Antithrombin Rouen IV mutation in Polish patient with deep vein thrombosis

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Case report/Kazuistyka

Antithrombin Rouen IV mutation in Polish patient with deep vein thrombosis

Kamil Brutkowski

¹

, Ewa Wypasek

^1,2

, Javier Corral

³

, Anetta Undas

^1,2,

*

1JohnPaulIIHospital,Cracow,Poland

2InstituteofCardiology,JagiellonianUniversitySchoolofMedicine,Cracow,Poland

3CentroRegionaldeHemodonación,UniversidaddeMurcia,Murcia,Spain

Introduction

Antithrombin (AT) is a 58kDa glycoprotein synthesized in the liver, which belongs to the serine protease inhibitors superfamily(serpins)andplayakeyroleasanaturalblood anticoagulant. AT forms inactive complex with thrombin and activated factor Xa that prevents blood clotting. Pre- senceofheparinincreasesthisactivity1000-fold[1].

Thehuman gene encoding AT (SERPINC1) is located on the chromosome 1q23-25 and consists of 7 exons and 6 introns [1, 2]. Currently, over 300 mutations causing AT

deficiency have been reported [3]. More than half of these genetic disorders are missense mutations, which lead to dysfunctional proteins.Theotherslikeinsertions,deletions and nonsense mutations, typically cause disruption or absence of geneproduct[4]. Hitherto, therewereidentified 14 different mutations corresponding to AT deficiency amongthePolishpatients[5].

Inherited AT deﬁciency is an autosomal dominant dis- orderthatconcernsbetween1:600and1:5000oftotalpopula- tion[6].TherearetwotypesofinheritedATdeﬁciency,both are associated with reduced AT activity but only type I is related to lower ATantigen levels inblood. TypeII can be actahaematologicapolonica 46(2015) 393–395

article info

Articlehistory:

Received:07.07.2015 Accepted:15.10.2015 Availableonline:26.10.2015

Keywords:

Antithrombindeﬁciency

SERPINC1mutation

RouenIV

Venousthromboembolism

abstract

Antithrombin(AT)deficiencyisarareautosomaldominantdisorderwhichincreasesthe riskofvenousthromboembolism(VTE).WereportherethecaseoftypeIIantithrombin deficiency in44-year-old man who developedleft leg deep veinthrombosis (DVT). All exons of SERPINC1, the gene encoding AT were amplified by PCR followed by direct sequencing.Aheterozygousmutationc.166C>Tinexon2causingtheaminoacidsubsti- tutionofArgtoCysatresidue56,wasfound.Thismutationdoesnotaffectthefolding andsecretionoftheprotein,butimpairstheheparinaffinity,reducingtheanticoagulant activityofAT.

Tothebestofourknowledge,thisisaﬁrstreportofATtypeIIHBSdeﬁciencyrelated toPolishpatientwhoexperiencedDVT.

*Correspondingauthorat:InstytutKardiologii,UniwersytetJagiellońskiCollegiumMedicum,ul.Prądnicka80,31-202Kraków,Poland.

Tel.:+48126143004;fax:+48124233900.

E-mailaddress:mmundas@cyf-kr.edu.pl(A.Undas).

ContentslistsavailableatScienceDirect

Acta Haematologica Polonica

journalhomepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2015.10.001

zo.o.Allrightsreserved.

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classiﬁedintothreegroupsdependingonthelocationofthe mutations:typeIIRSischaracterizedbyreactivesitedefects, type IIHBS isassociatedwith heparin-binding siteandtype IIPE is caused by mutations clustered in the C-terminus region ofATprotein[7]. ATdeﬁciency increases20-fold the riskofvenousthromboembolism(VTE)[8].

We report here the ﬁrst Polish case of AT type IIHBS deﬁciencycausedbyheterozygouspointmutationc.166C>T, p.Arg56Cys (Human Genome Variation Society numbering system)insecondexonofSERPINC1gene.

Case report

A 45-year-old overweight male patient 5 months after idiopathic left-sided deep vein thrombosis (DVT) was referredforthrombophiliascreening.Sixmonthsbeforethe incidenthesufferedalefttibiainjurybutwithouttheneed of plaster. Venous duplex ultrasound scans showed popli- tealandsuperﬁcialfemoralveinsreﬂuxandthrombiwithin thetibialveins.

Afteratraumathepatientwastreatedwithrivaroxaban (Xarelto) 20mg/d and this therapy is still continued. No signs of bleedingor DVT recurrence were observed during 12monthsoftherapy.

Theprobandwastheonlychild.Familyhistoryrevealed lowerlimbvariceswithoutdocumentedVTE inhismother.

The proband's father died of myocardial infarction (MI) before the age of 60. A 16-year proband's son remained asymptomatic.

Themostcommonthrombophilic factors,includingFac- tor V Leiden and prothrombin G20210A mutations, were absent. Plasma protein C, free protein S, antiphospholipid antibodies, factor FVIII and homocysteine were within normallimits.Lupusanticoagulantwasnegative.

Antithrombinanti-FXactivity,measuredbytwodifferent chromogenic assays: (Berichrom AT, Siemens, Marburg, Germany, reference range 75–125%, and HemosIL, Instru- mentation Laboratory SpA, Milano, Italy, reference range 80–120%),were56%and49%,respectively.ATantigenlevels were performed using a home-made sandwich ELISA kit (referencerange80–120%)andwas98%.

Moreover, plasma AT was evaluated by electrophoretic analysis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)performed in8% polyacryamide geland nativePAGE withthepresence andabsenceof 6M urea as described previously [9]. After separation, proteins weretransblottedontopolyvinylideneﬂuoride(PVDF)mem- brane.AT was immunostained with rabbit anti-human AT polyclonal antibody(Sigma,Madrid,Spain)andfollowed by donkeyanti-rabbit IgGhorseradishperoxidase(HRP) conju- gate(GEHealthcare,Barcelona,Spain)withdetectionbyECL kit(AmershamBioscences,Piscataway,NJ).Analysesdidnot identify any abnormal bands that might correspond to variantAT,exceptnativegels,whichshowstwobands,one withfasterelectrophoreticmobilityandsimilarintensityto the wild type, which is typical of mutations affecting arginineresiduesattheheparinbindingdomain.

After obtained informed written consent, the blood samplewascollectedinEDTAforDNAanalysis.The7exons

andﬂankingregionsofSERPINC1genewereampliﬁedusing polymerase chain reaction (PCR) followed by direct DNA sequencing. A heterozygous point mutation c.166C>T that causes a missense protein sequence change p.Arg56Cys (numberconsideringthewholeprotein)inexon2wasfound.

In addition, protein in silico analysis using PoliPhen-2 andSortingIntolerantFromTolerant(SIFT)wereperformed.

The obtained result classiﬁes this mutation as ‘probably damaging’withscore0.999.

Discussion

Tothebestofourknowledge,thisisaﬁrstreportofATtype II HBS deﬁciency detected in a Polish patient who experi- encedDVT.

The mutation identified in this patient was previously describedbyPerryandCarrellin1989ina25-year-oldman who developed an unexpected coronary thrombosis. This report demonstrated that the described mutation caused avariantknownas‘AntithrombinRouenIV’[10].Ithasbeen alsoreportedbyLuxembourgetal.that1outof272patients with reduced valuesof ATactivity/AT antigen and throm- boembolic event,and withpositive family history forDVT, carriedthep.Arg56Cysvariant[11].However,arecentcohort study shows that the carriers of IIHBSAT deficiency have a 80% lower risk of VTE and 6-fold higher risk of arterial thromboembolism than carriers of other type of AT deficiency[4].Thesemutationsareusuallycombinedwithother thrombotic riskfactorsinpatientswithVTE [12]. We show here uncommon clinical presentation of Antithrombin Rouen IV since our patient developed DVT without addi- tionalriskfactors.

Antithrombin Rouen IV occurs at CpG dinucleotides (CpGs) which are the hotspots for point mutations in SERPINC1 gene [10]. The cytosine (C) residue in the CpG region is methylated and the product of this process (5- methylocytosine) maybespontaneously deaminated form- ing thymine(T).Thischangeisnotenzymaticrepairedand results in a C to T transition on one DNA strand and consequently,changeGtoAonaoppositeDNAstrand.

It hasbeen shown thatthe CpGs mutations are mainly involved inthe developmentof ATtypeIIHBSdeﬁciencyin oppositiontotypeIIRSandIIPE[10,13].

Arginine atresidue56islocatedinN-terminalsequence and isinvolved inproteinconformational changesinduced by bindingof heparin[11].Replacement ofthis aminoacid bycysteinehastheinﬂuenceondecreasedheparinafﬁnity.

Itisimportanttopointoutthatcertainfunctionalmethods for measuringATactivityinthepresenceofheparinfailto detectthismutation[14].

Inconclusion,ourreportsupportstheneedforthrombo- philia screening in VTE patients below 50 years of age, especially in the absence of the established risk factors.

Similarstrategyshouldbeconsideredinarterialthrombosis at young age as evidenced for instance in our report on protein Cdeﬁciencydetectedafter MIinayoungman[15].

Appropriate and timely anticoagulation in symptomatic patientswithinheritedATdeﬁciencycanreducetheriskof recurrent thrombotic events. Life-long anticoagulant treat- acta haematologicapolonica 46 (2015) 393–395

394

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mentshould beconsideredinAT deﬁcient patientsifthey experienced a ﬁrst-ever idiopathic VTE episode as well as those with two episodes. Prophylactic anticoagulation in asymptomatic carriers, especially if family history for VTE is present may be used in certain high-risk state such as pregnancyorsurgery.

Authors’ contributions/Wkład autorów

EW – study design, data collection and interpretation, manuscript preparation, literature search. AU – study design,datacollection and interpretation,funds collection.

JC–datacollectionandinterpretation,literaturesearch.KB –datacollectionandinterpretation,manuscriptpreparation, literaturesearch.

Conﬂict of interest/Konﬂikt interesu

Nonedeclared.

Financial support/Finansowanie

ThisresearchwassupportedbythePolish NationalScience Center(NCN)GrantNo.UMO-2013/09/B/NZ5/00254.

Ethics/Etyka

Thework describedin this article has been carriedout in accordance with TheCode of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experi- ments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

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acta haematologicapolonica 46 (2015)393–395