CA2450902A1 - Method for the detection of risk factors associated with myocardial infarction - Google Patents
Method for the detection of risk factors associated with myocardial infarction Download PDFInfo
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Abstract
A method for determining whether an individual is at an increased risk for myocardial infarction, comprising screening for the presence of Factor II and Factor XIII
alleles associated with myocardial infarction. Also provided are kits and primers that specifically hybridize adjacent to the allele-specific regions of the Factor II and Factor XIII genes.
alleles associated with myocardial infarction. Also provided are kits and primers that specifically hybridize adjacent to the allele-specific regions of the Factor II and Factor XIII genes.
Description
FIEi~D OF THl~ INVEN'TiON
1 by present invel>tion relates to the field of myocardial inf:3rction, more particularly, the invention is directcil at a method fc~r determining the risk for myocardial infarction in an individual.
BACIs:.CROUND OF THE lIVVEi'~TIOI\
Liyacarclial infarctian (~ZIj is rlow among the most frcduent causes of illness arid death, esnevialiy in thi' industrial countries. Ii myocardial infarction is survimd, the vitality of the: patient is IinTit.ed 1:1 racist cases, by secondary symptoms such as paralysis or organ d<3rnagc. Thcrc ~rre also labor-intensive and cost-intensive follow-"-up tr~.atrneots; smh ,~s .{i coim~alc~si~enc.e, physiot:la~~raip~ and medicatiaTl to imprcn-a the health sittTation ~~.ld prevera W rthcr complicaxions.
(treat advances h;~vc been made iTT recent years, especially in research into the causes c)f myocardial infarction. and those iriclude cardiac tissue necrosis cau:;ed by an inadec)uate blood supply dae to the c>c:;lusion of arterial blood vessels either by cholesterol planue is guild-uh, or by i;tot hlnckage (thromhc>sis). Risk factors te)r thrombosis-ildtTCed Tn;'ocardial infaTCiioo r~rc~ thenl~fta to in~lucle both hereditar~~ an, KTC~uired can,iition~;.
liCllt;r~.i~y, t t.cildcili.V ii)~4ialilS tllyacarciial infarction would ariS~
~rGn1 fiyp~r~CLiVf:
coagulation pathways, hypaac~tive arlticoaguiant mechanisms, or hy-poacti.ve fibrinolysis.
1~-Zutatii:~:ns in gees that encode pratcins in these p;~thways arc thc~trght to l.~lay an zi> il~~pc)rtant role in tizc prtzdispa~ition to myaca.,.-dial infarction.
~vl'rc 5c'!'il'1C ~1'i)ti:~~>r t1:1"~;~7r11't1111 ~,~;iei'1'ACCI ~)' ~ll:iiVa~f: at 111f.TTlar7 l3rat~13't71'Ill)if't (<llsc) ~'.III.W'~"11 :i>
Hac;tv:~r 11 (loll}) ~:~teri.s a central actii>n to the proc.cssw ~~
thrumbo:;is ania haemc>stasi~a.
1. t-m. th.r~sTnbio lm~le~.H~ pl;y°s a T-olu in thz tinal 5t.~g~ of hli:~od coa~ulatii)n: the l:ortnr,,tion of art iT~aolul~lc ~ibrir~ ac:~t.
The hit:~.urtc~ ~:Tv:-»'r, con;;,znitttl disorders of prathrc~rnbirr art:
rare: arid invoh~e either lvdm.c_d synthc~si.s of ts.~a 1-11 crlr~lcctrfe (referred to a.~
hypoprc~thrurnbir?~litia or ypc I
prc,thramt,in defic;iencryt or the normal synthesis c~f a dysfuncticsllal rtt<>lout,le (referred to as dysprothrombinelnia or type II prothrombin deficiency). Patients with dysprc~thrombinemia have only ?°~o to 50% of the clotting activity of nc,rn~al prothrombin;
in these patients the severity of the bleedings correlates fairly well with the amount of hrothrombin activity in plasma. A number of dysprothrombinemias have been further characterized by amino acid sequence analysis of the isolated prothrombin molecule or by nucleotide sequence analysis of their prothrombin genes.
It is Jtno~4n that the gene variant Factor V Lviden (FVL-R~f)6t~) <tnd prothrombin ti20210A (FIfG20210A) are hvo of the most commonly recognized genetic prothrombic !o risk factors for venous thrombosis. Based on the increased thrombotic teaidency in ve:lous thrombosis studies, these two 8ene variants have also been examined for possible association with arterial thronabosi~ in myocardial infarction. 1"hv prothrombin variant (.;~7()? l UA, comprisirl~ a (l tc~ .=~ transition mutation at nucleotide 2021 U is a very good example. This point mutation is associated t~~ith increased prothrornbin levels that lend tt, I5 an increased rich of thrombosis (1'oortn, 131ood 199b; 88 (I0): 3698-703j.
i'ublications indis:ate an increased risk of cardiac. infarctions (I~oscndahl, Blood 1 ~~9?:
90(x) 174 i-Stl;
and. venous throrn'noses (t3ro~Tl, 13r. J. Ilaematoi: 9$(4): 907-~7).
fvowever. it has also been possibl4 to demonstrate that discrimination beru~een mutation carrier and the wild type is not possible on the basis of the prothrombin level, since tt:e two groups cannot be :?tr separated (Poortn, Blood I99c~: 88(10)' 3698-703).
~ev:rat studies ha~~e shuw~n higher prevalence of FIICi20,'1OA in patients with myocardial infarction compared to normal controls. however, most of the results from these studies !;tiled to achieve statistical significance, possible because c~f the extremely Io~.:E~ frequency of FIIG~O?l0A in the studied population and the use; of relatively small ?~ r.,i~mp!e sice5. ;~ieverilieh.~ss, a few studies have presented conf7ictin;_ results.
tllthough F~'l. tronglv i;orrclates with deep venous thrombosis, the majority of the previous studies have failea to show at correlation of FVI, to n~iyocardial infarction.
I~ecr~ntly, a te~.v studies have. suggested that i~ VI: may associate with early onset myocardial infarction artd myocardial infarction with nornial coronary angiography.
J
In contrast, a common gene val-iaJit, Factor XI11V34L (FXIIIV341,} has recently been suggested to confer a protcctiuc role against myocardial infarction bas::d on lower prevalence of I~X1I1V?4I_ it: myo~~ardial infarction patients coJnpared ~~~ith controls.
t-lowev,.:r, cc.rrllicting rLsS.;lts v.~ez': also reported. Furth;:rmore, results from function s sriadie:~ on the F.~IIV3=11_ allele clo not support tho hypothe=sis of a protective role abainst JJy~ocardial in faeetion. Therefore, the role W ~ these gene variants in tl'~.t. pathogenesis of myocardial intarttion remains unknown.
irJ-thernlore, no catls~ ezf Jnyc~:ardial infarction is detectable in a high proportion of all cases. Ii suc.ll delects etist, the helllostatic ecluilibritJrn is distmhed ar~J lire ratio bct,veen nt pro- and antiCOayJiatory factors is shifted in favor o,~one siut~, 3 o thi, ;ere added dzfects in tile f3hrinolvsis svstel~z that rLdur,e the izrea~:dovvn of clots forrtrc~i.
t3eing a multifactorial disorder. myocardial infarction may be a combined effect of a number i>f gems, with each playing only a small role. 'The l3redisl>osition irrlparled by individual gene. nuay act independently or interact r~rith other genes to result in an t~ a.ddiiive eFfcvct ,z;ic~l!ox a synergistic co-eFfect. Cc~uimon chailc:~ge~, fa;viJ~~~ case, control studies cjn possiole ~~~.rle-gene interactions include relatit-ely snit 1l sany~l~ si-rca. a to«
Ureclueocv of gene v.Jriazlts. arid ethnic heterogeneity of the investigated population.
This hackf~ro;~rici irzforniation is E:~l~~yl~idPd for the hul-pose cvf mahin~, kncs~~ti infornlation belit:ved by the applicant to tie of possibly: relevaaice to the pre~;ent invention. No Lrt admission is lzm:asarily icite;.lded. for should be construed, that .lliy-of the preceding iniornlation constitutes prior urt aEainst the present iuverJtion.
SU?~'!~I At~'Y O>p THE I?~V~:NTION
1u one aspect. Lh~ present inwntiort provides methods for determining a risk factor for rzlvlocarctial icil~~rtvti~arz. 1 h e~,t nw:tll~>ds c~:~ri~prisc~ assaying a suitahlc I~iolohical sampic. and 7s lCti;rr3l:nln~, tile InreSellW c71' iillsc~fl~e i>f Variant fG11et1G
elerrlentS, tll~~lr ~!c'tld. prot~liCL~, l)r altered phvsioclmmical activitic°:; c;f these gene products correlated with elc.v.ated risk of mvoL.ardi<ll infarction.
-i In cme embodiment, the iuventi;~n provides a method in which said c;enc.tic elements are a n~rEt~3tial~ in tlrt gene encoding FI1, na>re l~artict~larly a G to A
tra~rsiti;.~n mutation at nucleotide position 2t~~? I () (FIICi?()? 1 OA}, and a mutation in the FXtII
gene, more particularly a valine to Ieucine substitution mutation at amino acid position (FXIIlV341_.}. Ii is to be understood that other genetic elements, coding fur the components of the libr-inolvsis system, the clotting system, and the complement system, can be similas-ly analyzed to determine a correlation with an elevated risk for myocardial infarction. In another aspect the present invention provides a kit for use in said method.
hlce present invention also provides n first nucleotide sequence comprising at least part of tt~ the nucleotide sequence of the h~r:a~an FIIG202IOA allele, a second nucleotide sec;uetee conrpriaing at least p~irt of the nucleotide sequence of the human FXIllV34L
allele, and a third nucleotide sequence ccrrrprisin~; at least part of the nucleotide sequence of~ the human F'VL e, as well as the use ui such sequences for the detection of such mutations.
The present invention also provi:Ies primers for the allele specific detection of these mutatirns of the FII ger~c at nucleotide 20? 10. the FX11I gene. at amino :~~:i.~l 34. and the FV gore at amino acid 506.
In accordance with one aspect uf' t.lre present invention, there is provided a method of det~;rrnining a risk '.or myoc.wali~ri in iarction, or a pruperr>ity tlmi~iur io an individual curnpri:;inb:
?c~ (a} obtaining a biological sample tiom an individual; and rb} analysing said biological sample for tlm, presence of a variant of a Lene encod;nf3 Factt~r fl !'actor ~f; Factor XI1I or a combination th;.r-~.°ot.
In accordance with another aspect of the present invention, there is provided a method for thu detection ef defects in a rmulti-stage, nnulti-tactc;rial hiuchemical reaction system.
'~~ wherein the defects are associait~d with an increased risk. of myucaruiul iusfar~ctioo ivr atr individual., ~:.nmPrisin;~ the stets_< c,1:
11) screening for suitabli. rlatis.nts at lisle of myocardial infar<;tion, wherein said screening i5 conducted on the basis of a family history or individual case history;
nj obtaining a suitable biological sample from the individual;
c) determining the presence of variant genetic elements, the gene products of said variant I;enetic elerrlents, or altered physiochemical aLtivitics of said gene produias known to be correlated »rith n)yocardial infarctic>n; and d? determining the risk to the individual of myocardial infarction.
In accordance with mother aspect of the present invention, there is provided a method far to detc:rrtlining whetllet~ an individual is at an increased risk far myocardial infarction, cetmprising deteCtitlg, the pretence ~:ar ;~hscllc~: of mutations in grnctic elements, aberrant gene products of genetic. elements or altered physiochemical activity of tlzc gene products of genetic elements, wherein said genetic elements are ror~related with an elevated risk for lTh.'OC21rc:11a1 lntar vtlUn.
t s In accardanee with another ripe<< t of the pres;.nt invention, there is pr;)vided a method for determining whether an individual is at an increased risk for mtocardial infarction, CUIIIpI'lSlng dt',tC;riTllrlln~ Factor 11 su)d l~'aclor :X111 genetic element sequences of an individual, whereby tile presence of a CS2U2.IOA mutation in a Factor II gene seetuenee, and the presence of a u3~L r:ltltation in a Factor X111 gene sequence is indicative of an itlct-eased risk for rrl5~ts:arJial infal-ction in said indiv°idual.
In accordallc.e 4vith ancrtilcr aispect cli tile present ilivention, there is provided a rneihod fur determil-ling whether an individual is at an increased risk fc~r myocardial infarction, ctii111)1'istilb deteClilltllt7~ prenC ~~iudtiCCS Ot h~aCtai' 11 anti 1'ai;tOr ~illl getlvtiC 2itr1'WIliS Of2lri irldii iciual. vvhcr~:by Cllr preseitce ot~ t~'1 (iJ~~ C~21 UA and t~
XItI~3~l. gene products t ?7 JtiiiiC'atl~W . Ol';itl iil~it'rSC,CI Itsit tn!f iiiyi3~afdilil itlfc3r~t1fM1 Ill 3ald ti1t111'ldiicll.
I12 arcordarice :~:uil1 anotlret a.s~»-<:.t of the present irl~wlliic,n. there is lire>~~i.icd a tltethod for determining wh4ahPr nn individual is at an increased risk for 1~ yoctirdiai infarction, comprising determining physiochemical activity of gene products c~f the Factor II and hactt~r XIII genetic elements ~~1~ an individual, wh~:reby the presence .~.~t~
FIIG20? I OA and F~IIIV 34L gene products physiochemical activity is indicative of <rn increased risk for myocardial infarction in said individual.
a In accordance with another aspect of the present invention, them is provided a kit for determining whether ar-r individual is at an increased risk for n5yocardial infarction, comprising oligc~nurleotides sprvific to the ~~arianl region of the alleys of int;.rest or to sc.~uenc.e fiaoking the variant region and optionally instructions for a<:u.
13RIFF DESCRIPTION OF THE DRAWINt~~
Figure 1 shows a comparison between expected and observed prevalence of combined earners of mutations in myocardial infarction as described in Exampl1 (MI ---individuals, control == =;00 i.rrdividuals);
Figure 2 shows tho T~revaiGnc~. of (a) FhlIIV34L in MI pati~:nts and rrorn2al c;~nirols who carry the F'I1c-i~021()~ allele, and (t~I FXIIIV34L in Ml patiants ana nor:~~al i.:ontrols who is c,rrr~ them FVL: alleic, as descrsbed in Example 1 (h'II = 5(i0 indivi;luals, control = 500 individuals j:
Figure a shor~-s a comparison bettyten expected and observed prevalence of combined cvar~-i~xs of mutations in ioyoc,mdial infarction as described in Fxamipl~: ~
(ll~il = 73() individuals, control == 579 indi~~iduals); and Figure ~ shows the pre.valenc.e of (a) FhIIIV 4L in ~9I patterns and normal controls who carry the FIIfi2U?l0a allele and (bj FXtIIV34I, ira MI patients a.nci n<armal controls who carry the hVl, a!_tele, as descaibed in Lxample 2 (MI = 730 individuals, control = 579 individual s).
IJE'1':~li:)ED !)ESCVRii''t'iON Oi~ '1'!iE INVr:NT~fJN
'hhe pr~s~rlt invention providLs for methodss of identifying 1-isk factors associated with myc>cardial infarction. The invention further provides for the application of these identified risk i'actors in methods of identifyi.rlg individuals at risk for myocardial itlfarction or individuals having a propensity for myocardial infarction.
Thus, the priaent invwtion provides diagnostic arid prognostic tools valuable in a medical cvrttext. 1r the ec~~ntext of the present inven it~n, a ''risk facti>c" refers to a variant t;unetic ehment, typically a mutation, the prosz.nce of which in an individual's ~~ellOtlle provides an indication of increased risk lc~r nlyoeardial infwrction. 'I he preserECc o!~
such variant to genetic elements call bc~ d~ternrined at the genetic (nucleic acid) level oa at the protein (;poiypeptide) level. In accord;lnce with the invention, the risk factors comprise a variant of ;~nc or more genes encoding, proteins that are part of a mufti-stage, mufti-factorial biochemical rea ction system, sllc.h as the tibrinol~rsis systern, the clotting system, or the complement system.
t 5 I)ef in iiion.s t..lnless defined or_herwi<;e, all tecllllical ~uld scientific. terms used herein have the same meaning as cc>Ilnlomfy' understood h~~ tone of ordinary skill in the. art to which this invention belongs.
'f he tens "gene~:ie element-". as hsea_l herein, refers to a t,u~:l;:otic~le seduence present in the:
3t1 eenolxlc. of the c~raaltism iltldr~r ir~~~estigarion. Examples of genetic ~:lznl~ntcorrelated '.vlth tfCv''tlted rlll'OC~CCII~!1 ::lfelivti'J't rlSk C~:.Jm(lt7~L'. hllt elrl: llOt ri:stflWtd t;s. genes tildlll~
tear cotllponents afthe libl-incllysis system, the clotting sysrem, or the;
conlyletllent system, s,ucll as, tbr ex,~rtlple, the gene; cnclitlg fur .f'll, FS'. arlG FX111.
'~AMutations'' in these :genes oo:ltcmplated t~~y the invention inc-lode, but are not limited 1o, Z_~ Cl:,'letlOrlS, illsCrtltall~, ~;llr~Jr11C1W??Tlll d1s111Gc~t10I1S, t1~1I1510C~hOrlS, 1C1~'r'n3On5, ~tld Otlt~r EenetlG 171eGhan1Si115.
~lfethorl_f'or the identification oJ'risk factors associated witfi myocardial i~tJrarctiun Genetic elements and variants thereof associated evzth an elevated risk oI' myocardial infarction may be identified by methods known in the art. Ono strategy to identify said genetic elements and variants thereof encompasses the steps of sequencing a candidate gene in a panel of probands from families with documented myocardial infarction, s tollowcd by estimating the risk factor Ior myocardial infarction <associated with any observed sequence variation in a population based patient-control study.
:~ non-limiting example of such a strategy is provided as Examples 1 and 2, which set forth the inventors' case control study, simultaneously analyzing for the presence of the FV L, FIIG?(?'? ( OA and h~XIIIV.'i~rl, variants in myocardial infarcaion patients and noanul 1~.) individuals of a genetically i~oiated population of the island portion of Newfotmdland and I,abradc>r.
Thus, the present invention relates to a method for the identification of one or a plurality of variant genetic elrments that encode proteins that are part of a multi-stage, multi-factorial biochemical reaction system such as the tibrinolysis system, the clotting system, 15 :~r the complenjent system. wherein the defects are associated witlu an increased risk of tam cxat. dial into-~rctiotr in ;:.n i~ldividual. As used herein, ''a plurali!y" refers to two or more. In one i~mboditncni.. the iriv~ution provides methods for the clc:LeCttor2 c7f one or a plurality of variant genetic elert~nts that encode proteins that are hart of ih~w tibrinolysis system. the clotting systctn, moiior the complement system. In another embodiment, the 2t~ invention provides methods for the detection of a plurality of variant genetic elements chat encode proteins ths~t are hart of the fibrinolyrsis system. the clottinc system, and/or the corrzplemeaat system, wherein the plurality of variant gencaic a lensents indicate that an individual is at greater risk than any one of the variant genetic elements alone.
