U.S. patent application number 13/294802 was filed with the patent office on 2012-06-14 for genetic variants in the tcf7l2 gene as diagnostic markers for risk of type 2 diabetes mellitus.
This patent application is currently assigned to deCODE Genetics ehf.. Invention is credited to Struan F.A. Grant.
Application Number | 20120149016 13/294802 |
Document ID | / |
Family ID | 37036993 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120149016 |
Kind Code |
A1 |
Grant; Struan F.A. |
June 14, 2012 |
Genetic Variants in the TCF7L2 Gene as Diagnostic Markers for Risk
of Type 2 Diabetes Mellitus
Abstract
Polymorphisms in the gene TCF7L2 are shown by association
analysis to be a susceptibility gene for type II diabetes. Methods
of diagnosis of susceptibility to diabetes, of decreased
susceptibility to diabetes and protection against diabetes, are
described, as are methods of treatment for type II diabetes.
Inventors: |
Grant; Struan F.A.;
(Reykjavik, IS) |
Assignee: |
deCODE Genetics ehf.
Reykjavik
IS
|
Family ID: |
37036993 |
Appl. No.: |
13/294802 |
Filed: |
November 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12456381 |
Jun 15, 2009 |
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13294802 |
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11454296 |
Jun 16, 2006 |
7585630 |
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12456381 |
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60757155 |
Jan 6, 2006 |
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60692174 |
Jun 20, 2005 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12Q 2600/158 20130101;
A61P 3/10 20180101; C12Q 1/6886 20130101; C12Q 2600/172 20130101;
C12Q 2600/156 20130101; C12Q 1/6883 20130101; C12Q 2600/106
20130101; C12Q 2600/136 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of diagnosing a susceptibility to type II diabetes in
an individual, comprising analyzing at least one allele of at least
one marker associated with the exon 4 LD block of Transcription
Factor 7-Like 2 Gene (TCF7L2) in nucleic acid from the individual,
wherein the at least one marker is selected from the group
consisting of DG10S478, rs12255372, rs7895340, rs11196205,
rs7901695, rs7903146, rs12243326, rs4506565, and markers in linkage
disequilibrium, characterized by r.sup.2 greater than 0.2, with any
of said markers; and diagnosing a susceptibility to type II
diabetes in the individual from the presence or absence of the at
least one allele, wherein the presence of an at-risk allele for the
at least one marker in the nucleic acid is indicative of an
increased susceptibility to type II diabetes, and the absence of an
at-risk allele is indicative of a decreased susceptibility to type
II diabetes.
2. The method of claim 1, wherein the at least one marker comprises
at least one marker selected from the group consisting of
rs4074720, rs4074719, rs4074718, rs11196181, rs11196182, rs4603236,
rs7922298, rs17747324, rs7901695, rs11196185, rs4132115, rs4506565,
rs7068741, rs7069007, rs7903146, rs11196187, rs7092484, rs10885402,
rs12098651, rs6585198, rs7910244, rs12266632, rs6585199, rs7896811,
rs6585200, rs6585201, rs4319449, rs12220336, rs7896091, rs12354626,
rs7075199, rs7904519, rs13376896, rs10885405, rs10885406,
rs11196192, rs6585202, rs7924080, rs7907610, rs12262948,
rs12243326, rs12265110, rs7077039, rs11196198, rs12775336,
rs7904948, rs7100927, rs11196199, rs17685538, rs11592706,
rs7081912, rs7895340, rs11196200, rs11196201, rs11196202,
rs11196203, rs11196204, rs11196205, rs10885409, rs12255372,
rs12265291, rs7904443, rs11196208, rs7077247, rs11196209,
rs4077527, rs12718338, rs11196210, rs7907632, rs7071302,
rs12245680, rs11196213, rs4918789, rs7085785, rs7085989, rs7087006,
SG10S405, SG10S428, SG10S422, SG10S427, SG10S408, SG10S409,
SG10S406, SG10S407, DG10S2164, DG10S478, and DG10S479.
3. The method of claim 1, wherein the susceptibility is an
increased susceptibility characterized by a relative risk of at
least 1.2.
4. (canceled)
5. The method of claim 1, wherein the marker is selected from the
group consisting of DG10S478, rs12255372, rs7895340, rs11196205,
rs7901695, rs7903146, rs12243326, and rs4506565.
6. The method of claim 1, wherein the marker is marker DG10S478, or
a marker in linkage disequilibrium with DG10S478, characterized by
an r.sup.2 greater than 0.2, and wherein the presence of a non-0
allele in DG10S478 is indicative of increased susceptibility to
type II diabetes.
7. The method of claim 1, wherein the marker is marker rs7903146,
or a marker in linkage disequilibrium with rs7903146, characterized
by an r.sup.2 greater than 0.2, and wherein the presence of a T
allele in rs7903146 is indicative of increased susceptibility to
type II diabetes.
8. The method according to claim 1 of diagnosing a decreased
susceptibility to type II diabetes in an individual, comprising
detecting absence of the at-risk allele, wherein the absence of the
at-risk allele is indicative of a decreased susceptibility to type
II diabetes.
9. The method of claim 8, wherein the decreased susceptibility is
characterized by a relative risk of less than 0.8.
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. A method according to claim 1 of detecting an increased
susceptibility to type II diabetes in an individual, comprising
detecting an allele of at least one marker located within the exon
4 LD block of TCF7L2 in the individual, wherein identification of
said allele at the polymorphism that is indicative of increased
risk of type II diabetes in the individual.
19. (canceled)
20. (canceled)
21. The method of claim 18, wherein the at least one marker is
selected from the group consisting of rs4074720, rs4074719,
rs4074718, rs11196181, rs11196182, rs4603236, rs7922298,
rs17747324, rs7901695, rs11196185, rs4132115, rs4506565, rs7068741,
rs7069007, rs7903146, rs11196187, rs7092484, rs10885402,
rs12098651, rs6585198, rs7910244, rs12266632, rs6585199, rs7896811,
rs6585200, rs6585201, rs4319449, rs12220336, rs7896091, rs12354626,
rs7075199, rs7904519, rs13376896, rs10885405, rs10885406,
rs11196192, rs6585202, rs7924080, rs7907610, rs12262948,
rs12243326, rs12265110, rs7077039, rs11196198, rs12775336,
rs7904948, rs7100927, rs11196199, rs17685538, rs11592706,
rs7081912, rs7895340, rs11196200, rs11196201, rs11196202,
rs11196203, rs11196204, rs11196205, rs10885409, rs12255372,
rs12265291, rs7904443, rs11196208, rs7077247, rs11196209,
rs4077527, rs12718338, rs11196210, rs7907632, rs7071302,
rs12245680, rs11196213, rs4918789, rs7085785, rs7085989, rs7087006,
SG10S405, SG10S428, SG10S422, SG10S427, SG10S408, SG10S409,
SG10S406, SG10S407, DG10S2164, DG10S478, and DG10S479.
22. The method according to claim 1 of diagnosing a decreased
susceptibility to type II diabetes in an individual, comprising
detecting absence of the at-risk allele located within the exon 4
LD block of TCF7L2 wherein the absence of the at-risk allele is
indicative of decreased risk of type II diabetes in the
individual.
23. (canceled)
24. (canceled)
25. The method of claim 22, wherein the at least one marker is
selected from the group consisting of rs4074720, rs4074719,
rs4074718, rs11196181, rs11196182, rs4603236, rs7922298,
rs17747324, rs7901695, rs11196185, rs4132115, rs4506565, rs7068741,
rs7069007, rs7903146, rs11196187, rs7092484, rs10885402,
rs12098651, rs6585198, rs7910244, rs12266632, rs6585199, rs7896811,
rs6585200, rs6585201, rs4319449, rs12220336, rs7896091, rs12354626,
rs7075199, rs7904519, rs13376896, rs10885405, rs10885406,
rs11196192, rs6585202, rs7924080, rs7907610, rs12262948,
rs12243326, rs12265110, rs7077039, rs11196198, rs12775336,
rs7904948, rs7100927, rs11196199, rs17685538, rs11592706,
rs7081912, rs7895340, rs11196200, rs11196201, rs11196202,
rs11196203, rs11196204, rs11196205, rs10885409, rs12255372,
rs12265291, rs7904443, rs11196208, rs7077247, rs11196209,
rs4077527, rs12718338, rs11196210, rs7907632, rs7071302,
rs12245680, rs11196213, rs4918789, rs7085785, rs7085989, rs7087006,
SG10S405, SG10S428, SG10S422, SG10S427, SG10S408, SG10S409,
SG10S406, SG10S407, DG10S2164, DG10S478, and DG10S479.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/454,296, filed Jun. 16, 2006, which claims the benefit of
U.S. Provisional Application No. 60/757,155, filed on Jan. 6, 2006
and U.S. Provisional Application No. 60/692,174, filed on Jun. 20,
2005. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Diabetes mellitus, a metabolic disease wherein carbohydrate
utilization is reduced and lipid and protein utilization is
enhanced, is caused by an absolute or relative deficiency of
insulin. In the more severe cases, diabetes is characterized by
chronic hyperglycemia, glycosuria, water and electrolyte loss,
ketoacidosis and coma. Long term complications include development
of neuropathy, retinopathy, nephropathy, generalized degenerative
changes in large and small blood vessels and increased
susceptibility to infection. The most common form of diabetes is
Type II, non-insulin-dependent diabetes that is characterized by
hyperglycemia due to impaired insulin secretion and insulin
resistance in target tissues. Both genetic and environmental
factors contribute to the disease. For example, obesity plays a
major role in the development of the disease. Type II diabetes is
often a mild form of diabetes mellitus of gradual onset.
[0003] The health implications of Type II diabetes are enormous. In
1995, there were 135 million adults with diabetes worldwide. It is
estimated that close to 300 million will have diabetes in the year
2025. (King H., et al., Diabetes Care, 21(9): 1414-1431 (1998)).
The prevalence of Type II diabetes in the adult population in
Iceland is 2.5% (Vilbergsson, S., et al., Diabet. Med., 14(6):
491-498 (1997)), which comprises approximately 5,000 people over
the age of 34 who have the disease. The high prevalence of the
disease and increasing population affected shows an unmet medical
need to define the genetic factors involved in Type II diabetes to
more precisely define the associated risk factors. Also needed are
therapeutic agents for prevention of Type II diabetes.
SUMMARY OF THE INVENTION
[0004] The present invention relates to methods of diagnosing an
increased susceptibility to type II diabetes, as well as methods of
diagnosing a decreased susceptibility to type II diabetes or
diagnosing a protection against type II diabetes, by evaluating
certain markers or haplotypes relating to the TCF7L2 gene
(transcription factor 7-like 2 (T-cell specific, HMG-box),
previously referred to as the TCF4 gene (T-cell transcription
factor 4)). The methods comprise detecting a genetic marker
associated with the exon 4 LD block of TCF7L2 gene.
[0005] In a first aspect, the invention relates to a method of
diagnosing a susceptibility to type II diabetes in an individual,
comprising analyzing a nucleic acid sample obtained from the
individual for a marker or haplotype associated with the exon 4 LD
block of TCF7L2, wherein the presence of the marker or haplotype is
indicative of a susceptibility to type II diabetes. In one
embodiment, the marker or haplotype comprises at least one marker
selected from the markers listed in Table 6. In another embodiment,
the marker or haplotype is a marker.
[0006] In one preferred embodiment, the marker or haplotype is
indicative of increased susceptibility of type II diabetes. The
increased susceptibility is in one embodiment characterized by a
relative risk of at least 1.2, including a relative risk of at
least 1.3 and a relative risk of at least 1.4. In one embodiment,
the marker is selected from the group consisting of DG10S478,
rs12255372, rs7895340, rs11196205, rs7901695, rs7903146,
rs12243326, and rs4506565, and wherein the presence of a non-0
allele (e.g., -4, 4, 8, 12, 16, 20, or other non-0 allele) in
DG10S478, a T allele in rs12255372; an A allele in rs7895340; a C
allele in rs11196205; a C allele in rs7901695; a T allele in
rs7903146; a C allele in rs12243326; or an T allele in rs4506565,
is indicative of increased susceptibility to type II diabetes. In a
preferred embodiment, the marker is selected from the group
consisting of DG10S478 and rs7903146, and wherein the presence of a
non-0 allele in DG10S478 or a T allele in rs7903146 is indicative
of increased susceptibility to type II diabetes. In yet another
preferred embodiment, the marker is rs7903146, and wherein the
presence of a T allele in rs7903146 is indicative of increased
susceptibility to type II diabetes.
[0007] In another preferred embodiment, the marker or haplotype is
indicative of decreased susceptibility of type II diabetes. The
decreased susceptibility is in one embodiment characterized by a
relative risk of less than 0.8, including a relative risk of less
than 0.7. In one embodiment, the marker is selected from the group
consisting of DG10S478, rs12255372, rs7895340, rs11196205,
rs7901695, rs7903146, rs12243326, and rs4506565, and wherein the
presence of a 0 allele in DG10S478, a G allele in SNP rs12255372; a
G allele in rs7895340; a G allele in rs11196205; a T allele in
rs7901695; a C allele in rs7903146; a T allele in rs12243326; or an
A allele in rs4506565 is indicative of a decreased susceptibility
to type II diabetes. In a preferred embodiment, the marker is
DG10S478, and wherein the presence of a 0 allele in DG10S478 is
indicative of decreased susceptibility to type II diabetes. In
another preferred embodiment, the marker is rs7903146, and wherein
the presence of a C allele in rs7903146 is indicative of decreased
susceptibility to type II diabetes.
[0008] In a second aspect, the present invention relates to a kit
for assaying a sample from an individual to detect a susceptibility
to type II diabetes, wherein the kit comprises one or more reagents
for detecting one or more markers associated with the exon 4 LD
block of TCF7L2. In one embodiment, the one or more reagents
comprise at least one contiguous nucleotide sequence that is
completely complementary to a region comprising at least one marker
associated with the exon 4 LD block of TCF7L2. In one embodiment,
the one or markers is selected from the group consisting of
DG10S478, rs12255372, rs7895340, rs11196205, rs7901695, rs7903146,
rs12243326, and rs4506565. In a preferred embodiment, the one or
more marker is DG10S478 or rs7903146. In another preferred
embodiment, the marker is the C allele in rs7903146.
[0009] In another aspect, the present invention relates to a method
of assessing an individual for probability of response to a TCF7L2
therapeutic agent, comprising: detecting a marker associated with
the exon 4 LD block of TCF7L2, wherein the presence of the marker
is indicative of a probability of a positive response to a TCF7L2
therapeutic agent. In one embodiment, the marker is selected from
the group consisting of DG10S478, rs12255372, rs7895340,
rs11196205, rs7901695, rs7903146, rs12243326, and rs4506565. In
another embodiment, the marker is marker DG10S478 or marker
rs7903146, and wherein the presence of a non-0 allele in DG10S478
or a T allele in rs7903146 is indicative of a probability of a
positive response to a TCF7L2 therapeutic agent.
[0010] Another aspect of the invention relates to the use of a
TCF7L2 therapeutic agent for the manufacture of a medicament for
the treatment of type II diabetes. In one embodiment, the TCF7L2
therapeutic agent is an agent that alters activity in the Wnt
signaling pathway or in the cadherin pathway. In another
embodiment, the TCF7L2 therapeutic agent is an agent selected from
the group set forth in the Agent Table.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention. The patent
or application file contains at least one drawing executed in
color. Copies of this patent or patent application publication with
color drawings will be provided by the Office upon request and
payment of the necessary fee.
[0012] The FIGURE depicts the TCF7L2 region of interest with
respect to linkage disequilibrium (LD) of SNPs in HapMap project
Build 16. The 215.9 kb gene spans seven LD blocks as indicated by
the black arrow schematic (based on NCBI RefSeq) which shows the
direction of transcription; exons are indicated, with exon 4
highlighted. DG10S478 is located at 114.46 Mb on chromosome 10
(NCBI Build 34) in intron 3 of the TCF7L2 gene, within a 74.9 kb
block that incorporates part of intron 3, the whole of exon 4 and
part of intron 4 (herein referred to as the "exon 4 LD block of
TCF7L2"). The SNP markers are plotted equidistantly rather than
according to their physical positions. The FIGURE shows two
measures of LD--i.e. D' (upper left part of FIGURE) and r.sup.2
(lower right part).
DETAILED DESCRIPTION OF THE INVENTION
[0013] A description of preferred embodiments of the invention
follows.
Loci Associated with Type II Diabetes
[0014] Type II diabetes is characterized by hyperglycemia, which
can occur through mechanisms such as impaired insulin secretion,
insulin resistance in peripheral tissues and increased glucose
output by the liver. Most type II diabetes patients suffer serious
complications of chronic hyperglycemia including nephropathy,
neuropathy, retinopathy and accelerated development of
cardiovascular disease. The prevalence of type II diabetes
worldwide is currently 6% but is projected to rise over the next
decade (1). This increase in prevalence of type II diabetes is
attributed to increasing age of the population and rise in
obesity.
[0015] There is evidence for a genetic component to the risk of
type II diabetes, including prevalence differences between various
racial groups (2, 3), higher concordance rates among monozygotic
than dizygotic twins (4, 5) and a sibling relative risk
(.lamda..sub.2) for type II diabetes in European populations of
approximately 3.5 (6).
[0016] Two approaches have thus far been used to search for genes
associated with type II diabetes. Single nucleotide polymorphisms
(SNPs) within candidate genes have been tested for association and
have, in general, not been replicated or confer only a modest risk
of type II diabetes--the most widely reported being a protective
Pro12Ala polymorphism in the peroxisome proliferator activated
receptor gamma gene (PPARG2) (7) and an at risk polymorphism in the
potassium inwardly-rectifying channel, subfamily J, member 11 gene
(KIR6.2) (8).
[0017] Genome-wide linkage scans in families with the common form
of type II diabetes have yielded several loci, and the primary
focus of international research consortia has been on loci on
chromosomes 1, 12 and 20 observed in many populations (6). The
genes in these loci have yet to be uncovered. However, in Mexican
Americans, the calpain 10 (CAPN10) gene was isolated out of a locus
on chromosome 2q; this represents the only gene for the common form
of type II diabetes to date to be identified through positional
cloning (9). The rare Mendelian forms of type II diabetes, namely
maturity-onset diabetes of the young (MODY), have yielded six genes
by positional cloning (6).
[0018] We previously reported genome-wide significant linkage to
chromosome 5q for type II diabetes mellitus in the Icelandic
population (10); in the same study, we also reported suggestive
evidence of linkage to 10q and 12q. Linkage to the 10q region has
also been observed in Mexican Americans (11).
Transcription Factor 7-Like 2 Gene (TCF7L2) Association with Type
II Diabetes
[0019] The present invention relates to identification of a type II
diabetes-associated LD block ("exon 4 LD block of TCF7L2") within
the gene encoding T-cell transcription factor 4 (TCF4-- official
gene symbol TCF7L2). Several markers within the exon 4 LD block of
TCF7L2, including microsatellite DG10S478 and SNP markers rs7903146
and rs12255372, have been found to be associated with type II
diabetes.
The original observation, first found in an Icelandic cohort, of
the association of DG10S478 (P=1.3.times.10.sup.-9; Relative
risk=1.45; Population attributable risk=22.7%), has subsequently
been replicated in a Danish type II diabetes cohort and a United
States Caucasian cohort. DG10S478 is located in intron 3 of the
TCF7L2 gene on 10q25.2 and within a well defined LD block of 74.9
kb that encapsulates part of intron 3, the whole of exon 4 and part
of intron 4. The TCF7L2 gene product is a high mobility group (HMG)
box-containing transcription factor that plays a role in the Wnt
signaling pathway, also known as the APC3/.beta.-catenin/TCF
pathway. TCF7L2 mediates the cell type-specific regulation of
proglucagon gene expression (a key player in blood glucose
homeostasis) through the Wnt pathway members .beta.-catenin and
glycogen synthase kinase-3beta (12). In addition, Wnt signaling
maintains preadipocytes in an undifferentiated state through
inhibition of the adipogenic transcription factors CCAAT/enhancer
binding protein alpha (C/EBPalpha) and peroxisome
proliferator-activated receptor gamma (PPARgamma) (13). When Wnt
signaling in preadipocytes is prevented by overexpression of
dominant-negative TCF7L2, these cells differentiate into adipocytes
(13). In addition, it has been reported that the Wnt/.beta.-catenin
signaling pathway targets PPARgamma activity through physical
interaction with .beta.-catenin and TCF7L2 in colon cancer cells
(14). The multifunctional .beta.-catenin protein is also important
for mediating cell adhesion through its binding of cadherins
(15).
[0020] As a result of this discovery, methods are now available for
diagnosis of a susceptibility to type II diabetes, as well as for
diagnosis of a decreased susceptibility to type II diabetes and/or
a protection against type II diabetes. In preferred embodiments of
the invention, diagnostic assays are used to identify the presence
of particular alleles, including a 0 allele in marker DG10S478
(associated with a decreased susceptibility to type II diabetes and
is an allele that is protective against type II diabetes); a non-0
allele (e.g., -4, 4, 8, 12, 16 or 20, or other allele) in marker
DG10S478 (associated with susceptibility to type II diabetes); a G
allele in SNP rs12255372 (associated with a decreased
susceptibility to type II diabetes and is an allele that is
protective against type II diabetes): a T allele in SNP rs12255372
(associated with susceptibility to type II diabetes); a G allele in
SNP rs7895340 (associated with a decreased susceptibility to type
II diabetes and is an allele that is protective against type II
diabetes); an A allele in SNP rs7895340 (associated with
susceptibility to type II diabetes); a G allele in SNP rs11196205
(associated with a decreased susceptibility to type II diabetes and
is an allele that is protective against type II diabetes); a C
allele in SNP rs11196205 (associated with susceptibility to type II
diabetes); a T allele in SNP rs7901695 (associated with a decreased
susceptibility to type II diabetes and is an allele that is
protective against type II diabetes); a C allele in SNP rs7901695
(associated with susceptibility to type II diabetes); a C allele in
SNP rs7903146 (associated with a decreased susceptibility to type
II diabetes and is an allele that is protective against type II
diabetes); a T allele in SNP rs7903146 (associated with a
susceptibility to type II diabetes); a C allele in SNP rs12243326
(associated with a susceptibility to type II diabetes); and an T
allele in SNP rs4506565 (associated with a susceptibility to type
II diabetes). In additional embodiments of the invention, other
markers or SNPs, identified using the methods described herein, can
be used for diagnosis of a susceptibility to type II diabetes, and
also for diagnosis of a decreased susceptibility to type II
diabetes or for identification of an allele that is protective
against type II diabetes. The diagnostic assays presented below can
be used to identify the presence or absence of these particular
alleles.
Diagnostic Assays
[0021] Nucleic acids, probes, primers, and antibodies such as those
described herein can be used in a variety of methods of diagnosis
of a susceptibility to type II diabetes, as well as in kits (e.g.,
useful for diagnosis of a susceptibility to type II diabetes).
Similarly, the nucleic acids, probes, primers, and antibodies
described herein can be used in methods of diagnosis of a decreased
susceptibility to type II diabetes, as well as in methods of
diagnosis of a protection against type II diabetes, and also in
kits). In one aspect, the kit comprises primers that can be used to
amplify the markers of interest.
[0022] In one aspect of the invention, diagnosis of a
susceptibility to type II diabetes is made by detecting a
polymorphism in a TCF7L2 nucleic acid as described herein (e.g.,
the alleles in marker DG10S478 or in SNP rs12255372, rs7895340,
rs11196205, rs7901695, rs7903146, rs12243326, rs4506565). The
polymorphism can be a change in a TCF7L2 nucleic acid, such as the
insertion or deletion of a single nucleotide, or of more than one
nucleotide, resulting in a frame shift; the change of at least one
nucleotide, resulting in a change in the encoded amino acid; the
change of at least one nucleotide, resulting in the generation of a
premature stop codon; the deletion of several nucleotides,
resulting in a deletion of one or more amino acids encoded by the
nucleotides; the insertion of one or several nucleotides, such as
by unequal recombination or gene conversion, resulting in an
interruption of the coding sequence of the gene; duplication of all
or a part of the gene; transposition of all or a part of the gene;
or rearrangement of all or a part of the gene. More than one such
change may be present in a single gene. Such sequence changes cause
a difference in the polypeptide encoded by a TCF7L2 nucleic acid.
For example, if the difference is a frame shift change, the frame
shift can result in a change in the encoded amino acids, and/or can
result in the generation of a premature stop codon, causing
generation of a truncated polypeptide. Alternatively, a
polymorphism associated with a disease or condition or a
susceptibility to a disease or condition associated with a TCF7L2
nucleic acid can be a synonymous alteration in one or more
nucleotides (i.e., an alteration that does not result in a change
in the polypeptide encoded by a TCF7L2 nucleic acid). Such a
polymorphism may alter splicing sites, affect the stability or
transport of mRNA, or otherwise affect the transcription or
translation of the gene. A TCF7L2 nucleic acid that has any of the
changes or alterations described above is referred to herein as an
"altered nucleic acid."
[0023] In a first method of diagnosing a susceptibility to type II
diabetes, hybridization methods, such as Southern analysis,
Northern analysis, or in situ hybridizations, can be used (see
Current Protocols in Molecular Biology, Ausubel, F. et al., eds,
John Wiley & Sons, including all supplements through 1999). For
example, a biological sample (a "test sample") from a test subject
(the "test individual") of genomic DNA, RNA, or cDNA, is obtained
from an individual (RNA and cDNA can only be used for exonic
markers), such as an individual suspected of having, being
susceptible to or predisposed for, or carrying a defect for, type
II diabetes. The individual can be an adult, child, or fetus. The
test sample can be from any source which contains genomic DNA, such
as a blood sample, sample of amniotic fluid, sample of
cerebrospinal fluid, or tissue sample from skin, muscle, buccal or
conjunctival mucosa, placenta, gastrointestinal tract or other
organs. A test sample of DNA from fetal cells or tissue can be
obtained by appropriate methods, such as by amniocentesis or
chorionic villus sampling. The DNA, RNA, or cDNA sample is then
examined to determine whether a polymorphism in a TCF7L2 nucleic
acid is present, and/or to determine which splicing variant(s)
encoded by the TCF7L2 is present. The presence of the polymorphism
or splicing variant(s) can be indicated by hybridization of the
gene in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A
"nucleic acid probe", as used herein, can be a DNA probe or an RNA
probe; the nucleic acid probe can contain, for example, at least
one polymorphism in a TCF7L2 nucleic acid and/or contain a nucleic
acid encoding a particular splicing variant of a TCF7L2 nucleic
acid. The probe can be any of the nucleic acid molecules described
above (e.g., the gene or nucleic acid, a fragment, a vector
comprising the gene or nucleic acid, a probe or primer, etc.).
[0024] To diagnose a susceptibility to type II diabetes, a
hybridization sample can be formed by contacting the test sample
containing a TCF7L2 nucleic acid with at least one nucleic acid
probe. A preferred probe for detecting mRNA or genomic DNA is a
labeled nucleic acid probe capable of hybridizing to mRNA or
genomic DNA sequences described herein. The nucleic acid probe can
be, for example, a full-length nucleic acid molecule, or a portion
thereof, such as an oligonucleotide of at least 15, 30, 50, 100,
250 or 500 nucleotides in length and sufficient to specifically
hybridize under stringent conditions to appropriate mRNA or genomic
DNA. Suitable probes for use in the diagnostic assays of the
invention are described above (see e.g., probes and primers
discussed under the heading, "Nucleic Acids of the Invention").
[0025] The hybridization sample is maintained under conditions that
are sufficient to allow specific hybridization of the nucleic acid
probe to a TCF7L2 nucleic acid. "Specific hybridization", as used
herein, indicates exact hybridization (e.g., with no mismatches).
Specific hybridization can be performed under high stringency
conditions or moderate stringency conditions, for example, as
described above. In a particularly preferred aspect, the
hybridization conditions for specific hybridization are high
stringency.
[0026] Specific hybridization, if present, is then detected using
standard methods. If specific hybridization occurs between the
nucleic acid probe and TCF7L2 nucleic acid in the test sample, then
the TCF7L2 has the polymorphism, or is the splicing variant, that
is present in the nucleic acid probe. More than one nucleic acid
probe can also be used concurrently in this method. Specific
hybridization of any one of the nucleic acid probes is indicative
of a polymorphism in the TCF7L2 nucleic acid, or of the presence of
a particular splicing variant encoding the TCF7L2 nucleic acid and
can be diagnostic for a susceptibility to type II diabetes, or for
a decreased susceptibility to type II diabetes (or indicative of a
protective allele against type II diabetes).
[0027] In Northern analysis (see Current Protocols in Molecular
Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra)
the hybridization methods described above are used to identify the
presence of a polymorphism or a particular splicing variant,
associated with a susceptibility to type II diabetes or associated
with a decreased susceptibility to type II diabetes. For Northern
analysis, a test sample of RNA is obtained from the individual by
appropriate means. Specific hybridization of a nucleic acid probe,
as described above, to RNA from the individual is indicative of a
polymorphism in a TCF7L2 nucleic acid, or of the presence of a
particular splicing variant encoded by a TCF7L2 nucleic acid and is
therefore diagnostic for the susceptibility to type II diabetes or
the decreased susceptibility to type II diabetes (or indicative of
a protective allele against type II diabetes).
[0028] For representative examples of use of nucleic acid probes,
see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330.
[0029] Alternatively, a peptide nucleic acid (PNA) probe can be
used instead of a nucleic acid probe in the hybridization methods
described above. PNA is a DNA mimic having a peptide-like,
inorganic backbone, such as N-(2-aminoethyl) glycine units, with an
organic base (A, G, C, T or U) attached to the glycine nitrogen via
a methylene carbonyl linker (see, for example, Nielsen, P. E. et
al., Bioconjugate Chemistry 5, American Chemical Society, p. 1
(1994). The PNA probe can be designed to specifically hybridize to
a TCF7L2 nucleic acid. Hybridization of the PNA probe to a TCF7L2
nucleic acid can be diagnostic for a susceptibility to type II
diabetes or decreased susceptibility to type II diabetes (or
indicative of a protective allele against type II diabetes).
[0030] In another method of the invention, alteration analysis by
restriction digestion can be used to detect an alteration in the
gene, if the alteration (mutation) or polymorphism in the gene
results in the creation or elimination of a restriction site. A
test sample containing genomic DNA is obtained from the individual.
Polymerase chain reaction (PCR) can be used to amplify a TCF7L2
nucleic acid (and, if necessary, the flanking sequences) in the
test sample of genomic DNA from the test individual. RFLP analysis
is conducted as described (see Current Protocols in Molecular
Biology, supra). The digestion pattern of the relevant DNA fragment
indicates the presence or absence of the alteration or polymorphism
in the TCF7L2 nucleic acid, and therefore indicates the presence or
absence a susceptibility to type II diabetes or a decreased
susceptibility to type II diabetes (or indicative of a protective
allele against type II diabetes).
[0031] Sequence analysis can also be used to detect specific
polymorphisms in a TCF7L2 nucleic acid. A test sample of DNA or RNA
is obtained from the test individual. PCR or other appropriate
methods can be used to amplify the gene or nucleic acid, and/or its
flanking sequences, if desired. The sequence of a TCF7L2 nucleic
acid, or a fragment of the nucleic acid, or cDNA, or fragment of
the cDNA, or mRNA, or fragment of the mRNA, is determined, using
standard methods. The sequence of the nucleic acid, nucleic acid
fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is compared
with the known nucleic acid sequence of the gene or cDNA or mRNA,
as appropriate. The presence of a polymorphism in the TCF7L2
indicates that the individual has a susceptibility to type II
diabetes or a decreased susceptibility to type II diabetes (or
indicative of a protective allele against type II diabetes).
[0032] Allele-specific oligonucleotides can also be used to detect
the presence of a polymorphism in a TCF7L2 nucleic acid, through
the use of dot-blot hybridization of amplified oligonucleotides
with allele-specific oligonucleotide (ASO) probes (see, for
example, Saiki, R. et al., Nature 324:163-166 (1986)). An
"allele-specific oligonucleotide" (also referred to herein as an
"allele-specific oligonucleotide probe") is an oligonucleotide of
approximately 10-50 base pairs, preferably approximately 15-30 base
pairs, that specifically hybridizes to a TCF7L2 nucleic acid, and
that contains a polymorphism associated with a susceptibility to
type II diabetes or a polymorphism associated with a decreased
susceptibility to type II diabetes (or indicative of a protective
allele against type II diabetes). An allele-specific
oligonucleotide probe that is specific for particular polymorphisms
in a TCF7L2 nucleic acid can be prepared, using standard methods
(see Current Protocols in Molecular Biology, supra). To identify
polymorphisms in the gene that are associated with type II
diabetes, a test sample of DNA is obtained from the individual. PCR
can be used to amplify all or a fragment of a TCF7L2 nucleic acid
and its flanking sequences. The DNA containing the amplified TCF7L2
nucleic acid (or fragment of the gene or nucleic acid) is
dot-blotted, using standard methods (see Current Protocols in
Molecular Biology, supra), and the blot is contacted with the
oligonucleotide probe. The presence of specific hybridization of
the probe to the amplified TCF7L2 nucleic acid is then detected.
Hybridization of an allele-specific oligonucleotide probe to DNA
from the individual is indicative of a polymorphism in the TCF7L2
nucleic acid, and is therefore indicative of susceptibility to type
II diabetes or is indicative of decreased susceptibility to type II
diabetes (or indicative of a protective allele against type II
diabetes).
[0033] The invention further provides allele-specific
oligonucleotides that hybridize to the reference or variant allele
of a gene or nucleic acid comprising a single nucleotide
polymorphism or to the complement thereof. These oligonucleotides
can be probes or primers.
[0034] An allele-specific primer hybridizes to a site on target DNA
overlapping a polymorphism and only primes amplification of an
allelic form to which the primer exhibits perfect complementarity.
See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is
used in conjunction with a second primer, which hybridizes at a
distal site. Amplification proceeds from the two primers, resulting
in a detectable product, which indicates the particular allelic
form is present. A control is usually performed with a second pair
of primers, one of which shows a single base mismatch at the
polymorphic site and the other of which exhibits perfect
complementarity to a distal site. The single-base mismatch prevents
amplification and no detectable product is formed. The method works
best when the mismatch is included in the 3'-most position of the
oligonucleotide aligned with the polymorphism because this position
is most destabilizing to elongation from the primer (see, e.g., WO
93/22456).
[0035] With the addition of such analogs as locked nucleic acids
(LNAs), the size of primers and probes can be reduced to as few as
8 bases. LNAs are a novel class of bicyclic DNA analogs in which
the 2' and 4' positions in the furanose ring are joined via an
O-methylene (oxy-LNA), S-methylene (thio-LNA), or amino methylene
(amino-LNA) moiety. Common to all of these LNA variants is an
affinity toward complementary nucleic acids, which is by far the
highest reported for a DNA analog. For example, particular all
oxy-LNA nonamers have been shown to have melting temperatures of
64EC and 74EC when in complex with complementary DNA or RNA,
respectively, as opposed to 28EC for both DNA and RNA for the
corresponding DNA nonamer. Substantial increases in T.sub.m are
also obtained when LNA monomers are used in combination with
standard DNA or RNA monomers. For primers and probes, depending on
where the LNA monomers are included (e.g., the 3' end, the 5' end,
or in the middle), the T.sub.m could be increased considerably.
[0036] In another aspect, arrays of oligonucleotide probes that are
complementary to target nucleic acid sequence segments from an
individual can be used to identify polymorphisms in a TCF7L2
nucleic acid. For example, in one aspect, an oligonucleotide array
can be used. Oligonucleotide arrays typically comprise a plurality
of different oligonucleotide probes that are coupled to a surface
of a substrate in different known locations. These oligonucleotide
arrays, also described as "Genechips.TM.," have been generally
described in the art, for example, U.S. Pat. No. 5,143,854 and PCT
patent publication Nos. WO 90/15070 and 92/10092. These arrays can
generally be produced using mechanical synthesis methods or light
directed synthesis methods that incorporate a combination of
photolithographic methods and solid phase oligonucleotide synthesis
methods. See Fodor et al., Science 251:767-777 (1991), Pirrung et
al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO
90/15070) and Fodor et al., PCT Publication No. WO 92/10092 and
U.S. Pat. No. 5,424,186, the entire teachings are incorporated by
reference herein. Techniques for the synthesis of these arrays
using mechanical synthesis methods are described in, e.g., U.S.
Pat. No. 5,384,261; the entire teachings are incorporated by
reference herein. In another example, linear arrays can be
utilized.
[0037] Once an oligonucleotide array is prepared, a nucleic acid of
interest is hybridized with the array and scanned for
polymorphisms. Hybridization and scanning are generally carried out
by methods described herein and also in, e.g., published PCT
Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No.
5,424,186, the entire teachings are incorporated by reference
herein. In brief, a target nucleic acid sequence that includes one
or more previously identified polymorphic markers is amplified by
well-known amplification techniques, e.g., PCR. Typically, this
involves the use of primer sequences that are complementary to the
two strands of the target sequence both upstream and downstream
from the polymorphism. Asymmetric PCR techniques may also be used.
Amplified target, generally incorporating a label, is then
hybridized with the array under appropriate conditions. Upon
completion of hybridization and washing of the array, the array is
scanned to determine the position on the array to which the target
sequence hybridizes. The hybridization data obtained from the scan
is typically in the form of fluorescence intensities as a function
of location on the array.
[0038] Although primarily described in terms of a single detection
block, e.g., for detecting a single polymorphism, arrays can
include multiple detection blocks, and thus be capable of analyzing
multiple, specific polymorphisms. In alternative aspects, it will
generally be understood that detection blocks may be grouped within
a single array or in multiple, separate arrays so that varying,
optimal conditions may be used during the hybridization of the
target to the array. For example, it may often be desirable to
provide for the detection of those polymorphisms that fall within
G-C rich stretches of a genomic sequence, separately from those
falling in A-T rich segments. This allows for the separate
optimization of hybridization conditions for each situation.
[0039] Additional uses of oligonucleotide arrays for polymorphism
detection can be found, for example, in U.S. Pat. Nos. 5,858,659
and 5,837,832, the entire teachings of which are incorporated by
reference herein. Other methods of nucleic acid analysis can be
used to detect polymorphisms in a type II diabetes gene or variants
encoded by a type II diabetes gene. Representative methods include
direct manual sequencing (Church and Gilbert, Proc. Natl. Acad.
Sci. USA 81:1991-1995 (1988); Sanger, F. et al., Proc. Natl. Acad.
Sci. USA 74:5463-5467 (1977); Beavis et al., U.S. Pat. No.
5,288,644); automated fluorescent sequencing; single-stranded
conformation polymorphism assays (SSCP); clamped denaturing gel
electrophoresis (CDGE); denaturing gradient gel electrophoresis
(DGGE) (Sheffield, V. C. et al., Proc. Natl. Acad. Sci. USA
86:232-236 (1989)), mobility shift analysis (Orita, M. et al.,
Proc. Natl. Acad. Sci. USA 86:2766-2770 (1989)), restriction enzyme
analysis (Flavell et al., Cell 15:25 (1978); Geever, et al., Proc.
Natl. Acad. Sci. USA 78:5081 (1981)); heteroduplex analysis;
chemical mismatch cleavage (CMC) (Cotton et al., Proc. Natl. Acad.
Sci. USA 85:4397-4401 (1985)); RNase protection assays (Myers, R.
M. et al., Science 230:1242 (1985)); use of polypeptides which
recognize nucleotide mismatches, such as E. coli mutS protein;
allele-specific PCR, for example.
[0040] In one aspect of the invention, diagnosis of a
susceptibility to type II diabetes, or of a decreased
susceptibility to type II diabetes (or indicative of a protective
allele against type II diabetes), can also be made by expression
analysis by quantitative PCR (kinetic thermal cycling). This
technique, utilizing TaqMan.RTM. assays, can assess the presence of
an alteration in the expression or composition of the polypeptide
encoded by a TCF7L2 nucleic acid or splicing variants encoded by a
TCF7L2 nucleic acid. TaqMan.RTM. probes can also be used to allow
the identification of polymorphisms and whether a patient is
homozygous or heterozygous. Further, the expression of the variants
can be quantified as physically or functionally different.
[0041] In another aspect of the invention, diagnosis of a
susceptibility to type II diabetes or of a decreased susceptibility
to type II diabetes (or indicative of a protective allele against
type II diabetes), can be made by examining expression and/or
composition of a TCF7L2 polypeptide, by a variety of methods,
including enzyme linked immunosorbent assays (ELISAs), Western
blots, immunoprecipitations and immunofluorescence. A test sample
from an individual is assessed for the presence of an alteration in
the expression and/or an alteration in composition of the
polypeptide encoded by a TCF7L2 nucleic acid, or for the presence
of a particular variant encoded by a TCF7L2 nucleic acid. An
alteration in expression of a polypeptide encoded by a TCF7L2
nucleic acid can be, for example, an alteration in the quantitative
polypeptide expression (i.e., the amount of polypeptide produced);
an alteration in the composition of a polypeptide encoded by a
TCF7L2 nucleic acid is an alteration in the qualitative polypeptide
expression (e.g., expression of an altered TCF7L2 polypeptide or of
a different splicing variant). In a preferred aspect, diagnosis of
a susceptibility to type II diabetes or of a decreased
susceptibility to type II diabetes can be made by detecting a
particular splicing variant encoded by that TCF7L2 nucleic acid, or
a particular pattern of splicing variants.
[0042] Both such alterations (quantitative and qualitative) can
also be present. The term "alteration" in the polypeptide
expression or composition, as used herein, refers to an alteration
in expression or composition in a test sample, as compared with the
expression or composition of polypeptide by a TCF7L2 nucleic acid
in a control sample. A control sample is a sample that corresponds
to the test sample (e.g., is from the same type of cells), and is
from an individual who is not affected by a susceptibility to type
II diabetes. An alteration in the expression or composition of the
polypeptide in the test sample, as compared with the control
sample, is indicative of a susceptibility to type II diabetes.
