Tobacco and estrogen metabolic polymorphisms and risk of non-small cell lung cancer in women

To explore the potential role for estrogen in lung cancer susceptibility, candidate single-nucleotide polymorphism (SNPs) in tobacco and estrogen metabolism genes were evaluated. Population-based cases (n = 504) included women aged 18-74, diagnosed with NSCLC in metropolitan Detroit between November 2001 and October 2005. Population-based controls (n = 527) were identified through random digit dialing and matched on race and age. Eleven SNPs in 10 different genes were examined in relation to risk: CYP1A1 Msp1, CYP1A1 Ile462 Val, CYP1B1 Leu432Val, CYP17, CYP19A1, XRCC1 Gln399 Arg, COMT Val158Met, NQO1 Pro187Ser, GSTM1, GSTT1 and GSTP1 Ile105Val. Lung cancer risk associated with individual SNPs was seen for GSTP1 [A allele; odds ratio (OR) = 1.85; 95% confidence interval (CI), 1.04-3.27] and XRCC1 (A/A genotype; OR = 1.68; 95% CI, 1.01-2.79) in white women and CYP1B1 (G allele; OR = 11.1; 95% CI, 1.18-104) in black women smokers. White women smokers carrying two risk genotypes at the following loci were at increased risk of lung cancer compared with individuals not carrying risk alleles at these loci: CYP17 and GSTM1, COMT and GSTM1, CYP17 and GSTT1, XRCC1 and GSTP1, CYP1B1 and XRCC1 and COMT and XRCC1. The most parsimonious model of lung cancer risk in white smoking women included age, family history of lung cancer, history of chronic lung disease, pack-years, body mass index, XRCC1 A/A genotype, GSTM1 null and COMT A/G or G/G genotype. These findings support the need for continued study of estrogen in relation to lung cancer risk. Polymorphisms in the tobacco metabolism, estrogen metabolism and DNA repair pathways will be useful in developing more predictive models of individual risk. © The Author 2009. Published by Oxford University Press. All rights reserved

Improving genetic diagnosis in Mendelian disease with transcriptome sequencing

Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approachesThis project was supported by funding from the Broad Institute’s BroadIgnite and Broadnext10 programs. B.B.C. is supported by the NIH GM096911 training grant. T.T. is supported by the Academy of Finland, the Finnish Cultural Foundation, the Orion-Farmos Research Foundation, and the Emil Aaltonen Foundation. M.L. is supported by the Australian NHMRC (National Health and Medical Research Council) CJ Martin Fellowship, the Australian American Association Sir Keith Murdoch Fellowship, and a Muscular Dystrophy Association/American Association of Neuromuscular and Electrodiagnostic Medicine (MDA/AANEM) development grant. L.B.W., S.A.S., N.G.L., N.F.C., K.N.N., and E.C.O. are supported by the NHMRC of Australia (1080587, 1075451, 1002147, 1113531, 1022707, 1031893, and 1090428). K.J.K. is supported by a National Institute of General Medical Sciences (NIGMS) fellowship grant (F32GM115208). A.H.O.-L. is supported by an NIGMS fellowship grant (4T32GM007748). A.H.B. is supported by the NIH R01 HD075802 and R01 AR044345 and by MDA383249 from the Muscular Dystrophy Association. P.B.K., E.E., and H.K.M. are supported by NIH R01NS080929. Funding relevant to this research includes fellowship support of S.T.C. and a project grant supporting an Australian-wide program about gene discovery in inherited neuromuscular disorders performed in collaboration with D.G.M. [NHMRC APP1048816 (2013–2017) and NHMRC APP1080587 (2015–2019)]. The Broad CMG was funded by the National Human Genome Research Institute (NHGRI), the National Eye Institute, and the National Heart, Lung, and Blood Institute (NHLBI) grant UM1 HG008900 to D.G.M. and H. Rehm. The GTEx project was supported by the Common Fund of the Office of the Director of the NIH (http://commonfund.nih.gov/GTEx). Additional funds were provided by the National Cancer Institute (NCI), NHGRI, NHLBI, National Institute on Drug Abuse (NIDA), National Institute of Mental Health (NIMH), and National Institute of Neurological Disorders and Stroke (NINDS). Donors were enrolled at Biospecimen Source Sites that were funded by NCI/Science Applications International Corporation (SAIC)–Frederick Inc. (SAIC-F) subcontracts to the National Disease Research Interchange (10XS170) and the Roswell Park Cancer Institute (10XS171). The Laboratory, Data Analysis, and Coordinating Center (LDACC) was funded through a contract (HHSN268201000029C) to the Broad Institute Inc. Biorepository operations were funded through an SAIC-F subcontract to the Van Andel Institute (10ST1035). Additional data repository and project management were provided by SAIC-F (HHSN261200800001E). The Brain Bank was supported by a supplement to the University of Miami grant DA00622

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.