The methods of~ identifying the variant genetic elements provided by the present 2~ invention comprise the Steps of~ identifying a suitaole sample population of individuals t~:~
screen, obtaining a biological sample from each individual in the sample population, detent~ining gene sequences, ;,;tree products of genes, or activities oI' gene products to identify variants theret?f, and comparing the occuwence of the variants with the occurrc-nee in a suitable reference population. A suitable sample populaticm.
feet example, ~~uuld be made up of indis~iduals who have suffered myocardial infarction and a reference popuhttian »~or;ld bo made up of in<Iividuals who haci nu history (either individual or falmilia!) uh rhyoc<irdi<tl infarction. 'I'hc sample population may be geographlcailv restricted, restri~aed by age, gender or ethnicity if' desired.
Typically when a sample population is thus restricted the reference population is sin;ilarly restricted ;l.c.
matched) by geographical ref;icm, alg,e, grader or etllllicity.
()nee the sample and reference populations are selected. a suitable bioleyical sarnple must be obtainea in order :o carry out the analysis for variant ~,cnetic elements.
l3ioleyic.al sa.rrlples obtained ti~csnl a subjcca may comprise genomic ~)Na, fZN:I. or s o protein. 1~ or the purposes of physiochemical analysis, a suitable biological sample may be a blood or blood plasma saml.lle containing the protein of interest. 1~'or the purposes of nucleic acid analysis. a suitable biological sample may be a blood or blood plasm;i sample, urine, sali~~a, tissue biopsy, surgical specimen. tiw needle aspirates, amniocentesis samples, or any other material comprising the paiierlt's genomic DNA.
15 '~~f~thods tOr O~lliililltl~ suitable biological samples are known in the art.
Detection Of mutations rrlaybe l~erforrtled, inter olio. by methods known in the art, such as direct secluelice <~.ualvsis i}f asllhlified nucleic acid containing the aben--ltiun, by allele spLcilic anlpliflcatiull wllirll ciifferentially amplifies I)NA containing atnd DNA lackin~~
the aberration, or E~y restrict'tc~n fragtncnt analysis. I; may also he performed by 2il hybridization with a probe d~lat is able to differetltially hybridize under stringent mtlditions to the str~.tCh ui~ ;rtllplitied nucleic acid matc-rial r~~hich may contain the aberration.
~~clrlolll tl'.t,11n1C1t1~5 ii)r illill)lljyln~ rlllclClC al:ld at'e;
lvIlOVs'11 111 tllC iirt. t.~tlv e~2lml)1tG ~t a tc'.Chrll(lUe le'r tIIC aiillpl(tlC;ltlC)f" i)1';l l~:~11 t27.C~?ia 5~~nli:llt 1S the p01~'Illtril~e Chelln r~.~tt;t1C711 c;t'~:R). 1~-itll thz Pt:R te~:hni~.~~l~:. the copy number of a particular tar-_fet Segment i5 increased ehponentiai!y with ~: nulllher of cycles. :~ pair of pritllcrs is used and in each cycle a 1)NA pl-inler is annealr:cl to the 3' side of each uf~the two st(<~ulds of the doubled atr:uldcii Di~A~la~'lrei seiluance 'fTlli~ primers are e~aendoc:l ~..~ith a C~'.~.~ IrolytilLl'asc lI7 the presence oh the various monc>l~tncleotides to gener~.te double stranded I:INA.
The str~~ltds t ti of the double stranded DNA are separated from c;3ch other by thermal dcnaturation and each strand then serves as a ter_nplate for primer armealing and subsequent elongation in a following cycle. 'The PCR method has keen described in Saiki et al., Science 239, 487, 1988 and in Cwopean Patents no. I:P 200 3G2 and EP 201 18~. Other amplification techniques include mismatch PCR, the Ligase Chain Reaction (LCR). Repair Chain Reaction (Rf'R), TMA, rolling circle amplification, nucleic acid sequence based amplification {NASLiA), and strand displacement amplification tSI~A).
The term "oligonucleotide" as used herein refers to a linett.r~ pohrnucleotide molecule of !1p to ahc?ut 20l) nucleotide ba~es in length, for example a polynucleotide (such as DN.~1 l0 or RN.1) which is at least about 10 nucleotides, for example at Ieast 1 ~, 5ti, 100 or 200 nucleotides long. These oligonuc:leotides may function as primers and probes.
The term "primer" as used her;yin refers to an oligonucleotide either naturally occurring (e.g. as a restriction fragment) or produced symthetieally, which may act as a point of initiation of synthesis of a primer extension product and which is able to hybridize to a I s nucleic acid strand (tempate or target sequence) when played under Suitable conditions fc.g. buffer, salt temperature and p1-1) in the presence of nucleotides and an agent for nucleic acid pols-rnerization, such as DNA dcpenclent or R1'A dependent polylnerase. A
primer must be suffici.c.ntly l~,ng to prime the syniltcsis of exteT><siUn pro ducts in tr_e t71'tsCllCC; Ul an agent f<~r puIyrrlrir~tiun. A typrcal hrrmc;r contaua at least about 10 2o nucleotides of a sequence substantially complementary or homologous to the target sequence, but somewhat longer primers are preferred. Usually primers contain about 15-~6 nucleotides, but langer primers may also be employed. Normally a set of primers will consist of at bast two primers, one 'upstri:arrt' and one 'downstream primer which together define the sequznc~ that will be amplified using said primers. 'The primers used 2~ for am(7(ifrcation of tile varicJua refi.ons of the target genes may be sylrtl:esized by the methoxa~phosl:lw:~ramite mctho:i (Tetrahedron Letters 2?, 18>9-186, 198 l ) or other ~ulitabl:method Iwiilll-n lrl the art. Tlve sequcr;ccs of the primers are cl-losen such that they flank the aberrmt regions of the target gene. Suitable primers :~sr ampiifrcation of the various transcril-,ed ;tltd untm.nscribed regions of the alleles of interest ar-e those 3o detertrined to he most effiraci~u?s for the task.
,~
;qtr-urgent (hybridization) con;litiorts arc conditions under which a test nucleic acid molecule will hybridize to a t~.rget reference nucleotide sequence, to a detestably greater degree than other sequences (e.g., at least two-fold over bac~:ground).
Stringent conditions are sequence-dependent and wrill differ in experirrrental contexts.
F'or example, longer sequences hybridize specifically at higher temperatures. (.ienerally, stringent ~~onditions arc sc°lectccj to be ~rbout ~' C. to about 20° C.
lower, and preferably. s" C.
lower, than the therntal meltirl; point (Tnt) for the specific target sequence at a delined ionic strength arid pH. 'The T'nr is the temperature {under defined ionic strength and pH) at which 50°ro of a complementary target sequence hybridizes to a perfectly matched probe. 'l ypicallv, stringent conditions will be those in which the salt concentration is less titan about 1.0 '~~1 Na ion concr~ntration (or other salts), typically about 0.01 to 1.0 M Na iorr concentration (or other salts). at pH 7.0 to 8._i, and the temperature is at least about >() C. for slror2 probes (;e.g,., t 0 t:a 50 nucleotides) and at least about C;ii' C. for Long probes (e.g., greater than ~C~ nz.,~.~i~.otides;>. Stringent con:iitions nmy also be achieved with tlae addition of destal7iliaing rents such as fc.~noarrtido. 1=;x~:rnplary ;o~.v stringency conditions include hybridization with a buffer solution of >0°i°
formarnide, I M NaC_.'l, 1%
'~1~~5 at 37' ~.'.. and a w~a:;h in ? tunes ;~W ' at SO' C. I=xcrnplay high string.encv conditions include hybridization in ~(1°r~ fr~rmantide, 1 M NaC'1, 1 °,~o SDS at 37 C., and a wash in 0.7 times S5C at b0' C'.
Che term "probe" refers to av oligonucleotide, typically labeled, that forms a duplex structur:; with a sequence c~f a farces nucleic acid due t0 COmpleIIlent<ary base pairing. ~(~h~
probe will comprise a hybridizing region, prelerably consisting, of IO to ~0 nucleotides, snore preferabiv ?() to 3i) nucleotidea, corresponding to a region c~f the target sequence.
1 he hybridizing region of a probe is preferably identical or fully c~rnplernentary to the 2~ sequence of tho target rel;ion. hlte lrybridizirrg region rnay also eontairr a certain number of mismatches, those skilled in fhe art of nucleic acid technology :an c3eterntine duplex stability considering a muncac.r of variables including the ieng.th and base-pair composition of the prohG~, ionic: strength of the buffer, re-action ternl5eraturc and incidence of mismatched base pairs, seu. e.g. Sarnbrook et al., Moleciriar cloninf~: r~
laboratory 3c; n~~unu:xl, seccsnd edition ( I t)89) t:. olcl Spring Narbor Laboratory Press.
'~I'hi: term "label" as.usrd herein refers to anv atom or molecule which can be atiached to a nr.:cleic ~.cid and rrhich em be used either to provide a detectable signal or to interact with a sec-and molecule to modify Ehe detectable signal provided by said sec«nd mohc;ule.
hxarnples of suitable labels include, but are not limited to, radioisotopes, fluorescent s compounds, erraymes or chemiluminescent compounds.
Ilybridiaation of the probe v~ ith the target sequence may be detected by techniques known in the art of nucleic arid technology such as Northern or Southern blotting, see e.b. Satnbrook et al., supra. Detection systems that maybe used in conjunction with such methods include., for example, enhanced chemihrminescence (,ECL) based analysis or to onzyme linked get assay {E.LG n j based analysis.
Sequence analysis includes direct analysis of the DNA sequence tlankinc and constituting the cxons and uniranslated regions of the gene. This method involves any protocol that is currently available to any person skilled in the art for directly determining DNA- c.~r RN:~ 5eqllellc~s, su~,~h as the dide3xynucleotide method described by Singer ti (Pros. Natl. Acid. Sci. 1_lS:r, 7~ ~4G3-7, 1977}.
!t is also possible to analyze tl~e amplified material through restriction fragment analysis.
in this method the amplified material is digested v:-ith resirictior~ emytncs that recognize DNA sequences that are either present in I~NA sequences derived ti-om patients carrying an alet~ration in the exons or rmtranslated regions Of the g4tte, or that are present in the 2f) IlatlV~ sequence encoding the gene product.
It is also possible to analyze a known mutation by allele specific amplification (Trends in <ienetics, 12, 391--39?, 1 ~?96 at~d Mullis et al. cds, The polymerise chain reaction, I3itkhauser, I3c>ston, l.3as~l, llorlin, i')'~~1. ppI-13j r~llelc spL;;.itic PC'R fir the F'ltCi2()? I UA variation has been described in Thrombosis and hlaemostasis 78, 1157-1163 25 ( l ~>97j. 'this technique is based on the observation that under certain c«ndit.ions primer elongation caimot take place wlien the 3 tertninal nu~~leotids: of vj primer is neat complementary to the template. Vvith the else of two forward primers that differ only at their temtin:il :~' nucleotide it is possible to distingui>h between homozygous or heterozygous individuals, for instance with respect to their F'II(:i''U? 1 UA
allelc;s: analysis ;:f rnatcrial from lac~ioca:vg:~as i.ldi~~idual;, will result in a positive attihlificar.iou result with either one of the primers, material from heterozygous individuals will result in a positive amplification wiih bath primers. Similar analysis can be performed for the FVL, and F~IIIV341. alleles, as well as for alleles of any other genes involved in the fibrinolysis system, the clotting system, yr the complement system.
Other standard mutation analysis techniques known in the art may also be used {see, for example, Dracopoli, et al., Cuwent Protocols in Human Genetics, John Vv'iley &
Sons, Inc., I~i~"j. Examples of such techniques include, but are not limited to, restiiction-fi~agment-lengtO-polymorphistTi {EZI~Lf'j detection, hybridization using immobilized to oligvnucleotides or oligonuclevtide arrays, mismatch-repair detection (MRDj {I~aham and C:ox Genome Rcs ~:~74-48? (1995)). binding. of 1\~lutS protein {V'agner et al. Nucl Acids Res 23:3944-3948 (1~)~~~), denaturing-gradient gel clcctrvphvresis (DGC~F).
denaturing high-performance liquid chromatography {DHPI_C), agarose gel c>r capillary-based electropl:vresis, single-strand-confornzation-polymviphism {SSCP) detection, R?vAase cleavage at mismat.ehed base-pairs, chemical yr enzymatic cleavage of heteroditplex DNA, mass shectromety~ and radioactive andlor tluorescent DNA
sequencing using standard procedures well known in the art.
Physiochemical analysis of th4 products of candidate aliales comprises imrnunological or ?o physiochemical detection.
Immu~iological dctc-coon comprises the steps of obtaining a srunple of~the patient's blood, pl;;sma, or other bodily fluid in ~~s~hich the gene product may or m~3y not be present, adding a ki~o~,vn ~lu:mtit~r of a;n antibody specific to the variant gene product udder cu.;iditicatwhicri savor t~ir~c.-lii~g ~~f~ tire 5pecitiG ~antilm~dy to the vaiuant g,Cnr. product. aJld assessing the presence or absence of the binding of the antibody to the variant gene product. A pvsiuvv test result ivli;,rein such binding is observed is indicative of the presence c,f tljr: gene product, and a negative test result wherein sack birnlinb is not observed is indietaivce of the al~,ence of the gene product.
t,t For the purpose of this application, an antibody is an in~munoglubulir3 molecule and immurtologically active portions of an imnturtoglobulin molecule, l.c., a molecule that contitin an antigen binding site which specifically binds (itnmunoreacts with) an antigen.
A naturally occurring antibody: (e.g., IgG) includes four polypeptide chains, two heavy S (H) chains and iwo light (L) chains inter-connected by disulfide bonds.
However, the ~mtigen-binding fimctu~n of are antibody can be performed by frugment~ of a naturally occurring antibody. Thus, these antigen-binding fiagmc;nts are also intended to be designated hr- thv term antibodv. E~~trnples of binding Fragments encompassed ~~~thin the terns antibody- include (l) an Fab tra6ment consisting of the VL:, V1I.
C'L and C.'HI
l1 dorrtaitas; (ii) an Fd fragment consisting of the VH and CH 1 domains;
(iii) am fv fragment cUIISIJIInL Of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment ( Ward et ul . Nature 3~ 1:44-fi, 189) which consists of a VH dontain; (v) an isolated complimentarity determining region (CDR); and (vi) an F(ab') 2 fragment, a bivalent tiagrnent comprising two Fab fragments littlicd by a disulf de bridge at the hinge region.
15 hurthernnore, although the mc~ domains of the Fv fragment are coded tier by separate genes. a synthetic; linker cart hc: made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Hird et ul. Science ''4?:4? ~-G. 198H:
and I-Iustott rc al., I':~uc~. ,l~utf. :icoid. .~'~i. r.~.~~7~)-$3, 19~$) by recombinant methods. Such 5inglc chain ~ln:l~iV~.llvJ ~tre al<<~ ItlL:lllC~~:'~.
rct In one example. tttltibody fiagrltent; are capable of crosslinking tlueir target antigen, e.g., bivalent fragments such as F(ab')2 fragments. Alternatively, an antibody fragment which sloes nc?t itself crosslink its target antigort (e.~,., a Fab Iragment) can be used in conjunction with a seconchtry mtibody which serves to crosslirtti the antibody fragment, thereby crosslinkity ttze terror ~tntiyon. ,~~tttibodies can be fragmented t.rsirog cunverttional ''S teehnicfues and the fragments screened for utility in the same nmoner as described for whole :mtib~~dies. Arr anttbody° is further intended to include bispeciti~ and chimeric molecules that specifically bind the target antigen.
'vpecilicallsr binds" as useu hc~reitz with reference to an antibody, or antibody fi~artnent, r~;f~rs z.o the ability of an irtdi~-idttal antibody, or fragment. to specifically iiztmunoreact 1~
with ~uz azriigen. The binding is a non-random binding reaction between an antibody molecule and an antigenic determinant of the T cell surface znolecule. The desired binding specificity is tropically determined from the reference point of the ability of the antibody to differentially bind the T' cell surface molecule and an uruelated antigen, and therefore distinguish between two different antigens, particularly where the lwo antigens have unique epitopes. An antibody that specifically binds to a particular epitope is referred to as a "specific antibody".
Physiochemical detection comprises the steps of obtaining a sample of the patient's blood, plasma. or other bodily fluid in which the gene product may or may not be present, optionally isolating the gme product from the sample by inirmuzological or physical znc:ans, and assessing the presezzce or absence of the variant gene product.
In the ,:ontext of the: E~resent itt~~entiun, an ''isolated'' biological ccmpc>nent {such as a nucleic: acid molecule, polypeptide. or protein] has been substantially separated or purified away from other biolcgicai cotmponcnts in the cell of the c~rt;arrisrii in which the component naturally occurs {i.e. other c.liromoso:nal and cxtrachronzosomal D.N'~ and ItN.A). Nucleic acicjs and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The terra also embraces nucleic acids am:l proteins prepared 1-ry r~r:~ii~ibinant expression in a host cell to w~rli a~ chemically syntluesi~od nucleic; acids and proteins. The terns "isc>lated" does not require absolute ?u pu.ity; rather, it is intended as t relative. term. Thus, for exarnplt~, ci substa.niially purifie;i Frot.ein or nucleic acid preparation is one in which the protein or nu::leie acid rcfcn-cd to is more: pure than the protein in its nattua.l environment wit.hio a cell or within a production reaction chamber (as appropriate], For example, a preparation of a moditied protein is ptuified if the protein represents at least 50°,~0, for example at least 70°~0, of the 75 total pri>tein v~?nt~nt of ttie pret,aratic~rl. l~icthods for purificatiozz of protoiuis azid nucleic acids ~irc: well knr.mz~ in thL :irt. I'.~;azuplGs of methods that pan be usrrd try .l3urif~~ a protein include, but are not limited t~.~ the methods disctos..d in San~hrook et al.
{Alolecultrt-(..'.t,~rtin~: =1 L.~rbc~~'c'tcr_,: a-jurlurl. Cold Spring fiarbcsr, New York, 1~~~9, Cli. 1?~.
~H
13y "isolating the gene product fiom the sample by irnmunological means'' is meant, for example, that the variant gene product may be isolated from the patient sample by means of an antibody capable of detecting both the wild-type and the variant form of the gene product (that is, an antibody directed to an epitope conserved in both the wild-type and the variant gene product, but u~iique to the products of that gene locus). By "isolating the gene product from the sample by physical means" is meant, that the variant gene product may be isolated from the patient sample by means of centrifugation, chromatography.
electrophoresis, or other techniques known to the worker skilled in the art.