Similarly, the presence of one or more different splicing variants
in the test sample, or the presence of significantly different
amounts of different splicing variants in the test sample, as
compared with the control sample, is indicative of a susceptibility
to type II diabetes. Various means of examining expression or
composition of the polypeptide encoded by a TCF7L2 nucleic acid can
be used, including: spectroscopy, colorimetry, electrophoresis,
isoelectric focusing, and immunoassays (e.g., David et al., U.S.
Pat. No. 4,376,110) such as immunoblotting (see also Current
Protocols in Molecular Biology, particularly Chapter 10). For
example, in one aspect, an antibody capable of binding to the
polypeptide (e.g., as described above), preferably an antibody with
a detectable label, can be used. Antibodies can be polyclonal, or
more preferably, monoclonal. An intact antibody, or a fragment
thereof (e.g., Fab or F(ab').sub.2) can be used. The term
"labeled", with regard to the probe or antibody, is intended to
encompass direct labeling of the probe or antibody by coupling
(i.e., physically linking) a detectable substance to the probe or
antibody, as well as indirect labeling of the probe or antibody by
reactivity with another reagent that is directly labeled. Examples
of indirect labeling include detection of a primary antibody using
a fluorescently labeled secondary antibody and end-labeling a DNA
probe with biotin such that it can be detected with fluorescently
labeled streptavidin.
[0043] Western blotting analysis, using an antibody as described
above that specifically binds to a polypeptide encoded by an
altered TCF7L2 nucleic acid or an antibody that specifically binds
to a polypeptide encoded by a non-altered nucleic acid, or an
antibody that specifically binds to a particular splicing variant
encoded by a nucleic acid, can be used to identify the presence in
a test sample of a particular splicing variant or of a polypeptide
encoded by a polymorphic or altered TCF7L2 nucleic acid, or the
absence in a test sample of a particular splicing variant or of a
polypeptide encoded by a non-polymorphic or non-altered nucleic
acid. The presence of a polypeptide encoded by a polymorphic or
altered nucleic acid, or the absence of a polypeptide encoded by a
non-polymorphic or non-altered nucleic acid, is diagnostic for a
susceptibility to type II diabetes, as is the presence (or absence)
of particular splicing variants encoded by the TCF7L2 nucleic
acid.
[0044] In one aspect of this method, the level or amount of
polypeptide encoded by a TCF7L2 nucleic acid in a test sample is
compared with the level or amount of the polypeptide encoded by the
TCF7L2 in a control sample. A level or amount of the polypeptide in
the test sample that is higher or lower than the level or amount of
the polypeptide in the control sample, such that the difference is
statistically significant, is indicative of an alteration in the
expression of the polypeptide encoded by the TCF7L2 nucleic acid,
and is diagnostic for a susceptibility to type II diabetes.
Alternatively, the composition of the polypeptide encoded by a
TCF7L2 nucleic acid in a test sample is compared with the
composition of the polypeptide encoded by the TCF7L2 nucleic acid
in a control sample (e.g., the presence of different splicing
variants). A difference in the composition of the polypeptide in
the test sample, as compared with the composition of the
polypeptide in the control sample, is diagnostic for a
susceptibility to type II diabetes. In another aspect, both the
level or amount and the composition of the polypeptide can be
assessed in the test sample and in the control sample. A difference
in the amount or level of the polypeptide in the test sample,
compared to the control sample; a difference in composition in the
test sample, compared to the control sample; or both a difference
in the amount or level, and a difference in the composition, is
indicative of a susceptibility to type II diabetes.
[0045] The same methods can conversely be used to identify the
presence of a difference when compared to a control (disease)
sample. A difference from the control is indicative of a decreased
susceptibility to diabetes, and/or is indicative of a protective
allele against type II diabetes.
Assessment for Markers and Haplotypes
[0046] Populations of individuals exhibiting genetic diversity do
not have identical genomes. Rather, the genome exhibits sequence
variability between individuals at many locations in the genome; in
other words, there are many polymorphic sites in a population. In
some instances, reference is made to different alleles at a
polymorphic site without choosing a reference allele.
Alternatively, a reference sequence can be referred to for a
particular polymorphic site. The reference allele is sometimes
referred to as the "wild-type" allele and it usually is chosen as
either the first sequenced allele or as the allele from a
"non-affected" individual (e.g., an individual that does not
display a disease or abnormal phenotype). Alleles that differ from
the reference are referred to as "variant" alleles.
[0047] A "marker", as described herein, refers to a genomic
sequence characteristic of a particular variant allele (i.e.
polymorphic site). The marker can comprise any allele of any
variant type found in the genome, including SNPs, microsatellites,
insertions, deletions, duplications and translocations.
[0048] SNP nomenclature as reported herein refers to the official
Reference SNP (rs) ID identification tag as assigned to each unique
SNP by the National Center for Biotechnological Information
(NCBI).
[0049] A "haplotype," as described herein, refers to a segment of a
genomic DNA strand that is characterized by a specific combination
of genetic markers ("alleles") arranged along the segment. In a
certain embodiment, the haplotype can comprise one or more alleles,
two or more alleles, three or more alleles, four or more alleles,
or five or more alleles. The genetic markers are particular
"alleles" at "polymorphic sites" associated with the exon 4 LD
block of TCF7L2. As used herein, "exon 4 LD block of TCF7L2" refers
to the LD block on Chr10q whithin which association of variants to
type II diabetes is observed. NCBI Build 34 position of this LD
block is from 114,413,084-114,488,013 bp. The term
"susceptibility", as described herein, encompasses both increased
susceptibility and decreased susceptibility. Thus, particular
markers and/or haplotypes of the invention may be characteristic of
increased susceptility of type II diabetes, as characterized by a
relative risk of greater than one. Markers and/or haplotypes that
confer increased susceptibility of type II diabetes are furthermore
considered to be "at-risk", as they confer an increased risk of
disease. Alternatively, the markers and/or haplotypes of the
invention are characteristic of decreased susceptibility of type II
diabetes, as characterized by a relative risk of less than one.
[0050] A nucleotide position at which more than one sequence is
possible in a population (either a natural population or a
synthetic population, e.g., a library of synthetic molecules) is
referred to herein as a "polymorphic site". Where a polymorphic
site is a single nucleotide in length, the site is referred to as a
single nucleotide polymorphism ("SNP"). For example, if at a
particular chromosomal location, one member of a population has an
adenine and another member of the population has a thymine at the
same position, then this position is a polymorphic site, and, more
specifically, the polymorphic site is a SNP. Alleles for SNP
markers as referred to herein refer to the bases A, C, G or T as
they occur at the polymorphic site in the SNP assay employed. The
person skilled in the art will realise that by assaying or reading
the opposite strand, the complementary allele can in each case be
measured. Thus, for a polymorphic site containing an A/G
polymorphism, the assay employed may either measure the percentage
or ratio of the two bases possible, i.e. A and G. Alternatively, by
designing an assay that determines the opposite strand on the DNA
template, the percentage or ratio of the complementary bases T/C
can be measured.
[0051] Quantitatively (for example, in terms of relative risk),
identical results would be obtained from measurement of either DNA
strand (+ strand or - strand). Polymorphic sites can allow for
differences in sequences based on substitutions, insertions or
deletions. For example, a polymorphic microsatellite has multiple
small repeats of bases (such as CA repeats) at a particular site in
which the number of repeat lengths varies in the general
population. Each version of the sequence with respect to the
polymorphic site is referred to herein as an "allele" of the
polymorphic site. Thus, in the previous example, the SNP allows for
both an adenine allele and a thymine allele. SNPs and
microsatellite markers located within the exon 4 LD block of TCF7L2
found to be associated with type II diabes are described in Tables
2-7.
[0052] Typically, a reference sequence is referred to for a
particular sequence. Alleles that differ from the reference are
referred to as "variant" alleles. For example, the reference
genomic DNA sequence between positions 114413084 and 114488013 of
NCBI Build 34 (equals 74929 bp, or 74.9 kb), which refers to the
location within Chromosome 10, is described herein as SEQ ID NO:1.
A variant sequence, as used herein, refers to a sequence that
differs from SEQ ID NO:1 but is otherwise substantially similar.
The genetic markers that make up the haplotypes associated with the
exon 4 LD block of TCF7L2 are variants. Additional variants can
include changes that affect a polypeptide, e.g., a polypeptide
encoded by the TCF7L2 gene. These sequence differences, when
compared to a reference nucleotide sequence, can include the
insertion or deletion of a single nucleotide, or of more than one
nucleotide. Such sequence differences may result in a frame shift;
the change of at least one nucleotide, may result in a change in
the encoded amino acid; the change of at least one nucleotide, may
result in the generation of a premature stop codon; the deletion of
several nucleotides, may result in a deletion of one or more amino
acids encoded by the nucleotides; the insertion of one or several
nucleotides, such as by unequal recombination or gene conversion,
may result in an interruption of the coding sequence of a reading
frame; duplication of all or a part of a sequence; transposition;
or a rearrangement of a nucleotide sequence, as described in detail
herein. Such sequence changes alter the polypeptide encoded by the
nucleic acid. For example, if the change in the nucleic acid
sequence causes a frame shift, the frame shift can result in a
change in the encoded amino acids, and/or can result in the
generation of a premature stop codon, causing generation of a
truncated polypeptide. Alternatively, a polymorphism associated
with type II diabetes or a susceptibility to type II diabetes can
be a synonymous change in one or more nucleotides (i.e., a change
that does not result in a change in the amino acid sequence). Such
a polymorphism can, for example, alter splice sites, affect the
stability or transport of mRNA, or otherwise affect the
transcription or translation of an encoded polypeptide. It can also
alter DNA to increase the possibility that structural changes, such
as amplifications or deletions, occur at the somatic level in
tumors. The polypeptide encoded by the reference nucleotide
sequence is the "reference" polypeptide with a particular reference
amino acid sequence, and polypeptides encoded by variant alleles
are referred to as "variant" polypeptides with variant amino acid
sequences.
[0053] A polymorphic microsatellite has multiple small repeats of
bases that are 2-8 nucleotides in length (such as CA repeats) at a
particular site, in which the number of repeat lengths varies in
the general population. An indel is a common form of polymorphism
comprising a small insertion or deletion that is typically only a
few nucleotides long.
[0054] The haplotypes described herein are a combination of various
genetic markers, e.g., SNPs and microsatellites, having particular
alleles at polymorphic sites. The haplotypes can comprise a
combination of various genetic markers, therefore, detecting
haplotypes can be accomplished by methods known in the art for
detecting sequences at polymorphic sites. For example, standard
techniques for genotyping for the presence of SNPs and/or
microsatellite markers can be used, such as fluorescence-based
techniques (Chen, X. et al., Genome Res. 9(5): 492-98 (1999)), PCR,
LCR, Nested PCR and other techniques for nucleic acid
amplification. These markers and SNPs can be identified in at-risk
haplotypes. Certain methods of identifying relevant markers and
SNPs include the use of linkage disequilibrium (LD) and/or LOD
scores.
[0055] In certain methods described herein, an individual who is
at-risk for type II diabetes is an individual in whom an at-risk
marker or haplotype is identified. In one aspect, the at-risk
marker or haplotype is one that confers a significant increased
risk (or susceptility) of type II diabetes. In one embodiment,
significance associated with a marker or haplotype is measured by a
relative risk. In a further embodiment, the significance is
measured by a percentage. In one embodiment, a significant
increased risk is measured as a relative risk of at least about
1.2, including but not limited to: 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8 and 1.9. In a further embodiment, a relative risk of at least
1.2 is significant. In a further embodiment, a relative risk of at
least about 1.5 is significant. In a further embodiment, a
significant increase in risk is at least about 1.7 is significant.
In a further embodiment, a significant increase in risk is at least
about 20%, including but not limited to about 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a
further embodiment, a significant increase in risk is at least
about 50%.
[0056] In other embodiments of the invention, the marker or
haplotype confers decreased risk (decreased susceptibility) of type
II diabetes. In one embodiment, significant decreased risk is
measured as a relative risk at less than 0.9, including but not
limited to 0.9, 0.8, 0.7, 0.6, 0.5, and 0.4. In a further
embodiment, significant relative risk is less than 0.7. In another
embodiment, the decreased in risk (or susceptibility) is at least
about 20%, including but not limited to about 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a
further embodiment, a significant decrease in risk is at least
about 30%.
[0057] Thus, the term "susceptibility to type II diabetes"
indicates either an increased risk or susceptility or a decreased
risk or susceptibility of type II diabetes, by an amount that is
significant, when a certain allele, marker, SNP or haplotype is
present; significance is measured as indicated above. The terms
"decreased risk", "decreased susceptibility" and "protection
against," as used herein, indicate that the relative risk is
decreased accordingly when a certain other allele, marker, SNP,
and/or a certain other haplotype, is present. It is understood
however, that identifying whether an increased or decreased risk is
medically significant may also depend on a variety of factors,
including the specific disease, the marker or haplotype, and often,
environmental factors.
[0058] An at-risk marker or haplotype in, or comprising portions
of, the TCF7L2 gene, is one where the marker or haplotype is more
frequently present in an individual at risk for type II diabetes
(affected), compared to the frequency of its presence in a healthy
individual (control), and wherein the presence of the marker or
haplotype is indicative of susceptibility to type II diabetes. As
an example of a simple test for correlation would be a Fisher-exact
test on a two by two table. Given a cohort of chromosomes the two
by two table is constructed out of the number of chromosomes that
include both of the markers or haplotypes, one of the markers or
haplotypes but not the other and neither of the markers or
haplotypes.
[0059] In certain aspects of the invention, at-risk marker or
haplotype is an at-risk marker or haplotype within or near TCF7L2
that significantly correlates with type II diabetes. In other
aspects, an at-risk marker or haplotype comprises an at-risk marker
or haplotype within or near TCF7L2 that significantly correlates
with susceptibility to type II diabetes. In particular embodiments
of the invention, the marker or haplotype is associated with the
exon 4 LD block of TCF7L2, as described herein.
[0060] Standard techniques for genotyping for the presence of SNPs
and/or microsatellite markers can be used, such as fluorescent
based techniques (Chen, et al., Genome Res. 9, 492 (1999)), PCR,
LCR, Nested PCR and other techniques for nucleic acid
amplification. In a preferred aspect, the method comprises
assessing in an individual the presence or frequency of SNPs and/or
microsatellites in, comprising portions of, the TCF7L2 gene,
wherein an excess or higher frequency of the SNPs and/or
microsatellites compared to a healthy control individual is
indicative that the individual is susceptible to type II diabetes.
Such SNPs and markers can form haplotypes that can be used as
screening tools. These markers and SNPs can be identified in
at-risk haploptypes. For example, an at-risk haplotype can include
microsatellite markers and/or SNPs such as marker DG10S478 and/or
SNP rs12255372, rs7895340, rs11196205, rs7901695, rs7903146,
rs12243326 or rs4506565. The presence of an at-risk haplotype is
indicative of increased susceptibility to type II diabetes, and
therefore is indicative of an individual who falls within a target
population for the treatment methods described herein.
Identification of Susceptibility Variants
[0061] The frequencies of haplotypes in the patient and the control
groups can be estimated using an expectation-maximization algorithm
(Dempster A. et al., J. R. Stat. Soc. B, 39:1-38 (1977)). An
implementation of this algorithm that can handle missing genotypes
and uncertainty with the phase can be used. Under the null
hypothesis, the patients and the controls are assumed to have
identical frequencies. Using a likelihood approach, an alternative
hypothesis is tested, where a candidate at-risk-haplotype, which
can include the markers described herein, is allowed to have a
higher frequency in patients than controls, while the ratios of the
frequencies of other haplotypes are assumed to be the same in both
groups. Likelihoods are maximized separately under both hypotheses
and a corresponding 1-df likelihood ratio statistic is used to
evaluate the statistical significance.
[0062] To look for at-risk and protective markers and haplotypes
within a linkage region, for example, association of all possible
combinations of genotyped markers is studied, provided those
markers span a practical region. The combined patient and control
groups can be randomly divided into two sets, equal in size to the
original group of patients and controls. The marker and haplotype
analysis is then repeated and the most significant p-value
registered is determined. This randomization scheme can be
repeated, for example, over 100 times to construct an empirical
distribution of p-values. In a preferred embodiment, a p-value of
<0.05 is indicative of an significant marker and/or haplotype
association.
[0063] A detailed discussion of haplotype analysis follows.
Haplotype Analysis
[0064] One general approach to haplotype analysis involves using
likelihood-based inference applied to NEsted MOdels (Gretarsdottir
S., et al., Nat. Genet. 35:131-38 (2003)). The method is
implemented in the program NEMO, which allows for many polymorphic
markers, SNPs and microsatellites. The method and software are
specifically designed for case-control studies where the purpose is
to identify haplotype groups that confer different risks. It is
also a tool for studying LD structures. In NEMO, maximum likelihood
estimates, likelihood ratios and p-values are calculated directly,
with the aid of the EM algorithm, for the observed data treating it
as a missing-data problem.
Measuring Information
[0065] Even though likelihood ratio tests based on likelihoods
computed directly for the observed data, which have captured the
information loss due to uncertainty in phase and missing genotypes,
can be relied on to give valid p-values, it would still be of
interest to know how much information had been lost due to the
information being incomplete. The information measure for haplotype
analysis is described in Nicolae and Kong (Technical Report 537,
Department of Statistics, University of Statistics, University of
Chicago; Biometrics, 60(2):368-75 (2004)) as a natural extension of
information measures defined for linkage analysis, and is
implemented in NEMO.
Statistical Analysis
[0066] For single marker association to the disease, the Fisher
exact test can be used to calculate two-sided p-values for each
individual allele. All p-values are presented unadjusted for
multiple comparisons unless specifically indicated. The presented
frequencies (for microsatellites, SNPs and haplotypes) are allelic
frequencies as opposed to carrier frequencies. To minimize any bias
due the relatedness of the patients who were recruited as families
for the linkage analysis, first and second-degree relatives can be
eliminated from the patient list. Furthermore, the test can be
repeated for association correcting for any remaining relatedness
among the patients, by extending a variance adjustment procedure
described in Risch, N. & Teng, J. (Genome Res., 8:1273-1288
(1998)), DNA pooling (ibid) for sibships so that it can be applied
to general familial relationships, and present both adjusted and
unadjusted p-values for comparison. The differences are in general
very small as expected. To assess the significance of single-marker
association corrected for multiple testing we can carry out a
randomization test using the same genotype data. Cohorts of
patients and controls can be randomized and the association
analysis redone multiple times (e.g., up to 500,000 times) and the
p-value is the fraction of replications that produced a p-value for
some marker allele that is lower than or equal to the p-value we
observed using the original patient and control cohorts.
[0067] For both single-marker and haplotype analyses, relative risk
(RR) and the population attributable risk (PAR) can be calculated
assuming a multiplicative model (haplotype relative risk model)
(Terwilliger, J. D. & Ott, J., Hum. Hered. 42:337-46 (1992) and
Falk, C. T. & Rubinstein, P, Ann. Hum. Genet. 51 (Pt 3):227-33
(1987)), i.e., that the risks of the two alleles/haplotypes a
person carries multiply. For example, if RR is the risk of A
relative to a, then the risk of a person homozygote AA will be RR
times that of a heterozygote Aa and RR.sup.2 times that of a
homozygote aa. The multiplicative model has a nice property that
simplifies analysis and computations--haplotypes are independent,
i.e., in Hardy-Weinberg equilibrium, within the affected population
as well as within the control population. As a consequence,
haplotype counts of the affecteds and controls each have
multinomial distributions, but with different haplotype frequencies
under the alternative hypothesis. Specifically, for two haplotypes,
h.sub.i and h.sub.j,
risk(h.sub.i)/risk(h.sub.j)=(f.sub.i/p.sub.i)/(f.sub.j/p.sub.j),
where f and p denote, respectively, frequencies in the affected
population and in the control population. While there is some power
loss if the true model is not multiplicative, the loss tends to be
mild except for extreme cases. Most importantly, p-values are
always valid since they are computed with respect to null
hypothesis.
Linkage Disequilibrium Using NEMO
[0068] LD between pairs of markers can be calculated using the
standard definition of D' and R.sup.2 (Lewontin, R., Genetics
49:49-67 (1964); Hill, W. G. & Robertson, A. Theor. Appl.
Genet. 22:226-231 (1968)). Using NEMO, frequencies of the two
marker allele combinations are estimated by maximum likelihood and
deviation from linkage equilibrium is evaluated by a likelihood
ratio test. The definitions of D' and R.sup.2 are extended to
include microsatellites by averaging over the values for all
possible allele combination of the two markers weighted by the
marginal allele probabilities. When plotting all marker combination
to elucidate the LD structure in a particular region, we plot D' in
the upper left corner and the p-value in the lower right corner. In
the LD plots the markers can be plotted equidistant rather than
according to their physical location, if desired.
Statistical Methods for Linkage Analysis
[0069] Multipoint, affected-only allele-sharing methods can be used
in the analyses to assess evidence for linkage. Results, both the
LOD-score and the non-parametric linkage (NPL) score, can be
obtained using the program Allegro (Gudbjartsson et al., Nat.
Genet. 25:12-3 (2000)). Our baseline linkage analysis uses the
S.sub.pairs scoring function (Whittemore, A. S., Halpern, J.
Biometrics 50:118-27 (1994); Kruglyak L. et al., Am. J. Hum. Genet.
58:1347-63 (1996)), the exponential allele-sharing model (Kong, A.
and Cox, N. J., Am. J. Hum. Genet. 61:1179-88 (1997)) and a family
weighting scheme that is halfway, on the log-scale, between
weighting each affected pair equally and weighting each family
equally. The information measure that we use is part of the Allegro
program output and the information value equals zero if the marker
genotypes are completely uninformative and equals one if the
genotypes determine the exact amount of allele sharing by decent
among the affected relatives (Gretarsdottir et al., Am. J. Hum.
Genet., 70:593-603 (2002)). The P-values were computed two
different ways and the less significant result is reported here.
The first P-value can be computed on the basis of large sample
theory; the distribution of
Z.sub.lr=.quadrature.(2[log.sub.c(10)LOD]) approximates a standard
normal variable under the null hypothesis of no linkage (Kong, A.
and Cox, N. J., Am. J. Hum. Genet. 61:1179-88 (1997)). The second
P-value can be calculated by comparing the observed LOD-score with
its complete data sampling distribution under the null hypothesis
(e.g., Gudbjartsson et al., Nat. Genet. 25:12-3 (2000)). When the
data consist of more than a few families, these two P-values tend
to be very similar.
Haplotypes and "Haplotype Block" Definition of a Susceptibility
Locus
[0070] In certain embodiments, marker and haplotype analysis
involves defining a candidate susceptibility locus based on
"haplotype blocks" (also called "LD blocks"). It has been reported
that portions of the human genome can be broken into series of
discrete haplotype blocks containing a few common haplotypes; for
these blocks, linkage disequilibrium data provided little evidence
indicating recombination (see, e.g., Wall., J. D. and Pritchard, J.
K., Nature Reviews Genetics 4:587-597 (2003); Daly, M. et al.,
Nature Genet. 29:229-232 (2001); Gabriel, S. B. et al., Science
296:2225-2229 (2002); Patil, N. et al., Science 294:1719-1723
(2001); Dawson, E. et al., Nature 418:544-548 (2002); Phillips, M.
S. et al., Nature Genet. 33:382-387 (2003)).
[0071] There are two main methods for defining these haplotype
blocks: blocks can be defined, as regions of DNA that have limited
haplotype diversity (see, e.g., Daly, M. et al., Nature Genet.
29:229-232 (2001); Patil, N. et at, Science 294:1719-1723 (2001);
Dawson. E. et al., Nature 418:544-548 (2002); Zhang, K. et al.,
Proc. Natl. Acad. Sci. USA 99:7335-7339 (2002)), or as regions
between transition zones having extensive historical recombination,
identified using linkage disequilibrium (see, e.g., Gabriel, S. B.
et al., Science 296:2225-2229 (2002); Phillips, M. S. et al.,
Nature Genet. 33:382-387 (2003); Wang, N. et al., Am. J. Hum.
Genet. 71:1227-1234 (2002); Stumpf, M. P., and Goldstein, D. B.,
Curr. Biol. 13:1-8 (2003)). As used herein, the terms "haplotype
block" or "LD block" includes blocks defined by either
characteristic.
[0072] Representative methods for identification of haplotype
blocks are set forth, for example, in U.S. Published Patent
Application Nos. 20030099964, 20030170665, 20040023237 and
20040146870. Haplotype blocks can be used readily to map
associations between phenotype and haplotype status. The main
haplotypes can be identified in each haplotype block, and then a
set of "tagging" SNPs or markers (the smallest set of SNPs or
markers needed to distinguish among the haplotypes) can then be
identified. These tagging SNPs or markers can then be used in
assessment of samples from groups of individuals, in order to
identify association between phenotype and haplotype. If desired,
neighboring haplotype blocks can be assessed concurrently, as there
may also exist linkage disequilibrium among the haplotype
blocks.
Haplotypes and Diagnostics
[0073] As described herein, certain markers and haplotypes
comprising such markers are found to be useful for determination of
susceptibility to type II diabetes--i.e., they are found to be
useful for diagnosing a susceptibility to type II diabetes.
Particular markers and haplotypes are found more frequently in
individuals with type II diabetes than in individuals without type
II diabetes. Therefore, these markers and haplotypes have
predictive value for detecting type II diabetes, or a
susceptibility to type II diabetes, in an individual. Haplotype
blocks (i.e. the exon 4 LD block of TCF7L2) comprising certain
tagging markers, can be found more frequently in individuals with
type II diabetes than in individuals without type II diabetes.
Therefore, these "at-risk" tagging markers within the haplotype
block also have predictive value for detecting type II diabes, or a
susceptibility to type II diabetes, in an individual. "At-risk"
tagging markers within the haplotype or LD blocks can also include
other markers that distinguish among the haplotypes, as these
similarly have predictive value for detecting type II diabetes or a
susceptibility to type II diabetes. As a consequence of the
haplotype block structure of the human genome, a large number of
markers or other variants and/or haplotypes comprising such markers
or variants in association with the haplotype block (LD block) may
be found to be associated with a certain trait and/or phenotype.
Thus, it is possible that markers and/or haplotypes residing within
the exon 4 LD block of TCF7L2 as defined herein or in strong LD
(characterized by r.sup.2 greater than 0.2) with the exon 4 LD
block of TCF7L2 are associated with type II diabetes (i.e. they
confer increased or decreased susceptibility of type II diabetes).
This includes markers that are described herein (Table 6), but may
also include other markers that are in strong LD (characterized by
r.sup.2 greater than 0.2) with one or more of the markers listed in
Table 6. The identification of such additional variants can be
achieved by methods well known to those skilled in the art, for
example by DNA sequencing of the LD block A genomic region in
particular group of individuals, and the present invention also
encompasses such additional variants.
[0074] As described herein, certain markers within the exon 4 LD
block of TCF7L2 are found in decreased frequency in individuals
with type II diabetes, and haplotypes comprising two or more of
those markers listed in Tables 13, 20 and 21 are also found to be
present at decreased frequency in individuals with type II
diabetes. These markers and haplotypes are thus protective for type
II diabetes, i.e. they confer a decreased risk of individuals
carrying these markers and/or haplotypes developing type II
diabetes.
[0075] The haplotypes and markers described herein are, in some
cases, a combination of various genetic markers, e.g., SNPs and
microsatellites. Therefore, detecting haplotypes can be
accomplished by methods known in the art and/or described herein
for detecting sequences at polymorphic sites. Furthermore,
correlation between certain haplotypes or sets of markers and
disease phenotype can be verified using standard techniques. A
representative example of a simple test for correlation would be a
Fisher-exact test on a two by two table.
[0076] In specific embodiments, a marker or haplotype associated
with the exon 4 LD block of TCF7L2 is one in which the marker or
haplotype is more frequently present in an individual at risk for
type II diabetes (affected), compared to the frequency of its
presence in a healthy individual (control), wherein the presence of
the marker or haplotype is indicative of type II diabetes or a
susceptibility to type II diabetes. In other embodiments, at-risk
tagging markers in linkage disequilibrium with one or more markers
associated with the exon 4 LD block of TCF7L2, are tagging markers
that are more frequently present in an individual at risk for type
II diabetes (affected), compared to the frequency of their presence
in a healthy individual (control), wherein the presence of the
tagging markers is indicative of increased susceptibility to type
II diabetes. In a further embodiment, at-risk markers in linkage
disequilibrium with one or more markers associated with the exon 4
LD block of TCF7L2, are markers that are more frequently present in
an individual at risk for type II diabetes, compared to the
frequency of their presence in a healthy individual (control),
wherein the presence of the markers is indicative of susceptibility
to type II diabetes.
[0077] In certain methods described herein, an individual who is at
risk for type II diabetes is an individual in whom an at-risk
marker or haplotype is identified. In one embodiment, the strength
of the association of a marker or haplotype is measured by relative
risk (RR). RR is the ratio of the incidence of the condition among
subjects who carry one copy of the marker or haplotype to the
incidence of the condition among subjects who do not carry the
marker or haplotype. This ratio is equivalent to the ratio of the
incidence of the condition among subjects who carry two copies of
the marker or haplotype to the incidence of the condition among
subjects who carry one copy of the marker or haplotype. In one
embodiment, the marker or haplotype has a relative risk of at least
1.2. In other embodiments, the marker or haplotype has a relative
risk of at least 1.3, at least 1.4, at least 1.5, at least 2.0, at
least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least
5.0.
[0078] In other methods of the invention, an individual who has a
decreased risk (or deceased susceptibility) of type II diabetes is
an individual in whom a protective marker or haplotype is
identified. In such cases, the relative risk (RR) is less than
unity. In one embodiment, the marker or haplotype has a relative
risk of less than 0.9. In another embodiments, the marker or
haplotype has a relative risk of less than 0.8, less than 0.7, less
than 0.6, less than 0.5 or less than 0.4.
Utility of Genetic Testing
[0079] The knowledge about a genetic variant that confers a risk of
developing type II diabetes offers the opportunity to apply a
genetic-test to distinguish between individuals with increased risk
of developing the disease (i.e. carriers of the at-risk variant)
and those with decreased risk of developing the disease (i.e.
carriers of the protective variant). The core values of genetic
testing, for individuals belonging to both of the above mentioned
groups, are the possibilities of being able to diagnose the disease
at an early stage and provide information to the clinician about
prognosis/aggressiveness of the disease in order to be able to
apply the most appropriate treatment. For example, the application
of a genetic test for type II diabetes can provide an opportunity
for the detection of the disease at an earlier stage which may lead
to the application of therapeutic measures at an earlier stage, and
thus can minimize the deleterious effects of the symptoms and
serious health consequences conferred by type II diabetes.
Methods of Therapy
[0080] In another embodiment of the invention, methods can be
employed for the treatment of type II diabetes. The term
"treatment" as used herein, refers not only to ameliorating
symptoms associated with type II diabetes, but also preventing or
delaying the onset of type II diabetes; lessening the severity or
frequency of symptoms of type II diabetes; and/or also lessening
the need for concomitant therapy with other drugs that ameliorate
symptoms associated with type II diabetes. In one aspect, the
individual to be treated is an individual who is susceptible (at an
increased risk) for type II diabetes (e.g., an individual having
the presence of an allele other than a 0 allele in marker DG10S478;
the presence of a T allele in SNP rs12255372; the presence of an A
allele in SNP rs7895340; the presence of a C allele in SNP
rs11196205; the presence of a C allele in SNP rs7901695; the
presence of a T allele in SNP rs7903146; the presence of a C allele
in SNP rs12243326; or the presence of an T allele in SNP
rs4506565.
[0081] In additional embodiments of the invention, methods can be
employed for the treatment of other diseases or conditions
associated with TCF7L2. A TCF7L2 therapeutic agent can be used both
in methods of treatment of type II diabetes, as well as in methods
of treatment of other diseases or conditions associated with
TCF7L2.
[0082] The methods of treatment (prophylactic and/or therapeutic)
utilize a TCF7L2 therapeutic agent. A "TCF7L2 therapeutic agent" is
an agent that alters (e.g., enhances or inhibits) polypeptide
activity and/or nucleic acid expression of TCF7L2, either directly
or indirectly (e.g., through altering activity or nucleic acid
expression of a protein that interacts with TCF7L2, such as a
protein in the Wnt signaling pathway or in the cadherin pathway
(e.g., beta-catenin)). In certain embodiments, the TCF7L2
therapeutic agent alters activity and/or nucleic acid expression of
TCF7L2.
[0083] TCF7L2 therapeutic agents can alter TCF7L2 polypeptide
activity or nucleic acid expression by a variety of means, such as,
for example, by providing additional TCF7L2 polypeptide or by
upregulating the transcription or translation of the TCF7L2 nucleic
acid; by altering posttranslational processing of the TCF7L2
polypeptide; by altering transcription of TCF7L2 splicing variants;
or by interfering with TCF7L2 polypeptide activity (e.g., by
binding to a TCF7L2 polypeptide), or by binding to another
polypeptide that interacts with TCF7L2, by altering (e.g.,
downregulating) the expression, transcription or translation of a
TCF7L2 nucleic acid, or by altering (e.g., agonizing or
antagonizing) activity.
[0084] Representative TCF7L2 therapeutic agents include the
following: nucleic acids or fragments or derivatives thereof
described herein, particularly nucleotides encoding the
polypeptides described herein and vectors comprising such nucleic
acids (e.g., a gene, cDNA, and/or mRNA, such as a nucleic acid
encoding a TCF7L2 polypeptide or active fragment or derivative
thereof, or an oligonucleotide; or a complement thereof, or
fragments or derivatives thereof, and/or other splicing variants
encoded by a Type II diabetes nucleic acid, or fragments or
derivatives thereof); polypeptides described herein and/or splicing
variants encoded by the TCF7L2 nucleic acid or fragments or
derivatives thereof; other polypeptides (e.g., TCF7L2 receptors);
TCF7L2 binding agents; or agents that affect (e.g., increase or
decrease) activity, antibodies, such as an antibody to an altered
TCF7L2 polypeptide, or an antibody to a non-altered TCF7L2
polypeptide, or an antibody to a particular splicing variant
encoded by a TCF7L2 nucleic acid as described above;
peptidomimetics; fusion proteins or prodrugs thereof; ribozymes;
other small molecules; and other agents that alter (e.g., enhance
or inhibit) expression of a TCF7L2 nucleic acid, or that regulate
transcription of TCF7L2 splicing variants (e.g., agents that affect
which splicing variants are expressed, or that affect the amount of
each splicing variant that is expressed). Additional representative
TCF7L2 therapeutic agents include compounds that influence insulin
signaling and/or glucagons, GLP-1 or GIP signaling. More than one
TCF7L2 therapeutic agent can be used concurrently, if desired.
[0085] In preferred embodiments, the TCF7L2 therapeutic agent is an
agent that interferes with the activity of TCF7L2, such as, for
example, an agent that interferes with TCF7L2 binding or
interaction of TCF7L2 with beta-catenin (see, e.g., Fasolini, et
al., J. Biol. Chem. 278(23):21092-06 (2003)) or with other
proteins. Other TCF7L2 therapeutic agents include agents that
affect the Wnt signaling pathway or agents that affect the cadherin
pathway. Representative agents include agents such as those used
for cancer therapy, including, for example, proteins such as the
DKK proteins; the beta-catenin binding domain of APC, or Axin;
factors such as IDAX, AXAM and ICAT; antisense oligonucleotides or
RNA interference (RNAi), such as with the use of Vitravene;
oncolytic viral vectors; and other compounds (see, e.g., Luu et
al., Current Cancer Drug Targets 4:6530671 (2004)); small molecule
antagonists, including, for example, ZTM00990, PKF118-310,
PKF118-744, PKF115-584, PKF222-815, CGPO49090, NPDDG39.024, and
NPDDG1.024 as described by Lepourcelet et al. (see, e.g.,
Lepourcelet et al., Cancer Call 5:91-102 (2004)); compounds
described in U.S. Pat. No. 6,762,185; compounds described in US
Patent applications 20040005313, 20040072831, 20040247593, or
20050059628. Other representative TCF7L2 therapeutic agents include
gsk3 inhibitors, including, for example, those described in U.S.
Pat. Nos. 6,057,117; 6,153,618; 6,417,185; 6,465,231; 6,489,344;
6,512,102; 6,608,063; 6,716,624; 6,800,632; and published US Patent
applications 20030008866; 20030077798; 20030130289; 20030207883;
2000092535; and 200500851. The entire teachings of all of the
references, patents and patent applications recited in the
Specification are incorporated herein in their entirety.
[0086] Additional representative TCF7L2 therapeutic agents are
shown in the Agent Table, below.
TABLE-US-00001 AGENT TABLE Compound name (generated using Autonom,
ISIS Draw Compound version 2.5 from MDL Compound name(s)
Information Systems) Company Reference Indications AR-0133418
1-(4-Methoxy-benzyl)-3- AstraZeneca AD (SN-4521)
(5-nitro-thiazol-2-yl)- urea AR-025028 NSD AstraZeneca CT-98023
N-[4-(2,4-Dichloro- Chiron Corp Wagman et non-insulin
phenyl)-5-(1H-imidazol- al., Curr dependent 2-yl)-pyrimidin-2-yl]-
Pharm. Des diabetes N'-(5-nitro-pyridin-2- 2004:
yl)-ethane-1,2-diamine 10(10) 1105-37 CT-20026 NSD Chiron Corp
non-insulin dependent diabetes CT-21022 NSD Chiron Corp non-insulin
dependent diabetes CT-20014 NSD Chiron Corp non-insulin dependent
diabetes CT-21018 NSD Chiron Corp non-insulin dependent diabetes
CHIR-98025 NSD Chiron Corp Wagman et non-insulin al., Curr
dependent Pharm. Des diabetes 2004: 10(10) 1105-37 CHIR-99021 NSD
Chiron Corp WO- non-insulin CrystalGenomics 2004065370 dependent
and Yuyu diabetes mellitus (Korea) CG-100179 NSD Cyclacel Ltd.
non-insulin 4-[2-(4-Dimethylamino- dependent 3-nitro-phenylamino)-
diabetes, pyrimidin-4-yl]-3,5- among others. dimethyl-1H-pyrrole-2-
carbonitrile NP-01139, 4-Benzyl-2-methyl- Neuropharma SA CNS
disorders, NP-031112, [1,2,4]thiadiazolidine- AD NP-03112,
3,5-dione NP-00361 3-[9-Fluoro-2- Eli Lilly & Co non-insulin
(piperidine-1- dependent carbonyl)-1,2,3,4- diabetes tetrahydro-
[1,4]diazepino[6,7,1- hi]indo1-7-yl]-4- imidazo[1,2-a]pyridin-
3-yl-pyrrole-2,5-dione GW-784752x, Cyclopentanecarboxylic GSK
WO-03024447 non-insulin GW-784775, acid (6-pyridin-3-yl- (compound
dependent SB-216763, furo[2,3-d]pyrimidin-4- referenced: diabetes,
SB-415286 yl)-amide 4-[2-(2- neurodegenerative bromophenyl)-
disease 4-(4- fluorophenyl)- lH- imidazol-5- yl]pyridine
NNC-57-0511, 1-(4-Amino-furazan-3- Novo Nordisk non-insulin
NNC-57-0545, yl)-5-piperidin-1- dependent NNC-57-0588 ylmethyl-1H-
diabetes, [1,2,3]triazole-4- carboxylic acid [l-
pyridin-4-yl-meth-(E)- ylidene]-hydrazide CP-70949 NSD Pfizer
Hypoglycemic agent VX-608 NSD Cerebrovascular ischemia, non-insulin
dependent diabetes NSD Kinetek Nuclear factor kappa B modulator,
Anti- inflammatory, Cell cycle inhibitor, Glycogen synthase
kinase-3 beta inhibitor KP-403 class BYETTA Exenatide:
C.sub.184H.sub.282N.sub.50O.sub.60S- Amylin/Eli non-insulin
(exenatide) Amino acid Lilly & Co dependent
sequence:H-His-Gly-Glu- diabetes Gly-Thr-Phe-Thr-Ser-
Asp-Leu-Ser-Lys-Gln- Met-Glu-Glu-Glu-Ala- Val-Arg-Leu-Phe-Ile-
Glu-Trp-Leu-Lys-Asn- Gly-Gly-Pro-Ser-Ser- Gly-Ala-Pro-Pro-Pro-
Ser-NH.sub.2 Vildaglip- NSD Novartis non-insulin tin dependent
(LAF237) diabetes- DPP-4 inhibitor NSD = No Structure disclosed (in
Iddb3)
[0087] The TCF7L2 therapeutic agent(s) are administered in a
therapeutically effective amount (i.e., an amount that is
sufficient for "treatment," as described above). The amount which
will be therapeutically effective in the treatment of a particular
individual's disorder or condition will depend on the symptoms and
severity of the disease, and can be determined by standard clinical
techniques. In addition, in vitro or in vivo assays may optionally
be employed to help identify optimal dosage ranges. The precise
dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of a
practitioner and each patient's circumstances. Effective doses may
be extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0088] In one embodiment, a nucleic acid (e.g., a nucleic acid
encoding a TCF7L2 polypeptide); or another nucleic acid that
encodes a TCF7L2 polypeptide or a splicing variant, derivative or
fragment thereof can be used, either alone or in a pharmaceutical
composition as described above. For example, a TCF7L2 gene or
nucleic acid or a cDNA encoding a TCF7L2 polypeptide, either by
itself or included within a vector, can be introduced into cells
(either in vitro or in vivo) such that the cells produce native
TCF7L2 polypeptide. If necessary, cells that have been transformed
with the gene or cDNA or a vector comprising the gene, nucleic acid
or cDNA can be introduced (or re-introduced) into an individual
affected with the disease. Thus, cells which, in nature, lack
native TCF7L2 expression and activity, or have altered TCF7L2
expression and activity, or have expression of a disease-associated
TCF7L2 splicing variant, can be engineered to express the TCF7L2
polypeptide or an active fragment of the TCF7L2 polypeptide (or a
different variant of the TCF7L2 polypeptide). In certain
embodiments, nucleic acids encoding a TCF7L2 polypeptide, or an
active fragment or derivative thereof, can be introduced into an
expression vector, such as a viral vector, and the vector can be
introduced into appropriate cells in an animal. Other gene transfer
systems, including viral and nonviral transfer systems, can be
used. Alternatively, nonviral gene transfer methods, such as
calcium phosphate coprecipitation, mechanical techniques (e.g.,
microinjection); membrane fusion-mediated transfer via liposomes;
or direct DNA uptake, can also be used.
