Huntington, James A

Lisman, Ton

Meijer, Karina

Meijers, Joost CM

Mulder, André B

Mulder, René

English

Mulder, Rene

Meijers, Joost C. M.

Huntington, James A.

Mulder, Andre B.

Dissertations of the University of Groningen

   University of GroningenLinkage analysis combined with whole-exome sequencing identifies a novel prothrombin (F2)gene mutation in a Dutch Caucasian family with unexplained thrombosisMulder, Rene; Lisman, Ton; Meijers, Joost C. M.; Huntington, James A.; Mulder, Andre B.;Meijer, KarinaPublished in:HaematologicaDOI:10.3324/haematol.2019.232504IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:2020Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Mulder, R., Lisman, T., Meijers, J. C. M., Huntington, J. A., Mulder, A. B., & Meijer, K. (2020). Linkageanalysis combined with whole-exome sequencing identifies a novel prothrombin (F2) gene mutation in aDutch Caucasian family with unexplained thrombosis. Haematologica, 105(7), E370-E372.https://doi.org/10.3324/haematol.2019.232504CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Linkage analysis combined with whole-exomesequencing identifies a novel prothrombin (F2) genemutation in a Dutch Caucasian family with unexplained thrombosis Genetics play a major role in the development ofvenous thromboembolism (VTE). However, currentthrombophilia testing identifies a known heritable defectin only a minority of VTE patients.1 A part of this missingheritability for VTE may be caused by rare variants.2 Inthe current study we combined  genome-wide linkageanalysis with whole-exome sequencing (WES) and iden-tified a rare novel prothrombin (F2) gene mutation(c.1621C>T; p.Arg541Trp hereafter referred to asp.Arg173Trp according to chymotrypsin numbering) in aDutch family with unexplained thrombosis. The proband, from Dutch Caucasian ancestry, present-ed at the age of 31 years with a first deep vein thrombosis(DVT) during pregnancy. Shortly after, her father hadDVT after hospitalization at the age of 58. The familywas aware that they were prone for thrombosis, andoffered a family tree of the older generations. Theproband’s paternal uncle had had a spontaneous pul-monary embolism at the age of 38 and 42 years, a cousinhad a spontaneous DVT at the age of 44 years (dataobtained from the subjects themselves, VTE diagnoses ingenerations I, II and III were obtained from family mem-bers). Routine thrombophilia testing in the proband wasnegative, excluding deficiencies of antithrombin, proteinC, protein S, and factor V Leiden and the prothrombinG20210A mutation. In the years after, both the probandand her father had a spontaneous DVT recurrence, andthe proband’s younger sister presented with estrogen-related pulmonary embolism. We hypothesized that thisfamily carried a private, high risk mutation. Afterinformed consent was obtained we included a total of 19family members for testing. First, genome-wide single nucleotide polymorphismgenotyping was performed on 19 family members (fiveaffected and 14 unaffected; Figure 1A and OnlineSupplementary Data) and identified twelve chromosomeswith equally high significant linkage peaks (LOD >1).These chromosomes were: 3, 4, 7, 9, 11, 13, 14, 18, 19,20, 22, and X. Next, WES was performed on five familymembers (four affected: 26101, 26103, 26105, 26206;one unaffected: 26312;  Figure 1A and OnlineSupplementary Data). In total, 138,672 variants weredetected, of which, after rigorous filtering, 113 remainedcommon among affected family members. When wecombined these results with linkage data, e.g. filteringbased on their distance (~3 Mb) to the linkage peak, 30haematologica 2020; 105:e370LETTERS TO THE EDITORFigure 1. Identification and functional characterization of a novel prothrombin (F2) gene mutation in a Dutch family with unexplained thrombosis. (A) The pedi-gree. The arrow indicates the proband. Roman numbers denote the generation in the pedigree; the study number of included family members is depicted beloweach symbol; square symbols, male sex; round symbols, female sex; solid symbols represent VTE-affected family members, open symbols represent unaffectedfamily members. (B) Whole-exome