Thromb Haemost 2019; 119(09): 1441-1450
DOI: 10.1055/s-0039-1693130
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

Next-Generation Sequencing of 17 Genes Associated with Venous Thromboembolism Reveals a Deficit of Non-Synonymous Variants in Procoagulant Genes

Eric Manderstedt
1   Department of Environmental Science and Bioscience, Kristianstad University, Kristianstad, Sweden
,
Christina Lind-Halldén
1   Department of Environmental Science and Bioscience, Kristianstad University, Kristianstad, Sweden
,
Peter Svensson
2   Department of Coagulation Disorders, Skåne University Hospital, Lund University, Lund, Sweden
,
Bengt Zöller
3   Centre for Primary Health Care Research, Department of Clinical Sciences, Skåne University Hospital, Lund University, Malmö, Sweden
,
Christer Halldén
1   Department of Environmental Science and Bioscience, Kristianstad University, Kristianstad, Sweden
› Institutsangaben
Funding This work was supported by grants awarded to Dr. Bengt Zöller by the Swedish Heart-Lung Foundation, ALF funding from Skåne Region and by the Swedish Research Council. The funders had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
Weitere Informationen

Publikationsverlauf

04. April 2019

22. Mai 2019

Publikationsdatum:
28. Juli 2019 (online)

Abstract

Background The heritability of venous thromboembolism (VTE) is only partially explained by variants in 17 previously VTE-associated genes.

Objective This article screens for additional rare variants in the 17 genes and investigates the relative contributions of pro- and anticoagulant genes to VTE.

Patients and Methods Ninety-six VTE patients from the population-based Malmö Thrombophilia Study were analysed using an AmpliSeq strategy and Ion Torrent sequencing and the variant data were compared with data from public databases.

Results A total of 102 non-synonymous and 76 synonymous variants were identified. Forty-six non-synonymous variants were present in the human gene mutation database. Anticoagulant and procoagulant genes showed 14 and 22 rare non-synonymous variants, respectively. Individual patients showed varying numbers of risk factors; 13 patients had non-synonymous mutations in SERPINC1, PROC and PROS1 genes and 42 had factor V Leiden or prothrombin mutations generating a total of 47 patients with at least one of these risk factors. Ten common VTE-associated variants showed low level enrichments and no correlation to the other risk factors. The enrichment of previously identified risk factors was similar to previous studies. Determination of the nsyn/syn ratio (number of non-synonymous variants per non-synonymous site, nsyn, to the number of synonymous variants per synonymous site, syn) showed, as expected in patients, an increase of non-synonymous relative to synonymous anticoagulant variants compared with controls (nsyn/syn, 0.95 vs. 0.68). In contrast, non-synonymous procoagulant variants (nsyn/syn, 0.31 vs. 0.63) showed a decrease. We suggest that the deficit of non-synonymous variants in procoagulant genes is a novel mechanism contributing to VTE.