[0089] Alternatively, in another embodiment of the invention, a
nucleic acid of the invention; a nucleic acid complementary to a
nucleic acid of the invention; or a portion of such a nucleic acid
(e.g., an oligonucleotide as described below), can be used in
"antisense" therapy, in which a nucleic acid (e.g., an
oligonucleotide) which specifically hybridizes to the mRNA and/or
genomic DNA of a Type II diabetes gene is administered or generated
in situ. The antisense nucleic acid that specifically hybridizes to
the mRNA and/or DNA inhibits expression of the TCF7L2 polypeptide,
e.g., by inhibiting translation and/or transcription. Binding of
the antisense nucleic acid can be by conventional base pair
complementarity, or, for example, in the case of binding to DNA
duplexes, through specific interaction in the major groove of the
double helix.
[0090] An antisense construct of the present invention can be
delivered, for example, as an expression plasmid as described
above. When the plasmid is transcribed in the cell, it produces RNA
that is complementary to a portion of the mRNA and/or DNA which
encodes the TCF7L2 polypeptide. Alternatively, the antisense
construct can be an oligonucleotide probe that is generated ex vivo
and introduced into cells; it then inhibits expression by
hybridizing with the mRNA and/or genomic DNA of the polypeptide. In
one embodiment, the oligonucleotide probes are modified
oligonucleotides, which are resistant to endogenous nucleases,
e.g., exonucleases and/or endonucleases, thereby rendering them
stable in vivo. Exemplary nucleic acid molecules for use as
antisense oligonucleotides are phosphoramidate, phosphothioate and
methylphosphonate analogs of DNA (see also U.S. Pat. Nos.
5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in antisense therapy
are also described, for example, by Van der Krol et al.,
(BioTechniques 6:958-976 (1988)); and Stein et al., (Cancer Res.
48:2659-2668 (1988)). With respect to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation
site are preferred.
[0091] To perform antisense therapy, oligonucleotides (mRNA, cDNA
or DNA) are designed that are complementary to mRNA encoding the
TCF7L2 gene. The antisense oligonucleotides bind to TCF7L2 mRNA
transcripts and prevent translation. Absolute complementarity,
although preferred, is not required. A sequence "complementary" to
a portion of an RNA, as referred to herein, indicates that a
sequence has sufficient complementarity to be able to hybridize
with the RNA, forming a stable duplex; in the case of
double-stranded antisense nucleic acids, a single strand of the
duplex DNA may thus be tested, or triplex formation may be assayed.
The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid, as
described in detail above. Generally, the longer the hybridizing
nucleic acid, the more base mismatches with an RNA it may contain
and still form a stable duplex (or triplex, as the case may be).
One skilled in the art can ascertain a tolerable degree of mismatch
by use of standard procedures.
[0092] The oligonucleotides used in antisense therapy can be DNA,
RNA, or chimeric mixtures or derivatives or modified versions
thereof, single-stranded or double-stranded. The oligonucleotides
can be modified at the base moiety, sugar moiety, or phosphate
backbone, for example, to improve stability of the molecule,
hybridization, etc. The oligonucleotides can include other appended
groups such as peptides (e.g. for targeting host cell receptors in
vivo), or agents facilitating transport across the cell membrane
(see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA
86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA
84:648-652 (1987); PCT International Publication NO: WO 88/09810)
or the blood-brain barrier (see, e.g., PCT International
Publication NO: WO 89/10134), or hybridization-triggered cleavage
agents (see, e.g., Krol et al., BioTechniques 6:958-976 (1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549
(1988)). To this end, the oligonucleotide may be conjugated to
another molecule (e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent).
[0093] The antisense molecules are delivered to cells that express
TCF7L2 in vivo. A number of methods can be used for delivering
antisense DNA or RNA to cells; e.g., antisense molecules can be
injected directly into the tissue site, or modified antisense
molecules, designed to target the desired cells (e.g., antisense
linked to peptides or antibodies that specifically bind receptors
or antigens expressed on the target cell surface) can be
administered systematically. Alternatively, in a preferred
embodiment, a recombinant DNA construct is utilized in which the
antisense oligonucleotide is placed under the control of a strong
promoter (e.g., pol III or pol II). The use of such a construct to
transfect target cells in the patient results in the transcription
of sufficient amounts of single stranded RNAs that will form
complementary base pairs with the endogenous TCF7L2 transcripts and
thereby prevent translation of the TCF7L2 mRNA. For example, a
vector can be introduced in vivo such that it is taken up by a cell
and directs the transcription of an antisense RNA. Such a vector
can remain episomal or become chromosomally integrated, as long as
it can be transcribed to produce the desired antisense RNA. Such
vectors can be constructed by recombinant DNA technology methods
standard in the art and described above. For example, a plasmid,
cosmid, YAC or viral vector can be used to prepare the recombinant
DNA construct that can be introduced directly into the tissue site.
Alternatively, viral vectors can be used which selectively infect
the desired tissue, in which case administration may be
accomplished by another route (e.g., systemically).
[0094] Endogenous TCF7L2 polypeptide expression can also be reduced
by inactivating or "knocking out" the gene, nucleic acid or its
promoter using targeted homologous recombination (e.g., see
Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi,
Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989)).
For example, an altered, non-functional gene or nucleic acid (or a
completely unrelated DNA sequence) flanked by DNA homologous to the
endogenous gene or nucleic acid (either the coding regions or
regulatory regions of the nucleic acid) can be used, with or
without a selectable marker and/or a negative selectable marker, to
transfect cells that express the gene or nucleic acid in vivo.
Insertion of the DNA construct, via targeted homologous
recombination, results in inactivation of the gene or nucleic acid.
The recombinant DNA constructs can be directly administered or
targeted to the required site in vivo using appropriate vectors, as
described above. Alternatively, expression of non-altered genes or
nucleic acids can be increased using a similar method: targeted
homologous recombination can be used to insert a DNA construct
comprising a non-altered functional gene or nucleic acid in place
of an altered TCF7L2 in the cell, as described above. In another
embodiment, targeted homologous recombination can be used to insert
a DNA construct comprising a nucleic acid that encodes a Type II
diabetes polypeptide variant that differs from that present in the
cell.
[0095] Alternatively, endogenous TCF7L2 nucleic acid expression can
be reduced by targeting deoxyribonucleotide sequences complementary
to the regulatory region of a TCF7L2 nucleic acid (i.e., the TCF7L2
promoter and/or enhancers) to form triple helical structures that
prevent transcription of the TCF7L2 nucleic acid in target cells in
the body. (See generally, Helene, C., Anticancer Drug Des.,
6(6):569-84 (1991); Helene, C. et al., Ann. N.Y. Acad. Sci.
660:27-36 (1992); and Maher, L. J., Bioassays 14(12):807-15
(1992)). Likewise, the antisense constructs described herein, by
antagonizing the normal biological activity of one of the TCF7L2
proteins, can be used in the manipulation of tissue, e.g., tissue
differentiation, both in vivo and for ex vivo tissue cultures.
Furthermore, the anti-sense techniques (e.g., microinjection of
antisense molecules, or transfection with plasmids whose
transcripts are anti-sense with regard to a Type II diabetes gene
mRNA or gene sequence) can be used to investigate the role of
TCF7L2 or the interaction of TCF7L2 and its binding agents in
developmental events, as well as the normal cellular function of
TCF7L2 or of the interaction of TCF7L2 and its binding agents in
adult tissue. Such techniques can be utilized in cell culture, but
can also be used in the creation of transgenic animals.
[0096] In yet another embodiment of the invention, other TCF7L2
therapeutic agents as described herein can also be used in the
treatment of Type II diabetes gene. The therapeutic agents can be
delivered in a composition, as described above, or by themselves.
They can be administered systemically, or can be targeted to a
particular tissue. The therapeutic agents can be produced by a
variety of means, including chemical synthesis; recombinant
production; in vivo production (e.g., a transgenic animal, such as
U.S. Pat. No. 4,873,316 to Meade et al.), for example, and can be
isolated using standard means such as those described herein.
[0097] A combination of any of the above methods of treatment
(e.g., administration of non-altered polypeptide in conjunction
with antisense therapy targeting altered mRNA of TCF7L2;
administration of a first splicing variant encoded by a TCF7L2
nucleic acid in conjunction with antisense therapy targeting a
second splicing encoded by a TCF7L2 nucleic acid) can also be
used.
Methods of Assessing Probability of Response to TCF7L2 Therapeutic
Agents
[0098] The present invention additionally pertains to methods of
assessing an individual's probability of response to a TCF7L2
therapeutic agent. In the methods, markers or haplotypes relating
to the TCF7L2 gene are assessed, as described above in relation to
assessing an individual for susceptibility to type II diabetes. The
presence of an allele, marker, SNP or haplotype associated with
susceptibility (increased risk) for type II diabetes (e.g., an
allele other than a 0 allele in marker DG10S478; a T allele in SNP
rs12255372; an A allele in SNP rs7895340; a C allele in SNP
rs11196205; a C allele in SNP rs7901695; a T allele in SNP
rs7903146; a C allele in SNP rs12243326; an T allele in SNP
rs4506565; a marker associated with the exon 4 LD block of TCF7L2,
such as an at-risk haplotype associated with the exon 4 LD block of
TCF7L2); is indicative of a probability of a positive response to a
TCF7L2 therapeutic agent. "Probability of a positive response"
indicates that the individual is more likely to have a positive
response to a TCF7L2 therapeutic agent than an individual not
having an allele, marker, SNP or haplotype associated with
susceptibility (increased risk) for type II diabetes as described
herein. A "positive response" to a TCF7L2 therapeutic agent is a
physiological response that indicates treatment of type II
diabetes. As described above, "treatment" refers not only to
ameliorating symptoms associated with type II diabetes, but also
preventing or delaying the onset of type II diabetes; lessening the
severity or frequency of symptoms of type II diabetes; and/or also
lessening the need for concomitant therapy with other drugs that
ameliorate symptoms associated with type II diabetes.
Pharmaceutical Compositions
[0099] The present invention also pertains to pharmaceutical
compositions comprising agents that alter TCF7L2 activity or which
otherwise affect the Wnt signaling pathway or the cadherin pathway,
or which can be used as TCF7L2 therapeutic agents. The
pharmaceutical compositions can be formulated with a
physiologically acceptable carrier or excipient to prepare a
pharmaceutical composition. The carrier and composition can be
sterile. The formulation should suit the mode of
administration.
[0100] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions (e.g., NaCl), saline,
buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable
oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates
such as lactose, amylose or starch, dextrose, magnesium stearate,
talc, silicic acid, viscous paraffin, perfume oil, fatty acid
esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well
as combinations thereof. The pharmaceutical preparations can, if
desired, be mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
aromatic substances and the like which do not deleteriously react
with the active agents.
[0101] The composition, if desired, can also contain minor amounts
of wetting or emulsifying agents, or pH buffering agents. The
composition can be a liquid solution, suspension, emulsion, tablet,
pill, capsule, sustained release formulation, or powder. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, polyvinyl
pyrollidone, sodium saccharine, cellulose, magnesium carbonate,
etc.
[0102] Methods of introduction of these compositions include, but
are not limited to, intradermal, intramuscular, intraperitoneal,
intraocular, intravenous, subcutaneous, topical, oral and
intranasal. Other suitable methods of introduction can also include
gene therapy (as described below), rechargeable or biodegradable
devices, particle acceleration devises ("gene guns") and slow
release polymeric devices. The pharmaceutical compositions of this
invention can also be administered as part of a combinatorial
therapy with other agents.
[0103] The composition can be formulated in accordance with the
routine procedures as a pharmaceutical composition adapted for
administration to human beings. For example, compositions for
intravenous administration typically are solutions in sterile
isotonic aqueous buffer. Where necessary, the composition may also
include a solubilizing agent and a local anesthetic to ease pain at
the site of the injection. Generally, the ingredients are supplied
either separately or mixed together in unit dosage form, for
example; as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampule or sachette
indicating the quantity of active agent. Where the composition is
to be administered by infusion, it can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water,
saline or dextrose/water. Where the composition is administered by
injection, an ampule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0104] For topical application, nonsprayable forms, viscous to
semi-solid or solid forms comprising a carrier compatible with
topical application and having a dynamic viscosity preferably
greater than water, can be employed. Suitable formulations include
but are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, enemas, lotions, sols, liniments, salves,
aerosols, etc., which are, if desired, sterilized or mixed with
auxiliary agents, e.g., preservatives, stabilizers, wetting agents,
buffers or salts for influencing osmotic pressure, etc. The agent
may be incorporated into a cosmetic formulation. For topical
application, also suitable are sprayable aerosol preparations
wherein the active ingredient, preferably in combination with a
solid or liquid inert carrier material, is packaged in a squeeze
bottle or in admixture with a pressurized volatile, normally
gaseous propellant, e.g., pressurized air.
[0105] Agents described herein can be formulated as neutral or salt
forms. Pharmaceutically acceptable salts include those formed with
free amino groups such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with free carboxyl groups such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0106] The agents are administered in a therapeutically effective
amount. The amount of agents which will be therapeutically
effective depends in part on the nature of the disorder and/or
extent of symptoms, and can be determined by standard clinical
techniques. In addition, in vitro or in vivo assays may optionally
be employed to help identify optimal dosage ranges. The precise
dose to be employed in the formulation will also depend on the
route of administration, and the seriousness of the symptoms, and
should be decided according to the judgment of a practitioner and
each patient's circumstances. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0107] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use of
sale for human administration. The pack or kit can be labeled with
information regarding mode of administration, sequence of drug
administration (e.g., separately, sequentially or concurrently), or
the like. The pack or kit may also include means for reminding the
patient to take the therapy. The pack or kit can be a single unit
dosage of the combination therapy or it can be a plurality of unit
dosages. In particular, the agents can be separated, mixed together
in any combination, present in a single vial or tablet. Agents
assembled in a blister pack or other dispensing means is preferred.
For the purpose of this invention, unit dosage is intended to mean
a dosage that is dependent on the individual pharmacodynamics of
each agent and administered in FDA approved dosages in standard
time courses.
Screening Assays and Agents Identified Thereby
[0108] The invention also provides methods for identifying agents
(e.g., fusion proteins, polypeptides, peptidomimetics, prodrugs,
receptors, binding agents, antibodies, small molecules or other
drugs, or ribozymes) which alter (e.g., increase or decrease) the
activity of the TCF7L2, which otherwise interact with TCF7L2 or
with another member of the Wnt signaling pathway or the cadherin
pathway (e.g., beta-catenin). For example, in certain embodiments,
such agents can be agents which bind to TCF7L2; which have a
stimulatory or inhibitory effect on, for example, activity of
TCF7L2; or which change (e.g., enhance or inhibit) the ability of
TCF7L2 to interact with other members of the Wnt signaling pathway
or with members of the cadherin pathway, or which alter
posttranslational processing of TCF7L2. In other embodiments, such
agents can be agents which alter activity or function of the Wnt
signaling pathway or the cadherin pathway.
[0109] In one embodiment, the invention provides assays for
screening candidate or test agents that bind to or modulate the
activity of TCF7L2 protein (or biologically active portion(s)
thereof), as well as agents identifiable by the assays. Test agents
can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase or
solution phase libraries; synthetic library methods requiring
deconvolution; the `one-bead one-compound` library method; and
synthetic library methods using affinity chromatography selection.
The biological library approach is limited to polypeptide
libraries, while the other four approaches are applicable to
polypeptide, non-peptide oligomer or small molecule libraries of
compounds (Lam, K. S., Anticancer Drug Des. 12:145 (1997)).
[0110] In one embodiment, to identify agents which alter the
activity of TCF7L2, a cell, cell lysate, or solution containing or
expressing TCF7L2, or a fragment or derivative thereof, can be
contacted with an agent to be tested; alternatively, the protein
can be contacted directly with the agent to be tested. The level
(amount) of TCF7L2 activity is assessed (e.g., the level (amount)
of TCF7L2 activity is measured, either directly or indirectly), and
is compared with the level of activity in a control (i.e., the
level of activity of the TCF7L2 protein or active fragment or
derivative thereof in the absence of the agent to be tested). If
the level of the activity in the presence of the agent differs, by
an amount that is statistically significant, from the level of the
activity in the absence of the agent, then the agent is an agent
that alters the activity of TCF7L2. An increase in the level of
activity relative to a control, indicates that the agent is an
agent that enhances (is an agonist of) activity. Similarly, a
decrease in the level of activity relative to a control, indicates
that the agent is an agent that inhibits (is an antagonist of)
activity. In another embodiment, the level of activity of TCF7L2 or
a derivative or fragment thereof in the presence of the agent to be
tested, is compared with a control level that has previously been
established. A level of the activity in the presence of the agent
that differs from the control level by an amount that is
statistically significant indicates that the agent alters TCF7L2
activity.
[0111] The present invention also relates to an assay for
identifying agents which alter the expression of the TCF7L2 gene
(e.g., antisense nucleic acids, fusion proteins, polypeptides,
peptidomimetics, prodrugs, receptors, binding agents, antibodies,
small molecules or other drugs, or ribozymes) which alter (e.g.,
increase or decrease) expression (e.g., transcription or
translation) of the gene or which otherwise interact with TCF7L2,
as well as agents identifiable by the assays. For example, a
solution containing a nucleic acid encoding a TCF7L2 can be
contacted with an agent to be tested. The solution can comprise,
for example, cells containing the nucleic acid or cell lysate
containing the nucleic acid; alternatively, the solution can be
another solution that comprises elements necessary for
transcription/translation of the nucleic acid. Cells not suspended
in solution can also be employed, if desired. The level and/or
pattern of TCF7L2 expression (e.g., the level and/or pattern of
mRNA or of protein expressed, such as the level and/or pattern of
different splicing variants) is assessed, and is compared with the
level and/or pattern of expression in a control (i.e., the level
and/or pattern of the TCF7L2 expression in the absence of the agent
to be tested). If the level and/or pattern in the presence of the
agent differs, by an amount or in a manner that is statistically
significant, from the level and/or pattern in the absence of the
agent, then the agent is an agent that alters the expression of a
Type II diabetes gene. Enhancement of TCF7L2 expression indicates
that the agent is an agonist of TCF7L2 activity. Similarly,
inhibition of TCF7L2 expression indicates that the agent is an
antagonist of TCF7L2 activity. In another embodiment, the level
and/or pattern of TCF7L2 polypeptide(s) (e.g., different splicing
variants) in the presence of the agent to be tested, is compared
with a control level and/or pattern that have previously been
established. A level and/or pattern in the presence of the agent
that differs from the control level and/or pattern by an amount or
in a manner that is statistically significant indicates that the
agent alters TCF7L2 expression.
[0112] In another embodiment of the invention, agents which alter
the expression of TCF7L2 or which otherwise interact with TCF7L2 or
with another member of the Wnt signaling pathway or the cadherin
pathway, can be identified using a cell, cell lysate, or solution
containing a nucleic acid encoding the promoter region of the
TCF7L2 gene or nucleic acid operably linked to a reporter gene.
After contact with an agent to be tested, the level of expression
of the reporter gene (e.g., the level of mRNA or of protein
expressed) is assessed, and is compared with the level of
expression in a control (i.e., the level of the expression of the
reporter gene in the absence of the agent to be tested). If the
level in the presence of the agent differs, by an amount or in a
manner that is statistically significant, from the level in the
absence of the agent, then the agent is an agent that alters the
expression of TCF7L2, as indicated by its ability to alter
expression of a gene that is operably linked to the TCF7L2 gene
promoter. Enhancement of the expression of the reporter indicates
that the agent is an agonist of TCF7L2 activity. Similarly,
inhibition of the expression of the reporter indicates that the
agent is an antagonist of TCF7L2 activity. In another embodiment,
the level of expression of the reporter in the presence of the
agent to be tested is compared with a control level that has
previously been established. A level in the presence of the agent
that differs from the control level by an amount or in a manner
that is statistically significant indicates that the agent alters
expression.
[0113] Agents which alter the amounts of different splicing
variants encoded by TCF7L2 (e.g., an agent which enhances activity
of a first splicing variant, and which inhibits activity of a
second splicing variant), as well as agents which are agonists of
activity of a first splicing variant and antagonists of activity of
a second splicing variant, can easily be identified using these
methods described above.
[0114] In other embodiments of the invention, assays can be used to
assess the impact of a test agent on the activity of a polypeptide
in relation to a TCF7L2 binding agent. For example, a cell that
expresses a compound that interacts with a TCF7L2 polypeptide
(herein referred to as a "TCF7L2 binding agent", which can be a
polypeptide or other molecule that interacts directly or indirectly
with a TCF7L2 polypeptide, such as a member of the Wnt signaling
pathway or a member of the cadherin pathway) is contacted with
TCF7L2 in the presence of a test agent, and the ability of the test
agent to alter the interaction between the TCF7L2 and the TCF7L2
binding agent is determined. Alternatively, a cell lysate or a
solution containing the TCF7L2 binding agent, can be used. An agent
that binds to the TCF7L2 or the TCF7L2 binding agent can alter the
interaction by interfering with, or enhancing the ability of the
TCF7L2 to bind to, associate with, or otherwise interact with the
TCF7L2 binding agent. Determining the ability of the test agent to
bind to TCF7L2 or a TCF7L2 binding agent can be accomplished, for
example, by coupling the test agent with a radioisotope or
enzymatic label such that binding of the test agent to the
polypeptide can be determined by detecting the labeled with
.sup.125I, .sup.35S, .sup.14C or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively, test
agents can be enzymatically labeled with, for example, horseradish
peroxidase, alkaline phosphatase, or luciferase, and the enzymatic
label detected by determination of conversion of an appropriate
substrate to product. It is also within the scope of this invention
to determine the ability of a test agent to interact with the
polypeptide without the labeling of any of the interactants. For
example, a microphysiometer can be used to detect the interaction
of a test agent with TCF7L2 or a TCF7L2 binding agent without the
labeling of either the test agent, TCF7L2, or the TCF7L2 binding
agent. McConnell, H. M. et al., Science 257:1906-1912 (1992). As
used herein, a "microphysiometer" (e.g., Cytosensor.TM.) is an
analytical instrument that measures the rate at which a cell
acidifies its environment using a light-addressable potentiometric
sensor (LAPS). Changes in this acidification rate can be used as an
indicator of the interaction between ligand and polypeptide.
[0115] Thus, these receptors can be used to screen for compounds
that are agonists or antagonists, for use in treating or studying a
susceptibility to type II diabetes. Drugs could be designed to
regulate TCF7L2 activation that in turn can be used to regulate
signaling pathways and transcription events of genes
downstream.
[0116] In another embodiment of the invention, assays can be used
to identify polypeptides that interact with TCF7L2. For example, a
yeast two-hybrid system such as that described by Fields and Song
(Fields, S, and Song, O., Nature 340:245-246 (1989)) can be used to
identify polypeptides that interact with TCF7L2. In such a yeast
two-hybrid system, vectors are constructed based on the flexibility
of a transcription factor that has two functional domains (a DNA
binding domain and a transcription activation domain). If the two
domains are separated but fused to two different proteins that
interact with one another, transcriptional activation can be
achieved, and transcription of specific markers (e.g., nutritional
markers such as His and Ade, or color markers such as lacZ) can be
used to identify the presence of interaction and transcriptional
activation. For example, in the methods of the invention, a first
vector is used which includes a nucleic acid encoding a DNA binding
domain and also TCF7L2, splicing variant, or fragment or derivative
thereof, and a second vector is used which includes a nucleic acid
encoding a transcription activation domain and also a nucleic acid
encoding a polypeptide which potentially may interact with TCF7L2
or a splicing variant, or fragment or derivative thereof.
Incubation of yeast containing the first vector and the second
vector under appropriate conditions (e.g., mating conditions such
as used in the Matchmaker.TM. system from Clontech (Palo Alto,
Calif., USA)) allows identification of colonies that express the
markers of interest. These colonies can be examined to identify the
polypeptide(s) that interact with TCF7L2 or fragment or derivative
thereof. Such polypeptides can be used as agents that alter the
activity of expression of TCF7L2, as described in relation to
methods of treatment.
[0117] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either the
TCF7L2 gene, the TCF7L2 protein, the TCF7L2 binding agent (e.g.,
another member of the Wnt signaling pathway or member of the
cadherin pathway), or other components of the assay on a solid
support, in order to facilitate separation of complexed from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of a test agent to the
protein, or interaction of the protein with a binding agent in the
presence and absence of a test agent, can be accomplished in any
vessel suitable for containing the reactants. Examples of such
vessels include microtitre plates, test tubes, and micro-centrifuge
tubes. In one embodiment, a fusion protein (e.g., a
glutathione-S-transferase fusion protein) can be provided which
adds a domain that allows TCF7L2, TCF7L2 protein, or a TCF7L2
binding agent to be bound to a matrix or other solid support.
[0118] In another embodiment, modulators of expression of nucleic
acid molecules of the invention are identified in a method wherein
a cell, cell lysate, or solution containing TCF7L2 is contacted
with a test agent and the expression of appropriate mRNA or
polypeptide (e.g., splicing variant(s)) in the cell, cell lysate,
or solution, is determined. The level of expression of appropriate
mRNA or polypeptide(s) in the presence of the test agent is
compared to the level of expression of mRNA or polypeptide(s) in
the absence of the test agent. The test agent can then be
identified as a modulator of expression based on this comparison.
For example, when expression of mRNA or polypeptide is greater
(statistically significantly greater) in the presence of the test
agent than in its absence, the test agent is identified as a
stimulator or enhancer of the mRNA or polypeptide expression.
Alternatively, when expression of the mRNA or polypeptide is less
(statistically significantly less) in the presence of the test
agent than in its absence, the test agent is identified as an
inhibitor of the mRNA or polypeptide expression. The level of mRNA
or polypeptide expression in the cells can be determined by methods
described herein for detecting mRNA or polypeptide.
[0119] This invention further pertains to novel agents identified
by the above-described screening assays. Accordingly, it is within
the scope of this invention to further use an agent identified as
described herein in the methods of treatment described herein. For
example, an agent identified as described herein can be used to
alter activity of a protein encoded by a TCF7L2 gene, or to alter
expression of TCF7L2 by contacting the protein or the nucleic acid
(or contacting a cell comprising the polypeptide or the nucleic
acid) with the agent identified as described herein.
Nucleic Acids of the Invention
TCF7L2 Nucleic Acids, Portions and Variants
[0120] The present invention also pertains to isolated nucleic acid
molecules comprising human TCF7L2. The TCF7L2 nucleic acid
molecules of the present invention can be RNA, for example, mRNA,
or DNA, such as cDNA and genomic DNA. DNA molecules can be
double-stranded or single-stranded; single stranded RNA or DNA can
be the coding, or sense, strand or the non-coding, or antisense
strand. The nucleic acid molecule can include all or a portion of
the coding sequence of the gene and can further comprise additional
non-coding sequences such as introns and non-coding 3' and 5'
sequences (including regulatory sequences, for example).
[0121] Additionally, nucleic acid molecules of the invention can be
fused to a marker sequence, for example, a sequence that encodes a
polypeptide to assist in isolation or purification of the
polypeptide. Such sequences include, but are not limited to, those
that encode a glutathione-S-transferase (GST) fusion protein and
those that encode a hemagglutinin A (HA) polypeptide marker from
influenza.
[0122] An "isolated" nucleic acid molecule, as used herein, is one
that is separated from nucleic acids that normally flank the gene
or nucleotide sequence (as in genomic sequences) and/or has been
completely or partially purified from other transcribed sequences
(e.g., as in an RNA library). For example, an isolated nucleic acid
of the invention may be substantially isolated with respect to the
complex cellular milieu in which it naturally occurs, or culture
medium when produced by recombinant techniques, or chemical
precursors or other chemicals when chemically synthesized. In some
instances, the isolated material will form part of a composition
(for example, a crude extract containing other substances), buffer
system or reagent mix. In other circumstances, the material may be
purified to essential homogeneity, for example as determined by
PAGE or column chromatography such as HPLC. Preferably, an isolated
nucleic acid molecule comprises at least about 50, 80 or 90% (on a
molar basis) of all macromolecular species present. With regard to
genomic DNA, the term "isolated" also can refer to nucleic acid
molecules that are separated from the chromosome with which the
genomic DNA is naturally associated. For example, the isolated
nucleic acid molecule can contain less than about 5 kb but not
limited to 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotides
which flank the nucleic acid molecule in the genomic DNA of the
cell from which the nucleic acid molecule is derived.
[0123] The nucleic acid molecule can be fused to other coding or
regulatory sequences and still be considered isolated. Thus,
recombinant DNA contained in a vector is included in the definition
of "isolated" as used herein. Also, isolated nucleic acid molecules
include recombinant DNA molecules in heterologous host cells, as
well as partially or substantially purified DNA molecules in
solution. "Isolated" nucleic acid molecules also encompass in vivo
and in vitro RNA transcripts of the DNA molecules of the present
invention. An isolated nucleic acid molecule can include a nucleic
acid molecule or nucleic acid sequence that is synthesized
chemically or by recombinant means. Therefore, recombinant DNA
contained in a vector is included in the definition of "isolated"
as used herein. Also, isolated nucleic acid molecules include
recombinant DNA molecules in heterologous organisms, as well as
partially or substantially purified DNA molecules in solution. In
vivo and in vitro RNA transcripts of the DNA molecules of the
present invention are also encompassed by "isolated" nucleic acid
sequences. Such isolated nucleic acid molecules are useful in the
manufacture of the encoded polypeptide, as probes for isolating
homologous sequences (e.g., from other mammalian species), for gene
mapping (e.g., by in situ hybridization with chromosomes), or for
detecting expression of the gene in tissue (e.g., human tissue),
such as by Northern or Southern blot analysis.
[0124] The present invention also pertains to nucleic acid
molecules which are not necessarily found in nature but which
encode a TCF7L2 polypeptide, or another splicing variant of a
TCF7L2 polypeptide or polymorphic variant thereof. Thus, for
example, the invention pertains to DNA molecules comprising a
sequence that is different from the naturally occurring nucleotide
sequence but which, due to the degeneracy of the genetic code,
encode a TCF7L2 polypeptide of the present invention. The invention
also encompasses nucleic acid molecules encoding portions
(fragments), or encoding variant polypeptides such as analogues or
derivatives of a TCF7L2 polypeptide. Such variants can be naturally
occurring, such as in the case of allelic variation or single
nucleotide polymorphisms, or non-naturally-occurring, such as those
induced by various mutagens and mutagenic processes. Intended
variations include, but are not limited to, addition, deletion and
substitution of one or more nucleotides that can result in
conservative or non-conservative amino acid changes, including
additions and deletions. Preferably the nucleotide (and/or
resultant amino acid) changes are silent or conserved; that is,
they do not alter the characteristics or activity of a TCF7L2
polypeptide. In one aspect, the nucleic acid sequences are
fragments that comprise one or more polymorphic microsatellite
markers. In another aspect, the nucleotide sequences are fragments
that comprise one or more single nucleotide polymorphisms in a
TCF7L2 gene.
[0125] Other alterations of the nucleic acid molecules of the
invention can include, for example, labeling, methylation,
internucleotide modifications such as uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates,
carbamates), charged linkages (e.g., phosphorothioates,
phosphorodithioates), pendent moieties (e.g., polypeptides),
intercalators (e.g., acridine, psoralen), chelators, alkylators,
and modified linkages (e.g., alpha anomeric nucleic acids). Also
included are synthetic molecules that mimic nucleic acid molecules
in the ability to bind to a designated sequence via hydrogen
bonding and other chemical interactions. Such molecules include,
for example, those in which peptide linkages substitute for
phosphate linkages in the backbone of the molecule.
[0126] The invention also pertains to nucleic acid molecules that
hybridize under high stringency hybridization conditions, such as
for selective hybridization, to a nucleotide sequence described
herein (e.g., nucleic acid molecules which specifically hybridize
to a nucleotide sequence encoding polypeptides described herein,
and, optionally, have an activity of the polypeptide). In one
aspect, the invention includes variants described herein that
hybridize under high stringency hybridization conditions (e.g., for
selective hybridization) to a nucleotide sequence encoding an amino
acid sequence or a polymorphic variant thereof. In another aspect,
the variant that hybridizes under high stringency hybridizations
has an activity of a TCF7L2 polypeptide.
[0127] Such nucleic acid molecules can be detected and/or isolated
by specific hybridization (e.g., under high stringency conditions).
"Specific hybridization," as used herein, refers to the ability of
a first nucleic acid to hybridize to a second nucleic acid in a
manner such that the first nucleic acid does not hybridize to any
nucleic acid other than to the second nucleic acid (e.g., when the
first nucleic acid has a higher similarity to the second nucleic
acid than to any other nucleic acid in a sample wherein the
hybridization is to be performed). "Stringency conditions" for
hybridization is a term of art which refers to the incubation and
wash conditions, e.g., conditions of temperature and buffer
concentration, which permit hybridization of a particular nucleic
acid to a second nucleic acid; the first nucleic acid may be
perfectly (i.e., 100%) complementary to the second, or the first
and second may share some degree of complementarity which is less
than perfect (e.g., 70%, 75%, 85%, 90%, 95%). For example, certain
high stringency conditions can be used which distinguish perfectly
complementary nucleic acids from those of less complementarity.
"High stringency conditions", "moderate stringency conditions" and
"low stringency conditions", as well as methods for nucleic acid
hybridizations are explained on pages 2.10.1-2.10.16 and pages
6.3.1-6.3.6 in Current Protocols in Molecular Biology (Ausubel, F.
et al., "Current Protocols in Molecular Biology", John Wiley &
Sons, (1998)), and in Kraus, M. and Aaronson, S., Methods Enzymol.,
200:546-556 (1991),
[0128] The percent homology or identity of two nucleotide or amino
acid sequences can be determined by aligning the sequences for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first sequence for optimal alignment). The
nucleotides or amino acids at corresponding positions are then
compared, and the percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=# of identical positions/total # of
positions.times.100). When a position in one sequence is occupied
by the same nucleotide or amino acid residue as the corresponding
position in the other sequence, then the molecules are homologous
at that position. As used herein, nucleic acid or amino acid
"homology" is equivalent to nucleic acid or amino acid "identity".
In certain aspects, the length of a sequence aligned for comparison
purposes is at least 30%, for example, at least 40%, in certain
aspects at least 60%, and in other aspects at least 70%, 80%, 90%
or 95% of the length of the reference sequence. The actual
comparison of the two sequences can be accomplished by well-known
methods, for example, using a mathematical algorithm. A preferred,
non-limiting example of such a mathematical algorithm is described
in Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993).
Such an algorithm is incorporated into the NBLAST and XBLAST
programs (version 2.0) as described in Altschul et al., Nucleic
Acids Res. 25:389-3402 (1997). When utilizing BLAST and Gapped
BLAST programs, the default parameters of the respective programs
(e.g., NBLAST) can be used. In one aspect, parameters for sequence
comparison can be set at score=100, wordlength=12, or can be varied
(e.g., W=5 or W=20).
[0129] Another preferred non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS 4(1): 11-17 (1988). Such an algorithm
is incorporated into the ALIGN program (version 2.0) which is part
of the GCG sequence alignment software package (Accelrys,
Cambridge, UK). When utilizing the ALIGN program for comparing
amino acid sequences, a PAM120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4 can be used. Additional
algorithms for sequence analysis are known in the art and include
ADVANCE and ADAM as described in Torellis and Robotti, Comput.
Appl. Biosci. 10:3-5 (1994); and FASTA described in Pearson and
Lipman, Proc. Natl. Acad. Sci. USA 85:2444-8 (1988).
[0130] In another aspect, the percent identity between two amino
acid sequences can be accomplished using the GAP program in the GCG
software package using either a BLOSUM63 matrix or a PAM250 matrix,
and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3,
or 4. In yet another aspect, the percent identity between two
nucleic acid sequences can be accomplished using the GAP program in
the GCG software package using a gap weight of 50 and a length
weight of 3.
[0131] The present invention also provides isolated nucleic acid
molecules that contain a fragment or portion that hybridizes under
highly stringent conditions to a nucleotide sequence of TCF7L2, or
the complement of such a sequence, and also provides isolated
nucleic acid molecules that contain a fragment or portion that
hybridizes under highly stringent conditions to a nucleotide
sequence encoding an amino acid sequence or polymorphic variant
thereof. The nucleic acid fragments of the invention are at least
about 15, preferably at least about 18, 20, 23 or 25 nucleotides,
and can be 30, 40, 50, 100, 200 or more nucleotides in length.
Longer fragments, for example, 30 or more nucleotides in length,
which encode antigenic polypeptides described herein, are
particularly useful, such as for the generation of antibodies as
described below.
Probes and Primers
[0132] In a related aspect, the nucleic acid fragments of the
invention are used as probes or primers in assays such as those
described herein. "Probes" or "primers" are oligonucleotides that
hybridize in a base-specific manner to a complementary strand of
nucleic acid molecules. Such probes and primers include polypeptide
nucleic acids, as described in Nielsen et al., Science
254:1497-1500 (1991).
[0133] A probe or primer comprises a region of nucleotide sequence
that hybridizes to at least about 15, for example about 20-25, and
in certain aspects about 40, 50 or 75, consecutive nucleotides of a
nucleic acid molecule comprising a contiguous nucleotide sequence
of TCF7L2 or polymorphic variant thereof. In other aspects, a probe
or primer comprises 100 or fewer nucleotides, in certain aspects
from 6 to 50 nucleotides, for example from 12 to 30 nucleotides. In
other aspects, the probe or primer is at least 70% identical to the
contiguous nucleotide sequence or to the complement of the
contiguous nucleotide sequence, for example at least 80% identical,
in certain aspects at least 90% identical, and in other aspects at
least 95% identical, or even capable of selectively hybridizing to
the contiguous nucleotide sequence or to the complement of the
contiguous nucleotide sequence. Often, the probe or primer further
comprises a label, e.g., radioisotope, fluorescent compound,
enzyme, or enzyme co-factor.
[0134] The nucleic acid molecules of the invention such as those
described above can be identified and isolated using standard
molecular biology techniques and the sequence information provided
herein. For example, nucleic acid molecules can be amplified and
isolated by the polymerase chain reaction using synthetic
oligonucleotide primers designed based on the sequence of TCF7L2 or
the complement of such a sequence, or designed based on nucleotides
based on sequences encoding one or more of the amino acid sequences
provided herein. See generally PCR Technology: Principles and
Applications for DNA Amplification (ed. H. A. Erlich, Freeman
Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and
Applications (Eds. Innis et al., Academic Press, San Diego, Calif.,
1990); Mattila et al., Nucl. Acids Res. 19: 4967 (1991); Eckert et
al., PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson
et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. The
nucleic acid molecules can be amplified using cDNA, mRNA or genomic
DNA as a template, cloned into an appropriate vector and
characterized by DNA sequence analysis.
[0135] Other suitable amplification methods include the ligase
chain reaction (LCR) (see Wu and Wallace, Genomics 4:560 (1989),
Landegren et al., Science 241:1077 (1988), transcription
amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173
(1989)), and self-sustained sequence replication (Guatelli et al.,
Proc. Nat. Acad. Sci. USA 87:1874 (1990)) and nucleic acid based
sequence amplification (NASBA). The latter two amplification
methods involve isothermal reactions based on isothermal
transcription, which produce both single stranded RNA (ssRNA) and
double stranded DNA (dsDNA) as the amplification products in a
ratio of about 30 or 100 to 1, respectively.
[0136] The amplified DNA can be labeled, for example, radiolabeled,
and used as a probe for screening a cDNA library derived from human
cells, mRNA in zap express, ZIPLOX or other suitable vector.
Corresponding clones can be isolated, DNA can obtained following in
vivo excision, and the cloned insert can be sequenced in either or
both orientations by art recognized methods to identify the correct
reading frame encoding a polypeptide of the appropriate molecular
weight. For example, the direct analysis of the nucleotide sequence
of nucleic acid molecules of the present invention can be
accomplished using well-known methods that are commercially
available. See, for example, Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al.,
Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).
Additionally, fluorescence methods are also available for analyzing
nucleic acids (Chen et al., Genome Res. 9, 492 (1999)) and
polypeptides. Using these or similar methods, the polypeptide and
the DNA encoding the polypeptide can be isolated, sequenced and
further characterized.
[0137] Antisense nucleic acid molecules of the invention can be
designed using the nucleotide sequence of TCF7L2 and/or the
complement or a portion, and constructed using chemical synthesis
and enzymatic ligation reactions using procedures known in the art.
For example, an antisense nucleic acid molecule (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Alternatively, the
antisense nucleic acid molecule can be produced biologically using
an expression vector into which a nucleic acid molecule has been
subcloned in an antisense orientation (i.e., RNA transcribed from
the inserted nucleic acid molecule will be of an antisense
orientation to a target nucleic acid of interest).
[0138] The nucleic acid sequences can also be used to compare with
endogenous DNA sequences in patients to identify one or more of the
disorders described above, and as probes, such as to hybridize and
discover related DNA sequences or to subtract out known sequences
from a sample. The nucleic acid sequences can further be used to
derive primers for genetic fingerprinting, to raise
anti-polypeptide antibodies using DNA immunization techniques, and
as an antigen to raise anti-DNA antibodies or elicit immune
responses. Portions or fragments of the nucleotide sequences
identified herein (and the corresponding complete gene sequences)
can be used in numerous ways, such as polynucleotide reagents. For
example, these sequences can be used to: (i) map their respective
genes on a chromosome; and, thus, locate gene regions associated
with genetic disease; (ii) identify an individual from a minute
biological sample (tissue typing); and (iii) aid in forensic
identification of a biological sample. Additionally, the nucleotide
sequences of the invention can be used to identify and express
recombinant polypeptides for analysis, characterization or
therapeutic use, or as markers for tissues in which the
corresponding polypeptide is expressed, either constitutively,
during tissue differentiation, or in diseased states. The nucleic
acid sequences can additionally be used as reagents in the
screening and/or diagnostic assays described herein, and can also
be included as components of kits (e.g., reagent kits) for use in
the screening and/or diagnostic assays described herein.
