Whole Exome Sequencing in Atrial Fibrillation.

PLoS Genet. 2016 Sep;12(9):e1006284

Authors: Lubitz SA, Brody JA, Bihlmeyer NA, Roselli C, Weng LC, Christophersen IE, Alonso A, Boerwinkle E, Gibbs RA, Bis JC, NHLBI GO Exome Sequencing Project, Cupples LA, Mohler PJ, Nickerson DA, Muzny D, Perez MV, Psaty BM, Soliman EZ, Sotoodehnia N, Lunetta KL, Benjamin EJ, Heckbert SR, Arking DE, Ellinor PT, Lin H

Abstract
Atrial fibrillation (AF) is a morbid and heritable arrhythmia. Over 35 genes have been reported to underlie AF, most of which were described in small candidate gene association studies. Replication remains lacking for most, and therefore the contribution of coding variation to AF susceptibility remains poorly understood. We examined whole exome sequencing data in a large community-based sample of 1,734 individuals with and 9,423 without AF from the Framingham Heart Study, Cardiovascular Health Study, Atherosclerosis Risk in Communities Study, and NHLBI-GO Exome Sequencing Project and meta-analyzed the results. We also examined whether genetic variation was enriched in suspected AF genes (N = 37) in AF cases versus controls. The mean age ranged from 59 to 73 years; 8,656 (78%) were of European ancestry. None of the 99,404 common variants evaluated was significantly associated after adjusting for multiple testing. Among the most significantly associated variants was a common (allele frequency = 86%) missense variant in SYNPO2L (rs3812629, p.Pro707Leu, [odds ratio 1.27, 95% confidence interval 1.13-1.43, P = 6.6x10-5]) which lies at a known AF susceptibility locus and is in linkage disequilibrium with a top marker from prior analyses at the locus. We did not observe significant associations between rare variants and AF in gene-based tests. Individuals with AF did not display any statistically significant enrichment for common or rare coding variation in previously implicated AF genes. In conclusion, we did not observe associations between coding genetic variants and AF, suggesting that large-effect coding variation is not the predominant mechanism underlying AF. A coding variant in SYNPO2L requires further evaluation to determine whether it is causally related to AF. Efforts to identify biologically meaningful coding variation underlying AF may require large sample sizes or populations enriched for large genetic effects.

PMID: 27589061 [PubMed - indexed for MEDLINE]

A genetic risk factor for thrombophilia in a Han Chinese family.

Mol Med Rep. 2017 Apr;15(4):1668-1672

Authors: Sun G, Jia Y, Meng J, Ou M, Zhu P, Cong S, Luo Y, Sui W, Dai Y

Abstract
Thrombophilia is a multifactorial disorder involving environmental and genetic factors. Well‑known factors that result in predisposition to congenital disorders associated with thrombophilia include antithrombin deficiency, protein C and S deficiency, Factor V Leiden mutation, abnormal prothrombin and antiphospholipid syndrome. The present study revealed an association between a mutation of the F2 gene, which codes for coagulation factor II, thrombin, and the risk of thrombophilia in a Han Chinese family, of which four members (I‑2, II‑2, II‑3 and III‑1) had a history of deep venous thromboembolism. The disease was measured in this family using laboratory measurements and computed tomography angiography. To identify the abnormality underlying the increased thrombophilia risk, whole‑exome sequencing technology was used to analyze two affected individuals (II‑2 and III‑1). An exonic missense F2 mutation, T165M (NM_000506:c.C494T:p.T165M;rs5896), was identified from a total of 2,222 and 2,203 genetic variations observed in the two affected individuals, respectively, which were subsequently filtered and confirmed using Sanger sequencing. I‑2, II‑3 and III‑1 shared this mutation with the proband (II‑2), and II‑6 had a heterozygous form of the mutation. This deleterious mutation was not identified in normal controls. The present study may improve understanding of the function of the F2 gene.

PMID: 28259966 [PubMed - indexed for MEDLINE]

Developmental dysplasia of the hip: usefulness of next generation genomic tools for characterizing the underlying genes - a mini review.

Clin Genet. 2016 Jul;90(1):16-20

Authors: Basit S, Hannan MA, Khoshhal KI

Abstract
Developmental dysplasia of the hip (DDH) is one of the most common skeletal anomalies. DDH encompasses a spectrum of the disorder ranging from minor acetabular dysplasia to irreducible dislocation, which may lead to premature arthritis in later life. Involvement of genetic factors underlying DDH became evident when several studies reported chromosomal loci linked to DDH in families with multiple affected individuals. Moreover, using association studies, variants in genes involved in chondrogenesis and joint formation have been shown to be associated with DDH. At least, one study identified a pathogenic variant in the chemokine receptor gene in DDH. No genetic analysis has been reported or carried out in DDH patients from the Middle East. Here, we review the literature related to genetics of DDH and emphasized the usefulness of new generation technologies in identifying genetic variants underlying DDH in consanguineous families.