Acknowledgements: We acknowledge H. Luque, L. Phillips, J. Casement, O. Magnuson, D. Nguyen and Y. Hu for technical support; R. García-Tercero and C. Díaz for sample collection; E. Zorio, M.E. Leach, D. Bharucha-Goebel, J. Dastgir and C. Konersman for clinical expertise and M. Gautel for helpful advice. We also thank CureCMD for their help in patient recruitment and the patients for donating their samples. The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013; 2012-305121) ‘Integrated European—omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)’ (to A. Töpf, V.S., I.T.Z. and F.M.); the European Union’s Horizon 2020 research and innovation program (Solve-RD project; 779257 to A. Töpf); Muscular Dystrophy UK and Muscular Dystrophy Association US (mda577346 to F.M.); Päulon Säätiö (to M. Savarese); Academy of Finland, Sigrid Juselius Foundation (to B.U.); core funding to the Sanger Institute by the Wellcome Trust (098051 and 206194 to E.M.B.-N., J.P. and N.W.); EURO-NMD and Fundación Gemio (to J.J.V., N.M. and P.M.); Intramural Research Grant (2-5, 29-4) for Neurological and Psychiatric Disorders of NCNP and AMED (JP20ek0109490h0001 to I.N.); Inserm, CNRS, University of Strasbourg, Labex INRT (ANR-10-LABX-0030 and ANR-10-IDEX-0002-02), France Génomique (ANR-10-INBS-09) and Fondation Maladies Rares for the ‘Myocapture’ sequencing project, AFM-Téléthon (22734), the European Joint program (EJPRD2019-126 IDOLS-G and ANR-19-RAR4-0002 to J.L., X.L. and V.B.); Intramural funds from the NIH National Institute of Neurological Disorders and Stroke (to C.G.B.); the Dutch Princess Beatrix Muscle Fund and the Dutch Spieren voor Spieren Muscle fund (to C.E.E.); PI16/00316 supported by the Instituto de Salud Carlos III (ISCIII), Madrid and the Generalitat Valenciana (grant PROMETEO/2019/075 to N.M.); Australian NHMRC Neil Hamilton Fairley Early Career Research Fellowship (GNT1090428 to E.C.O.); Starship Foundation A+7340 (to G.L.O.); Early Career Award from the Thrasher Research Fund (to S.S.); U54 HD090255 from the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (to A.H.B.); Wellcome Center for Mitochondrial Research (203105/Z/16/Z), the Mitochondrial Disease Patient Cohort (UK; G0800674), the Medical Research Council International Center for Genomic Medicine in Neuromuscular Disease (MR/S005021/1), the Medical Research Council (MR/W019027/1), the Lily Foundation, Mito Foundation, the Pathological Society, the UK NIHR Biomedical Research Center for Ageing and Age-related Disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust and the UK NHS Highly Specialized Service for Rare Mitochondrial Disorders of Adults and Children (to R.W.T.). MYO–SEQ was funded by Sanofi Genzyme, Ultragenyx, LGMD2I Research Fund, Samantha J Brazzo Foundation, LGMD2D Foundation, Kurt+Peter Foundation, Muscular Dystrophy UK and Coalition to Cure Calpain 3. Sequencing and analysis for relevant families (Supplementary Note) were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and were funded by the National Human Genome Research Institute, the National Eye Institute and the National Heart, Lung and Blood Institute under grant UM1 HG008900 and the National Human Genome Research Institute under grants U01HG0011755 and R01 HG009141. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DNA samples for NeurOmics and MYO–SEQ were provided by the John Walton Muscular Dystrophy Research Center Biobank. This facility is supported by the NIHR Newcastle Biomedical Research Center. Newcastle University’s Electron Microscopy Research Services and equipment Hitachi HT7800 120 kV TEM microscope are funded by BBSRC grant reference BB/R013942/1.Funder: Genzyme (Genzyme Corporation); doi: https://doi.org/10.13039/100004329Funder: Ultragenyx Pharmaceutical (Ultragenyx Pharmaceutical Inc.); doi: https://doi.org/10.13039/100013220Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): 2012-305121In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3−/−; ttn.1+/−) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.Peer reviewe

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.