[0139] Kits (e.g., reagent kits) useful in the methods of diagnosis
comprise components useful in any of the methods described herein,
including for example, hybridization probes or primers as described
herein (e.g., labeled probes or primers), reagents for detection of
labeled molecules, restriction enzymes (e.g., for RFLP analysis),
allele-specific oligonucleotides, antibodies which bind to altered
or to non-altered (native) TCF7L2 polypeptide, means for
amplification of nucleic acids comprising a TCF7L2 nucleic acid or
for a portion of TCF7L2, or means for analyzing the nucleic acid
sequence of a TCF7L2 nucleic acid or for analyzing the amino acid
sequence of a TCF7L2 polypeptide as described herein, etc. In one
aspect, the kit for diagnosing a susceptibility to type II diabetes
can comprise primers for nucleic acid amplification of a region in
the TCF7L2 nucleic acid comprising the marker DG10S478, the SNP
rs12255372, rs895340, rs11196205, rs7901695, rs7903146, rs12243326
and/or rs4506565, or an at-risk haplotype that is more frequently
present in an individual having type II diabetes or who is
susceptible to type II diabetes. The primers can be designed using
portions of the nucleic acids flanking SNPs that are indicative of
type II diabetes.
Vectors and Host Cells
[0140] Another aspect of the invention pertains to nucleic acid
constructs containing a nucleic acid molecules described herein and
the complements thereof (or a portion thereof). The constructs
comprise a vector (e.g., an expression vector) into which a
sequence of the invention has been inserted in a sense or antisense
orientation. As used herein, the term "vector" refers to a nucleic
acid molecule capable of transporting another nucleic acid to which
it has been linked. One type of vector is a "plasmid", which refers
to a circular double stranded DNA loop into which additional DNA
segments can be ligated. Another type of vector is a viral vector,
wherein additional DNA segments can be ligated into the viral
genome. Certain vectors are capable of autonomous replication in a
host cell into which they are introduced (e.g., bacterial vectors
having a bacterial origin of replication and episomal mammalian
vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Expression vectors are capable of directing the expression
of genes to which they are operably linked. In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids. However, the invention is intended to include
such other forms of expression vectors, such as viral vectors
(e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses) that serve equivalent functions.
[0141] In certain aspects, recombinant expression vectors of the
invention comprise a nucleic acid molecule of the invention in a
form suitable for expression of the nucleic acid molecule in a host
cell. This means that the recombinant expression vectors include
one or more regulatory sequences, selected on the basis of the host
cells to be used for expression, which is operably linked to the
nucleic acid sequence to be expressed. Within a recombinant
expression vector, "operably linked" or "operatively linked" is
intended to mean that the nucleotide sequence of interest is linked
to the regulatory sequence(s) in a manner which allows for
expression of the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). The term "regulatory sequence"
is intended to include promoters, enhancers and other expression
control elements (e.g., polyadenylation signals). Such regulatory
sequences are described, for example, in Goeddel, "Gene Expression
Technology", Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1990). Regulatory sequences include those which direct
constitutive expression of a nucleotide sequence in many types of
host cell and those which direct expression of the nucleotide
sequence only in certain host cells (e.g., tissue-specific
regulatory sequences). It will be appreciated by those skilled in
the art that the design of the expression vector can depend on such
factors as the choice of the host cell to be transformed and the
level of expression of polypeptide desired. The expression vectors
of the invention can be introduced into host cells to thereby
produce polypeptides, including fusion polypeptides, encoded by
nucleic acid molecules as described herein.
[0142] The recombinant expression vectors of the invention can be
designed for expression of a polypeptide of the invention in
prokaryotic or eukaryotic cells, e.g., bacterial cells such as E.
coli, insect cells (using baculovirus expression vectors), yeast
cells or mammalian cells. Suitable host cells are discussed further
in Goeddel, supra. Alternatively, the recombinant expression vector
can be transcribed and translated in vitro, for example using T7
promoter regulatory sequences and T7 polymerase.
[0143] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0144] A host cell can be any prokaryotic or eukaryotic cell. For
example, a nucleic acid molecule of the invention can be expressed
in bacterial cells (e.g., E. coli), insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0145] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing a foreign nucleic acid molecule (e.g., DNA) into a host
cell, including calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofection,
or electroporation. Suitable methods for transforming or
transfecting host cells can be found in Sambrook, et al., (supra),
and other laboratory manuals.
[0146] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
for resistance to antibiotics) is generally introduced into the
host cells along with the gene of interest. Preferred selectable
markers include those that confer resistance to drugs, such as
G418, hygromycin and methotrexate. Nucleic acid molecules encoding
a selectable marker can be introduced into a host cell on the same
vector as the nucleic acid molecule of the invention or can be
introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid molecule can be identified by drug
selection (e.g., cells that have incorporated the selectable marker
gene will survive, while the other cells die).
[0147] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture can be used to produce (i.e.,
express) a polypeptide of the invention. Accordingly, the invention
further provides methods for producing a polypeptide using the host
cells of the invention. In one aspect, the method comprises
culturing the host cell of invention (into which a recombinant
expression vector encoding a polypeptide of the invention has been
introduced) in a suitable medium such that the polypeptide is
produced. In another aspect, the method further comprises isolating
the polypeptide from the medium or the host cell.
Antibodies of the Invention
[0148] Polyclonal antibodies and/or monoclonal antibodies that
specifically bind one form of the gene product but not to the other
form of the gene product are also provided. Antibodies are also
provided which bind a portion of either the variant or the
reference gene product that contains the polymorphic site or sites.
The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain antigen-binding sites that
specifically bind an antigen. A molecule that specifically binds to
a polypeptide of the invention is a molecule that binds to that
polypeptide or a fragment thereof, but does not substantially bind
other molecules in a sample, e.g., a biological sample, which
naturally contains the polypeptide. Examples of immunologically
active portions of immunoglobulin molecules include F(ab) and
F(ab').sub.2 fragments which can be generated by treating the
antibody with an enzyme such as pepsin. The invention provides
polyclonal and monoclonal antibodies that bind to a polypeptide of
the invention. The term "monoclonal antibody" or "monoclonal
antibody composition", as used herein, refers to a population of
antibody molecules that contain only one species of an antigen
binding site capable of immunoreacting with a particular epitope of
a polypeptide of the invention. A monoclonal antibody composition
thus typically displays a single binding affinity for a particular
polypeptide of the invention with which it immunoreacts.
[0149] Polyclonal antibodies can be prepared as described above by
immunizing a suitable subject with a desired immunogen, e.g.,
polypeptide of the invention or a fragment thereof. The antibody
titer in the immunized subject can be monitored over time by
standard techniques, such as with an enzyme linked immunosorbent
assay (ELISA) using immobilized polypeptide. If desired, the
antibody molecules directed against the polypeptide can be isolated
from the mammal (e.g., from the blood) and further purified by
well-known techniques, such as protein A chromatography to obtain
the IgG fraction. At an appropriate time after immunization, e.g.,
when the antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein, Nature 256:495-497
(1975), the human B cell hybridoma technique (Kozbor et al.,
Immunol. Today 4: 72 (1983)), the EBV-hybridoma technique (Cole et
al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985,
Inc., pp. 77-96) or trioma techniques. The technology for producing
hybridomas is well known (see generally Current Protocols in
Immunology (1994) Coligan et al., (eds.) John Wiley & Sons,
Inc., New York, N.Y.). Briefly, an immortal cell line (typically a
myeloma) is fused to lymphocytes (typically splenocytes) from a
mammal immunized with an immunogen as described above, and the
culture supernatants of the resulting hybridoma cells are screened
to identify a hybridoma producing a monoclonal antibody that binds
a polypeptide of the invention.
[0150] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating a monoclonal antibody to a polypeptide of the
invention (see, e.g., Current Protocols in Immunology, supra;
Galfre et al., Nature 266:55052 (1977); R. H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980); and Lerner, Yale J.
Biol. Med. 54:387-402 (1981)). Moreover, the ordinarily skilled
worker will appreciate that there are many variations of such
methods that also would be useful.
[0151] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody to a polypeptide of the invention
can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage
display library) with the polypeptide to thereby isolate
immunoglobulin library members that bind the polypeptide. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog No. 27-9400-01; and the Stratagene SurfJZAP.TM. Phage
Display Kit, Catalog No. 240612). Additionally, examples of methods
and reagents particularly amenable for use in generating and
screening antibody display library can be found in, for example,
U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT
Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT
Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT
Publication No. WO 90/02809; Fuchs et al., Bio/Technology 9:
1370-1372 (1991); Hay et al., Hum. Antibod Hybridomas 3:81-85
(1992); Huse et al., Science 246: 1275-1281 (1989); and Griffiths
et al., EMBO J. 12:725-734 (1993).
[0152] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art.
[0153] In general, antibodies of the invention (e.g., a monoclonal
antibody) can be used to isolate a polypeptide of the invention by
standard techniques, such as affinity chromatography or
immunoprecipitation. A polypeptide-specific antibody can facilitate
the purification of natural polypeptide from cells and of
recombinantly produced polypeptide expressed in host cells.
Moreover, an antibody specific for a polypeptide of the invention
can be used to detect the polypeptide (e.g., in a cellular lysate,
cell supernatant, or tissue sample) in order to evaluate the
abundance and pattern of expression of the polypeptide. Antibodies
can be used diagnostically to monitor protein levels in tissue as
part of a clinical testing procedure, e.g., to, for example,
determine the efficacy of a given treatment regimen. The antibody
can be coupled to a detectable substance to facilitate its
detection. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin, and examples
of suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S or .sup.3H.
[0154] The present invention is now illustrated by the following
Exemplification, which is not intended to be limiting in any
way.
Exemplification
[0155] Described herein is the identification of transcription
factor 7-like 2 (TCF7L2-formerly TCF4) as a gene conferring risk of
type II diabetes through single-point association analysis using a
dense set of microsatellite markers within the 10q locus.
Methods
Icelandic Cohort
[0156] The Data Protection Authority of Iceland and the National
Bioethics Committee of Iceland approved the study. All participants
in the study gave informed consent. All personal identifiers
associated with blood samples, medical information, and genealogy
were first encrypted by the Data Protection Authority, using a
third-party encryption system (18).
[0157] For this study, 2400 type II diabetes patients were
identified who were diagnosed either through a long-term
epidemiologic study done at the Icelandic Heart Association over
the past 30 years or at one of two major hospitals in Reykjavik
over the past 12 years. Two-thirds of these patients were alive,
representing about half of the population of known type II diabetes
patients in Iceland today. The majority of these patients were
contacted for this study, and the cooperation rate exceeded 80%.
All participants in the study visited the Icelandic Heart
Association where they answered a questionnaire, had blood drawn
and a fasting plasma glucose measurements taken. Questions about
medication and age at diagnosis were included. The type II diabetes
patients in this study were diagnosed as described in our
previously published linkage study (10). In brief, the diagnosis of
type II diabetes was confirmed by study physicians through previous
medical records, medication history, and/or new laboratory
measurements. For previously diagnosed type II diabetes patients,
reporting of the use of oral glucose-lowering agent confirmed type
II diabetes.
[0158] Individuals who were currently treated with insulin were
classified as having type II diabetes if they were also using or
had previously used oral glucose-lowering agents. In this cohort
the majority of patients on medication take oral glucose-lowering
agents and only a small portion (9%) require insulin. For hitherto
undiagnosed individuals, the diagnosis of type II diabetes and
impaired fasting glucose (IFG) was based on the criteria set by the
American Diabetes Association (Expert Committee on the Diagnosis
and Classification of Diabetes Mellitus 1997). The average age of
the type II diabetes patients in this study was 69.7 years.
Replication Cohorts
[0159] The Danish study group was selected from the PERF
(Prospective Epidemiological Risk Factors) study in Denmark (19).
228 females had been diagnosed previously with type II diabetes
and/or measured >=7 mM glucose. As controls, 539 unaffected
(with respect to type II diabetes) females were randomly drawn from
the same study cohort.
[0160] The PENN CATH study in the US is a cross sectional study of
the association of biochemical and genetic factors with coronary
atherosclerosis in a consecutive cohort of patients undergoing
cardiac catheterization at the University of Pennsylvania Medical
Center between July 1998 and March 2003. Type II diabetes was
defined as history of fasting blood glucose .gtoreq.126 mg/dl,
2-hour post-prandial glucose .gtoreq.200 mg/dl, use of oral
hypoglycemic agents, or insulin and oral hypoglycemic in a subject
greater than age 40. The University of Pennsylvania Institutional
Review Board approved the study protocol and all subjects gave
written informed consent. Ethnicity was determined through
self-report. 361 Caucasian type II diabetes cases were derived from
this cohort. 530 unaffected (with respect to type II diabetes and
myocardial infarction) Caucasian controls were randomly drawn from
the same study.
[0161] The DNA used for genotyping was the product of whole-genome
amplification, by use of the GenomiPhi Amplification kit
(Amersham), of DNA isolated from the peripheral blood of the Danish
and US type II diabetes patients and controls.
Genotyping
[0162] New sequence repeats (i.e. dinucleotide, trinucleotide, and
tetronucleotide repeats) were identified using the Tandem repeats
finder software (20) and tested for polymorphicity in 94 controls.
The size in basepairs of the lower allele of the CEPH sample
1347-02 (CEPH genomics repository) was subtracted from the size of
the microsatellite amplicon and used as a reference. SNP genotyping
was carried using direct DNA sequencing (Applied BioSystems) or the
Centaurus platform (Nanogen).
Statistical Methods for Association Analysis
[0163] For single marker association to type II diabetes, we used a
likelihood ratio test to calculate a two-sided p-value for each
allele. We present allelic frequencies rather than carrier
frequencies for the microsatellites employed.
[0164] We calculated relative risk (RR) and population attributable
risk (PAR) assuming a multiplicative model (16, 17). For the CEPH
Caucasian HapMap data, we calculated LD between pairs of SNPs using
the standard definition of D' (21) and R.sup.2 (22). When plotting
all SNP combinations to elucidate the LD structure in a particular
region, we plotted D' in the upper left corner and p-values in the
lower right corner. In the LD plot we present, the markers are
plotted equidistantly rather than according to their physical
positions.
Results
Locus-Wide Association Study
[0165] We previously reported genome-wide significant linkage to
chromosome 5q for type II diabetes mellitus in the Icelandic
population (10); in the same study, we also reported suggestive
evidence of linkage to 10q and 12q. To follow up the 10q locus, we
used an association approach employing a high density of genotyped
microsatellite markers across a 10.5 Mb region (NCBI Build 34:
Chr10:114.2-124.7 Mb) corresponding to this locus. We identified
and typed 228 microsatellite markers--i.e. to an average density of
one marker every 46 kb (Table 1). All the markers were typed in
1185 Icelandic type II diabetes patients and 931 unrelated
population controls.
TABLE-US-00002 TABLE 1 Location of the 228 genotyped
microsatellites on chromosome 10 in NCBI Build 34 of the human
genome assembly. END: Build 34 Chr10 Alias START: Build 34 Chr10
location location D10S1269 114186051 114186276 DG10S475 114389853
114390116 D10S168 114410102 114410266 DG10S478 114460845 114461228
DG10S479 114475488 114475632 DG10S480 114507574 114507829 DG10S481
114542657 114542924 DG10S1624 114545990 114546237 DG10S1625
114568323 114568715 DG10S488 114713594 114714008 DG10S1630
114770344 114770609 DG10S1631 114778307 114778598 DG10S492
114811884 114812269 DG10S494 114852114 114852280 DG10S495 114879344
114879474 DG10S496 114919414 114919678 DG10S498 114964123 114964270
DG10S500 115024471 115024854 DG10S501 115045332 115045710 DG10S508
115241356 115241602 DG10S1634 115267106 115267460 DG10S512
115357290 115357439 DG10S514 115400157 115400338 DG10S17 115463773
115464048 DG10S1635 115519619 115519900 DG10S520 115536945
115537130 D10S554 115695920 115696071 D10S1237 115784580 115784977
DG10S535 115858565 115858720 D10S1158 115937134 115937433 DG10S1636
115966165 115966382 DG10S540 115983225 115983471 DG10S1637
116025219 116025491 DG10S542 116054130 116054255 DG10S1638
116062921 116063264 D10S1776 116140681 116140897 DG10S546 116141340
116141590 DG10S547 116173634 116173887 DG10S1639 116184720
116184898 DG10S548 116202775 116203174 DG10S550 116288175 116288560
D10S562 116304948 116305132 DG10S1640 116344030 116344279 DG10S1641
116638155 116638540 DG10S566 116866173 116866431 D10S468 116869582
116869674 DG10S567 116904174 116904433 D10S1731 117001692 117001870
DG10S573 117070087 117070192 DG10S576 117153566 117153823 DG10S578
117196538 117196813 DG10S1644 117206992 117207391 DG10S579
117226056 117226234 DG10S580 117240674 117240858 DG10S584 117336471
117336821 DG10S585 117364742 117364845 DG10S586 117385650 117385816
DG10S589 117481892 117482165 DG10S590 117508690 117508966 DG10S591
117520912 117521057 DG10S593 117567541 117567800 D10S1748 117589638
117589885 DG10S596 117629981 117630119 DG10S597 117654759 117654928
DG10S523 117691905 117692329 DG10S598 117691905 117692156 D10S1773
117708786 117708989 DG10S599 117713714 117714115 DG10S524 117713997
117714115 DG10S600 117742602 117743019 DG10S525 117742701 117742986
DG10S1250 117861226 117861405 DG10S604 117867801 117868010
DG10S1293 117932494 117932721 DG10S1144 117950298 117950606
DG10S609 118014503 118014752 DG10S610 118041410 118041787 DG10S1252
118085912 118086081 DG10S612 118092869 118093247 DG10S613 118126058
118126312 DG10S614 118150018 118150178 D10S544 118164684 118164979
D10S1683 118211053 118211180 D10S1657 118287426 118287695 D10S545
118299618 118299851 DG10S1649 118306954 118307121 D10S187 118317655
118317730 DG10S1295 118375973 118376205 DG10S624 118401694
118402073 DG10S1203 118440472 118440835 DG10S627 118514695
118515072 DG10S1650 118521021 118521210 DG10S1681 118522946
118523333 DG10S628 118553693 118553836 DG10S634 118566844 118567191
DG10S639 118712208 118712596 DG10S640 118743450 118743821 D10S221
118766458 118766560 DG10S1686 118766464 118766561 DG10S641
118788135 118788401 DG10S1651 118794961 118795267 DG10S1255
118834290 118834438 DG10S644 118857362 118857745 DG10S1652
118862172 118862311 DG10S1654 118954536 118954869 DG10S1688
118972583 118972717 DG10S1689 118987319 118987480 DG10S1690
119004704 119004986 D10S1425 119004742 119004920 DG10S651 119030166
119030595 DG10S1655 119044005 119044188 DG10S1691 119078576
119078943 DG10S1207 119094382 119094722 D10S1693 119109493
119109731 DG10S1258 119131611 119131788 DG10S656 119177278
119177672 DG10S1694 119177430 119177614 DG10S1695 119204432
119204655 DG10S657 119204769 119205174 DG10S658 119223917 119224102
DG10S1696 119243071 119243408 DG10S1657 119282299 119282586
DG10S1658 119290241 119290632 DG10S661 119305067 119305226 DG10S662
119317406 119317660 DG10S663 119330718 119331131 DG10S1699
119364904 119365188 DG10S665 119396863 119397144 DG10S1659
119412611 119412992 DG10S667 119448478 119448736 DG10S1701
119473676 119473914 D10S1236 119473739 119473870 DG10S669 119485378
119485552 DG10S670 119505799 119505905 D10S190 119510348 119510554
DG10S1702 119510362 119510479 DG10S1153 119526060 119526329
DG10S673 119606691 119606963 DG10S1305 119615268 119615484 DG10S675
119659153 119659532 DG10S1661 119663175 119663453 DG10S1662
119700563 119700948 DG10S1306 119703996 119704204 DG10S1663
119783538 119783739 DG10S1704 119783569 119783694 DG10S631
119788517 119788678 D10S1148 119803465 119803663 D10S1150 119803465
119803662 D10S503 119803476 119803653 DG10S632 119811193 119811621
DG10S681 119811347 119811621 DG10S633 119833701 119833987 D10S2473
119833724 119833869 DG10S682 119838539 119838806 DG10S683 119853558
119853862 DG10S684 119880412 119880572 DG10S685 119909682 119910062
DG10S686 119923527 119923790 DG10S687 119954835 119955083 DG10S1212
119972358 119972707 DG10S1261 119995566 119995727 DG10S1350
120004924 120005036 DG10S1 120030830 120031131 DG10S693 120100794
120101005 DG10S1263 120132349 120132528 D10S542 120417003 120417230
DG10S1664 120444685 120444808 DG10S1163 120506796 120507066
DG10S703 120538236 120538484 DG10S704 120570334 120570593 DG10S706
120642052 120642312 DG10S708 120699520 120699811 DG10S709 120723780
120724158 D10S1701 120849161 120849428 DG10S716 120893782 120894153
DG10S1669 120969521 120969659 DG10S720 121016792 121017048 D10S1792
121042408 121042574 DG10S722 121070320 121070693 DG10S1181
121101362 121101685 DG10S724 121117025 121117286 DG10S1670
121162511 121162898 DG10S726 121217327 121217580 DG10S1167
121247552 121247838 DG10S729 121283257 121283429 DG10S730 121318865
121319131 DG10S731 121342622 121342893 DG10S1278 121384227
121384464 DG10S734 121425229 121425633 DG10S735 121446549 121446695
DG10S1185 121466936 121467248 DG10S1129 121472295 121472600
DG10S1085 121494260 121494657 DG10S1327 121526700 121526830
DG10S1271 121559895 121560066 DG10S741 121638254 121638391
DG10S1087 121647884 121648273 DG10S1359 121713760 121713892
DG10S1120 121726128 121726519 DG10S1671 121750886 121750993
DG10S1673 121823695 121823925 DG10S749 121841816 121841997
DG10S1134 121901381 121901668 DG10S1674 121931406 121931809
DG10S755 121976143 121976435 D10S1757 121989325 121989539 D10S209
121995173 121995376 DG10S757 122029990 122030248 DG10S1283
122045222 122045429 DG10S1191 122071761 122072115 DG10S761
122141102 122141322 DG10S1678 122146312 122146535 DG10S762
122167889 122168135 DG10S763 122185793 122185925 DG10S1284
122207287 122207508 DG10S1137 122220809 122221073 DG10S766
122257534 122257929 DG10S767 122283871 122284250 DG10S1361
122318975 122319081 DG10S1680 122390160 122390294 D10S1230
122407279 122407403 DG10S772 122421708 122421845 DG10S775 122463781
122463941 DG10S777 122524358 122524547 DG10S779 122580228 122580603
DG10S784 122719087 122719236 D10S1483 122948181 122948324 D10S587
124728937 124729112
[0166] Single marker association analysis with the microsatellite
markers identified association with DG10S478 (Table 2 and the
FIGURE).
TABLE-US-00003 TABLE 2 DG10S478 Association to Type II Diabetes in
Iceland Affected freq Control freq Allele (n = 1185) (n = 931) RR
[95% CI] Two sided P 0 0.636 0.724 0.67 2.1 .times. 10.sup.-9 4
0.005 0.002 2.36 0.12 8 0.093 0.078 1.21 0.090 12 0.242 0.178 1.48
4.6 .times. 10.sup.-7 16 0.022 0.015 1.53 0.076 20 0.001 0.003 0.39
0.17 X 0.364 0.276 1.50 [1.31, 1.71] 2.1 .times. 10.sup.-9
[0167] Six alleles are observed with this tetra-nucleotide repeat,
with alleles 0, 8 and 12 accounting for 98% of chromosomes in the
population controls. Allele 0 showed a protective association
(Relative Risk (RR)=0.67; P=2.1.times.10.sup.-9) relative to the
other alleles combined. This P-value is two-sided and takes into
account that some of the patients are related to each other.
DG10S478 is located in intron 3 of the transcription factor 7-like
2 (TCF7L2--formerly TCF4) gene on 10q25.2. This marker is within a
well defined LD block of 74.9 kb (based on the CEPH Caucasian
HapMap Phase II) that encapsulates part of intron 3, the whole of
exon 4 and part of intron 4 (the FIGURE).
[0168] When DG10S478 was genotyped in the CEPH Caucasian HapMap
families, it became clear that allele G of SNP rs12255372, is
observed to be nearly perfectly correlated with allele 0 of
DG10S478 (r.sup.2=0.95, P=5.53.times.10.sup.-38), and allele T of
rs12255372 is correlated with other alleles of DG10S478. Moreover,
the risk conferred by alleles 8 and 12 of DG10S478 do not differ
(P=0.3). Hence it is natural to collapse all the non-0 alleles of
DG10S478 into a composite allele which will be referred to as
allele X. Allele X has frequency of 27.6% and 36.4% in controls and
patients respectively. Assuming a multiplicative model (16, 17),
compared to the risk for non-carriers, allele X has an estimated RR
of 1.50 per copy carried.
Replication of the DG10S478 Association to Type II Diabetes
[0169] To verify the association of DG10S478 to type II diabetes,
the microsatellite was genotyped in a Danish type II diabetes
cohort of 228 cases and 539 controls. The Danish cohort was
selected from the PERF (Prospective Epidemiological Risk Factors)
study in Denmark (19). This female type II diabetes cohort had been
diagnosed previously with type II diabetes. The association
observed in Iceland was replicated (Table 3).
TABLE-US-00004 TABLE 3 DG10S478 Association to Type II Diabetes in
Denmark Affected freq Control freq Allele (n = 228) (n = 539) RR
[95% CI] Two sided P 0 0.669 0.740 0.71 0.0048 4 0.002 0.004 0.59
0.62 8 0.070 0.048 1.49 0.091 12 0.239 0.190 1.34 0.032 16 0.020
0.018 1.12 0.78 X 0.331 0.260 1.41 [1.11, 1.79] 0.0048
[0170] The composite at-risk allele X has a frequency of 26.0% in
controls and 33.1% in type II diabetes cases, giving an estimated
RR of 1.41 (P=0.0048).
[0171] Subsequently, the microsatellite was genotyped in a US
Caucasian type II diabetes cohort of 361 cases and 530 controls
from the PENN CATH study. This study is a cross sectional study of
the association of biochemical and genetic factors with coronary
atherosclerosis in a consecutive cohort of patients undergoing
cardiac catheterization at the University of Pennsylvania Medical
Center. Type II diabetes was defined as a history of fasting blood
glucose .gtoreq.126 mg/dl, 2-hour post-prandial glucose .gtoreq.200
mg/dl, use of oral hypoglycemic agents, or insulin and oral
hypoglycemic in a subject greater than age 40. The association
observed in Iceland was also replicated in this population (Table
4).
TABLE-US-00005 TABLE 4 DG10S478 Association to Type II Diabetes in
the United States Affected freq Control freq Allele (n = 361) (n =
530) RR [95% CI] Two sided P -4 0.001 0.000 -- -- 0 0.615 0.747
0.54 3.3 .times. 10.sup.-9 4 0.003 0.004 0.73 0.72 8 0.085 0.049
1.79 0.0029 12 0.256 0.180 1.57 1.2 .times. 10.sup.-4 16 0.040
0.020 2.07 0.012 X 0.385 0.253 1.85 [1.51, 2.27] 3.3 .times.
10.sup.-9
[0172] The composite at-risk allele X has a frequency of 25.3% in
controls and 38.5% in type II diabetes cases, giving an estimated
RR of 1.85 (P=3.3.times.10.sup.-9). Combining the results from all
3 cohorts using a Mantel-Haneszel model (NOTE 3) yields an overall
two-sided P of 4.7.times.10.sup.-18.
[0173] The association of the composite at-risk allele to type II
diabetes in three populations constitutes strong evidence that
variants of the TCF7L2 gene contribute to the risk of type II
diabetes.
[0174] After establishing beyond doubt the association of the
allele X to type II diabetes, we investigated the mode of
inheritance more closely. The dominant model and recessive model
can be rejected as the heterozygous carriers clearly have increased
risk relative to the non-carriers (P<1.times.10.sup.-6) and
reduced risk compared to the homozygous carriers (P<0.0001). The
multiplicative model provides a better fit, but there is evidence
that the risk of the homozygous carriers relative to the
heterozygous carriers is greater than that of the risk of the
heterozygous carriers relative to the non-carriers. Table 5
provides model-free estimates of the relative risks of the
heterozygous carriers and homozygous carriers compared to the
non-carriers,
TABLE-US-00006 TABLE 5 Model-free estimates of the relative risks
Genotype Relative Risk Cohort 00 0X [95% CI] XX [95% CI] PAR
Iceland 1 1.41 [1.17, 1.70] 2.27 [1.70, 3.04] 0.21 Denmark 1 1.37
[0.98, 1.90] 1.92 [1.13, 3.26] 0.17 USA 1 1.64 [1.23, 2.19] 3.29
[2.13, 5.07] 0.28 Combined 1 1.45 [1.26, 1.67] 2.41 [1.94, 3.00]
0.21
[0175] The three cohorts have similar population frequency for the
at-risk allele, but the RR estimates vary; with the strongest
effect seen in the US cohort and the weakest in the Danish cohort.
While there is no reason for the RR to be identical in the cohorts,
it is noted that the differences in the estimated relative risks do
not quite reach statistical significance (P>0.05). Combining the
results from the cohorts assuming common relative risks, the
heterozygous carriers and homozygous carriers are estimated to have
relative risks of 1.45 and 2.41 respectively compared to the
non-carriers (Table 5). Assuming a population frequency of 26% for
the at-risk allele, heterozygous and homozygous carriers make up
38% and 7% of the population respectively. Hence, this variant has
enough predictive value to be of clinical use. The corresponding
population attributed risk is 21%, which is substantial from a
public health point of view.
[0176] It should also be noted that allele X is in excess in
impaired fasting glucose (IFG) individuals (fasting serum glucose
between 6.1 and 6.9 mM). The composite at-risk allele X has a
frequency of 27.7% in 1393 controls and 37.1% in 278 IFG cases,
giving an estimated RR of 1.54 (P=1.36.times.10.sup.-5).
Association of SNP Markers within Exon 4 LD Block of TCF7L2 with
Type 2 Diabetes.
[0177] In Table 6 we list microsatellite and SNP markers residing
within the exon 4 LD block of TCF7L2. The table contains publically
available SNPs, as well as SNPs discovered by sequencing the entire
LD block region. The table furthermore provides polymorphic
microsatellite markers residing within the block.
TABLE-US-00007 TABLE 6 Polymorphic markers residing within the exon
4 LD block of TCF7L2 (between markers rs4074720 and rs7087006,
positions in Build 34 co-ordinates: rs4074720 (B34: 114413084) -
rs7087006 (B34: 114488013) = 74929bp. Sequence identification
references are indicated as appropriate, referring in each instance
to the SEQ ID number for the amplimer containing the polymorphism,
and forward and reverse primers, as disclosed in the Sequence
listing. A. Public SNPs (including all HapMap ethnicities) Public
Alias Chromosome 10 B34 location Base Change Sequence ID NO:
rs4074720 114413084 A/G rs4074719 114413145 C/T rs4074718 114413204
C/T rs11196181 114413605 A/G rs11196182 114414744 C/T rs4603236
114414765 G/T rs7922298 114414856 C/T rs17747324 114417090 C/T
rs7901695 114418675 C/T 17-19 rs11196185 114420079 C/T rs4132115
114420083 A/C rs4506565 114420628 A/T 14-16 rs7068741 114420845 C/T
rs7069007 114420872 C/G rs7903146 114422936 C/T 11-13 rs11196187
114424032 A/G rs7092484 114425520 A/G rs10885402 114426284 A/C
rs12098651 114426306 A/G rs6585198 114426824 A/G rs7910244
114427209 C/G rs12266632 114429546 C/G rs6585199 114429758 A/G
rs7896811 114431304 C/T rs6585200 114433196 A/G rs6585201 114433370
A/G rs4319449 114433993 G/T rs12220336 114434854 A/G rs7896091
114436550 A/G rs12354626 114437016 A/G rs7075199 114437307 C/G
rs7904519 114438514 A/G rs13376896 114441336 A/C rs10885405
114442257 C/T rs10885406 114442311 A/G rs11196192 114446874 G/T
rs6585202 114447390 C/T rs7924080 114451599 C/T rs7907610 114451677
A/G rs12262948 114452313 C/G rs12243326 114453402 C/T 8-10
rs12265110 114453606 C/T rs7077039 114453664 C/T rs11196198
114456472 A/G rs12775336 114459590 G/T rs7904948 114459672 A/T
rs7100927 114460635 A/G rs11196199 114460704 A/G rs17685538
114462058 C/G rs11592706 114463573 C/T rs7081912 114463678 A/G
rs7895340 114466112 A/G 23-25 rs11196200 114466525 C/G rs11196201
114467894 A/T rs11196202 114470254 A/G rs11196203 114470447 A/C
rs11196204 114470518 A/G rs11196205 114471634 C/G 20-22 rs10885409
114472659 C/T rs12255372 114473489 G/T 5-7 rs12265291 114474827 C/T
rs7904443 114475774 A/G rs11196208 114475903 C/T rs7077247
114476658 C/T rs11196209 114477314 A/G rs4077527 114477628 A/G
rs12718338 114477634 C/T rs11196210 114478558 C/T rs7907632
114481823 A/G rs7071302 114482114 G/T rs12245680 114484778 C/T
rs11196213 114486141 C/T rs4918789 114486394 G/T rs7085785
114487050 C/T rs7085989 114487326 A/G rs7087006 114488013 A/G B.
Novel SNPs discovered and subsequently validated in the exon 4 LD
block of TCF7L2 (amplimers below): Sequence ID deCODE Alias
Chromosome 10 B34 location Base Change NO: SG10S405 114418658 C/T
26-28 SG10S428 114421901 A/C 29-31 SG10S422 114457824 A/G 32-34
SG10S427 114463480 A/T 35-37 SG10S408 114466074 A/T 38-40 SG10S409
114471574 A/C 41-43 SG10S406 114471618 C/G 42-44 SG10S407 114473534
C/G 45-47 C. Polymorphic microsatellites within the exon 4 LD block
of TCF7L2 (amplimers below): Sequence ID Microsatellite C10 B34
Start C10 B34 End NO: DG10S2164 114460344 114460627 48-50 DG10S478
114460845 114461228 2-4 DG10S479 114475487 114475632 51-53
TABLE-US-00008 TABLE 7 Amplimers and primers for selected markers
within the exon 4 LD block of TCF7L2 >DG10S478
TTCAGGCCATTGGTGTTGTATATATTTCAAGATTTGCTCACAGGTCCAAA
GCTTAACTTAAGCTCCCTGAGACATATCATAAAATATGATTTGGGGAAAA
ACCCTAATGGGCCATGATCAGAACATTATTATTCAACAAAGGATGAAATG
CTTAAGCCAAGATGGCCTTCTTTCTTTCTTTCTTTCTTICTTTTTTTTTA
ATGAAAGTTGAGCAGACTCCCGTCCAACAGTTTTCAATGTAGGAATTCCC
ACAGCCCCATTTGATTGCAGTTTGTTGAAAAGTTTAATGTTTTTGTAGGC
AATTCATAATTTCCACATTGAACAGCCTGAGAGGAAGAGAGCTGGAGCCC
ACTGTTGTTTTTGTAGTGGGATGGTGGGAACTTT (SEQ ID NO: 2) Primers: F:
TTCAGGCCATTGGTGTTGTA (SEQ ID NO: 3) R: AAAGTTCCCACCATCCCACT (SEQ ID
NO: 4) >rs12255372
TTGTCCCTTGAGGTGTACTGGAAACTAAGGCGTGAGGGACTCATAGGGGT
CTGGCTTGGAAAGTGTATTGCTATGTCCAGTTTACACATAAGGATGTGCA
AATCCAGCAGGTTAGCTGAGCTGCCCAGGAATATCCAGGCAAGAAT K
ACCATATTCTGATAATTACTCAGGCCTCTGCCTCATCTCCGCTGCCCCCC
CGCCCCCTGACTCTCTTCTGAGTGCCAGATTCAGCCTCCATTTGAATGCC
AAATAGACAGGAAATTAGCATGCCCAGAATCCACGTCTTTAGTGCACTCT
CTCCCCAGCTCCAAACCTGTTACTGCTTGTGITCAACATCTCAGTAAAGC
TCAACAACATCGACCCATT (SEQ ID NO: 5) Primers: F:
TTGTCCCTTGAGGTGTACTGG (SEQ ID NO: 6) R: AATGGGTCGATGTTGTTGAG (SEQ
ID NO: 7) >rs12243326
GCTGTGAAATCCCCTGTGTAGTGGGAAGAAGAAATAGCAAATCTTAGCTG
CCTTGGACCTGATATAATTATTTGTCTTCATTTACATGGTT Y
ATCCTTCAAGGTTGAATAAATGATGTGGGAGCTAGTCAAGGGGCTTTAGG
TATGTGATTTCATGCCTACTTTTTTTTAGGTAGAGAAACTGAGGTCACAG
GGTACTAGAGAATGGACTCTAAGATTCAGGTTTCTGAATTGCCTGTGGTT
TTGTTGACTCAACTGCTCTTCTGTTGTTTTTTAGCCACATGCCTTGAAAC
AGTCCTCTTTCCCATGTTTCTTCATCAGCACCATTAACCCAAGGTATACT
GTCCTCTCTTATCTTTCACAAGGTCTTGGAGTTCCCATGCCTTTGTAAGC
ATCCCTCCCCGAGATTCAGCACCAACCAAAATCACATTTGGAAAAATTGC
TTGTTTCCCAAGAAGCTTTGGAGGATATGATTTTGTATAGAACGGGTTCA
CAGGTTTTCTGTTCATTCTTCTATGGTGGAGTGTGTGTGTATGTGACTCT GTCTTCTCTCCATTCC
(SEQ ID NO: 8) Primers: F: GCTGTGAAATCCCCTGTGTAG (SEQ ID NO: 9) R:
GGAATGGAGAGAAGACAGAGTCA (SEQ ID NO: 10) >rs7903146
AAGGGAGAAAGCAGGATTGAGCAGGGGGAGCCGTCAGATGGTAATGCAGA
TGTGATGAGATCTCTGCCGGACCAAAGAGAAGATTCCTTTTTAAATGGTG
ACAAATTCATGGGCTTTCTCTGCCTCAAAACCTAGCACAGCTGTTATTTA
CTGAACAATTAGAGAGCTAAGCACTTTTTAGATA Y
TATATAATTTAATTGCCGTATGAGGCACCCTTAGTTTTCAGACGAGAAAC
CACAGTTACAGGGAAGGCAAGTAACTTAGTCAATGTCAGATAACTAGGAA
AAGGTTAGAGGGGCCCTGGACACAGGCCTGTGTGACTGAGAAGCTTGGGC
ACTTCACTGCTACATTTCATCTCTTCGCT (SEQ ID NO: 11) Primers: F:
AAGGGAGAAAGCAGGATTGA (SEQ ID NO: 12) R: AGCGAAGAGATGAAATGTAGCA (SEQ
ID NO: 13) >rs4506565
CTGATGAGGGTAGGGAGCATCTGTCTGCAGCTTCATCTTCATTGTCTAGG
GGCTCCAGAAATATCTGTGAGTAAATAAGTTATTTAATCTTTGCCTCAAA
TTTCCAGTGACTGTAGGGATATAGCTGTGAGCCTCTAGGAGCTGAGATTT
TTTAAATTTCCCACTTAAACATTTATTTAAAAATTTTGTGCTCAGCATGG
ACTAAGGACTTTACATTCATTAACTCATTTACAGCTTGATCCTATGCGGT
GGGCATTCATTTACAGAGGATCCCATTTTACAGGTGAGGAAGAGGCCAGC
TAGGGGTGCAGCCTAGGTTAGTATTCTAGAGCTCATCAGGCTGTGTTGTC
CCCAGTGAAAGAATAAGCAAAGAAGTGAATGTTGTGCATTGAGAAAAATG
ACTCTCGGAGGAGGATGAGCCTCTCGGATATGGCGACCGAAGTGAT W
TGGGGCCCTTGTCAAGGGTCTCTATTATGGCATCAAGAAAAGATGCTGCT
TTCGGTGATGCCCGAGGAGAGCCTCAATATTTTACATGGGAAACCTAAAA
AAGGGGCCATGTTGTGGTCTCTGCACCTAAGA (SEQ ID NO: 14) Primers: F:
CTGATGAGGGTAGGGAGCA (SEQ ID NO: 15) R: TCTTAGGTGCAGAGACCACAAC (SEQ
ID NO: 16) >rs7901695
TATTTAGAAACCATAAAATCCACCTATTTGAGGIGTACAATTGAGTGATT
TTCTGTATAGTCACAGATCTGTGCAGTCATCCACACCCTCTAACTCCAGG
ACATTTTCCTCACCCCCGAGGAGAAACCTCCCTTACCCATTAGCAGTCAC
TCCTCATTTCCTCTCCCCCCAGCCCCTGGCAATCACTGTGGATTTGCCTG
TTCTTGACATTTCATATAAATGGTATCATAAAATCTA Y
GGGCTTTTGTGTCTGTCTGCTTTCACTTAGCATACGGTTCTCAAGGTTCA
TCCAGTATTGTAGCATCTATCAGTATGTCATTCCITTTTATGGCCAAATA
ATATTTTATTGTATGGATAGACATTTTGTTTATTCATTTATCTGTTTTTG
GTTATTATGAGTAACACTACTATGAACATTTTGCACAAATTTTTGTATTG
ACATGTTTTCATTTCTCCTGGGTATAGTCCTATGAGTGGAATTGCTGG (SEQ ID NO: 17)
Primers: F: TATTTAGAAACCATAAAATCCACCTAT (SEQ ID NO: 18) R:
CCAGCAATTCCACTCATAGGAC (SEQ ID NO: 19) >rs11196205
TTGTCTCCTTTTGTTTCTGCTACTGTGAATGATCCTGTGATGATCATCTT
TGTGTGTAAATCTTTGTCCCCTCGCCCCCTCCCCTTTTATTATTTTCTTG
GGATAGACCCCAGGACAAAAGGTAGAAAAGAACAAAGTGTTAAAAAATTT
CTTGATACATAGCCACAGATTATTTTCCTGAAAGTTCTCAACATTTATAA CTAC S
AGCAGTATGTAAGAGAGTTATGGTTGGAATGATTTTAATGTCTCTGGGGA
ATTTAACAACAAAAAAACTTTAGGCTTCTTTGGAGAGAGACATGCCCTTA
ACTCCACCCCGCCCTAGAACAGAGACCCAGCCCATCCAAGTCAGCCTCCC
CAGGTCCTCCACCTTCAAAACAGGCAAACGAAATCATTTCTTGAATAATT
GGTAGGCTTCAAGGTCAGATGTT (SEQ ID NO: 20) Primers: F:
TTGTCTCCTTTTGTTTCTGCTAC (SEQ ID NO: 21) R: AACATCTGACCTTGAAGCCTAC
(SEQ ID NO: 22) >rs7895340
TCAGGGACAGTGCATAGGTGTAAAGAAGTTGCTGGTTGGGGGTTCTAATG
CAGGTTTCTCCAAAAGTGAATGCCCTGTTAAAAAAAAATTCTTAACAAAT
ATACAGAGATTTTTTTTTTAAAAAAGTGTGACAGTTCTAGACACCTAGAG AGTAAA R
TGAAGAAGCCTGTTTTCAGGTTTCCCGCCTCCCTGAATTTCCCAGCATGG
TCCAGGCTTTGAAATTTATTTATCTGCTTTTGGCAATGGTTGATGGGAAT
TTCCCACATTTATTTTTTAGCTACAGAGAAAGGACATTATCTTTAAAATC
TCTTCGTTGTTCTCTCTCTTTGA (SEQ ID NO: 23) Primers: F:
TCAGGGACAGTGCATAGGTG (SEQ ID NO: 24) R: TCAAAGAGAGAGAACAACGAAGA
(SEQ ID NO: 25) >SG10S405
TATTTAGAAACCATAAAATCCACCTATTTGAGGTGTACAATTGAGTGATT
TTCTGTATAGTCACAGATCTGTGCAGTCATCCACACCCTCTAACTCCAGG
ACATTTTCCTCACCCCCGAGGAGAAACCTCCCTTACCCATTAGCAGTCAC
TCCTCATTTCCTCTCCCCCCAGCCCCTGGCAATCACTGTGGATTTGCCTG
TTCTTGACATTTCATATAAA Y
GGTATCATAAAATCTATGGGCTTTTGTGTCTGTCTGCTTTCACTTAGCAT
ACGGTTCTCAAGGTTCATCCAGTATTGTAGCATCTATCAGTATGTCATTC
CTTTTTATGGCCAAATAATATTTTATTGTATGGATAGACATTTTGTTTAT
TCATTTATCTGTTTTTGGTTATTATGAGTAACACTACTATGAACATTTTG
CACAAATTTTTGTATTGACATGTTTTCATTTCTCCTGGGTATAGTCCTAT
GAGTGGAATTGCTGGGTCATATAATAAATAACTGTTTAACATTTTGGGGA
GCTGCCAAACTTTTAAAACCTTGGGTTCTGTGATGTACCAGTTGTGTTAG GCA (SEQ ID NO:
26) Primers: F: TATTTAGAAACCATAAAATCCACCTAT (SEQ ID NO: 27) R:
TGCCTAACACAACTGGTACATC (SEQ ID NO: 28) >SG10S428
TGCCAGGGGTTTTATGGTTAATTTTCCTCCATTATGAGGGTTGACTCAGC
CTTGGGTATTAGATGTCTTTGAGAATCCAGGGTTCAAATACCACAGCTGG
TAGAATGTTTCTCAACTTGGAGCCAATCTCCATCTACTGAAGGTACGCTG
GTTTAGACAGACAACAGGGACATCAGCATTTTAAAAAGCGGTGGAAAAAG
TTTGCTTGTCTTGATTGGAGCCATGACATTTTATTTTGAAATTTCAAATA
ACATGAAGGGAGGTTTGGAGCGGTTTTTGGTTTATCCAAAGGGCAGTGGA
TTGAAGGCTGAGAAACACCAGGCTGAATGGGAGAGGGGTTGGGGTCCCCC
TGTGAGATAGTGAAACAATGGTAGTGCCATCCAATGATAGGCACTTTTCT
GTCATTCAGAAGCAGAAAGGGGGCCAGAGGCCCATTGGCCTTACTGGG M
AGTAAGCTGTAGAGCTGCTGCCTTTTCGTGAAAGGGTTGACACCAACCTT
CTCCCCCAGGAAGAGTGACCAGGGACCTGAGGGGCATGGTCGAGCAGATG
ACAGCCTTTGTAAAACATCTCC (SEQ ID NO: 29) Primers: F:
TGCCAGGGGTTTTATGGTTA (SEQ ID NO: 30) R: GGAGATGTTTTACAAAGGCTGTC
(SEQ ID NO: 31) >SG10S422
TTGGTAGAGATGGGGTCTCCTAGGCTGGTCTTGAACTCCTGG R
CTCAAGCAATCTTCCTGCCTCAGCCTTCCAAAGTACTGGGATTACTGGCG
TGGGCCACCATGCCTGGCTTGAAATTTTTCTATGGCTTTATTCTTTCTCC
AAGTACAGAGTCTACCCAACCTICTGAGATCTTTGGTTTTCTTTTCCTAG
GTAACTATAGTACATACTTATTTATGTTAAACAACAGCAATCACACATTT
CTTTTTCTATACAGTCATGCTTTATAGGCAAATAAAGCCTCCGTCTTAGG
CTTTCTGGATTTTTTCAAAAGATGCAATTCCTGGAGTATGTTTTTACTTA
GAGCAAAGCAGCCTAGTCTCCTATACCTTCTGCATCTGCAGAAAAGTTGG
TTAAACAGACTTTGTAATGATGCCCCTTACAATTCTGAAGGGACTTGTGA
AATAGTTTCACAGAGTTTCAGTGTTAGGTATATTTGATCAATGCTAACTT
TTGGAAAACTTTGGTGCCTGTATGATTCAGAGGGTAGGGCAGAATATTAA
ATTAATCACAACTTCTTGTATTTTAACCATTCTGGGTAAATTGGGATTCC
GTGACGCCCAGGCAAAATTAT (SEQ ID NO: 32) Primers: F:
TTGGTAGAGATGGGGTCTCC (SEQ ID NO: 33) R: ATAATTTTGCCTGGGCGTCA (SEQ
ID NO: 34) >SG10S427
TATCTTATATCCCCTCCAAGCATTCATTAACTGATGGATTAGTGAGTTGG
CCTTGAGAAGCATAAAGGCTCGTCTCCATGTGCTTCTAAGCATTGTGTCT
AAGTTCTGTTTGGTTTCCTGAGTGAAACTGTCTTAATGTTACCAACAGAA GTTAAATGCCTAAGAG
W TTCTTATACATGGGCTGAGTACCTCTGTGACTGGGCAAGCCACCTCACCT
CATTTTACCTTGTCTGCAAAATGAGGAACTGGGTCAACTCATCGTTCAAA
TCTCACTGAAAGCTAATTGATCGCTTTTGACAGAAGTAGCTCCCTTGGGC
CGTATATTTATTTCCTAGCTTGGAGGAAGGIGGGGACAGACAGAATTGAT
GTACACCTITATTTTTATCTCTATGGTAAACCTGTGCATACTAAAGCATT
CCTCTGGTCTTTTGAGATGAGTGTATACATTGTGTCTGGCCCTGTGCATT
TTTTACCAAGAAGTAAGTTTTGTTGAGTAAACTTGGGTTGTATGAAGAAC
TGCATGCTCACCGTACTCAAGTAGCTTTTGCTACCTAAAGGACAGCTGCT
CATATGTACTTGACTTCCTTTAAAGTGAAGGATGATGACATTTGAAAAAC GGAGGTTGAAAAGGAG
(SEQ ID NO: 35) Primers: F: TATCTTATATCCCCTCCAAGCATTC (SEQ ID NO:
36)
R: CTCCTTTTCAACCTCCGTTTT (SEQ ID NO: 37) >SG10S408
TTGAGCATGTGTTATTTAATGAGTTATACCTCTGTCATATGTGTGTGTTT
ATATCACAAAATAACTTATTTTTATAAAACCATATTTTGAGTCATCATTT
GTGACAATGTCTTCTTTTCTCTGGTATAAATGAGGCATGTAGAAAGAAGA
TTGACATTTGCTAGAAGCTTCCCCTTTCCTCTAACTCCACAATAAAATGG
ATGCTCATAATTACATCTGCTCCTATAAGGTCAAGATTTCAGGGCTGGAA
GTGACCTTAGATCATTTAGGCCCAACTTGCCCTCAGGAAAGGAAACTGAG
GCCCAGAGATGCCTTAAGTGAATTGCCCAATGTCACACGCTGAGTCAGTG
GCCAGAGCAAGGCTTGGATCCAGTTCTCTGCTCCCTTTCCAGAGCCTTGT
GATGTCTTCTCTCCTACAGGAGGTGAAAATAACTGCTGTGGCTGGTTCTG
TTTTGCTGACTGTAAATTGGGTCATGGTCAGGGACAGTGCATAGGTGTAA
AGAAGTTGCTGGTTGGGGGITCTAATGCAGGTTTCTCCAAAAGTGAATGC
CCTGTTAAAAAAAAATTCTTAACAAATATACAGAGATTTTTTTTT W
AAAAAAGTGTGACAGTTCTAGACACCTAGAGAGTAAAGTGAAGAAGCCTG
TTTTCAGGTTTCCCGCCTCCCTGAATTTCCCAGCATGGTCCAGGCTTTGA
AATTTATTTATCTGCTTTTGGCAATGGTTGATGGGAATTTCCCACATTTA
TTTTTTAGCTACAGAGAAAGGACATTATCTTTAAAATCTCTTCGTTGTTC
TCTCTCTTTGAGTGAGGAGAGAAGATGTGAATCCTGGCAGTGGTTCAGAG
TGGACACAGCCCCTGTGTTTGTGGCATAGGCTCTGTGGGCCCCATGCCAG
GGAGCAGTACCCCCGTGTAAAGGAGTGGGGGTTTGTCCATTTGGATAGAG
CAAAGATCCTCCACCTCAAATCCCACAAGAACAGTTGCCACAACCTGGGC
CCTAAGCATCTCATTTTCCTATGTAGAAATTAATGATCTGGAGGAGATGG
CAAAACATTCCTTCCAGAGCCTGTGTGGATTTTGG (SEQ ID NO: 38) Primers: F:
TTGAGCATGTGTTATTTAATGAGTTA (SEQ ID NO: 39) R: CCAAAATCCACACAGGCTCT
(SEQ ID NO: 40) >SG10S409
TAGTGCTCAGTATTTCCAACGTTCTGTTTATTTAAGATGAAAATTGCTGT
AGTTAATAAGCACTTCCCCATGTCATTAAAATGCTTAAGGATTTTTAATG
ACCACATAACAGTCCATAATATGATTAAACCCCAATTTACTGAATCAATG
CCATATTGTTGGGTCTTTAGATTGTCTCCTTTTGTTTCTGCTACTGTGAA
TGATCCTGTGATGATCATCTTTGTGTGTAAATCTTTGTCCCCTCGCCCCC
TCCCCTTTTATTATTTTCTTGGGATAGACCCCAGGACAAAAGGTAGAAAA GAACAAAGTGTTAAA
M AATTTCTTGATACATAGCCACAGATTATTTTCCTGAAAGTTCTCAACATT
TATAACTACGAGCAGTATGTAAGAGAGTTATGGTTGGAATGATTTTAATG
TCTCTGGGGAATTTAACAACAAAAAAACITTAGGCTTCTTTGGAGAGAGA
CATGCCCTTAACTCCACCCCGCCCTAGAACAGAGACCCAGCCCATCCAAG
TCAGCCTCCCCAGGTCCTCCACCTTCAAAACAGGCAAACGAAATCATTTC
TTGAATAATTGGTAGGCTTCAAGGTCAGATGTT (SEQ ID NO: 41) Primers: F:
TAGTGCTCAGTATTTCCAACGTTCT (SEQ ID NO: 42) R:
AACATCTGACCTTGAAGCCTACC (SEQ ID NO: 43) >SG10S406
TAGTGCTCAGTATTTCCAACGTTCTGTTTATTTAAGATGAAAATTGCTGT
AGTTAATAAGCACTTCCCCATGTCATTAAAATGCTTAAGGATTTTTAATG
ACCACATAACAGTCCATAATATGATTAAACCCCAATTTACTGAATCAATG
CCATATTGTTGGGTCTTTAGATTGTCTCCTTTTGTTTCTGCTACTGTGAA
TGATCCTGTGATGATCATCTTTGTGTGTAAATCTTTGTCCCCTCGCCCCC
TCCCCTTTTATTATTTTCTTGGGATAGACCCCAGGACAAAAGGTAGAAAA
GAACAAAGTGTTAAAAAATTTCTTGATACATAGCCACAGATTATTTTCCT GAAAGTTCT S
AACATTTATAACTACGAGCAGTATGTAAGAGAGTTATGGTTGGAATGATT
TTAATGTCTCTGGGGAATTTAACAACAAAAAAACTTTAGGCTTCTTTGGA
GAGAGACATGCCCTTAACTCCACCCCGCCCTAGAACAGAGACCCAGCCCA
TCCAAGTCAGCCTCCCCAGGTCCTCCACCTTCAAAACAGGCAAACGAAAT
CATTTCTTGAATAATTGGTAGGCTTCAAGGICAGATGTT (SEQ ID NO: 44) Primers: F:
TAGTGCTCAGTATTTCCAACGTTCT (SEQ ID NO: 42) R:
AACATCTGACCTTGAAGCCTACC (SEQ ID NO: 43) >SG10S407
TGCTATGTCCAGTTTACACATAAGGATGTGCAAATCCAGCAGGTTAGCTG
AGCTGCCCAGGAATATCCAGGCAAGAATGACCATATTCTGATAATTACTC
AGGCCTCTGCCTCATCTCCGCTG S
CCCCCCGCCCCCTGACTCTCTTCTGAGTGCCAGATTCAGCCTCCATTTGA
ATGCCAAATAGACAGGAAATTAGCATGCCCAGAATCCACGTCTTTAGTGC
ACTCTCTCCCCAGCTCCAAACCTGTTACTGCTTGTGTTCAACATCTCAGT
AAAGCTCAACAACATCGACCCATTACTTAGGCCTCAAACCTTGGGTGGCA
TCGTCGATTGCTCTTTTCTTTCATACCCCACATTCAACCCATCAGCCCAT
CCCACAGGCCCAAGTGIGTCCICTCTACCTTCAAAGCGTGTGTGGCATCC
ACCGCTTATCACCACCTCTGCCATTACCACTGGAGTCCAGTGCCATCATC
TCTCACTTGGATGTGGCCAGAGTGTCTTTGCTGGTCTCCTTCTTGCTTCC
TACCTTTGTAACAGCCTATCATCTATCTCTGGICTCCATAGCTCACTCCC
ATACTTTGAGAGGGCCTTTGAAAGCCTTAGACAGATCATATCACAGACCT
CTATACTGAAAGTCGGG (SEQ ID NO: 45) Primers: F:
TGCTATGTCCAGTTTACACATAAGG (SEQ ID NO: 46) R:
CCCGACTTTCAGTATAGAGGTCTG (SEQ ID NO: 47) >DG10S2164
CCATCTGTGGAGCAGAGTCACTGAAAGGAAATACTGGAAATACTGGAAGC
CACTTGGTGTTTTATCAAGGATGTGAGGTTTCCTGGCAACTTTGTCGCCA
TATCATCATCATCATCACCATCATCATCATCATCATCATCATCATCATCA
TCATCATCATCATCATCTGCCCTTTAAGTTTTCTGCTTGTTTAGAAAAGA
AATTTATACAGAGCCCCCAGTAGCAGCTGTAAGGGGGCAGGTTCTTGGAG
CAGCCCATCCTCAACATTCTTGCTGCTGATGGAA (SEQ ID NO: 48) Primers: F:
CCATCTGTGGAGCAGAGTCA (SEQ ID NO: 49) R: TTCCATCAGCAGCAAGAATG (SEQ
ID NO: 50) >DG10S479
TCCACGCAGAGAGGATCTAAATCTGGCTCTTTCAATTGCCTTCATACAT
GTGCATACACACCACACACACACACACACACACACACACACACACACACA
CAGACACATACATATGCACACACCCCGACTCAATGGAGGACCCTC (SEQ ID NO: 51)
Primers: F: TCCACGCAGAGAGGATCTAAA (SEQ ID NO: 52) R:
GAGGGTCCTCCATTGAGTCG (SEQ ID NO: 53)
[0178] To further investigate the possibility that other marker
alleles in the exon 4 LD block of TCF7L2 exhibit a higher
correlation with type II diabetes than allele X, we used the
DG10S478 genotype data generated in the HapMap CEU samples. The
five SNPs from HapMap Phase I with strongest correlation to
DG10S478 were, in descending order, rs12255372 (r.sup.2=0.95),
rs7903146 (r.sup.2=0.78), rs7901695 (r.sup.2=0.61), rs11196205
(r.sup.2=0.43), and rs7895340 (r.sup.2=0.42). We genotyped these
five SNPs in the three cohorts and the correlations between the
five SNPs and DG10S478, the latter treated as a biallelic marker,
were very similar to that observed in the CEU samples. All five
SNPs showed association to type II diabetes. While some SNPs showed
slightly higher estimated relative risks and lower p-values in one
or two of the cohorts, none exhibited stronger association to type
II diabetes than DG10S478 when the results for all three cohorts
were combined using the Mantel-Haenszel model. However, although
rs11196205 and rs7895340 clearly have weaker association to type II
diabetes, compared to allele X (RR=1.56, P=4.7.times.10.sup.-18),
the strength of the association to type II diabetes for allele T of
rs12255372 (RR=1.52, P=2.5.times.10.sup.-16) and for allele T of
rs7903146 (RR=1.54, P=2.1.times.10.sup.-17) are comparable.
[0179] Following the subsequent release of HapMap Phase II in
October 2005, two additional SNPs were identified that show strong
correlation to microsatellite DG10S478--rs12243326 (r.sup.2=0.961)
and rs4506565 (r.sup.2=0.716). The alleles associated with
susceptibility to type 2 diabetes will be C for rs12243326 (C/T
SNP) and T for rs4506565 (A/T SNP).
[0180] It should be noted that among those haplotypes that carry
the C allele of rs7903146, those that carry the A allele of
rs10885406 have an estimated relative risk of 1.06 compared to
those that carry the G allele of rs10885406, but the difference is
not statistically significant (P=0.22).
[0181] In an attempt to replicate and refine this association with
type 2 diabetes, we genotyped DG10S478, rs12255372 and rs7903146 in
a large additional Danish cohort, consisting of 1111 cases and 2315
controls and in a more genetically diverse West African cohort,
consisting of 618 cases and 434 controls derived from the Africa
America Diabetes Mellitus study (23). In the Danes, all three
variants were strongly associated with disease risk, as previously
observed in Iceland. However, the association of allele T of
rs7903146 (Relative Risk=1.53, P=4.06.times.10.sup.-14, PAR=24.4%)
was noticeably stronger than that provided by the other two
variants. In the West African study group, after adjustment for
relatedness and ethnic origin, we replicated the association of
allele T of rs7903146 to type 2 diabetes (Relative Risk=1.45, 95%
C.I.=1.20-1.76, P=0.000146, PAR=22.2%), but not in the case of the
other two variants. This suggests that allele T of rs7903146 is
either the risk variant itself or the closest known correlate of an
unidentified risk variant. The exclusion of the markers DG10S478
and rs12255372 as at-risk markers in the West African group was
possible because unlike in populations of European ancestry, where
the T allele of rs7903146 occurs almost exclusively on chromosomes
carrying both allele X of DG10S478 and allele T of rs12255372, in
West Africans the T allele of rs7903146 occurs with both alleles of
DG10S478 and rs12255372. This is consistent with the observation
that T is the ancestral allele of rs7903146, whereas allele X of
DG10S478 and allele T of rs12255372 are both different from the
chimpanzee reference sequence. More generally, this finding is also
consistent with the expectation that relatively diverse
populations, such as those of West Africa, provide the means to
refine association signals detected in regions of strong linkage
disequilibrium in more homogeneous populations.
Discussion
[0182] In this study we describe the identification of a novel
candidate gene for type II diabetes within the previously reported
10q linkage region (10), encoding transcription factor 7-like 2
(TCF7L2--formerly TCF4) on 10q25.2. We show that it confers risk of
type II diabetes in Iceland, Denmark and the US with similar
frequency and relative risks. While the variant does not explain a
substantial fraction of the familial clustering of type II
diabetes, the population attributed risk of at least 20% is
significant from a public health point of view. Compared to the
non-carriers, the relative risks of heterozygous carrier of the
at-risk composite allele (approximately 38% of the population) and
homozygous carriers (about 7% of the population) are 1.45 and 2.41,
respectively. Hence, this variant has enough predictive value to be
of clinical use.
[0183] We report the variant as a type II diabetes-associated
microsatellite, DG10S478, within the third intron of the TCF7L2
gene. The TCF7L2 gene product is a high mobility group (HMG)
box-containing transcription factor which plays a role in the Wnt
signalling pathway. This pathway is considered one of the key
developmental and growth regulatory mechanisms of the cell; it is
mediated by secreted glycoproteins, known as Wnts, which initiate
many signalling cascades within target cells upon binding to a
cognate receptor complex, consisting of a member of the Frizzled
family and a member of the LDL receptor family, Lrp5/6 (24). Wnt
signaling uncouples the central player in this pathway,
.beta.-catenin, from the degradation complex and translocates it to
the nucleus where it transiently converts TCF factors from
repressors into transcriptional activators (25). The .beta.-catenin
protein is also important for mediating cell adhesion through its
binding of cadherins (15).
[0184] The NCBI RefSeq for TCF7L2 contains 14 exons. However, Duval
et al (26) showed that TCF7L2 has 17 exons, of which 5 are
alternative; in addition, it was reported that three alternative
splice acceptor sites are used. This study also demonstrated the
alternative use of three consecutive exons located in the 3' end of
the TCF7L2 gene which change the reading frames used in the last
exon, leading to the synthesis of a large number of TCF7L2 isoforms
with short, medium, or long COOH-terminal ends.
[0185] Similar to TCF7L2, five of the six positionally cloned genes
for the rare Mendelian forms of Type II Diabetes, namely
maturity-onset diabetes of the young (MODY), are transcription
factors (27). Additional transcription factors have been implicated
in the pathogenesis of type II diabetes, including peroxisome
proliferator-activated receptor gamma (PPAR.gamma.) (7) and the
forkhead gene family (28, 29). Noble et al described a missense
mutation (C883A) in the related TCF7 gene in type 1 diabetes (30).
However, it is not clear if TCF7 and TCF7L2 operate in the same
pathway with respect to the pathogenesis of diabetes.
[0186] Mutations have been described in the TCF7L2 gene, including
the deletion of an A in an (A).sub.9 coding repeat (exon 17) (26,
31-33) and a number of mutations in colorectal cell lines (26).
DG10S478 resides within a clearly defined 74.9 kb LD block (CEPH
Caucasian HapMap Phase II) that encapsulates exon 4 and flanking
intronic sequences 5' and 3' to the exon. It is possible that
DG10S478 is the causative variant itself; it is also possible that
DG10S478 is a surrogate for an underlying variant that affects
transcription, splicing or message stability. Such a variant is
likely to be in strong LD with DG10S478, i.e. the variant resides
within the exon 4 LD block of TCF7L2
[0187] Several lines of evidence suggest an enteroendocrine role of
this gene in the pathogenesis of type II diabetes. Firstly, TCF7L2
has been implicated in the development of colorectal cancer (34)
and small-molecule antagonists of the oncogenic TCF/.beta.-catenin
protein complex have been already described (35). In addition,
TCF7L2-/- mice, which die within 24 hours after birth, lack an
intestinal epithelial stem-cell compartment (36). Variants of the
TCF7L2 gene could influence the susceptibility to type II diabetes
through altering levels of the insulinotropic hormone glucagon-like
peptide 1 (GLP-1), one of the peptides encoded by the proglucagon
gene whose expression in enteroendocrine cells is transcriptionally
regulated by TCF7L2. In concert with insulin, GLP-1 exerts crucial
effects on blood glucose homeostasis (12). GLP-1 analogs and
inhibitors of dipeptidyl peptidase IV are currently in clinical
development.
[0188] The references cited in this specification are incorporated
herein in their entirety.
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[0225] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
53174930DNAHomo sapiens 1cttgtgtagg aactcacgct ttgtttattc
agcaatcatt cctccagaaa taaccttaat 60agcaacaaga aaaaagaata ggtgtttttt
gagctctatc tgccagtttc tctatatatg 120gacattatat attgcaacat
aacactcaca atgcctttaa acatcatccc cgttatacag 180ataagaaaac
agaatttcaa agaaggtagg ggacttgccc agggatacat agctagcaag
240tggcagcgct ggattgagtc tgggccttgt ctgaggctcg ggtcctgtca
tgctctgcgg 300ttgctatgtt gacatgcaaa gggagaggca gctgctggga
gtctaggtgg gtttctcttt 360gagaatgcta acgtgaaccc tcaaggtgaa
tcagaatcct tttgcaagtg aataatcaga 420tgtaggttcc tgtgtctccc
tgtaaaatga aagcctcttt tttccaaggt ccagtataga 480cctgaagctg
ggttactctg gaatttccct ctctggctgg agtgactgag gccttgcacg
540tgacattggt gaggactcgc agcctcaggt ctggcttccc ttagcaaccc
ccctttcctg 600tctctgcctc tggagttcac cattaaaaaa aaaaaaagaa
aaaaagccaa aacactttat 660aaagttacat gctgggtttc ttctatgtcc
tagaaactgt cttaattcat cttccccttt 720actcttatat gagcaggaag
aaaaaaaaat tgctagtcaa tgctaataat tatggcatgt 780aatgtaattg
gaagtgtttc actgacatgc tcatgagagt ttgcggcttc atcttcaggc
840tgggatgtag cactagactt gccttgagtg tctgcacaag cctttgatgc
aggtagacca 900tattataaat aggcgcgttg ctatggtgag gatggcagtc
cttgcttgct gtgggtaacc 960ttttctacct tctcggacac tgttttaaaa
cacagcagcg tgatagcatt tcatttaatt 1020tggaccaagg tggggtagat
gaaatgttga gatttagatc taaaatgttg ttgtggtgtt 1080tcagggggtt
ctggctcacc tagtactatg gaagattttg cagattgggc ttcctcatga
1140tttatttaga aatagatttt ctaatagatg gggtgagggg agggtggtgg
gcagaaggct 1200gggctttctt ctcttccccc tcctcctttc attgagcgct
tctgcgaatg tgttggcttt 1260gatgccccag gagctcatac agtgaaatgg
aagttcaggt tggcacgttg cagaaatgat 1320tattcctggt agtacgtttc
ccattactgt taataatata aagacaattg cctgcctctc 1380aggactcctg
cacgtggcta cagtcatttc ttcatggaat tagacacata gcagtgggga
1440ccaggagtgt tttattagtg attgtcctcc tgcaagtttc cagggtatct
cagcttagac 1500acatgaatta ttttttcctg ttgcttggag ggtatacttt
taattatatt cattcaataa 1560cagagcagtt caggtttgta aaatattttt
tctcccccaa ccttttcccc agcatacatc 1620cccgtcccgt aagtttctgg
gcagagacaa tctcaggaac ctaaaggttg ctaaaaaatt 1680agctagttgg
ccaggcgcat gactcatgcc agtaatccca gcactttggg aggctgaggt
1740gggtggatcg cttgagccca gaaattcgag accagcctag acaacatggc
aaaaccctgt 1800ctctacaaac aaaacaaaat ctagctgggc atggtggtgc
atgcctgtag tcccagctac 1860tggggaggct gaggtgggcg ggcgattgag
ctcaggaggt ccaggctgca gtgagccgtg 1920attgtgccac tgcactgcag
cctggatgac tgagtgggac cctgtctcaa taataaataa 1980ataaataaat
aaaaaataaa aaaaattagc tagccaagct gcttataggt cttttacatg
2040gccaagccac tttctcacct ttaaaatggt aataacgttt ccgtactcat
ctcaatgggt 2100tttgagtgcc aagacagacc gtttgatgga agccctctgg
ggagaaaaat gctacccaag 2160acaggctttt caattggaga ctgatccatt
ggtgttttgg tcagttggtg ttgaaatccc 2220tatttttcca gctcaggact
gcctctctcc ctggaactct tcccgaggtg agttctgcag 2280ccttccttgg
gaactctcag cctctggatc ccttcttgcc aggtggagtg gacatgccaa
2340agttgtgggc cagactcgga ctgcctggct tgtctcagca cctttgggga
cccacttccc 2400ctctctggga actggggaag ctaacagaga tcttgctagg
ggggtggaat cctgtatcca 2460tgtgaggttg tacccccagg ctcctgagtg
gtttgaaagt ggggaaccct ggccgggcgc 2520ggtggctcat gcctataatc
ccagcacttt gggaggctga ggcgggcgga tcacaaggtc 2580aggagatcga
aaccatcctg gctaacacga tgaaaccccg tctctatgtg cgtggtggct
2640ggcacctgta gtcccagctg ctcgggagtc tgaggcagga gaatggcgtg
aacccgggag 2700gcggagcttg cagtgagccg agatcgcccc actgcactcc
agcctgggcg acagagcgag 2760actccatctc aaaaaaaaaa agaaagaaaa
aaaaagaaag tggggaaccc ctcccccagg 2820atgagaagag ccatggggtg
agtctctgcc accgccaagg ggagtcaggc tcagaggctg 2880ctacagggac
agccagctct ctttagatgg tccccaccat ctagtcaggg cttgttacat
2940atggagcaga gacagcgcag gctgctgctg ttttcctgga gaaggcccct
gtcggtctgt 3000tcagctgtag ctgacctttc ctccttgtgc tttttgggga
gggagccttg gaaggagtag 3060ggcacgtggg gcactctgct tcccggcccc
acactggcga acctatggat tctgcctctg 3120attcctgagg aaacatcact
gtgaaggtgg aatgagccac atacagaggt ggctgttggg 3180gccggggagg
ggtgaaacgc ccccagggtg tacattgcac caaaagccag gctgcatata
3240gacctcagga tgggctggct tttctattta tttagaagta tttccagagg
gtaacctcat 3300tggctacaaa gcatgtctga acaagagctc cgttgttcat
tcccagccct gttaccctgg 3360caggatgcag actccaggcg gcctgttggt
caggccttgg actcagagag cagtgaagcc 3420tgaggagggg tggggggcag
aggcgtgagt ggtctagggc ctcagtccct ccaggacacc 3480ccttgccaag
cgcagagaaa gctctgccca tccgtcccct caggcagtgg gattgggcaa
3540cctgggaagc agtgaatgtg cgtcggtagc atagattcca ttccgcacgc
caccctcgcc 3600tccgcccccc agccctggga gggatgcatg ccctccggga
gacacccaga cccgacagag 3660aggcctttgt tggagctgga ggtgagaatc
tgtgggcgtt gggattcctg ggttcgagtt 3720ccagctcact gccaattgcc
cgagtgctgg gcgaacattt ctggaatcaa aaggagtgca 3780gcctgcccag
cagggcctac gggagccgga ggctgcaggg tgctaagatt gcgttatctt
3840taccaagtgc ccggagctcc tgggagggaa gagagagtcc taggactcag
gataggaggt 3900ggttggagtt tctcgaggaa gactccatgc tttggttctg
gcccctggaa acccctcctg 3960aggactggac ctccaagcag accccctctg
tgactccgga atgcagtgtt actctcttat 4020atttttcttt cttttttttt
ttttgagacg gagtctcact ctgtcaccca ggctggagtg 4080cagtggcacg
atctcggctc actgcaacct ccgccctccg agttcaagcg attctcctgc
4140ctcagcctcc caagtagctg ggattacagg tgcctgacac cgcgcctggc
taattttttg 4200tatttttagt agagatgggg ttttaccatc ttggccaggc
tggtcttgaa ctcctgacct 4260cataatccac ccgcctcggc ctccgaaagt
gctgggatca caggcgtgag ccaccgcacc 4320cggccactgt cttgtatttc
taacgtcccc ctgacttttc tgatcatgta attcttaact 4380ttctcaaaac
tgagatttgt cacgtgtcct ctccccactc cattttgtga atcagagtct
4440tccaggggca ggacctggag aatgggtctt tattaacaca catgtgaaaa
tgcttttgcc 4500agcaaggcgc ggtggctcat gcatgtaatc ccggcacttt
gggaggccga ggcaggcgga 4560tcacttgagg tcagcctggc caacatggta
aaaccctgtc tctactaaaa atacaaaaat 4620tagctgggtg tggtcgtggg
cacctgtagt cccacctact cgagaggctg aggcatgaga 4680atcactggaa
cccaggaggt agaagttgca gtgagccgag atcacaccac tggactccag
4740cttgggtgat agagtgagac tctgtctcaa aaaaagaaaa aaaaaagaaa
atgcttttgc 4800catgggctgt ctcctgcttc tgctttgcat tgggcctctg
tacctaggtt gcaagattcc 4860tcagggtgca cctgggctta tcgttatctg
taagttatcc cagcaagcac ttaaaacaca 4920gtgttggacg atgaatcccc
tctacaagag agggacaggg caaaaacgac acctcttgcc 4980tcgcaagctg
tcttgggcca aacctcaggt ctattctttc ttttttttga aagtagtggc
5040tgggcacggt ggcttacgcc tgtaatccta gcactttggg aggccaaggc
gggcggatct 5100tgaggtcagg agttcgagac cagcttggcc aacatggtaa
aactccatct ctactaaaaa 5160tacaaaaatt agctgggcgt ggtggcgcat
gcctgtagac ccagctactc aggaggctga 5220ggcaggagaa tcacttgaac
ctgagaggca gaggttgcag ttagctgaga ccatgccatt 5280gcactccagc
ctgggcggca gagcgagact ctgtctcaaa aaaaaaaaaa aagaaagtag
5340cagctctact gagatattta gaaaccataa aatccaccta tttgaggtgt
acaattgagt 5400gattttctgt atagtcacag atctgtgcag tcatccacac
cctctaactc caggacattt 5460tcctcacccc cgaggagaaa cctcccttac
ccattagcag tcactcctca tttcctctcc 5520ccccagcccc tggcaatcac
tgtggatttg cctgttcttg acatttcata taaatggtat 5580cataaaatct