PMID: 26842108 [PubMed - indexed for MEDLINE]

Unfolding the pathogenesis of scleroderma through genomics and epigenomics.

J Autoimmun. 2017 May 16;:

Authors: Tsou PS, Sawalha AH

Abstract
With unknown etiology, scleroderma (SSc) is a multifaceted disease characterized by immune activation, vascular complications, and excessive fibrosis in internal organs. Genetic studies, including candidate gene association studies, genome-wide association studies, and whole-exome sequencing have supported the notion that while genetic susceptibility to SSc appears to be modest, SSc patients are genetically predisposed to this disease. The strongest genetic association for SSc lies within the MHC region, with loci in HLA-DRB1, HLA-DQB1, HLA-DPB1, and HLA-DOA1 being the most replicated. The non-HLA genes associated with SSc are involved in various functions, with the most robust associations including genes for B and T cell activation and innate immunity. Other pathways include genes involved in extracellular matrix deposition, cytokines, and autophagy. Among these genes, IRF5, STAT4, and CD247 were replicated most frequently while SNPs rs35677470 in DNASE1L3, rs5029939 in TNFAIP3, and rs7574685 in STAT4 have the strongest associations with SSc. In addition to genetic predisposition, it became clear that environmental factors and epigenetic influences also contribute to the development of SSc. Epigenetics, which refers to studies that focus on heritable phenotypes resulting from changes in chromatin structure without affecting the DNA sequence, is one of the most rapidly expanding fields in biomedical research. Indeed extensive epigenetic changes have been described in SSc. Alteration in enzymes and mediators involved in DNA methylation and histone modification, as well as dysregulated non-coding RNA levels all contribute to fibrosis, immune dysregulation, and impaired angiogenesis in this disease. Genes that are affected by epigenetic dysregulation include ones involved in autoimmunity, T cell function and regulation, TGFβ pathway, Wnt pathway, extracellular matrix, and transcription factors governing fibrosis and angiogenesis. In this review, we provide a comprehensive overview of the current findings of SSc genetic susceptibility, followed by an extensive description and a systematic review of epigenetic research that has been carried out to date in SSc. We also summarize the therapeutic potential of drugs that affect epigenetic mechanisms, and outline the future prospective of genomics and epigenomics research in SSc.

PMID: 28526340 [PubMed - as supplied by publisher]

Next-generation sequencing for D47N mutation in Cx50 analysis associated with autosomal dominant congenital cataract in a six-generation Chinese family.

BMC Ophthalmol. 2017 May 19;17(1):73

Authors: Shen C, Wang J, Wu X, Wang F, Liu Y, Guo X, Zhang L, Cao Y, Cao X, Ma H

Abstract
BACKGROUND: Congenital cataract is the most frequent cause of blindness during infancy or early childhood. To date, more than 40 loci associated with congenital cataract have been identified, including at least 26 genes on different chromosomes associated with inherited cataract. This present study aimed to identify the genetic mutation in a six-generation Chinese family affected with congenital cataract.
METHODS: A detailed six-generation Chinese cataract family history and clinical data of the family members were recorded. A total of 27 family members, including 14 affected and 13 unaffected individuals were recruited. Whole exome sequencing was performed to determine the disease-causing mutation. Sanger sequencing was used to confirm the results.
RESULTS: A known missense mutation, c. 139G > A (p. D47N), in Cx50 was identified. This mutation co-segregated with all affected individuals and was not observed in the unaffected family members or in 100 unrelated controls. The homology modeling showed that the structure of the mutant protein was different with that wild-type Cx50.
CONCLUSIONS: The missense mutation c.139G > A in GJA8 gene is associated with autosomal dominant congenital cataract in a six-generation Chinese family. The result of this present study provides further evidence that the p. D47N mutation in CX50 is a hot-spot mutation.

PMID: 28526010 [PubMed - in process]

Mutations in CRADD Result in Reduced Caspase-2-Mediated Neuronal Apoptosis and Cause Megalencephaly with a Rare Lissencephaly Variant.