Acknowledgements: We acknowledge H. Luque, L. Phillips, J. Casement, O. Magnuson, D. Nguyen and Y. Hu for technical support; R. García-Tercero and C. Díaz for sample collection; E. Zorio, M.E. Leach, D. Bharucha-Goebel, J. Dastgir and C. Konersman for clinical expertise and M. Gautel for helpful advice. We also thank CureCMD for their help in patient recruitment and the patients for donating their samples. The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7/2007-2013; 2012-305121) ‘Integrated European—omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)’ (to A. Töpf, V.S., I.T.Z. and F.M.); the European Union’s Horizon 2020 research and innovation program (Solve-RD project; 779257 to A. Töpf); Muscular Dystrophy UK and Muscular Dystrophy Association US (mda577346 to F.M.); Päulon Säätiö (to M. Savarese); Academy of Finland, Sigrid Juselius Foundation (to B.U.); core funding to the Sanger Institute by the Wellcome Trust (098051 and 206194 to E.M.B.-N., J.P. and N.W.); EURO-NMD and Fundación Gemio (to J.J.V., N.M. and P.M.); Intramural Research Grant (2-5, 29-4) for Neurological and Psychiatric Disorders of NCNP and AMED (JP20ek0109490h0001 to I.N.); Inserm, CNRS, University of Strasbourg, Labex INRT (ANR-10-LABX-0030 and ANR-10-IDEX-0002-02), France Génomique (ANR-10-INBS-09) and Fondation Maladies Rares for the ‘Myocapture’ sequencing project, AFM-Téléthon (22734), the European Joint program (EJPRD2019-126 IDOLS-G and ANR-19-RAR4-0002 to J.L., X.L. and V.B.); Intramural funds from the NIH National Institute of Neurological Disorders and Stroke (to C.G.B.); the Dutch Princess Beatrix Muscle Fund and the Dutch Spieren voor Spieren Muscle fund (to C.E.E.); PI16/00316 supported by the Instituto de Salud Carlos III (ISCIII), Madrid and the Generalitat Valenciana (grant PROMETEO/2019/075 to N.M.); Australian NHMRC Neil Hamilton Fairley Early Career Research Fellowship (GNT1090428 to E.C.O.); Starship Foundation A+7340 (to G.L.O.); Early Career Award from the Thrasher Research Fund (to S.S.); U54 HD090255 from the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (to A.H.B.); Wellcome Center for Mitochondrial Research (203105/Z/16/Z), the Mitochondrial Disease Patient Cohort (UK; G0800674), the Medical Research Council International Center for Genomic Medicine in Neuromuscular Disease (MR/S005021/1), the Medical Research Council (MR/W019027/1), the Lily Foundation, Mito Foundation, the Pathological Society, the UK NIHR Biomedical Research Center for Ageing and Age-related Disease award to the Newcastle upon Tyne Foundation Hospitals NHS Trust and the UK NHS Highly Specialized Service for Rare Mitochondrial Disorders of Adults and Children (to R.W.T.). MYO–SEQ was funded by Sanofi Genzyme, Ultragenyx, LGMD2I Research Fund, Samantha J Brazzo Foundation, LGMD2D Foundation, Kurt+Peter Foundation, Muscular Dystrophy UK and Coalition to Cure Calpain 3. Sequencing and analysis for relevant families (Supplementary Note) were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and were funded by the National Human Genome Research Institute, the National Eye Institute and the National Heart, Lung and Blood Institute under grant UM1 HG008900 and the National Human Genome Research Institute under grants U01HG0011755 and R01 HG009141. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. DNA samples for NeurOmics and MYO–SEQ were provided by the John Walton Muscular Dystrophy Research Center Biobank. This facility is supported by the NIHR Newcastle Biomedical Research Center. Newcastle University’s Electron Microscopy Research Services and equipment Hitachi HT7800 120 kV TEM microscope are funded by BBSRC grant reference BB/R013942/1.Funder: Genzyme (Genzyme Corporation); doi: https://doi.org/10.13039/100004329Funder: Ultragenyx Pharmaceutical (Ultragenyx Pharmaceutical Inc.); doi: https://doi.org/10.13039/100013220Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): 2012-305121In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases