CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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.
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CA 02450902 2003-12-12
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.
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CA 02450902 2003-12-12
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
34
(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:
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CA 02450902 2003-12-12
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,
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CA 02450902 2003-12-12
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 ---
500
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).
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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
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CA 02450902 2003-12-12
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,
9
CA 02450902 2003-12-12
~~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
CA 02450902 2003-12-12
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.
,~
CA 02450902 2003-12-12
;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.
12
CA 02450902 2003-12-12
'~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
13
CA 02450902 2003-12-12
;: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
CA 02450902 2003-12-12
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~
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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]
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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.
23
CA 02450902 2003-12-12
.~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
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
(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,
FXIIIV34L
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
CA 02450902 2003-12-12
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
27
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
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
FX1II-A L34
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
20210A
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
29
CA 02450902 2003-12-12
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
CA 02450902 2003-12-12
'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
CA 02450902 2003-12-12
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
108 6
<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
1
CA 02450902 2003-12-12
<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
Image