 
  • References

  • 1 Heit JA, Spencer FA, White RH. The epidemiology of venous thromboembolism. J Thromb Thrombolysis 2016; 41 (01) 3-14
  • 2 Rosendaal FR. Venous thrombosis: a multicausal disease. Lancet 1999; 353 (9159): 1167-1173
  • 3 Zöller B, García de Frutos P, Hillarp A, Dahlbäck B. Thrombophilia as a multigenic disease. Haematologica 1999; 84 (01) 59-70
  • 4 Mannucci PM, Franchini M. Classic thrombophilic gene variants. Thromb Haemost 2015; 114 (05) 885-889
  • 5 Zöller B, Li X, Ohlsson H, Ji J, Sundquist J, Sundquist K. Family history of venous thromboembolism as a risk factor and genetic research tool. Thromb Haemost 2015; 114 (05) 890-900
  • 6 Morange PE, Suchon P, Trégouët DA. Genetics of venous thrombosis: update in 2015. Thromb Haemost 2015; 114 (05) 910-919
  • 7 Manolio TA, Collins FS, Cox NJ. , et al. Finding the missing heritability of complex diseases. Nature 2009; 461 (7265): 747-753
  • 8 Subramanian S. Quantifying harmful mutations in human populations. Eur J Hum Genet 2012; 20 (12) 1320-1322
  • 9 Cunha ML, Meijers JC, Middeldorp S. Introduction to the analysis of next generation sequencing data and its application to venous thromboembolism. Thromb Haemost 2015; 114 (05) 920-932
  • 10 Lotta LA, Wang M, Yu J. , et al. Identification of genetic risk variants for deep vein thrombosis by multiplexed next-generation sequencing of 186 hemostatic/pro-inflammatory genes. BMC Med Genomics 2012; 5: 7
  • 11 Halvorsen M, Lin Y, Sampson BA. , et al. Whole exome sequencing reveals severe thrombophilia in acute unprovoked idiopathic fatal pulmonary embolism. EBioMedicine 2017; 17: 95-100
  • 12 Lotta LA, Tuana G, Yu J. , et al. Next-generation sequencing study finds an excess of rare, coding single-nucleotide variants of ADAMTS13 in patients with deep vein thrombosis. J Thromb Haemost 2013; 11 (07) 1228-1239
  • 13 Pagliari MT, Lotta LA, de Haan HG. , et al. Next-generation sequencing and in vitro expression study of ADAMTS13 single nucleotide variants in deep vein thrombosis. PLoS One 2016; 11 (11) e0165665
  • 14 Rühle F, Witten A, Barysenka A. , et al. Rare genetic variants in SMAP1, B3GAT2, and RIMS1 contribute to pediatric venous thromboembolism. Blood 2017; 129 (06) 783-790
  • 15 Cunha MLR, Meijers JCM, Rosendaal FR, Vlieg AVH, Reitsma PH, Middeldorp S. Whole exome sequencing in thrombophilic pedigrees to identify genetic risk factors for venous thromboembolism. PLoS One 2017; 12 (11) e0187699
  • 16 Lee EJ, Dykas DJ, Leavitt AD. , et al. Whole-exome sequencing in evaluation of patients with venous thromboembolism. Blood Adv 2017; 1 (16) 1224-1237
  • 17 de Haan HG, van Hylckama Vlieg A, Lotta LA. , et al; INVENT consortium. Targeted sequencing to identify novel genetic risk factors for deep vein thrombosis: a study of 734 genes. J Thromb Haemost 2018; 16 (12) 2432-2441
  • 18 Lindström S, Brody JA, Turman C. , et al; INVENT Consortium. A large-scale exome array analysis of venous thromboembolism. Genet Epidemiol 2019; 43 (04) 449-457
  • 19 Isma N, Svensson PJ, Gottsäter A, Lindblad B. Prospective analysis of risk factors and distribution of venous thromboembolism in the population-based Malmö Thrombophilia Study (MATS). Thromb Res 2009; 124 (06) 663-666
  • 20 Saemundsson Y, Sveinsdottir SV, Svantesson H, Svensson PJ. Homozygous factor V Leiden and double heterozygosity for factor V Leiden and prothrombin mutation. J Thromb Thrombolysis 2013; 36 (03) 324-331
  • 21 Kroll KW, Eisfeld AK, Lozanski G, Bloomfield CD, Byrd JC, Blachly JS. MuCor: mutation aggregation and correlation. Bioinformatics 2016; 32 (10) 1557-1558
  • 22 Karczewski KJ, Weisburd B, Thomas B. , et al; The Exome Aggregation Consortium. The ExAC browser: displaying reference data information from over 60 000 exomes. Nucleic Acids Res 2017; 45 (D1): D840-D845
  • 23 Ameur A, Dahlberg J, Olason P. , et al. SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population. Eur J Hum Genet 2017; 25 (11) 1253-1260
  • 24 Zöller B, Melander O, Svensson PJ, Engström G. Factor V Leiden paradox in a middle-aged Swedish population: a prospective study. Vasc Med 2018; 23 (01) 52-59
  • 25 Hillarp A, Zöller B, Svensson PJ, Dahlbäck B. The 20210 A allele of the prothrombin gene is a common risk factor among Swedish outpatients with verified deep venous thrombosis. Thromb Haemost 1997; 78 (03) 990-992
  • 26 Richards S, Aziz N, Bale S. , et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17 (05) 405-424
