (P-value ), with the best-fit estimate being of growth speed 12% faster than exponential.

Abstract 2756: Whole-exome sequencing reveals a novel germline variant in CEBPA-associated familial acute myeloid leukemia: 45-year follow-up of a large family

Abstract Familial acute myeloid leukemia (AML) is rare and linked to germline mutations in RUNX1 or CCAAT/enhancer binding protein-α (CEBPA). We conducted whole-exome sequencing on a large family with AML originally evaluated at NIH in 1969 and uncovered a CEBPA Q311P mutation in all tested, affected members. The Q311P variant, located in the C-terminal bZip domain of CEBPA, was predicted to be highly deleterious by in silico algorithms; the Q311 position was highly conserved among CEBPA orthologs and paralogs. This variant has not been observed in the NHLBI's Exome Sequencing Project or the Broad Institute's Exome Aggregation Consortium. Protein structural modeling suggested that the Q311P mutation alters the ability of the CEBPA dimer to bind DNA. Electrophoretic mobility shift assays showed that the Q311P mutant had attenuated binding to DNA, as predicted by the protein modeling. Consistent with these findings, we found that the Q311P mutation has reduced transactivation of a promoter with a tetramer of CEBP sites, consistent with a loss-of-function mutation. In addition, there was decreased colony survival in fibroblasts with CEBPA Q311P, associated with the clinical radiosensitivity postulated in one carrier. From 45 years of follow-up, we observed incomplete penetrance (46%) of CEBPA Q311P; we did not observe other cancers or leukemias segregating with the mutation. Thus, we present a comprehensive clinical and molecular characterization of a novel AML-associated variant in CEBPA in a NCI family with nearly half a century of follow-up. This study reveals that a germline mutation in the C-terminal bZip domain can alter the ability of CEBPA to bind DNA and reduces transactivation, leading to AML, though with lower penetrance than the canonical N-terminal frameshift germline mutations. Citation Format: Anand Pathak, Katja Seipel, Alexander Pemov, Ramita Dewan, Christina Brown, Sarangan Ravichandran, Brian T. Luke, Meredith Yeager, Richard A. Gatti, Neil Caporaso, John J. Mulvihill, Lynn Goldin, Thomas Muller Pabst, Mary Lou McMaster, Douglas R. Stewart. Whole-exome sequencing reveals a novel germline variant in CEBPA-associated familial acute myeloid leukemia: 45-year follow-up of a large family. [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 2756. doi:10.1158/1538-7445.AM2015-2756

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.

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.