Am J Hum Genet. 2016 Nov 03;99(5):1117-1129

Authors: Di Donato N, Jean YY, Maga AM, Krewson BD, Shupp AB, Avrutsky MI, Roy A, Collins S, Olds C, Willert RA, Czaja AM, Johnson R, Stover JA, Gottlieb S, Bartholdi D, Rauch A, Goldstein A, Boyd-Kyle V, Aldinger KA, Mirzaa GM, Nissen A, Brigatti KW, Puffenberger EG, Millen KJ, Strauss KA, Dobyns WB, Troy CM, Jinks RN

Abstract
Lissencephaly is a malformation of cortical development typically caused by deficient neuronal migration resulting in cortical thickening and reduced gyration. Here we describe a "thin" lissencephaly (TLIS) variant characterized by megalencephaly, frontal predominant pachygyria, intellectual disability, and seizures. Trio-based whole-exome sequencing and targeted re-sequencing identified recessive mutations of CRADD in six individuals with TLIS from four unrelated families of diverse ethnic backgrounds. CRADD (also known as RAIDD) is a death-domain-containing adaptor protein that oligomerizes with PIDD and caspase-2 to initiate apoptosis. TLIS variants cluster in the CRADD death domain, a platform for interaction with other death-domain-containing proteins including PIDD. Although caspase-2 is expressed in the developing mammalian brain, little is known about its role in cortical development. CRADD/caspase-2 signaling is implicated in neurotrophic factor withdrawal- and amyloid-β-induced dendritic spine collapse and neuronal apoptosis, suggesting a role in cortical sculpting and plasticity. TLIS-associated CRADD variants do not disrupt interactions with caspase-2 or PIDD in co-immunoprecipitation assays, but still abolish CRADD's ability to activate caspase-2, resulting in reduced neuronal apoptosis in vitro. Homozygous Cradd knockout mice display megalencephaly and seizures without obvious defects in cortical lamination, supporting a role for CRADD/caspase-2 signaling in mammalian brain development. Megalencephaly and lissencephaly associated with defective programmed cell death from loss of CRADD function in humans implicate reduced apoptosis as an important pathophysiological mechanism of cortical malformation. Our data suggest that CRADD/caspase-2 signaling is critical for normal gyration of the developing human neocortex and for normal cognitive ability.

PMID: 27773430 [PubMed - indexed for MEDLINE]

Mutations in CIT, encoding citron rho-interacting serine/threonine kinase, cause severe primary microcephaly in humans.

Hum Genet. 2016 Oct;135(10):1191-7

Authors: Shaheen R, Hashem A, Abdel-Salam GM, Al-Fadhli F, Ewida N, Alkuraya FS

Abstract
Primary microcephaly is a clinical phenotype in which the head circumference is significantly reduced at birth due to abnormal brain development, primarily at the cortical level. Despite the marked genetic heterogeneity, most primary microcephaly-linked genes converge on mitosis regulation. Two consanguineous families segregating the phenotype of severe primary microcephaly, spasticity and failure to thrive had overlapping autozygomes in which exome sequencing identified homozygous splicing variants in CIT that segregate with the phenotype within each family. CIT encodes citron, an effector of the Rho signaling that is required for cytokinesis specifically in proliferating neuroprogenitors, as well as for postnatal brain development. In agreement with the critical role assigned to the kinase domain in effecting these biological roles, we show that both splicing variants predict variable disruption of this domain. The striking phenotypic overlap between CIT-mutated individuals and the knockout mice and rats that are specifically deficient in the kinase domain supports the proposed causal link between CIT mutation and primary microcephaly in humans.

PMID: 27503289 [PubMed - indexed for MEDLINE]

Whole-Exome Sequencing Suggests LAMB3 as a Susceptibility Gene for Morbid Obesity.

Diabetes. 2016 Oct;65(10):2980-9

Authors: Jiao H, Kulyté A, Näslund E, Thorell A, Gerdhem P, Kere J, Arner P, Dahlman I

Abstract
Identification of rare sequencing variants with a larger functional impact has the potential to highlight new pathways contributing to obesity. Using whole-exome sequencing followed by genotyping, we have identified a low-frequency coding variant rs2076349 (V527M) in the laminin subunit β3 (LAMB3) gene showing strong association with morbid obesity and thereby risk of type 2 diabetes. We exome-sequenced 200 morbidly obese subjects and 100 control subjects with pooled DNA samples. After several filtering steps, we retained 439 obesity-enriched low-frequency coding variants. Associations between genetic variants and obesity were validated sequentially in two case-control cohorts. In the final analysis of 1,911 morbidly obese and 1,274 control subjects, rs2076349 showed strong association with obesity (P = 9.67 × 10(-5); odds ratio 1.84). This variant was also associated with BMI and fasting serum leptin. Moreover, LAMB3 expression in adipose tissue was positively correlated with BMI and adipose morphology (few but large fat cells). LAMB3 knockdown by small interfering RNA in human adipocytes cultured in vitro inhibited adipogenesis. In conclusion, we identified a previously not reported low-frequency coding variant that was associated with morbid obesity in the LAMB3 gene. This gene may be involved in the development of excess body fat.