atgggctttt gtgtctgtct gctttcactt agcatacggt tctcaaggtt
5640catccagtat tgtagcatct atcagtatgt cattcctttt tatggccaaa
taatatttta 5700ttgtatggat agacattttg tttattcatt tatctgtttt
tggttattat gagtaacact 5760actatgaaca ttttgcacaa atttttgtat
tgacatgttt tcatttctcc tgggtatagt 5820cctatgagtg gaattgctgg
gtcatataat aaataactgt ttaacatttt ggggagctgc 5880caaactttta
aaaccttggg ttctgtgatg taccagttgt gttaggcagc acagcaaaat
5940gtgacttttg attgccagaa acaatattta aaaagtggtt ataaaaagtg
gtttgggagg 6000ctgaggcagg aggatcactt gagcccagga gtttgagacc
agcctgggca acatagtgag 6060accctgttaa aaaaaaagaa ggccaggcac
agtggctcat gcctgtaatc ccagcacttt 6120gggagactga ggcgagcaga
tcacctaagg tcaggagttc cagaccagcc tggccaacat 6180ggcgaaaccc
catctctact aaaaatacaa aaattagcca ggcctggtgg tgggcgcctg
6240taatcccagc tactcaggag gcttgaggca ggagaatcgc ttgaacctgg
gagactgagg 6300ttgcagtgag cggagatcat gccattgcac tccagcctgg
gcaacaagag cgaaactgtg 6360tctcaaaaca aatgaaaaga aaaggctgtc
atgttagatc caccctcctc ctcaggggaa 6420cccctgggct gctctctggg
tagagatggg aacccaggcc tcgggccagt gagtggaagg 6480aaactttggg
atgattgact tgggactggg ctagaggtga agaatctccc agtaggcaaa
6540gttcggcctt acgttttttt gtttcaagca aaccacatca ttacccacag
aggccattgg 6600tgagatattt gtaagtctcc tgacagtggc tggagttcgt
tgcttggttg ttgtttctct 6660gtctcagccc tggagatggg agtgaccacc
tgctctctct ggacagaggc tgtccacgtt 6720catgcaattc cttggacacc
ggtggtgcag cgggaggcgt aactgggagt gggagaccct 6780gaactgtgcc
ggttcttgca gagtatcact gtgacttcag gcgagtcacc ccacatcagg
6840cagctcagaa caagggattg atctagaagg acctttcacc tgggctattc
tgtgactcaa 6900attatcttct cctaagccca ctactgcctg gtgtgttggt
taaattagcc taaaggtcat 6960tccctcggag aggccctctg ggaaacctcc
ctttcctgag agtcactgct tgctggcgcc 7020tgcccctggg gttccttcag
agtcgtgatc atgccctggc ctcttccttt atttggcagt 7080cccttccctt
ccccatccct gatgagggta gggagcatct gtctgcagct tcatcttcat
7140tgtctagggg ctccagaaat atctgtgagt aaataagtta tttaatcttt
gcctcaaatt 7200tccagtgact gtagggatat agctgtgagc ctctaggagc
tgagattttt taaatttccc 7260acttaaacat ttatttaaaa attttgtgct
cagcatggac taaggacttt acattcatta 7320actcatttac agcttgatcc
tatgcggtgg gcattcattt acagaggatc ccattttaca 7380ggtgaggaag
aggccagcta ggggtgcagc ctaggttagt attctagagc tcatcaggct
7440gtgttgtccc cagtgaaaga ataagcaaag aagtgaatgt tgtgcattga
gaaaaatgac 7500tctcggagga ggatgagcct ctcggatatg gcgaccgaag
tgatatgggg cccttgtcaa 7560gggtctctat tatggcatca agaaaagatg
ctgctttcgg tgatgcccga ggagagcctc 7620aatattttac atgggaaacc
taaaaaaggg gccatgttgt ggtctctgca cctaagatac 7680taaaggaaat
attttatgga gagatgcaac atgtcaggcc ttggagggaa accccaggat
7740ccagatggtt gcactctcaa accagggccc ccctcacctt ggccttcagc
atttagtgtt 7800ggaaccaata gcataagctt tggtcaggac ctttgatgga
agccacagtg ctcattagtg 7860accacggttg actaccttct ctctcctaag
ctgacttctg gagggcacct gggatttccg 7920gccagtgatc agtgctggtg
aagcctgaag gccaatgtgt aggtttagct gttcagtcag 7980aacccaaaag
gggccaaaga gatggtttcc ttcaacctcc actgagggaa gtgaaagtca
8040tggttcgtta aaaggctgag ctgggaccag agtctagggt tctagaggtg
ggaatttcta 8100cagctttggg ggaccttgca agggcatttg ctcttctggg
actgcaggga gactgtgctt 8160ctcagagatg ttagcatttg gcttggggag
agagaggaaa ggagaggttc atgctccgcc 8220atgatggtgg aaagtgatgt
tggtgtggtg aggagctgag ctgaattcta agtggttcca 8280gggaattaac
aatgttcctg cccaagtgtc ctgttccccc acaaactaat gaggcagcag
8340gtgtctgaag agaaacattg cagaatgtct gccaggggtt ttatggttaa
ttttcctcca 8400ttatgagggt tgactcagcc ttgggtatta gatgtctttg
agaatccagg gttcaaatac 8460cacagctggt agaatgtttc tcaacttgga
gccaatctcc atctactgaa ggtacgctgg 8520tttagacaga caacagggac
atcagcattt taaaaagcgg tggaaaaagt ttgcttgtct 8580tgattggagc
catgacattt tattttgaaa tttcaaataa catgaaggga ggtttggagc
8640ggtttttggt ttatccaaag ggcagtggat tgaaggctga gaaacaccag
gctgaatggg 8700agaggggttg gggtccccct gtgagatagt gaaacaatgg
tagtgccatc caatgatagg 8760cacttttctg tcattcagaa gcagaaaggg
ggccagaggc ccattggcct tactgggcag 8820taagctgtag agctgctgcc
ttttcgtgaa agggttgaca ccaaccttct cccccaggaa 8880gagtgaccag
ggacctgagg ggcatggtcg agcagatgac agcctttgta aaacatctcc
8940ctggtctcat cagcgatatt cgtcctgcct tccttctgag taatttccat
cttaggactg 9000gagtcaggtg gagcaagatt ccatgttggt ttctgttggg
cctagagtgt cacactgaga 9060cctaatttca tactttatga attctagtac
tgctctcgaa ggtaagagcc gtcctctttg 9120gctgaaggtt tttgcctgca
accttgcatt gtaatccagt gacacctgac gtatctgtaa 9180atttcttcaa
atttctaagt gtattacaac cccgtgtgca aaagatgatt aattaattgc
9240cttgacagta aaacaaaaaa caaaaaaaag gtgtgggggt atatggtatc
cctgatttac 9300tatagaagat gcagagagtg aagggagatg aggtggggag
gaggggccca ggttctggtc 9360ctactttttt tttttttttt ctaaagagat
ggagtcttac catgttggcc agtctaggct 9420tgaactcctg gcctcaagag
gtgctctcac ctcagcctcc caaagtgctg ggattatagg 9480cgtgagccac
cgagtttagc ccaggttctg tttcttgctt agtcactttc tgtttgaaca
9540aaattggaat ttcctttttg gatctgtttc tttaattgta aattgaatcg
gactaaaacc 9600tttccaattt tttcacatgt gaagacatac acaaaagttt
tattggaggg ttgcacatgt 9660gaaagaaaaa gggagaaagc aggattgagc
agggggagcc gtcagatggt aatgcagatg 9720tgatgagatc tctgccggac
caaagagaag attccttttt aaatggtgac aaattcatgg 9780gctttctctg
cctcaaaacc tagcacagct gttatttact gaacaattag agagctaagc
9840actttttaga tactatataa tttaattgcc gtatgaggca cccttagttt
tcagacgaga 9900aaccacagtt acagggaagg caagtaactt agtcaatgtc
agataactag gaaaaggtta 9960gaggggccct ggacacaggc ctgtgtgact
gagaagcttg ggcacttcac tgctacattt 10020catctcttcg ctataaacat
tttagctttt tgtgtttgct gactggcaac aatacatagt 10080gaaagttcta
ataatttgta atgcttttgc atgtctttgt atttttcttg gttatcacat
10140cacatcaaat taagatactg atcagcagtg tgagaggtta tttttccatg
tcctcttcat 10200tagtgttagc ttgtggatgg atttgaggct ctctgtgctt
tccccccagc aaagtgaata 10260ccagactttc ctattaaaaa aagtatttta
tttttcagag acagggtctc attctgtctc 10320ccaggctgga gtgcagtggc
acaatcatag cccactgcag cctccaactc ttgggttcaa 10380atgatcctcc
tgcctcagcc tctcttaagc agtgcctttc cccattctca tgggactttc
10440caatccatga gatactttgc tgcagggaag ccctgtctgt ccaggcctgt
gtaatagacg 10500acttcacatg gtcctgtgtt gttgtttgcc ttctgtgtgg
ctaagtttcc atgacctggt 10560ggcttggaag ccccatccct gatttgtggg
agaggcaggg aggcaccttg tagcgcacta 10620ggcgttgggc ctgaacaagt
ctgtgtgctt ccaatgtctt tgtggggagg tttacgagtc 10680cttcttatta
tataatagta tcttgtctta gcttggtgcc tttcttctca gaagcttgag
10740gcactctgca gataccatct caatttgctt tctgggagga ggagaggaag
ctacccaaaa 10800gatgaagttc tctgtgaggg gcttgaacac aggttgatag
cgttgctggt tagttattct 10860catggtgtgg atgaaaaatg gaatacgctg
aaatttcagt tactcgtcac aaaaataagg 10920cgtatgtaga aaacatcctg
ggctaagggt ttgcatgctt ctagaacttc ctgttactta 10980atggctgttg
agtataaacc tcgggaacag tggggatcct tggagacccc aaataacttg
11040tatttgtggt tactcctgtc ttgtctatca atacccctgt ctatatcgtg
ttagaactag 11100gacacacaga ctggattcag aagctggcct ggggtttagg
agaacatggg acctaatcct 11160ggccatctcg atttacctcc tggatcttgt
tttctcatct gtaaaatgaa ttggggtgtg 11220gactgtttat ggcctgtagg
atgctagccc tgagaatttt ctccagatat tctacggtta 11280agtaatttta
ggggacactg tctaagcagt tgcctcttgg agaatgaaga tgttcattag
11340gatattgaag gctctgagaa gtcctaaagt taaagaaaat ctgcaatgtt
ctttgtggga 11400ccgaataatg caacctggga aatgagggat tagatgacac
ttgagtagcc ttccagatct 11460gagacgagtc tcactctgtt tgtttactcc
atctgtgatg ggtgtaggca ccatcttggg 11520gagcaagctg tgatagagag
ggaacaatac cttgttaatg tttgtctaat tcactaccca 11580ggtgcatggt
agtgaattag acactacttt gtaggttctg gagggaagaa gaaaagacga
11640gacctgcctg gactggggct tgagaccact gtcaaataca agtacagttg
tacaactggt 11700agggagtggg tcatagtatg gccggtcttt ttaaaggtga
ggaattctta ggcccagaaa 11760ggcaaagtga cagatcctgg atttaaccag
cagcccagat ttgaggccta gcacatagca 11820aagcaccata gctattcaat
agctgccaag tgggagtttg gatgatggct ttcctggaca 11880gcgaaagcag
tgatgtttgc ttaggatggc ctttggcagt gctgctgtta tccttaccac
11940tggcaagcca tctcacgggc ccggagggga gggcaaggaa tcctaattct
gtgagaaggc 12000tctgggtaca tgagtgtgag atatggatac cctaggctct
gcccctgaag acagtggcat 12060cggatttact gcactattcc agtcggacag
gcaccttaat ttttctcttt ctgggtgttt 12120gatatggttg ggtcctattt
cttctcctcc aaaccccgct agggccattc ccccaccctt 12180cacttcccgg
ccttccactg cagtctctaa ggattctgct tcatctttat gtgtgaacag
12240ggttttgaca aacatgatta actgggtatt tttggaaggc tcaggaggaa
cgcagagtgc 12300tccggagggc aggcctggag tcaggaatgc ttcctgcaac
ctgttcgtgc agtgagcgtg 12360tcttcctcgc cctgcccttg gctggggaat
gtgctggctt ggagggcagg agagtgacag 12420gcggtttgag aactccgggc
tctcccgtct tcggatggct cctgtgaaag cagggcctga 12480aacttttatc
gtcactgctg caggtgaaag actttcattt ggctgtagtg gtccaacaaa
12540gagtatttta tttatgtgtt tccaagccct taaaaattct tttagggcac
atcagtgggg 12600agttaataga aactttgaaa taagaaaaat gcctgcaggg
taagtagaac cccagccagc 12660cagctccgag ttctgtgctg ttagctggta
ggttggttct cagagaagtg gctggctggc 12720tgggttacgg agcccacatc
tctaatgcct tagtgttcaa tcattaagtg gatttttttt 12780tttcccttct
cttcttttgg tttggaggga ggactactct aaactttact cagggcaggg
12840tagctcctga aagggctccc taacctttct ggtttatgac acaaagaaag
tttggaggta 12900ctgggataag agatggcttg ggtgaccccc ctatcatgcc
ccctaacaca tacacagcaa 12960accaaaccaa ctcacccttg atcatactcg
ttgtttacac gaagggaatt tttattgtct 13020tgtgagtgtt gagtgatgat
taaacagaag agatgtgact ccaagcctgg cttcactaag 13080atagtcttgt
ttgtttcttt tcctccaaag taatttccta aagaattaaa agcccctttg
13140aaacccagca ctaccttgtc tctgattatc agcataggca ggaagggctt
ttaaggtctg 13200agcccagctg tttagaggct acgagacgtg aggcaaatcc
tggtatctct ctttgggcct 13260cagtttcttc atctgtgaaa tggcacagta
ctaccctcca ccaaggatga tgatgagaat 13320taaatgggat gacaggtttc
atccccagct cctgttctta ggaaggaaaa actgtgactt 13380atgaagcctg
taggttgtgt tcaggtttgt atgaggcctc ggacttcata caaaggtatc
13440aaagtggcaa accctgatcc agatgttttc agttcagtca gctggtcctt
gagcctgttg 13500tgtgccagat atcctgacca aagaagctag atgggagctg
ctgtgttgtt ccttggggct 13560gctggatgca agttgtttag gtcggcggtt
ttcaaatgct ggtgattttg ctcaccagag 13620gacatttggc aatgtctaga
gacatttagc atggccagtc attgggaggt actcctggca 13680tctcgtgggc
agaggctaag gatgctattg aacatcctgc aatgcccagg acagccccct
13740gtgacaggag tcatccagcc caacatgtca ctagtgctgc agtggagaag
ccctggctgt 13800gtgtgggggt gtgtgtgtgt cctcttctac atttgataag
gtaactcaca cttgctgccc 13860ccatgatcgc tgtgggggat gcttatctat
gccccagtcc tggtgttggt tgatgggaac 13920atcaagattc aggcaagatg
gaaaatagcc cttagaacta gcaggaaaag aatctccttt 13980catttgtcta
gaggttctgt taaagtgcct ttgcttctat tttgagactt gttcttaaaa
14040aaaatgcgga tatgaaagaa aataaaaacc acattatccc tccacttttt
cttggaggag 14100gatgtgttga agaagtcaaa gttcaccatc cctttagata
gaatcatttt gaacaatttc 14160atatgtcaat acattttgct catctctaaa
tttcatttta gagcctgtgg tgttctgtgc 14220atggatatgt gtgcgtgtat
gcacacaaaa ataaaaggaa atatttattc ttatgaataa 14280gtatagaaat
aaattaattt ttggaatctc aaactatcag agacttatgt aataaccaga
14340ggcaggcctg attatgtatg ggcaaagcat ttgtgaacaa tgtctccatt
gtataacata 14400caaaacaagc ttttcttcca cattggatat gcaagtcggc
cttctccaat aagggcctgt 14460ctctttccaa ctccccccac ctcccacctt
tgagcaaaca ttatttattg tggctgatgt 14520gtgatcaggt cttgatttgg
ggcctctttt tgatgccttc tctttgtggg atctcaccca 14580cgtgcccctg
gagacccttt ggctgccagg gcctttgttt cccagccacc catgtggtgc
14640cagtagtgtc tgctttgtag cgagctgtcc ccagagcctc agcatggctt
ggggatggtc 14700tctgaggttg ggcttggatc cctcccactt ttgggctcag
aaagaatgac tgccctctat 14760ttccctgtcc ctgccctctc ttatcctgtt
tcccagcccg catcatgtta tctttgcttc 14820ttgtaactta ccaaacgatt
tatgggcaag taggggaggt gaagagggaa ctcatctatc 14880aagataacct
actttgtgcc aaccactgag catagcaatt gtcccttctc cagccctctg
14940aggaccgtgg atgggattct catgaaatga aacaggtgag gaacttttct
tttagggaac 15000ttgcttgagg tcccacaggc agcgagtatc aatcaacgtc
aggatctgag ccctgttctg 15060ttcggctgaa aatatactcc ctgagatggt
gtaggccacc atggctttca gcaggctctg 15120tgcttggtgg aaggaagctg
gaagctgtgt acacacccac ggggaacagg gaccatagag 15180gagcaccttt
tgagtgcaga acctggcgaa acatacacct ttagagggat tttaggtacc
15240cttgaggctg ggagaatcaa gcagagctaa gtttcccatt ggggtgtcac
agactgaaga 15300aacagagccc taggtagcac agggaagttg attgcccagt
atcagttagt ttggctttaa 15360tgactgagaa gagattccac cagttcattg
aagagagggc ggacttttta ttggaggaaa 15420gaagagtgcc tgtaagtaga
gaagtctccg gggtgtagtg ctgtttgggg caggaagaac 15480agtgtgagcc
actgtggaga gaaagcccaa agagtcttgg cagggcaggg agtaggatgg
15540atttgaagcc agaggaagta tggggtctct gtagactcca ggcaagccat
gttaatattt 15600taggaagccg tgatggagct gcagatgggt gtggaagtta
aagtttaact gttcattcac 15660cagtccttcc cctggagaat gtgcagcacg
tggacagtgg aactttaagg tccttggctt 15720gtatttcaca cccaagagat
gaataggtcc aggtatgtca tagaccagac taatgaaata 15780acaaatttct
tttcaaaaat tttacttttt gtaggaaagc ttctctgtct ggcatttttc
15840ttctcccagt tgtgactcaa tcttaaacgt cttcagacaa ttagcataaa
atttcccaca 15900gtgaattgac gtatactttt gagggttcca tttctttttt
attttttttt tcttttgaga 15960tggagtttct cgtcacccag gttggagtgc
aatggtgcca tcttggctcg ctgcaacctc 16020cgcctcccgg gttcaagcga
ttctcctgcc tcagcctcct gagtagctgg gatgtcaggc 16080acccgccacc
atgcccggct aattttcgtg tgttttagta gagatggggt tccaccgtgt
16140tggccaggct ggtcacaaac tcccgacctc aggcaatccg cccgcctcgg
cctcccaaag 16200gctgtgatta caggtgtgag ccactgtgcc cagcctaggg
ttccatttct taacccctcc 16260ttctgatgcc tcagaaagtc ttgctctgta
agcctcttgt agctgcctcg gttcagggga 16320agggggaggc ttttgtttta
ggaccgtcca gaccatagac acatttcctg gcacctagca 16380cgtgttgggt
caaacaggaa tgatgaatgc atgcatgaat gaggttctta gcgctgaaga
16440cggtgtcata ggtggtctac cacgccgcct gatcattcca atggcccatt
atgaatgtgt 16500gtgctgcagg gccctcccac gatcccgtca gcactgtgca
tgttgtgggg aggtgctggg 16560agaaagactg ggtctcagaa gatgggttag
aggtgggtcc ttctctgctg ctggctagca 16620gggtagctgt ggaggggtgc
cccatcttgc tggtcttaaa ttttctcact gtaggcaggg 16680agcatgacct
ggctgaattc taagtccttt tctactctga ggttcattgt gggtgtgacc
16740tgctgggctc agctctggct ttgggagaca ccctctcccc ttgatctcga
caacccctta 16800gcagagccca gtggctccta cagtgccctg agctgcttgc
ccgaaggatg cggttgtggt 16860tatctcaccc cctgccaccc tgtttgcgca
agggtttgag attgtgtggc ccctccttgt 16920acttcggggt gaggcttgct
ccagaaaggt ggtctgcaaa ggggttggct gggggggagg 16980aggaagtcat
tctccaagtg tttgtcctca tcgttatccc aaattgcttg cctggaataa
17040ggaaggaaag aaaaaaaaat actcttgagt ggtttgggcc aggattttag
ctgatggatc 17100tggtagttcc ctctgtcaga tttgttttct ttgaactgtc
tgggccggtc acagtgtcat 17160tgtttaaatg tggaatgtag gtgttctgtg
ttctgggaaa taaaaaccaa aactggtcca 17220ggggatccac agaggtaaga
aaagaacatt ccaataggaa tgtttcagaa ccaggagggg 17280aggagagaaa
aacggctctg ttggtctcct agaggaagaa cttgttagat ttggggagag
17340tcaggataaa tttgacccta agagtctctg attcctttta gagacttttc
ttataagaaa 17400taaaatggaa cttgggagag gcggcaactt gggaaacagc
acattctgcc gtaatgaaag 17460tcgtcccata agaatttctc tatcccttta
gccaaatttc tgtttctaaa aggggaaaag 17520gggctagaga taggcttgtt
tgttttctta gttgaatctt actttttgta tttccagccc 17580attctgcagg
gtaagaacaa gcacagcccg agggctcact cagtgtgatg ttctagagcc
17640tggctctgcc tcaatccctc acgctggagg atcaggcagc aggggccagt
gatggatttt 17700tttttcttcc tttcctcccc tattaatatt tactgaggta
taaattacag caaagtgcgc 17760agacctaggt atctaggact gtgaggtttt
cctgtgttac ctgtgtaacc acgacccaga 17820tcaagataag gaacttttct
ggcatctcag aggctcttcc tgctcccttt cagactccgt 17880ctcccagaag
gaactacttc tgattcctat agccatagac tgaattttct tttccaactt
17940catatgcata ggatcatcat gggtgtttta attttaattt catgtctgct
tgccactccc 18000aaatggaaat gtgttggcat ctctggatgt ttcttcataa
gaaacatgcc ctgtggggca 18060aagcccagga cagggctgtg ctgctgctgg
aagtcctgtg cagctggcca gcctctgctc 18120acccctccgg ccacgctggc
actttcagct tctccagcct cctgcccttc ccacttccag 18180tcctgcacct
gctgtcctca ctgatgcacc tgcccttttc cttccgtcct ttatgtggca
18240cacccttaag ggagacatct tcctgtctgt gttttgcacc ctcttaaaac
tacattcctt 18300tcccttcagc attggcatct ctgtccttgt gtattacctg
ggatgactat tcagttaaca 18360aatgctttct tcctaggctg tgagcccaag
tttgttggat gattggatgg gggcacgttg 18420tgtgagagaa ggatcatggg
gtagcatctg gctctcttag aggtgtgtgg gggcgtgtga 18480tgcctgccaa
ggcgctttcg ttctgggggg ttctgtgtgt ttgaagcact tgggttgtgt
18540gtccctgagg cctccgtcac gggcaacctc attccttctc tagcctccat
cccctgcccc 18600ctgcccaccc caggcctctg gagctggctc cctttcctgc
tcactctctt ttggccagga 18660ttttaacata tatcacaggc tggtaggcta
agagcttggg acttcccctc accacactca 18720aagcctttga tcttttgctt
tggaggtaac atcaaaagga aggctgagga agacagccag 18780gctgtgaagt
tcaacgttca agttaatagc ttgactgaag gttgtgctgc gttgtggcag
18840catcaccgag gctggagtaa acagagtgat tctgccacat tttcctggaa
atgcacccca 18900atattggaag agggcttctt ttacattcgg aatgaattca
ggctgtagtc agagctgctt 18960ttccctttcc ccattttcct tggaagtgtg
aaaacttggg ggagaagatg tttgtaggag 19020ggcatgatga ggggtagagg
aagcccaaag agaggatctg gggaggggaa gccccatggg 19080atgagactct
gaagttatcc ttgccccgat tccgggactt gctatctgcc tgccttttgg
19140cgtggtgtct ctgtgcccct gactgttcct gatttagcga ggtgtttctg
aattctgatg 19200gaattcaaag aagcctgggc aggcaggcag cttgacttgg
ggcttgggga agcgtgcagc 19260ccagacatag cagcgatgag agggcctcag
ggctgagggc tgagatgaga atttcatcac 19320atgcaaaagt gaaagcgacc
catcgtcttc tccacttgat ctcttgctga gctttgcaga 19380cactttggtt
gttgtttaat ttaacatttt ctgcaatgct ccttttttca gattttcatc
19440caaagctctg tatgagaggt tttcaaaccc attttggccc tgattctatt
tggcatacga 19500ttcaactctg gggatggtca tcttccccac acctgcgttg
ggtacctttt tggtgtatgc 19560tcagagcatc cttggacatc ttcctggtca
gtgtccagca tcgtgaagct gccctttagc 19620ctctcagtgc ccccagatac
acctgtctct ctgcgtagcg gcactcagcg tcacctttct 19680gtggggtctt
gagaccctga tgatatcagc actatgctgc cagaattccc cttggattct
19740ttagtgtggc ttctcaagca tcccttatcg ctataacgcc ttcatggttt
ttggcataac 19800tgtatactac ctgtgctatt atttatttga tgcattcaaa
catttgattc atttatttaa 19860actcagtctc actgtaatcc ttaattaaca
cctgtgaaat tataggtttg atgtgctact 19920tatttattta ttttttaata
cacattagta taatcccgta acggctaaag taacactttg 19980tactgcctaa
aaccatgctt gggagcgcca cagtttgaga aagtgcttag ccttcctttc
20040cctcctttag tgacttgtgg tttggggcat ctgttgactc ctagggctcc
cttgttcatc 20100tttctgttcc taagctcagg gattagttgc tcaacccagg
tgtggcctca aaattctgct 20160catggaatag cctcaggctt ctataaatct
catctttttt gttttgtttt gtttttgttt 20220ttgagactga gtcttgctct
gttgcccagg ctggagcaca gtggcgcaat ccactgtaac 20280cattgcgttc
tgggttcaag cgatcctccc atctcagact cccaagtagc tgggactgta
20340ggctggtacc accaggcccg actaattttt aaattttttg aagagatggg
gtctcactat 20400attgcccatg ccggaagtct agttttatag tgatgagaat
tcatctgggg tccaaggggc 20460cctcctgtgt tgcttcctgt gctcccctct
aaataaagat actccttcca agttgtcctg 20520attttcaggt catcaccatt
ttttgagctg gatggggaag ttggcctgga gcagccttcc 20580ctgtctccga
gttgcattac ctcctgagag gtctcagcaa atcactgcca tctcttgatc
20640agagttgctg gcaagagtcc tctgtggttc taggttttca gccctggaga
ctctcgcctg 20700cattcattat acatgtcctt ttggtgcctt gttgaaaggc
atctcctgcc accgaagggt 20760gtgggcttct ggaaattctc agaaaacaca
atatgccagc ctccagggat gggtctccaa 20820agcttcagga acatatcctg
gggtgttgag gaaacaccca ccttaaaatg ttcctcaagg 20880gggaatgtta
ctgcttgccc taaccctctt gagctgatgc tcacatgacg tccctgagat
20940gggcttcttt tttgcccgta cttaaagctg taaagggcca ttgtcaaatt
tgtttagctt 21000ctcaattcat gttccttaga ggatggtaaa ttaaagttag
cattcctgga cagagccttt 21060catacattga agacaacccg gtgagtctca
aggggagagg taagggagag atgaaaggtt 21120ttctccaggc ctgttcggca
gcatggactg ttcttttagg taattaaggg agaccataaa 21180agacaattgt
gtgagtccat ttacctttca cttgggggtc ttaagtcttt ggttgggctt
21240ctttaaccct gtgtgtcacc cacgggctcc tatgggtgct gttttcattg
ttccgttatc 21300tagttggctg gaacacacct ttggggattg gagaatggag
ttctgggggc tttgggaact 21360ttgagttttc ctgcaatgtc ctatagaagc
ttgagtctgt gattcctggg cagggccttc 21420tcctagttga gtgagattgg
tggggcaggg cagccagtta gggggtcatg ggagcaggtg 21480tggaaaaggt
tatatgtctt agtaattctt tgtgacaatc accctcattc attgatatct
21540tcttcctatc atgtattagg gcagtggttc ccccaatgtg ctgcacatta
ggttcacctg 21600gagagctttt ataaaaatgc caatgcccgg ggcccacttt
gggaggagcc aggcatcagt 21660aatttcaaag gtctctaaat gatttacagt
ttgggaatca ccgtatgagg atagtaagct 21720ctgagtccta tgcgttctgt
gccgaacacc catgaagcag tcttccaagc attttacctg 21780catcatctca
attctcacac tgttaaggag atagacagta tcatctccat tttgtagaca
21840agacaactga atctcagaga ggtttaagtc tcaggacacc aaggtcatta
ttaatcaggg 21900ggactgtgat tgctcccttt ataaaatgta ggagatattg
tggagtacgg ttgagaaacc 21960attgcaatag ttttcttact ttgttaagaa
attaggctgg gcgtggtggc tcaggcctat 22020aatcccagca cattgggaat
ccgaggtgga cagatctctt gagctcggga gttccagacc 22080agcttgggca
acagggtgaa accccatctc gactaaaaat acaaaaatat tagccgggcc
22140tggtggtgtg cacctgtagt ctcagctact tgagaggctg aggtgggagg
atcacctgag 22200tccggctgca gtgagctggc attgtgccac tgtactccag
cctgggcaat gagagtgaga 22260tcctgtctca aaaaaaagaa aaaaaaggaa
attagtggtg gaaggtgact ttgcatctgg 22320gcgtatctgc ctgcagagtt
ggtgtcctta ccttgaagaa accctgcttt agttggagta 22380tccttaatgg
ttagtggcag gaggggagga gtggttcctg ggagactgga acaaaatatg
22440gtacctgaat gcttaaggct tggcagatga gcagtcattt tcttacacag
agcttaggaa 22500agggcatcca ggtagaggaa tcagcatgaa caaaagcaca
gggccataga gttctcagaa 22560ggaaagatgg ggttaaccgg agccaagcca
gagatctggt ggtagtgggg ggtttccaag 22620ctagaatggt tgtgtggtat
tctgtcctca ggggctttga actctgtgtg ctaatgaggc 22680ctcaaattct
ctggggctct ggttaaaatg tagattctga tatcagttgg cttgggtggg
22740gccttgcatt tctgtaagcc cttagcagtt gcactgctgc tactaccgtg
agtattgctg 22800ttgagcatta ctaccttgag tattgctgtc aagtgttact
accttgagta ttgctgttga 22860gtattactgt cgaattttac taccttgagt
gttgctgttg agtattacta ccttgagtgt 22920tgctgttgaa tattactact
ttgagtatta ctgttgagca taaccacttt gagtattgct 22980cttgagtatt
accaccttga gtattgcttt tgagtgctac tgccttgagt atcgctgttg
23040agtattgcta ccttgaatat tactgttgag tattaccacc ttgagtattg
ctcttgagta 23100ttaccacctt gagttttgtt cttgagtatt gctaccttga
gtattgctgt tgagcattac 23160taccttgagt attgctgttg agcattacta
ccttgagtat tgctgttgag cattactacc 23220tcaaggattg ctcttgagct
ttaccgcctc aagtattgct cttgagcgtt actgcctcga 23280gtattgccgt
tgagtattac tcccttgagt attgccattg agtttagtcc tgtgagtatt
23340gctgctactg cgccttggca atggttttca aactttgcaa cacatcagaa
tcacttggga 23400aacctttaaa attctaacgc ccaggtcaca tcccattcca
actagatcag aacatctggg 23460gaatgcgagc catgcaccag tagttataaa
acctgcccag gtgattccaa agtgtgggaa 23520cctttgagaa gcactgcttt
aggggttgga atagtcctgg ctgaatttta atcagggaag 23580actgactgct
ccgtttatga aacgtaggag agtggagcag ggttgagaaa ccatcgggat
23640agtgttctta ctttgttacg tgagcaatat ttgttgagtc tctgtggtgg
gttctagggg 23700ttcagaggac agcagtgtgc tgctaggatg gtggtctgaa
ctagtggaaa ggcactcaaa 23760ggaagaaaga cagaattcta agaggagagg
aattttagga aggagatacc caggactttt 23820gaattacagg taatttgatc
agaacccaaa actgaaatgt ctctgctctg tgatgaaagg 23880gtttgctggc
attgagtaag gagctgcagg aaggccttta acttgtctcc aggtctctta
23940acagctttgt catttacata caagcacctg cctggctaaa ccattcattt
ctgtagcttc 24000cttctggatc tgtctaggga atatttgctt tgcatatttt
ggggttatct taagtgtttg 24060aaggaaccaa aatatttttc ttaaaaataa
cactcaaatg tagttcacat gattaatttt 24120gactgatttg tgagaatcag
taagtgctga ctgactgagg cgccccacac atccggcttc 24180cttctgttac
tctacgcgtg ttgctgaaac ttaacgaacc catgtggggt cttctcgcct
24240ggtgcagtcc ggcccagtat tcatactgag gtttgcagtg ggagaaagga
aggtatttat 24300ttgtaggtca ccaagcaggg caaatccagc agctcacgct
taagacctga cctctcccat 24360ggtttataag caagtggttt tttttttttt
tttttttttc agactgagtc ttgctctgtc 24420acccaggctg gagtgcagtg
gcgtgatctc agctcactgc aacctccgcc tcccaggttc 24480aagcgattct
cctgcctcag cctcctgaat agctgggact acaggcgtgc gcccccacac
24540ctggctaagt tttgtctttt tagtagagat ggggtttcac catgttgccc
aggctagttt 24600ccagctcctg acctcaagtg atcctcctgc cttgacctcc
cagagtgctg ggattacggg 24660catgagccac agtgcctggc ctgtaagcaa
gtgtttttaa agaaaggggt aaattttagg 24720gaaacagaag ttctaggcaa
aatggtaaat taatacaggg aggtaagaca ttggtttggc 24780ctaaaaagat
gggatatttt gaagtggggg ctcataggtc ataagtggat ttaaagattt
24840ttttggtttg taattggtta aggaagataa gctttgatta aagatttggg
gtcagcagaa 24900agaaatgtta ggtctggctc gtgggcatgt ctttttctag
gcccctcctt ggaaagaact 24960ttagagcaaa gaaaggcagt tggagcttag
tccccacttt ctcctgatct gaggtctacg 25020gaccactgga tccatttggt
ggggtccatc tttctgaaaa acaagtcagg gacatgtatt 25080gagatgatat
tattggtatt tatagggaac caaacaacgc cccatgactc ttttttggct
25140attgttttaa gccactgttt ttttttgttt attgagttgt taacttattt
tttaaagcta 25200gctagctgcc tggaatttct ttagaaggaa ctgaagtttt
taaaaatttt tatgttgggg 25260ggtattgccc tgcaggcccc taaaaggggt
ccctgcgctg tctcaaaact tggatgcaaa 25320aagaagttga gttaacacag
gaggacaggg gtagacgcac caagggcatg tgcctcgagt 25380gcgtggtcct
tattaagaag ggtggttaga cagggaatgg gttagttccc aggtcggcat
25440tcagctgaaa cagtgatggt taaaattctg aaaaatgtcc acgctctgca
ttctcttcct 25500aacacccagg acccagtaac tataaagccc cctaccctgg
ggcatagcag ggggcttcag 25560ggacccatga gaaggtcatc tgctgctagt
tacactcctt ctgggacctg atttagacag 25620tttggtggta gttttgcgag
ggttaatttc agggccaagg atgcttctag aatggaaata 25680ccttcttgac
attgggagct ttattggttg attatgtcaa tgtgagaatt caggaagccc
25740agtgctaatc ctccatccta aaaggagtag attggctggg cgtggtggcg
catgcctgta 25800atcccagcac tttgggaggc cgagggggcg cggatcacct
gaggtcagga gttcaagacc 25860aacatggcga aaccccgtct ctactaaaaa
tacataaatt agccaggtgt ggtggtgggc 25920gcctgtaatg ccacctactc
gggaggctga ggcagggaga attgcttgat cccaggaggc 25980ggaggctgca
gtgagccaag attgtgccac tgccctccag cctgggcgac agagcgagac
26040ttcatctcac aaaaacaaac aaacaaacaa acaaaaacta aaaggagatt
tcctccttct 26100gtcctttatg ggagacttca accttgggaa agtctggaat
ccttggacat tagaaattct 26160gaagttttgg ctggctgtag tggctcatgc
ctataatccc agcacgctgg gaggccgagg 26220caggtggtca cttaggccag
gagtttgaga ccagcctggc caacatggtg aaaccccatc 26280tctactaaaa
atacaaaaat tagctgggcg tggtagcgga cgcctgtaag cccagctact
26340tgggaggctg aggcaggaga atctccagaa cctatgaggt ggaggttgca
gtgagctgag 26400atcacaccat tgcactccag cctgggcaac agaacaagat
tccgtttcaa gaaagcagaa 26460actctgaaat ttttgcctgt ccaggccaca
tcaatcccat tcctctgctg tctctgcagg 26520attctgtgag gaataattag
ttaatgtttg cagagcactt tgaaatcctc agatgaaagg 26580caccggagaa
gcacaaagta ttattattta ttattagctt gccccagaat ggaggcgcat
26640gaggccctgg cagctccctg cctcgtgcca ggtgtgatcc tcctgctggg
cttttcctgc 26700ctgatgagct tttttttttt tttttttttt gagatcaggt
tcagctctgt cgcccaggct 26760ggagtgcagt ggcatgaaaa cagttcactg
cacacagctc actgcactgc agcctcaaac 26820acctgggctc aagcaatccc
cctgcctcag cctcccaggt aactgggact atatactaca 26880ggcatgcgcc
accactcctg gctaattaaa aaaaattttt ttttgtagag atgggggtct
26940cactatgttg cccaggctgg tctcaaactc ctgggcctca aagatgccaa
aggttcacac 27000cttggcctct caaagtgctg agatgacagg cgtgagccac
tgtgcctgtg ctcaattgat 27060tttctttatt aaagaaacat ggaagaaagt
gaaggatgag aatcagtaac gtaacgtgtg 27120cttcagattg tggacaagtg
atgtgaagga aacacattgg tcccactgtg gtgacagagc 27180aggggtttcc
ttacctggca aggttgcggc tgccattcct tggggtctgg ggttaagacc
27240atctgcctga gggtaacgca gtaataaatc agtactaaag ggcgtactaa
agtactgtat 27300tgctaggcta ggccatgctt ggtgtatttt tttttttttt
taattgagac ggagtcttgc 27360tttgttgccc aggctggagt gcagtggtgt
gatctcggct cactacaacc tctgctgccc 27420agtttcaagt gattctcctg
ccttagcctc ctgagtagct gggattacag gcacgtgcta 27480ccatgcttgg
ctagttttaa aatattttta gtagagattg ggttttgccg tgttgtccaa
27540gctggtctca aactcctgac ctcaagggat cagcccacct cggcctcccg
aagtgctggg 27600attacaggca tgagcctggc tggtgtattt gttttaaatt
taaagtttac taaatttaat 27660gatatctggg gaatcagctt gcttcctggg
gatctggatg tacttgaggt gagagggtgg 27720ggattcagaa ttatcctttc
tatcgcagca tgttctggat tgattcatgt aggtctcaag 27780tgtgtgtaat
atttcatttc tttgtgcaat tttggcatgc cgaggcgggc accctgaagc
27840tccggcagag cctggagaca gagtggggag ctctccgctc tttcccttcc
ttcatcccag 27900ctgacttcga ctggaattga attcatcagc tgctggagag
ttgttttatt tgccctgctg 27960gtggagaggg aggaaaggaa catcatgggg
ccaggctttt tttttttaaa ggaaagattt 28020gatttacttt cccccttagt
agcatgatgg gcacctgcac ccgccagcta atcagaagcc 28080actgtcccct
gaatgcctcc gctgcccacc agatcctgac agcatcccac gcgggagcac
28140tctcgtgtgc ccctggcagc ttctgctgcc tggcagttct ctaaacttgc
tggtgtctct 28200ctgcccggag gctcagaaac ccagaggact gaccacttct
tgaggctcat gtccagtttg 28260caaagagccc ccagcaagca gagaagggga
tttttgtacc agcgatatct cttctccact 28320cctcaacaca ctcctttcca
ctctgtctcc tataaacatg gaacagccag gaatactcaa 28380atcctagcct
gtcatgaagc caaaaattga tagagatcta ctgtccagaa tgatttctta
28440tagtgaccct gtgtttagtt ggtaagactt tcttaaacca tgagggattc
tggtcccaca 28500gggcagtaat atctggggca gagcctgaga cttttctcat
tgatttcctc tgtgagccag 28560gagtgactgc tctgatgcag ggtgctgtgt
ggttggtaga agctggcgtt atcccatttt 28620acccacgagg aaacaaatga
ccagtggtgg agcgggagct cagcatccca tgtgcccact 28680tcctcctcgg
gtggactttt cacctgccca tgccgtcttc tttgcaaact ttactgcagt
28740gacggagaca tctttaaata caaattcttg ggggaaccct gtgttccttg
gctggagcct 28800ggctgggaag gaggagggag cagagggctc tcttgggtgt
ggcctattgc agttgagcca 28860gggaaaggct ggtccactgg agacaccctc
tctggtcacc gcagacttcc tgccctccat 28920ccagtgtcct tctacttgca
ggatgtgtgc ccagcagaga gaatctctga agccatgtca 28980ttattgggat
aacattcctg tcccagtcac cttatttctc agaaaaagga caatgggaaa
29040caagttttta ttgaatccta tgctgggcct attaatgggg tctcttactt
ttcatagcag 29100cactgcaaac agagttacgt ttctattcat tttatggatt
agaaagctga gatccagagc 29160gggcagatgt aaacctgggg tctttaaaat
gcatcctttt tgcaaacaaa taaacttagt 29220gtattaaaag ggctggagag
agcagagtaa ggtaacattt gggtggtcag catgtagttc 29280tgggtcccca
cagtggagat ggcacagtgc tgggtgctgg gggaactatg gtcactaaga
29340gacactgaat aatttaatgc atgcccctga ttccatcact gactgttgag
gtaacacata 29400catttatatt gtcagtggtg gtgatgatta catgagctgc
gtaaagcgtt tgaccagtgc 29460ctgcacataa catagtaggt gctcaataaa
gatcacccac tcttaagagg tgggaggagg 29520tgaagtcatc tttctgggga
gtgttgccct gttgttctct gctgcattct ttctgtcctt 29580tgggctccga
gaatgctggg ttgggcagtg tgagtggtct tctcaggcct ctgtgacatg
29640ttgctttcat gaaaggttcc cctctagcca aagactgagt ggtccttgca
ggctttctcc 29700tgagtccttt tttttttttt tttttttttt ttaaagacag
agactctgtt gccagattgg 29760agtgcagtga cgcggtctcg gctcactgca
accgctgcct cccaggttca agcaatctac 29820aaaatgcatc tataaaatga
tgcatcagcc tcctgagtat ttgggatcac aggtgcccac 29880taccatgcct
gggtattttt ttgtattttt agtagagaca gggtttcacc ctgttgacca
29940gtttggtctc aaactcctga cctcgagtga tccgcctgtc ttgacctccc
aaagtgctgg 30000gattacaggc gtgagccact gcacctggcc tctctcctga
gtccttttgt ttgtgcctgc 30060tttggggatt ccctctggct ggggtggact
gccgggatct
gtttgtccag tgtacatttc 30120ctggtcacct agcaccggcc agctgcggtg
ctgggaggaa cagggcctgg ctctgggagg 30180cagctgggag agtcaggaag
tgaagaaagt tcttgtgggt gtgatggtgg aaacccaagc 30240agcgtccaga
gggagcacaa gagggaggga caaatcttgg gagggtcccg ggccaatggg
30300acccagtgta agaaattgca cctgtcctgg cagatagaga aggtggaagc
agtgaatggt 30360agagcatcct cactcttctc tctgccagca agcacctttg
gggaagtcct cacggacagg 30420aatgtcgtgt gtcttggctt gagatgtcaa
agaaacatgt tggacacacc atggtgacag 30480agcaggagtc tcttaacccc
ggcgtggttg aggctgccgt tctggtggga tctggggtca 30540gtcaggggtt