sequencing (WES) combined with linkage analysis. The diagram depicts effects of filtering WES data based on specificaspects including among other things such as type of mutation and location to linkage peak. (C) Direct sequencing results of exon 12 of F2 gene. The prothrom-bin (F2) gene mutation (c.1621C>T; p.Arg173Trp) is highlighted. (D) This figure shows the genotyping results of a custom made Taqman quantitative PCR assayfor 19 included family members. The green lines represent the carriers of the prothrombin (F2) gene mutation (26101, 26103, 26105, 26206, and 26209),whereas red lines represent the non-carriers of the mutation (26102, 26104, 26207, 26208, 26210, 26211, 26312, 26313, 26314, 26315, 26316, 26317,26418, and 26419). (E) Thrombin generation in prothrombin depleted plasma supplemented with wild-type (WT) and mutant protein in the presence or absenceof 0.1 U/mL unfractionated heparin. ABCDEvariants remained. In the final step, in silico predictionwas performed with MetaSVM and MetaLR therebyidentifying 1 potentially pathogenic novel prothrombin(F2) gene mutation (p.Arg173Trp) (Figure 1B) located onchromosome 11. Using Sanger sequence analysis we con-firmed the heterozygous presence of a novel prothrom-bin (F2) gene mutation (Figure 1C). Next, with a custommade TaqMan quantitative PCR assay (AppliedBiosystems) we confirmed the presence of the prothrom-bin (F2) gene mutation p.Arg173Trp in four affected fam-ily members (26101, 26103, 26105, 26206), whereas oneunaffected family member (216209) also carried thismutation (Figure 1D). Furthermore, one family memberwho did not carry the mutation (216418) had a history ofDVT.  Functional testing was complicated due to the useof vitamin K antagonists in the carriers of the prothrom-bin mutation with available plasma. We therefore per-formed functional assays with recombinant prothrombinmolecules (U-Protein Express BV Utrecht, theNetherlands). A hydroxyapatite column was used toobtain fully γ-carboxylated wild-type (WT) and mutantprothrombin. Prothrombin levels were measured by one-stage clotting assay on an automated coagulationanalyser (ACL 300 TOP) with reagents and protocolsfrom the manufacturer (Instrumentation Laboratory,Breda, the Netherlands). At a final concentration of 90μg/mL in prothrombin deficient plasma, the recombinantWT and mutant prothrombins had prothrombin activityof 82% and 67%, respectively. Furthermore, thrombingeneration was performed using calibrated automatedthrombography and Thrombinoscope software accordingto the manufacturer’s instruction (Thrombinoscope BV,Maastricht, the Netherlands). Recombinant WT orp.Arg173Trp prothrombin was added to prothrombindeficient plasma (Siemens) at a final concentration of 90 µg/mL in the absence or presence of 0.1 U/mL unfrac-tionated heparin (LEO Pharma BV) and in vitro thrombingeneration was assessed. Figure 1E depicts representativethrombin generation curves. In the absence of unfrac-tionated heparin, plasma containing p.Arg173Trp pro-thrombin had a substantially higher endogenous throm-bin potential  compared to plasma to which WT pro-thrombin was added (2,400±113 vs. 797±42 nM IIa*min,P<0.0001, n=3). Addition of unfractionated heparinreduced thrombin generation in plasma containing thep.Arg173Trp prothrombin by only 7.3±3.9% comparedto a 31.4±9.3% reduction in thrombin generation in plas-ma containing WT prothrombin (P=0.01, n=3).Finally, we performed molecular dynamics in order tosimulate the effect of the p.Arg173Trp mutation (OnlineSupplementary Data). The prothrombin (F2) gene muta-tion is located in the 170s loop, near the non-primed sideof the active site (Figure 2A). The results of moleculardynamics are consistent with a conformational alterationin the non-prime side of the active site due to the reposi-tioning of the Trp173 side chain to minimize its solventaccessibility by packing up against Trp215 (Figure 2B). Venous thrombosis is a disorder in which genetic pre-disposition plays an important role. However, in a largeportion of patients with VTE no genetic defect can bedetected. Several aspects contribute to the missing heri-tability for VTE, including rare variants. To identify suchvariants we combined linkage analysis with WES andfound a novel prothrombin (F2) gene mutationp.Arg173Trp.This mutation is assumed to be rare as it was absent intwo control groups, e.g. in 24 probands of families withunexplained thrombosis and 100 healthy volunteerswithout VTE (selection from GoNL; http://www.nlgenome.nl/) (data not shown). Furthermore, this mutationwas not present in double stranded single nucleotidepolymorphism (https://www.ncbi.nlm. nih.gov/snp) orHGMD (http://www. hgmd.cf.ac.uk/ac/index.php).   