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Patent 2522815 Summary

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(12) Patent Application: (11) CA 2522815
(54) English Title: ANALYSIS AND USE OF PAR 1 POLYMORPHISMS FOR THE RISK ESTIMATION OF CARDIOVASCULAR DISEASES
(54) French Title: ANALYSE ET UTILISATION DE POLYMORPHISMES DE PAR 1 DANS L'EVALUATION DE RISQUES POUR DES MALADIES CARDIOVASCULAIRES
Status: Withdrawn
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/57 (2006.01)
  • C07K 14/705 (2006.01)
  • C12N 15/10 (2006.01)
  • C12N 15/12 (2006.01)
  • C12P 19/34 (2006.01)
(72) Inventors :
  • KOZIAN, DETLEF (Germany)
  • CZECH, JOERG (Germany)
  • SIEGLER, KARL-ERNST (Germany)
  • DELEUZE, JEAN-FRANCOIS (France)
  • RICARD, SYLVAIN (France)
  • MACE, SANDRINE (France)
(73) Owners :
  • SANOFI-AVENTIS DEUTSCHLAND GMBH
(71) Applicants :
  • SANOFI-AVENTIS DEUTSCHLAND GMBH (Germany)
(74) Agent: SMART & BIGGAR LP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2004-04-16
(87) Open to Public Inspection: 2004-11-04
Examination requested: 2009-04-16
Availability of licence: N/A
Dedicated to the Public: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/EP2004/004035
(87) International Publication Number: WO 2004094470
(85) National Entry: 2005-10-19

(30) Application Priority Data:
Application No. Country/Territory Date
103 18 496.1 (Germany) 2003-04-24

Abstracts

English Abstract


The invention relates to polynucleotide sequences comprising genetic
variations of PAR 1 gene on positions 3090 and/or 3329. Surprisingly, the
presence of said variants in humans correlates with specific cardiovascular
diseases. The invention also relates to methods for the detection of said
genetic variations.


French Abstract

L'invention concerne des séquences polynucléotidiques comprenant des variations génétiques du gène PAR 1 au niveau des positions 3090 et/ou 3329. Très étonnamment, l'occurrence de ces variantes chez l'homme est en corrélation avec certaines maladies cardiovasculaires. Cette invention se rapporte en outre à des procédés pour identifier ces variations génétiques.

Claims

Note: Claims are shown in the official language in which they were submitted.


28
What is claimed is:
1. An isolated polynucleotide sequence of the PAR1 gene, which
comprises a C for T substitution at position 3090 of the sequence
according to NM-001992.
2. The isolated polynucleotide sequence as claimed in claim 1, wherein
the polynucleotide sequence of the PAR1 gene encompasses a
sequence according to SEQ ID NO: 2.
3. The isolated polynucleotide sequence as claimed in claim 1, wherein
the polynucleotide sequence of the PAR1 gene comprises a
sequence according to SEQ ID NO: 2.
4. An isolated polynucleotide sequence of the PAR1 gene, which
comprises a C for A substitution at position 3329 of the sequence
according to NM-001992.
5. The isolated polynucleotide sequence as claimed in claim 4, wherein
the polynucleotide sequence of the PAR1 gene encompasses a
sequence according to SEQ ID NO: 3.
6. The isolated polynucleotide sequence as claimed in claim 4, wherein
the polynucleotide sequence of the PAR1 gene comprises a
sequence according to SEQ ID NO: 3.
7. An isolated polynucleotide sequence of the PAR1 gene, which
comprises a C for T substitution at position 3090 and a C for A
substitution at position 3329, in each case based on NM-001992.
8. The isolated polynucleotide sequence as claimed in claim 7, wherein
the polynucleotide sequence of the PAR1 gene encompasses a
sequence according to SEQ ID NO: 4.
9. The isolated polynucleotide sequence as claimed in claim 7, wherein
the polynucleotide sequence of the PAR1 gene comprises a
sequence according to SEQ ID NO: 4.

29
10. An isolated part of the polynucleotide sequence of the PAR1 gene,
comprising a sequence according to SEQ ID NO: 5.
11. An isolated part of the polynucleotide sequence of the PAR1 gene,
comprising a sequence according to SEQ ID NO: 6.
12. An isolated part of the polynucleotide sequence of the PAR1 gene,
comprising a sequence according to SEQ ID NO: 7.
13. An isolated part of the polynucleotide sequence of the PAR1 gene,
comprising a sequence according to SEQ ID NO: 8.
14. A method for preparing a polynucleotide sequence as claimed in any
of claims 1 to 9 by means of the following method steps:
a] Providing human cDNA comprising a PAR1 sequence
according to SEQ ID NO: 2 and/or a PAR1 sequence
according to SEQ ID NO: 3 and/or a PAR1 sequence
according to SEQ ID NO: 4,
b] Providing a primer pair according to SEQ ID NO: 9 and SEQ
ID NO: 10.
c] Amplifying the PAR1 polynucleotide sequence by the
polymerase chain extension reaction (PCR),
d] Isolating and/or purifying the 3.56 kb fragment obtained from
c],
e] Sequencing the fragment from d].
15. A method for preparing a polynucleotide sequence as claimed in any
of claims 10 to 13 by means of the following method steps:
a] Providing human genomic DNA comprising a PAR1 sequence
according to SEQ ID NO: 1 and/or a PAR1 sequence
according to SEQ ID NO: 2 and/or a PAR1 sequence
according to SEQ ID NO: 3 and/or a PAR1 sequence
according to SEQ ID NO: 4
b] Providing a primer pair according to SEQ ID NO: 11 and SEQ
ID NO: 12
c] Amplifying the fragment of the PAR1 polynucleotide
sequence by the polymerase chain extension reaction (PCR),
d] Isolating and/or purifying the fragment obtained from c],
e] Sequencing the fragment from d].

30
16. A method for detecting whether or not there is in a PAR1 gene a T to
C substitution at position 3090 of the sequence according to NM-
001992 and/or an A to C substitution at position 3329 of the
sequence according to NM-001992, which method comprises the
following method steps:
a] Providing biological material comprising human cells,
b] Obtaining chromosomal DNA from the material of a],
c] Amplifying a polynucleotide fragment by means of the primers
according to SEQ ID NO: 11 and SEQ ID NO: 12, using a
PCR reaction,
d] Sequencing the polynucleotide fragment from c].
17. A method for detecting whether or not there is in a PAR1 gene a T to
C substitution at position 3090 of the sequence according to NM-
001992 and/or an A to C substitution at position 3329 of the
sequence according to NM-001992, which method comprises the
following method steps:
a] Providing biological material comprising human cells,
b] Obtaining RNA from the material of a],
c] Transcribing said RNA into cDNA by means of reverse
transcriptase,
d] Possibly amplifying a polynucleotide fragment by means of
the primers according to SEQ ID NO: 10 and SEQ ID NO: 11,
using the PCR reaction,
e] Sequencing the cDNA from c] and/or the polynucleotide
fragment from d].
18. A method for detecting whether or not there is in a PAR1 gene a T to
C substitution at position 3090 of the sequence according to NM-
001992 and/or an A to C substitution at position 3329 of the
sequence according to NM-001992, which method comprises the
following method steps:
a] Providing biological material comprising human cells,
b] Obtaining chromosomal DNA from the material of a],
c] Southern blotting the chromosomal DNA from b],
d] Providing a probe according to SEQ ID NO: 5 and/or SEQ ID
NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8,

31
e] Hybridizing the Southern blot from c] with the probe from d]
under stringent hybridization conditions,
f] Determining the presence or absence of a genetic variation in
the PAR1 gene at position 3090 and/or 3329 according to
NM-001992 by comparing the results of the hybridization from
a].
19. A method for detecting, whether or not there is in a PAR1 gene a T
to C substitution at position 3090 of the sequence according to NM-
001992 and/or an A to C substitution at position 3329 of the
sequence according to NM-001992, which method comprises the
following method steps:
a] Providing biological material comprising human cells,
b] Obtaining RNA from the material of a],
c] Providing a probe according to SEQ ID NO: 5 and/or SEQ ID
NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8,
e] Hybridizing the Northern blot form c] with the probe from d]
under stringent hybridization conditions,
f] Determining the presence or absence of a genetic variation in
the PAR1 gene at position 3090 and/or 3329 according to
NM-001992 by comparing the results of the hybridization.
20. An isolated polynucleotide sequence having from 21 to 50
nucleotides, which comprises a sequence according to SEQ ID NO:
11.
21. An isolated polynucleotide sequence comprising SEQ ID NO: 11.
22. An isolated polynucleotide sequence having from 21 to 50
nucleotides, which comprises a sequence according to SEQ ID NO:
12.
23. An isolated polynucleotide sequence comprising SEQ ID NO: 12.
24. The use of an isolated polynucleotide sequence as claimed in claim
20 or 21 in combination with an isolated polynucleotide sequence as
claimed in claim 22 or 23 for amplifying a fragment of the PAR1
gene by means of the PCR reaction.

32
25. The use as claimed in 24, wherein the PAR1 gene has a T to C
substitution at position 3090 of the sequence according to NM-
001992 and/or an A to C substitution at position 3329 of the
sequence according to NM-001992.
26. A kit of parts, comprising
a] a polynucleotide sequence as claimed in claim 20 or 21,
b] a polynucleotide sequence as claimed in claim 22 or 23,
c] at least one enzyme for carrying out the PCR reaction
d] and possibly substances and/or solutions for carrying out the
polymerase chain extension reaction (PCR),
e] and possibly furthermore polynucleotide sequences as
claimed in one or more of claims 1 to 17
f] and possibly reagents for carrying out a sequencing.
27. A method for preparing the kit of parts as claimed in claim 26, which
method comprises
a] preparing a polynucleotide sequence as claimed in claim 20
or 21,
b] providing a polynucleotide sequence as claimed in claim 22
or 23,
c] providing an enzyme for carrying out the PCR reaction,
d] providing, where appropriate, reagents for carrying out a
sequencing
e] possibly providing substances and/or solutions for carrying
out the polymerase chain extension reaction (PCR),
f] possibly providing polynucleotide sequences as claimed in
one or more of claims 1 to 13,
g] introducing the components from a] to e] in each case
separately into suitable containers,
h] combining, where appropriate, the containers from g] in one
or more pack-units.
28. The use of the kit of parts as claimed in claim 26 or 27 for amplifying
a fragment of the PAR1 gene and possibly for the further analysis,
as to whether genetic variations are present in the PAR1 gene at
positions 3090 and/or 3329 according to NM-001992.

Description

Note: Descriptions are shown in the official language in which they were submitted.


CA 02522815 2005-10-19
WO 2004/094470 PCT/EP2004/004035
Description
Analysis and use of PAR1 polymorphisms for evaluating the risk of
cardiovascular disorders.
The invention relates to polynucleotide sequences comprising genetic
variations of the PAR1 gene at positions 3090 and/or 3329.
The protease-activated receptor 1 (PAR1) is a thrombin receptor which
belongs to the class of G protein-coupled receptors (GCPR). The gene for
PAR1 is located on chromosome 5q13, consists of two exons and covers a
region of approx. 27 kb. PAR1 is expressed in, inter alia, endothelial cells,
smooth muscles cells, fibroblasts, neurons and human platelets. In
platelets, PAR1 is an important signal transduction receptor which is
involved in the initiation of platelet aggregation.
PARs are activated via proteolytic removal of a part of the N terminus of
said PARs, whereby a new N-terminal sequence is exposed which then
activates the receptor.
PAR1 and PAR4 play a central part in the activation of platelets; the
activation of these receptors in platelets leads to morphological changes,
release of ADP and aggregation of said platelets.
A connection of coronary heart diseases with single nucleotide
polymorphisms (SNP) in the promoter region of PAR1 in a group of Korean
patients was not confirmed. In another study, a PAR1 promoter variant was
shown to have a protective action for the development of venous
thromboembolisms.
The sequence of the human PAR1 gene is known. The polynucleotide
sequence of this gene can be accessed under the number NM-001992 at
the NCB/ nucleotide database. Likewise, the protein sequence is available
under the number NP-001983 at the NCB/ protein database. NCB/ is the
National Center for Biotechnology Information (postal address: National
Center for Biotechnology Information, National Library of Medicine, Building
38A, Bethesda, MD 20894, USA; Web address: www.ncbi.nhm.nih.gov).
The cloning of the PAR1 gene has been described, inter alia, in "Schmidt et
al., J. Biol. Chem. 271, 9307-9312, 1996".