aacagtcgct cctgcttgcc tgattgacac agtaataaag gcagtgacac
30600caaactaggt ctcaggaatg tgtcctcgtt agaaagactc actaatggtt
gtgggggggt 30660ggcccatgag tccttctggg tggtggcgag aagtagggga
ccctttgggc tttgcccttt 30720ttggtcatag gacttcactc cacagacata
attgaaccgt tgggtttctg cagccaaatt 30780caaatgtcac caatcttggt
cacccctttc atctcttggg tcctctgtaa gttatagcta 30840tctgatagtt
tactgaaaaa taaactgaaa atatgtttta aattgtactt tcgatttaaa
30900ataatgttta gagacaaaaa aaaagggtcc aatccacttg gagaaaagca
ttgtcaaagg 30960tggttgattt ttttcttttg ctgttttaaa gtggtaagtg
gatgagtgtt ttggatatat 31020tgatttttca ggtgtgcagg cggtcacatg
aacagctgac attttttttt ttttcatgtg 31080gacttcagcc agtcttgaca
cctgcccctt aacgaaaagt aaaccatcgc cttgtttgac 31140agtttaagtg
cagtgatacg gatggaggca ggtttacgtt atgttaaagg cttgacaacc
31200cagaaccccc ctgttggttt ctttgttgta acctttgagc cggtggcctg
ctgaaatgtc 31260acctttgccc ttctttaaaa gcaggaataa taggtggtga
gtgggtggat gcctcttaaa 31320atactggaaa gtgctgtggc ccgagggtaa
gctttttaga agtgagtgtg tgtgttgtgt 31380tgttttaatt aatgaatctt
ctgggcctga agataatgag gtcagtgagg gcagccatgc 31440tgcctcacag
ctcaccttag ggtccttgtt gtccagaacg tgcctgacct actggagggg
31500cctgggaatg cttctttgat tgacgtgggt aggaagacag atgtggcggc
ctccatgctg 31560atgggaggca gctgggaaga aggtcatggg caccatctca
ggagtggcag agccacctcc 31620ccctctcctc accccgtgtg tctggattct
tccagctgtg tggtccttct tcctgcctgg 31680aaatgagcat cctgcagagc
tcggctcctg ttcacaccct cctcctaacc ccctactctc 31740cctctccctt
tcatccaggg ctggaggacc agatgggctt tacctgatgg agtgtgcttt
31800gctgacatgg tgcaaagagc caattcctgg ttgcaaagag gcagctgggt
gcagaggcgg 31860ggtgcattcc tgtaataata ataacttgtg tttttataat
actttacagt ctaagtactt 31920ttcaaatact tgacctcatt tagttctcac
cacagccctc tgaagggata ttactattac 31980cttcatttta tagatgcgtt
aaccagggct tgttttggga ggtagagggg gtgtgagggg 32040gacagagggg
agggaaccag tgttgaatga attctgaggc cctgccaaag cagccagcta
32100gctaggtgtt gtttaatgga ctctttgcat ctacagaatg agggaggtgg
gatgagggga 32160aattatttca caataactga aggtcggaga gactaactct
ctgctcattg tcacacagca 32220gttgagtgcc agctgggatt tgtcacccag
gtcacctgac tcctaagccc tgtgatcgtt 32280ctcttctgtc tctagtatac
ccagcataat gcccggcaaa gtgctggcat caataaatat 32340ttgtcgaatg
ttaaatgagg cttaaagaga accattcatg cttggcacag gggcacagtg
32400agacaaacat gtttcctgcc ctcgtaacct tcgcttccaa attctgtgac
cttgggcggg 32460ttgcctgagc tcttttccag ctcagtttcc tgaaaactca
tccggaaaat gggtaaaata 32520tcagagtgca ttctgtatgg taagactgca
aatgttagat gattctgcta cttattattg 32580ttttcttttt ctcaccacac
accttccctt tttatgtaca cctcctagga agtaagtttc 32640tgataacata
ctgcattgtt ggataacagc aacaaaaagc acttcctgac attattgccc
32700aaatcaccaa atgaggcaat taccaacttt ggaataagaa tagcaacggt
ggtaagagct 32760gacatttctt gagcgcttgc catatgctgg gtgtactact
ataagctgct gcctgcaatt 32820atgatttggg aacaactctg aaagtagtta
cctcccattt tatagaagag taaactgagg 32880ttcagagagg ttaagtaacc
cccccagggt ctctcaggaa gtagttggtg gcctgggatt 32940caaacaccag
aattggtctg acttcccact ctttaaacca cactcaaaac tgaactctcc
33000acgtgtgtgt gttctgggca ttttcgcatc tcccttggct tgttgacagc
gtggactttt 33060gctttcccat tctcatagaa catggccagt gcaggaggag
gaaatccaca ctggtctttg 33120gactgaacca gaggctggcg atggtcccga
aacagggtgc caagtggctg acccttgttt 33180ttatgccttg cgctggtaag
cttggggcac caggagttct ttgaatttct ctttcttgac 33240tgtccacgcc
ttctttaggc aatcttttaa caggctatgt tttaaatctt attgacatct
33300ctgaagaaag aggaggaaaa aaaaatcaag acatggcctt agagaagtga
caggttttct 33360ttgagatttt gttttctgtt ttccttttta ttttgtgcac
attgcaaaac ctctttggga 33420tgatgatttc gtgtggtttc ttggtagccc
ttgggcagct gctgccaggt ttcacccaaa 33480tgcattgtga ccccctgttt
cgtgggacgg ctttgcctcc acatggctga ttgtgctctg 33540tgtgtccgct
gtgggcagag tgtgattgta agaatcagaa ttctgctggg cttgcaagca
33600tttaaaaaat ctctataagt ttgagaactg gttggaaggg agagatgcag
cgacttagaa 33660cacccggcct gcagctgagc ttccgtgtgc ctgggaggag
cccatatgga gaaacaggaa 33720aattccactt caccagaaag ctggggaaat
gagtgggagt aggggccagg ctggttaact 33780aggaagactt ttggtcactc
tgctttactt agcctaaagt gttcatttcc cccttaagcg 33840gtggttaata
cgcgtatctg cagatttact ttttggatga tttaaaatct tgcaacatct
33900caagggattg tatcctgatg atactgatta tattaataac aagataatag
cttataatat 33960aatagctagc aattaccaag cacttacctt acaccaggta
caatgccagg catttgatcc 34020ttacacgaac tctgagagat ggctgtttgt
attcccattt tacagatgag ggaagctgtg 34080ctgagagagg tgagttcatt
tgcccaagct cacccacttt gagatggtct gcttttatat 34140cctcttagcc
caattctttt ggatgtcagc acttggcatg tattaggcac tggataagtg
34200ttttgttgaa tgaacaaaat gatggaactt ggatttgaac ccaggtctga
gctggctttg 34260agtcttcaga atagtaggtc caattagtgg agtgggggct
cagtagtcca aagggaaagg 34320agcaaggaga cattgtgggg gccgagaaga
gggcttctgg ggtgtttcct gggcacattg 34380gcattaaagg catagtgtga
agtgccattc aagaccatgt gctggattag tgtttttcct 34440ctccacttga
ggtgcttgcg attggctttg tgccccggtg tctgcaaagt gagttgggct
34500gaactcagga agaccttttg gttgggatga ctgtgtattc acctgcacct
gagtagggac 34560tgagtttcac ttgccagttt taccgcagca agacctcgtt
aagttggctt cctctcattt 34620aggcatttgg gaaactttag gcggctggag
tttaattctc aaggcaaagc cccttttcaa 34680gggacatgaa gaaaggcaga
gggatatatt taaaatacct gaatgaactg tttctttttc 34740tttttatttt
ttgagatgga gtctccctct gtcacccaag ctggagtgca gtggcacgat
34800ctcagctcac tgcaaccttc acctcccagg ttcaagcgat tctcctgcct
cagcctcccc 34860agtagctggg actgcaggtg tgcaccacca cacccagcta
atttttctat tgttttattt 34920tattttattt attttttaat tttttttttg
agacggagtc tcgctctgtt gcccaggctg 34980gagtgcaatg gcgtgatctc
ggctcactgc aagctccacc tcccgggttc atgccattct 35040cctgaatcag
cctcccaagt agctgggact acaggcacct gccaccacac ccggctcatt
35100ttttgtattt ttagtagaga tggggtttca ccatgttggc caggtctctt
gaccttgtga 35160tccgcccgcc tcggcctccc aaagtgctgg gattacaggc
gtgagccact gcgacttgca 35220tgtagacagt aatggcaggt cactatcagt
gggtctgtta atcaggtgtc caacctggtg 35280ctgggcttgg tggctcatgc
ctgtaatcct agcactctgg gaggccaagg cgagtggatc 35340atctgaggtc
aggagtacaa gaccagcctg gccaacatag taaaacccca tctctactaa
35400aaatacaaaa attagctagg catggtggga tgcatctgta gtcccagcta
ctcaagaaga 35460tgaggcagga gaatggcttg aacctgggag gcggagattg
cagtgagcca agatcatgcc 35520actgcactcc atccagcctg gacaacaaag
cgagactctc acaacaaaac aaaacaaaca 35580aacaaacaaa caaaaagtca
cttgcttctt tttttgcttg cttatggaca taaaccctgt 35640aaactatctc
atacatcatg ggagtgagtt tgcagtgggt agactgctat tcacaaactc
35700atatacatcc tatgaaggag tacaggttaa ataaccatta tccaaaatgc
ttggggctga 35760aagtgttttg gatttttaat ttttttcaga ttttggaata
ttcgcatata cataatgaga 35820tatcctgggg atgggaccca aatctgaaca
ggaaattcat ttatgtttta tataaaccct 35880tttttttttt tttttttttg
agacagagtt tcacgcttgt tccccaggct ggaatgcagt 35940ggtgtgatct
cggctcactg caacctctac ctcccaggtc gaaacgattc tcctgcctca
36000gcctcctgag tagctgagat tacaggggct tcccaccacg cacagctaat
ttttgtattt 36060ttagtagaga tgaggtttca ccctgttagc caggctggtc
tcgaactcct gacctcaagt 36120gatccacccg ctttggcctc ccaaagtgct
gggattacaa tgtgagccac tgtgcatggc 36180cttatataaa ccttaggtaa
ttttatacaa tattttaaat aatttttgtg catgaaacag 36240agttttgact
gcattttgac tgtgactcct cacttgaggt caggtgtaga cttttccact
36300tgtggtgtca aatttcagat tttgaagctt tatataatga gatagggtct
tgctctgttg 36360cccaggctga agtacggtgg cacaatcaca gctcactgca
accatgacct cctgggctca 36420agtgatcctc ccatctcagc cacctgagta
gctgggacta caggcatgca ctgtgactgg 36480attttttttt tttttttttt
ttttgagacg gagtctggaa tctcaagtct cgctctggtg 36540cccaggctgg
agtgcaagtg gcgcgttctt ggctcactgc aatctccgcc tcctgggttc
36600aagtgattct cctgtctcag cctcctgagt agctgggatt ataggcgtgt
gccaccactt 36660ctggctaatt tttgtagttt tagtagggtc ggagtttcac
tgtgttggcc aggttggtct 36720tgaactcctg aactgaagtg atctgcccac
cttggcctcc cagagtgatg ggattatagg 36780catgagccac cgtgcccagc
cttggctaat tttttatatt ttttgtagag acagggtttc 36840gctatgttgc
ccaggttggt cttgaattcc tggactcaag caatctgccc accttggcct
36900cgcaaagtgc tgggattaca ggtgtgagtc accgctcctg gcctgaagca
ttttggattt 36960ttgggttaga gttgcacagc ctttactgtt attatcctga
tgttattatc cacattttac 37020aggcaaggat ctggaggcgt agagaggtaa
aatcattttt tcaaagcccc agaagtacta 37080agccgcagat tctgaatttg
aactcaggca ttctgggtca gaattagtga ggttttaagt 37140taattttttt
ttttttttta gatagagtct tgctctgtta cccagggtgg agtgacagtg
37200gtgctatctc ggctcactgc aacctctgcc tcccgggttc aagtgattct
cctgcctcag 37260cttctagagt agctgggact acagacatgt gccaccacgc
ctggctaatt tttgtatttt 37320tattagagat ggtgtttcgc cacgttggcc
aggctggtct tgaactcctg acctcaggtg 37380atctaaccac ctcggcctcc
agaagtgctg ggattacagg cgtgagccac tgcgcctggc 37440ctaccccctg
tcgcccaggc tggagtgcaa gtggcacagt ctcggctcac tgcaacctct
37500gcctcccagg ttcaagcgat tctcctgcct cagcctcctg agtagctggg
attacagata 37560cccaccacca tgcctggcta attttttttt tttaagtatt
tttagtagag acagagtttc 37620aacaagttgg tcaggctcct cttgaacttc
tgacctcatg atctgcctgc ctcggcctcc 37680caaagtgctg ggattacagg
catgagccac catgcctggc ctaagtttgg tttttaacca 37740tgctgctttt
tctagaccct tctgtcagcc agctccacaa tggtgaatca gggagttagg
37800tccgtctgta agagagggcc caggagctgg gtcagatagg taaagagaca
ttccttagtt 37860cttatcctct gctaccaagc catttcttgg gatcaagccc
tctttggcct gtgtcattcc 37920acctccatta agttcagcct tctttccttc
tttccacatc tttccactgc tgttgaaaac 37980ttgagacctg aaatcccatc
tcctgaattc ctgggagctc tagaagtgga gatggccagg 38040ttctgtggtc
agagctggtt gggattacaa ataaaccaaa gcctgggaaa ctttcttgct
38100attaatagcg cagacctttt ggggagggaa tacccaaact cgatgctgtt
ggaattgatt 38160ttgcctgtct agatgacata ctaatgagct aagtggttag
cttcggatca ttattgctct 38220ttcccaagcc aagttctttt aaagactaaa
accacaaaag cagagaacga gttgggttag 38280agaggcatag tggctgggtc
cagagaaggg agagtggtca gccctggcct taaacatgag 38340aaaataaagg
tggtccttgc tttggaatga gttagtggtg ctgactaatt caactggttt
38400ttctttttct ttttggaagt ggaatttgat ttggtgtctg gattttgata
gggccattta 38460tatttcttca gcactttttg gttcttgcag aaagttacat
tcctagttcc tcaactgctt 38520atttcttttt ggtttttgaa gcaggaattt
gatttggtgt ctgggttttg ataggggtgt 38580ttatgtttca tcaatgtctt
ttggttcttt cagcgtttct ctccttgtct gtcatgtgtc 38640agagagggtg
cctgtcaacg attctctctc tccagggaga aagtctttct taaaacagcc
38700ctaagatcct tatctcctca aatcacccac tttgatgata atatccattg
ttctcttctc 38760tgcttcttgg catattctag tcagtctatg tatacaatta
aaaaaacaaa acagccctct 38820gtgtccaaag tgcttggaat atcccagtgt
ttcacaggag actttggaag tggacaaaac 38880tgtatttcct tccccaaatg
aggttattgt gctcgaaata tctcctggta gtttattaaa 38940ggaaaccgca
ggcaggggta agagaggcag tttctacagc ctgcaaccct attatcttgc
39000ctcttctttt cgaccctcct tcctccctct cctcttctct ccccccctca
ccctattcaa 39060cccagcccca catgtcatgc cgtccccagg aggtagccct
gcagccctgc ttctctggga 39120tggtctgttc ttgccacccg tcccatggaa
cgtgaagaag gaatttgggg tgtggacttc 39180cttgagtgac taggattaga
cccgtcgggt ctgcagtcag acgagaagcg tgtgggcaaa 39240gggaactatt
gtgtgaggct tctctggaca gaaagcctgc cttcatcttt tactgtgcct
39300aatggacaat tgagacattc agcttatgtc tgaaaggaaa gtgggccggg
atggtctagc 39360agacctccca gatgaaggct tgtaggagga gcaaatagag
acaaggatta ccaacagggg 39420gaacaactgg ggcagagtcc tgggagagaa
tgtttatttc cttgctctct aggagggatt 39480tggaaagagc cataatcctg
ggttaggagt aatttgttac agcaagatga tacttgagtg 39540acaggctgct
tctggctgag gcagcaagac ttgcatgcag gggggtcgtg gggcctccag
39600aaggtcagcc tcccgtaaat cttcaccctg gctttggggt ttgttcctcc
ccaagcaaaa 39660ttaaccagag gcactgctga cctttgggct tcctgggtgt
agcgttacga agcatctcca 39720catgtttgtc acagctagaa tttgacaata
aaaatttgga cagggagacc ctgccagagc 39780cactgacctc tttccaatgt
gacaagggga aaaaaaacaa aaggaaaacg cagcacgggg 39840tgcggtttca
gttgaagttg gaggacacgg agcccagcct gtctcgcatt tgctgtctat
39900gtagactcac taaagcaagt taattcattg ctctttgacc gccaagtctt
tcgttgtctt 39960ttgttgttgt ggaatggggg aaagaaatac agaatgggga
ggagaaccta attagaaaaa 40020tcaagccttg agagctccca gccatggaga
aagaaaggga ttttttagaa gttgtgattt 40080taatatctgc tgcaatctga
tgattcatgg atttaaaata acccttccag gtcaccagga 40140ccctgttact
tgctggcttt gtacctctca aaggtcattt gttggcttcg tctcttaaca
40200atttccatgg tagacctaaa atttctggct gtgaaatccc ctgtgtagtg
ggaagaagaa 40260atagcaaatc ttagctgcct tggacctgat ataattattt
gtcttcattt acatggttta 40320tccttcaagg ttgaataaat gatgtgggag
ctagtcaagg ggctttaggt atgtgatttc 40380atgcctactt ttttttaggt
agagaaactg aggtcacagg gtactagaga atggactcta 40440agattcaggt
ttctgaattg cctgtggttt tgttgactca actgctcttc tgttgttttt
40500tagccacatg ccttgaaaca gtcctctttc ccatgtttct tcatcagcac
cattaaccca 40560aggtatactg tcctctctta tctttcacaa ggtcttggag
ttcccatgcc tttgtaagca 40620tccctccccg agattcagca ccaaccaaaa
tcacatttgg aaaaattgct tgtttcccaa 40680gaagctttgg aggatatgat
tttgtataga acgggttcac aggttttctg ttcattcttc 40740tatggtggag
tgtgtgtgta tgtgactctg tcttctctcc attcctcttt tttttttttt
40800ttttttgaga tggaatttcg cttttgtggc ccaggcttga gtgcaatggc
gtgatctcgg 40860ctcactgcaa cctccacctc ctgggttcaa gcgattctcc
tgtctcagcc tcgcaagtag 40920ctaggattac aggcatgcgc caccacgtcc
agctaatttt tgtattttta gtagagatgg 40980agtttcatca ctttggtcag
tctggtcaca caaactcctg acctcaggtg atccaaccgc 41040ctcggcctcc
caaagtgctg ggattacagg tgtgagccac cgcgccaagc ctccccatcc
41100ccttttatct cttaaatgaa tgtggtcacc atcaaagatg gtgcctgact
cttttttgtt 41160ttcagttcat cttaaattca catataattc acacgtcata
aaatgtaccc atttaaggtg 41220tacagttcag tggtttttta gtctatttag
tatatttaca agattgtaca aacataccag 41280tatcttaata tttttatcat
ccccaaaaga aacactgtaa ccctagcagc cagtctctac 41340ccgccttccc
catagctcct ggcaatcact aatttacttc ctgtctctat gaatttgcct
41400attttggtta tttcatataa aaagaatcat acaccatgaa actttcttca
tctgccttga 41460agttagcata ttttcaaggg ctaccatgtt gtggcatgtg
tcagtactcc atttgttttt 41520attactgaat agtattccat tttatggctg
taccgcattt tagttatcca gctatcggtt 41580gagacttggt gcattcttat
cccagaacat accatattca gctcccagtg acacccacat 41640tcattcctgg
gctgctcctt gtcttccagc tattttcctg gtctcctgtt gcctctgcct
41700acttcagcat gctgtagaga catgggtagt aactaaaaca ttccaattaa
ctgcattgta 41760cttggccttt ttataagaag cagtaattag aaaatatggt
ggccacaaga ttgatattaa 41820agtgaaagat tgtaaatact tttctgcctg
aaggtagatg gcctctggcc tgcctcttag 41880tgggaggttc ttccaggagc
ttgcaagcat ccattatttg ttagtcatca gcttagcggc 41940caaggagcat
tagcctgtct tgctctgtct gctgaagact ctgagagaca tgggagggca
42000agggctgctc cttttgaatt cttccaatgt cttcatgtcc tttaacctcc
tggcttaggg 42060acttgtgtgc tggtggtgga gctgacattt gtttggaatc
cacagccctt tgggtgggac 42120tcaatcttgg ggttgcctga agactttgag
atggctaggt ctgggcctct tttggtcact 42180atggaacaag actgtctcag
aggccagagt ctgtctcacc agctccctgt cttgggactg 42240caccattgca
gggtctttgc cctcccctgg agatttctct tcctgcctgg gcacccattg
42300gccattctgc ccgtaagctc agtagggtgt aggcaaaaga gttctggcct
ggaagtacca 42360aagtcctgcg ttctggtttc agtccctcat aactgtgtat
aactaagtca cttagttttc 42420tgtgcctcac tttcttctgt tttaagatgg
atttggagat tattggcttt gaccacctaa 42480aaaggatgta gtgacaatca
atttagaggt ctaaaagagc ctttgaggaa gtaaaatgga 42540atcttcaaat
ggactacatg ctgattattg acactgccct agcactgata gttgatgttg
42600actgatggtc agaattgctt ggcaagttgg aaaaaagtac gtacagatcc
tgggccacta 42660ccaagtttca tttaacagat ctggagtgca tcaggaaaaa
agtccctcta aacaagccag 42720caaggtttgg atactgtgca accttttttt
tttttttttt ttccttttga gatggagtct 42780ggctctgttg cccaagctgg
agtgcagttg cacaatcttg gctcactata acctctgcct 42840cccaggttca
agcaattctc ctgctttagc ctcccgagta gctggcataa caggcgcctg
42900ccaccacacc cagctaattt ttatattttt tggagagatg gggtctcacc
atgttggcca 42960ggctggtctc gaactactga cctcaaagtg atccgcccac
ctctgcctct taaagtgctg 43020ggattacagg catgagccac tgtgtctggc
cctacttacc ttctttgtgt taattcctgc 43080accattgatt agcttattgt
cccattgact gtgtctttag atgacttctc tgggcctcag 43140aatatctagt
ccatagctga cacagagcat ctgtttaatg gtaaatgctg caggaatcca
43200tgcattggag tagaaagagt tttagatcat gttcctcatt tcttgctaca
gacttaggca 43260aagcgtggag aagaggttgt ccaatgaaga aatgaagtga
catgccaggt cagtggcaga 43320gctaggcctg gaaaataggt ttccagactc
ttccctttct accatacttt tcctgggagt 43380acgcactcgt aatttgaaga
gcgacttttg ggagagggtg gaaggaaggc ctgggcctca 43440gcctaagggg
cccattggtt gtgagaggag ggtctggtga aattccatac cgattgtccg
43500tgtgtgagct gctgtaccat agcctccctg cagaaccact aacctgtcaa
atgcagaaat 43560agttcaggga cagagctgtt aaaggattgg cgggttaaag
aaaacagtga atcccaagtt 43620ttgttaattg gatttttttg tttgttagtt
atttgttttg cttcattgtc ttcatcacac 43680caggggcctc cttaaatctg
gtggaaaaat ttccttggaa aacaattcag tgtttgtcca 43740tagacttggg
agggagagat gctagatgct ggaaagtctt gcttattact ttggggacac
43800tgagatgttc ccttcaccat gtactttgag acacacatcc tggttgagtt
caggcaagga 43860tgcctaacag ttgataagaa aactgggaaa gatagaaggg
atttgtaagg taagtcaggg 43920tgagtgaaaa cacatccggt atgctggaga
cctagatgct tgactgccac tcgctcctgt 43980cacctcagtc aatctgggtc
ttgctctgtt ggccttagtt tcctccttgc taacaggtta 44040gttccacctt
tctgcccatt tattttgtag ggttattgtg gatgtcattc tgaactctaa
44100aataccctct aaatatgaag tgatattagt gctctttaca ttgttatgat
taaaaatatt 44160tatgagaaaa aggttaactg taaggatttc attgaaaatc
ttataacaac caactgatag 44220agatagaaga taaggctatt aaattgttca
cacagatgcc ttgatatcct acctttttcc 44280ccctatattc cttttatgtg
agaaatgaga tagtgattta agggaaaaac ttaaaagagt 44340tccgactatg
ttggtttttt ttcccccaag tcaaccttaa tatcttactt aaatcttttt
44400cttttttatc ttttcttttc tttttttctt ttccctccct ccctcctttc
ctcctcctcc 44460ttcccttcct cctcctcctt ctgctgcttc tctctctctc
tctcgtttcc ttttcttttc 44520tattcttcct ttttcttttg agaccaggtc
ttgctctgtt gctcaggctg gagtgcagtg 44580gcaccttctt ggcttattgc
aacctctgcc tcctgggctc aagtgatcct cccacctcag 44640cctcccaagt
agctgggacc acaggcacgc gccaccacac tcagctaatt tttttttttt
44700ggtagagatg gggtctccta ggctggtctt gaactcctgg actcaagcaa
tcttcctgcc 44760tcagccttcc aaagtactgg gattactggc gtgggccacc
atgcctggct tgaaattttt 44820ctatggcttt attctttctc caagtacaga
gtctacccaa ccttctgaga tctttggttt 44880tcttttccta ggtaactata
gtacatactt atttatgtta aacaacagca atcacacatt 44940tctttttcta
tacagtcatg ctttataggc aaataaagcc tccgtcttag gctttctgga
45000ttttttcaaa agatgcaatt cctggagtat gtttttactt agagcaaagc
agcctagtct 45060cctatacctt ctgcatctgc agaaaagttg gttaaacaga
ctttgtaatg atgcccctta 45120caattctgaa gggacttgtg aaatagtttc
acagagtttc
agtgttaggt atatttgatc 45180aatgctaact tttggaaaac tttggtgcct
gtatgattca gagggtaggg cagaatatta 45240aattaatcac aacttcttgt
attttaacca ttctgggtaa attgggattc cgtgacgccc 45300aggcaaaatt
atttgtttat agaagatggg ctgaattttc catcgtccat ttctgagaaa
45360tgaggtaggt ttagaaagag acaatcaggc ctcttcttta acagaaatgt
ttgtgtctac 45420taggtgtgtg tcacaatatg agttcctgaa gaaataagtg
tccgctattg ggttgtatac 45480ttgtacttcc tattttctta ttttgcacat
ttttctggta tttccctttc tatggtgagt 45540ggcttctgat cgtctttcct
tttgtaaagt gtaatgatat gagaatcata atcgtggtgc 45600ggtcttttgt
gttgcatatt tgtagggggt cagtatgaat ggcccgtggt gaggctgcac
45660tgaaagatta ggagcagcca ccttgatgcg gaggaggctt agtgactttg
gacatgatgg 45720gctatggctg gctatactct cagctttggg cgcataagca
gagtattgat tttgtatttg 45780gttaaaacca gaagtacaac tttctggcac
cagaggatta ggaaaattta acagcggaaa 45840gccatcatga ggatagtaac
caattaattc gatttttttg gtcagacatg gctcccacct 45900gtaatcccag
cactttggga ggctgaggtg ggagggtcat ctgaggtcag gagtttgaga
45960ccagcctgac caacatggta aaacccgatc tctactaaaa atacaaaaat
tagcctggcg 46020tggtgatacg cgcctgtaat cccagctact cgggaggctg
aggcaggaga atcacttgaa 46080gctggaaggt agaggttgca gtgagtcgag
cttgcgtcac tgcactccag cctaggcaac 46140agagtaagac tgtatctcaa
aaataccata attcgttttg tcttttcttt acttttttct 46200ttccttttcc
ttcccctctc ccctcccctc cttccctttc ctcccctttc cttccctttc
46260catctctttc cttcctttct tttctctctt tctctctttc tttcaacagg
gtctcgctct 46320gaaccttttc cagtcagaat tgctcaggga tttttagact
tccattctgg aaaagagggg 46380gtagttattt tggtgagatt gtggtcttgt
ggttagacct tgtgatgggg gcctcagcca 46440aagggttcag gatttttttc
caagcttttc cctcacaact tgagttaatc cgaaacgttg 46500ctattaggcc
accggacatg cttttctgca tgcctgtgtt gggctgtttg gattgaaggc
46560ccagcaaggg aaggcaccct cgcccatctg acacaggcag gcctctacaa
ttttattccc 46620taaccagggc atgacaaact atggcccata gaccaaaatt
ggcttgccac gtgctttttt 46680ctggccagtg agttaagaat gactttttat
tatcattatt attaatattt tttgagccag 46740gttctcattt tgtcacccag
gctggagtgc agtggtgcaa tcacggctcc tgcagcgtga 46800aactcctggg
ctctagcaat cctcctgcta acttttttta tttttgtaca gtcttgctgt
46860tgttgcccag gctggtctgg aactcctggc cttaagcaat cttccggcct
tggccttcca 46920aaatgttggg actacaggcc tgagccgctg catccagcac
ttttattatt tttaaatggt 46980tgaaacacat caagagagga ataatatttt
ctgacacagg aaaatgatat gaaattcaca 47040tttcagtatc tgtaaataag
cttttattgg agcacagcca tgatacaaga catatactga 47100ctgcctgtgg
ctgctttcga gttacaatgg ctgagtcgag tagttatgac agagattgtg
47160tgggccgcaa agcctaagat atttgctgtc tggcactttg cagaaaaagt
ttgccaaccc 47220tgccctgaac aaataaaggg acaaattcca cttgccccgt
ccatctgtgg agcagagtca 47280ctgaaaggaa atactggaaa tactggaagc
cacttggtgt tttatcaagg atgtgaggtt 47340tcctggcaac tttgtcgcca
tatcatcatc atcatcacca tcatcatcat catcatcatc 47400atcatcatca
tcatcatcat catcatctgc cctttaagtt ttctgcttgt ttagaaaaga
47460aatttataca gagcccccag tagcagctgt aagggggcag gttcttggag
cagcccatcc 47520tcaacattct tgctgctgat ggaagattct caaggatgaa
ggcccctcta tgggagcagg 47580atcagtctgg ctttagtaga tgccaatttc
tgctaagact atttcctaaa ggagcctctc 47640ctcatttgcc ttttctccct
gttttcattg ggggaggtgg aagaggagaa aaataattag 47700agatgctcac
ctttttcttt ttgctggcaa tttaacagtc ttttcagctg ctttgattcc
47760tttcaggcca ttggtgttgt atatatttca agatttgctc acaggtccaa
agcttaactt 47820aagctccctg agacatatca taaaatatga tttggggaaa
aaccctaatg ggccatgatc 47880agaacattat tattcaacaa aggatgaaat
gcttaagcca agatggcctt ctttctttct 47940ttctttcttt cttttttttt
aatgaaagtt gagcagactc ccgtccaaca gttttcaatg 48000taggaattcc
cacagcccca tttgattgca gtttgttgaa aagtttaatg tttttgtagg
48060caattcataa tttccacatt gaacagcctg agaggaagag agctggagcc
cactgttgtt 48120tttgtagtgg gatggtggga actttttttt tccctccccc
aaaaggatat aaaactaagt 48180cagatggttg ggaaaacgtg gcacagggtt
ccagcccttt tgtaaatctg agatgccccc 48240tcctttaggt cttcctttag
gacccaacag aatagaaatt cctgctgctt aatgtctcca 48300ggaaggaaaa
aaattttcct ctaggctgta atagtaccta atttcctttt tcttctcttt
48360atttatttat tttccctatt aataagcacc aattgtagaa gatgaaggaa
gctgggaaac 48420ccatcacttt tggagaaggt taatagcttc ctttagaaaa
tcctgacata atacttattt 48480ccccaaaagg cacttcatca gcctgaatgc
cagttaagat tcaaggaatg ggcttggatt 48540tgtgtgtacc cagcggttct
gtggcatcaa gttgcactgg gaaggagagt ttggggctgt 48600cactgtggag
tccctgcaag tcagcaggac cagggctgtc ttcctgcacc atctggattt
48660ggttagctct ctctgggcag tggggccgag tctcatttcc tccaacaata
atgttatata 48720ggcaatgatc ctgggctgcc ctaacataat tgaaaattat
gtgtattgta ggcttggagt 48780gctgaaatgt gggctcataa aaatatgtgg
tgcaggtagc ctatggagat tggatgtggc 48840acacaatgaa gcttttatgt
aaagtaagaa ttataagtct ccatgttaat attgtattat 48900gagtatgaca
gttcttgggt gggtcctcag ggcaggtctg tcaccttcaa caaagcccga
48960gtttcctaat tctacagagc tggtatttgg atgtaatcaa atcggttttg
caggtggcca 49020aagatgaaaa cttgtccacc aatccagctc tccccactga
gggatagcat gggatgtaga 49080tgggtttgac tccatttggc atttttgttc
acgggttttt atgagatgga gaggtgagtg 49140ttggtgggtg tccattttgg
ttggcctcaa ggaaatgact ctattgagtg gttttgacca 49200atgcagctca
tatagttatg tggtaagtga gaatgggaag aagttgggat gagatggggc
49260agtttagatt cccagagccc tctggcctgg gttacagatg gagactggaa
atatttactt 49320tagtggttct caacttgaga tgatactgct cccagagaag
gtatttggaa gtgatgagat 49380ggtaaggata accaaggggg ttcctgttgg
tatttactgt ctgggggctt ggagtcctac 49440aagtccttca gtgtttgggg
cagactcccc acctaatacc ctgtcgcaga taggacaact 49500cattcagtac
acagatgaaa aaaacagaga tcactgaagc aaggggagtc gatgcagggt
49560cttgtggcaa gatgcagaca caaccggact aataactagg ttgctcacca
cgggaggcct 49620ctaggtgaaa gctctgaatt tgtagcagac acacccacct
cgtatagatc ctagacgtca 49680tgggaaaatc gactgtgtac tttggcaagt
agttcttggg caatgatctt ccagctttag 49740gtataaccaa atttggtttg
aatttgccaa gcagtcgtat cttcgaggaa ctccgtcggc 49800tggcttgtgg
atggctttgg cacttctgtc tctcgtggga tttgtgcaaa cccttctttc
49860tgtattatcc tttcctgtct tttttctttc tattgaaatt gttctgacca
tcaagaccta 49920actctgtgca gccttcccca gtctattgtc ccagaaattc
tgtcatcttt cttggcattt 49980cctgagtccc tgagtctctg tcacagtgtc
accatgttct gtcttgattt acctgtgtct 50040gtaaggctcc tcatgctggc
aaaactcccc gagagcggac atctttgtct ctcctagtgc 50100ttgtcacagc
ctgtacacaa agcaagtagt actcagtgtt cattgagtaa agttttctat
50160agaattaata ttaaaaccag ccatttattt tgcttgagga ggtctccgaa
atgaccaagg 50220tgtctcctta tatcttatat cccctccaag cattcattaa
ctgatggatt agtgagttgg 50280ccttgagaag cataaaggct cgtctccatg
tgcttctaag cattgtgtct aagttctgtt 50340tggtttcctg agtgaaactg
tcttaatgtt accaacagaa gttaaatgcc taagagtttc 50400ttatacatgg
gctgagtacc tctgtgactg ggcaagccac ctcacctcat tttaccttgt
50460ctgcaaaatg aggaactggg tcaactcatc gttcaaatct cactgaaagc
taattgatcg 50520cttttgacag aagtagctcc cttgggccgt atatttattt
cctagcttgg aggaaggtgg 50580ggacagacag aattgatgta cacctttatt
tttatctcta tggtaaacct gtgcatacta 50640aagcattcct ctggtctttt
gagatgagtg tatacattgt gtctggccct gtgcattttt 50700taccaagaag
taagttttgt tgagtaaact tgggttgtat gaagaactgc atgctcaccg
50760tactcaagta gcttttgcta cctaaaggac agctgctcat atgtacttga
cttcctttaa 50820agtgaaggat gatgacattt gaaaaacgga ggttgaaaag
gagcagattt ggaattgatg 50880gtttcctagg acacttctgg cttgagattt
gtgttttact ttcttccttt ggaatagctc 50940tatattcttt cctctccctc
cccacctctc ccactcccct ccagccccca ccaagttaag 51000gtagtagtaa
tgaaatcatt ttttctgaag ctaccctgta ctttgaatgc aaagacaaaa
51060aatacagttg ctagtaacat taatcttcta tatgtgtact tactgaactt
gagctctgag 51120gaagacccta ttggaattgc atgctttttt atttttttaa
tgattatttg catgcttgta 51180tgtttttcag tttctgaccc atgtcacagt
tatttcttgg gctagttgtt ctgcatttac 51240tttctgaatt cattgttttt
catttcactt ttgtttcctc tcgccagtat ctccagatga 51300aatggccact
gcttgatgtc caggcaggga gcctccagag tagacaagcc ctcaaggatg
51360cccggtcccc atcaccggca cacattgtcg taagtaacct cccagagatg
atggcttcct 51420ttattgaggg ggtgaaaaag aaaatgcttt tttgatgata
acaggcctta tttgtcattt 51480ttttctttct ttaaacacat tttctttgga
aatattgttg ggtatagttt atatctataa 51540ggtattcatt ttctgctatt
ggaccttaat gattgtaacc tacctggaaa ttttacaaac 51600ctttcctcca
ctcttttcca tgtatttggt taaaatctag ccttgtgggc tctagtttat
51660aggacacaat caccatggta tggaggagac tagaggtggt atcaaagcag
ttataaaaat 51720acattcaggg caggtgaagt gaagaagagg gaattagaaa
actcaaaagg gggtcctgga 51780tttgaaactt gcctattatc ctctccccca
atttatctta atatttgttg gcaacattct 51840acactaacat tagaaaaatt
tcatctgggc tggctgactt gtaaacctag agtagaaatg 51900aactttgaaa
ggctaaaatg gaatttaatc tatacatcca tggctttgaa agtatgtagg
51960tttgatagag aaagcatttg tttttagtac taagagacta caagtgtgtg
tctacatata 52020tttttaatgt attttcttag ggttttgtag gctctaagag
tggaatttat aaattaacct 52080cttgagaaga tagctcagcc ttatttgaag
attcccttct atgtatttat atcatgagct 52140ggacttcata cttttgaaat
aattaatgga aggcatattt ttataatgaa tccatccatg 52200acaggtagaa
ttatgcaaag catgaatcaa tcatgggttt ttcatttgag tatcacaaaa
52260tgttaatcat aaatacattt tgcctctata ttgtaatttc taaaaattgc
aaaataagtt 52320tcttaagtag aaaaatctta agatgcattc tgccattttg
ggctaactgc ctccttattt 52380tggagcttgc tgtaattgag catgtgttat
ttaatgagtt atacctctgt catatgtgtg 52440tgtttatatc acaaaataac
ttatttttat aaaaccatat tttgagtcat catttgtgac 52500aatgtcttct
tttctctggt ataaatgagg catgtagaaa gaagattgac atttgctaga
52560agcttcccct ttcctctaac tccacaataa aatggatgct cataattaca
tctgctccta 52620taaggtcaag atttcagggc tggaagtgac cttagatcat
ttaggcccaa cttgccctca 52680ggaaaggaaa ctgaggccca gagatgcctt
aagtgaattg cccaatgtca cacgctgagt 52740cagtggccag agcaaggctt
ggatccagtt ctctgctccc tttccagagc cttgtgatgt 52800cttctctcct
acaggaggtg aaaataactg ctgtggctgg ttctgttttg ctgactgtaa
52860attgggtcat ggtcagggac agtgcatagg tgtaaagaag ttgctggttg
ggggttctaa 52920tgcaggtttc tccaaaagtg aatgccctgt taaaaaaaaa
ttcttaacaa atatacagag 52980attttttttt taaaaaagtg tgacagttct
agacacctag agagtaaagt gaagaagcct 53040gttttcaggt ttcccgcctc
cctgaatttc ccagcatggt ccaggctttg aaatttattt 53100atctgctttt
ggcaatggtt gatgggaatt tcccacattt attttttagc tacagagaaa
53160ggacattatc tttaaaatct cttcgttgtt ctctctcttt gagtgaggag
agaagatgtg 53220aatcctggca gtggttcaga gtggacacag cccctgtgtt
tgtggcatag gctctgtggg 53280ccccatgcca gggagcagta cccccgtgta
aaggagtggg ggtttgtcca tttggataga 53340gcaaagatcc tccacctcaa
atcccacaag aacagttgcc acaacctggg ccctaagcat 53400ctcattttcc
tatgtagaaa ttaatgatct ggaggagatg gcaaaacatt ccttccagag
53460cctgtgtgga ttttggccag gggtgcagca agggggctta ggcacctttt
tcctctgctg 53520tgtcttagca ggcgtgttga ccatagcaac tcccctgggg
catacacacc ctcttgtaga 53580tggagacctt tgtccaaagc agccacagct
ggcaactgtc tacaatcttt tgggctttct 53640gctgtgctca aggggatctg
ggaatggcca ttgcctagag gggatgggct ggtggaggaa 53700ggtgggctct
gggagccggg gagaagggaa aagccatgaa tttggacaaa aggacaaatg
53760tggtttacat ttgtgaaata cttgaatgct tgtcatgaat ggtgactttg
gttctatgag 53820tcagccctgt gatggggtat ttctgcagtc ttcacctgac
accaggggtg agaaggagga 53880tttctgggga ggaggaaaga gttgagggag
ataggaaagt agagtggaag aaaggccttg 53940cgttgttgac ctctatccac
ctggtcacct atagtttttg ggattgagga tgcatacacc 54000ttgagactac
aaatttatga ttatattttt gctgaacata aggcaatgtg ccaaccaaaa
54060ccagctgttc tttggctggt acagtgtgtc tttgtttgta aagggtgcat
tctgaatggt 54120ggctgataca tcatttgggt ctttgtacag ttaaacattg
gccagagggt ctggttcgtg 54180tttagagtcg ccgatgaagg gctaactttt
ctccagacac ttggggctct tgttcacact 54240ttgcttttca ctcttttaag
taagacatag tcacatcaca gtgtttcatc agacatgttt 54300caaaataatt
gtctaaggat tgcttcttaa tttccccgaa atttggaatt gttgtaactt
54360ttgggccaag ctatttcata attatttcta atgtctcgct tgaagaatag
ggatgtattc 54420agtgttgatt attaatcatt cgaaactaca actttacaga
ttgctaagaa gaataacttc 54480ttccagtacc catatggggc agaatcttca
cgtgggaatt cagagcattt tgttggacta 54540ttttaatctg attggattat
tttcatgtgg tatgtgggtt accacattag aaacgattga 54600tgtgtagaat
aaatgttctt aacaagtgga ggtcaactta tcaaatgata tttacattaa
54660gaatagactc cacaaatttt agttcctgta gctgatatag catctcattt
gttatataat 54720ccagtgattc ctaatctgtg ttcagaggag agaggaaatc
gattgcaaca gggacgatgc 54780cttcattggc tggcccaaaa ctgggagttt
atacaaggcg tcagtctttg ccttcctcct 54840ccctgccttc cctcttcctt
cttccttccc catactcccc aacaaattca tggacttctt 54900aacaactcag
agacattagc cacaagttcc aagacacccc caccccccag cctccccagt
54960cctattttcg cattcatata actaaactct ttttctttct tggtggagtt
ttgaaattta 55020tatttttaat tctttgctcc cttttttcct cttacaaaat
gagtgccaag cagctaagtt 55080gtgctgagtg gtagagtttg agtcagtctt
ggctggtaag ctgtggggtt aggagccgct 55140ccctggatac cacctctggt
gtctttgcta tacaaagact ttcatttagc ctcctttgta 55200tccagcaaaa
aaagattcag tacccaaaat ggtggtattt tggtatagta tgtatcttac
55260aaaacggcaa aagacttcaa aagttcctac aattttatct tgggggtttc
cttttgaagt 55320cgatgtagaa ttttaccttg gggtggattt tttgtacttc
ttggtctggt gtgttttgtt 55380gtgtaatgag catggaggtg tgggataaga
aagcagactg aatcccgagg aacaaagcct 55440gccagactgt ggtggtgtac
ttttcttgtt gttattgctt aaatgctgca agagagtgga 55500aaactcttac