PMID: 27431458 [PubMed - indexed for MEDLINE]

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

Am J Hum Genet. 2016 Jul 07;99(1):125-38

Authors: Fenwick AL, Kliszczak M, Cooper F, Murray J, Sanchez-Pulido L, Twigg SR, Goriely A, McGowan SJ, Miller KA, Taylor IB, Logan C, WGS500 Consortium, Bozdogan S, Danda S, Dixon J, Elsayed SM, Elsobky E, Gardham A, Hoffer MJ, Koopmans M, McDonald-McGinn DM, Santen GW, Savarirayan R, de Silva D, Vanakker O, Wall SA, Wilson LC, Yuregir OO, Zackai EH, Ponting CP, Jackson AP, Wilkie AO, Niedzwiedz W, Bicknell LS

Abstract
DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

PMID: 27374770 [PubMed - indexed for MEDLINE]

Platelet-Related Variants Identified by Exomechip Meta-analysis in 157,293 Individuals.

Am J Hum Genet. 2016 Jul 07;99(1):40-55

Authors: Eicher JD, Chami N, Kacprowski T, Nomura A, Chen MH, Yanek LR, Tajuddin SM, Schick UM, Slater AJ, Pankratz N, Polfus L, Schurmann C, Giri A, Brody JA, Lange LA, Manichaikul A, Hill WD, Pazoki R, Elliot P, Evangelou E, Tzoulaki I, Gao H, Vergnaud AC, Mathias RA, Becker DM, Becker LC, Burt A, Crosslin DR, Lyytikäinen LP, Nikus K, Hernesniemi J, Kähönen M, Raitoharju E, Mononen N, Raitakari OT, Lehtimäki T, Cushman M, Zakai NA, Nickerson DA, Raffield LM, Quarells R, Willer CJ, Peloso GM, Abecasis GR, Liu DJ, Global Lipids Genetics Consortium, Deloukas P, Samani NJ, Schunkert H, Erdmann J, CARDIoGRAM Exome Consortium, Myocardial Infarction Genetics Consortium, Fornage M, Richard M, Tardif JC, Rioux JD, Dube MP, de Denus S, Lu Y, Bottinger EP, Loos RJ, Smith AV, Harris TB, Launer LJ, Gudnason V, Velez Edwards DR, Torstenson ES, Liu Y, Tracy RP, Rotter JI, Rich SS, Highland HM, Boerwinkle E, Li J, Lange E, Wilson JG, Mihailov E, Mägi R, Hirschhorn J, Metspalu A, Esko T, Vacchi-Suzzi C, Nalls MA, Zonderman AB, Evans MK, Engström G, Orho-Melander M, Melander O, O'Donoghue ML, Waterworth DM, Wallentin L, White HD, Floyd JS, Bartz TM, Rice KM, Psaty BM, Starr JM, Liewald DC, Hayward C, Deary IJ, Greinacher A, Völker U, Thiele T, Völzke H, van Rooij FJ, Uitterlinden AG, Franco OH, Dehghan A, Edwards TL, Ganesh SK, Kathiresan S, Faraday N, Auer PL, Reiner AP, Lettre G, Johnson AD

Abstract
Platelet production, maintenance, and clearance are tightly controlled processes indicative of platelets' important roles in hemostasis and thrombosis. Platelets are common targets for primary and secondary prevention of several conditions. They are monitored clinically by complete blood counts, specifically with measurements of platelet count (PLT) and mean platelet volume (MPV). Identifying genetic effects on PLT and MPV can provide mechanistic insights into platelet biology and their role in disease. Therefore, we formed the Blood Cell Consortium (BCX) to perform a large-scale meta-analysis of Exomechip association results for PLT and MPV in 157,293 and 57,617 individuals, respectively. Using the low-frequency/rare coding variant-enriched Exomechip genotyping array, we sought to identify genetic variants associated with PLT and MPV. In addition to confirming 47 known PLT and 20 known MPV associations, we identified 32 PLT and 18 MPV associations not previously observed in the literature across the allele frequency spectrum, including rare large effect (FCER1A), low-frequency (IQGAP2, MAP1A, LY75), and common (ZMIZ2, SMG6, PEAR1, ARFGAP3/PACSIN2) variants. Several variants associated with PLT/MPV (PEAR1, MRVI1, PTGES3) were also associated with platelet reactivity. In concurrent BCX analyses, there was overlap of platelet-associated variants with red (MAP1A, TMPRSS6, ZMIZ2) and white (PEAR1, ZMIZ2, LY75) blood cell traits, suggesting common regulatory pathways with shared genetic architecture among these hematopoietic lineages. Our large-scale Exomechip analyses identified previously undocumented associations with platelet traits and further indicate that several complex quantitative hematological, lipid, and cardiovascular traits share genetic factors.