CA 02522815 2005-10-19
2
There are various new polymorphisms of the PAR1 gene, by means of
which it is possible to determine a relatively strong disposition of an
individual for coronary heart diseases. The affected individuals are thus
enabled to counteract this risk factor in time by adapting their life style
accordingly, for example by compensating via increased control of other
damaging influences such as smoking, alcohol consumption, cholesterol-
rich food, high blood pressure etc.
Such health-related preventive mechanisms would not be possible without
knowledge of the PAR1 polymorphisms which are explained in more detail
below and the use thereof in corresponding methods.
Variants of a particular nucleotide sequence with substitutions at individual
positions are known to the skilled worker under the term SNP (_ single
nucleotide polymorphism).
The invention relates to an isolated polynucleotide sequence of the PAR1
gene, which comprises a C for T substitution at position 3090 of the PAR1
sequence according to NM-001992 which, as prior art, is publicly available.
In a preferred embodiment, the polynucleotide sequence of the PAR1 gene
having a T to C substitution at position 3090 encompasses a sequence
according to SEQ ID NO: 2 and, in a particularly preferred embodiment of
said polynucleotide sequence, the latter comprises a sequence of SEQ ID
NO: 2.
The invention furthermore relates to an isolated polynucleotide sequence of
the PAR1 gene, which comprises an C for A substitution at position 3329 of
the PAR1 sequence according to NM-001992 which, as prior art, is publicly
available. In a preferred embodiment, the polynucleotide sequence of the
PAR1 gene having an A to C substitution at position 3329 encompasses a
sequence according to SEQ ID NO: 3 and, in a particularly preferred
embodiment of said polynucleotide sequence, the latter comprises a
sequence of SEQ ID NO: 3.
The invention also relates to an isolated polynucleotide sequence of the
PAR1 gene, which comprises a C for T substitution at position 3090 of the
PAR1 sequence according to NM-001992 and, simultaneously, a V for A
substitution at position 3329 of said PAR1 sequence. In a preferred
embodiment, the polynucleotide sequence of the PAR1 gene having a T to

CA 02522815 2005-10-19
3
C substitution at position 3090 and a simultaneous A to C substitution at
position 3329 encompasses a sequence according to SEQ ID NO: 4 and, in
a particularly preferred embodiment of said polynucleotide sequence, the
latter comprises a sequence of SEQ ID NO; 4.
The invention also relates to an isolated part of the polynucleotide
sequence of the PAR1 gene, which comprises a sequence according to
SEQ ID NO: 5.
The invention also relates to an isolated part of the polynucleotide
sequence of the PAR1 gene, which sequence comprises a C for T
substitution at position 3090, based on the PAR1 sequence according to
NM-001992, which part comprises a sequence according to SEQ ID NO: 6.
The invention also relates to an isolated part of the polynucleotide
sequence of the PAR1 gene, which sequence comprises a C for A
substitution at position 3329, based on the PAR1 sequence according to
NM-001992, which part comprises a sequence according to SEQ ID NO: 7.
The im. :o relates to an isolated part of the polynucleotide
sequenL PAR1 gene, which sequence comprises a C for T
substitution; ~ition 3090, based on the PAR1 sequence according to
NM-001992, and simultaneously a C for A substitution at position 3329 of
said PAR1 sequence, which part comprises a sequence according to SEQ
ID NO: 8.
The invention furthermore comprises the preparation of a 3592 base pair
polynucleotide sequence of the PAR1 cDNA gene, which sequence may or
may not comprise the polymorphisms at positions 3090 and 3329, as
defined above, individually or in combination, which preparation comprises
the following method steps:
a] Providing human DNA comprising a PAR1 sequence according to
SEQ ID NO: 2 and/or a PAR1 sequence according to SEQ ID NO: 3
and/or a PAR1 sequence according to SEQ ID N0: 4,
b] Providing a primer pair having a sequence according to SEQ ID NO:
9 and SEQ ID NO: 10 .
c] Amplifying the PAR1 polynucleotide sequence by the polymerase
chain extension reaction (PCR),
d] Isolating and/or purifying the 3.56 kb fragment obtained from c],

CA 02522815 2005-10-19
4
e] Sequencing the fragment from d].
The invention also relates to the preparation of a polynucleotide sequence
according to SEQ ID NO: 5, SEQ ID N0: 6, SEQ ID NO: 7 or SEQ ID NO:
8, which preparation comprises the following method steps:
a] Providing human genomic DNA comprising a PAR1 sequence
according to SEQ ID NO: 1 and/or a PAR1 sequence according to
SEQ ID NO: 2 and/or a PAR1 sequence according to SEQ ID NO: 3
and/or a PAR1 sequence according to SEQ ID NO: 4
b] Providing a primer pair according to SEQ ID NO: 11 and SEQ ID
NO: 12
c] Amplifying the fragment of the PAR1 polynucleotide sequence by
the polymerase chain extension reaction (PCR),
d] Isolating and/or purifying the fragment obtained from c],
e] Sequencing the fragment from d].
The invention furthermore relates to a method for detecting whether or not
there is in a PAR1 gene a T to C substitution at position 3090 of the
sequence according to NM-001992 and/or an A to C substitution at position
3329 of the sequence according to NM-001992, which method comprises
the following method steps:
a] Providing biological material comprising human cells,
b] Obtaining chromosomal DNA from the material of a],
c] Amplifying a polynucleotide fragment by means of the primers
according to SEQ ID NO: 11 and SEQ ID NO: 12, using a PCR
reaction,
d] Sequencing the polynucleotide fragment from c].
The invention furthermore relates to a method for detecting, whether or not
there is in a PAR1 gene a T to C substitution at position 3090 of the
sequence according to NM-001992 and/or an A to C substitution at position
3329 of the sequence according to NM-001992, which method comprises
the following method steps:
a] Providing biological material comprising human cells,
b] Obtaining RNA from the material of a],
c] Transcribing said RNA to cDNA by means of reverse transcriptase,
d] Possibly amplifying a polynucleotide fragment by means of the
primers according to SEQ ID NO: 10 and SEQ ID NO: 11, using said
PCR reaction,

CA 02522815 2005-10-19
e] Sequencing the cDNA from c] and/or the polynucleotide fragment
from d].
The invention also relates to a method for detecting whether or not there is
5 in a PAR1 gene a T to C substitution at position 3090 of the sequence
according to NM-001992 and/or an A to C substitution at position 3329 of
the sequence according to NM-001992, which method comprises the
following method steps:
a] Providing biological material comprising human cells,
b] Obtaining chromosomal DNA from the material of a],
c] Southern blotting the chromosomal DNA from b],
d] Providing a probe according to SEQ ID NO: 5 and/or SEQ ID NO: 6
and/or SEQ ID NO: 7 and/or SEQ ID NO: 8,
e] Hybridizing the Southern blot from c] with the probe form d] under
stringent hybridization conditions,
f] Determining the presence or absence of a genetic variation in the
PAR1 gene at position 3090 and/or 3329 according to NM-001992
by comparing the results of the hybridization from a].
The invention furthermore relates to a method for detecting whether or not
there is in a PAR1 gene a T to C substitution at position 3090 of the
sequence according to NM-001992 and/or an A to C substitution at position
3329 of the sequence according to NM-001992, which method comprises
the following method steps:
a] Providing biological material comprising human cells,
b] Obtaining RNA from the material of a],
c] Northern blotting the RNA from b],
d] Providing a probe according to SEQ ID NO: 5 and/or SEQ ID NO: 6
and/or SEQ ID NO: 7 and/or SEQ ID NO: 8,
e] Hybridizing the Northern blot form c] with the probe from d] under
stringent hybridization conditions,
f] Determining the presence or absence of a genetic variation in the
PAR1 gene at position 3090 and/or 3329 according to NM-001992
by comparing the results of the hybridization.
Detection of the genetic variations or polymorphisms in the PAR1 gene at
positions 3090 and/or 3329 may be used as (a) genetic marker for
evaluating the risk of atrial fibrillation, acute coronary syndrome,
cardiomyopathy and/or unstable angina, as (b) marker for preventive

CA 02522815 2005-10-19
6
treatment for atrial fibrillation, acute coronary syndrome, cardiomyopathy
and/or stable angina of the carriers of the corresponding genetic variants,
as (c) marker for adjusting the dose of a pharmaceutically active substance
to be administered for atrial fibrillation, acute coronary syndrome,
cardiomyopathy and/or unstable angina, as (d) marker for determining the
high throughput-screening strategy for identifying a pharmaceutically active
substance for atrial fibrillation, acute coronary syndrome, cardiomyopathy
andlor unstable angina, as (e) marker for identifying the relevant individuals
or patients for clinic studies in order to test the tolerability, safety and
efficacy of a pharmaceutical substance for atrial fibrillation, acute coronary
syndrome, cardiomyopathy and/or unstable angina, and as (f) basis for
developing assays systems for analyzing the genetic variation in the PAR1
gene at the DNA, RNA or protein level.
The invention also relates to an isolated polynucleotide sequence having
from 21 to 50 nucleotides, which comprises a sequence according to SEQ
ID NO: 11. Said sequence preferably comprises SEQ ID NO: 11. The
invention furthermore relates to an isolated polynucleotide sequence
having from 20 to 50 nucleotides, which comprises a sequence according
to SEQ ID NO: 12. Said sequence preferably comprises SEQ ID NO: 12.
The invention also relates to the use of an isolated polynucleotide
sequence having from 21 to 50 nucleotides, which encompasses or
comprises a sequence according to SEQ ID NO: 11, in combination with an
isolated polynucleotide sequence having from 20 to 50 nucleotides, which
encompasses or comprises a sequence according to SEQ ID NO: 12, for
amplifying a corresponding fragment of the PAR1 gene by means of the
polymerase chain extension reaction (PCR). This use preferably relates to
the amplification of a fragment of a PAR1 gene having a T to C substitution
at position 3090 of the sequence according to NM-001992 and/or having an
A to C substitution at position 3329 of the sequence according to NM-
001992.
Moreover, the invention comprises a kit of parts which comprises
a] an isolated polynucleotide sequence of from 21 to 50 nucleotides in
length, which encompasses or comprises a sequence according to
SEQ ID NO: 11,
b] an isolated polynucleotide sequence of from 20 to 50 nucleotides in
length, which encompasses or comprises a sequence according to

CA 02522815 2005-10-19
7
SEQ ID NO: 12,
c] at least one enzyme for carrying out the polymerase chain extension
reaction (PCR),
d] possibly substances and/or solutions for carrying out the polymerase
chain extension reaction,
e] possibly polynucleotide sequences encompassing the PAR1 gene
with or without substitution at position 3090 of the PAR1 sequence
according to NM-001992 and/or position 3329 according to
NM-001992 in full length and/or parts thereof
f] and possibly reagents for carrying out the sequencing reaction.
Kit of parts here and below means the combination of said components
which have been combined into a functional unit in spatial juxtaposition to
each other.
The invention furthermore relates to the preparation of the above-described
kit of parts, which comprises
a] providing an isolated polynucleotide sequence of from 21 to 50
nucleotides in length, which encompasses or comprises a sequence
according to SEQ ID NO: 11,
b] providing an isolated polynucleotide sequence of from 20 to 50
nucleotides in length, which encompasses or comprises a sequence
according to SEQ ID NO: 12,
c] providing an enzyme for carrying out the polymerase chain
extension reaction (PCR),
d] providing, where appropriate, reagents for carrying out a sequencing
e] possibly providing substances and/or solutions for carrying out said
polymerase chain extension reaction (PCR)
f] possibly providing polynucleotide sequences comprising the PAR1
gene with or without a T to C substitution at position 3090 of the
PAR1 sequence according to NM-001992 and/or an A to C
substitution at position 3329 according to NM-001992, in each case
in the full length, or parts thereof,
g] introducing the components from a] to f] in each case separately into
suitable containers,
h] combining, where appropriate, the containers from g] in one or more
pack units.
The above-described kit of parts may be used for amplifying a fragment of

CA 02522815 2005-10-19
the PAR1 gene.
The technical aspects of the invention are discussed in more detail in the
following embodiments.
Isolated polynucleotide sequences of the PAR1 gene may be prepared, for
example, by amplification by means of the polymerise chain extension
reaction (PCR). Suitable primers for this purpose are described in
SEQ ID NO: 9 and SEQ ID NO: 10.
The PCR is an in-vitro technique which may be used to selectively
duplicate polynucleotide sections which are flanked by two known
sequences. Amplification requires short, single-stranded DNA molecules
which are complementary to the ends of a defined sequence of a DNA or
RNA template (primers). A DNA polymerise extends the primers, under the
correct reaction conditions and in the presence of deoxynucleotide
triphosphates (dNTPs), along the single-stranded and denatured
polynucleotide template and thus synthesizes new DNA strands whose
sequence is complementary to said template. During this process, the
temperature is changed at regular intervals so that, time after time, the
polynucleotide strands are denatured and the primers can be attached and
extended. Heat-stable DNA polymerises, for example Taq polymerise, are
used. A typical PCR reaction mixture contains, apart from a polynucleotide
template, two suitable primer nucleotides, for example at concentrations
between 0.2 to 2 ~M, furthermore dNTPs, for example at concentrations of
200 pM per dNPT, furthermore MgCl2 having a concentration of 1 - 2 mM,
and 1 -10 units of a heat-stable DNA polymerise such as, for example,
Taq polymerise (Thermus aquaticus polymerise). Heat-stable DNA
polymerise and the components for carrying out the same, and also
protocols, are commercially supplied by numerous companies such as, for
example, Roche Diagnostics, Clontech, Life Technologies, New England
Biolabs, Promega, Stratagene, etc.
The polynucleotide template for amplifying the polynucleotide sequence to
be isolated may be present in the form of RNA or DNA. If the
polynucleotide template is RNA, then the latter is transcribed to DNA by
means of reverse transcriptase, prior to the actual PCR reaction. The
amount of polynucleotide template for carrying out the PCR reaction may
be from 0.01 to 20 ng, for example.

CA 02522815 2005-10-19
9
The polynucleotide template is obtained using techniques known to the
skilled worker for obtaining DNA and/or RNA from biological material.
Biological material should include here, inter alia, the cells of a tissue or
organ (e.g. brain, blood, liver, spleen, kidney, heart, blood vessels) of a
vertebrate, including humans, or cells from a eukaryotic cell culture (e.g.
Hela cells, CHO cells, 3T3 cells) or cells comprising bacteria or yeasts in
which the DNA sequence to be isolated is present in cloned form.
Cells of a tissue assemblage or organ of a vertebrate, including humans,
may be obtained by taking blood, tissue puncture or surgical techniques. A
polynucleotide template may be obtained therefrom, for example, by
disrupting the cells, possibly concentrating individual organelles, in
particular the nucleus, and recovering the DNA or RNA by precipitation and
centrifugation.
Another method for preparing isolated polynucleotide sequences of the
PAR1 gene comprises cloning the PAR1 gene, subsequently expressing it
in bacteria or yeast and purifying the expressed polynucleotide. The
previously mentioned PCR reaction, for example, is suitable for preparing a
polynucleotide fragment which is clonable. It is advantageous to use, for a
fragment to be cloned, primers which carry the recognition sequence of a
reaction enzyme 5' of the complementary sequence. The two primers may
use in each case the same or different recognition sequences for restriction
enzymes.
Examples of common restriction enzymes are: BamHl (GGATCC), Clal
(ATCGAT), EcoRl (GAATTC), EcoRV (GATATC), Hindlll (AAGCTT) Ncol
(CCATGG) Sall (GTCGAC), Xbal (TCTAG1 ).
For cloning, a vector is treated with the restriction enzymes which
correspond to the recognition sequences attached to the primers. The
fragment is connected to the vector by means of ligase by isolation and
treatment with the same restriction enzymes. Vector means a DNA
molecule such as, for example, a plasmid, bacteriophage or a cosmid, with
the aid of which it is possible to clone genes or other DNA sequences and
to introduce them into a bacterial or eukaryotic cell for replication.
Examples of vectors are DNA molecules such as pBR322, pUC18/19,
pBluescript, pcDNA3.1. Vectors are commercially available from specialist
companies for biotechnological material, such as Roche Diagnostics, New
England Biolabs, Promega, Stratagene etc.

CA 02522815 2005-10-19
The instructions required for carrying out the PCR reaction, for providing
polynucleotides or for carrying out cloning procedures can be found by the
skilled worker in the form of recipes and protocols in standard manuals
5 such as, for example, in a] "Current Protocols in Molecular Biology by
Frederick M. Ausubel (Editor), Roger Brent (Editor), Robert E. Kingston
(Editor), David D. Moore (Editor), J. G. Seidman (Editor), Kevin Struhl
(Editor), loose leaf edition, continuously updated, John Wiley & Sons, Inc.,
New York or in b] Short Protocols in Molecular Biology, 5th edition, by
10 Frederick M. Ausubel (Editor), Roger Brent (Editor), Robert E. Kingston
(Editor), David D. Moore (Editor), J. G. Seidman (Editor); John A. Smith
(Editor), Kevin Struhl (Editor), October 2002, John Wiley & Sons, Inc., New
York" or in c] "Molecular Cloning by J. Sambrock, E. F. Fritsch, T. Maniatis;
Cold Spring Harbor Laboratory Press".
Suitable primer sequences are provided, for example, via chemical
synthesis thereof which may be carried out commercially to order by
companies such as MWG Biotech, etc.
Human cDNA from different organs is commercially available from
companies such as, for example, Promega, Stratagene or others.
The sequencing of a polynucleotide is carried out by means of routine
methods known to the skilled worker by using, for example, laboratory
robots from companies such as, for example, Life Technologies, Applied
Biosystems, BioRad or others.
Isolated polynucleotide sequences of the PAR1 variant and fragments
therefrom may also be used for hybridization at different stringencies.
Stringency describes reaction conditions which influence the specificity of
hybridization or attachment of two single-stranded nucleic acid molecules.
The stringency and thus also specificity of a reaction can be increased by
increasing the temperature and lowering the ionic strength. Low stringency
conditions are present, for example, if the hybridization is carried out at
room temperature in 2 x SSC solution. High stringency conditions are
present, for example, if hybridization is carried out at 68°C in
0.1 x SSC/0.1 % SDS solution.
Hybridization under stringent hybridization conditions in accordance with

CA 02522815 2005-10-19
11
the present application means:
1] Hybridizing the labeled probe with the sample to be studied at 65°C
(or, in the case of oligonucleotides, 5°C below the melting
temperature) overnight in 50 mM Tris pH 7.5, 1 NaCI, 1 % SDS, 10%
dextran sulfate, 0.5 mglml denatured salmon sperm DNA.
2] Washing at room temperature in 2 x SSC for 10 min.
3] Washing at 65°C (or, in the case of oligonucleotides, 5°C
below the
melting temperature) in 1 x SSC/1 % SDS for 30 min.
4] Washing at 65°C (or, in the case of oligonucleotides, 5°C
below the
melting temperature) in 0.2 x SSC/0.1 % SDS for 30 min.
5] Washing at 65°C (or, in the case of oligonucleotides, 5°C
below the
melting temperature) in 0.1 % SSC/0.1 % SDS for 30 min.
DNA fragments of 20 nucleotides in overall length are to be regarded as
being oligonucleotides for this purpose. The melting temperature results
from the formula Tm = 2 (number of A+T) + 4 (number of G+C)C°.
A 2 x SSC or 0.1 x SSC solution is prepared by diluting a 20 x SSC solution
accordingly. The 20 x SSC solution comprises a 3M NaCI/0.3 sodium
citrate 2 H20 solution. SDS is sodium dodecyl sulfate.
The hybridization is carried out by transferring the polynucleotides to be
studied to a nylon or nitrocellulose membrane (Southern blot - DNA;
Northern blot - RNA), after electrophoretic fractionation and subsequent
denaturation. The hybridization is carried out using a probe which is radio-
labeled or has been labeled in another way, for example with the aid of
fluorescent dyes. The probe comprises a usually single-stranded and/or
denatured DNA or RNA polynucleotide sequence which binds to the
complementary nucleotide sequence of the once again single-stranded
and/or denatured DNA or RNA polynucleotide sequence to be studied.
Single nucleotide polymorphisms of the PAR1 gene may be detected with
the aid of the primers of the invention, also by SSCP analysis. SSCP
stands for Single Stranded Conformation Polymorphism which is an
electrophoretic technique for identifying individual base pair substitutions.
The polynucleotides to be studied are amplified by PCR by means of
labeled primers and, after denaturation into single strands, fractionated in a
polyacrylamide gel electrophoresis (PAGE). If the DNA fragments to be

CA 02522815 2005-10-19
12
studied exhibit individual base pair substitutions, they then possess
different conformations and thus migrate in the PAGE at different rates.
Examples of substances for carrying out the PCR are buffers such as
Hepes or Tris, furthermore dAPP, dGTP, dTTP, dCTP, and Mg2+ and
possibly further divalent or monovalent irons. Solutions contain these
substances in dissolved form.
Examples
Amplification of genomic regions of the PAR1 gene
The T to C nucleotide substitution at position 3090 and the A to C
substitution at position 3329 in the PAR1 sequence were detected using
the following primers:
Primer 1: 5'-ACAGAGTGGAATAAGACAGAG-3' (SEQ ID NO: 11)
Primer 2: . 5'-CCAGTGCTAGCTTCTACTTAC-3 (SEQ ID NO: 12)
Primer 1 (SEQ ID NO: 11) corresponds to positions 2767 to 2789 of the
NM-001992 reference sequence. Primer 2 is derived from Exon No. 1 of
the PAR1 gene.
PCR protocol for the amplification:
The reagents used are from Applied Biosystems (Foster City, USA):
20 ng of genomic DNA; 1 unit of TaqGold DNA polymerase; 1 x Taq
polymerase buffer; 500 ~M of dNTPs; 2.5 mM MgCl2: 200 nM of each
amplification primer pair; H20 to 5 ~,I.
PCR amplification program for the genotyping
95°C for 10 min x 1 cycle
95°C for 30 sec
70°C for 30 sec x 2 cycles
95°C for 30 sec
65°C for 30 sec x 2 cycles;

CA 02522815 2005-10-19
13
95°C for 30 sec
60°C for 30 sec x 2 cycles;
95°C for 30 sec
56°C for 30 sec
72°C for 30 sec x 40 cycles;
72°C for 10 min
4°C for 30 sec x 1 cycle;
Identification of SNPs
Protocol for the minisequencing and detection of the SNPs.
All reagents are from Applied Biosystems (Foster City, USA). 2 p1 of
purified PCR product, 1.5 ~L of BigDye Terminator Kit, 200 nM sequencing
primer; H20 to 10 p1.
Amplification program for the sequencing:
96°C for 2 min x 1 cycle
96°C for 10 sec
55°C for 10 sec
65°C for 4 min x 30 cycles
72°C for 7 min
4°C for 30 sec x 1 cycle;
Analysis of the sequencing products:
The sequences were first analyzed using the sequence analysis software
(Applied Biosystems, Foster City, USA) to obtain the raw data, then
processed using Phred, Phrap, Polyphred and Consed. Phred, Phrap,
Polyphred and Consed are software written by Phil Green at Washington
University
(http://www.genome.washington.edu).
Assigning PAR1 SNPs to coronary disorders

CA 02522815 2005-10-19
14
In a clinical study, two PAR1 polymorphisms from the 3'-noncoding region
of the gene were studied for a connection with thrombotic and
cardiovascular complications in a cohort of patients.
The following abbreviations are used below (all positions indicated refer to
the nucleotide positions in the reference sequence NM-001992).
PAR1 T3090T describes the group of individuals whose alleles of the PAR1
gene both have a thymidine (T) at position 3090. These individuals are
homozygous with respect to this PAR1 variant.
PAR1 T3090C describes the group of individuals whose one allele of the
PAR1 gene has a cytidine (C) at position 3090 and whose other allele of
the PAR1 gene has a thymidine (T) at position 3090. These individuals are
heterozygous with respect to this PAR1 variant.
PAR1 C3090C describes the group of individuals whose alleles of the
PAR1 gene both have a cytidine (C) at position 3090. These individuals are
homozygous with respect to this PAR1 variant.
PAR1 A3329A describes the group of individuals whose alleles of the
PAR1 gene both have an adenosine (A) at position 3329. These individuals
are homozygous with respect to this PAR1 variant.
PAR1 A3329C describes the group of individuals whose one allele of the
PAR1 gene has a cytidine (C) at position 3329 and whose other allele of
the PAR1 gene has an adenosine (A) at position 3329. These individuals
are heterozygous with respect to this PAR1 variant.
PAR1 C3329C describes the group of individuals whose alleles of the
PAR1 gene both have a cytidine (C) at position 3329. These individuals are
homozygous with respect to this PAR1 variant.
In the group of patients analyzed (Fig. 1), statistically significant
associations of the homozygous carriers of the PAR1 variant C3090C with
atrial fibrillation and cardiomyopathy were observed. After carrying out a
logistic regression, a 1.97 fold increased risk of atrial fibrillation and a
1.84
fold increased risk of cardiomyopathy were found in homozygous carriers
of the PAR1 variant C3090C compared to carriers of the PAR1 variants
T3090/T3090T (Fig. 3).
It was shown that, for carriers of the PAR1 variant C3329C, said variant is
associated with a 2.35 fold increased risk of atrial fibrillation compared to
carriers of the PAR1 variants C3329A/A3329A. In carriers of the PAR1
variant C3329C, said variant seems, in addition, to be protective with

CA 02522815 2005-10-19
respect to the appearance of acute coronary syndrome and unstable
angina. Carriers of the PAR1 variant C3329C have a 2.78 fold reduced risk
of the appearance of acute coronary syndrome and/or unstable angina
compared to carriers of the PAR1 variants A3329C/A3329A (Fig. 4).
5
It is therefore possible, by means of a method of the invention and using an
isolated PAR1 sequence of the particular SNP type or a fragment thereof,
to determine for human individuals whether there is as assignment a risk
group in accordance with the results presented.
Preparation of plasmid DNA
1 ml of a bacterial overnight culture is transferred to an Eppendorf tube and
centrifuged (5 000 rpm for 5 min) in a Heraeus Biofuge. The bacterial cell
pellet is to be resuspended in 100 ~I of cooled solution I and then to be
placed on ice for 5 min.
Solution I: 25 mM tris-HCI, pH 8.0, 50 mM glucose (sterile-filtered)
10 mM EDTA 100 ~g/ml Rnase A.
After addition of 200 p1 of solution II, the entire mixture is mixed well,
resulting in alkaline denaturation of the DNA.
Solution I I: 200 mM NaOH, 1 % SDS.
After subsequent incubation for 5 min on ice, 150 p.1 of solution III are
added to the mixture. This is followed by mixing once more and incubating
on ice for a further 15 min.
Solution III: 3 M sodium acetate (pH 4.8).
Centrifugation in the Heraeus Biofuge at 12 000 rpm for 15 minutes
removes the cell debris, the genomic DNA and the denatured proteins. The
supernatant produced, which contains the plasmid DNA, is decanted into a
second Eppendorf tube and admixed with 1 ml of 96% strength EtOH (or
300 ~,I of isopropanol). The precipitation mixture is mixed thoroughly and
again centrifuged (15 min at 12 000 rpm in Heraeus Biofuge). This results
in precipitation of the plasmid DNA. The plasmid DNA sediment is washed
with ice-cold 70% strength EtOH and then dried in air. Finally, the dry

CA 02522815 2005-10-19
16
sediment is taken up in 50 ~,I of sterile distilled water.
Alcohol precipitation of DNA
Precipitation mixture: DNA solution, 1/10 volume of 3 M sodium
acetate (pH 5.4), 2 to 3 volumes of 96% EtOH (1 volume of isopropanol).
The mixture is mixed well and can be stored at -20°C, although
this does
not increase the precipitation yield. The plasmid DNA is sedimented by
centrifugation at 12 000 rpm for 20 minutes.
In order to remove residues of the sodium acetate used, the plasmid DNA
must be washed once more with 1 ml of 70% strength EtOH after
precipitation.
Phenol extraction of DNA
A DNA solution is admixed with the same volume of phenol (Rotiphenol~,
equilibrated with TE buffer, pH 7.6, Roth, Karlsruhe, Germany), shaken for
5 min and centrifuged at 5000 rpm. Most of the now denatured proteins
accumulate in the interface. The upper, aqueous phase contains the DNA
and is carefully removed by suction, and then mixed with a
chloroform/isoamyl alcohol mixture (24:1) in order to remove phenol
residues. This is followed by another centrifugation, after which the
aqueous supernatant is removed and the DNA is isolated from the solution
by alcohol precipitation.
Purification of amplified DNA molecules
DNA amplicons are purified using a PCR purification kit (Qiagen). This
removes the starter molecules, nucleotides (dNTPs), polymerases and
salts. For this purpose, the PCR reaction mixture is admixed with five times
the volume of PB buffer, mixed well and applied to the Qiaquick column.
The amplified DNA is then selectively bound to the column material, and
the dNPTs are removed by washing twice with 750 p.1 of PE buffer. The
amplified DNA is then eluted with the desired volume of water, with the best
volume being the same as that of the PCR reaction mixture starting
material.

CA 02522815 2005-10-19
17
DNA cleavage with restriction enzymes
Mix: 3 p1 of DNA, 2 p1 of 10 x cleavage buffer, 2.5-5 U of restriction
enzyme (e.g. EcoRl, BamHl, Sall, Xbal, Xhol etc.), add distilled water to a
volume of 20 p,1.
Depending on the restriction enzyme, the cleavage reaction runs at
25-55°C for 1-2 h. For analysis, the fragments are electrophoretically
fractionated in an agarose or polyacrylamide gel in parallel with a length
standard. If the reaction is a double cleavage, then first one enzyme is
added to the mixture. After 1 hour, an aliquot is applied to an appropriate
gel, and, if the cleavage has occurred, the second enzyme can be added. If
the second enzyme does not cleave in the same cleavage buffer, then an
alcohol precipitation is required first.
Agarose gel electrophoresis of DNA
The agarose (Roth) is dissolved in 1 x agarose buffer at the desired
concentration and boiled in a microwave oven, until the agarose has
completely dissolved. The solution is then poured into a sealed Plexiglass
flat bed gel chamber.
The DNA samples are admixed with 1/10 volume of loading blue (50% v/v
glycerol; 50 mM EDTA; 0.005% w/v BPB [Merck, Darmstadt, Germany] and
0.005% xylene cyanol) and pipetted into the gel pockets which are
generated by means of a comb.
The electrophoresis is carried out horizontally in 1 x agarose buffer as
running buffer at a constant voltage of 80-140 V, depending on the size of
the gel and the distance between the electrodes.
1 x agarose buffer: 40 mM Tris-HCI (pH 7.8), 5 mM sodium acetate, 1 mM
EDTA.
Polyacrylamide gel electrophoresis of DNA
7.5% polyacrylamide gel solution; 0.94 ml of 40% strength acrylamide-
bisacrylamide stock solution, 0.5 ml of 10 x TBE buffer (400 mM Tris-HCI,
pH 8.3; 200 mM sodium acetate, 20 mM EDTA), 0.25 ml of 1 % AMPS,
10 u1 of TEMED, 3.33 ml of distilled water.

CA 02522815 2005-10-19
18
This mixture is poured between well-cleaned, vertical glass plates mounted
in vertical apparatuses for polymerization (approx. 10-20 min). The gel is
run in 1 x TBE buffer at a constant voltage of 140 V.
DNA sequencing
1-2 ~g of DNA are to be dissolved in 81 p1 of distilled H20 and 9 p1 of
NaOH (2 N) is to be added for denaturation. After incubation at room
temperature for 10 minutes, the mixture is precipitated, with thorough
washing of the resulting DNA sediment with ice-cold 80% strength ethanol
being important for the subsequent sequencing reactions. 2 p1 of 5 x
Sequenase buffer (200 mM Tris-CI pH 7.5/100 mM MgCl2/250 mM NaCI),
1 ~,I of oligonucleotide (1 pM/~I) and, finally, distilled H20 are to be added
to
the sediment to a total volume of 10 p,1. During the subsequent incubation
in a 37°C water bath for 30 minutes, the starter oligonucleotide
hybridizes
to the D NA.
Reagents added to the hybridization mixture for the sequencing reaction:
1.0 p,1 of DTT (0.1 M), 2.0 ~I of labeling mixture (diluted 1:5), 0.5 p,1 of
[a-35S]dATP, 2 p1 of Sequenase~" (13 U/p,l, United States Biochemical),
(diluted 1:8 with enzyme dilution buffer).
During the subsequent incubation at room temperature for 5 minutes, the
counter strand is synthesized, with the synthetic DNA being tabled by
incorporation of the radiolabeled dATP. This is followed by adding in each
case 3.5 pt of the labeling mixture to 2.5 ~I of the four different
termination
mixtures. Another incubation at 37°C for 5 minutes results in the
randomly
distributed termination reactions of counter stand synthesis. The reactions
are stopped by adding 4 ~I of stop buffer, after which the mixtures are
denatured at 80-90°C and then applied to a 6% strength denatured
sequencing gel. After loading the samples, the main run is carried out at
30-50 W and, respectively, 1300-1600 V for 2-5 h. The gel is then fixed in a
10% strength acetic acid bath (15 min), freed of urea residues under
running water and then dried (for 45 min, using a heat gun, or for 2 h, in a
70°C incubator). The subsequent autoradiography is carried out at
4°C for
16-24 h (Fuji Medical X-ray-Film RX, 30 x 40; Kodak Scientific Imaging Film
X-omat AR).

CA 02522815 2005-10-19
19
Labeling-mixture stock solution: in each case 7.5 pM dATP, dTTP, dGTP,
dCTP
Termination mixtures: in each case 80 ~M dATP, dTTP, dGTP, dCTP and
in each case 8 ~M of the respective ddNTP
Sequenase dilution buffer: 10 mM Tris/HCI; pH 7.5, 5 mM DTT, 0.5 mg/ml
BSA
Stop buffer: 95% formamide, 20 mM EDTA, 0.005% (w/v) xylene cyanol FF
Automated DNA sequencing
Mix: 1 ~g of plasmid DNA (in the case of PCR fragments, for example,
100ng/500 nucleotides), 3-5 pmol of starter molecule (PCR primer, Tm of
55°C, if possible), 4 ~I of Dye Terminator ready-mix (FddNTPs-Ampli-
TaqFS mixture), add distilled water to a volume of 20 ~,I.
The PCR reaction [25 x (15 sec at 94°C, 15 sec. at 50°C, 4
min at 60°C] is
precipitated with alcohol and taken up in 4 ~I of loading buffer. The
samples are then denatured at 95°C for 3 min, removed by centrifugation
and applied to a vertical polyacrylamide gel (34 cm in length, provided with
24 parallel lanes).
After excitation by an argon laser beam at 488 nm, the dyes emit light of
different wavelengths of between 525 nm and 605 nm which is separated
into its spectral colors via a grating, a "spectrograph". The spectral colors
are subsequently detected simultaneously with the aid of the high-
resolution pixel field of a CCD camera. The data are recorded with the aid
of a computer (Macintosh Quadra/650 Macllcx Apple Share) and the
corresponding data analysis software (PE Biosystems, Weiterstadt,
Germany).
Sequencing gel: 30 g of urea (Sigma), 21.5 ml of distilled H20, 6 ml of
10 x TBE
The mixture is dissolved in a wide-necked flask on a heating block at
50°C,
with the following being added: 9 ml of 40% bisacrylamide (filtered), 180 p1
of 10% APS, 24 ~.I of Temed.

CA 02522815 2005-10-19
Polymerase chain reaction (PCR reaction)
The following DNA polymerases may be used:
Taq (Thermus aquaticus) DNA polymerase (recombinant, Gibco/BRL) and
5 10 x PCR buffer
[200 mM Tris/HCI (pH 8.4), 500 mM KCIJ
Tfl (Thermus flavus) DNA polymerase (Master Amp', Biozym, Oldendorf,
Germany) and 20 x PCR
Buffer [20 mM (NH2)S04, 1 M Tris/HCI (pH 9.0)
PCR reaction mixture:
PCR com onents Amount
DNA tem late 10-100 n
Starter molecule 1 25 M
Starter molecule 2 25 M
Nucleotide mixture (dNTPs) 20 mM (from a mixturecontaining
10 mM of each dNTP
DNA polymerase buffer 1 x: 5.0 ~I in the
case of Taq DNA
polymerase buffer
2.5 ~.I in the case of Tfl
DNA
pol merase buffer
M CIZ 75 mM
DNA polymerase 2 U in the case of Taq DNA
polymerase
1 U in the case of Tfl DNA
of merase
Distilled H20 to 50 I total volume
The following applies here: 1 U catalyses the conversion of 10 nM
deoxyribonucleoside triphosphates, at 74°C within 30 min, to an acid-
insoluble DNA product .The PCR reaction usually commences with the "hot
start": the mixture is incubated first without the polymerase at 94°C
in order
to enable the DNA to be denatured for the first time. After the temperature
has reached 80°C, the DNA polymerase is added to the mixture in order
to
avoid nonspecific amplification at a still low temperature. Thereafter, the
actual PCR reaction is carried out over 25-35 cycles.
For each cycle, the following reaction conditions apply:

CA 02522815 2005-10-19
21
Reaction Tem erature Time
Denaturation 94C 30-60 sec
Hybridization Tm-5C 30-60 sec
annealin
Extension 72C 1 min/1 kb
Finally and in addition, the chain extension is carried out at 72°C
for 10
min, finally followed by cooling.
Isolation of total RNA
All centrifugation steps are carried out at 13 000 rpm and 16°C.
Cells are lysed with 600 ~I of lysis buffer (100 RLT buffer:
1 mercaptoethanol). The cell lysate is applied to a QiaSchredder column
and removed by centrifugation for 2 min.
The eluate is admixed with 600 ~I of 70% ethanol, mixed well, and the DNA
is applied to an RNAeasy mini spin column and centrifuged for 15 s
(binding of RNA to the silica matrix). The column is washed three times
(once with 700 ml of RW1 buffer and twice with 500 p,1 of RPE buffer). The
column is then transferred to an autoclaved 1.5 ml Eppendorf tube and the
RNA is eluated with 15 p1 of distilled H20_ The average concentration of
total RNA obtained in this way is 1 pg/pl.
RNA fractionation via agarose gel electrophoresis
Denaturing aragrose gel:
1 g of agraose, 37 ml of distilled water, 10 ml of 10 x MOPS (0.2 mM
MOPS, 10 mM EDTA, 100 mM NaAc),
the mixture is boiled and cooled to 60°C
16 ml of 37% strength formaldehyde are added.
After it has solidified, the gel is inserted with RNA gel running buffer into
the
electrophoresis apparatus. The RNA is applied together with a special
sample buffer.
RNA gel running buffer: 40 ml of 10 x MOPS, 65 ml of 37% strength
formaldehyde, 295 ml of distilled water

CA 02522815 2005-10-19
22
RNA sample buffer: 1-5 pg of RNA, 5 ~I of RNA-NEW buffer (7.5 p,1 37%
strength formaldehyde, 4.5 ~I of 10 x MOPS, 25.9 ~,I of formamide, 7.5 ~.I of
distilled water), 2 p.1 of formamide dye marker [50% (v/v) glycerol, 1 mM
EDTA (pH 8.0), 0.25% (v/v) bromophenol blue, 0.25% (v/v) xylene cyanol].
The gel runs at 80 V for approx. 3 h. Since this work uses only eukaryotic
RNA isolates, the dominant bands visible on the gel should be those of 28S
and 18S rRNA.
Reverse Transcriptase with MMLV-RT
(Moloney murine leukemia virus - Reverse Transcriptase)
Reverse transcriptase mixture: 5 ~.g of RNA, 100 p,M of starter molecule
The RNA preparation and the starter molecule are incubated at 75°C
for 10
min, in order to avoid possible formation of secondary structures in the
RNA template as factors interfering with the transcriptase. However, even
without this step, a transcription reaction usually takes place.
Reverse transcriptase reaction mixture: 28 U of Rnasin~ (Promega), 25 mM
dNTPS, 5 ~I of 10 x reverse transcriptase buffer [10 m Tris/HCI (pH 8.3), 75
mM KCI, 3 mM MgCl2], 50 U of reverse transcriptase (StrataScript~"~,
Stratagene), to 50 p,1 with distilled water.
The reverse transcription is carried out by incubating the mixture at
42°C
for 15 min and at 37°C for 45 min. A longer incubation of 2 h at
42°C with a
30-sec interruption at 55°C is recommended for relatively long RNA
templates. Subsequent incubation of the mixture at 95°C for 5 minutes
results in inactivation of said reverse transcriptase. Subsequently, 5-20 p1
of the reverse transcription mixture are used for a PCR reaction.
Preparation of genomic DNA from tissue
100 mg of tissue are crushed in liquid nitrogen to give a powder. The tissue
powder is introduced into a Falcon tube containing 6 ml of reaction buffer
(30 p1 of proteinase K [20 mg/ml] are added freshly to the buffer) and
incubated with careful shaking at 56°C overnight (12-18 hours). After
incubation, 100 p,1 of RNase A (10 p,g/pl) are added and the mixture is
incubated with further shaking at 37°C for one hour.
This is followed by adding 4 ml of phenol and turning the tube manually

CA 02522815 2005-10-19
23
upside down and up again for approximately 5 min. 4 ml of CI
(chloroform/isoamyl alcohol) are added immediately and the tube is turned
upside down and up again for another 5 minutes and then centrifuged for
15 min (3 000 rpm). The supernatant is carefully removed and transferred
to 10 ml Falcon tubes. If the supernatant is still not clear, the phenol
extraction must be repeated, otherwise another 4 ml of CI are added and
the tube is manually turned upside down and up for 5 min and then
centrifuged for 15 min (3 000 rpm). The supernatant is carefully removed
and the CI extraction repeated. The final supernatant obtained is admixed
with 1/10 volumes of sodium acetate solution (3 M, pH 6) and 2.5 volumes
of ethanol (99.8%). The tube is carefully rotated, until the DNA precipitates
as a tangle. This DNA tangle is transferred to approximately 25 ml of
ethanol (70%) with the aid of a glass hook and left resting for 3 min. The
washing was repeated twice. The DNA was then dried in air and dissolved
in 0.5 ml of double-distilled water at room temperature.
Southern Blot
DNA fractionation via an agarose gel
Leave the gel on short-wave UV for approx. 5 min for strand breaks to
occur in the larger DNA molecules (> 6kBp).
Continuously tilt the gel in denaturating solution for 30 min for DNA
denaturation.
Continuously tilt the gel in neutralizing solution for 30 min for
neutralization.
Blot construction (from bottom to top): gel, nylon membrane, dry filter
paper, blotting paper, plate, weight (approx. 1 kg).
Blotting with 20 x SSC overnight.
Wash membrane in 2 x SSC for 10 min
Dry membrane on filter paper
Fixing of nucleic acid by baking at 80°C for 1 h or UV crosslinking
(e.g. in
"Stratalinker", automatic position). The membrane may then be stored until
hybridization.
Prehybridization of membrane in hybridization solution for approx. 1 - 2 h
Covering of nonspecific binding sites on the membrane.
Hybridization solution: 5 x SSC, 5 x Denhardt's solution, 0.5% SDS,

CA 02522815 2005-10-19
24
100 ~g/ml herring sperm DNA
Denaturing solution: 0.5 M NaOH (20 g), 1 M NaCI
Neutralizing solution: 1.5 M NaCI/0.5 M Tris pH 7.4
20 x SSC is 3 M NaCI, 0.3 M Na-citrate: 175.3 g of NaCI, 88.2 g of sodium
citrate X 2 H20, to 1 I with double-distilled water, adjust pH to 7.0 with
HCI.
50 x Denhard's solution: 5 g of Ficoll 400, 5 g of PVP (polyvinyl
pyrrolidone), 5 g of BSA, to 500 ml with double-distilled water
Northern Blot
RNA fractionation using a formaldehyde agarose gel
Blot construction (from bottom to top): gel, nylon membrane, dry filter
paper, blotting paper, plate, weight (appox. 1 kg).
Blotting with 20 x SSC overnight.
Fix RNA on filter by baking at 80°C (1 h)
Introduce filter into boiling 20 mM Tris pH 8 for RNA deglyoxylation and let
cool to RT.
Description of the Figures
Fig. 1
Characteristics of study group
Fig.2
Distribution of PAR1 variants T3090C and A3329C in 1362 individuals
analyzed
Fig.3
Association of PAR1 variants C3090C with atrial fibrillation and
cardiomyopathy

CA 02522815 2005-10-19
Fig. 4
Association of PAR1 variants C3329C with atrial fibrillation, acute coronary
syndrome and unstable angina.
5
Fig. 5
Polynucleotide sequence of the cDNA of the human PAR1 gene in 5'/3'
orientation. The sequence corresponds to the sequence made publicly
10 available by the NCBI Nucleotide Database under number NM-001992.
The prepared sequence is identical to SEQ ID NO: 1.
Fig. 6
15 Polynucleotide sequence of the cDNA of the human PAR1 gene in 5'/3'
orientation with a polymorphism at position 3090 of the sequence according
to NM-001992, which polymorphism comprises a T to C substitution. The
depicted sequence is identical to SEQ ID NO: 2.
20 Fig.7
Polynucleotide sequence of the cDNA of the human PAR1 gene in 5'/3'
orientation with a polymorphism at position 3329 of the sequence according
to NM-001992, which polymorphism comprises an A to C substitution. The
25 depicted sequence is identical to SEQ ID NO: 3.
Fig. 8
Polynucleotide sequence of the cDNA of the human PAR1 gene in 5'/3'
orientation with a polymorphism at position 3090 of the sequence according
to NM-001992, which polymorphism comprises a T to C substitution, and
with a simultaneous second polymorphism at position 3329 of the
sequence according to NM-001992, which polymorphism comprises an A
to C substitution. The depicted sequence is identical to SEQ ID NO: 4.

CA 02522815 2005-10-19
26
Fig. 9
Polynucleotide sequence of a fragment of the human PAR1 gene in 5'/3'
orientation. The depicted sequence is identical to SEQ ID NO: 5.
Fig. 10
Polynucleotide sequence of a fragment of the human PAR1 gene in 5'/3'
orientation with a polymorphism at position 3090 of the sequence according
to NM-01992, which polymorphism comprises a T to C substitution. The
depicted sequence is identical to SEQ ID NO: 6.
Fig. 11
Polynucleotide sequence of a fragment of human PAR1 gene in 5'/3'
orientation with a polymorphism at position 3329 of the sequence according
to NM-001992, which polymorphism comprises an A to C substitution. The
depicted sequence is identical to SEQ ID NO: 7.
Fig. 12
Polynucleotide sequence of a fragment of the human PAR1 gene in 5'/3'
orientation with a polymorphism at position 3090 of the sequence according
to NM-001992, which polymorphism comprises a T to C substitution, and
with a simultaneous second polymorphism at position 3329 of the
sequence according to NM-001992, which polymorphism comprises an A
to C substitution. The depicted sequence is identical to SEQ ID NO: 8.
Fig. 13
Polynucleotide sequence in 5'/3' orientation of the 5' end of the cDNA of
the human PAR1 gene. The depicted sequence is identical to SEQ ID NO;
9.
Fig. 14
Polynucleotide sequence in 5'/3' orientation of the 3' end of the cDNA of
the human PAR1 gene. The depicted sequence is identical to SEQ ID NO;
10.

CA 02522815 2005-10-19
27
Fig. 15
Polynucleotide sequence in 5'/3' orientation of the cDNA of the human
PAR1 gene, relating to positions 2767 to 2789 according to NM-001992.
The depicted sequence is identical to SEQ ID N0; 11.
Fig. 16
Polynucleotide sequence in 5'/3' orientation of the Exon No. 1 of the human
PAR1 gene. The depicted sequence is identical to SEQ ID NO; 12.
Fig. 17
Protein sequence of the human PAR1 receptor. The sequence corresponds
to the sequence made publicly available by the NCBI Protein Database
under number NP-001983. The depicted sequence is identical to
SEQ ID NO: 13.

CA 02522815 2005-10-19
WO 20041094470 1/11 PCT/EP2004/004035
SEQUENCE LISPING
<110> Aventis Pharma Deutschland GmbH
<120> Analysis and use of PAR1 polymorphism for
evaluating the risk of cardiovascular disorders
<130> DEAV2003/0030
<140>
<141>
<lso> 13
<170> PatentIn Vez. 2.1
<210> 1
<211> 3592
<212> DNA
<213> Homo Sapiens
<400> 1
ggcggggggc gcacagagcc agaggggctt gcgagcggcg gctgagggac cgcggggagg 60
gggcgccgag cggctccagc gcagagactc tcactgcacg ccggaggccc cttcctcgct 120_
ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc cagacacagc 180
gctcgccgag ggtcgcttgg accctgatct tacccgtggg caccctgcgc tctgcctgcc 240
gcgaagaccg gctccccgac ccgcagaagt caggagagag ggtgaagcgg agcagcccga 300
ggcggggcag cctcccggag cagcgccgcg cagagcccgg gacaatgggg ccgcggcggc 360
tgctgctggt ggccgcctgc ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg 420
cccgcaggcc agaatcaaaa gcaacaaatg ccaccttaga tccccggtca tttcttctca 480
ggaaccccaa tgataaatat gaaccatttt gggaggatga ggagaaaaat gaaagtgggt 540
taactgaata cagattagtc tccatcaata aaagcagtcc tcttcaaaaa caacttcctg 600
cattcatctc agaagatgcc tccggatatt tgaccagctc ctggctgaca ctctttgtcc 660
catctgtgta caccggagtg tttgtagtca gcctcccact aaacatcatg gccatcgttg 720
tgttcatcct gaaaatgaag gtcaagaagc cggcggtggt gtacatgctg cacctggcca 780
cggcagatgt gctgtttgtg tctgtgctcc cctttaagat cagctattac ttttccggca 840
gtgattggca gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt tactgtaaca 900
tgtacgcctc tatcttgctc atgacagtca taagcattga ccggtttctg gctgtggtgt 960
atcccatgca gtccctctcc tggcgtactc tgggaagggc ttccttcact tgtctggcca 1020
tctgggcttt ggccatcgca ggggtagtgc ctctcgtcct caaggagcaa accatccagg 1080
tgcccgggct caacatcact acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct 1140
actatgccta ctacttctca gccttctctg ctgtcttctt ttttgtgccg ctgatcattt 1200
ccacggtctg ttatgtgtct atcattcgat gtcttagctc ttccgcagtt gccaaccgca 1260
gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt ctgcatcttc atcatttgct 1320
tcggacccac aaacgtcctc ctgattgcgc attactcatt cctttetcac acttccacca 1380
cagaggctgc ctactttgcc tacctcctct gtgtctgtgt cagcagcata agctcgtgca 1440
tcgaccccct aatttactat tacgcttcct ctgagtgcca gaggtacgtc tacagtatct 1500
tatgctgcaa agaaagttcc aatcccagca gttataacag cagtgggcag ttgatggcaa 1560

CA 02522815 2005-10-19
2/11
gtaaaatgga tacctgctct agtaacctga ataacagcat atacaaaaag ctgttaactt 1620
aggaaaaggg actgctggga ggttaaaaag aaaagtttat aaaagtgaat aacctgagga 1680
ttctattagt ccccacccaa actttattga ttcacctcct aaaacaacag atgtacgact 1740
tgcatacctg ctttttatgg gagctgtcaa gcatgtattt ttgtcaatta ccagaaagat 1800
aacaggacga gatgacggtg ttattccaag ggaatattgc caatgctaca gtaataaatg 1860
aatgtcactt ctggatatag ctaggtgaca tatacatact tacatgtgtg tatatgtaga 1920
tgtatgcaca cacatatatt atttgcagtg cagtatagaa taggcacttt aaaacactct 1980
ttccccgcac cccagcaatt atgaaaataa tctctgattc cctgatttaa tatgcaaagt 2040
ctaggttggt agagtttagc cctgaacatt tcatggtgtt catcaacagt gagagactcc 2100
atagtttggg cttgtaccac ttttgcaaat aagtgtattt tgaaattgtt tgacggcaag 216D
gtttaagtta ttaagaggta agacttagta ctatctgtgc gtagaagttc tagtgttttc 2220
aattttaaac atatccaagt ttgaattcct aaaattatgg aaacagatga aaagcctctg 2280
ttttgatatg ggtagtattt tttacatttt acacactgta cacataagcc aaaactgagc 2340
ataagtcctc tagtgaatgt aggctggctt tcagagtagg ctattcctga gagctgcatg 2400
tgtccgcccc cgatggagga ctccaggcag cagacacatg ccagggccat gtcagacaca 2460
gattggccag aaaccttcct gctgagcctc acagcagtga gactggggcc actacatttg 2520
ctccatcctc ctgggattgg ctgtgaactg atcatgttta tgagaaactg gcaaagcaga 2580
atgtgatatc ctaggaggta atgaccatga aagacttctc tacccatctt aaaaacaacg 264D
aaagaaggca tggacttctg gatgcccatc cactgggtgt aaacacatct agtagttgtt 270D
ctgaaatgtc agttctgata tggaagcacc cattatgcgc tgtggccact ccaataggtg 2760
ctgagtgtac agagtggaat aagacagaga cctgccctca agagcaaagt agatcatgca 2820
tagagtgtga tgtatgtgta ataaatatgt ttcacacaaa caaggcctgt cagctaaaga 2880
agtttgaaca tttgggttac tatttcttgt ggttataact taatgaaaac aatgcagtac 2940
aggacatata ttttttaaaa taagtctgat ttaattgggc actatttatt tacaaatgtt 3000
ttgctcaata gattgctcaa atcaggtttt cttttaagaa tcaatcatgt cagtctgctt 3060
agaaataaca gaagaaaata gaattgacat tgaaatctag gaaaattatt ctataatttc 3120
catttactta agacttaatg agactttaaa agcatttttt aacctcctaa gtatcaagta 3180
tagaaaatct tcatggaatt cacaaagtaa tttggaaatt aggttgaaac atatctctta 3240
tcttacgaaa aaatggtagc attttaaaca aaatagaaag ttgcaaggca aatgtttatt 3300
taaaagagca ggccaggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggctga 3360
ggcgggtgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg tgaaacccgt 3420
ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg caggcacctg tagtcccagc 3480
tactcgggag gctgaggcag gagactggcg tgaacccagg aggcggacct tgtagtgagc 3540
cgagatcgcg ccactgtgct ccagcctggg caacagagca agactccatc tc 3592
<210> 2
<211> 3592
<212> DNA
<213> Homo Sapiens
<400> 2
ggcggggggc gcacagagcc agaggggctt gcgagcggcg gctgagggac cgcggggagg 60
gggcgccgag cggctccagc gcagagactc tcactgcacg ccggaggccc cttcctcgct 120
ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc cagacacagc 180
gctcgccgag ggtcgcttgg accctgatct tacccgtggg caccctgcgc tctgcctgcc 240
gcgaagaccg gctccccgac ccgcagaagt caggagagag ggtgaagcgg agcagcccga 300
ggcggggcag cctcccggag cagcgccgcg cagagcccgg gacaatgggg ccgcggcggc 360

CA 02522815 2005-10-19
3/11
tgctgctggt ggccgcctgc ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg 420
cccgcaggcc agaatcaaaa gcaacaaatg ccaccttaga tccccggtca tttcttctca 480
ggaaccccaa tgataaatat gaaccatttt gggaggatga ggagaaaaat gaaagtgggt 540
taactgaata cagattagtc tccatcaata aaagcagtcc tcttcaaaaa caacttcctg 600
cattcatctc agaagatgcc tccggatatt tgaccagctc ctggctgaca ctctttgtcc 660
catctgtgta caccggagtg tttgtagtca gcctcccact aaacatcatg gccattgttg 720
tgttcatcct gaaaatgaag gtcaagaagc cggcggtggt gtacatgctg cacctggcca 780
cggcagatgt gctgtttgtg tctgtgctcc cctttaagat cagctattac ttttccggca 840
gtgattggca gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt tactgtaaca 900
tgtacgcctc tatcttgctc atgacagtca taagcattga ccggtttctg gctgtggtgt 960
atcccatgca gtccctctcc tggcgtactc tgggaagggc ttccttcact tgtctggcca 1020
tc2gggcttt ggccatcgca ggggtagtgc ctctcgtcct caaggagcaa accatccagg 1080
tgcccgggct caacatcact acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct 1140
actatgccta ctatttctca gccttctctg ctgtcttctt ttttgtgccg ctgatcattt 1200
ccacggtctg ttatgtgtct'atcattcgat gtcttagctc ttccgcagtt gccaaccgca 1260
gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt ctgcatcttc atcatttgct 1320
tcggacccac aaacgtccto ctgattgcgc attactcatt cetttctcac acttccacca 1380
cagaggctgc ctactttgcc tacctcctct gtgtctgtgt cagcagcata'agctcgtgca 1440
tcgaccccct aatttactat tacgcttcct ctgagtgcca gaggtacgtc tacagtatct 1500
tatgctgcaa agaaagttcc gatcccagca gttataacag cagtgggcag ttgatggcaa 1560
gtaaaatgga tacctgctct agtaacctga ataacagcat atacaaaaag ctgttaactt 1620
aggaaaaggg actgctggga ggttaaaaag aaaagtttat aaaagtgaat aacctgagga 1680
ttctattagt ccccacccaa actttattga ttcacctcct aaaacaacag atgtacgact 1740
tgcatacctg ctttttatgg gagctgtcaa gcatgtattt ttgtcaatta ccagaaagat 1800
aacaggacga ~gatgacggtg ttattccaag ggaatattgc caatgctaca gtaataaatg 1860
aatgtcactt ctggatatag ctaggtgaca tatacatact tacatgtgtg tatatgtaga 1920
tgtatgcaca cacatatatt atttgcagtg cagtatagaa taggcacttt aaaacactct 1980
ttccccgcac cccagcaatt atgaaaataa tctctgattc cctgatttaa tatgcaaagt 2040
ctaggttggt agagtttagc cctgaacatt tcatggtgtt catcaacagt gagagactcc 21'00
atagtttggg cttgtaccac ttttgcaaat aagtgtattt tgaaattgtt tgacggcaag 2160
gtttaagtta ttaagaggta agacttagta ctatctgtgc gtagaagttc tagtgttttc 2220
aattttaaac atatccaagt ttgaattcct aaaattatgg aaacagatga aaagcctctg 2280
ttttgatatg ggtagtattt tttacatttt acacactgta cacataagcc aaaactgagc 2340
ataagtcctc tagtgaatgt aggctggctt tcagagtagg ctattcctga gagctgcatg 2400
tgtccgcccc cgatggagga ctccaggcag cagacacatg ccagggccat gtcagacaca 2460
gattggccag,aaaccttcct gctgagcctc acagcagtga gactggggcc actacatttg 2520
ctccatcctc ctgggattgg ctgtgaactg atcatgttta tgagaaactg gcaaagcaga 2580
atgtgatatc ctaggaggta atgaccatga aagacttctc tacccatctt aaaaacaacg 2640
aaagaaggca tggacttctg gatgcccatc cactgggtgt aaacacatct agtagttgtt 2700
ctgaaatgtc agttctgata tggaagcacc cattatgcgc tgtggccact ccaataggtg 2760
ctgagtgtac agagtggaat aagacagaga cctgccctca agagcaaagt agatcatgca 2820
tagagtgtga tgtatgtgta ataaatatgt ttcacacaaa caaggcctgt cagctaaaga 2880
agtttgaaca tttgggttac tatttcttgt ggttataact taatgaaaac aatgcagtac 2940
aggacatata ttttttaaaa taagtctgat ttaattgggc actatttatt tacaaatgtt 3000
ttgctcaata gattgctcaa atcaggtttt cttttaagaa tcaatcatgt cagtctgctt 3060
agaaataaca gaagaaaata gaattgacac tgaaatctag gaaaattatt ctataatttc 3120
catttactta agacttaatg agactttaaa agcatttttt aacctcctaa gtatcaagta 3180
tagaaaatct tcatggaatt cacaaagtaa tttggaaatt aggttgaaac atatctctta 3240

CA 02522815 2005-10-19
4/11
tcttacgaaa aaatggtagc attttaaaca aaatagaaag ttgcaaggca aatgtttatt 3300
taaaagagca ggccaggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggctga 3360
ggcgggtgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg tgaaacccgt 3420
ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg caggcacctg tagtcccagc 3480
tactcgggag gctgaggcag gagactggcg tgaacccagg aggcggacct tgtagtgagc 3540
cgagatcgcg ccactgtgct ccagcctggg caacagagca agactccatc tc 3592
<210> 3
<211> 3592
< 212 > DDTA
<213> Homo sapiras
<400> 3
ggcggggggc gcacagagcc agaggggctt gcgagcggcg gctgagggac cgcggggagg 60
gggcgccgag cggctccagc gcagagactc tcactgcacg ccggaggccc cttcctcgct 120
ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc cagacacagc 180
gctcgccgag ggtcgcttgg accctgatct tacccgtggg caccctgcgc tctgcctgcc 240
gcgaagaccg gctccccgac ccgcagaagt caggagagag ggtgaagcgg agcagcccga 300
ggcggggcag cctcccggag cagcgccgcg cagagcccgg gacaatgggg ccgcggcggc 360
tgctgctggt ggccgcctgc ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg 420
cccgcaggcc agaatcaaaa~gcaacaaatg ccaccttaga tccccggtca tttcttctca 480
ggaaccccaa tgataaatat gaaccatttt gggaggatga ggagaaaaat gaaagtgggt 540
taactgaata cagattagtc tccatcaata aaagcagtcc tcttcaaaaa caacttcctg 600
cattcatctc agaagatgcc tccggatatt tgaccagctc ctggctgaca ctctttgtcc 660
catctgtgta caccggagtg tttgtagtca gcctcccact aaacatcatg gccatcgttg 720
tgttcatcct gaaaatgaag gtcaagaagc cggcggtggt gtacatgctg cacctggcca 780
cggcagatgt gctgtttgtg tctgtgctcc cctttaagat.cagctattac ttttccggca 840
gtgattggca gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt tactgtaaca 900
tgtacgcctc tatcttgctc atgacagtca taagcattga ccggtttctg. gctgtggtgt 960
atcccatgca gtccctctcc tggcgtactc tgggaagggc ttccttcact tgtctggcca 1020
tctgggcttt ggccatcgca ggggtagtgc ctctcgtcct caaggagcaa accatccagg 1080
tgcccgggct caacatcact acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct 1140
actatgccta ctacttctca gccttctctg ctgtcttctt ttttgtgccg ctgatcattt 1200
ccacggtctg ttatgtgtct atcattcgat gtcttagctc ttccgcagtt gccaaccgca 1260
gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt ctgcatcttc atcatttgct 1320
tcggacccac aaacgtcctc ctgattgcgc attactcatt cctttctcac acttccacca 1380
cagaggctgc ctactttgcc tacctcctct gtgtctgtgt cagcagcata agctcgtgca 1440
tcgaccccct aatttactat tacgcttcct ctgagtgcca gaggtacgtc tacagtatct 1500
tatgctgcaa agaaagttcc gatcccagca gttataacag cagtgggcag ttgatggcaa 1560
gtaaaatgga tacctgctct agtaacctga ataacagcat atacaaaaag ctgttaactt 1620
aggaaaaggg actgctggga ggttaaaaag aaaagtttat aaaagtgaat aacctgagga 1680
ttctattagt ccccacccaa actttattga ttcacctcct aaaacaacag atgtacgact 1740
tgcatacctg ctttttatgg gagctgtcaa gcatgtattt ttgtcaatta ccagaaagat 1800
aacaggacga gatgacggtg ttattccaag ggaatattgc caatgctaca gtaataaatg 1860
aatgtcactt ctggatatag ctaggtgaca tatacatact tacatgtgtg tatatgtaga 1920
tgtatgcaca cacatatatt atttgcagtg cagtatagaa taggcacttt aaaacactct 1980
ttccccgcac cccagcaatt atgaaaataa tctctgattc cctgatttaa.tatgcaaagt 2040

CA 02522815 2005-10-19
5/11
ctaggttggt agagtttagc cctgaacatt tcatggtgtt ~catcaacagt gagagactcc 21D0
atagtttggg cttgtaccac ttttgcaaat aagtgtattt tgaaattgtt tgacggcaag 2160
gtttaagtta ttaagaggta agacttagta ctatctgtgc gtagaagttc tagtgttttc 2220
aattttaaac atatccaagt ttgaattcct aaaattatgg aaacagatga aaagcctctg 2280
ttttgatatg ggtagtattt tttacatttt acacactgta cacataagcc aaaactgagc 2340
ataagtcctc tagtgaatgt aggctggctt tcagagtagg ctattcctga gagctgcatg 240-0
tgtccgcccc cgatggagga ctccaggcag cagacacatg ccagggccat gtcagacaca 2460
gattggccag aaaccttcct gctgagcctc acagcagtga gactggggcc actacatttg 2520
ctccatcctc ctgggattgg ctgtgaactg atcatgttta tgagaaactg gcaaagcaga 2580
atgtgatatc ctaggaggta atgaccatga aagacttctc tacccatctt aaaaacaacg 2640
aaagaaggca tggacttctg gatgcccatc cactgggtgt aaacacatct agtagttgtt 2700
ctgaaatgtc agttctgata tggaagcacc cattatgcgc tgtggccact ccaataggtg 2760
ctgagtgtac agagtggaat aagacagaga cctgccctca agagcaaagt agatcatgca 2820
tagagtgtga tgtatgtgta ataaatatgt ttcacacaaa caaggcctgt cagctaaaga 2880
agtttgaaca tttgggttac tatttcttgt ggttataact taatgaaaac aatgcagtac 2940
aggacatata ttttttaaaa taagtctgat ttaattgggc actatttatt tacaaatgtt 3000
ttgctcaata gattgctcaa atcaggtttt cttttaagaa tcaatcatgt cagtctgctt 3060
agaaataaca gaagaaaata gaattgacat tgaaatctag gaaaattatt ctataatttc 3120
catttactta agacttaatg agactttaaa agcatttttt aacctcctaa gtatcaagta 3180
tagaaaatct tcatggaatt'cacaaagtaa tttggaaatt aggttgaaac~atatctctta 3240
tcttacgaaa aaatggtagc attttaaaca aaatagaaag ttgcaaggca aatgtttatt 3300
taaaagagca ggccaggcgc ggtggctccc gcctgtaatc ccagcacttt gggaggctga 3360
ggcgggtgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg tgaaacccgt 3420
ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg caggcacctg tagtcccagc 3480
tactcgggag gctgaggcag gagactggcg tgaacccagg aggcggacct tgtagtgagc 3540
cgagatcgcg ccactgtgct ccagcctggg caacagagca .agactccatc tc 3592
<210> 4
c211> 3592
<212> ANA
<213> Homo Sapiens
<400> 4
ggcggggggc gcacagagcc agaggggctt gcgagcggcg gctgagggac cgcggggagg 60
gggcgccgag cggctccagc gcagagactc tcactgcacg ccggaggccc cttcctcgct 120
ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc cagacacagc 180
gctcgccgag ggtcgcttgg accctgatct tacccgtggg caccctgcgc tctgcctgcc 240
gcgaagaccg gctccccgae ccgcagaagt caggagagag ggtgaagcgg agcagcccga 300
ggcggggcag cctcccggag cagcgccgcg cagagcccgg gacaatgggg ccgcggcggc 360
tgctgctggt ggccgcctgc ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg 420
cccgcaggcc agaatcaaaa gcaacaaatg ccaccttaga tccccggtca tttcttctca 480
ggaaccccaa. tgataaatat gaaccatttt gggaggatga ggagaaaaat gaaagtgggt 540
taactgaata cagattagtc tccatcaata aaagcagt,,cc tcttcaaaaa caacttcctg 600
cattcatctc agaagatgcc tccggatatt tgaccagctc ctggctgaca ctctttgtcc 660
catctgtgta caccggagtg tttgtagtca gcctcccact aaacatcatg gccatcgttg 720
tgttcatcct gaaaatgaag gtcaagaagc cggcggtggt gtacatgctg cacctggcca 780
cggcagatgt gctgtttgtg tctgtgctcc cctttaagat cagctattac ttttccggca 840

CA 02522815 2005-10-19
6/11
gtgattggca gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt tactgtaaca 900
tgtacgcctc tatcttgctc atgacagtca taagcattga ccggtttctg gctgtggtgt 960
atcccatgca gtccctctcc tggcgtactc tgggaagggc ttccttcact tgtctggcca 1020
tctgggcttt ggccatcgca ggggtagtgc ctctcgtcct caaggagcaa accatccagg 1080
tgcccgggct caacatcact acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct 1140
actatgccta ctacttctca gccttctctg ctgtcttctt ttttgtgccg ctgatcattt 1200
ccacggtctg ttatgtgtct atcattcgat 9tcttagctc ttccgcagtt gccaaccgca 1260
gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt ctgcatcttc atcatttgct 1320
tcggacccac aaacgtcctc ctgattgcgc attactcatt cctttctcac acttccacca 1380
cagaggctgc ctactttgcc tacctcctct gtgtctgtgt cagcagcata agctcgtgca 1440
tcgaccccct aatttactat tacgcttcct ctgagtgcca gaggtacgtc tacagtatct 1500
tatgctgcaa agaaagttcc gatcccagca gttataacag cagtgggcag ttgatggcaa 1560
gtaaaatgga tacctgctct agtaacctga ataacagcat atacaaaaag ctgttaactt 1620
aggaaaaggg actgctggga ggttaaaaag aaaagtttat aaaagtgaat aacctgagga 1680
ttctattagt ccccacccaa actttattga ttcacctcct aaaacaacag atgtacgact 1740
tgcatacctg ctttttatgg gagctgtcaa gcatgtattt ttgtcaatta ccagaaagat 1800
aacaggacga gatgacggtg ttattccaag ggaatattgc caatgctaca gtaataaatg 1860
aatgtcactt ctggatatag ctaggtgaca tatacatact tacatgtgtg tatatgtaga 1920
tgtatgcaca cacatatatt atttgcagtg cagtatagaa taggcacttt aaaacactct 1980
ttccccgcac cccagcaatt atgaaaataa tctctgattc cctgatttaa tatgcaaagt 2040
ctaggttggt agagtttagc cctgaacatt tcatggtgtt catcaacagt gagagactcc 2100
atagtttggg cttgtaccac ttttgcaaat aagtgtattt tgaaattgtt tgacggcaag 2160
gtttaagtta ttaagaggta agacttagta ctatctgtgc gtagaagttc tagtgttttc 2220
aattttaaac atatccaagt ttgaattcct aaaattatgg aaacagatga aaagcctctg 2280
ttttgatatg ggtagtattt tttacatttt acacactgta cacataagcc aaaactgagc 2340
ataagtcctc tagtgaatgt aggctggctt tcagagtagg ctattcctga gagctgcatg 2400
tgtccgcccc cgatggagga ctccaggcag cagacacatg ccagggccat gtcagacaca 2460
gattggccag aaaccttcct gctgagcctc acagcagtga gactggggcc actacatttg 2520
ctccatcctc ctgggattgg ctgtgaactg atcatgttta tgagaaactg gcaaagcaga 2580
atgtgatatc ctaggaggta atgaccatga aagacttctc~tacccatctt aaaaacaacg 2640
aaagaaggca tggacttctg gatgcccatc cactgggtgt aaacacatct agtagttgtt 2700
ctgaaatgtc agttctgata tggaagcacc cattatgcgc tgtggccact ccaataggtg 2760
ctgagtgtac agagtggaat aagacagaga cctgccctca agagcaaagt agatcatgca 2820
tagagtgtga tgtatgtgta ataaatatgt ttcacacaaa caaggcctgt cagctaaaga 288'0
agtttgaaca tttgggttac tatttcttgt ggttataact taatgaaaac aatgcagtac 2940
aggacatata ttttttaaaa taagtctgat ttaattgggc actatttatt,tacaaatgtt 3000
ttgctcaata gattgctcaa atcaggtttt cttttaagaa tcaatcatgt cagtctgctt 3060
agaaataaca gaagaaaata gaattgacac tgaaatctag gaaaattatt ctataatttc 3120
catttactta agacttaatg agactttaaa agcatttttt aacctcctaa gtatcaagta 3180
tagaaaatct tcatggaatt cacaaagtaa tttggaaatt aggttgaaac atatctctta 3240
tcttacgaaa aaatggtagc attttaaaca aaatagaaag ttgcaaggca aatgtttatt 3300
taaaagagca ggccaggcgc ggtggctccc gcctgtaatc ccagcacttt gggaggctga 3360
ggcgggtgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg tgaaacccgt 3420
ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg caggcacctg tagtcccagc 3480 .
tactcgggag gctgaggcag gagactggcg tgaacccagg aggcggacct tgtagtgagc 3540
cgagatcgcg ccactgtgct ccagcctggg caacagagca agactccatc tc 3592

CA 02522815 2005-10-19
7/11
<210> s
<211> 939
<212> DNA
<213> Homo Sapiens
<400> 5
acagagtgga ataagacaga gacctgccct caagagcaaa gtagatcatg catagagtgt 60
gatgtatgtg taataaatat gtttcacaca aacaaggcct gtcagctaaa gaagtttgaa 120
catttgggtt actatttctt gtggttataa cttaatgaaa acaatgcagt acaggacata 180
tattttttaa aataagtctg atttaattgg gcactattta tttacaaatg ttttgctcaa 240
tagattgctc aaatcaggtt.ttcttttaag aatcaatcat gtcagtctgc ttagaaataa 300
.czgaagaaaa tagaattgac attgaaatct aggaaaatta ttctataatt tccatttact 360
taagacttaa tgagacttta aaagcatttt ttaacctcct aagtatcaag tatagaaaat-420
cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa acatatctct tatcttacga 480
aaaaatggta gcattttaaa caaaatagaa agttgcaagg caaatgttta tttaaaagag 540
caggccaggc gcggtggctc acgcctgtaa tcccagcact ttgggaggct gaggcgggtg 600
gatcacgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc gtctctacta~660
aaaatgcaaa aaaaattagc cgggcgtggt ggcaggcacc tgtagtccca gctactcggg 720
aggctgaggc aggagactgg cgtgaaccca ggaggcggac cttgtagtga gccgagatcg 780
cgccactgtg ctccagcctg ggcaacagag caagactcca tctcaaaaaa taaaaataaa 840
taaaaaataa aaaaataaaa gagcaaacta tttccaaata ccatagaata acttacataa 900
aagtaatata actgtattgt aagtagaagc tagcactgg 939
<210> 6
<211> 939
<212> DNA
<213> Homo Sapiens
<400> 6
acagagtgga.ataagacaga gacctgccct caagagcaaa gtagatcatg catagagtgt 60
gatgtatgtg taataaatat gtttcacaca aacaaggcct gtcagctaaa gaagtttgaa 120
catttgggtt actatttctt gtggttataa cttaatgaaa acaatgcagt acaggacata 180
tattttttaa aataagtctg atttaattgg gcactattta tttacaaatg ttttgctcaa 240
tagattgctc aaatcaggtt ttcttttaag aatcaatcat gtcagtctgc ttagaaataa 300
cagaagaaaa tagaattgac actgaaatct aggaaaatta ttctataatt tccatttact 360
taagacttaa tgagacttta aaagcatttt ttaacctcct aagtatcaag tatagaaaat 420
cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa acatatctct tatcttacga 480
aaaaatggta gcattttaaa caaaatagaa agttgcaagg caaatgttta tttaaaagag 540
caggccaggc gcggtggctc acgcctgtaa tcccagcact ttgggaggct gaggcgggtg 600
gatcacgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc gtctctacta 660
aaaatgcaaa aaaaattagc cgggcgtggt ggcaggcacc tgtagtccca gctactcggg 720
aggctgaggc aggagactgg cgtgaaccca ggaggcggac cttgtagtga gccgagatcg 780
cgccactgtg ctccagcctg ggcaacagag caagactcca tctcaaaaaa taaaaataaa B40
taaaaaataa aaaaataaaa gagcaaacta tttccaaata ccatagaata acttacataa 900
aagtaatata actgtattgt aagtagaagc tagcactgg ~ 93g

CA 02522815 2005-10-19
8/11
<zlo> 7
<211> 939
<212 > DNA
<213> Homo Sapiens
<40b> 7
acagagtgga ataagacaga gacctgccct caagagcaaa gtagatcatg catagagtgt 60
gatgtatgtg taataaatat gtttcacaca aacaaggcct gtcagctaaa gaagtttgaa 120
catttgggtt actatttctt gtggttataa cttaatgaaa acaatgcagt~acaggacata 180
tattttttaa aataagtctg atttaattgg gcactattta tttacaaatg ttttgctcaa 240
tagattgctc aaatcaggtt ttcttttaag aatcaatcat gtcagtctgc ttagaaataa 300
cagaagaaaa tagaattgac attgaaatct aggaaaatta ttctataatt tccatttact 360
taagacttaa tgagacttta aaagcatttt ttaacctcct aagtatcaag tatagaaaat 420
cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa acatatctct tatcttacga 480
aaaaatggta gcattttaaa-caaaatagaa agttgcaagg caaatgttta tttaaaagag 540
caggccaggc gcggtggctc ccgcctgtaa tcccagcact ttgggaggct.gaggcgggtg 600
gatcacgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc gtctctacta 660
aaaatgcaaa aaaaattagc cgggcgtggt ggcaggcacc tgtagtccca gctactcggg 720
aggctgaggc aggagactgg cgtgaaccca ggaggcggac cttgtagtga gccgagatcg 780
cgccactgtg ctccagcctg ggcaacagag caagactcca tctcaaaaaa taaaaataaa~840
taaaaaataa aaaaataaaa gagcaaacta tttccaaata ccatagaata acttacataa 900
aagtaatata actgtattgt aagtagaagc tagcactgg 939
<210> 8
<211> 939
<212> DNA
<213> Homo Sapiens
<400> 8
acagagtgga ataagacaga gacctgccct caagagcaaa gtagatcatg catagagtgt 60
gatgtatgtg taataaatat gtttcacaca aacaaggcct gtcagctaaa gaagtttgaa 120
catttgggtt actatttctt gtggttataa cttaatgaaa acaatgcagt acaggacata 180
tattttttaa aataagtctg atttaattgg'gcactattta tttacaaatg ttttgctcaa 240
tagattgctc aaatcaggtt ttcttttaag aatcaatcat~gtcagtctgc ttagaaataa 300
cagaagaaaa tagaattgac actgaaatct aggaaaatta ttctataatt tccatttact 360
taagacttaa tgagacttta aaagcatttt ttaacctcct aagtatcaag tatagaaaat 420
cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa acatatctct tatcttacga 480
aaaaatggta gcattttaaa caaaatagaa agttgcaagg caaatgttta tttaaaagag 540
caggccaggc gcggtggctc ccgcctgtaa tcccagcact ttgggaggct gaggcgggtg 600
gatcacgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc gtctctacta 660
aaaatgcaaa aaaaattagc cgggcgtggt ggcaggcacc tgtagtccca gctactcggg 720
aggctgaggc aggagactgg cgtgaaccca ggaggcggac cttgtagtga gccgagatcg 780
cgccactgtg ctccagcctg ggcaacagag caagactcca tctcaaaaaa taaaaataaa 840
taaaaaataa aaaaataaaa gagcaaacta tttccaaata ccatagaata acttacataa 900
aagtaatata actgtattgt aagtagaagc tagcactgg 939

CA 02522815 2005-10-19
9/11
<210> s
<211> 20
<212> DNA
c213> Homo Sapiens
<400> 9
ggcggggggc gcacagagcc 20
<210> 10
<211> 22
c212> DNA
<213> Homo Sapiens
<400> 10
gagatggagt cttgctctgt tg 22
<210> 11
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 11
acagagtgga ataagacaga g 21
<210> 12
<211> 21
<212> DNA
<213> Homo Sapiens
<400> 12
ccagtgctag cttctactta c 21
<210> 13
<211> 425
<212> PRT
<213> Homo Sapiens
<400> 13
Met Gly Pro Arg Arg Leu Leu Leu Val Ala Ala Cys Phe Ser Leu Cys
1 . 5 10 15
Gly Pro Leu Leu Ser Ala Arg Thr Arg Ala Arg Arg Pro Glu Ser Lys
20 25 34

CA 02522815 2005-10-19
10/11
Ala Thr Asn A1a Thr Leu Asp Pro Arg Ser Phe Leu Leu Arg Asn Pro
35 40 45
Asn Asp Lys Tyr Glu Pro Phe Trp Glu Asp Glu Glu Lys Asn Glu Ser
50 55 60
Gly Leu Thr Glu Tyr Arg Leu Val Ser Ile Asn Lys Ser Ser Pro Leu
65 70 75 . 80
Gln Lys Gln Leu Pro Ala Phe Ile Ser Glu Asp Ala Ser Gly Tyr Leu
85 90 95
Thr Ser Ser Trp Leu Thr Leu Phe Val Pro Ser Val Tyr Thr Gly Val
100 105 110
Phe Val Val Ser Leu Pro Leu Asn Ile Met Ala Ile Val Val Phe Ile
115 120 125
Leu Lys Met Lys Val Lys Lys Pro Ala Val Val Tyr Met Leu His Leu
130 135 140
Ala Thr Ala Asp Val Leu Phe Val Ser Val Leu Pro Phe.Lys Ile Ser
145 150 155 160
Tyr Tyr Phe Ser Gly Ser Asp Trp Gln Phe Gly Ser Glu Leu Cys Arg
165 ~ 170 175
Phe Val Thr Ala Ala Phe Tyr Cys Asn Met Tyr Ala Ser Ile Leu Leu
180 185 190
Met Thr Val Ile Ser Ile Asp Arg Phe Leu Ala Val Val Tyr Pro Met
195 200 205

CA 02522815 2005-10-19
11/11
Gln Ser Leu Ser Trp Arg Thr Leu Gly Arg Ala Ser Phe Thr Cys Leu
210 215 220
Ala Ile Trp Ala Leu Ala Ile Ala Gly Val Val Pro Leu Val Leu Lys
225 230 235 240
Glu Gln Thr Ile Gln Val Pro Gly Leu Asn Ile Thr Thr Cys His Asp
245 250 255
Val Leu Asn Glu Thr Leu Leu Glu Gly Tyr Tyr Ala Tyr Tyr Phe Ser
260 265 270
Ala Phe Ser Ala Val Phe Phe Phe Val Pro Leu Ile Ile Ser Thr Val
275 280 285
Cys Tyr Val Ser Ile Ile Arg Cps Leu Ser 5er Ser Ala Val Ala Asn
290 295 300
Arg Ser Lys Lys Ser Arg Ala Leu Phe Leu Ser Ala Ala Val Phe Cps
305 310 315 320
Ile Phe Ile Ile Cys Phe Gly Pro Thr Asn Val Leu Leu Ile Ala His
325 330 335
Tyr Ser Phe Leu Ser His Thr Ser Thr Thr Glu Ala Ala Tyr Phe Ala
340 345 350
Tyr Leu Leu Cys Val Cys Val Ser Ser Ile Ser Ser Cys Ile Asp Pro
355 360 365
Leu Ile Tyr Tyr Tyr Ala Ser Ser Glu Cys Gla Arg Tyr Val Tyr Ser
370 375 380
Ile Leu Cys Cys Lys Glu Ser 5er Asp Pro Ser 5er Tyr Asn Ser Ser
385 390 395 400
Gly Gln Leu Met Ala Ser Lys Met Asp Thr.Cys Ser Ser Rsn Leu Asn
405 410 415
Asn 5er Ile Tyr Lys Lys Leu Leu Thr
420 425

Representative Drawing

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Administrative Status

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Event History

Description Date
Inactive: IPC expired 2018-01-01
Inactive: Office letter 2012-01-05
Inactive: Withdraw application 2011-12-22
Inactive: Withdraw application 2011-12-22
Amendment Received - Voluntary Amendment 2011-07-27
Inactive: S.30(2) Rules - Examiner requisition 2011-02-01
Letter Sent 2009-05-21
Request for Examination Requirements Determined Compliant 2009-04-16
Request for Examination Received 2009-04-16
All Requirements for Examination Determined Compliant 2009-04-16
Letter Sent 2007-01-23
Letter Sent 2007-01-23
Letter Sent 2007-01-23
Letter Sent 2007-01-23
Inactive: Correspondence - Formalities 2006-12-06
Inactive: Single transfer 2006-12-06
Inactive: IPC removed 2006-11-09
Inactive: IPC assigned 2006-11-09
Inactive: IPC assigned 2006-11-09
Inactive: IPC assigned 2006-11-09
Inactive: IPC assigned 2006-11-09
Inactive: IPC assigned 2006-11-09
Inactive: First IPC assigned 2006-11-09
Inactive: Sequence listing - Amendment 2006-08-16
Amendment Received - Voluntary Amendment 2006-08-16
Inactive: Office letter 2006-07-14
Inactive: Cover page published 2005-12-13
Inactive: Courtesy letter - Evidence 2005-12-13
Inactive: First IPC assigned 2005-12-11
Inactive: Notice - National entry - No RFE 2005-12-09
Application Received - PCT 2005-11-22
National Entry Requirements Determined Compliant 2005-10-19
Application Published (Open to Public Inspection) 2004-11-04

Abandonment History

There is no abandonment history.

Maintenance Fee

The last payment was received on 2011-03-18

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  • the late payment fee; or
  • additional fee to reverse deemed expiry.

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Fee History

Fee Type Anniversary Year Due Date Paid Date
MF (application, 2nd anniv.) - standard 02 2006-04-18 2005-10-19
Basic national fee - standard 2005-10-19
Registration of a document 2006-12-06
MF (application, 3rd anniv.) - standard 03 2007-04-16 2007-03-21
MF (application, 4th anniv.) - standard 04 2008-04-16 2008-03-28
MF (application, 5th anniv.) - standard 05 2009-04-16 2009-03-27
Request for examination - standard 2009-04-16
MF (application, 6th anniv.) - standard 06 2010-04-16 2010-03-25
MF (application, 7th anniv.) - standard 07 2011-04-18 2011-03-18
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
SANOFI-AVENTIS DEUTSCHLAND GMBH
Past Owners on Record
DETLEF KOZIAN
JEAN-FRANCOIS DELEUZE
JOERG CZECH
KARL-ERNST SIEGLER
SANDRINE MACE
SYLVAIN RICARD
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Description 2011-07-27 38 1,785
Description 2005-10-19 38 1,732
Drawings 2005-10-19 12 514
Abstract 2005-10-19 1 70
Claims 2005-10-19 5 209
Cover Page 2005-12-13 1 30
Description 2006-08-16 39 1,775
Claims 2006-08-16 5 211
Claims 2011-07-27 6 197
Notice of National Entry 2005-12-09 1 192
Request for evidence or missing transfer 2006-10-23 1 101
Courtesy - Certificate of registration (related document(s)) 2007-01-23 1 127
Courtesy - Certificate of registration (related document(s)) 2007-01-23 1 127
Courtesy - Certificate of registration (related document(s)) 2007-01-23 1 127
Courtesy - Certificate of registration (related document(s)) 2007-01-23 1 127
Reminder - Request for Examination 2008-12-17 1 117
Acknowledgement of Request for Examination 2009-05-21 1 175
PCT 2005-10-19 3 102
Correspondence 2005-12-09 1 27
PCT 2005-10-19 1 44
PCT 2004-04-16 1 47
Correspondence 2006-07-14 1 28
Correspondence 2006-12-06 1 46
Correspondence 2011-12-22 1 31

Biological Sequence Listings

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