Prothrombin deficiency has long been considered toonly be associated with bleeding tendency untilMiyawaki et al. reported in 2012 on a family with dyspro-thrombinemia and venous thrombosis.3 This dyspro-thrombinemia was caused by a substitution of arginine toleucine at position 221a in  the F2 gene (prothrombinYukuhashi).3 In the following years, two additional muta-tions at position 221a were reported, e.g. prothrombinBelgrade4 and prothrombin Padua 2.5 Apart from pro-thrombin Belgrade, these mutations were all associatedwith a strong risk of VTE at a young age.3,5 The associa-tion with VTE was explained by antithrombin resistancecaused by mutations at position 221a.3-5  All carriers of the prothrombin (F2) gene mutationp.Arg173Trp, with the exception of 216209 have hadvenous thrombosis. One family member who did notcarry the mutation (216418) also had a history of DVT.We cannot exclude that additional unknown throm-bophilic factors are present in the family. We have alsoobserved in studies of protein C, S and antithrombin defi-ciency that non-affected family members have a higherhaematologica 2020; 105:e371LETTERS TO THE EDITORFigure 2. Structural characterization of the novel prothrombin (F2) gene mutation. (A) A ribbon diagram of the heavy chain of thrombin in the standard orien-tation showing the position of the mutation (side chain of Arg173 as sticks) and the Na+ (purple ball) binding site (colored from N-to-C terminus, blue to red). (B)Surface representation of the resulting structures after molecular dynamics simulation of wild-type (WT) (left) and the p.Arg173Trp mutant variant (right) throm-bin coloured according to electrostatics. The oval indicates the non-primed side of the active site cleft, and the arrow indicates the position of the mutation.A Bincidence of VTE than the normal population.6 However,venous thromboembolism is a common disorder and thisone case in a family member without the mutation mightalso have been due to chance.Our functional studies showed a clear heparin resist-ance that is suggestive for antithrombin resistance. In linewith this, Tamura et al. performed an in vitro exploratorystudy using prothrombin mutants and showed that arecombinant p.Arg173Trp mutation exhibited a mildantithrombin resistance.7The side chain of p.Arg173 is not fully modeled in thestructure of the antithrombin-thrombin Michaelis com-plex, suggesting that it is not making a single distinct con-tact with antithrombin. It is in a position where it couldexert a favorable long-range electrostatic influence ondocking, since it is in the vicinity of glutamates 232, 377and 378 on antithrombin. However, such an influencewould be overwhelmed by the magnitude of the effect ofheparin on the association rate. The heparin resistance ofthe p.Arg173Trp thrombin variant therefore suggestssomething more profound than the loss of a favorableinteraction or electrostatic influence. A structuralrearrangement of the active site caused by the introduc-tion of a large hydrophobic residue to a solvent-exposedloop  is more likely (Figure 2A). The 170s loop has beenimplicated in the zymogen to protease conformationalchange and allosteric activation. The molecular dynamicsresults suggest a distinct conformational change in thenon-prime side of the active site due to the Trp173 bury-ing itself as much as possible thereby interfering withantithrombin inhibition (Figure 2B). However, more evi-dence is still needed to conclude that the prothrombinmutation p.Arg173Trp leads to antithrombin resistance. Finally, at a final concentration of 90 µg/mL in deficientplasma, the