PMID: 27346686 [PubMed - indexed for MEDLINE]

Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis.

Hum Mol Genet. 2017 May 17;:

Authors: Yang C, Xu Y, Yu M, Lee D, Alharti S, Hellen N, Ahmad Shaik N, Banaganapalli B, Sheikh HAM, Ramu E, Przyborski S, Tenin G, Williams S, O'Sullivan J, Al-Radi OO, Atta J, Harding SE, Keavney B, Lako M, Armstrong L

Abstract
Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.

PMID: 28521042 [PubMed - as supplied by publisher]

GeMSTONE: orchestrated prioritization of human germline mutations in the cloud.

Nucleic Acids Res. 2017 May 18;:

Authors: Chen S, Beltrán JF, Esteban-Jurado C, Franch-Expósito S, Castellví-Bel S, Lipkin S, Wei X, Yu H

Abstract
Integrative analysis of whole-genome/exome-sequencing data has been challenging, especially for the non-programming research community, as it requires simultaneously managing a large number of computational tools. Even computational biologists find it unexpectedly difficult to reproduce results from others or optimize their strategies in an end-to-end workflow. We introduce Germline Mutation Scoring Tool fOr Next-generation sEquencing data (GeMSTONE), a cloud-based variant prioritization tool with high-level customization and a comprehensive collection of bioinformatics tools and data libraries (http://gemstone.yulab.org/). GeMSTONE generates and readily accepts a shareable 'recipe' file for each run to either replicate previous results or analyze new data with identical parameters and provides a centralized workflow for prioritizing germline mutations in human disease within a streamlined workflow rather than a pool of program executions.

PMID: 28521008 [PubMed - as supplied by publisher]

VCF.Filter: interactive prioritization of disease-linked genetic variants from sequencing data.

Nucleic Acids Res. 2017 May 17;:

Authors: Müller H, Jimenez-Heredia R, Krolo A, Hirschmugl T, Dmytrus J, Boztug K, Bock C

Abstract
Next generation sequencing is widely used to link genetic variants to diseases, and it has massively accelerated the diagnosis and characterization of rare genetic diseases. After initial bioinformatic data processing, the interactive analysis of genome, exome, and panel sequencing data typically starts from lists of genetic variants in VCF format. Medical geneticists filter and annotate these lists to identify variants that may be relevant for the disease under investigation, or to select variants that are reported in a clinical diagnostics setting. We developed VCF.Filter to facilitate the search for disease-linked variants, providing a standalone Java program with a user-friendly interface for interactive variant filtering and annotation. VCF.Filter allows the user to define a broad range of filtering criteria through a graphical interface. Common workflows such as trio analysis and cohort-based filtering are pre-configured, and more complex analyses can be performed using VCF.Filter's support for custom annotations and filtering criteria. All filtering is documented in the results file, thus providing traceability of the interactive variant prioritization. VCF.Filter is an open source tool that is freely and openly available at http://vcffilter.rarediseases.at.

PMID: 28520890 [PubMed - as supplied by publisher]

Using high-resolution variant frequencies to empower clinical genome interpretation.