)3y "physiochemically assessing the presence or absence of the variant gene product" is meant the use of techniques such as protein kinetics, spectroscopy, crystallography or other techniques known to workers skilled in the art to distinguish the unique physiochemical signature of thv variarnt gene product from that of the native or wild-type protein.
Finally. the frequency of occurrence of a particular variant within the sample population t 5 can be compared with the lreduency of occurrence in thu reference population and the statistical significance of any difference can be calculated by standard methods. A
statistically significant difference in the frcquenc:y of occurrence between the two populations indicates that the variant is a risk factor for myocardial infarction.
l)eterntining the risk of »iyvcardirrl infarction tv the ijtdividua~
?o 'hhe present invention further provides for methods of determining the cist~ for myocardial infarction to an individual andlor the propensity of an individual towards myocardial infarction. 'Thus, the C~resent invention relates to a method for the detection of a plurality of defects in a mufti-stage, tnulti-factorial biochemical reaction system such as the tibrinolvsis system, the clotting system, or the complement system, wherein the defects 2~ are associated with an increased risk of rr~yocardial infarction in an individual. The method comprises the steps of obtaining a suitable biological sample from the individual, analyzing for the presence of v~wiant gene sequences, gene products of genes, or activities of gene products known to bE associated with myocardial infarction, and determining the i risk to the individual of myocardial infarction. The method may optionally further comprise a preliminary step of screening for suitable individuals at risk of myocardial infarction.
In one embodiment of the present invention, the variant genetic eler~ients associated with myocardial infarction being analyzed for are one or more variant gene selected from the group of; genes encoding Factor II, Factor V, Factor XIII, or a combination thereof. In a further embodiment. the variant genetic elements associated with myocardial infarction being analyzed for are one or more variant gene selected from the group of:
FIIG202I OA, FVL, IXIfIV34L, or a combination thereof. In another embodiment. the variant genetic to elements associated with myocardial infarction being analyzed for are two or more variant gene selected from the group of: genes encoding Factor I1, Factor V, Factor XIII, or a combination thereof.
It was tutherto unrecognized ti,at a combined carrier status of the FIIG20210A
and FXIIIV34L genetic elements is a strong risk factor for myocardial infarction.
With the present invention such a correlation has now been establisheii. Therefore, in another embodiment of the present rnventron, the variant genetic elements associated with myocardial infarction being amrlyzc:d for are FIIG20210A and FXIIIV34L.
I. ,S'c reen lo~~ risk, fiictors to .select patieru.s In general, in such a preliminary screening step, if one is underta;;en, individuals are ?o selected either according to a family history of myocardial infarction, indicative of a genetic predisposition to the c:mdition, or on the basis of an individual case study. In a typical individrr:l case study a patient may either present with a prior history of myocardial infarction, or global l,arameters may determine the interaction of several components of the clotting sy:~tem. For example, the prothrombin time (,PT) is a global 2s parameter which determines tl;e scale of the exogenous cloning system and the partial thromboplastin tune (a1 r Tj is a global pararneier which determines the state of the endogenous clotting system. 'The protllrombin level can be measured in a hmrran plasma sample or other suitable biological sample using techniques known in the art.
Measuring ~n prothrombin levels in a statistically sufficient number of healthy individuals can define a normal value.
It~ a positive family history is recorded, or a positive finding is obtained from a personal history or a global parameter such a_s PT or aPTT, individuals parameters can be analysed s to discover the underlying genetic hasis for the condition.
?. ()btair~ing a suitable biological sample A suitable biological sample is obtained from the individual under study in order to carry out the analysis. As described above, biological samples obtained ,from a subject may contiiin genomic DNA, R;vA, or protein, and can be a blood or blood plasma sample containing a protein of interesor a blood or blood plasma sample, urine, saliva, tissue biopsy, surgical specimen, fine needle aspirates, amniocentesis sarnples, or other material comprising tlje patient's genomic DN A. Meth<xis For obtaining suitable biological samples are known in the are.
1 by present invel>tion relates to the field of myocardial inf:3rction, more particularly, the invention is directcil at a method fc~r determining the risk for myocardial infarction in an individual.
BACIs:.CROUND OF THE lIVVEi'~TIOI\
Liyacarclial infarctian (~ZIj is rlow among the most frcduent causes of illness arid death, esnevialiy in thi' industrial countries. Ii myocardial infarction is survimd, the vitality of the: patient is IinTit.ed 1:1 racist cases, by secondary symptoms such as paralysis or organ d<3rnagc. Thcrc ~rre also labor-intensive and cost-intensive follow-"-up tr~.atrneots; smh ,~s .{i coim~alc~si~enc.e, physiot:la~~raip~ and medicatiaTl to imprcn-a the health sittTation ~~.ld prevera W rthcr complicaxions.
(treat advances h;~vc been made iTT recent years, especially in research into the causes c)f myocardial infarction. and those iriclude cardiac tissue necrosis cau:;ed by an inadec)uate blood supply dae to the c>c:;lusion of arterial blood vessels either by cholesterol planue is guild-uh, or by i;tot hlnckage (thromhc>sis). Risk factors te)r thrombosis-ildtTCed Tn;'ocardial infaTCiioo r~rc~ thenl~fta to in~lucle both hereditar~~ an, KTC~uired can,iition~;.
liCllt;r~.i~y, t t.cildcili.V ii)~4ialilS tllyacarciial infarction would ariS~
~rGn1 fiyp~r~CLiVf:
coagulation pathways, hypaac~tive arlticoaguiant mechanisms, or hy-poacti.ve fibrinolysis.
1~-Zutatii:~:ns in gees that encode pratcins in these p;~thways arc thc~trght to l.~lay an zi> il~~pc)rtant role in tizc prtzdispa~ition to myaca.,.-dial infarction.
~vl'rc 5c'!'il'1C ~1'i)ti:~~>r t1:1"~;~7r11't1111 ~,~;iei'1'ACCI ~)' ~ll:iiVa~f: at 111f.TTlar7 l3rat~13't71'Ill)if't (<llsc) ~'.III.W'~"11 :i>
Hac;tv:~r 11 (loll}) ~:~teri.s a central actii>n to the proc.cssw ~~
thrumbo:;is ania haemc>stasi~a.
1. t-m. th.r~sTnbio lm~le~.H~ pl;y°s a T-olu in thz tinal 5t.~g~ of hli:~od coa~ulatii)n: the l:ortnr,,tion of art iT~aolul~lc ~ibrir~ ac:~t.
The hit:~.urtc~ ~:Tv:-»'r, con;;,znitttl disorders of prathrc~rnbirr art:
rare: arid invoh~e either lvdm.c_d synthc~si.s of ts.~a 1-11 crlr~lcctrfe (referred to a.~
hypoprc~thrurnbir?~litia or ypc I
prc,thramt,in defic;iencryt or the normal synthesis c~f a dysfuncticsllal rtt<>lout,le (referred to as dysprothrombinelnia or type II prothrombin deficiency). Patients with dysprc~thrombinemia have only ?°~o to 50% of the clotting activity of nc,rn~al prothrombin;
in these patients the severity of the bleedings correlates fairly well with the amount of hrothrombin activity in plasma. A number of dysprothrombinemias have been further characterized by amino acid sequence analysis of the isolated prothrombin molecule or by nucleotide sequence analysis of their prothrombin genes.
It is Jtno~4n that the gene variant Factor V Lviden (FVL-R~f)6t~) <tnd prothrombin ti20210A (FIfG20210A) are hvo of the most commonly recognized genetic prothrombic !o risk factors for venous thrombosis. Based on the increased thrombotic teaidency in ve:lous thrombosis studies, these two 8ene variants have also been examined for possible association with arterial thronabosi~ in myocardial infarction. 1"hv prothrombin variant (.;~7()? l UA, comprisirl~ a (l tc~ .=~ transition mutation at nucleotide 2021 U is a very good example. This point mutation is associated t~~ith increased prothrornbin levels that lend tt, I5 an increased rich of thrombosis (1'oortn, 131ood 199b; 88 (I0): 3698-703j.
i'ublications indis:ate an increased risk of cardiac. infarctions (I~oscndahl, Blood 1 ~~9?:
90(x) 174 i-Stl;
and. venous throrn'noses (t3ro~Tl, 13r. J. Ilaematoi: 9$(4): 907-~7).
fvowever. it has also been possibl4 to demonstrate that discrimination beru~een mutation carrier and the wild type is not possible on the basis of the prothrombin level, since tt:e two groups cannot be :?tr separated (Poortn, Blood I99c~: 88(10)' 3698-703).
~ev:rat studies ha~~e shuw~n higher prevalence of FIICi20,'1OA in patients with myocardial infarction compared to normal controls. however, most of the results from these studies !;tiled to achieve statistical significance, possible because c~f the extremely Io~.:E~ frequency of FIIG~O?l0A in the studied population and the use; of relatively small ?~ r.,i~mp!e sice5. ;~ieverilieh.~ss, a few studies have presented conf7ictin;_ results.
tllthough F~'l. tronglv i;orrclates with deep venous thrombosis, the majority of the previous studies have failea to show at correlation of FVI, to n~iyocardial infarction.
I~ecr~ntly, a te~.v studies have. suggested that i~ VI: may associate with early onset myocardial infarction artd myocardial infarction with nornial coronary angiography.
J
In contrast, a common gene val-iaJit, Factor XI11V34L (FXIIIV341,} has recently been suggested to confer a protcctiuc role against myocardial infarction bas::d on lower prevalence of I~X1I1V?4I_ it: myo~~ardial infarction patients coJnpared ~~~ith controls.
t-lowev,.:r, cc.rrllicting rLsS.;lts v.~ez': also reported. Furth;:rmore, results from function s sriadie:~ on the F.~IIV3=11_ allele clo not support tho hypothe=sis of a protective role abainst JJy~ocardial in faeetion. Therefore, the role W ~ these gene variants in tl'~.t. pathogenesis of myocardial intarttion remains unknown.
irJ-thernlore, no catls~ ezf Jnyc~:ardial infarction is detectable in a high proportion of all cases. Ii suc.ll delects etist, the helllostatic ecluilibritJrn is distmhed ar~J lire ratio bct,veen nt pro- and antiCOayJiatory factors is shifted in favor o,~one siut~, 3 o thi, ;ere added dzfects in tile f3hrinolvsis svstel~z that rLdur,e the izrea~:dovvn of clots forrtrc~i.
t3eing a multifactorial disorder. myocardial infarction may be a combined effect of a number i>f gems, with each playing only a small role. 'The l3redisl>osition irrlparled by individual gene. nuay act independently or interact r~rith other genes to result in an t~ a.ddiiive eFfcvct ,z;ic~l!ox a synergistic co-eFfect. Cc~uimon chailc:~ge~, fa;viJ~~~ case, control studies cjn possiole ~~~.rle-gene interactions include relatit-ely snit 1l sany~l~ si-rca. a to«
Ureclueocv of gene v.Jriazlts. arid ethnic heterogeneity of the investigated population.
This hackf~ro;~rici irzforniation is E:~l~~yl~idPd for the hul-pose cvf mahin~, kncs~~ti infornlation belit:ved by the applicant to tie of possibly: relevaaice to the pre~;ent invention. No Lrt admission is lzm:asarily icite;.lded. for should be construed, that .lliy-of the preceding iniornlation constitutes prior urt aEainst the present iuverJtion.
SU?~'!~I At~'Y O>p THE I?~V~:NTION
1u one aspect. Lh~ present inwntiort provides methods for determining a risk factor for rzlvlocarctial icil~~rtvti~arz. 1 h e~,t nw:tll~>ds c~:~ri~prisc~ assaying a suitahlc I~iolohical sampic. and 7s lCti;rr3l:nln~, tile InreSellW c71' iillsc~fl~e i>f Variant fG11et1G
elerrlentS, tll~~lr ~!c'tld. prot~liCL~, l)r altered phvsioclmmical activitic°:; c;f these gene products correlated with elc.v.ated risk of mvoL.ardi<ll infarction.
-i In cme embodiment, the iuventi;~n provides a method in which said c;enc.tic elements are a n~rEt~3tial~ in tlrt gene encoding FI1, na>re l~artict~larly a G to A
tra~rsiti;.~n mutation at nucleotide position 2t~~? I () (FIICi?()? 1 OA}, and a mutation in the FXtII
gene, more particularly a valine to Ieucine substitution mutation at amino acid position (FXIIlV341_.}. Ii is to be understood that other genetic elements, coding fur the components of the libr-inolvsis system, the clotting system, and the complement system, can be similas-ly analyzed to determine a correlation with an elevated risk for myocardial infarction. In another aspect the present invention provides a kit for use in said method.
hlce present invention also provides n first nucleotide sequence comprising at least part of tt~ the nucleotide sequence of the h~r:a~an FIIG202IOA allele, a second nucleotide sec;uetee conrpriaing at least p~irt of the nucleotide sequence of the human FXIllV34L
allele, and a third nucleotide sequence ccrrrprisin~; at least part of the nucleotide sequence of~ the human F'VL e, as well as the use ui such sequences for the detection of such mutations.
The present invention also provi:Ies primers for the allele specific detection of these mutatirns of the FII ger~c at nucleotide 20? 10. the FX11I gene. at amino :~~:i.~l 34. and the FV gore at amino acid 506.
In accordance with one aspect uf' t.lre present invention, there is provided a method of det~;rrnining a risk '.or myoc.wali~ri in iarction, or a pruperr>ity tlmi~iur io an individual curnpri:;inb:
?c~ (a} obtaining a biological sample tiom an individual; and rb} analysing said biological sample for tlm, presence of a variant of a Lene encod;nf3 Factt~r fl !'actor ~f; Factor XI1I or a combination th;.r-~.°ot.
In accordance with another aspect of the present invention, there is provided a method for thu detection ef defects in a rmulti-stage, nnulti-tactc;rial hiuchemical reaction system.
'~~ wherein the defects are associait~d with an increased risk. of myucaruiul iusfar~ctioo ivr atr individual., ~:.nmPrisin;~ the stets_< c,1:
11) screening for suitabli. rlatis.nts at lisle of myocardial infar<;tion, wherein said screening i5 conducted on the basis of a family history or individual case history;
nj obtaining a suitable biological sample from the individual;
c) determining the presence of variant genetic elements, the gene products of said variant I;enetic elerrlents, or altered physiochemical aLtivitics of said gene produias known to be correlated »rith n)yocardial infarctic>n; and d? determining the risk to the individual of myocardial infarction.
In accordance with mother aspect of the present invention, there is provided a method far to detc:rrtlining whetllet~ an individual is at an increased risk far myocardial infarction, cetmprising deteCtitlg, the pretence ~:ar ;~hscllc~: of mutations in grnctic elements, aberrant gene products of genetic. elements or altered physiochemical activity of tlzc gene products of genetic elements, wherein said genetic elements are ror~related with an elevated risk for lTh.'OC21rc:11a1 lntar vtlUn.
t s In accardanee with another ripe<< t of the pres;.nt invention, there is pr;)vided a method for determining whether an individual is at an increased risk for mtocardial infarction, CUIIIpI'lSlng dt',tC;riTllrlln~ Factor 11 su)d l~'aclor :X111 genetic element sequences of an individual, whereby tile presence of a CS2U2.IOA mutation in a Factor II gene seetuenee, and the presence of a u3~L r:ltltation in a Factor X111 gene sequence is indicative of an itlct-eased risk for rrl5~ts:arJial infal-ction in said indiv°idual.
In accordallc.e 4vith ancrtilcr aispect cli tile present ilivention, there is provided a rneihod fur determil-ling whether an individual is at an increased risk fc~r myocardial infarction, ctii111)1'istilb deteClilltllt7~ prenC ~~iudtiCCS Ot h~aCtai' 11 anti 1'ai;tOr ~illl getlvtiC 2itr1'WIliS Of2lri irldii iciual. vvhcr~:by Cllr preseitce ot~ t~'1 (iJ~~ C~21 UA and t~
XItI~3~l. gene products t ?7 JtiiiiC'atl~W . Ol';itl iil~it'rSC,CI Itsit tn!f iiiyi3~afdilil itlfc3r~t1fM1 Ill 3ald ti1t111'ldiicll.
I12 arcordarice :~:uil1 anotlret a.s~»-<:.t of the present irl~wlliic,n. there is lire>~~i.icd a tltethod for determining wh4ahPr nn individual is at an increased risk for 1~ yoctirdiai infarction, comprising determining physiochemical activity of gene products c~f the Factor II and hactt~r XIII genetic elements ~~1~ an individual, wh~:reby the presence .~.~t~
FIIG20? I OA and F~IIIV 34L gene products physiochemical activity is indicative of <rn increased risk for myocardial infarction in said individual.
a In accordance with another aspect of the present invention, them is provided a kit for determining whether ar-r individual is at an increased risk for n5yocardial infarction, comprising oligc~nurleotides sprvific to the ~~arianl region of the alleys of int;.rest or to sc.~uenc.e fiaoking the variant region and optionally instructions for a<:u.
13RIFF DESCRIPTION OF THE DRAWINt~~
Figure 1 shows a comparison between expected and observed prevalence of combined earners of mutations in myocardial infarction as described in Exampl1 (MI ---individuals, control == =;00 i.rrdividuals);
Figure 2 shows tho T~revaiGnc~. of (a) FhlIIV34L in MI pati~:nts and rrorn2al c;~nirols who carry the F'I1c-i~021()~ allele, and (t~I FXIIIV34L in Ml patiants ana nor:~~al i.:ontrols who is c,rrr~ them FVL: alleic, as descrsbed in Example 1 (h'II = 5(i0 indivi;luals, control = 500 individuals j:
Figure a shor~-s a comparison bettyten expected and observed prevalence of combined cvar~-i~xs of mutations in ioyoc,mdial infarction as described in Fxamipl~: ~
(ll~il = 73() individuals, control == 579 indi~~iduals); and Figure ~ shows the pre.valenc.e of (a) FhIIIV 4L in ~9I patterns and normal controls who carry the FIIfi2U?l0a allele and (bj FXtIIV34I, ira MI patients a.nci n<armal controls who carry the hVl, a!_tele, as descaibed in Lxample 2 (MI = 730 individuals, control = 579 individual s).
IJE'1':~li:)ED !)ESCVRii''t'iON Oi~ '1'!iE INVr:NT~fJN
'hhe pr~s~rlt invention providLs for methodss of identifying 1-isk factors associated with myc>cardial infarction. The invention further provides for the application of these identified risk i'actors in methods of identifyi.rlg individuals at risk for myocardial itlfarction or individuals having a propensity for myocardial infarction.
Thus, the priaent invwtion provides diagnostic arid prognostic tools valuable in a medical cvrttext. 1r the ec~~ntext of the present inven it~n, a ''risk facti>c" refers to a variant t;unetic ehment, typically a mutation, the prosz.nce of which in an individual's ~~ellOtlle provides an indication of increased risk lc~r nlyoeardial infwrction. 'I he preserECc o!~
such variant to genetic elements call bc~ d~ternrined at the genetic (nucleic acid) level oa at the protein (;poiypeptide) level. In accord;lnce with the invention, the risk factors comprise a variant of ;~nc or more genes encoding, proteins that are part of a mufti-stage, mufti-factorial biochemical rea ction system, sllc.h as the tibrinol~rsis systern, the clotting system, or the complement system.
t 5 I)ef in iiion.s t..lnless defined or_herwi<;e, all tecllllical ~uld scientific. terms used herein have the same meaning as cc>Ilnlomfy' understood h~~ tone of ordinary skill in the. art to which this invention belongs.
'f he tens "gene~:ie element-". as hsea_l herein, refers to a t,u~:l;:otic~le seduence present in the:
3t1 eenolxlc. of the c~raaltism iltldr~r ir~~~estigarion. Examples of genetic ~:lznl~ntcorrelated '.vlth tfCv''tlted rlll'OC~CCII~!1 ::lfelivti'J't rlSk C~:.Jm(lt7~L'. hllt elrl: llOt ri:stflWtd t;s. genes tildlll~
tear cotllponents afthe libl-incllysis system, the clotting sysrem, or the;
conlyletllent system, s,ucll as, tbr ex,~rtlple, the gene; cnclitlg fur .f'll, FS'. arlG FX111.
'~AMutations'' in these :genes oo:ltcmplated t~~y the invention inc-lode, but are not limited 1o, Z_~ Cl:,'letlOrlS, illsCrtltall~, ~;llr~Jr11C1W??Tlll d1s111Gc~t10I1S, t1~1I1510C~hOrlS, 1C1~'r'n3On5, ~tld Otlt~r EenetlG 171eGhan1Si115.
~lfethorl_f'or the identification oJ'risk factors associated witfi myocardial i~tJrarctiun Genetic elements and variants thereof associated evzth an elevated risk oI' myocardial infarction may be identified by methods known in the art. Ono strategy to identify said genetic elements and variants thereof encompasses the steps of sequencing a candidate gene in a panel of probands from families with documented myocardial infarction, s tollowcd by estimating the risk factor Ior myocardial infarction <associated with any observed sequence variation in a population based patient-control study.
:~ non-limiting example of such a strategy is provided as Examples 1 and 2, which set forth the inventors' case control study, simultaneously analyzing for the presence of the FV L, FIIG?(?'? ( OA and h~XIIIV.'i~rl, variants in myocardial infarcaion patients and noanul 1~.) individuals of a genetically i~oiated population of the island portion of Newfotmdland and I,abradc>r.
Thus, the present invention relates to a method for the identification of one or a plurality of variant genetic elrments that encode proteins that are part of a multi-stage, multi-factorial biochemical reaction system such as the tibrinolysis system, the clotting system, 15 :~r the complenjent system. wherein the defects are associated witlu an increased risk of tam cxat. dial into-~rctiotr in ;:.n i~ldividual. As used herein, ''a plurali!y" refers to two or more. In one i~mboditncni.. the iriv~ution provides methods for the clc:LeCttor2 c7f one or a plurality of variant genetic elert~nts that encode proteins that are hart of ih~w tibrinolysis system. the clotting systctn, moiior the complement system. In another embodiment, the 2t~ invention provides methods for the detection of a plurality of variant genetic elements chat encode proteins ths~t are hart of the fibrinolyrsis system. the clottinc system, and/or the corrzplemeaat system, wherein the plurality of variant gencaic a lensents indicate that an individual is at greater risk than any one of the variant genetic elements alone.
The methods of~ identifying the variant genetic elements provided by the present 2~ invention comprise the Steps of~ identifying a suitaole sample population of individuals t~:~
screen, obtaining a biological sample from each individual in the sample population, detent~ining gene sequences, ;,;tree products of genes, or activities oI' gene products to identify variants theret?f, and comparing the occuwence of the variants with the occurrc-nee in a suitable reference population. A suitable sample populaticm.
feet example, ~~uuld be made up of indis~iduals who have suffered myocardial infarction and a reference popuhttian »~or;ld bo made up of in<Iividuals who haci nu history (either individual or falmilia!) uh rhyoc<irdi<tl infarction. 'I'hc sample population may be geographlcailv restricted, restri~aed by age, gender or ethnicity if' desired.
Typically when a sample population is thus restricted the reference population is sin;ilarly restricted ;l.c.
matched) by geographical ref;icm, alg,e, grader or etllllicity.
()nee the sample and reference populations are selected. a suitable bioleyical sarnple must be obtainea in order :o carry out the analysis for variant ~,cnetic elements.
l3ioleyic.al sa.rrlples obtained ti~csnl a subjcca may comprise genomic ~)Na, fZN:I. or s o protein. 1~ or the purposes of physiochemical analysis, a suitable biological sample may be a blood or blood plasma saml.lle containing the protein of interest. 1~'or the purposes of nucleic acid analysis. a suitable biological sample may be a blood or blood plasm;i sample, urine, sali~~a, tissue biopsy, surgical specimen. tiw needle aspirates, amniocentesis samples, or any other material comprising the paiierlt's genomic DNA.
15 '~~f~thods tOr O~lliililltl~ suitable biological samples are known in the art.
Detection Of mutations rrlaybe l~erforrtled, inter olio. by methods known in the art, such as direct secluelice <~.ualvsis i}f asllhlified nucleic acid containing the aben--ltiun, by allele spLcilic anlpliflcatiull wllirll ciifferentially amplifies I)NA containing atnd DNA lackin~~
the aberration, or E~y restrict'tc~n fragtncnt analysis. I; may also he performed by 2il hybridization with a probe d~lat is able to differetltially hybridize under stringent mtlditions to the str~.tCh ui~ ;rtllplitied nucleic acid matc-rial r~~hich may contain the aberration.
~~clrlolll tl'.t,11n1C1t1~5 ii)r illill)lljyln~ rlllclClC al:ld at'e;
lvIlOVs'11 111 tllC iirt. t.~tlv e~2lml)1tG ~t a tc'.Chrll(lUe le'r tIIC aiillpl(tlC;ltlC)f" i)1';l l~:~11 t27.C~?ia 5~~nli:llt 1S the p01~'Illtril~e Chelln r~.~tt;t1C711 c;t'~:R). 1~-itll thz Pt:R te~:hni~.~~l~:. the copy number of a particular tar-_fet Segment i5 increased ehponentiai!y with ~: nulllher of cycles. :~ pair of pritllcrs is used and in each cycle a 1)NA pl-inler is annealr:cl to the 3' side of each uf~the two st(<~ulds of the doubled atr:uldcii Di~A~la~'lrei seiluance 'fTlli~ primers are e~aendoc:l ~..~ith a C~'.~.~ IrolytilLl'asc lI7 the presence oh the various monc>l~tncleotides to gener~.te double stranded I:INA.
The str~~ltds t ti of the double stranded DNA are separated from c;3ch other by thermal dcnaturation and each strand then serves as a ter_nplate for primer armealing and subsequent elongation in a following cycle. 'The PCR method has keen described in Saiki et al., Science 239, 487, 1988 and in Cwopean Patents no. I:P 200 3G2 and EP 201 18~. Other amplification techniques include mismatch PCR, the Ligase Chain Reaction (LCR). Repair Chain Reaction (Rf'R), TMA, rolling circle amplification, nucleic acid sequence based amplification {NASLiA), and strand displacement amplification tSI~A).
The term "oligonucleotide" as used herein refers to a linett.r~ pohrnucleotide molecule of !1p to ahc?ut 20l) nucleotide ba~es in length, for example a polynucleotide (such as DN.~1 l0 or RN.1) which is at least about 10 nucleotides, for example at Ieast 1 ~, 5ti, 100 or 200 nucleotides long. These oligonuc:leotides may function as primers and probes.
The term "primer" as used her;yin refers to an oligonucleotide either naturally occurring (e.g. as a restriction fragment) or produced symthetieally, which may act as a point of initiation of synthesis of a primer extension product and which is able to hybridize to a I s nucleic acid strand (tempate or target sequence) when played under Suitable conditions fc.g. buffer, salt temperature and p1-1) in the presence of nucleotides and an agent for nucleic acid pols-rnerization, such as DNA dcpenclent or R1'A dependent polylnerase. A
primer must be suffici.c.ntly l~,ng to prime the syniltcsis of exteT><siUn pro ducts in tr_e t71'tsCllCC; Ul an agent f<~r puIyrrlrir~tiun. A typrcal hrrmc;r contaua at least about 10 2o nucleotides of a sequence substantially complementary or homologous to the target sequence, but somewhat longer primers are preferred. Usually primers contain about 15-~6 nucleotides, but langer primers may also be employed. Normally a set of primers will consist of at bast two primers, one 'upstri:arrt' and one 'downstream primer which together define the sequznc~ that will be amplified using said primers. 'The primers used 2~ for am(7(ifrcation of tile varicJua refi.ons of the target genes may be sylrtl:esized by the methoxa~phosl:lw:~ramite mctho:i (Tetrahedron Letters 2?, 18>9-186, 198 l ) or other ~ulitabl:method Iwiilll-n lrl the art. Tlve sequcr;ccs of the primers are cl-losen such that they flank the aberrmt regions of the target gene. Suitable primers :~sr ampiifrcation of the various transcril-,ed ;tltd untm.nscribed regions of the alleles of interest ar-e those 3o detertrined to he most effiraci~u?s for the task.
,~
;qtr-urgent (hybridization) con;litiorts arc conditions under which a test nucleic acid molecule will hybridize to a t~.rget reference nucleotide sequence, to a detestably greater degree than other sequences (e.g., at least two-fold over bac~:ground).
Stringent conditions are sequence-dependent and wrill differ in experirrrental contexts.
F'or example, longer sequences hybridize specifically at higher temperatures. (.ienerally, stringent ~~onditions arc sc°lectccj to be ~rbout ~' C. to about 20° C.
lower, and preferably. s" C.
lower, than the therntal meltirl; point (Tnt) for the specific target sequence at a delined ionic strength arid pH. 'The T'nr is the temperature {under defined ionic strength and pH) at which 50°ro of a complementary target sequence hybridizes to a perfectly matched probe. 'l ypicallv, stringent conditions will be those in which the salt concentration is less titan about 1.0 '~~1 Na ion concr~ntration (or other salts), typically about 0.01 to 1.0 M Na iorr concentration (or other salts). at pH 7.0 to 8._i, and the temperature is at least about >() C. for slror2 probes (;e.g,., t 0 t:a 50 nucleotides) and at least about C;ii' C. for Long probes (e.g., greater than ~C~ nz.,~.~i~.otides;>. Stringent con:iitions nmy also be achieved with tlae addition of destal7iliaing rents such as fc.~noarrtido. 1=;x~:rnplary ;o~.v stringency conditions include hybridization with a buffer solution of >0°i°
formarnide, I M NaC_.'l, 1%
'~1~~5 at 37' ~.'.. and a w~a:;h in ? tunes ;~W ' at SO' C. I=xcrnplay high string.encv conditions include hybridization in ~(1°r~ fr~rmantide, 1 M NaC'1, 1 °,~o SDS at 37 C., and a wash in 0.7 times S5C at b0' C'.
Che term "probe" refers to av oligonucleotide, typically labeled, that forms a duplex structur:; with a sequence c~f a farces nucleic acid due t0 COmpleIIlent<ary base pairing. ~(~h~
probe will comprise a hybridizing region, prelerably consisting, of IO to ~0 nucleotides, snore preferabiv ?() to 3i) nucleotidea, corresponding to a region c~f the target sequence.
1 he hybridizing region of a probe is preferably identical or fully c~rnplernentary to the 2~ sequence of tho target rel;ion. hlte lrybridizirrg region rnay also eontairr a certain number of mismatches, those skilled in fhe art of nucleic acid technology :an c3eterntine duplex stability considering a muncac.r of variables including the ieng.th and base-pair composition of the prohG~, ionic: strength of the buffer, re-action ternl5eraturc and incidence of mismatched base pairs, seu. e.g. Sarnbrook et al., Moleciriar cloninf~: r~
laboratory 3c; n~~unu:xl, seccsnd edition ( I t)89) t:. olcl Spring Narbor Laboratory Press.
'~I'hi: term "label" as.usrd herein refers to anv atom or molecule which can be atiached to a nr.:cleic ~.cid and rrhich em be used either to provide a detectable signal or to interact with a sec-and molecule to modify Ehe detectable signal provided by said sec«nd mohc;ule.
hxarnples of suitable labels include, but are not limited to, radioisotopes, fluorescent s compounds, erraymes or chemiluminescent compounds.
Ilybridiaation of the probe v~ ith the target sequence may be detected by techniques known in the art of nucleic arid technology such as Northern or Southern blotting, see e.b. Satnbrook et al., supra. Detection systems that maybe used in conjunction with such methods include., for example, enhanced chemihrminescence (,ECL) based analysis or to onzyme linked get assay {E.LG n j based analysis.
Sequence analysis includes direct analysis of the DNA sequence tlankinc and constituting the cxons and uniranslated regions of the gene. This method involves any protocol that is currently available to any person skilled in the art for directly determining DNA- c.~r RN:~ 5eqllellc~s, su~,~h as the dide3xynucleotide method described by Singer ti (Pros. Natl. Acid. Sci. 1_lS:r, 7~ ~4G3-7, 1977}.
!t is also possible to analyze tl~e amplified material through restriction fragment analysis.
in this method the amplified material is digested v:-ith resirictior~ emytncs that recognize DNA sequences that are either present in I~NA sequences derived ti-om patients carrying an alet~ration in the exons or rmtranslated regions Of the g4tte, or that are present in the 2f) IlatlV~ sequence encoding the gene product.
It is also possible to analyze a known mutation by allele specific amplification (Trends in <ienetics, 12, 391--39?, 1 ~?96 at~d Mullis et al. cds, The polymerise chain reaction, I3itkhauser, I3c>ston, l.3as~l, llorlin, i')'~~1. ppI-13j r~llelc spL;;.itic PC'R fir the F'ltCi2()? I UA variation has been described in Thrombosis and hlaemostasis 78, 1157-1163 25 ( l ~>97j. 'this technique is based on the observation that under certain c«ndit.ions primer elongation caimot take place wlien the 3 tertninal nu~~leotids: of vj primer is neat complementary to the template. Vvith the else of two forward primers that differ only at their temtin:il :~' nucleotide it is possible to distingui>h between homozygous or heterozygous individuals, for instance with respect to their F'II(:i''U? 1 UA
allelc;s: analysis ;:f rnatcrial from lac~ioca:vg:~as i.ldi~~idual;, will result in a positive attihlificar.iou result with either one of the primers, material from heterozygous individuals will result in a positive amplification wiih bath primers. Similar analysis can be performed for the FVL, and F~IIIV341. alleles, as well as for alleles of any other genes involved in the fibrinolysis system, the clotting system, yr the complement system.
Other standard mutation analysis techniques known in the art may also be used {see, for example, Dracopoli, et al., Cuwent Protocols in Human Genetics, John Vv'iley &
Sons, Inc., I~i~"j. Examples of such techniques include, but are not limited to, restiiction-fi~agment-lengtO-polymorphistTi {EZI~Lf'j detection, hybridization using immobilized to oligvnucleotides or oligonuclevtide arrays, mismatch-repair detection (MRDj {I~aham and C:ox Genome Rcs ~:~74-48? (1995)). binding. of 1\~lutS protein {V'agner et al. Nucl Acids Res 23:3944-3948 (1~)~~~), denaturing-gradient gel clcctrvphvresis (DGC~F).
denaturing high-performance liquid chromatography {DHPI_C), agarose gel c>r capillary-based electropl:vresis, single-strand-confornzation-polymviphism {SSCP) detection, R?vAase cleavage at mismat.ehed base-pairs, chemical yr enzymatic cleavage of heteroditplex DNA, mass shectromety~ and radioactive andlor tluorescent DNA
sequencing using standard procedures well known in the art.
Physiochemical analysis of th4 products of candidate aliales comprises imrnunological or ?o physiochemical detection.
Immu~iological dctc-coon comprises the steps of obtaining a srunple of~the patient's blood, pl;;sma, or other bodily fluid in ~~s~hich the gene product may or m~3y not be present, adding a ki~o~,vn ~lu:mtit~r of a;n antibody specific to the variant gene product udder cu.;iditicatwhicri savor t~ir~c.-lii~g ~~f~ tire 5pecitiG ~antilm~dy to the vaiuant g,Cnr. product. aJld assessing the presence or absence of the binding of the antibody to the variant gene product. A pvsiuvv test result ivli;,rein such binding is observed is indicative of the presence c,f tljr: gene product, and a negative test result wherein sack birnlinb is not observed is indietaivce of the al~,ence of the gene product.
t,t For the purpose of this application, an antibody is an in~munoglubulir3 molecule and immurtologically active portions of an imnturtoglobulin molecule, l.c., a molecule that contitin an antigen binding site which specifically binds (itnmunoreacts with) an antigen.
A naturally occurring antibody: (e.g., IgG) includes four polypeptide chains, two heavy S (H) chains and iwo light (L) chains inter-connected by disulfide bonds.
However, the ~mtigen-binding fimctu~n of are antibody can be performed by frugment~ of a naturally occurring antibody. Thus, these antigen-binding fiagmc;nts are also intended to be designated hr- thv term antibodv. E~~trnples of binding Fragments encompassed ~~~thin the terns antibody- include (l) an Fab tra6ment consisting of the VL:, V1I.
C'L and C.'HI
l1 dorrtaitas; (ii) an Fd fragment consisting of the VH and CH 1 domains;
(iii) am fv fragment cUIISIJIInL Of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment ( Ward et ul . Nature 3~ 1:44-fi, 189) which consists of a VH dontain; (v) an isolated complimentarity determining region (CDR); and (vi) an F(ab') 2 fragment, a bivalent tiagrnent comprising two Fab fragments littlicd by a disulf de bridge at the hinge region.
15 hurthernnore, although the mc~ domains of the Fv fragment are coded tier by separate genes. a synthetic; linker cart hc: made that enables them to be made as a single protein chain (known as single chain Fv (scFv); Hird et ul. Science ''4?:4? ~-G. 198H:
and I-Iustott rc al., I':~uc~. ,l~utf. :icoid. .~'~i. r.~.~~7~)-$3, 19~$) by recombinant methods. Such 5inglc chain ~ln:l~iV~.llvJ ~tre al<<~ ItlL:lllC~~:'~.
rct In one example. tttltibody fiagrltent; are capable of crosslinking tlueir target antigen, e.g., bivalent fragments such as F(ab')2 fragments. Alternatively, an antibody fragment which sloes nc?t itself crosslink its target antigort (e.~,., a Fab Iragment) can be used in conjunction with a seconchtry mtibody which serves to crosslirtti the antibody fragment, thereby crosslinkity ttze terror ~tntiyon. ,~~tttibodies can be fragmented t.rsirog cunverttional ''S teehnicfues and the fragments screened for utility in the same nmoner as described for whole :mtib~~dies. Arr anttbody° is further intended to include bispeciti~ and chimeric molecules that specifically bind the target antigen.
'vpecilicallsr binds" as useu hc~reitz with reference to an antibody, or antibody fi~artnent, r~;f~rs z.o the ability of an irtdi~-idttal antibody, or fragment. to specifically iiztmunoreact 1~
with ~uz azriigen. The binding is a non-random binding reaction between an antibody molecule and an antigenic determinant of the T cell surface znolecule. The desired binding specificity is tropically determined from the reference point of the ability of the antibody to differentially bind the T' cell surface molecule and an uruelated antigen, and therefore distinguish between two different antigens, particularly where the lwo antigens have unique epitopes. An antibody that specifically binds to a particular epitope is referred to as a "specific antibody".
Physiochemical detection comprises the steps of obtaining a sample of the patient's blood, plasma. or other bodily fluid in which the gene product may or may not be present, optionally isolating the gme product from the sample by inirmuzological or physical znc:ans, and assessing the presezzce or absence of the variant gene product.
In the ,:ontext of the: E~resent itt~~entiun, an ''isolated'' biological ccmpc>nent {such as a nucleic: acid molecule, polypeptide. or protein] has been substantially separated or purified away from other biolcgicai cotmponcnts in the cell of the c~rt;arrisrii in which the component naturally occurs {i.e. other c.liromoso:nal and cxtrachronzosomal D.N'~ and ItN.A). Nucleic acicjs and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The terra also embraces nucleic acids am:l proteins prepared 1-ry r~r:~ii~ibinant expression in a host cell to w~rli a~ chemically syntluesi~od nucleic; acids and proteins. The terns "isc>lated" does not require absolute ?u pu.ity; rather, it is intended as t relative. term. Thus, for exarnplt~, ci substa.niially purifie;i Frot.ein or nucleic acid preparation is one in which the protein or nu::leie acid rcfcn-cd to is more: pure than the protein in its nattua.l environment wit.hio a cell or within a production reaction chamber (as appropriate], For example, a preparation of a moditied protein is ptuified if the protein represents at least 50°,~0, for example at least 70°~0, of the 75 total pri>tein v~?nt~nt of ttie pret,aratic~rl. l~icthods for purificatiozz of protoiuis azid nucleic acids ~irc: well knr.mz~ in thL :irt. I'.~;azuplGs of methods that pan be usrrd try .l3urif~~ a protein include, but are not limited t~.~ the methods disctos..d in San~hrook et al.
{Alolecultrt-(..'.t,~rtin~: =1 L.~rbc~~'c'tcr_,: a-jurlurl. Cold Spring fiarbcsr, New York, 1~~~9, Cli. 1?~.
~H
13y "isolating the gene product fiom the sample by irnmunological means'' is meant, for example, that the variant gene product may be isolated from the patient sample by means of an antibody capable of detecting both the wild-type and the variant form of the gene product (that is, an antibody directed to an epitope conserved in both the wild-type and the variant gene product, but u~iique to the products of that gene locus). By "isolating the gene product from the sample by physical means" is meant, that the variant gene product may be isolated from the patient sample by means of centrifugation, chromatography.
electrophoresis, or other techniques known to the worker skilled in the art.
)3y "physiochemically assessing the presence or absence of the variant gene product" is meant the use of techniques such as protein kinetics, spectroscopy, crystallography or other techniques known to workers skilled in the art to distinguish the unique physiochemical signature of thv variarnt gene product from that of the native or wild-type protein.
Finally. the frequency of occurrence of a particular variant within the sample population t 5 can be compared with the lreduency of occurrence in thu reference population and the statistical significance of any difference can be calculated by standard methods. A
statistically significant difference in the frcquenc:y of occurrence between the two populations indicates that the variant is a risk factor for myocardial infarction.
l)eterntining the risk of »iyvcardirrl infarction tv the ijtdividua~
?o 'hhe present invention further provides for methods of determining the cist~ for myocardial infarction to an individual andlor the propensity of an individual towards myocardial infarction. 'Thus, the C~resent invention relates to a method for the detection of a plurality of defects in a mufti-stage, tnulti-factorial biochemical reaction system such as the tibrinolvsis system, the clotting system, or the complement system, wherein the defects 2~ are associated with an increased risk of rr~yocardial infarction in an individual. The method comprises the steps of obtaining a suitable biological sample from the individual, analyzing for the presence of v~wiant gene sequences, gene products of genes, or activities of gene products known to bE associated with myocardial infarction, and determining the i risk to the individual of myocardial infarction. The method may optionally further comprise a preliminary step of screening for suitable individuals at risk of myocardial infarction.
In one embodiment of the present invention, the variant genetic eler~ients associated with myocardial infarction being analyzed for are one or more variant gene selected from the group of; genes encoding Factor II, Factor V, Factor XIII, or a combination thereof. In a further embodiment. the variant genetic elements associated with myocardial infarction being analyzed for are one or more variant gene selected from the group of:
FIIG202I OA, FVL, IXIfIV34L, or a combination thereof. In another embodiment. the variant genetic to elements associated with myocardial infarction being analyzed for are two or more variant gene selected from the group of: genes encoding Factor I1, Factor V, Factor XIII, or a combination thereof.
It was tutherto unrecognized ti,at a combined carrier status of the FIIG20210A
and FXIIIV34L genetic elements is a strong risk factor for myocardial infarction.
With the present invention such a correlation has now been establisheii. Therefore, in another embodiment of the present rnventron, the variant genetic elements associated with myocardial infarction being amrlyzc:d for are FIIG20210A and FXIIIV34L.
I. ,S'c reen lo~~ risk, fiictors to .select patieru.s In general, in such a preliminary screening step, if one is underta;;en, individuals are ?o selected either according to a family history of myocardial infarction, indicative of a genetic predisposition to the c:mdition, or on the basis of an individual case study. In a typical individrr:l case study a patient may either present with a prior history of myocardial infarction, or global l,arameters may determine the interaction of several components of the clotting sy:~tem. For example, the prothrombin time (,PT) is a global 2s parameter which determines tl;e scale of the exogenous cloning system and the partial thromboplastin tune (a1 r Tj is a global pararneier which determines the state of the endogenous clotting system. 'The protllrombin level can be measured in a hmrran plasma sample or other suitable biological sample using techniques known in the art.
Measuring ~n prothrombin levels in a statistically sufficient number of healthy individuals can define a normal value.
It~ a positive family history is recorded, or a positive finding is obtained from a personal history or a global parameter such a_s PT or aPTT, individuals parameters can be analysed s to discover the underlying genetic hasis for the condition.
?. ()btair~ing a suitable biological sample A suitable biological sample is obtained from the individual under study in order to carry out the analysis. As described above, biological samples obtained ,from a subject may contiiin genomic DNA, R;vA, or protein, and can be a blood or blood plasma sample containing a protein of interesor a blood or blood plasma sample, urine, saliva, tissue biopsy, surgical specimen, fine needle aspirates, amniocentesis sarnples, or other material comprising tlje patient's genomic DN A. Meth<xis For obtaining suitable biological samples are known in the are.
3. :T rral~.~si.,~
is Suitable methods of analysis comprise physiochemical analysis such as enzyme assay, an immunoassay to determine th~.~ presence: of a variant gene product. or a nucleic acid ac alysis to deternline the presence of a variant gene sequence. Such methods are described in detail above.
The physiochernical analysis comprises individual parameters, each of which deter-rnines 2o the presence or absence of only an individual genetic product, such as variants of Factors 1(, ~', or X111, or other components involved it the fibrinolysis system, the clotting system, or the complement sysaetn. For c;xample, the presence of the gene product of the F?tIlIV3~L allele van be determined by reaction tvith au antibt~dy which specifically birds to the epitope comprising the valine to leucine substitution at amino arid position ~1.
l'hc, nucleic acid analysis co=uprises assessing individual parameter. each of which determines the presence or absf~nce of only an individz~al genetic element, such as FVL, j4 F lIG''U? l U:'1 or FXIII V 3~L alleles, or any other genetic element involved in the fibrinolysis system, the clotting system, or the complement system. For example, the presence of the FVL allele can be dctcrn~ined by the use of sequence-specific oligonucleotides.
s In Example 1, the FII, FV, and IrXlll genes were analyzed as candidate genes in a search for genetic elements that may contribute to elevated risk of myocardial infarction. F1I is encoded by a ?1-kb-long gene localized on chromosome 11. position l lpl l-qI?.
The gene is organized in 14 exuns, :>f which exon 1 comprises the 5' untranslated (LET) region and exon 14 comprises the 3'-~,T region. The nucleotide sequence of the FII
gene, its n3 flanking sequences as well as the position of~ the various exons has been described previously (Biochemistry 26, 616-6177, I987). 'fhe G202IOA sequence variation is loc;3ted at the last position ot~ the 3'-LET at or near the cleavage site in the mItNA
precursor to which poly A is added. Three conserved sequences in mRNA
precursors, located in the vi;.inity of this site. are required for cleavage and palyadenylation: the ~ 5 AAU.~1..~~A seauence, the nucleotide to which poly A is added, and the region downstmnm of this mtcleotide. Generally, the nucleotide to which poly A is added is an A, mostly preceded by a (:. As a consequence of the G to A transition at position 20210, a CA dinucleotide (rather than a CrA dinucleotide) has been introduced at or near the cleavage and polyadenylation site.
2o FV and FXIII are encoded by genes w.~hose nucleotide sequence, flanking sequences, and position of the Var10li3 i,'XOilS have been described previously.
Genetic analysis of the candidate genes comprises obtaining from the patient a biological sample, selectively amplifting from said sample of nucleic acid coding regions or other regulatory elements of tl-,e c;uzdidatv genes comprising the genetic aberration, and anulyza:g the sequence for the press°ncc of signature substiti.ttions indicative ot~ the gene variant of interest. Biological samples are those containing genomic DNA, cDNA, RNA, or protein obtained from the cells of a aubject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, flrle needle aspirates, anuliacentesis samples arld autopsy material.
2(l As is illustrated in e~:ample 1, :lte G''021 OA mutation in the FII gene, combined with the V3~L. mutation in the FXlll i:ene, has L~een demonstrated to be present in a group of patients exhibiting a t~isk for myocardial infarction without the cause thereof laving been previously determined. Example 1, which illustrates the general use. of the methods for the detection of mutations indicative of an elevated risk for myocardial infarction, is provided for illustrative purposes only.
The study in Example 1 shows that the presence of sequence variation G?0210A
in the FII gene, CUIIlblned with the sequence variation V34L in the FXIII gene, is a risk factor for myocardial infarction.
]() t~. ~YIEI')Ylll?l)1L; t'tS~i Of7T11'L~i:C7T11~1L1j 1)1,(I)'L't10)1 Once the genetic material frc.~m the patient's sample has been analyzed for the presence of the sequence variation in the target gene, or the biological material from the patient's sample has been analyzed for the presance of the variant product of the target gene, the risk for myocardial infarction cart be determined on the basis of the risk established in a t5 case-control study such as. for example, the Newfoundland study of Exaanple 1.
Kit~or tf:e detection o/'risk fuctars associated with m)~ocardfal i~efarctior~
Kits for the detection of risk factors associated with myocardial it>farction may be kits for the genetic, immi.:nolo~ical or physiochemical dctc:ction of risk factors associated »kith nlvocardial infarction.
2o hits fc,r the geneti:; detection: oi~ risk factors associated with myocardial infarction COfIIpI'iSt', olieonucleotides specitic to the variant region of the alleles of interest c>r to scduence flanking the: variant legion, and optionally butters. nucleotides.
enzymes such as polymerises, lit;ases or endonucleases as appropriate to the specific method of genetic analysis knows in the art. ;end other reagent useful in performing such analysis.
2~ Kits for the immtlnological detection of risk factors associated with myocardial infarction comprise primary arnibodies (monoclonal, polyclonal or purified) slaecific to the variant 2]
epitopes of the gene products of interest, and optionally buffers, membranes, secondary antibodies, and preferably labeled secondary antibodies specific to the primary antibodies, and other reagent useful in performing such analysis Kits for the physiochemical detection of risk factors associated with myocardial a infarction comprise electrophoresis buffers, centrifugation buffers, substrates specific to the variant gene products, and preferably chromogenic substrates, and other reagent useful in perfornling such analysis.
Te gain a better underst<zrrding of the invention described herein, the following examples are set forth. It should be understood that these ex~unples are for illustrative purposes Ollly. Therefore, they should not limit the scope of this invention in any way.
EXAMPLES
~:xamnle 1 Sl:'I3,~E~:TS, ~1IATERL~LS.-1~VD ufETHODS
S'uhjects:
t s 131ood samples were collected from 500 consecutive myocardial infarction patients and SUO normal controls of the genetically isolated lvewdoundland population. 'The population consists mainly of descendants of English and Irish settlers who arrived in the 18't' and 19" centuries. Tl:e geographic and social isolation of the island has ensured vend little inward migration for several hundred years, and thus has lead to a small population {30.000 individuals) with a relatively homogenous genetic background, ideal for the sW dy of complex multilactorial diseases such as myocardial infarction.
Patients categorized in the myocardial infarction group represented those presenting to the emergency department or mithin one of the Health Care Corporation of St.
John's hCSpital5 Vvlth symptorr~s and rio;;henucal evidence suggestit.e of _~.yocardial infarction.
25 Unly patients with cardiac Truponin I values greater than 2.0 yg/L (Axsym, Abbott I)iagnosticsj or greater than 0.~~ Ey~L (Access II, Beckman-Coulter Corp.) were used in this group. Control subjects were selected from consecutive individuals without prior history of myocardial iniarction or thrombosis presenting to the emergency department for trauma, accidental injun~, or other non-cardiac and non-thrombotic related events, Discarded blood samples c-ollected for complete blood count were used far DNA
extraction and analysis. Ethica approval for this study was granted by the Human Investigations O:ommittce of Memorial C.'niversity and by the Health Care Corporation of St. John's.
Genotylaiug ry~F'lIG2li~ I f~.~, Fl'G, arid F~'lllb'34L:
Cienoniic DNA was isolated fr om the peripheral blood using standard methods {Miller 1o S.A., Dykes D.D., Polensky H.P., A simple salting out procedure for extracting DNA
from human nucleated cells.Nucleir Acid Research 1988; 1 b(3 ):1'? 1 S).
Genotyping of the FVL., F1IG20210A, and 1'XIIIV3~1L vvas performed br~ PCR amplification of each of the target ;alleles frorn SeIlOIIIIC DNA followed by restriction digestion with each of corresponding enzymes alnll, flind.lll and I~del respectively, as previously described (Linfert D.R., Rezulcc \~'.N., Tsungalis G.1., Rapici multiplex analysis fbr the factor VC
Lkeiden and prothroWbin tJ20:? 1 OA mutations associated with hereditary thrombophilia.
Conn. Med. 1998: fit{9): Slr)-2~). The digested PCR products were separated by electrophoresis in 10°i° polyarrylamide gels and visualized by staining with etl-udium bromide.
2o Prevalence c~uternrinatio.=z urrcl css.~~nciation .slarclj.~:
The prevalence of each gene variant was calculated by counting the total carrier freduencv including heterozygotes and homoZ,~gotes. 'fllc allele frequencies were determined by gene cowving, f'earson C: hi Square statistical analysis was performed using SPSS x10.0 to test the association between genotypes and the prevalence of 25 nlvoeardial mfarctlon. Odds ratios (OR) were calculated as a measure of the relative risk fur myocardial infarction and were given with 95% confidence intervals.
.~IIClIyST.S f)f f;BiT(?-~BYtG' TnIC'YaLIIC)Yl:
Gene-gene interactions were detc.miined, first by comparing the prevalence of combined carrier for two of the three gene variants in patients and controls; and second by analysis of the distribution of one chosen gene variant in sub-grouped patients and controls who cal-ty another gene variant as genetic background.
RF.,St~'LTS
Geravtyping FIIG'?031<1:~, FYL, and F.klllt'3~L:
'hhe genotype distributions. c<i:-rier frequencies, and allele frequencies of FTIG2U210A, i~VL, and FXIlIV34L in both the myocardial infarction patient and the control to populations are given in Table 1: distributions of genotypes, arid carrier and allele frccvluencies of FI1G4()?10~1, FfI, and FXIIT-A V341, in MI patient and nornial control populations. (A-91: myocardial infarction, NC: Ilormai control, UR: odds ratio) The FIIG2U?l0A allele was detected in 3.2°~0 of patleIltS ~'~'171C11 ~VaS
~l~nltlCantlV higher than the 1 °~~ observed in controls (OR 3.3. 95°~'° C( 2.6-4.0; P=U.OI 5).
t _ _ _ ...~~..
_ Genotype Mt (n = lYC (n = app)-_ ~~ _-.._ P-.__ S00) _ value _~-____.__~_ {'~/,G _ 495 (99.0!0) ... _ _.__..__ ___~84 (~6.8%i FlICi20210A Cu''A 16 (3.2v) 5 (l.0io) A/A 0 ((I,' 0 (0%j j C:'arrier l~ . 3.2% ~ .0% 3.3 0.015 Alle!e F. 1 6% 0.5.fo R/R 4 7719.4%) 477 (95.4%) FVL(R506Q) l,'Q ' 3 (4.5%) 23 (4.6,~~) QiQ o (or~) 0 (o~~a~
Carrier F. 4 ti".%o 4.6.~0 l .00 !~'S
Allele: 1~. :~ 0 2.3,-0 y:'%y :'.05 (i i.0%) 261 (~2.?,'n) -x111-A v34L vu_ 103 (3s.6i) zo7 (41.4r~
LiI. 4? (8.4:0) 32 (6.4%) C.'arrier F. 4 %.0!a 47.80 0.97 NS
Alle~~ 1~. 2 ~.7.~~ 27.1,~0 ?4 An identical prevalence of FV L was observed in both patient and control populations (4.6°/° vs. 4.6%). No homozygotes for either FIIG20210A or FVL
allele were found in either population. In both patient and control populations, FXIIIV34L had similar prevalence (47.0% vs. 47.8°.'0) and allele frequency (27.7% vs. 27.1 %). The prevalence of homozygous V34I. was higher in patients compared wish controls (8.4% vs.
6.4%), but the difference did not reach statistical significance. It is to be understood that, because the statistical significance of the prevalence of a genetic element as it relates to myocardial infarction risk is dependent on the sample size, an increase in the sample size will conclusively determine said statistical significance.
1o The distribution of the three gene variations was further analyzed by sub-grouping patients and controls according to age, as shown in Tabic 2: distribution of genotypes among Ml patients with different onset ages and compared with age-matched normal controls. Myocardial intar~tion patients were divided into those with an early age of onset (less than 50 years) and those with a later age of onset (greater than SU
years).
iii yC oR p F1I 20210A C;arricrs 161500 (3.2%) 5/500 (1%) 3.3 0.015 Age <_ SOY 2/-16 (=t.3~ o) 3.373 (0.8°ro) ~.6 0.04 Age:> SOY 14:'454 ~,3.1%) 2/127 (l.6%) 2.0 NS
F1-'L Carriers 23,500 (a.6%) 23/500 (4.6%) 1.0 NS
Age < SOY ti;~(i (1~~.U°,%) 18!373 (4.8%) 3.0 0.007 Age > 'fly' 17.''454 ,'i,8°io) 51127 (3.9°io) 1.0 NS
F~XIIi 34LCarriers 235!50G 2391500 {47.8%) 1.0 NS
(47.0% ) Age. < s0Y 19146 (~~l .3%) 1761373 (-17.2%) 0.8 fJS
Age > >()'~' 2 ! ~i (:17.6%;i 6:1/127 (=19.G°,%o) O.~l NS
The control population was also divided into the two corresponding age groups.
hnctesti~igly, ii iiia~lll~lliUlilfil illa:~li3iitiVii eli ihC' ~'~'1., 3liC:li; 1'4'11: Ours;?"v2d tli illi; ~~ll'ly onset patient Group. 'fhe FVL allele was detected in 13.0% of patients v~ith early age of ?o onset, which was significantly 'tugher than the 3.8% in patients with a later age of onset (OR: 3.9, 95 % CI 3.3-4.4: P =11.004) and the 4.8 % in the age matched controls (OR: 3.0, 95 °,'o CI 2.2-~.7: Y=0.007 . The prevalence of FIIG~O:?10.A was alsc7 significantly higher iru the early onset group of myocardial infarction (4.3'%) compared to the controls under age 50 (0.8%). Although, the prevalence of FIIG20? l0A was slightly higher in the early onset group compared to the later onset group, this difference did not achieve statistical significance.. he FXII ~' 34L avowed a slight but statistically insignificant difference in prevalence beriveen the patient:c with early and later onset age.
Gene-gene Interactions:
Interaction between FXIIIV34~. and FIIG2()210A was first analyzed by comparing the f~) prevalence of combined carriers (individuals carrying both PIIG20210A and FXIIIV34L) in the total patient and control populations with their corresponding theoretical prevalence of combined carrier;
Using carrier trequencies described in Table 1. the theoretical prevalence for combined carriers of F'lIC~20210A and FhIIIV34L is 1.5% (47.0°~o x 3.2%) in myocardial infarction 2 5 patients and 0.48°'0 (47.8% ~ 1"'0) in controls (Figure la).
The observed prevalence of combined carriers in the myocardial infarction patient population (2.4°r; 12 c>l~ X00) ,.vas 1.6-find higher than its theoretic prevalence (1.5°~0), and in normal control population (0.2'%; 1 of 500) was 2.4-fold lower than its theoretic expected prevalence (0.48%). ~t~he observed prevalence of combined carriers was 12-fold 2o higher in myocardial infarction patient compared with the control population (P = 0.002).
'hhe interaction betwE:en the FII(i20210A and the FXIII~'34L was fm°ther examined by analysis of the distribution of FXIIIV34I. in sub-grouped patients and cotarols cvho carry FIIG202I0A as a genetic backl;round. Although the FXIIIV34I. showed an almost equal distribution in our myocardial infarction patient and i;untrul populations, 25 alleles ~.~~ere deteclecl in '75.0'e~ ( 12 of 16) patients with a genetic background of FIIG20?IOA ba.tt only in 20.01°,~ (1 of 5) of controls with the same genetic background (OR 3.7. 95 °1o C:I 2.4-5.1; P---0.013) (higure 2: (a) Prevalence of FYIIIV34I, in MI
?6 patients and normal controls who carry the Fiig20210A allele (b) Prevalence of FXIIIV34L in MI patients and normal controls who carry the FVL allele).
Of 13 combined carriers of FIIG20210A and FXIIIV34L identified from the studied population (500 patients and 500 controls), 12 subjects (92.3 °,~o) belonged to the myocardial infarction patient population but only 1 (0.7%) from the control population.
'the co-existence of these two gene variants imparts a strong predisposition for myocardial infarction with high penetrance.
the prevalence of combined carriers of FVL and FXI1134L were similar in both myocardial iltfarction patient (12 of 500, 2.4%) and control (9 of a00, 1.8%) groups and to was consistent with their expected t~equencies {2.1G% in patients and 2_2%
in controls;
Figure 1b). We further analyzed the prevalence of FXIIIV34L in patients and controls wlio carry the FVL allele as a genetic background. The prevalence of FXIIIV34L
was slightly higher in the sub-grouped controls (13 of 23, 56.2%) than in the sub-grouped patients (9 oC23. 39.1%) but the ditlerence was not statistically SlgIll~Cant.
t s There were no combined carriers of FIIG20210A and FVL in the myocardial infarction patient or control populations. 'This is expected considering a calculated expected frequency of combined carriers of 0.13 % in myocardial infarction patients and O.Oi % in normal controls, respectively.
Figure 1 (a) comparison of'the f,~xpected and observed prevalence of combined carrier for 2o FIIG20210A and hXtliV34L among MI patients and controls; (b) comparison of the expected and observed prevale,jco of combined c;u-rier for l'VL and FXiIIV34L
among X11 patients and Ci)lltr~lS. U1': observed prwalence: E.P: expacted prevalence(prevalence of FIICi20210A x prevalence of FXIlIV34I.).
E.~r~mple 2 s SC% l3.lL'(:' I:S. :t2-4 ? ~ RLflI.S' : I N~~ !~fE? HUMS' Blood samples were collected From 230 additional patients with MI and 79 additional nc,rrrlal controls, using tine metl-:ods and rn,aterials set out in Fxaxnple I. The cumulative summary of the results based total 730 patients with M1 and 579 normal controls are given in the Following tables and figures:
Rrsults CJenvty~ing FIICT20210r1, Ft'L and FXIll-.~4 l'3~L
The genotype distributions. carrier frequencies, and allele frequencies of FII
20210A, FVL, and E'Xlll-A L3:~ in both the IvII patient and the control populations are given in Table 3: distributions of genotypes, and carrier and allele frequencies of F11G202IOA, FVL and FXIII-;'~ V34L in hMl patient and noumal control populations. (MI:
myocardial infarction, NC: normal control, Oh:: odds ratio).
to Genotvp MI (n = NC (n ~ 579) 4R (95% CI) Y value 730) a _ -_-___._ ____.
CfIC7 7l (' (97.3%)572 (911.8%) r-lIG?0m0A c~,a 20 ~~.~%) 7 (1.2~0) :'i!.~ 0 (O';-o) 0 (0%) Carrir_r F. 2. 7'v, 1.2% 2.3U?(0.~?67, 0.053 5.48:_' ) Allele F. 1.4'ro 0.6%
R/R 693 (94.9ro) 553 (9:i.5./) FVL.(R506Q) R~Q 38 (5.2%) 26 (4.5%) 0 {Ci) 0 (0%) l'.arrier F. S.2"% 4.S% 1.16(U.69~,1.926) O.S7 i Allele F. 2.6'ro 2.3,/0 '~:'!V 391 (53.6./) 303 (52.3,/0) FX.III-A V34L: V,''L 22h 207 (:10.8ro) (31.2%) 1.~'I, ~7 i7.8,'7 3? (6.9~0) Carrier F. ?9~' :~ 4 l .3/a Q. 9-~8~~'. ? -'.~?-U. 59?
1.159 AIICI: F, 73,_l~/p 7 ~.ijr~
The F11 2021 OA allele was detected in ?.7% of patients. which was higher than the 1.2° o observed in controls (UR 2.26, 95% C:I 0.95-S.S.t), P - O.OS9). Prevalence of FVL was observed in 5.2° o patient and ~.5% control populations. No homozygotes for either the 2b FII 20210A or hVL alleles were found in either population. In both patielt and control populations, the FXIII-A L34 allele had similar prevalence (39.0°~~ vs.
41.3','%) and allele frequency (23.4°io vs. 23.4%). 'The prevalence of homozygosity for the allele was higher in patients compared with controls (7.8% vs. 6.9°~0), but the difference dil not reach statistical slgnlficance.
Vei2e-~,'C'Yle IY11C'TC1C'11t71~:
The observed prevalence of combined carrier in die MI patient population (1.92%, 14 of 730) Gvas 1.8-fold higher than it's theoretical prevalence (1.1%?, and in normal control population (0.17° ~; 1 of 79) was 2.9-fold lower than its theoretic expected prevalence to (0.5%). 'The relative risk for the combined carriers to development Ml is 11.3.
'fhe interaction between the FII 20210A and the FX111-A I,34 alleles was ftirther examined by analysia of the distribution of the FXIII-A L34 allele in sub-grouped patients and controls who carry the FII 20210A allele as a genetic background.
Although the FX11I-A L34 allele showed an almost equal distribution in otu' MI patient 1;39%j and ~ 5 control populations (;41 %). it .vas detected in 70% ( I 4 of 20) patients w7th a genetic backgrotuld of the FIl 20210A , llele but only in 14.3% (1 of 7) of controls with the same genetic background (Oft 4.9, 95°~° CI 0.8-30.8, P = 0.011 ) (Figure 4: Prevalence of the FX1II-A L.34 allele in MI patients and normal controls {NC) who carry the FII
allele {a) and FVL (b) {Ml=730. NC=579)).
2o Of 1 ~ combined carriers of the f I1 20210A and I-'XIII-A L34 alleles identified from the Studied population (730 patients and X79 controls), 14 subjects (93.3'%) had MI. The co-e~:iatence of these hvo gene variants imparts a strong predisposition For IV1I
with high penetrance.
7~Ile prevalence of cc»nbined c~u-riers of FVL. and the FXIII-A L34 allele was 1.78°io (13 25 of 730) in MI patients, urhich is lower than its expected frequency, 2.03°~0. V~lhile, the prevalence of combined c~irricrs in healthy controls was 2.42% in control groups (,14 of 79), which is higher than its exp.:;cted prevalence, 1.85°,~0 (Figure 3: Comparison between the expected and observed prevalence of combined carriers of mutations irr MI
patients and controls. (MI=73f~, T~TC= 579). {a) Comparison of the expected and observed prevalence of combined carrier for the FII 2U210A -~- FXIII-A L,34 alleles in MI patients anti control subjects. respectively. (b) Comparison between the expected and observed prevalence of combincci carrier for FVL + PXIII-A L3=1 alleles in MI patients and control subjects, respectively. Filled oar (r) represents the observed prevalence of combined carriers. Empty bar {o) represents the c?~pected prevalence of combined carriers.).
However, all of these differences did not reach statistical significance.
The prevalence of the FXIII-.~~ L34 allele in patients and controls who carry the FVL
allele as a genetic backgrrotuzd was further analyzed. ~fhe prevalence of the FXIII-A 1,34 l0 allele was higher in the sub-gr~~uped controls {14 of 26, 53.$5°%) than in the. sub-grouped patients (13 of'37, 3.14°~0) but the difference was not statistically significant.
M'here was only one combined carrier of the F1I 2U21UA allele and FVL
identified from each of the Ml patient ~=roup and control populations. 'this is expected considering a calculated expected frequency of combined carriers of 0.13% in Wi patients and 0.01'% in 1 s norntal controls, respec.ti vely.
~l'able 4: Distribution of genorypes among MI patients with different onset ages and cumpar~.d with age-matchod normal controls {NC).
1\1)( NC OR (95%CI) P
FII 20210A 201732 (2.73%)SI579 {0.86%)3.164(1.180, 0.016 Carriers 8.481) Ag,e < :>(IY 2193 (2.1''~u)3i-123 {0.71'0)3.1165(0.05,18.606)0.201 Aae > SOY 18/640 (2.81'0)2/156 (1.28%)2.194(0.504, 0.283 9.554) E~ 6Z C'arrier.s38/732 (5.19%)261579 (4.49%)1.156(0.694, 0.577 1.926) Age < 50Y 8.%c)2 (8. 2(}!423 ('1.73';%)1.8_19(0.'186, 0.1 7(~~0) =1.3(>4) ~S
AL,z > SC)5' 30640 (4.69,~)6! t 56 (3.85~0?1.2 I ~~(,0.-1<>9,(>.66~~
'?.979) FXIII 34Lcarrier5 3411732 (46.58%) 2771579 0.974(0.803, 1.180} 0.786 (47.84%) Age < SOY 43192 (:16.7a%) 1991423 0.994(0.666, 1.481 j 0.974 __ (47.o4°i°) A~e > 501' 298/640 (=16.56%) 781156 (50.0°io) 0.931(0.687, 1.263) 0.647 'Table 5: Comparison of genot.-pes distribution in different genders among NlI
patients normal controls Males Females OR (95°~o C1) P
FII Z0, l r7A
lVf1 9J353 (2.55°io} 10 /339 (2.95%) 0.864('0.347, 2.153) U.7~4 NC 5/255 (1.~?6°,~0) ?:'324 (0.G2%) 3.176(0.611, 16.507) 0.147 FVI.
Mt 261353 (7.37°~0) 10/339 (2.95%) 2.497(1.186, 5.256) 0.013 NC 10/2» (3.92%) 16/324 (4.94%) 0.794 (0.354, l.l8ll) 0.5 IS
f Xltl-A 134 ?01I353 MI , 1201339 (35.40%) 1.609 (1.227, 2.109} 0.001 (56.9=°%~i r 19/255 NC i26i'324 (38.89%) 1.200 (0.$89, i.61=ij O.G33 (46.67°'0) ~a«.
The disclosure of all patents, tmublications, including published patent applications, and database entries referenced in the specification are specii'ically mcosporat::d by reference i0 in their entirety to the same extent as if each such individual patent.
publication, and database entry were specifically and individually indicated to be incorporated by reference.
fhe invention being thus dcscribed_ it will be obvious that the same may be varied in many ways. Such varranons armor to be regarded as a departure from tile spirit and 1 ~ scope of the invention, and all such modifications as u.~ould be obvious ti> one skilled in the art are intended to be included within the scape of the following claims.
3t SEQUENCE LISTING
<110> Genesis Group .inc.
Xie, Ya-Gang <120> Method for the Detection of Risk Factors ,4ssociated with Myocardial Infarction <130> 365-127 <140> n/a <141>
<:150> 2,414,301 <151> 2002-12-13 160> 9 <170> FastSEQ for windows version 4.0 <210> 1 <211> 1086 <2:12> DNn <213> Homo Sapiens <400> 1 ctcaccagct gtgtctcgtg aaggggcgtg gctgggctat gagctatgct cctgagcaca 60 gacggctgtt ctctttcaag gttacaagc:~ tgatgaaggg aaacgagggg atgcctgtga 120 aggtgacagt gggggaccct ttgtcatgaa ggtaagcttc tctaaagccc agggcctggt 180 gaacacatct tctgggggtg gggagaaact ctagtatcta gaaacagttg cctggca.ggg 240 gaatactgat gtgaccttga acttgactc~ attggaaacc tcatctttct tcttcagagc 300 ccctttaaca accgctggta tcaaatgggc atcgtctcat ggggtgaagg ctgtgaccgg 360 gatgggaaat atggcttcta cacacatgtg ttccgcctga agaagtggat acagaaggtc 420 attgatcagt ttggagagta gggggccact catattctgg gctcctggaa ccaatcccgt 480 gaaagaatta tttttgtgtt tctaaaacta tggttcccaa taaaagtgac tctcagcaag 54U
cctcaatgct~cccagtgcta ttcatgggca gctctctggg ctaggaagag ccagtaatac 600 tactggataa agaagactta agaatccacc acctggtgca cgctggtagt ccgagcactc 660 gggaggctga ggtgggagga tr_gcttgagc ccaggaggtg gaggctgcag tgagccactg 720 caccccagcc tgggtgacag agtgagaccc tgtcccaaaa gaatccacta tctatcttca 780 gagcagggcc aggtgagagg aaagatggc.~ ggttgaat~tt acaggcatta aagatgttcc 840 accctctggg ttttaatgga ttatctcat~ taatcctcac aagaggtagg tgagtaaact 900 gagatttgga gaagtaeett gr_ceaaagte aeatggetaa gaaageteaa agtaggactt 960 caaatataga aaatattgag tgaggacggt gcttttttag ttaactccct acatcttccc 1020 ttgtatcatt aaaatgatat cagatcaggt agggcatggt ggctcacacc tgtaatctca 1080 gcaatt <210> 2 <211> 20 <212> DN:a <?.13> Primer <400> 2 tctagaaaca gttgcctggc 20 <210> 3 ~-211> 2?
<2:12> oN.a <213> Primer <400> 3 atagcar_tgg c3agcattgaa gc Z2 <210> 4 <211> 480 <212> DN.a <213> Homo Sapiens <400> 4 gcaaatgaaa acaattttga atatattttL tttcaggcag gaacaacacc atgatcagag 60 cagttcaacc aggggaaacc tatacttat:a agtggaacat ct~tagagttt gatgaaccca 120 cagaaaatga tgcccagtgc ttaacaagac catactacag tgacgtggac atcatgagag 180 acatcgcctc tgggctaata ggactactt~ taatctgtaa gagcagatcc ctggacaggc 240 aaggaataca ggtattttgt ccttgaagta acctttcaga aattctgaga atttcttctg 300 gctagaacat gttaggtctc ctggctaaat aatggggcat ttccttcaag agaacagtaa 360 ttgtcaagta gtccttttta gcaccagtg: gataacattt attctttttt tttttttgtc 420 ttgtctattt ttatcagtac catcactgc~= gaaggcaagt ctagagtgtg ataacatatt 480 <210> S
<211> Z1 <212> DIVA
<213> Primer <400> 5 acccacagaa aatgatgccc a 21 <210> 6 <211> 21 <212> DNA
<213> Nrimer <400> 6 tgccccatto tttagccagg a 21 <210> 7 <211> 653 <212> ~~A
<7.13> Homo Sapiens <400> i ttccatatgt tttgacacat acaaaaatcf~ ccccaagatc cttggggaac tgtattccat 60 cattagacta atccttgctg ccacttctca gtttttattt atgcaaacgg caaaatgtgt 120 tgctcaagtg ctatcacaca cagatai;atc tgtt:ctcta ttttggaatc cttgtctcaa 180 atgttactca ctttacatgc cttttcrgtt gtcttctttt tttttttttt. ctgaa.gy acc 240 ttgtaaagtc aaaaatgtca gaaacttcca ggaccgcctt tgc2aggcaga agacjcagttc 300 cacccaataa ctctaatgca gcggaagatc~ acctgcccac agtggagctt cagggcrtgg 360 tgccccgggg cgtcaacctg caaggtatg~~ gcataccccc cttccccacc actctgc3gtc 420 caggcacagc cgggccct.gg cccctcttcr ctgcaggtaa acatcctctg tctccactgg 480 ggttcccaca aaaggaagcc ccctgccaa~ ctctggttt: ataaaggaag aaagcaaaag 54 0 ctttctttta agtggtgaaa gcaccgaagi cccagagcct tgtcccagtt ctgcccctta 640 ctgaccttgt aacctcagag aagttgctt~ gtctttctgc ctctcaacta ttt 653 <Z10>
<211> 21 <212> DNA
<213> Primer <400> 8 catgcctttt ctgttgtctt c Z1 < 2 ~. 0> ~~
<211> 30 <212> DNA
<213> Primer <400> 9
is Suitable methods of analysis comprise physiochemical analysis such as enzyme assay, an immunoassay to determine th~.~ presence: of a variant gene product. or a nucleic acid ac alysis to deternline the presence of a variant gene sequence. Such methods are described in detail above.
The physiochernical analysis comprises individual parameters, each of which deter-rnines 2o the presence or absence of only an individual genetic product, such as variants of Factors 1(, ~', or X111, or other components involved it the fibrinolysis system, the clotting system, or the complement sysaetn. For c;xample, the presence of the gene product of the F?tIlIV3~L allele van be determined by reaction tvith au antibt~dy which specifically birds to the epitope comprising the valine to leucine substitution at amino arid position ~1.
l'hc, nucleic acid analysis co=uprises assessing individual parameter. each of which determines the presence or absf~nce of only an individz~al genetic element, such as FVL, j4 F lIG''U? l U:'1 or FXIII V 3~L alleles, or any other genetic element involved in the fibrinolysis system, the clotting system, or the complement system. For example, the presence of the FVL allele can be dctcrn~ined by the use of sequence-specific oligonucleotides.
s In Example 1, the FII, FV, and IrXlll genes were analyzed as candidate genes in a search for genetic elements that may contribute to elevated risk of myocardial infarction. F1I is encoded by a ?1-kb-long gene localized on chromosome 11. position l lpl l-qI?.
The gene is organized in 14 exuns, :>f which exon 1 comprises the 5' untranslated (LET) region and exon 14 comprises the 3'-~,T region. The nucleotide sequence of the FII
gene, its n3 flanking sequences as well as the position of~ the various exons has been described previously (Biochemistry 26, 616-6177, I987). 'fhe G202IOA sequence variation is loc;3ted at the last position ot~ the 3'-LET at or near the cleavage site in the mItNA
precursor to which poly A is added. Three conserved sequences in mRNA
precursors, located in the vi;.inity of this site. are required for cleavage and palyadenylation: the ~ 5 AAU.~1..~~A seauence, the nucleotide to which poly A is added, and the region downstmnm of this mtcleotide. Generally, the nucleotide to which poly A is added is an A, mostly preceded by a (:. As a consequence of the G to A transition at position 20210, a CA dinucleotide (rather than a CrA dinucleotide) has been introduced at or near the cleavage and polyadenylation site.
2o FV and FXIII are encoded by genes w.~hose nucleotide sequence, flanking sequences, and position of the Var10li3 i,'XOilS have been described previously.
Genetic analysis of the candidate genes comprises obtaining from the patient a biological sample, selectively amplifting from said sample of nucleic acid coding regions or other regulatory elements of tl-,e c;uzdidatv genes comprising the genetic aberration, and anulyza:g the sequence for the press°ncc of signature substiti.ttions indicative ot~ the gene variant of interest. Biological samples are those containing genomic DNA, cDNA, RNA, or protein obtained from the cells of a aubject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, flrle needle aspirates, anuliacentesis samples arld autopsy material.
2(l As is illustrated in e~:ample 1, :lte G''021 OA mutation in the FII gene, combined with the V3~L. mutation in the FXlll i:ene, has L~een demonstrated to be present in a group of patients exhibiting a t~isk for myocardial infarction without the cause thereof laving been previously determined. Example 1, which illustrates the general use. of the methods for the detection of mutations indicative of an elevated risk for myocardial infarction, is provided for illustrative purposes only.
The study in Example 1 shows that the presence of sequence variation G?0210A
in the FII gene, CUIIlblned with the sequence variation V34L in the FXIII gene, is a risk factor for myocardial infarction.
]() t~. ~YIEI')Ylll?l)1L; t'tS~i Of7T11'L~i:C7T11~1L1j 1)1,(I)'L't10)1 Once the genetic material frc.~m the patient's sample has been analyzed for the presence of the sequence variation in the target gene, or the biological material from the patient's sample has been analyzed for the presance of the variant product of the target gene, the risk for myocardial infarction cart be determined on the basis of the risk established in a t5 case-control study such as. for example, the Newfoundland study of Exaanple 1.
Kit~or tf:e detection o/'risk fuctars associated with m)~ocardfal i~efarctior~
Kits for the detection of risk factors associated with myocardial it>farction may be kits for the genetic, immi.:nolo~ical or physiochemical dctc:ction of risk factors associated »kith nlvocardial infarction.
2o hits fc,r the geneti:; detection: oi~ risk factors associated with myocardial infarction COfIIpI'iSt', olieonucleotides specitic to the variant region of the alleles of interest c>r to scduence flanking the: variant legion, and optionally butters. nucleotides.
enzymes such as polymerises, lit;ases or endonucleases as appropriate to the specific method of genetic analysis knows in the art. ;end other reagent useful in performing such analysis.
2~ Kits for the immtlnological detection of risk factors associated with myocardial infarction comprise primary arnibodies (monoclonal, polyclonal or purified) slaecific to the variant 2]
epitopes of the gene products of interest, and optionally buffers, membranes, secondary antibodies, and preferably labeled secondary antibodies specific to the primary antibodies, and other reagent useful in performing such analysis Kits for the physiochemical detection of risk factors associated with myocardial a infarction comprise electrophoresis buffers, centrifugation buffers, substrates specific to the variant gene products, and preferably chromogenic substrates, and other reagent useful in perfornling such analysis.
Te gain a better underst<zrrding of the invention described herein, the following examples are set forth. It should be understood that these ex~unples are for illustrative purposes Ollly. Therefore, they should not limit the scope of this invention in any way.
EXAMPLES
~:xamnle 1 Sl:'I3,~E~:TS, ~1IATERL~LS.-1~VD ufETHODS
S'uhjects:
t s 131ood samples were collected from 500 consecutive myocardial infarction patients and SUO normal controls of the genetically isolated lvewdoundland population. 'The population consists mainly of descendants of English and Irish settlers who arrived in the 18't' and 19" centuries. Tl:e geographic and social isolation of the island has ensured vend little inward migration for several hundred years, and thus has lead to a small population {30.000 individuals) with a relatively homogenous genetic background, ideal for the sW dy of complex multilactorial diseases such as myocardial infarction.
Patients categorized in the myocardial infarction group represented those presenting to the emergency department or mithin one of the Health Care Corporation of St.
John's hCSpital5 Vvlth symptorr~s and rio;;henucal evidence suggestit.e of _~.yocardial infarction.
25 Unly patients with cardiac Truponin I values greater than 2.0 yg/L (Axsym, Abbott I)iagnosticsj or greater than 0.~~ Ey~L (Access II, Beckman-Coulter Corp.) were used in this group. Control subjects were selected from consecutive individuals without prior history of myocardial iniarction or thrombosis presenting to the emergency department for trauma, accidental injun~, or other non-cardiac and non-thrombotic related events, Discarded blood samples c-ollected for complete blood count were used far DNA
extraction and analysis. Ethica approval for this study was granted by the Human Investigations O:ommittce of Memorial C.'niversity and by the Health Care Corporation of St. John's.
Genotylaiug ry~F'lIG2li~ I f~.~, Fl'G, arid F~'lllb'34L:
Cienoniic DNA was isolated fr om the peripheral blood using standard methods {Miller 1o S.A., Dykes D.D., Polensky H.P., A simple salting out procedure for extracting DNA
from human nucleated cells.Nucleir Acid Research 1988; 1 b(3 ):1'? 1 S).
Genotyping of the FVL., F1IG20210A, and 1'XIIIV3~1L vvas performed br~ PCR amplification of each of the target ;alleles frorn SeIlOIIIIC DNA followed by restriction digestion with each of corresponding enzymes alnll, flind.lll and I~del respectively, as previously described (Linfert D.R., Rezulcc \~'.N., Tsungalis G.1., Rapici multiplex analysis fbr the factor VC
Lkeiden and prothroWbin tJ20:? 1 OA mutations associated with hereditary thrombophilia.
Conn. Med. 1998: fit{9): Slr)-2~). The digested PCR products were separated by electrophoresis in 10°i° polyarrylamide gels and visualized by staining with etl-udium bromide.
2o Prevalence c~uternrinatio.=z urrcl css.~~nciation .slarclj.~:
The prevalence of each gene variant was calculated by counting the total carrier freduencv including heterozygotes and homoZ,~gotes. 'fllc allele frequencies were determined by gene cowving, f'earson C: hi Square statistical analysis was performed using SPSS x10.0 to test the association between genotypes and the prevalence of 25 nlvoeardial mfarctlon. Odds ratios (OR) were calculated as a measure of the relative risk fur myocardial infarction and were given with 95% confidence intervals.
.~IIClIyST.S f)f f;BiT(?-~BYtG' TnIC'YaLIIC)Yl:
Gene-gene interactions were detc.miined, first by comparing the prevalence of combined carrier for two of the three gene variants in patients and controls; and second by analysis of the distribution of one chosen gene variant in sub-grouped patients and controls who cal-ty another gene variant as genetic background.
RF.,St~'LTS
Geravtyping FIIG'?031<1:~, FYL, and F.klllt'3~L:
'hhe genotype distributions. c<i:-rier frequencies, and allele frequencies of FTIG2U210A, i~VL, and FXIlIV34L in both the myocardial infarction patient and the control to populations are given in Table 1: distributions of genotypes, arid carrier and allele frccvluencies of FI1G4()?10~1, FfI, and FXIIT-A V341, in MI patient and nornial control populations. (A-91: myocardial infarction, NC: Ilormai control, UR: odds ratio) The FIIG2U?l0A allele was detected in 3.2°~0 of patleIltS ~'~'171C11 ~VaS
~l~nltlCantlV higher than the 1 °~~ observed in controls (OR 3.3. 95°~'° C( 2.6-4.0; P=U.OI 5).
t _ _ _ ...~~..
_ Genotype Mt (n = lYC (n = app)-_ ~~ _-.._ P-.__ S00) _ value _~-____.__~_ {'~/,G _ 495 (99.0!0) ... _ _.__..__ ___~84 (~6.8%i FlICi20210A Cu''A 16 (3.2v) 5 (l.0io) A/A 0 ((I,' 0 (0%j j C:'arrier l~ . 3.2% ~ .0% 3.3 0.015 Alle!e F. 1 6% 0.5.fo R/R 4 7719.4%) 477 (95.4%) FVL(R506Q) l,'Q ' 3 (4.5%) 23 (4.6,~~) QiQ o (or~) 0 (o~~a~
Carrier F. 4 ti".%o 4.6.~0 l .00 !~'S
Allele: 1~. :~ 0 2.3,-0 y:'%y :'.05 (i i.0%) 261 (~2.?,'n) -x111-A v34L vu_ 103 (3s.6i) zo7 (41.4r~
LiI. 4? (8.4:0) 32 (6.4%) C.'arrier F. 4 %.0!a 47.80 0.97 NS
Alle~~ 1~. 2 ~.7.~~ 27.1,~0 ?4 An identical prevalence of FV L was observed in both patient and control populations (4.6°/° vs. 4.6%). No homozygotes for either FIIG20210A or FVL
allele were found in either population. In both patient and control populations, FXIIIV34L had similar prevalence (47.0% vs. 47.8°.'0) and allele frequency (27.7% vs. 27.1 %). The prevalence of homozygous V34I. was higher in patients compared wish controls (8.4% vs.
6.4%), but the difference did not reach statistical significance. It is to be understood that, because the statistical significance of the prevalence of a genetic element as it relates to myocardial infarction risk is dependent on the sample size, an increase in the sample size will conclusively determine said statistical significance.
1o The distribution of the three gene variations was further analyzed by sub-grouping patients and controls according to age, as shown in Tabic 2: distribution of genotypes among Ml patients with different onset ages and compared with age-matched normal controls. Myocardial intar~tion patients were divided into those with an early age of onset (less than 50 years) and those with a later age of onset (greater than SU
years).
iii yC oR p F1I 20210A C;arricrs 161500 (3.2%) 5/500 (1%) 3.3 0.015 Age <_ SOY 2/-16 (=t.3~ o) 3.373 (0.8°ro) ~.6 0.04 Age:> SOY 14:'454 ~,3.1%) 2/127 (l.6%) 2.0 NS
F1-'L Carriers 23,500 (a.6%) 23/500 (4.6%) 1.0 NS
Age < SOY ti;~(i (1~~.U°,%) 18!373 (4.8%) 3.0 0.007 Age > 'fly' 17.''454 ,'i,8°io) 51127 (3.9°io) 1.0 NS
F~XIIi 34LCarriers 235!50G 2391500 {47.8%) 1.0 NS
(47.0% ) Age. < s0Y 19146 (~~l .3%) 1761373 (-17.2%) 0.8 fJS
Age > >()'~' 2 ! ~i (:17.6%;i 6:1/127 (=19.G°,%o) O.~l NS
The control population was also divided into the two corresponding age groups.
hnctesti~igly, ii iiia~lll~lliUlilfil illa:~li3iitiVii eli ihC' ~'~'1., 3liC:li; 1'4'11: Ours;?"v2d tli illi; ~~ll'ly onset patient Group. 'fhe FVL allele was detected in 13.0% of patients v~ith early age of ?o onset, which was significantly 'tugher than the 3.8% in patients with a later age of onset (OR: 3.9, 95 % CI 3.3-4.4: P =11.004) and the 4.8 % in the age matched controls (OR: 3.0, 95 °,'o CI 2.2-~.7: Y=0.007 . The prevalence of FIIG~O:?10.A was alsc7 significantly higher iru the early onset group of myocardial infarction (4.3'%) compared to the controls under age 50 (0.8%). Although, the prevalence of FIIG20? l0A was slightly higher in the early onset group compared to the later onset group, this difference did not achieve statistical significance.. he FXII ~' 34L avowed a slight but statistically insignificant difference in prevalence beriveen the patient:c with early and later onset age.
Gene-gene Interactions:
Interaction between FXIIIV34~. and FIIG2()210A was first analyzed by comparing the f~) prevalence of combined carriers (individuals carrying both PIIG20210A and FXIIIV34L) in the total patient and control populations with their corresponding theoretical prevalence of combined carrier;
Using carrier trequencies described in Table 1. the theoretical prevalence for combined carriers of F'lIC~20210A and FhIIIV34L is 1.5% (47.0°~o x 3.2%) in myocardial infarction 2 5 patients and 0.48°'0 (47.8% ~ 1"'0) in controls (Figure la).
The observed prevalence of combined carriers in the myocardial infarction patient population (2.4°r; 12 c>l~ X00) ,.vas 1.6-find higher than its theoretic prevalence (1.5°~0), and in normal control population (0.2'%; 1 of 500) was 2.4-fold lower than its theoretic expected prevalence (0.48%). ~t~he observed prevalence of combined carriers was 12-fold 2o higher in myocardial infarction patient compared with the control population (P = 0.002).
'hhe interaction betwE:en the FII(i20210A and the FXIII~'34L was fm°ther examined by analysis of the distribution of FXIIIV34I. in sub-grouped patients and cotarols cvho carry FIIG202I0A as a genetic backl;round. Although the FXIIIV34I. showed an almost equal distribution in our myocardial infarction patient and i;untrul populations, 25 alleles ~.~~ere deteclecl in '75.0'e~ ( 12 of 16) patients with a genetic background of FIIG20?IOA ba.tt only in 20.01°,~ (1 of 5) of controls with the same genetic background (OR 3.7. 95 °1o C:I 2.4-5.1; P---0.013) (higure 2: (a) Prevalence of FYIIIV34I, in MI
?6 patients and normal controls who carry the Fiig20210A allele (b) Prevalence of FXIIIV34L in MI patients and normal controls who carry the FVL allele).
Of 13 combined carriers of FIIG20210A and FXIIIV34L identified from the studied population (500 patients and 500 controls), 12 subjects (92.3 °,~o) belonged to the myocardial infarction patient population but only 1 (0.7%) from the control population.
'the co-existence of these two gene variants imparts a strong predisposition for myocardial infarction with high penetrance.
the prevalence of combined carriers of FVL and FXI1134L were similar in both myocardial iltfarction patient (12 of 500, 2.4%) and control (9 of a00, 1.8%) groups and to was consistent with their expected t~equencies {2.1G% in patients and 2_2%
in controls;
Figure 1b). We further analyzed the prevalence of FXIIIV34L in patients and controls wlio carry the FVL allele as a genetic background. The prevalence of FXIIIV34L
was slightly higher in the sub-grouped controls (13 of 23, 56.2%) than in the sub-grouped patients (9 oC23. 39.1%) but the ditlerence was not statistically SlgIll~Cant.
t s There were no combined carriers of FIIG20210A and FVL in the myocardial infarction patient or control populations. 'This is expected considering a calculated expected frequency of combined carriers of 0.13 % in myocardial infarction patients and O.Oi % in normal controls, respectively.
Figure 1 (a) comparison of'the f,~xpected and observed prevalence of combined carrier for 2o FIIG20210A and hXtliV34L among MI patients and controls; (b) comparison of the expected and observed prevale,jco of combined c;u-rier for l'VL and FXiIIV34L
among X11 patients and Ci)lltr~lS. U1': observed prwalence: E.P: expacted prevalence(prevalence of FIICi20210A x prevalence of FXIlIV34I.).
E.~r~mple 2 s SC% l3.lL'(:' I:S. :t2-4 ? ~ RLflI.S' : I N~~ !~fE? HUMS' Blood samples were collected From 230 additional patients with MI and 79 additional nc,rrrlal controls, using tine metl-:ods and rn,aterials set out in Fxaxnple I. The cumulative summary of the results based total 730 patients with M1 and 579 normal controls are given in the Following tables and figures:
Rrsults CJenvty~ing FIICT20210r1, Ft'L and FXIll-.~4 l'3~L
The genotype distributions. carrier frequencies, and allele frequencies of FII
20210A, FVL, and E'Xlll-A L3:~ in both the IvII patient and the control populations are given in Table 3: distributions of genotypes, and carrier and allele frequencies of F11G202IOA, FVL and FXIII-;'~ V34L in hMl patient and noumal control populations. (MI:
myocardial infarction, NC: normal control, Oh:: odds ratio).
to Genotvp MI (n = NC (n ~ 579) 4R (95% CI) Y value 730) a _ -_-___._ ____.
CfIC7 7l (' (97.3%)572 (911.8%) r-lIG?0m0A c~,a 20 ~~.~%) 7 (1.2~0) :'i!.~ 0 (O';-o) 0 (0%) Carrir_r F. 2. 7'v, 1.2% 2.3U?(0.~?67, 0.053 5.48:_' ) Allele F. 1.4'ro 0.6%
R/R 693 (94.9ro) 553 (9:i.5./) FVL.(R506Q) R~Q 38 (5.2%) 26 (4.5%) 0 {Ci) 0 (0%) l'.arrier F. S.2"% 4.S% 1.16(U.69~,1.926) O.S7 i Allele F. 2.6'ro 2.3,/0 '~:'!V 391 (53.6./) 303 (52.3,/0) FX.III-A V34L: V,''L 22h 207 (:10.8ro) (31.2%) 1.~'I, ~7 i7.8,'7 3? (6.9~0) Carrier F. ?9~' :~ 4 l .3/a Q. 9-~8~~'. ? -'.~?-U. 59?
1.159 AIICI: F, 73,_l~/p 7 ~.ijr~
The F11 2021 OA allele was detected in ?.7% of patients. which was higher than the 1.2° o observed in controls (UR 2.26, 95% C:I 0.95-S.S.t), P - O.OS9). Prevalence of FVL was observed in 5.2° o patient and ~.5% control populations. No homozygotes for either the 2b FII 20210A or hVL alleles were found in either population. In both patielt and control populations, the FXIII-A L34 allele had similar prevalence (39.0°~~ vs.
41.3','%) and allele frequency (23.4°io vs. 23.4%). 'The prevalence of homozygosity for the allele was higher in patients compared with controls (7.8% vs. 6.9°~0), but the difference dil not reach statistical slgnlficance.
Vei2e-~,'C'Yle IY11C'TC1C'11t71~:
The observed prevalence of combined carrier in die MI patient population (1.92%, 14 of 730) Gvas 1.8-fold higher than it's theoretical prevalence (1.1%?, and in normal control population (0.17° ~; 1 of 79) was 2.9-fold lower than its theoretic expected prevalence to (0.5%). 'The relative risk for the combined carriers to development Ml is 11.3.
'fhe interaction between the FII 20210A and the FX111-A I,34 alleles was ftirther examined by analysia of the distribution of the FXIII-A L34 allele in sub-grouped patients and controls who carry the FII 20210A allele as a genetic background.
Although the FX11I-A L34 allele showed an almost equal distribution in otu' MI patient 1;39%j and ~ 5 control populations (;41 %). it .vas detected in 70% ( I 4 of 20) patients w7th a genetic backgrotuld of the FIl 20210A , llele but only in 14.3% (1 of 7) of controls with the same genetic background (Oft 4.9, 95°~° CI 0.8-30.8, P = 0.011 ) (Figure 4: Prevalence of the FX1II-A L.34 allele in MI patients and normal controls {NC) who carry the FII
allele {a) and FVL (b) {Ml=730. NC=579)).
2o Of 1 ~ combined carriers of the f I1 20210A and I-'XIII-A L34 alleles identified from the Studied population (730 patients and X79 controls), 14 subjects (93.3'%) had MI. The co-e~:iatence of these hvo gene variants imparts a strong predisposition For IV1I
with high penetrance.
7~Ile prevalence of cc»nbined c~u-riers of FVL. and the FXIII-A L34 allele was 1.78°io (13 25 of 730) in MI patients, urhich is lower than its expected frequency, 2.03°~0. V~lhile, the prevalence of combined c~irricrs in healthy controls was 2.42% in control groups (,14 of 79), which is higher than its exp.:;cted prevalence, 1.85°,~0 (Figure 3: Comparison between the expected and observed prevalence of combined carriers of mutations irr MI
patients and controls. (MI=73f~, T~TC= 579). {a) Comparison of the expected and observed prevalence of combined carrier for the FII 2U210A -~- FXIII-A L,34 alleles in MI patients anti control subjects. respectively. (b) Comparison between the expected and observed prevalence of combincci carrier for FVL + PXIII-A L3=1 alleles in MI patients and control subjects, respectively. Filled oar (r) represents the observed prevalence of combined carriers. Empty bar {o) represents the c?~pected prevalence of combined carriers.).
However, all of these differences did not reach statistical significance.
The prevalence of the FXIII-.~~ L34 allele in patients and controls who carry the FVL
allele as a genetic backgrrotuzd was further analyzed. ~fhe prevalence of the FXIII-A 1,34 l0 allele was higher in the sub-gr~~uped controls {14 of 26, 53.$5°%) than in the. sub-grouped patients (13 of'37, 3.14°~0) but the difference was not statistically significant.
M'here was only one combined carrier of the F1I 2U21UA allele and FVL
identified from each of the Ml patient ~=roup and control populations. 'this is expected considering a calculated expected frequency of combined carriers of 0.13% in Wi patients and 0.01'% in 1 s norntal controls, respec.ti vely.
~l'able 4: Distribution of genorypes among MI patients with different onset ages and cumpar~.d with age-matchod normal controls {NC).
1\1)( NC OR (95%CI) P
FII 20210A 201732 (2.73%)SI579 {0.86%)3.164(1.180, 0.016 Carriers 8.481) Ag,e < :>(IY 2193 (2.1''~u)3i-123 {0.71'0)3.1165(0.05,18.606)0.201 Aae > SOY 18/640 (2.81'0)2/156 (1.28%)2.194(0.504, 0.283 9.554) E~ 6Z C'arrier.s38/732 (5.19%)261579 (4.49%)1.156(0.694, 0.577 1.926) Age < 50Y 8.%c)2 (8. 2(}!423 ('1.73';%)1.8_19(0.'186, 0.1 7(~~0) =1.3(>4) ~S
AL,z > SC)5' 30640 (4.69,~)6! t 56 (3.85~0?1.2 I ~~(,0.-1<>9,(>.66~~
'?.979) FXIII 34Lcarrier5 3411732 (46.58%) 2771579 0.974(0.803, 1.180} 0.786 (47.84%) Age < SOY 43192 (:16.7a%) 1991423 0.994(0.666, 1.481 j 0.974 __ (47.o4°i°) A~e > 501' 298/640 (=16.56%) 781156 (50.0°io) 0.931(0.687, 1.263) 0.647 'Table 5: Comparison of genot.-pes distribution in different genders among NlI
patients normal controls Males Females OR (95°~o C1) P
FII Z0, l r7A
lVf1 9J353 (2.55°io} 10 /339 (2.95%) 0.864('0.347, 2.153) U.7~4 NC 5/255 (1.~?6°,~0) ?:'324 (0.G2%) 3.176(0.611, 16.507) 0.147 FVI.
Mt 261353 (7.37°~0) 10/339 (2.95%) 2.497(1.186, 5.256) 0.013 NC 10/2» (3.92%) 16/324 (4.94%) 0.794 (0.354, l.l8ll) 0.5 IS
f Xltl-A 134 ?01I353 MI , 1201339 (35.40%) 1.609 (1.227, 2.109} 0.001 (56.9=°%~i r 19/255 NC i26i'324 (38.89%) 1.200 (0.$89, i.61=ij O.G33 (46.67°'0) ~a«.
The disclosure of all patents, tmublications, including published patent applications, and database entries referenced in the specification are specii'ically mcosporat::d by reference i0 in their entirety to the same extent as if each such individual patent.
publication, and database entry were specifically and individually indicated to be incorporated by reference.
fhe invention being thus dcscribed_ it will be obvious that the same may be varied in many ways. Such varranons armor to be regarded as a departure from tile spirit and 1 ~ scope of the invention, and all such modifications as u.~ould be obvious ti> one skilled in the art are intended to be included within the scape of the following claims.
3t SEQUENCE LISTING
<110> Genesis Group .inc.
Xie, Ya-Gang <120> Method for the Detection of Risk Factors ,4ssociated with Myocardial Infarction <130> 365-127 <140> n/a <141>
<:150> 2,414,301 <151> 2002-12-13 160> 9 <170> FastSEQ for windows version 4.0 <210> 1 <211> 1086 <2:12> DNn <213> Homo Sapiens <400> 1 ctcaccagct gtgtctcgtg aaggggcgtg gctgggctat gagctatgct cctgagcaca 60 gacggctgtt ctctttcaag gttacaagc:~ tgatgaaggg aaacgagggg atgcctgtga 120 aggtgacagt gggggaccct ttgtcatgaa ggtaagcttc tctaaagccc agggcctggt 180 gaacacatct tctgggggtg gggagaaact ctagtatcta gaaacagttg cctggca.ggg 240 gaatactgat gtgaccttga acttgactc~ attggaaacc tcatctttct tcttcagagc 300 ccctttaaca accgctggta tcaaatgggc atcgtctcat ggggtgaagg ctgtgaccgg 360 gatgggaaat atggcttcta cacacatgtg ttccgcctga agaagtggat acagaaggtc 420 attgatcagt ttggagagta gggggccact catattctgg gctcctggaa ccaatcccgt 480 gaaagaatta tttttgtgtt tctaaaacta tggttcccaa taaaagtgac tctcagcaag 54U
cctcaatgct~cccagtgcta ttcatgggca gctctctggg ctaggaagag ccagtaatac 600 tactggataa agaagactta agaatccacc acctggtgca cgctggtagt ccgagcactc 660 gggaggctga ggtgggagga tr_gcttgagc ccaggaggtg gaggctgcag tgagccactg 720 caccccagcc tgggtgacag agtgagaccc tgtcccaaaa gaatccacta tctatcttca 780 gagcagggcc aggtgagagg aaagatggc.~ ggttgaat~tt acaggcatta aagatgttcc 840 accctctggg ttttaatgga ttatctcat~ taatcctcac aagaggtagg tgagtaaact 900 gagatttgga gaagtaeett gr_ceaaagte aeatggetaa gaaageteaa agtaggactt 960 caaatataga aaatattgag tgaggacggt gcttttttag ttaactccct acatcttccc 1020 ttgtatcatt aaaatgatat cagatcaggt agggcatggt ggctcacacc tgtaatctca 1080 gcaatt <210> 2 <211> 20 <212> DN:a <?.13> Primer <400> 2 tctagaaaca gttgcctggc 20 <210> 3 ~-211> 2?
<2:12> oN.a <213> Primer <400> 3 atagcar_tgg c3agcattgaa gc Z2 <210> 4 <211> 480 <212> DN.a <213> Homo Sapiens <400> 4 gcaaatgaaa acaattttga atatattttL tttcaggcag gaacaacacc atgatcagag 60 cagttcaacc aggggaaacc tatacttat:a agtggaacat ct~tagagttt gatgaaccca 120 cagaaaatga tgcccagtgc ttaacaagac catactacag tgacgtggac atcatgagag 180 acatcgcctc tgggctaata ggactactt~ taatctgtaa gagcagatcc ctggacaggc 240 aaggaataca ggtattttgt ccttgaagta acctttcaga aattctgaga atttcttctg 300 gctagaacat gttaggtctc ctggctaaat aatggggcat ttccttcaag agaacagtaa 360 ttgtcaagta gtccttttta gcaccagtg: gataacattt attctttttt tttttttgtc 420 ttgtctattt ttatcagtac catcactgc~= gaaggcaagt ctagagtgtg ataacatatt 480 <210> S
<211> Z1 <212> DIVA
<213> Primer <400> 5 acccacagaa aatgatgccc a 21 <210> 6 <211> 21 <212> DNA
<213> Nrimer <400> 6 tgccccatto tttagccagg a 21 <210> 7 <211> 653 <212> ~~A
<7.13> Homo Sapiens <400> i ttccatatgt tttgacacat acaaaaatcf~ ccccaagatc cttggggaac tgtattccat 60 cattagacta atccttgctg ccacttctca gtttttattt atgcaaacgg caaaatgtgt 120 tgctcaagtg ctatcacaca cagatai;atc tgtt:ctcta ttttggaatc cttgtctcaa 180 atgttactca ctttacatgc cttttcrgtt gtcttctttt tttttttttt. ctgaa.gy acc 240 ttgtaaagtc aaaaatgtca gaaacttcca ggaccgcctt tgc2aggcaga agacjcagttc 300 cacccaataa ctctaatgca gcggaagatc~ acctgcccac agtggagctt cagggcrtgg 360 tgccccgggg cgtcaacctg caaggtatg~~ gcataccccc cttccccacc actctgc3gtc 420 caggcacagc cgggccct.gg cccctcttcr ctgcaggtaa acatcctctg tctccactgg 480 ggttcccaca aaaggaagcc ccctgccaa~ ctctggttt: ataaaggaag aaagcaaaag 54 0 ctttctttta agtggtgaaa gcaccgaagi cccagagcct tgtcccagtt ctgcccctta 640 ctgaccttgt aacctcagag aagttgctt~ gtctttctgc ctctcaacta ttt 653 <Z10>
<211> 21 <212> DNA
<213> Primer <400> 8 catgcctttt ctgttgtctt c Z1 < 2 ~. 0> ~~
<211> 30 <212> DNA
<213> Primer <400> 9
Claims (19)
1. A method of determining a risk for myocardial infarction, or a propensity therefor in an individual comprising:
(c) obtaining a biological sample from an individual; and (d) analysing said biological sample for the presence of a variant of a gene encoding Factor II, Factor V, Factor XIII or a combination thereof.
(c) obtaining a biological sample from an individual; and (d) analysing said biological sample for the presence of a variant of a gene encoding Factor II, Factor V, Factor XIII or a combination thereof.
2. The method according to claim 1, wherein the sequence of the Factor II gene comprises SEQ ID NO:1.
3. The method according to claim 1, wherein the sequence of the Factor V gene comprises SEQ ID NO:2.
4. The method according to claim 1, wherein the sequence of the Factor XIII
gene comprises SEQ ID NO:3.
gene comprises SEQ ID NO:3.
5. The method according to claim 1, wherein said variant of Factor II is FIIG20210A.
6. The method according to claim 1, wherein said variant of Factor V is FVL.
7. The method according to claim 1, wherein said variant of Factor XIII is FXIIIV34L.
8. A method for the detection of defects in a multi-stage, multi-factorial biochemical reaction system, wherein the defects are associated with an increased risk of myocardial infarction in an individual, comprising the steps of:
e) screening for suitable patients at risk of myocardial infection, wherein said screening is conducted on the basis of a family history or individual case history;
f) obtaining a suitable biological sample from the individual;
g) determining the presence of variant genetic elements, the gene products of said variant genetic elements, or altered physiochemical activities of said gene products known to be correlated with myocardial infarction; and h) determining the risk to the individual of myocardial infarction.
e) screening for suitable patients at risk of myocardial infection, wherein said screening is conducted on the basis of a family history or individual case history;
f) obtaining a suitable biological sample from the individual;
g) determining the presence of variant genetic elements, the gene products of said variant genetic elements, or altered physiochemical activities of said gene products known to be correlated with myocardial infarction; and h) determining the risk to the individual of myocardial infarction.
9. The method as in claim 1, wherein the multistage, multifactorial biochemical reaction system is selected from the group comprising the fibrinolysis system, the clotting system, and the complement system.
10. A method for determining whether an individual is at an increased risk for myocardial infarction, comprising detecting the presence or absence of mutations in genetic elements, aberrant gene products of genetic elements or altered physiochemical activity of the gene products of genetic elements, wherein said genetic elements are correlated with an elevated risk for myocardial infarction.
11. The method according to claim 8, wherein the genetic elements known to be correlated with myocardial infarction are at least two genes selected from the group comprising the genetic elements encoding Factor II, Factor V, and Factor XIII.
12. The method according to claim 9, wherein the sequence of the factor II
gene comprises SEQ ID NO:~.
gene comprises SEQ ID NO:~.
13. The method according to claim 9, wherein the sequence of the Factor V gene comprises SEQ ID NO:2.
14. The method according to Claim 9, wherein the sequence of the Factor XIII
gene comprises SEQ ID NO:3.
gene comprises SEQ ID NO:3.
15. The method according to claim 9, wherein the presence of at least two of SEQ ID
NO:1, SEQ ID NO:2, and SEQ ID NO:3, is indicative of an increased risk for myocardial infarction in said individual.
NO:1, SEQ ID NO:2, and SEQ ID NO:3, is indicative of an increased risk for myocardial infarction in said individual.
16. A method for determining whether an individual is at an increased risk for myocardial infarction, comprising determining Factor II and Factor XIII
genetic element sequences of ar individual, whereby the presence of a G20210A mutation in a Factor II gene sequence, and the presence of a V34L mutation in a Factor XIII gene sequence is indicative of an increased risk for myocardial infarction in said individual.
genetic element sequences of ar individual, whereby the presence of a G20210A mutation in a Factor II gene sequence, and the presence of a V34L mutation in a Factor XIII gene sequence is indicative of an increased risk for myocardial infarction in said individual.
17. A method for determining whether an individual is at an increased risk for myocardial infarction, comprising determining gene products of Factor II and Factor XIII genetic elements of an individual, whereby the presence of FIIG20210A and FXIIIV34L gene products is indicative of an increased risk for myocardial infarction in said individual.
18. A method for determining whether an individual is at an increased risk for myocardial infarction, comprising determining physiochemical activity of gene products of the Factor II and Factor XIII genetic elements of an individual, whereby the presence of FIIG20210A and FXIIIV34L gene products' physiochemical activity is indicative of an increased risk for myocardial infarction in said individual.
19. A kit for determining whether an individual is at an increased risk for myocardial infarction, comprising oligonucleotides specific to the variant region of the alleles of interest or to sequence flanking the variant region and optionally instructions for use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2450902 CA2450902A1 (en) | 2002-12-13 | 2003-12-12 | Method for the detection of risk factors associated with myocardial infarction |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2414301 | 2002-12-13 | ||
| CA2,414,301 | 2002-12-13 | ||
| CA 2450902 CA2450902A1 (en) | 2002-12-13 | 2003-12-12 | Method for the detection of risk factors associated with myocardial infarction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2450902A1 true CA2450902A1 (en) | 2004-06-13 |
Family
ID=32597868
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2450902 Abandoned CA2450902A1 (en) | 2002-12-13 | 2003-12-12 | Method for the detection of risk factors associated with myocardial infarction |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2450902A1 (en) |
-
2003
- 2003-12-12 CA CA 2450902 patent/CA2450902A1/en not_active Abandoned
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