  • 27 Stenson PD, Mort M, Ball EV. , et al. The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum Genet 2017; 136 (06) 665-677
  • 28 Flanagan SE, Patch AM, Ellard S. Using SIFT and PolyPhen to predict loss-of-function and gain-of-function mutations. Genet Test Mol Biomarkers 2010; 14 (04) 533-537
  • 29 Schwarz JM, Rödelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods 2010; 7 (08) 575-576
  • 30 Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 1986; 3 (05) 418-426
  • 31 Millar DS, Wacey AI, Ribando J. , et al. Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis. Hum Genet 1994; 94 (05) 509-512
  • 32 Reitsma PH, Bernardi F, Doig RG. , et al. Protein C deficiency: a database of mutations, 1995 update. On behalf of the Subcommittee on Plasma Coagulation Inhibitors of the Scientific and Standardization Committee of the ISTH. Thromb Haemost 1995; 73 (05) 876-889
  • 33 Gandrille S, Alhenc-Gelas M, Gaussem P. , et al. Five novel mutations located in exons III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity. Blood 1993; 82 (01) 159-168
  • 34 Conard J, Horellou MH, van Dreden P. , et al. Homozygous protein C deficiency with late onset and recurrent coumarin-induced skin necrosis. Lancet 1992; 339 (8795): 743-744
  • 35 Reitsma PH, Poort SR, Allaart CF, Briët E, Bertina RM. The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects. Blood 1991; 78 (04) 890-894
  • 36 Zheng YZ, Sakata T, Matsusue T, Umeyama H, Kato H, Miyata T. Six missense mutations associated with type I and type II protein C deficiency and implications obtained from molecular modelling. Blood Coagul Fibrinolysis 1994; 5 (05) 687-696
  • 37 Duchemin J, Gandrille S, Borgel D. , et al. The Ser 460 to Pro substitution of the protein S alpha (PROS1) gene is a frequent mutation associated with free protein S (type IIa) deficiency. Blood 1995; 86 (09) 3436-3443
  • 38 Tang L, Jian XR, Hamasaki N. , et al. Molecular basis of protein S deficiency in China. Am J Hematol 2013; 88 (10) 899-905
  • 39 Wu CM, Dwivedi DJ, Zarin W. , et al. Targeted gene sequencing to identify polymorphisms in the protein C and EPCR genes in patients with unprovoked venous thromboembolism. Blood 2009; 114: 454
  • 40 Medina P, Navarro S, Estellés A, Vayá A, Bertina RM, España F. Influence of the 4600A/G and 4678G/C polymorphisms in the endothelial protein C receptor (EPCR) gene on the risk of venous thromboembolism in carriers of factor V Leiden. Thromb Haemost 2005; 94 (02) 389-394
  • 41 Chegeni R, Kazemi B, Hajifathali A, Pourfathollah A, Lari GR. Factor V mutations in Iranian patients with activated protein C resistance and venous thrombosis. Thromb Res 2007; 119 (02) 189-193
  • 42 Castaman G, Lunghi B, Missiaglia E, Bernardi F, Rodeghiero F. Phenotypic homozygous activated protein C resistance associated with compound heterozygosity for Arg506Gln (factor V Leiden) and His1299Arg substitutions in factor V. Br J Haematol 1997; 99 (02) 257-261
  • 43 Smith NL, Hindorff LA, Heckbert SR. , et al. Association of genetic variations with nonfatal venous thrombosis in postmenopausal women. JAMA 2007; 297 (05) 489-498
  • 44 Bezemer ID, Bare LA, Arellano AR, Reitsma PH, Rosendaal FR. Updated analysis of gene variants associated with deep vein thrombosis. JAMA 2010; 303 (05) 421-422
  • 45 Bertina RM, Koeleman BP, Koster T. , et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369 (6475): 64-67
  • 46 Terasawa F, Okumura N, Kitano K. , et al. Hypofibrinogenemia associated with a heterozygous missense mutation gamma153Cys to arg (Matsumoto IV): in vitro expression demonstrates defective secretion of the variant fibrinogen. Blood 1999; 94 (12) 4122-4131
  • 47 Brennan SO, Fellowes AP, Faed JM, George PM. Hypofibrinogenemia in an individual with 2 coding (gamma82 A-->G and Bbeta235 P-->L) and 2 noncoding mutations. Blood 2000; 95 (05) 1709-1713
  • 48 Board PG, Shaw DC. Determination of the amino acid substitution in human prothrombin type 3 (157 Glu leads to Lys) and the localization of a third thrombin cleavage site. Br J Haematol 1983; 54 (02) 245-254
  • 49 Goodeve A, Eikenboom J, Castaman G. , et al. Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD). Blood 2007; 109 (01) 112-121
  • 50 James PD, Notley C, Hegadorn C. , et al. The mutational spectrum of type 1 von Willebrand disease: results from a Canadian cohort study. Blood 2007; 109 (01) 145-154