recombinant WT and mutant showed bor-derline low prothrombin activity levels, which is in con-trast with the increased thrombin potential observedwith the thrombin generation assay. Such a discrepancyhas also been observed for four antithrombin-resistantmutations in position 221a.7 This discrepancy could beexplained by the fact that clotting assays measure onlythe clotting time corresponding to the initiation phase ofthe coagulation process, whereas thrombin generationassays also provide information about the amplificationand propagation phases of the hemostatic system. In conclusion, in this study we identified a rare novelprothrombin (F2) gene mutation p.Arg173Trp that causesheparin resistance which is suggestive for antithrombinresistance. Evidently further investigations are needed tofully disentangle the mechanism responsible for thepotential association between the prothrombin (F2) genemutation p.Arg173Trp and venous thrombosis.  René Mulder,1 Ton Lisman,2 Joost C.M. Meijers,3James A. Huntington,4 André B. Mulder1 and Karina Meijer51Department of Laboratory Medicine, University of Groningen,University Medical Center Groningen, Groningen, the Netherlands;2Surgical Research Laboratory and Section of Hepatobiliary Surgeryand Liver Transplantation, Department of Surgery, University ofGroningen, University Medical Center Groningen, Groningen, theNetherlands; 3Department of Molecular and Cellular Hemostasis,Sanquin Research, Amsterdam and Amsterdam UMC, University ofAmsterdam, Department of Experimental Vascular Medicine,Amsterdam Cardiovascular Sciences, Amsterdam, the Netherlands;4Department of Haematology, Cambridge Institute for MedicalResearch, University of Cambridge, Cambridge, UK and 5Departmentof Hematology, University of Groningen, University Medical CenterGroningen, Groningen, the Netherlands Correspondence: RENÉ MULDER - r.mulder01@umcg.nl.   doi:10.3324/haematol.2019.232504Information on authorship, contributions, and financial & other dis-closures was provided by the authors and is available with the onlineversion of this article at www.haematologica.org.References1. Morange PE, Tregouet DA. Deciphering the molecular basis ofvenous thromboembolism: where are we and where should we go?Br J Haematol. 2010;148(4):495-506.2. McCarthy MI, Abecasis GR, Cardon LR, et al. Genome-wide associ-ation studies for complex traits: consensus, uncertainty and chal-lenges. Nat Rev Genet. 2008;9(5):356-369.3. Miyawaki Y, Suzuki A, Fujita J, et al. Thrombosis from a prothrom-bin mutation conveying antithrombin resistance. N Engl J Med.2012;366(25):2390-2396.4. Djordjevic V, Kovac M, Miljic P, et al. A novel prothrombin mutationin two families with prominent thrombophilia--the first cases ofantithrombin resistance in a Caucasian population. J ThrombHaemost. 2013;11(10):1936-1939.5. Bulato C, Radu CM, Campello E, et al. New Prothrombin mutation(Arg596Trp, prothrombin Padua 2) associated with venous throm-boembolism. Arterioscler Thromb Vasc Biol. 2016;36(5):1022-1029.6. Mahmoodi BK, Brouwer JL, Ten Kate MK, et al. A prospective cohortstudy on the absolute risks of venous thromboembolism and predic-tive value of screening asymptomatic relatives of patients withhereditary deficiencies of protein S, protein C or antithrombin. JThromb Haemost. 2010;8(6):1193-1200.7. Tamura S, Murata-Kawakami M, Takagi Y, et al. In vitro explorationof latent prothrombin mutants conveying antithrombin resistance.Thromb Res. 2017;159:33-38.haematologica 2020; 105:e372LETTERS TO THE EDITOR

Linkage analysis combined with whole-exome sequencing identifies a novel prothrombin (F2) gene mutation in a Dutch Caucasian family with unexplained thrombosis

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Linkage analysis combined with whole-exome sequencing identifies a novel prothrombin (F2) gene mutation in a Dutch Caucasian family with unexplained thrombosis.

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Linkage analysis combined with whole-exome sequencing identifies a novel prothrombin (F2) gene mutation in a Dutch Caucasian family with unexplained thrombosis.

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