Genet Med. 2017 May 18;:

Authors: Whiffin N, Minikel E, Walsh R, O'Donnell-Luria AH, Karczewski K, Ing AY, Barton PJR, Funke B, Cook SA, MacArthur D, Ware JS

Abstract
PurposeWhole-exome and whole-genome sequencing have transformed the discovery of genetic variants that cause human Mendelian disease, but discriminating pathogenic from benign variants remains a daunting challenge. Rarity is recognized as a necessary, although not sufficient, criterion for pathogenicity, but frequency cutoffs used in Mendelian analysis are often arbitrary and overly lenient. Recent very large reference datasets, such as the Exome Aggregation Consortium (ExAC), provide an unprecedented opportunity to obtain robust frequency estimates even for very rare variants.MethodsWe present a statistical framework for the frequency-based filtering of candidate disease-causing variants, accounting for disease prevalence, genetic and allelic heterogeneity, inheritance mode, penetrance, and sampling variance in reference datasets.ResultsUsing the example of cardiomyopathy, we show that our approach reduces by two-thirds the number of candidate variants under consideration in the average exome, without removing true pathogenic variants (false-positive rate<0.001).ConclusionWe outline a statistically robust framework for assessing whether a variant is "too common" to be causative for a Mendelian disorder of interest. We present precomputed allele frequency cutoffs for all variants in the ExAC dataset.GENETICS in MEDICINE advance online publication, 18 May 2017; doi:10.1038/gim.2017.26.

PMID: 28518168 [PubMed - as supplied by publisher]

DNA Sequence Analysis in Clinical Medicine, Proceeding Cautiously.

Front Mol Biosci. 2017;4:24

Authors: Smith M

Abstract
Delineation of underlying genomic and genetic factors in a specific disease may be valuable in establishing a definitive diagnosis and may guide patient management and counseling. In addition, genetic information may be useful in identification of at risk family members. Gene mapping and initial genome sequencing data enabled the development of microarrays to analyze genomic variants. The goal of this review is to consider different generations of sequencing techniques and their application to exome sequencing and whole genome sequencing and their clinical applications. In recent decades, exome sequencing has primarily been used in patient studies. Discussed in some detail, are important measures that have been developed to standardize variant calling and to assess pathogenicity of variants. Examples of cases where exome sequencing has facilitated diagnosis and led to improved medical management are presented. Whole genome sequencing and its clinical relevance are presented particularly in the context of analysis of nucleotide and structural genomic variants in large population studies and in certain patient cohorts. Applications involving analysis of cell free DNA in maternal blood for prenatal diagnosis of specific autosomal trisomies are reviewed. Applications of DNA sequencing to diagnosis and therapeutics of cancer are presented. Also discussed are important recent diagnostic applications of DNA sequencing in cancer, including analysis of tumor derived cell free DNA and exosomes that are present in body fluids. Insights gained into underlying pathogenetic mechanisms of certain complex common diseases, including schizophrenia, macular degeneration, neurodegenerative disease are presented. The relevance of different types of variants, rare, uncommon, and common to disease pathogenesis, and the continuum of causality, are addressed. Pharmogenetic variants detected by DNA sequence analysis are gaining in importance and are particularly relevant to personalized and precision medicine.

PMID: 28516087 [PubMed - in process]

Carrier frequency of Wilson's disease in the Korean population: a DNA-based approach.

J Hum Genet. 2017 May 18;:

Authors: Jang JH, Lee T, Bang S, Kim YE, Cho EH

Abstract
Wilson's disease (WD) is an autosomal recessive disorder caused by ATP7B gene mutation. The frequency of WD is about 1 in 30 000 worldwide. In the present study, we screened 14 835 dried blood spots (DBSs) from asymptomatic Korean neonates and retrospectively reviewed massively parallel sequencing of 1090 control individuals to estimate carrier frequency. TaqMan real-time PCR assays were conducted to detect six mutations that account for 58.3% of mutations in Korean WD patients: c.2333G>T (p.Arg778Leu), c.2621C>T (p.Ala874Val), c.3086C>T (p.Thr1029Ile), c.3247C>T (p.Leu1083Phe), c.3556G>A (p.Gly1186Ser) and c.3809A>G (p.Asn1270Ser). We also retrospectively reviewed data from 1090 individuals with various indications other than WD for whom whole-exome or panel sequencing data were available. Mutant allele frequency based on the six most common mutations was 0.0067 among the total of 14 835 DBSs screened. Given that these six mutations account for 58.3% of mutations in Korean WD patients, the corrected mutant allele frequency is 0.0115 (95% confidence interval (CI): 0.0103-0.0128). Corresponding incidence (q(2)) and carrier frequency (2pq) were estimated to be 1:7561 and 1:44, respectively. In retrospective data analysis of 1090 control individuals, allele frequency of pathogenic or likely pathogenic variants was 0.0096 (95% CI: 0.0063-0.0146). Corresponding carrier frequency was estimated to be 1:53. Estimated allele and carrier frequencies based on DNA screening were relatively higher than those reported previously based on clinical ascertainment.Journal of Human Genetics advance online publication, 18 May 2017; doi:10.1038/jhg.2017.49.

