Inherited Thrombophilia: Part 1

Thrombosis and Haemostasis © F K Schattauer Verlagsgesellschaft mbH (Stuttgart) 76 (<>) 651-62 (1996)

Inherited Thrombophilia*: Part l

David A. Lane1, Pier M. Mannucci2, Kenneth A, Bauer3, Regier M. Bertina4,

Nikoiay P. Bochkov5, Victor Boulyjenkov6, Mammen Chandy7, Björn Dahlbäck8,

E. K. Ginter9, Joseph P Miletich10, Frits R. Rosendaal4, Un Seligsohn11

From the 'Channg Cross and Westminster Medical School London UK 2IRCCS Maggiore Hospital, Milan Italy 3Beth Israel Hospital, Boston USA, "University Hospital Leiden The Netherlands 5Research Centre of Medical Genetics Moscow The Russian Federation, 6WHO, Switzerland, 7Chnstian Medical College Hospital Vellore India 8University of Lund, Malmo Sweden 'Institute of Clinical Genetics Moscow The Russian Federation, toWashmgton University, St Louis, USA, "Institute of Thrombosis and Hemostasis Tel Hashomer, Israel

Introduction

Although familial clustenng of venous thiomboembohc events was observed at the statt of this Century, it was not until the late nmeteen-seventies before our insight mto the orgamzation of the haemostatic and fibnnolytic Systems was sufficiently complete to enable a Start to be made on a systematic seatch for genetic defects associated with familial thrombosis The discovery that hetei ozygosity of antithrombm deficiency co-segregdted with the thiombotic tendency m affected families seemed to make a strong case foi the association between single gene detects and thrombosis The results of these systematic studies had been m part very fuutful and could be considered in the context of two prmciple endogenous anticoagulant pathways (Fig 1). the antithiombin-heparan sulphate pathway and the protem C/protem S (PC/PS) pathway However only three single gene disorders had been identified that were associated with a significant increase in the nsk for venous thrombembohsm m tamihes identified through a symptomatic deficient patient. with one of antithrombm, PC or PS deficiency

The awaieness that m 85% of tamihes piedisposed to thiombosis no explanation could be found foi the clustenng of this disorder sümulated the search for alternative approaches A major bieakthrough m the study of familial thrombosis has been achieved dunng the past two years Fustly, the concept and investigation of activated protem C tesistance (APC-R) was mtioduced and, secondly, a mutation in the factoi V gene (1691 G -» A m exon 10, leadmg to 506Aig to Gin) was identified äs the moleculai basis tor the phenotype of APC-R in the large majonty of aftected individuals (2,3) This mutation, which is associated with a significant increase m thtombotic nsk (3-5), has been tound in about 50% of selected tamihes with thrombophiha and in 20% of consecutive patients with thiombosis A consequence ot this advance has been a conceptual change in how thiombophiha is viewed, which has imphcations tor diagnosis and tieatment of the disoider This leview [published m two parts, see also (6)] attempts to summanse lecent progress and to present lecommendations toi diagnosis, treat-ment and reseatch m developed and developmg countnes

Coirespondence to Di D A Lane Department ot Haematology, Channg Cross and Westminster Medital Sohool Hammeismith London W6 8RF U K - F A X + 1 8 1 8 4 6 7 1 1 1 e mail d lane@cxwms ac uk

1 Bascd upon the Woild Health Organisation rcpoit ot a Jomt Woild Health Oiganisation/Internaüonal Society on Thrombosis and Haemostasis Meeting held in Geneva 6-8 Novembei 1991 Published \ \ i t h peimission ot llie Woild Health Oigamsation 'Meeting Rapportcur IS1H ScucUnal "WHO Secielanat

Pathogenesis of Thrombophilia and Definition of Inherited Thrombophilia

Thrombophilia is a tendency to thrombosis The predisposmg defects do not necessanly cause contmuous climcal impairment they need only weaken the abihty to cope with fluctuations mduced by mteractions with the envuonment Clmicians usually apply the term thrombophiha only to a subset ot patients with atypical thrombosis Fiequently cited features mclude (1) eaily age of onset (2) hequent recurrence. (3) stiong family history (4) unusual. migraton 01 widespread locations, and (5) seventy out of propoition to any lecogmzed Stimulus Theie aie patients (see below) with fulminant thiombophiha who, without theiapy. thiombose contmuoush But m most patients thiombosis is episodic, separated by often piolonged asymptomaüc penods The discontmuity suggests that theie is some tnggei foi each event, peihaps a duect Stimulus a tempoiary detenora tion of intnnsic tesistance, 01 some combmation of these tactors

The term mhented thiombophiha acknowledges the piesence of an mhented factoi that by itself predisposes towaids thrombosis but due to the episodic nature of thiombosis, requnes mteraction with othei components (mhented or acquned) bei01 e onset of the climcal disorder. see Fig 2 Undoubtedly the concept of mhented thiombophiha is an opeiational one, the dehmtion of which has undeigone contmuous lermement It was ongmally based upon early piesentation of thiombo-sis. usually coupled with mhented phenotypic abnormality ot one öl the mhibitory piotems, antithiombm, PC or PS Progress m the molecular basis of thrombosis has enabled a moie genetically based dehmtion to be foimulated Inherited thiombophiha h a genettcalh deteinnned

tendenc\ to venous thiomboemboliiin Dominant abnoimahnes 01 combmatwns of leis were defects ma^ be chmcalh appaient fwm eaih age of onset fieqiient lecunence 01 famih Inston Mildei tiatts mav be ditcovered only b} laboiaton imeitigatwn All genetic mfluences and theu mteraction aie not \et undei itood

The identified and potential genetic factoi s predisposmg toi thiom-bophiha discussed fully below aie summansed m Table l In its tootnote aie hsted some ot the many potential mteiacting acquned nsk tactois

Coagulation Regulation By Antithrombin FXa Antithrombin Complex Anticoagulant Antithrombin +· Heparln or EC Heparan Sulphate Anticoagulant Thrombm-Antithrombin Complex Fibrinogen Fibrin Coagulation Regulation by PC/PS Protein S Protein C Fibrinogen Fibrin Thrombomodulm Fig l Representation of the two prmciple anticoagulant pathways known to

be important m the regulation of Coagulation protemase activity To the left of each diagram is a simplified view of the Coagulation cascade illustratmg the positive 'procoagulant' feedback loops by which thrombin activates factors V and VIII To the nght are the 'anttcoagulant" pathways bv which excessive activation of Coagulation is prevented These pathways imolve antithrombm (which directly mhibits the coaguiaüon piotemases such äs factor Xa and thrombin), and PC/PS (which mactivate factor Va and factor Villa) PS normally forms a complex with C4bBP and it is only the free form of PS that acts äs a cofactor for PC

Risk Factors Genetic + Genetic

Risk Factors Genetic + Acquired

Venous thromboembolism

Λ? 2 Venous thrombosis can be caused by interactmg genetic and acquired nsk factors

commonly referred to äs the P l-P Γ bond) Inhibition of most of the blood coaguiaüon protemases is relattvely slow, but can be accelerated at least 1000-fold by the bmdmg of hepann (and heparm-hke com pounds, such äs endothelial cell hepann sulphate) to antithrombm The mteraction between hepann and hepann-bmding domams m anti-thrombm results m a conformational change of the molecule which facihtates its mteraction with the protemase Inacme antithrombm senne protemases complexes are rapidly cleared from the circulation More Information on the structure, biochemistry and mechamsm of action of antithrombm can be found m a number of iccent reviews (7-9)

Human antithrombm cDNA clones have been isolated and se quenced (10 12) The gene codmg for antithrombm is locahzed on chromosome l between Iq23 and Iq25 (13), it is 13 480 bp long and containssevenexons(l,2 3A, 3B 4,5,6)(14), itsnucleotidesequence has been lecently completed (15) Several sequence \auations or poh-morphisms have been descnbed withm the human gene [for reviews see (7-9)], mcludmg a highly polymoiphic trmucleotide repeat sequence m mtron 4 The latter, particularly, seems useful for haplotype anaiv sis m the study of recurrent mutations or Imkage analysis (16)

Antithrombin deficiency is a heteiogenous disorder The subclassifi-cation of antithrombm deticiency was ongmally based mamly on the results of functional and Immunologie assays m plasma Later after more Information had become available on the actual mutations m the antithrombm gene, the nomenclature was modified (17, 18) Presentlv we recogmze type I antithrombm deficiency (identified by a concordant

reviewed for which the available studies still do not permit definitive Statements to be made on their association with a thiombotic nsk

Antithrombm Deficiency

Antithrombm is a smgle chain plasma glycoprotem (58 kDa) which belongs to the superfamily of the Senne Protease Inhibitois (serpms) It is synthesized in the hver and its concentration m plasma is 2 5 μΜ Antithrombin is the pnmary mhibitor of thrombin and also mhibits most of the other activated senne protemases m\olved m blood Coagulation (factor Xa, factor fXa, factor XIa. factor Xlla, kalhkrein) It is therefore one of the most important physiological regulators ot fibnn tormation

Inactivation ot protemases by antithrombm occuis \ ui the tormation of an irreversible l l molar complex, m which -\ig393 toi ms a stabihsed bond with the active site of the protemase The stable bond totms äs the protemase attempts to cleavc the mhibitoi Aig 393-Ser 394 bond (this bond is at the reactive centie ot antithiombm and is

Tobte l Possible causes of mherited thrombophilia (I) Inhei ited

Antithrombm deficiency PC deiiciency PS deficiencv APC R/factor V 506Arg Dysfibrmogenaemia Thrombomodulm toGln

(u) Acquired/Inhented(precise relative contnbution äs \et uncertain) Hyperhomocystememia Elevated factor VIII Elevated fibnnogen1 (in) Potentmllv Inhented (butfirm e\ideiue lackmq)

Plasmmogen deticiency Hepann cotactor II Elevated histidme detiuencj rieh glycopiotem Plasmmogen activator Elevated plasmmogen

deiiciency' activator mhibitoi'

As discussed m the text there is yet no hrm evidence that conditions listed im dei (m) are hnked to mherited thrombophilia The development ot thiombosis is thought otten to be uiused by mtei action between genelic and nquired laüors the best iec.ogmsed öl the lattci bemg advanung age immobilisation mapr surgery orthopaedic suigery and neurosuigery pregnancy puerpertum use ot oestrogen-contammg hoimones malignancies and the antiphospholipid syndiome

reduction ot both functional and immunological antithrombm) and type II antithrombm deficiency [also identihed b> a vanant antithrombm molecule, which has a detect m the Reactive Site (II RS), a detect atfecting the Hepaun Bmdmg Site (II HBS) or multiple tunctional detects (Pleiotropic Etfect) (II PE)] From a clmical pomt ot view anti-thiombm deticiency is heterogenous. see belovv with mutations caus-mg type II HBS deficiency being ot much less nsk than those causcaus-mg the other subtypes( 18. 19)

In 1991 the tust database ot antithrombm gene mutations was pub-lished (20) A revision ot this database became available m 1993 äs a leport of the Thrombm and its Inhibitors Subcommittee of the Scien-titic and Standardization Committee ot the International Society on Thrombosis and Haemostasis (SSC ISTH) (18) In the 1993 database 39 distmct mutations and nme whole or partial gene deletions (>30 bp) are hsted (hat have been tound in type I antithrombm deficiency These mutations will mtroduce a trameshift (plus prematwe termmation codon), a dnect teimmation codon. a change in mRNA piocessmg or unstable translation products In the type II deticiencies 11 diffeient mutations have been repoited m the type II RS gioup, 1 1 m the type II HBS group and 9 m the type II PE group Most of the mutations found m type II RS patients concern the leactive site of antithrombm (P12. ΡΙΟ, Ρ2, Pl and ΡΓ) Type II HBS mutations are located mamly

on the A, C and D α hehces, Sites that have been proposed to be mvolved m hepaim bmding (21) Mutations pioducing pleiotiopic effects (type II PE) mterestmgly all are located in stiand IC dose to the C-teimmal end ot the piotein, this legion has been shown to be essential both toi the stability of the protem and tor the tiansmission of conformational changes that endow the protem with its antithiombm activity (both m the piesence and absence of hepaim) (22) Smce the publication of the fust levision of the mutation database several leports on novel mutations in the antithrombm gene have been published (23-27)

Recunent mutations are tound in all types of antithiombm deticien-cy, but especially m the type II HBS From the 21 distmct repeat mutations 9 mvolved a CpG dmucleotide (hotspot tor mutation) Only prelimmary infoimation is available (16) that addresses the issue of whethei these recurrent mutations are the result of mdependent mutations or of a founder effect (identity by descent)

PC is a vitamm K dependent plasma glycoprotem which is the piecmsor of the senne protemase Acüvated Protein C (APC) PC is

synthesized m the hver äs a smgle cham molecule (62 kDa) Smgle cham PC is converted mto a two cham molecule by lemoval of a dipeptide (Aigl57 - Thrl58) probably m the Golgi In plasma most ot the PC is in the two cham form (41 kDa heavy cham and 21 kDa light cham), the concentration ot PC m plasma is 65 nM and is reduced dm mg tieatment with oial anticoagulants

PC is a multimodulai piotein the ammo termmal light cham contams a 7-carboxyglutamic acid rieh domain (Gla-domam) and two Epideimal Growth Factor hke domams (EGF domams) These domams have been tound to be mvolved m the tormation ot Ca:+-mduced

contoimational changes, bmding to phosphohpid suitaces, the actna tion of the molecule and its mteiaction with other piotems (such äs its cotactoi PS), while the carboxyteimmal heavy cham contams the sei me piotemasc moiety ot the molecule

Dm mg coagulation PC can bc activated by thiombm via cleavage ot the Argl69-Lcul70 bond This reaction (which is noimally very slow) can be gieatly accclciated by the bmding öl thrombin to

thiombo-modulm, a transmembiane piotein receptor present on the membrane of endothelial cclls The APC. thus tormed, mactivates the cofactors, tactor Va and Villa, by selectue proteolytic cleavages To do this etficiently APC needs to torm a complex with PS on a suitable mem-brane surface Apart from these anticoagulant properties. APC also has antitibrmoKtic ptoperties and anti-mflammatoiy etfects More detailed infoimation on structural. biochemical and tunctional aspects ot PC can be tound in recent review aiticles (28-30)

Human cDNA clones have been isolated and sequenced (31. 32) Also the structme of the gene (PROC) has been tesohed (33. 34) u contams 9 exons and 8 mtions on 11 kb of genomic DNA The gene transcnpt is 1795 bp It contams a 5' untianslated region of 74 bp a protem codmg region (exons 2-9) and a 3' untranslated region ot 294 bp The gene has been mapped to the cmomosome 2ql3-ql4 region (35)

A numbei ot DNA sequence polymorphisms are known to occur m the PC gene, both m the piomotor region and m the codmg region (36, 37) None of these vanations leads to an ammo acid dimorphism All these polymorphisms have been foimatted for detecüon by PCR This is also tiue for the two restnction fragment length polymorphisms (RFLPs), reported by te Lmtel-Hekkert et al that are located ~7 kb 5' to the PROC gene (38)

PC deficiency is a heteiogenous disorder (37, 39) A phenotypic subclassification has been proposed that is based on the results of func-tional and Immunologie PC assays In type I PC deficiency there is a concordant reduction m PC activity and PC antigen, while m type II PC deficiency there is eudence tor the piesence of an abnormal PC mole cule (leduced PC actn ity normal PC antigen) A furthei classification of the type II PC deficiency can be made by compaimg the results of diffeient functional tests (clottmg lest versus chromogenic test)

In May 1995, the first Update of the data base of PROC gene mutations was published on behalt of the Subcommittee on Plasma Coagulation Inhibitois ot the SSC ISTH (37) The database iontams 331 entnes denved fiom 315 umelated probands In total 160 different mutations were reported that had resulted in type I or type II PC deficiency Suipusingly -60% (n = 132) of the mutations (n = 28) causmg type I PC deticiency are missense mutations Probably these ammo acid substitutions lead to changes in the mteractions with other lesidues and thus mteifeie with protem foldmg, a condition associated with rapid mtracellulai degradation of the protem Missense mutations leadmg to type II PC deticiency predommantly are located m surface and solvent accessible aieas ot the protem that are lelevant tor the function of the protem (hke propeptide cleavage, calcium bmding, acti-vation, mteraction with thrombomodulm. active site. Substrate bmding) (40,41) Further, theie are mutations m the promoter and 5' untranslat-ed tegion, sphce site abnormahties. small deletions and msertions (eventually leadmg to a prematme termmation codon) and nonsense mutations that also aie responsible for type I deticiency

About 30% of the umque events occur m CpG dmucleotides and are C ->· T and G -»· A transitions Interestmgly, 18 tiansitions m 16 differ-ent CpG dmucleotides foim about 40% of all the differ-entnes m the 1995 database At present theie is msutficient Information to distmguish recuirent e\ents tiom events that aie identical by descent

Τίώΐί'2 PROS gene sequence \anations in patients with phenoUpii. PS

dehcienc\

Exon Codon. sequ mutat Predicted mutation Ref 1 - 2 5 , Ins T -25, Leu -» Leu (64)

frameshift, stop

2 - 2, G->T -2, Arg -> Leu (68) 2 - 1, G->A -1, Arg -> His (68)

2 22, C->A 22, Cys -» stop (68)

2 26, A-»C 26, Glu -4 Ala (68)

2 31, T-*G 3 1 , P h e - > C y s (68)

2 37, C^T 37, Thr -> Met (68) 3 43, de! A 43, Lys -> Asn (144)

frameshift, stop

4 49, G->A 49, Arg -> His (68) 5 82, del T 82, Pro -» Pro, (145)

' frameshift stop

5 103, C->A 103, Thr -> Asn (68) 6 155, A-»G 155, Lys -4 Glu (69) 8 204, A-»G 204, Asp -> Gly (68) 8 208, G -»A 208, Glu -> Lys (68) 8 217, A-»G 217, Asn -» Ser (146)

8 220, delG 220, Gly-» Glu (146) frameshift, stop

8 224, C^G 224, Cys -» Trp (68) 8 224, T-»C 224, Cys -4 Arg (68)

9 2 6 1 , delT 261, Leu -»Trp (65) frameshift, stop

9 267, delG 267, Gly -» Gly (65) frameshift, stop

10 335, G^A 335, Asp -» Asn (68) 10 340, G-^T 340, Gly ^ Val (65)

12 448, G ^ T 448, Gly -> stop (146)

13 467, T->G 467 Val -» Gly (65)

14 547-548, del AC frameshift, stop ( 1 4 6 )

14 565, ms T 565, Val -> Val

frameshift, stop

14 570, T-»C 570, Met -> Thr (146) 14 578, m s C 578 Pro -» Leu (65)

frameshift, stop

15 636, A-+T 636 stop -» Tyr (64) new stop at 649 mtron d G -> A exon 4+1 ( 1 4 6 ) mtron e G -» A exon 5+5 (68) mtron j G -» A exon 10+1 (146) mtron j G -» A exon 10+5 (64) PS Defiticncy

PS is a vitamm K dependent plasma glycoprotem (70 kDa) It is svnthesized m the hver, but also in endothehal cells. megakaryocytes and Leydig cells m the testis The concentration ot PS m plasma is 25 μι/ml and is leduced durmg treatment with oral anticoagulants

PS is a multimodulai protem, it contams a •γ-caiboxyglutarmc ι ich domam, a thrombin sensitive region, tour epidermal giowth tactor like domains and a carboxytermmal legion which is highly homologous to the sex hormone bmdmg globuhn (SHBG) PS seives äs a non-enzy-matic cotactor ot activated protem C (APC) m the mactivation ot tactois Va and Villa probably by tacilitatmg the loimation öl

cnzyme-substrate complexes on the surtace ot phosphohpid/platelet rhem-branes This APC-cotactoi activity is destroyed by one ot three proteo-lytic cleavages m the so-called "thrombin-sensitive region' (Aig49 ArgoO. Arg 70) In plasma PS circulates both tree (40%) and in a 1 1 stoichiometnc complex with C4b-bindmg protem (60%) The latter compnses 7 identical α chams (70 kDa) and one single β cham (45 kDa) which are hnked to each other m the carboxytermmal region by disulphide bonds The β cham contams the PS bmdmg site Two legions m PS have been repoited to be m\olved m bmdmg of the C4b bmdmg protem (Gly605 to Ile614 and Gly420 to His434) Only the tree form ot PS has APC cofactor activity More recently it has been reported that PS itself also has anticoagulant activity under well-detmed conditions it may tnhibit (m an APC-mdependent way) the activity of both the tenase (IXa- Villa) and piothrombmase (Xa-Va) complexes, some reactions bemg mdependent of the presence of C4b-bmding protem (42-44)

A novel functional aspect of PS has been levealed recently tiom studies teportmg on its bmdmg to the receptor tyrosme kmase known äs Rse/Tyro 3 (45) and to a specific receptor on vascular smooth muscle cells This might mdicate that PS also is mvolved in the regulation of cell prohferation More detailed Information on structural. biochemical and functional aspects of PS can be tound m a recent levtew aiücle

l A H\

(47)

Human PS cDNAs have been isolated and sequenced (48-50) From the cDNA nucleotide sequence the ammo acid sequence of human PS has been denved Two highly homologous PS genes have been identi-fied and sequenced (51-53) The PROS 01 PS« gene is the acttve gene it consists of 15 exons which ate spiead over 80 kb of genomic DNA and has been mapped to the chromosome 3 pl 1 1 -3 pl 1 2 legion (54) The PSß gene shows 96 5% homology with PROS m exon sequences and the positions ot the mtions are virtually identical to those m the PROS gene However the PSß gene is a pseudogene. it contams onh exons 2-15 and contams a laige vanety ot detnmental abenations (a sphce site mutation, a hame shitt mutation and three stop codons) With the use of a raie RFLP the pseudogene has been located withm 4 cM of the PROS gene (55) Several DNA sequence polymoiphisms have been reported m the PROS gene (48 49,56) Some ofthese have been very useful for tracking PS deticiency through famihes, foi prenatal diagnosis and for evaluatmg the possibility of allelic exclusion m the case of the study of reveise transcupts of platelet PROS mRNA (57. 58)

consensus sequence Ιοί the N-lmked glycosylation ot Asn458 (PS Heeilen) (62) The licquency ot the PS Heerlen allele in the general Population is 05% and not ditteient trom that m thrombophihc patients (0 79c) At present it is not clear \vhy this genotype is so tiequent among type III PS dehciencies

The genetiL analysis oi the PROS genes ot symptomatic PS deticient piobands has been hampeied and delayed bv the stmctuial complexity ot the PROS gene and the existence ot the highly homologous pseudo-gene \ tuithei eomphcation is the unexpectedly lovv yield ot success-lul genetic analyses In thiee sepaiate studies mutations weie only tound in 50 60% ot the patients. although all coding and flankmg legions had been amphtied and sequenced (63-65)

Onl) two laige deletions m the PROS gene have been lepoited äs a

cause of a type I PS detiueney (66, 67) Agam. the majonty ot genetic lesions causmg a type I deticiencv aie smgle nucleotide substitutions msertions and deletions So tar 33 unique e\ents have been lepoited see Table 2, but this number will mciease lapidly m the neai futuie At piesent these numbeis aie too low to make any futther analysis Four diffeient mutations have been repoited that cause a type II PS deficien-cy (68,69), two m the propeptide, one m the fast EGF domam and one m the second EGF domam Table 2 gives a summaiy of all the PROS gene mutations lepoited to date m symptomatic PS deficient patients In 1995, m Jeiusalem, the Subcommittee on Plasma Coagulation Inhibitois of the SSC ISTH dectdcd to publish the first database ot PROS gene mutations m 1996/1997

Faitoi V AI $506 to Gin

Factoi V is a smglc chain plasma glycoprotem (300.000 kDa) It is synthesized m the hver and in megakaiyocytes Human plasma has a concenttation ot 20 nM ot this piocofactot, while its concentiation m platelets ts 4 μg/109 platelets Dunng blood coagulation factoi V is

convetted mto factoi Va by (meizo) thiombm and/οι tactoi Xa Throm-bm-activated tactoi V is formed attei cleavages of the Aig709-Sei710, Aigl01S-Thil019 and Aigl545-Serl546 bonds It is composed of an amino teimmal tiagment (heavy chain 105 kDa) and a caiboxy termmal tragment (light cham 74 kDa) non-covalently Imked via a tightly bound

Ca:+ lon Factoi Va seives äs a nonenzymatic cotactoi m

piothrom-bmase (factot Xa. phosphohpids, Ca2+) by mcreasmg the catalytic

efficiency approximately 2000-told Factoi Va light chain is lepoited to contam the phosphohpid bmdmg site, while the heavy cham site is mainly lesponsible toi the cofactot activity

Factoi Va is mactivated by pioteolytic degradation ot its heavy chain by APC, this mactivation is moie etficient in the presence of phospho-hpids and Ca2+ and is accoidmg to some an oideied and sequential

event with a tirst cleavage at Aig506 and subsequent mactivatmg cleavages at Aig306 and Aig679 (70) Otheis demonstiated that two tandom cleavages (mainly at Aig506 and Aig306) aie mvolved and that both conüibute to the mactivation of tactoi Va (71)

Moie rccently it was lepoited that factoi V is not only a piocotactor m the piothiombmase leaction but also a colactoi m the mactivation of lactot Villa by APC (72) More mtoimation on the stiucture and tunc-tion ot human tactoi V can be tound m a lecent leview (73)

Paitial and oveilappmg human tactoi V cDNAs have been isolatcd hom HepG2 and human (letal) hvei cDNA libiancs (74-76) Fiom the nucleotide sequence, the completc amino acid sequcnce ot factoi V was denved It consists öl 2196 amino acids and shows a chaiactenstic do-mam stiuctuic (Α,-Α-,-Β-Α,-^-Ο,) that is also lound m lactoi VIII

The chaiactenzation öl the human tactoi V gene was lepoited m 1992

h\ Cupe et al (77) Twenty live cxons and 24 intions span

appioxi-matelv 80 kb genomic DNA The nucleotide sequence ot all coding and flankmg regions has been deteimmed The factor V gene has been mapped to chiomosome l (lq21-25) and is closeh Imked to the anti-thiombm gene (78) A number ot nucleotide sequence \anations m human tactoi V cDNAs ha\e been identitied (74-76 79) but \vith some exceptions (79 80) the allele trequencies ha\e not been reported The microsatelhte maikei D1S61 has been mapped \\ithin 4 cVI ot the tactor V gene locus and has been used successtulh toi trackmg the tactor V gene m a tamil} with hereditary APC-R (})

In 1994 the smgle pomt mutation m the factor \ gene was identitied äs the genetic detect causmg the phenotype ot APC-R m the \ äst majoi-ity ot atfected mdividuals (3 5 81) It m\ol\es a G ->· A transition ot nucleotide 1691 in exon 10. which piedicts the s\nthesis ot a \anant tactoi V moiecule (factor V 506Aig to Gin 01 tactor V Leiden)

The mechamsm by which the mutation leads to the phenoupe ot APC-R is still subject of detailed biochemical studies Hov,e\er it is clear that the replacement of Aig506 by Gin will pie\ent clea\age ot factoi V(a) at this site by APC and by that dela\ the mactnation ot tactoi Va (82. 83) either by pieventmg the contoimaüonal change necessary foi the mhibitoiy cleavage at Arg306 01 bv preventmg the kmetically moie tavourable mhibitory cleavage at \ig506

So far the factoi V 506Aig to Gin mutation is the only genetic defect identified in APC-R families It has a lelativeh high fiequenq m Caucasian populations (up to -6%) but a much lower fiequenc\ m the Japanese and othei Eastem populations (~09c) (84) Prehminan evidence toi a foundei etfect in the spiead ot this disoidei was obtamed trom the lesults ot haplotype analysis of 53 Dutch cameis ot the mutation (3)

Other Canclidates'

Theie aie a numbei ot othei genetic detects 01 isolatcd deticiencies that ha\e been imphcated m contitbuting to the iisk ot thiombosis m tamihes with thiombophiha In most cases these ha\e been based on obseivations m case-tamihes Sometimes genetic defects ha\e been identitied but no data on genotype-phenotype relaüonships aie available äs yet (85) In the next paiagraphs the vanous candidates will be buefly discussed

Heiediian chsfibnnogenemm is detected by a piolonged plasma

thiombm time Clmical Symptoms vaiy hom none mild bleeding to venous 01 aiteual thiombosis The phenotype ma\ follow iecessue 01 dominant inhentanie Recentlv the eudence toi a causal lelattonship between an isolated dystibimogenemia and venous thrombosis has been cntically leviewed and discussed bv a woiking paity ot the SSC Subcommittee on Fibi mögen ot the ISTH (86) In that mvestigation 5 tamihes (tiom 5 diffeient countnes) weie identified m which apait of the pioband. two 01 more tamily membeis had both the detect and thiombosis In all tive cases the genetic detect had been identitied (two mutations m the Aa cham two mutations in the B β cham and one

mutation m the γ chain ot hbnnogen) In one tamih only homozvgotes foi the mutation (tibrmogen Naples) weie chnicalh atfected The lela-tionship between the dclect m the tibimögen moiecule and the

phenotype ot thiombophiha is still pooily undeistood Fuithei studies aie hampeied bv the low tiequencv öl dvslibimogenemia m cohoits ot palients with thiombosis (0 8%) (86)

Thioinlwmocliilin (TM) is anothei component öl the PC anti

moclulm may be associated with an mcreased nsk ot thiombosis Unfortunately plasma is not an abundant source ot TM and theretore cannot be used tor the laboratory diagnosis ot TM detects Analysis ot the TM genes m cohorts ot symptomatic probands with a tamilv history of thrombophiha did not reveal any alteration m the coding and flankmg regions of the gene (0/30) in one studv (Reitsma PH & Berti-na RM unpubhshed observations) while tour ditferent mutations (4/87) vvere identitied by a second group (85 87) To date there is still very limited mtormation ot the co-segregation ot these mutations with thrombophiha m the famihes of the piobands

Recently mild fape>hoino<.\stein<nnia was found in 19% of patients with juvemle venous thrombosis and family studies showed that in most cases this phenotype was mhented (88) Two large patient-control studies have shown that hyperhomocystememia is a nsk factor for lecurrent thiombosis (89) and for thiombosis m unselected patients, among whorn elevated levels were present in 10% (90) Seveie hypei-homocystememia has a population prevalence of ~1 300 000 and is most frequently caused by homozygous c\statluomne β svnthase deficiency Homocysteme is a sulphydryl amino acid denved üom metabolic conversion of methiomne Its mtracellular metabohsm occurs through remethylation to methiomne or transulphuration to cysteine Cystathiomne ß-synthase is involved in the tiansulphmation pathway A small number of cases of severe hyperhomocystememia are caused by homozygous deficiency of methyleneteti ahydi ofolate reditc

tcife Gene defects m both of these metabolic enzymes are implicated m

mild hyperhomocystememia (91, 92) A recenth descubed mutation with theimolabihty of methylenetetrafolate reductase may in the homo-zygous state be a sigmficant and frequent cause for mild hypeihomo cystememia (93)

Plasmmogen deficiency and d\5plasmmoqenemia have been repoit

ed frequently to be associated with thrombophiha Howevei, family studies reveal that m most famihes with a type I plasmmogen deficien-cy (parallel reduction of plasmmogen activity and antigen) only the proband suffers from thrombotic disease (94) In a lecent retrospective analysis of 20 famihes, heteiozygotes of a type I plasmmogen deficien-cy weie found to expenence sigmficantly more thrombotic events (although rather late m life) than their normal family members (95) The frequency of plasmmogen deficiency m the general population seems to be slighter lower (0 4%) to that m cohorts of patients with thrombosis (l 3%) (96,97) Dysplasmmogenemia or type II plasmmo gen deficiency (reduced plasmmogen activity, normal antigen) asso ciated with Substitution of AlaoOl by threomne is a common vanant in the Japanese population and seems not to be associated with thrombo sis (98) Studies of plasmmogen deficiency at the DNA level are still very tare

In a model in which it is expected that teduced plasmmogen levels may cause thrombophilia it seems reasonable to propose that an mher-ited elevated Hntidme Rieh Ghcopiotem (HRG) level m plasma is al-so a nsk tactor tor thrombosis HRG (a non enzymatic protem) forms a l l complex with plasmmogen m plasma (via bmding to its lysme bmdmg sites) and thus leduces the free plasmmogen concentration to aiound 50% (99) Complex tormation with HRG mteiferes with the bmdmg ot plasmmogen to tibnn Although several tamihes with thrombophiha and high HRG levels have been repoited (100 101) theie is still no tormal evidente loi its association Surpnsmgly, recent-ly two famihes have been leported where a partial deticiency öl HRG seems to be associated with thrombophiha (102 103)

Anothcr potential nsk tactot ot thrombosis, deticiency of the /mifi

Fiictoi Ρί/f/nwn Inlubitoi (TFPl) has been mvestigated but no

muta-tion m TFPl genes öl 30 symptomaiic piobands of famihes with

ihmm-bophiha (Reitsma PH & Bertina unpublished obsenations) could be found Other potential candidate nsk tactors tor thrombosis are hepa

nn Lofactoi II defiuenc\ and ß-, gl\u>piotem I deficienc\ Laboiatorv

analysis ot large groups ot patients with thiombophiha and ot controls revealed however, that the hequency ot heterozygotes tor these detects among patients and controls is very similar (0 6% and 6% respectively) (104 105) Nevertheless a tew famihes ha\e been leport ed in which an isolated heparm cofactoi II deticienc\ seems to segre-gate with the thiombophiha (106 107) In two famihes with a type II heparm II cotactor deficiency the lesion m the gene was identified (lepiacement ot Arg 189 by His) (108) This mutation is thought to etfect the dermatan sulphate bmding site and is analagous to ceitam detects m type II HBS antithrombm deticiency that effect the heparm bmdmg

A last genetic defect that has been discussed dunng the last years with respect to its possible association with thrombophiha is a partial

factoi XII deficiency Original studies from Mannhalter et al suggested

a high fiequency of heterozygotes for factoi XII deticiency m cohorts ot thrombophilic patients (109) Subsequent studies have not suppoited these fmdmgs (110)

Fmally there is a phenotype that recently has been identified äs a nsk factor for thrombosis m large patient-control studies elevated factoi

VIII levels (111) The hereditabihty of this phenotype and the eventual

underlymg molecular defects have not been reported so far Epidemiology of Inhented Thrombophiha

Pie\alence ofHeieditan Thiombophiha

Venous thrombosis has an overall annual mcidence of <1 m 1000 It is lare m the young, and becomes more frequent with advancing age The true pievalence of hereditary thrombophiha is not yet known It seems cleai that we do not know all genetic abnormahties causmg a tendency to venous thrombosis, since even m patients fiom famihes selected on the basis of a high numbei of unexplamed thromboses m only about half an underlymg defect will be tound(112) This indicates that the prevalence of hereditaiy thrombophiha m the general popula tion will be higher, possibly up to two fold than estimates from large prevalence studies on the known genetic defects A high prevalence of hereditary thrombophiha will make this an important factor m the over-all mcidence of thiombosis This mav also be assessed by takmg the piesence of a positive family histoiy mto account Among unselected consecutive patients with deep vein thrombosis a tamily histoiy was reported by one out ot every four patients (113) Even though a positive family history may occur by chance äs venous thiombosis is not a laie disorder, these figures mdicate that genetic causes are prominent m the etiology of venous thiombosis

The prevalence ot deficiencies of PC and antithrombm has been mvestigated m a veiy laige study ot almost 10,000 blood donors (l 14-117), Table 3 Repeated testmg ot the le\els of these protems coupled with tamily studies and DNA analvsis led to estimates of l m 500 toi PC deficiency and l m 5000 toi type I antithrombm deficiency This is m the same lange ot the fmdmgs öl a pievious study among over 5000 blood donois where l m 250 weie consideied PC deficient (118) Although some caution is needed m mteipietmg data based on studies among blood donois who are a seil selected sample from the general population these seem to be fair estimates Vitcptmg the approximate prevalence ot PC deticiency to be l 350 the prevalence öl severe (homozygous 01 compound heteiozygous) deticiency will be l 700 X l 7()0 = 4 9 Χ 101 Ottspimg liom iclated paients will ot couisc have much highci nsk ot sevcic cleliucni\ The pie\alence ot se\erc

rablc 3 Pre\alence ot the major thromboplulic clotting ibnormalitits

Hialth\ i

T a i t e t a l (n = 9669) (l 14 117) M i l c t t c h e t a l (n = 5422) (l 18) S\ensson and Dahlb ick (n = l 30) ( Rosendaal et al (n = 474) (4) Ridkeretal (n = 704) (l 20) C()iiKcuti\( patienft uithfnst DVT Heijbocretal (n = 277) (111) Koster et al (n = 474) (123) Rostndaal et al (n = 471) (4) Tluomboplulic patients Briet et ai (n = 1 13) (147) Schaneretal ( n = 1581(148) B e n T a l e t a l ( n = 107) (149) Tabernoetal (n = 204) (l 50) Griffin et al (n = 25) (l 12) 119) PC PS Anmhrombin APC R detiuenc\ dehuencv defiuencs

02rc+ 04%

3%+

thiombm deficiency is hkel} to be 100 1000 less than se\ete PC deficiency There are no studies of sufficient size on PS deficiency among healthy individuals on which a reliable estimate ot its prevalence can be based

Fot APC-R, the groups that have been studied are not äs large äs m the blood donoi studies Howevei smce the prevalence of this abnormahty ts an oidei ot magnitude htghet than the other mhibitoi deficiencies the estimates aie ab tehable The estimates toi Caucastans tange hom 3 to 7 percent (4119 120), and foi several studies aie based on genetic mvestigation (4, 120) Smce these estimates aie also based on seit selected individuals \uthoul a history of caidiovascular disease or venous thiombosis they are under mthei than o\ei-estimates The prevalence at birth ot homozygous factor V 506Gln mutatton has been estimated at ~ l 5000 (4)

Among consecutive patients with objectively confumed deep vem thiombosis, deüciencies of PC PS and antithrombm combmed account for -5% APC-R is present m 20 percent ot consecutive patients with deep-vem thiombosis (4, 121)

Seveial studies have tocused on selected patients with venous thiombosis and usually tound a highei pie\alence of deficiencies of PC, PS and antithrombm than has been lepoited among unselected consecutive patients Diffeient cnteiia were used in selectmg the patients for these series some authors mcluded patients who expen-enced a first thrombosis at a young age others studied individuals with tecurrent thrombotic events or thiombotic events that occuned m the absence of any of the classical nsk factots foi venous thrombosis, while some mcluded mdiuduals with a positive tatnily histoiy Because ot these heterogeneous inclusion critena the prevalences that have been leported m these studies should not be compaied too closely The pievalences that ha\e been lepoited among selected patient groups foi deticiencies ot PC PS and antithrombm aie mostly between 5 and 10%, much higher than the pre\ alenccs lound in Ihc population studies, and also somewhat highei than the tiequency among consecutive unse lected patients These highei prevalences in patients with thiombosis than m healthy mdniduals and in thrombophilic individuals äs com pared to unselected patients also mdicate that these deticiencies indeed lead to venous thiombosis and \enous thiombophiha APC R appcais to account tor halt ot all cases öl heieclitarv thiombophiha and cleaily

emetges tiom Table 3 äs the most important cause of heieditarv throm-bosis and peihaps ot venous thromthrom-bosis m general

Risk of Venous Tluombosis

The nsk of venous thrombosis toi mdmduals with clottmg factor gene abnormahties has laigeh been mvestigated bv two appioaches tirst by studies in tamily membeis of piobands with one of these abnormahties and second b\ population-based studies (case contiol studies) These diffeient appioaches do not necessanly vield the same Information The foimei aie based on tamihes in which the hereditabil-ity of the abnormahty has been shov n bv mcludmg only tamihes \\ ith one 01 more individuals with the clottmg abnoimahty apart fiom the proband who is both symptomatic tor thrombosis and has the clottmg defect In the analysis typicallv the occurrence of thrombosis is compaied between the famih members with and without the clottmg factoi abnoimahty while the proband is excluded from the analvsis Smce hereditabihty is a preiequistte in studies of this design thev aie most fitting to dnectly answei questions concerning nsks ot genetic disordeis In population based studies patients with thrombosis aie compaied to healthv mdmduals with legaid to the pievalence ot clottmg tactor abnoimahties These case control studies yield ielati\e nsk estimates which mdicate how much highei the nsk of thrombosis is tor an mdividual with a pamculai nsk tactoi compared to an mdi vidual without that factor Smce onh indiMduals aie mcluded and not famihes, no direct Statements about hereditabilttv lesult from these studies Furtheimoie case control studies can only yield estimates ot ielati\e nsks not of absolute (hte time) nsks

When consecutive patients are mcluded m a population-based stud\ (with population-controls) the lesults associatmg a particular nsk lactoi with thrombosis apph indiscnminateiv to all individuals v\ith that abnoimahty m the population The results iiom famih studies aie based on lamilies that stood out and \vcre lecogmsed because ot a conspicuous high frequenu ot thiombosis stnctly speakmg these lesults only apply to tamihes detected m a smiilai wav In othci words population-based studies yield an a\eiage nsk lor individuals with a paiticulai abnoimahtv wheicas lamih studies are conductcd among those with highct nsks Smce it is likch and has now been shov\n to be

true tor APC-R that many tamihes vvith thiombophilia displav more than one genetic abnoimalitv one should exercise extreme caution m apphmg results fiom familv studies to unselected mdividuals 01 results trom population-based studies to thrombophihc tamihes It an mdividual is identified äs tairymg a thiombogenic abnormahtv by means ot a study among the general population m all hkehhood he onK cames that one abnoimalit) if he is tound äs pioband ot a tamih \vith thrombophiha he might well caiT} two 01 moie and it he is an unselected patient vvith thiombosis he mav well be one of both

Antitliiombin Dejicienc\

When the data tiora all the available family leports of antithrombm deticiencv are combmed it appeais to confer a highei nsk ot thiombo sis than deticiencies ot PC and PS Thiombosis is not uncommon betoie age 16 and about halt of the patients from these lepoited tamihes expeuenced a first thrombotic event before age 25 (122 123) Antithrombm deticiency especially has a rauch highei nsk ot thrombo sis in pregnancy than deficiencies of PC or PS (124) The tifty fold diffeience m the prevalence among patients with a tirst event ot deep venous thiombosis and the prevalence m a healthy population (113 114 125) also suggests a highei thrombotic nsk m antithrombm defi ciencv than in PC deficiency, although such a difference could not be substantiated m a population-based studv (125) There is some debate uhethei the moie severe form ot thrombophiha caused by anti-thiombm deficiency might also lesult m mcieased moitahty (126 127) in some pedigrees fatal thromboses have been obseived (128) but m histouc studies of Dutch pedigrees no excess moitaht) was evident (24 129)

PC Deficienc\

Smce 1981(130) many tamilies with heieditaiv piotem C deficien cy have been leported The nsk ot thrombosis appeais not to be diffei-ent tor the ditfeidiffei-ent types of piotem C deiiciency (t\pe I low plasma le\el and type II low activity) noi foi the laige numbei of rautations identihed m PC deficiency (37) In family studies it has been shown that tamih members who are PC dehcient have an increased nsk of \enous thrombosis (about 8 10 fold) and that by age 40 about half ot them will have expenenced at least one thrombotic event (131, 132) In a population-based study (125), a lelative nsk estimate of 7 has been repoited which (surpnsmgly in view of the comments made above) is \eiy similar to the relative nsk denved fiom familv studies The preva lences leported m unselected patients with fast thiombotic event (3%) (113 125) and healthy mdividuals fiom the geneial population (02%) (117) also suppoit a telative nsk of this size or slightlv highei

These data all apply for heterozygous PC deficiencv m which PC activitv on aveiage is 50 peicent of noimal The laie homozygous patients with no PC activity m plasma has a much highei nsk of thiombosis and develop puipuia tulmmans shoitlv atter birth (see below)

PS Deficicnc\

Smce 1984 (133 134) many tamihes have been lepoited with venous thrombophiha and PS dchciencv It is not cleai whethei the Ihice different subtypes that have been desuibed (tvpes I II and III) conlcr similar nsks ot thiombosis It is \ e i y dithuilt to ainve at nsk cstimates foi PS deliuency smce the pievalcnce in the geneial popula tion is unknown theie aie no lepoits tiom lamih studies lormalh

assessing the relative nsk the moleculai basis m many cases remains unclear and several tamihes with APC-R appeai to ha\e been misclas sitied äs PS deficient (type II) (135) In one population based case con-trol studv the pievalence ot mdividuals vvith lepeatedl) lovv levels ot PS did not diffei between cases and tontrols which does not support an association ot PS deficiency and venous thrombosis (125) It is difficult to leconcile this findmg with the reports trom seveial tamihes äs well äs the highei pievalence of PS deficiencv in selected patients vvith thiombophilia than m unselected patients with a tust event (Table 2) Although the available evidence generallv leads to the conclusion that PS deficiency mcreases the nsk of thrombosis this evidence is rauch less solid than for PC deficiency Homozvgous PS deficiencv has been lepoited and vvhile extremely rare appeais to be äs seveie äs homo zygous PC deficiency (see below)

APC RJFactoi V506Aig to Gin

In a study in 34 tamihes with APC R the life time nsk of thiombo-sis was clearly highei m family membeis with this phenotvpe than m those who did not (119) At age 50, about 25 percent of mdividuals with APC-R had expenenced at least one thiombotic event (119) This is lower than the figures reported foi famihes with PC deficiencv (132) it should be borne in mmd, however, that these famihes with APC R weie not selected on the seventy of thrombophiha äs was the case m familv studies of PC deficiency In a population based case contiol study APC-R was found in 21 peicent of patients with a first episode ot deep vem thiombosis and m üve percent of controls which led to an estimate öl the lelative nsk associated with APC-R of 7 (121) This nsk estimate is veiy similar foi that found for PC deficiencv

Because of its high allele frequencv homozygous cainers of the factoi V Aig506 to Gin alleles are not uncommon The homozygous abnoimahly appears much less seveie than homozygous PC deticiencv smce seveial of the homozygous patients have remamed thiombosis fiee well mto adult life (4 136) Still the nsk of thiombosis toi those homozygous foi the mutation is higher than ior those heteiozvgous (10-fold higher), estimated at 90 fold increased compared to mdividu als without the mutation (4)

Combmed Abnormalities

than m the gcneral population (132) many ot them had APC R (137) In this study it was shown that the combination of PC deticienc\ and APC-R contened a higher nsk than eath ot these abnormahties separately Similar conclusions have been drawn tegarding the m creased nsk ot thrombosis following mteraction ot tactor V 506 Arg to Gin mutation with the othei mhibitordetiuencies PS and antithrombm deticiencies (138 140) The Situation with combmed tactor V 506Aig to Gin and antithiombm mutation is lurther comphtated b\ their dose genetic pioximitv In some tamilies co segiegation ot tvvo genetic detects can occur lesultmg m high thrombotic usk to all arfeued mdi-Mduals(139)

Atquired nsk factors aie important in the development ot thrombo-sis Unfoitunately theie are tew reported studies that contam quan-titative Information It has been shown that the nsk ot thrombosis is turthei mcreased among women with PC deficiency who use oial contiaceptives (141), and is gieatly incieased among APC-R women who use oral contraceptives (142) The nsk is leported to van with the t)pe of progestagen used (143)

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