PMID: 28515472 [PubMed - as supplied by publisher]

Identification of somatic mutations using whole-exome sequencing in Korean patients with acute myeloid leukemia.

BMC Med Genet. 2017 Mar 01;18(1):23

Authors: Heo SG, Koh Y, Kim JK, Jung J, Kim HL, Yoon SS, Park JW

Abstract
BACKGROUND: Acute myeloid leukemia (AML) is a biologically and clinically heterogeneous cancer of the bone marrow that is characterized by the rapid growth of abnormal myeloid cells.
METHODS: We performed a mutational analysis to identify AML somatic mutations using the whole-exome sequencing data of 36 tumor-normal sample pairs from Korean patients with de novo AML. We explored the functional impact of the genes identified in the mutational analyses through an integrated Gene Ontology (GO) and pathway analysis.
RESULTS: A total of 11 genes, including NEFH (p = 6.27 × 10(-13) and q = 1.18 × 10(-8)) and TMPRSS13 (p = 1.40 × 10(-10) and q = 1.32 × 10(-6)), also demonstrated q values less than 0.1 in 36 Korean AML patients. Five out of the 11 novel genes have previously been reported to be associated with other cancers. Two gene mutations, CEBPA (p = 5.22 × 10(-5)) and ATXN3 (p = 9.75 × 10(-4)), showed statistical significance exclusively in the M2 and M3 subtypes of the French-American-British classifications, respectively. A total of 501 genes harbored 478 missense, 22 nonsense, 93 frameshift indels, and/or three stop codon deletions and these gene mutations significantly enriched GO terms for signal transduction (GO:0007165, p = 1.77 × 10(-3)), plasma membrane (GO:0005886, p = 3.07 × 10(-4)), and scaffold protein binding (GO:0097110, p = 8.65 × 10(-4)). The mitogen-activated protein kinase (hsa04010, 7.67 × 10(-4)) was the most enriched Kyoto Encyclopedia of Genes and Genomes pathway.
CONCLUSIONS: Morphological AML subtypes may in part reflect subtype specific patterns of genomic alterations. Following validation, future studies to evaluate the usefulness of these genes in genetic testing for the early diagnosis and prognostic prediction of AML patients would be worthwhile.

PMID: 28249600 [PubMed - indexed for MEDLINE]

Identification of novel candidate variants including COL6A6 polymorphisms in early-onset atopic dermatitis using whole-exome sequencing.

BMC Med Genet. 2017 Jan 26;18(1):8

Authors: Heo WI, Park KY, Jin T, Lee MK, Kim M, Choi EH, Kim HS, Bae JM, Moon NJ, Seo SJ

Abstract
BACKGROUND: The prevalence of atopic dermatitis has increased over the last 10 years. Atopic dermatitis tends to run in families and commonly begins to manifest in childhood. The prevalence of atopic dermatitis is as high as 20% in children. Thus, early diagnosis and treatment of atopic dermatitis are important. Understanding its genetic basis is also needed to facilitate early detection.
METHODS: To identify family-specific candidate genetic variants associated with early-onset atopic dermatitis in Koreans, we carried out whole-exome sequencing of three separate families with this condition. Additional validation was performed in 112 AD patients and 61 controls using Sanger sequencing.
RESULTS: We focused on both common functional variants with a minor allele frequency higher than 1% and rare variants with a minor allele frequency less than 1%. The relevance of the respective variants was supported by a program that could predict whether the mutations resulted in damaged protein function. Fourteen overlapping genes were identified during exome sequencing. Three variants of the COL6A6 gene appeared in all three families and were in close proximity to atopic dermatitis-related loci on chromosome 3q21. The homozygous frequency for the rs16830494 minor allele (AA) and the rs59021909 (TT) allele and the rs200963433 heterozygous (CT) frequency were all higher in AD cases compared to controls in a population-based case-control study.
CONCLUSION: Identifying family-specific COL6A6 polymorphisms and genetic variants of other candidate genes associated with AD using WES is a novel approach. Our study suggests that COL6A6 variants may be risk factors for atopic dermatitis. This study provides a genetic basis for early-onset AD diagnosis in Korean patients and the development of new therapies.
TRIAL REGISTRATION: Trial registration number: IRB NO. C2008030 (133); Name of registry: The collection research of clinical data and patient blood to identify genetic and protein biomarker of atopic dermatitis; Date of registration: 09-July-2008.
TRIAL REGISTRATION NUMBER: IRB NO. C2015258 (1716); Name of registry: The collection study of patient blood and clinical data for the development of the prognosis prediction and early diagnosis of atopic dermatitis; Date of registration: 15-jan-2016.

PMID: 28125976 [PubMed - indexed for MEDLINE]

Identification of a de novo variant in CHUK in a patient with an EEC/AEC syndrome-like phenotype and hypogammaglobulinemia.

Am J Med Genet A. 2017 May 17;:

Authors: Khandelwal KD, Ockeloen CW, Venselaar H, Boulanger C, Brichard B, Sokal E, Pfundt R, Rinne T, van Beusekom E, Bloemen M, Vriend G, Revencu N, Carels CEL, van Bokhoven H, Zhou H

Abstract
The cardinal features of Ectrodactyly, Ectodermal dysplasia, Cleft lip/palate (EEC), and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndromes are ectodermal dysplasia (ED), orofacial clefting, and limb anomalies. EEC and AEC are caused by heterozygous mutations in the transcription factor p63 encoded by TP63. Here, we report a patient with an EEC/AEC syndrome-like phenotype, including ankyloblepharon, ED, cleft palate, ectrodactyly, syndactyly, additional hypogammaglobulinemia, and growth delay. Neither pathogenic mutations in TP63 nor CNVs at the TP63 locus were identified. Exome sequencing revealed de novo heterozygous variants in CHUK (conserved helix-loop-helix ubiquitous kinase), PTGER4, and IFIT2. While the variant in PTGER4 might contribute to the immunodeficiency and growth delay, the variant in CHUK appeared to be most relevant for the EEC/AEC-like phenotype. CHUK is a direct target gene of p63 and encodes a component of the IKK complex that plays a key role in NF-κB pathway activation. The identified CHUK variant (g.101980394T>C; c.425A>G; p.His142Arg) is located in the kinase domain which is responsible for the phosphorylation activity of the protein. The variant may affect CHUK function and thus contribute to the disease phenotype in three ways: (1) the variant exhibits a dominant negative effect and results in an inactive IKK complex that affects the canonical NF-κB pathway; (2) it affects the feedback loop of the canonical and non-canonical NF-κB pathways that are CHUK kinase activity-dependent; and (3) it disrupts NF-κB independent epidermal development that is often p63-dependent. Therefore, we propose that the heterozygous CHUK variant is highly likely to be causative to the EEC/AEC-like and additional hypogammaglobulinemia phenotypes in the patient presented here.

PMID: 28513979 [PubMed - as supplied by publisher]

Exome sequencing of two Italian pedigrees with non-isolated Chiari malformation type I reveals candidate genes for cranio-facial development.

Eur J Hum Genet. 2017 May 17;:

Authors: Merello E, Tattini L, Magi A, Accogli A, Piatelli G, Pavanello M, Tortora D, Cama A, Kibar Z, Capra V, De Marco P

Abstract
Chiari malformation type I (CMI) is a congenital abnormality of the cranio-cerebral junction with an estimated incidence of 1 in 1280. CMI is characterized by underdevelopment of the occipital bone and posterior fossa (PF) and consequent cerebellar tonsil herniation. The presence for a genetic basis to CMI is supported by many lines of evidence. The cellular and molecular mechanisms leading to CM1 are poorly understood. The occipital bone formation is dependent on complex interactions between genes and molecules with pathologies resulting from disruption of this delicate process. Whole-exome sequencing of affected and not affected individuals from two Italian families with non-isolated CMI was undertaken. Single-nucleotide and short insertion-deletion variants were prioritized using KGGSeq knowledge-based platform. We identified three heterozygous missense variants: DKK1 c.121G>A (p.(A41T)) in the first family, and the LRP4 c.2552C>G (p.(T851R)) and BMP1 c.941G>A (p.(R314H)) in the second family. The variants were located at highly conserved residues, segregated with the disease, but they were not observed in 100 unaffected in-house controls. DKK1 encodes for a potent soluble WNT inhibitor that binds to LRP5 and LRP6, and is itself regulated by bone morphogenetic proteins (BMPs). DKK1 is required for embryonic head development and patterning. LRP4 is a novel osteoblast expressed receptor for DKK1 and a WNT and BMP 4 pathways integrator. Screening of DKK1 in a cohort of 65 CMI sporadic patients identified another missense variant, the c.359G>T (p.(R120L)), in two unrelated patients. These findings implicated the WNT signaling in the correct development of the cranial mesenchyme originating the PF.European Journal of Human Genetics advance online publication, 17 May 2017; doi:10.1038/ejhg.2017.71.

PMID: 28513615 [PubMed - as supplied by publisher]