gaaataatgc acgatgggta gaacttcaga gaaaatctct gccgtctacc
55560ctgtgcattt tcgaggaagc tcagagggca tgctgaacct ttgctttttg
tttctgaaga 55620gttcagggga acctacccat aattaatttt ttaaaacact
acctagagag caccctcttg 55680gttattaaac acatgcgctg tttcgatggg
atgtttgacc tggattgtgg atgcttgctg 55740ggacgtggca tgtgttggga
ggctctgtgc tgcctgctga gcaccagcaa agccacagtg 55800gcccctacct
ctgtgggagg ccctgtgcca ggtgccctca aagagtaggg ggcccatgag
55860ggtatgacca gggggacctg atttcggctg agaagttggc ggggattaca
ggcctgggcg 55920gctccctgag gaaattgcat taaaaatgag atctgaaggc
ttgattgggg ttggcccaat 55980gaagggatag gagaagggat ggggagtggg
cagaaggaaa cacatgtgtg aaggtcctca 56040agggaaaagt gcttggcttg
gacagaggca ggaaatcagg taggaggcta gaggtcgggc 56100agggctccgg
gagagtgact tggggtgcag catatggtga ggatctgaca ctggggagtc
56160atttgagcag gttggctgtt tctgtaggag cgtgtgttaa gctgctggca
gtggggatgg 56220tgaaaataga gatgtggagg aaacagcagc ggaacttgct
gacaggttag atattggcat 56280tgagggagaa aggagagtca aaggtaggta
gatggagatg cttcactgag tggggagtat 56340tggaggagga gcaggtttgg
ggtggaagcg ttgtcctttt agagagattg tatttgccat 56400tgattgattc
attcattgtt tctgcaaata tttagtgtgg gaaaaagcat gctagacacc
56460aagagagagt ggagtcaatg aagaacgata acagcaacaa agactgtagc
gcttcctatg 56520cgaggcttgt tccagttgct tcagaggctg tgttacccct
gttctagaga ggaggaacta 56580ggcccaggga ggtggggatt tgcccagtcg
tgggagtcag gatgtgaaac aaggcaccct 56640ggctccagag cacaccgtcc
tctcaaccac tgcagagaag ctgggaaaga gacaaataag 56700tgggtgctta
gagcacaatg tgtgtggtgt gccaagagca gctgggagcc ctgggacccc
56760cagggaaccc cagccccacc tgggcatggt gggcatggct ggaggaggcc
tgctggcttt 56820gctggagagt gggacatgca tcaaggtggc cagagactgg
gcttctgggt gtcgtgctgt 56880gactgctgca aagggctcat tgacatatgg
tggggagggc cagcgtattt tctgcgggca 56940ggacatttgg gggatatggg
gtgtgaccct gtactatcta aaatctttta cttctggatt 57000atctccactt
tctctactgc atatatactt tgtttttatt tattttattc atttatctat
57060gactcagcca gactctctaa aagagttgac ttgtgtttcc tagcagccac
tgagtcagaa 57120ctttcccatt tcgcagtcag ggctgtggtc agggtgtctg
tgttgtctaa ggatataaag 57180caagccttcg ggcactacca aaacattatt
ttataaggag aactatgagt acctaatagg 57240aagaaccagg caatcaggtt
atcttttggt gaggaagaag tggtagatgg gatcattggt 57300gctttgaagg
gagtgggtgg tgtagactcc aaagtgtaca tggggccatg atagagtcta
57360tgtcagatgt ccaaagcttc cttctctcct cccagaaact ctgtcctctg
gtgaagagtt 57420ttgaagtttc ctgaggtttg ggttcatggt gtggcaggtg
ataccatggc aatagaaaat 57480atcccatcaa gaaggattgt gtgacctcag
ttgtagcccc tgcatgttgg aatcacaaca 57540atttgcaggg ccttaaaatc
aaatgccatt tcaccaactg ccctcccccg tttttttcag 57600cactgtttgg
tagctatctg tttcccctga tattcttgga cacttccaga gatgggggct
57660ctatctcctg gtggtagact gtttcttttt ggtacaatat gaactcttaa
gagagttcta 57720cctttaggga gctgcagtct ctctcctgga aatgctcaac
tccttaattc atgttttgct 57780gttaaattct gctaatgcct caccttacat
gtcttgacaa tttgaaggta gctattgtat 57840tccccgcaac cccaagtctt
ctcttcaaaa tgattattaa ttgtaattca aatcatcagt 57900gactggtatc
ttagactact taaggatggg aattgctaat tttgtattta aaagttgtac
57960ctctaaagta agtgaaattt atttttaaac gtagctttct tcattcataa
agtttatgtt 58020cattgtaggc agtttggaaa acagcccata atctcaccac
tcggagatta cattgtgaat 58080aatttggtat atttcctttt agaaatatac
caaattatcc ttttttcctc tgagtgtatg 58140aatatttata tttgttttta
acatacttga gctcatagtg ctcagtattt ccaacgttct 58200gtttatttaa
gatgaaaatt gctgtagtta ataagcactt ccccatgtca ttaaaatgct
58260taaggatttt taatgaccac ataacagtcc ataatatgat taaaccccaa
tttactgaat 58320caatgccata ttgttgggtc tttagattgt ctccttttgt
ttctgctact gtgaatgatc 58380ctgtgatgat catctttgtg tgtaaatctt
tgtcccctcg ccccctcccc ttttattatt 58440ttcttgggat agaccccagg
acaaaaggta gaaaagaaca aagtgttaaa aaatttcttg 58500atacatagcc
acagattatt ttcctgaaag ttctcaacat ttataactac gagcagtatg
58560taagagagtt atggttggaa tgattttaat gtctctgggg aatttaacaa
caaaaaaact 58620ttaggcttct ttggagagag acatgccctt aactccaccc
cgccctagaa cagagaccca 58680gcccatccaa gtcagcctcc ccaggtcctc
caccttcaaa acaggcaaac gaaatcattt 58740cttgaataat tggtaggctt
caaggtcaga tgtttatttt agataattca cagcataaat 58800ttatatgttt
taggtacctt agcccctgaa tatactcagt tcatttagga ctattttaga
58860ggtcttgagt ttactcttat aacctcacat ttttttgtga atttttagtt
ctattatctt 58920tgttttcatg gcatattatt gggcaaagat actatttatt
cgatgctatg tgtgagctgg 58980gtcaggatta tgaccctgag ttatgtttct
gggaaaatgt acccacttgt caaagatgcc 59040gttggctcct gtgattaagg
tcagcccaca atgaatgtgg ggagggctgg cagcctctca 59100aatcagctct
tgaccatttc tcaagctggg gcctgttgtg cttgggggaa gagtctttgg
59160cagctcagct cggggctagc gtttcctgac atttgtttcg ctgaatgtta
acaaggttac 59220tggaaaaaag ggttctctcc taaaataggt ttagggaagc
actgggatat gcgaagtgaa 59280tgagtttctt tagggcagga tcttgactct
gcagggggct tggaggcctt ccctagagtg 59340gggcttccta acactgcaga
gctcttccca ggacgagggg caagattggg acctactttg 59400gaaggttgtt
tttgtttcgg cacctgctct gtttacgaag cgtgggagcc tgttttaaat
59460taatgtgcgc ctacttagag ctacactcat ggttttgact atgtttatct
ttccagtaaa 59520taaaacaaaa ttgttcattt ggcacccagc ctgtcctgct
tgtcatttct tgtcttgctg 59580attaactcta tggatggggc atgtttctcc
aaccagattg taagtttctt gaagccaagg 59640agccctgtgg ttgatttctt
cacatgtggc tctctctcct cccacaatgg tgcttcgtta 59700attaagcaga
aaacccatct ctggttaggg actggagttg atttcgtttg gaatgagtgt
59760gacttcatca tgacctgaaa gtgttcagaa ccatcttggt tagcacaagg
gcgtggacgt 59820gtgtctactt tctacctgat gggatagcat gtttaatttg
gggttatgac actgaatggt 59880ttgccagtaa cttgctaatc caaccttata
cattccagct cacagtggag cgtgtctaat 59940tgccacagca gcatttatgt
ggaacgtggt tgcacaaaag ctccagaaag tcaggctgag 60000ggctcctatc
tctcctcaat cttggtttac gatgtctgtt tctgaggaat cctgggatgg
60060ggccactggc tctttaagag agagcccgat ttggaaatct aggacttgat
tgttgattat 60120gggcaataga tacattttaa gaatgatgtt gtaggctgta
tgaagtcatt tgatgattgt 60180tttgttaatg gcttgcaggt cagattttca
tctttttaaa
ttaattatca tagaaggaga 60240aaacaactgg atttcagaat tgtcccttga
ggtgtactgg aaactaaggc gtgagggact 60300cataggggtc tggcttggaa
agtgtattgc tatgtccagt ttacacataa ggatgtgcaa 60360atccagcagg
ttagctgagc tgcccaggaa tatccaggca agaatgacca tattctgata
60420attactcagg cctctgcctc atctccgctg cccccccgcc ccctgactct
cttctgagtg 60480ccagattcag cctccatttg aatgccaaat agacaggaaa
ttagcatgcc cagaatccac 60540gtctttagtg cactctctcc ccagctccaa
acctgttact gcttgtgttc aacatctcag 60600taaagctcaa caacatcgac
ccattactta ggcctcaaac cttgggtggc atcgtcgatt 60660gctcttttct
ttcatacccc acattcaacc catcagccca tcccacaggc ccaagtgtgt
60720cctctctacc ttcaaagcgt gtgtggcatc caccgcttat caccacctct
gccattacca 60780ctggagtcca gtgccatcat ctctcacttg gatgtggcca
gagtgtcttt gctggtctcc 60840ttcttgcttc ctacctttgt aacagcctat
catctatctc tggtctccat agctcactcc 60900catactttga gagggccttt
gaaagcctta gacagatcat atcacagacc tctatactga 60960aagtcgggat
aaattttatc tctggaaaga gtcccaaagc agcgatgaac agatattttg
61020tcctgtcact tgatgaagag gtggggcttt gagacccaag agcttagaat
ggagagccta 61080gatgccacta agcccaggca ctggccatgc ttcgagtgga
gcttttgtgc tggtggagga 61140gagatggctg ggggacacct gtaggctgag
caagtccccg ttcatcagac cctggctcat 61200ccagcagggc gtggctgatg
ttttcaatgt tgtatcctga gtgggaccca gatgcttccc 61260aactgtgcca
catctgagcc ctgcatgcca tctgtccagt tgcagcctga ctgcaatgtg
61320aggctgctga agagctctgg atggtgtgaa gcaatctgtt ttctagcccg
agcctgcata 61380gctggtggat cctggaccgt gattaagtgc atcacctagg
cttcaatgag atggagtcac 61440tgtgtgtcca aacagtggga taaaggcttt
actctttgtc ttcctgctct gagggcacaa 61500gctgcttgtt tctctcacaa
ggacaccgtc tgtgttgctc aggtgctggg gtgaaaaaaa 61560cagcaagcat
ttgaaaaggc tgaagaagga aagaaagctg agagcggtac agccttgggg
61620actgagccat cccattgtcc cagaggtggg ggtgttatca agacctgttt
ttgagccata 61680cctctgactc ttcctggaaa gttagaccca actcaagaac
acactaagag aagtgtttcc 61740ccctagccct ttcagattga aaggagacgc
caaccttgat gggtggaggt agaaaataaa 61800gtcccaaaac agtgtcttgt
aagcgaaggg gaacatggct gggcagaggg cttctggtga 61860aacttttggg
agtattcagt tggaactcag gaaaaaaaaa ttgttttttt ggaaagaggt
61920agcagccccc ttcagccaaa gctcataaat gaaggaatgt ctgagactca
gaattacagt 61980gaccaaggca agacattgtc aaaggctgaa taagtgagtt
tgactgacag aggccatctc 62040catttttagt atatggccaa gcatctttcc
cacagtcttc cttgagcccc ttcccatccc 62100acttctgaaa agcactgagt
tggccattat tatgcttttt tcttaaatta tgaagttgtt 62160ttcaggtatt
gagaataaca cccaggtgct gaactcccag cataagaaat caaacattca
62220aaatggagta aggttctgaa gctgacatct gtctctacac attttttttt
ttctgataat 62280ggcatttcct atctccaccc tcactctttt tgttgtggtg
aactacactt cccttgttcc 62340actcggttct gttgcacatg tgattaggca
aggggcagat atgtgatatt tattatgagt 62400cttttccacg cagagaggat
ctaaatctgg ctctttgcaa ttgccttcat acatgtgcat 62460acacaccaca
cacacacaca cacacacaca cacacacaca cacacagaca catacatatg
62520cacacacccc gactcaatgg aggaccctca tttgtagaag ggtaaaatgg
gtgaggcgga 62580aatgcctgta tggcaccatg gagttctgtg tagccagttc
taatcctggg ctatttggta 62640aggaatgaag ttggagatag tcttctgtcc
cttacaacca aaggaattct aactaatagt 62700ttgccaagtt ttatgtttat
aataaaaaat gacatgcttt ttcttttgga tttttaatgc 62760ttttgaatta
aaaatgctag aacatgaact gattcttcta tcgctattta gatagagcct
62820tgcaagagca gagcacgcat gctttcttta agaacaggtt ggtttgtggt
cgtctgagga 62880ctgttttaag gagacttatt atacacaatc atcccccaca
aatgatttct aaagagaggc 62940tggtatgaaa gaaggagttt ccatgattct
gtcctgtggt tctggggaat tctgaaaatg 63000aactttagat atttttgtga
aattcttatt ttcatatttt tggtatctca gagttttctt 63060ttctggcttc
tgtttaacat actcttcttt gccctaaatc tctcttattt ttgctccttg
63120ggacaactga agaatcctta gataattaat agtatgaaat actgcccttt
tagttgaaaa 63180atgtcacaat aatgtaataa gataaataag gaggtgtcgc
tttaacctgt atcgtgtagt 63240ctcctctact tactaacact tacttgtatt
actagaagca ttatttttta aatcatggaa 63300aattggtggc aagctgagca
tacagttgtt tatttctgtt tgactgatta ttacaacttc 63360attatttgat
gaaggttctg tacgttttcc tttaagacac atagaaattg tgagaagatc
63420ctgcagcccc gaaaggctac agtgttgatc caaggactct gagccgagtg
cagggtttgt 63480acttggacct gcaggctggg tggcgtctgt gggagcagtg
tgttgagaga gattctgagg 63540ctgtatgtgt cagggcctcc aggggaagga
tgcattgatg gattaatttc tgccaaggct 63600gaaagaggag agagtaagag
gctgtagagg tgtcacagct gtcattgctg ttttaggcag 63660tcaagctttt
gggaaagtgt cagaaattga gccccctact ggatctatcg gagccctgtc
63720aaatgtccat ttagatgtcc tggtgaacaa aagttctctg actcaccatt
taaaaacttg 63780ttccaaatga aattatggga gaaaggaaca tttttcatcc
gaacccagaa tgaggatgta 63840cccaaggaaa aggacgtagg ctcaggagct
ggactgtggc tcagctggcc tgatgtatcc 63900cactttgttc ctcccatggc
tgggatgtct ctttgctctc catgacccat gtatcttgag 63960gacatgacac
atggaccaag cttgaactgc ggattcattt ttatgcattc tacctgtgaa
64020tgattgcagc ggatctagtc gtatttctga gagttactca aactggactt
cagcagtgaa 64080ctctacagtt ctcttttcct cccacctttc tattagacat
tgcatgatac aaaaatcaag 64140atatttctaa gagggtgata acttcaatgt
tatctaaact tttaatttgg aagaagaggg 64200gttctttgtt ctttttaaaa
agatacaaac gaacttcttt atctgattct ttttttggtg 64260caaacccatg
atgccttctt cctgattcat ctgctacact gtgagttcaa gcctggcgtg
64320ggacacaggc acagctctca tgccaacgat ctcatggtta agttttggaa
cataatttga 64380aaaatgtaac ccattgagag gcagtaagga catacggtga
gctagtgcgt gtttggacgt 64440ctgtgtggaa taagtgagtg ggtagagagg
acatttgtca aggagcggga gggcgggcca 64500ttggcttggg ggaaatgggc
tgagactcta ggggtggcca gcaccgcata cggaggccag 64560cagggttggg
cttggctaag tgctgtggtg tctggatgcc tatgtgagtt tcctccagaa
64620gttttcagtt ggcaaagtag aacctgctgg atatgtagca agggtgtgga
ttgtcgggat 64680cctgctgggc gcaggcgtgt gataccagag gtcagaacag
aagctgaggg atgaggcttt 64740gggagctttt tgtcatgcac tgtcctggag
cctcagttac tacaaagtct gcaaatgata 64800gaccggagct ttggttctgc
ctgatgctag ctcccctgtt cctgattttt cttttcaata 64860ttagacttaa
tcccagaatt cacatgttga aagaaaactt agaggtctag tgacataaaa
64920gcctcatttt gatcgttaca gaactgatgc cttgagaaat ggagagagaa
gtacacgatc 64980atggtaatac tggatgttca ctgagcactc actagctcca
ggccttttct aagtaattta 65040tgaagttgtc aggtttaatc ctcacaacgc
ccttatgaat gagctattgt tattatcccg 65100atttggcaga tgaggaaact
gaggcttgag gggaggatga cgtactcaag gtcacacagc 65160tgggaggcgg
caagctggaa gttgaaccca aggagtctca catcggagcc aggactctca
65220cccttcagtg ttatgctgcc ttaatcaggc acacatacag gcggggagag
gcaggtttcc 65280ggacaccaga ctaggctggt gccggtcagg ctacaccagg
gaacctggag gcctgtcatt 65340cttttgtgat gctgttagtt cctgttgagg
aagtgaggct ttgtgggttc ccaggaggaa 65400aaggtatgaa ctcatggcaa
aagaaaggaa ccaaaaaagg gagatttgca tcacaatgag 65460ccttctattc
atcctaaatt atacctcctt ttataccatg tgtgtctgca aacttgtggg
65520taaatcacaa atctttctgg taagttacaa tggatggaag gtttttgcat
ttctctcaaa 65580tcaccaacca tttaatgcta tgtgtagtca ctccctaatc
tatcttttgt ataaatttgg 65640atctttgagt attggggttt tccatgatgt
ttggcagttc cccttagggt gtctatctca 65700aagtttgtca cactgacaag
ctttggggag agaagttaga ggtgggcttc cctgttttta 65760gtggctgtgt
ctgattgttc tgtctgttct ccaggacagg agagattgat tgctttctag
65820ctttttttaa aattaaaaca acaacaacaa aaaaatacag aaaggtacaa
aggataacaa 65880acacattcat gtacctgcca cctaaaataa caattactaa
tcttttcacc ctcctagccc 65940atgatcttcc ctcccaggct gttattaata
tgaaaaccga gttcaggttt ttatactttt 66000cgacatctat ttatattaac
gtatgtatta taaataatct tagtagtttt taactttgac 66060ataagtggct
tcacattcca cataacattc tgcagcatgt tttcttttat ttttattttt
66120ttctttattt ttaaattttt attttgcagc atgcttttct tattcaacat
tacatttgaa 66180ttttttcaac attgtacatt gaaatttagc tcattctttt
taactgctct gtagtattta 66240ttgtatgcat atactacagc tttctatttc
tgtattgatg gttaattagg ttgcttacag 66300ttttttaaga ttacagattc
tgctgtaata accatccttt gggcaagtgt atgtaggtac 66360ctatatatga
gtttctctag gattcatacc aaagtagagg aattggtagg gcattggttt
66420gctggtttta attttaattc acatgctatt gtcaagctct ccagaacaac
tggatgagtt 66480gattggatca atgagtattt ccatcaccag catataaact
ctttcctcat aatcacacca 66540atgcttgatc ctgttggact taaaattttt
gccaatttgc tgggtatgca acggcatctt 66600acctaatttg cctttatttg
atgactcctg aggttgaaca tctggtcata tgtttatttt 66660ctcctctgtg
gcttgcctgg tttaatgcct tcttcatttt aaagaatcag atagttttct
66720gttattgatt tataggaact ctttatataa gttgaaaact tgattatatg
tgttggaaat 66780actttttcta ggctgtgatg ttttaaaata ttgctttaga
tgggttttca tttttacctt 66840ttattttaga gatggagtct cactgcattg
cccaggctgg attgcagtgg ctattcacag 66900gaaagagcat agtatgttac
agcctccacc tcctggtacc aagaggtcct cctgccccag 66960cctcctgaat
aggtgggacc acaggtgcac atcactgtgc ctagctttgg atgggttttg
67020aaagaaagaa gttttaaatt ttaatgccct caaattcatc tgtattttcc
tctgtgcttt 67080tattttgtac ccactctaag tagctccgaa ttctgcagat
agttggtgca ggaattctga 67140ttttgagtgg acatctgctc tctaacagtc
acattgaagg aaattaggtt tttttggtag 67200gaatctaagc aaggggttga
tttgtaaact aggctttaaa tatgatttta agcaactcac 67260ttagaacaag
atacaaaaat tgtggactgg acctatatct ggaaaacttg aaagtgctag
67320ggcaataaat aattcttggt cacatacagc cgagatcctg ggctcctgac
tctgggacag 67380aagctttcta tattttatct catcagtctt tgcaacaggc
tccttgaagc aattttatcc 67440ccattttaga gataagaaaa ccagagctta
aagcagttag ataatttatg aagtaagtgg 67500cagagccaag attcaaatcc
agacctttct gaccacaaag ctcgttgctg aataccgcgc 67560ctcattgcct
tcttgcgaat tacttgggat ttgtttgaat cccaaaatct ttatatgtta
67620ttttaaattt gaatctaatt ggaagtgggg cagtgagggt agaggacaga
aagaagggga 67680agagcttgag actcaataat agaaacaaaa aacccgtctc
caggagggcg gttcaaaagg 67740aagaattcca tatttcatgt aactgaaacg
ttaaaagccc aaataattgc atcatgcaag 67800tctgatgctg agtaatcacc
ctcccccata ttattgggga gagggggcaa gaagtctggg 67860aagctgtttt
tgcctaagga attacattcc aggggactct gaggatttag gtaaccacaa
67920aagccattta tttcgagtac actgagattt ctaccacttt gatccctaat
ccatagcata 67980attaataaat gaaatgtgct gtagcatggg ttttttacaa
agtgtacttt taaaatggct 68040tttggtctga catgattcat ttgccacttg
gaaaagcgtc atcgcctcag atgggcaggc 68100tgggagaggc tgcctggtgg
gtagctgagg gcggtttcct ggggcacagt tcctgccttg 68160ggcctctaca
gagcggtctc atccaaacat ctcccagact ctgcgttttc caggaagcgt
68220gcagaaatag gaggccagta ctgaaatgct atctgctctg tgtatgtcag
aagaccacaa 68280accacttata acaaatgaag atctttttat ttgttcttat
ccctttatgt cacttgagga 68340aagttgctgt gagtaggtga tgatcattac
agtgatcact ggttgcccaa actgagaagc 68400cagacatttg gcttggtttc
tctcccttcc tcttgtctct cctaccctgt aaacacatac 68460ttggtgatta
cccatgggga gacaagacag gctgggaata tatacttctg caacttcagc
68520ctcctgggtt ccagcgattc tcctgcctca gtctccagaa gagctcggat
tacaggtgtg 68580caccaccagg cccagctaac tttttgtatt tttagtaaag
atggggtttc atcatgttgg 68640ccaggctggt ctcgaactcc tgacctcagg
tgatctgccc ttctcggcct cccgaagtgg 68700tgggattata ggcgtgagtc
accgagcctg gccccaggca ataatatacc agtgggcaag 68760aaaatattct
tgctctcatg ggacttctgt tgggggtcag ggtataggga ggaaggcata
68820gagatgaaaa ccagtaaata agtaacaggg gaaaacattt taaatacatt
aataactaat 68880aaaatagaaa taaatctgtt ggctacttaa caggatgtgc
cacattccag atacattacg 68940ttaatcctta tgatctttgg gggctaagta
ttagtattcc attttacgga tgaagagact 69000gaggctcaga gggaagggag
gtggcttttc tcaggtggaa agccagacct tttcagtggt 69060cattcagttc
atagctaagg tcttattttc tgtgctctct gtcggctgaa aatgggcaag
69120gtaatttcac atagtgacag gagccatgtc agagaaagag caggacagtg
ggacagagag 69180ggaccaggct gggggctgtt tgagatggag ggtcaggaag
aaccaaacta agatgtgaac 69240agtgggaggt gttggagctg tggtgcttgc
ctagaaggac cctcatcgag caaatagaag 69300cttctggcag gaagaagtta
atgtcttgcg tgtgccctat gtaggttcat tagggccttt 69360aaagggggaa
gaaggtggtg gctataaatg ttacaatctt acctttggcc cctagggatt
69420ctgtctttca accttggttc agtaacaact tgtgactgcc caacagggct
tcctttcggg 69480agagaatggc ttgttacatt caaatatgcc atgaaagtat
caccatttat ttcagtgtct 69540gatgccccag cttgggcagc ctgagcaggc
tctgaatggg tctgaagagg ccctttagag 69600tagagatgaa gagggggtgg
ggaatcctca attctaaaca aagagtctgc aatgggaaga 69660tggccaaatg
ctgtttttgg agtgggtgag agggaaaaga aaggtataga tggttcgttg
69720gaaaatgtgg ttttataccg ggttttggtg tcaggtcccc gagggcaaca
tggactccac 69780actgtgatcc tccgggcagc tcatagcccc agccccttcc
ttttgcttcc tggtcagttt 69840gtgagaagga ggggttgtgt ctccaatctg
agcaataagg ggtctgaggg gggttggatc 69900catgtggctt tcctgtgtct
tgttccttgt aaaagttcca ggttttgggt cgtgagctgt 69960gtgtgtgtgt
gtgcgtgtgt gtgcgctgta cgttaatatg gagagatggg cttgggccag
70020tgggaaatag agagacccgc aagcacagag tgacagggtt tgatagtaag
cagcaggcca 70080gcgttgctgc ttttattcct cggtaaatcc ttgcacaatg
ccatatgctc ttgcattccg 70140tagctgctgc atagggtgtg atttagttaa
tgcccgctct gcaaacagga aacggtgctc 70200actgctgtgt atgcttttca
tggagataaa gtgtcaggag caagacccca aacctgcgaa 70260atcactaatg
caaccgcccc ccatgcccca aaaggtggga gtgggggata aaaagagtag
70320gaaagtggtg tggggagggg aagctttagg gccataactc agacaatttg
tcaggcagtg 70380gcatcggttg ggaggaaaat attgatgtac actttttgtt
tttgaacctg aagtttgggt 70440tttttcggat gcattggagg acttttaaat
gttttcggag tgccagagtt tggactgtta 70500ggtcaccgta ggtaccggct
tgcatatcat ttcagaggaa tattttcaaa actccataaa 70560aacatgcggc
tttcaaggct ggaccacttg ttcaggtcct cctcccaccc cccacccttt
70620ttggcaaaac catgcaaaca ttggtattca aaaatatttt gttacttttc
ttggcaaagt 70680gttccaagaa ggaattgcaa cacagtctca gagttaggag
gcaactttct ggggaaaagg 70740cgggggttgg ggaggtttgg agtttgaatc
aaaaacagac accgaagctt taataaaata 70800aatgaagcgg agccctttca
gctcacggtg gactgtgttg gtgcgcgggt caggctttaa 70860cgtgcctagt
ggaaattgac agtctgagaa ctgggacata aacaaaaatg tcagtccctg
70920ggagtcttgt tcactggaca atgtctcaat tgttcctttg gttttcaagg
cagcagggag 70980agtggaatat taactgttta ctgcccaaag ctggctcgga
aattgcttgg agaaggggag 71040aaaaaagaca gaaaatcaca ttttttattt
agaaactatt aaacatgtca gtaagagata 71100ggaaaagagc agattgtttt
ctccttaatt atctgccatt cacttccata tttctgcata 71160ccatttttgg
ggtgtgtgtg tgtgaaggaa cagcagggtg tttcttttta aatttgaatg
71220ttagccttgc atattgtcag tttttaaagc ttgctggcat gtagattatc
cgcccccggt 71280ggatatgaca gtgggcttta ggaaaggaag tgtgatttct
gataacattt acatcttagc 71340tgttcagcgg ataccctgtt agtgtttgtt
cttcagaatg ctcagataga acaaaaatca 71400agtggttgga attttaaaaa
acaaaatgta tttggctctc cataaaaatg catttagtga 71460taaagggggg
cagcaagtaa ctatgtctga gagaaggaat tgcaggcaca gaggagatcc
71520agaattctgt tcacacttga atttacttga ttcgagaaac aaacagcaaa
gcctggtgta 71580ttggccttta tctgggcaaa gttcaaaact caactggtaa
ttatgtcctt agaagcctta 71640aaaggactgt gttgttacaa aagcagtgac
tgagcttact tcttcaggac cgaatgcact 71700cgagttgttt gttagataaa
cttgttttaa taaatggggg ggtcagggga gaggtttctg 71760ttcttggaag
attccctgat aagtagcttt cttctcttgg agaacttcag gctttctctc
71820caagcgaggg gtttgcaggc agctaaagtc agcttcggct tctgcttcct
gtcagtcagg 71880aagtcacttc cttaacccaa attacaagct agagcacaac
tccccagcca taccgaaaag 71940agcaggtttt tcccagaaga ctgtgtttct
agatgcggaa gtgtaaattg gtacgctgtg 72000tgatcatgga atgcccaaaa
tacataggga acagtgttgt tggaaagagg cgctgtgtcc 72060ccaaggagaa
gacgccgccc agaatggctg gatcgcctgt tgtggctgag tgcgaggcag
72120ctgtggctgg ctgctgtgtg acgatgacct agtagccacc catgtggagt
cctggctgcc 72180tcagaaccct atcacatcta ggcaaaatct tgcatttttt
atctgggagg cctgaggact 72240tcagggctgg tggatagtaa gctccttggt
tatctcacag atacaagagg tcttgggaat 72300ccacgatcaa acttgatgtg
tgcgtttacc ctcctccctt tgaatctgtt attcaaatat 72360ttaagcctcc
aaccttgtgg cccctacctg caccacccct cacccccccg acaaaaatca
72420agctcttgac ctcatggctt ctttcagtga cccttggggg acagggtttc
ccaaggctgg 72480ttgccagctg gcatggtccc ccgttggtga agtggagacc
tgtgtttttt tggtcatttt 72540gcaaagagct tatggatgac agcagttctc
tgtgcctcgc tgggacagag tgtattctga 72600ggtccagcgt ctgcatggag
atctgcctat ccttcacttg gggtgctcag tagataacgc 72660ggccactttc
ctatacattt ccttaattta agggaacagc gtaaactcag cccaggtgga
72720ttaatctctc cagtgacttt tgaaacttca atttccaatt tccctcttat
gtctaggtgt 72780gagtgaggat acgtgtagta attgtcgcag gtattagtga
gaaagggtgc agatcacaca 72840aatatttcac acgttattag ttggaccaga
ctttggaggc aagggagggc cgtgtcacct 72900aggaaatttg ctcttccgtg
gagatgaaag ggcagtgaat taagtgcctg ctttttctcc 72960ctttttccct
ctgacggtta ttgatcctcc cctggaactg tacagttcac gttctgatct
73020ttttcttgac aaagggaatt cccagtttgt tcgctggcga acgcactagc
aggtgaggag 73080ttaaaagttg gcaacgcctg ccctctcgag agtgtcagga
tttttagtct cttccttgag 73140agctagaaga tgtttctaaa agaatctctt
tggtgactta gaagtggaga gagctttaga 73200agcatggcac aaataaaagg
aaagaggcaa acaccgtcat tctacatctg tttattttgt 73260tattaacaaa
aggcaaggcg attttcatta aagttttgct ggggttgggg ttgagggtgt
73320agagagcaaa agtgtgagtt gtacaccatg actggaatcg cttggacata
ctcttcagca 73380gacatcgtgt gactgtggaa gaaatgagtt tcatgaagat
gactgataga aggaagccac 73440tgaaccagtc ctctatcacc tcttccaagg
ctaaagtttg gagccacttg cagaaggctc 73500tcctcaaacc cctgtgttct
ttgcctaccc ctgctgttgc cacatcatct tggagagctg 73560gctgcttccc
tcctcaacta gaagttccta gtgcctgctt agttcttgtc tcttgcttcc
73620caagtgctca caaaatacat ccatgttcgc tacgaggaaa tggaccacat
aaggtttccg 73680tgaaaacctt agcccttagg tctaacacag taggaacaga
agttaatgtt ttcctgacgt 73740agaagtttct cttgctgctt ctggtcacat
ttctttcttg tgtggttctt ctatggctac 73800tgcacttttt tttttttctt
actgtctccc ccttccccca cacaccacct tttggggata 73860gggtggcagg
tgagaatata aacagataat ggttaagaga tagtttagtc tttctaggcc
73920agattattta gtttttgcca tctaggtaaa attcggtcca attaagcgtc
cattaagtgt 73980tttaatataa gctggagaag gagttgaacc tggaggtcag
ggctctgtgg tctattacag 74040tccccctggg gtctctagcc caagggagac
tccagggtct taataaatga ctgggggttt 74100cattttgagg cctttactac
caaagactga ataatacatt gggcatgatg gttttgtcct 74160aaacattaac
agccacaaaa ggtagagagt gtgtctgttt atagatacac atgtatcatg
74220aataattagt tggggactgt gcatcaggtc tctcatttta cattcgagga
agcaatgcac 74280ggaatgaatt ctggacctgc gaactctgaa tttcaattct
ctgtctccta cttttactgg 74340agtgcttgca aacagtacag tgtttttgtt
gtgaagttat accgtgcctg taatctctct 74400gcgggtggcc ctcctaagcc
ctacttcaag aaatagctct aagctcatga cacccgcccc 74460acccgatgcc
tacatatgtc ttatatcctt ggagtagtgt ttggggttgc aaatttgact
74520ttagggagac atactctctg atgataggct aatgcttata tttactgata
aacttccttt 74580ttgacggtca tgggcttcgg gggccaccca accaaactgt
gtggctgctt ttatgttggg 74640ccaaaagaca ggctccttgt gtcctcccag
tttcttaaac aatgaagtca tggcatttta 74700cagtgctggt gaatggattg
agattgtggt ggccctggaa tgtggcactg ctctggctgg 74760agggaagatg
agagtgaggg atggagagga gaggagagcg ggagatggga acctggtgga
74820cacaggaggg agtgtgagtt ctgagggcca aaggaaactt gacaccggat
gggacattaa 74880tctgattctg ttatctgagg ctgtcaccag tcctccctgt
cctcctggca 749302384DNAHomo sapiens 2ttcaggccat tggtgttgta
tatatttcaa gatttgctca caggtccaaa gcttaactta 60agctccctga gacatatcat
aaaatatgat ttggggaaaa accctaatgg gccatgatca 120gaacattatt
attcaacaaa ggatgaaatg cttaagccaa gatggccttc tttctttctt
180tctttctttc ttttttttta atgaaagttg agcagactcc cgtccaacag
ttttcaatgt 240aggaattccc acagccccat ttgattgcag tttgttgaaa
agtttaatgt ttttgtaggc 300aattcataat ttccacattg
aacagcctga gaggaagaga gctggagccc actgttgttt 360ttgtagtggg
atggtgggaa cttt 384320DNAHomo sapiens 3ttcaggccat tggtgttgta
20420DNAHomo sapiens 4aaagttccca ccatcccact 205366DNAHomo sapiens
5ttgtcccttg aggtgtactg gaaactaagg cgtgagggac tcataggggt ctggcttgga
60aagtgtattg ctatgtccag tttacacata aggatgtgca aatccagcag gttagctgag
120ctgcccagga atatccaggc aagaatkacc atattctgat aattactcag
gcctctgcct 180catctccgct gcccccccgc cccctgactc tcttctgagt
gccagattca gcctccattt 240gaatgccaaa tagacaggaa attagcatgc
ccagaatcca cgtctttagt gcactctctc 300cccagctcca aacctgttac
tgcttgtgtt caacatctca gtaaagctca acaacatcga 360cccatt 366621DNAHomo
sapiens 6ttgtcccttg aggtgtactg g 21720DNAHomo sapiens 7aatgggtcga
tgttgttgag 208558DNAHomo sapiens 8gctgtgaaat cccctgtgta gtgggaagaa
gaaatagcaa atcttagctg ccttggacct 60gatataatta tttgtcttca tttacatggt
tyatccttca aggttgaata aatgatgtgg 120gagctagtca aggggcttta
ggtatgtgat ttcatgccta ctttttttta ggtagagaaa 180ctgaggtcac
agggtactag agaatggact ctaagattca ggtttctgaa ttgcctgtgg
240ttttgttgac tcaactgctc ttctgttgtt ttttagccac atgccttgaa
acagtcctct 300ttcccatgtt tcttcatcag caccattaac ccaaggtata
ctgtcctctc ttatctttca 360caaggtcttg gagttcccat gcctttgtaa
gcatccctcc ccgagattca gcaccaacca 420aaatcacatt tggaaaaatt
gcttgtttcc caagaagctt tggaggatat gattttgtat 480agaacgggtt
cacaggtttt ctgttcattc ttctatggtg gagtgtgtgt gtatgtgact
540ctgtcttctc tccattcc 558921DNAHomo sapiens 9gctgtgaaat cccctgtgta
g 211023DNAHomo sapiens 10ggaatggaga gaagacagag tca 2311364DNAHomo
sapiens 11aagggagaaa gcaggattga gcagggggag ccgtcagatg gtaatgcaga
tgtgatgaga 60tctctgccgg accaaagaga agattccttt ttaaatggtg acaaattcat
gggctttctc 120tgcctcaaaa cctagcacag ctgttattta ctgaacaatt
agagagctaa gcacttttta 180gataytatat aatttaattg ccgtatgagg
cacccttagt tttcagacga gaaaccacag 240ttacagggaa ggcaagtaac
ttagtcaatg tcagataact aggaaaaggt tagaggggcc 300ctggacacag
gcctgtgtga ctgagaagct tgggcacttc actgctacat ttcatctctt 360cgct
3641220DNAHomo sapiens 12aagggagaaa gcaggattga 201322DNAHomo
sapiens 13agcgaagaga tgaaatgtag ca 2214579DNAHomo sapiens
14ctgatgaggg tagggagcat ctgtctgcag cttcatcttc attgtctagg ggctccagaa
60atatctgtga gtaaataagt tatttaatct ttgcctcaaa tttccagtga ctgtagggat
120atagctgtga gcctctagga gctgagattt tttaaatttc ccacttaaac
atttatttaa 180aaattttgtg ctcagcatgg actaaggact ttacattcat
taactcattt acagcttgat 240cctatgcggt gggcattcat ttacagagga
tcccatttta caggtgagga agaggccagc 300taggggtgca gcctaggtta
gtattctaga gctcatcagg ctgtgttgtc cccagtgaaa 360gaataagcaa
agaagtgaat gttgtgcatt gagaaaaatg actctcggag gaggatgagc
420ctctcggata tggcgaccga agtgatwtgg ggcccttgtc aagggtctct
attatggcat 480caagaaaaga tgctgctttc ggtgatgccc gaggagagcc
tcaatatttt acatgggaaa 540cctaaaaaag gggccatgtt gtggtctctg cacctaaga
5791519DNAHomo sapiens 15ctgatgaggg tagggagca 191622DNAHomo sapiens
16tcttaggtgc agagaccaca ac 2217486DNAHomo sapiens 17tatttagaaa
ccataaaatc cacctatttg aggtgtacaa ttgagtgatt ttctgtatag 60tcacagatct
gtgcagtcat ccacaccctc taactccagg acattttcct cacccccgag
120gagaaacctc ccttacccat tagcagtcac tcctcatttc ctctcccccc
agcccctggc 180aatcactgtg gatttgcctg ttcttgacat ttcatataaa
tggtatcata aaatctaygg 240gcttttgtgt ctgtctgctt tcacttagca
tacggttctc aaggttcatc cagtattgta 300gcatctatca gtatgtcatt
cctttttatg gccaaataat attttattgt atggatagac 360attttgttta
ttcatttatc tgtttttggt tattatgagt aacactacta tgaacatttt
420gcacaaattt ttgtattgac atgttttcat ttctcctggg tatagtccta
tgagtggaat 480tgctgg 4861827DNAHomo sapiens 18tatttagaaa ccataaaatc
cacctat 271922DNAHomo sapiens 19ccagcaattc cactcatagg ac
2220428DNAHomo sapiens 20ttgtctcctt ttgtttctgc tactgtgaat
gatcctgtga tgatcatctt tgtgtgtaaa 60tctttgtccc ctcgccccct ccccttttat
tattttcttg ggatagaccc caggacaaaa 120ggtagaaaag aacaaagtgt
taaaaaattt cttgatacat agccacagat tattttcctg 180aaagttctca
acatttataa ctacsagcag tatgtaagag agttatggtt ggaatgattt
240taatgtctct ggggaattta acaacaaaaa aactttaggc ttctttggag
agagacatgc 300ccttaactcc accccgccct agaacagaga cccagcccat
ccaagtcagc ctccccaggt 360cctccacctt caaaacaggc aaacgaaatc
atttcttgaa taattggtag gcttcaaggt 420cagatgtt 4282123DNAHomo sapiens
21ttgtctcctt ttgtttctgc tac 232222DNAHomo sapiens 22aacatctgac
cttgaagcct ac 2223330DNAHomo sapiens 23tcagggacag tgcataggtg
taaagaagtt gctggttggg ggttctaatg caggtttctc 60caaaagtgaa tgccctgtta
aaaaaaaatt cttaacaaat atacagagat ttttttttta 120aaaaagtgtg
acagttctag acacctagag agtaaartga agaagcctgt tttcaggttt
180cccgcctccc tgaatttccc agcatggtcc aggctttgaa atttatttat
ctgcttttgg 240caatggttga tgggaatttc ccacatttat tttttagcta
cagagaaagg acattatctt 300taaaatctct tcgttgttct ctctctttga
3302420DNAHomo sapiens 24tcagggacag tgcataggtg 202523DNAHomo
sapiens 25tcaaagagag agaacaacga aga 2326574DNAHomo sapiens
26tatttagaaa ccataaaatc cacctatttg aggtgtacaa ttgagtgatt ttctgtatag
60tcacagatct gtgcagtcat ccacaccctc taactccagg acattttcct cacccccgag
120gagaaacctc ccttacccat tagcagtcac tcctcatttc ctctcccccc
agcccctggc 180aatcactgtg gatttgcctg ttcttgacat ttcatataaa
yggtatcata aaatctatgg 240gcttttgtgt ctgtctgctt tcacttagca
tacggttctc aaggttcatc cagtattgta 300gcatctatca gtatgtcatt
cctttttatg gccaaataat attttattgt atggatagac 360attttgttta
ttcatttatc tgtttttggt tattatgagt aacactacta tgaacatttt
420gcacaaattt ttgtattgac atgttttcat ttctcctggg tatagtccta
tgagtggaat 480tgctgggtca tataataaat aactgtttaa cattttgggg
agctgccaaa cttttaaaac 540cttgggttct gtgatgtacc agttgtgtta ggca
5742727DNAHomo sapiens 27tatttagaaa ccataaaatc cacctat
272822DNAHomo sapiens 28tgcctaacac aactggtaca tc 2229571DNAHomo
sapiens 29tgccaggggt tttatggtta attttcctcc attatgaggg ttgactcagc
cttgggtatt 60agatgtcttt gagaatccag ggttcaaata ccacagctgg tagaatgttt
ctcaacttgg 120agccaatctc catctactga aggtacgctg gtttagacag
acaacaggga catcagcatt 180ttaaaaagcg gtggaaaaag tttgcttgtc
ttgattggag ccatgacatt ttattttgaa 240atttcaaata acatgaaggg
aggtttggag cggtttttgg tttatccaaa gggcagtgga 300ttgaaggctg
agaaacacca ggctgaatgg gagaggggtt ggggtccccc tgtgagatag
360tgaaacaatg gtagtgccat ccaatgatag gcacttttct gtcattcaga
agcagaaagg 420gggccagagg cccattggcc ttactgggma gtaagctgta
gagctgctgc cttttcgtga 480aagggttgac accaaccttc tcccccagga
agagtgacca gggacctgag gggcatggtc 540gagcagatga cagcctttgt
aaaacatctc c 5713020DNAHomo sapiens 30tgccaggggt tttatggtta
203123DNAHomo sapiens 31ggagatgttt tacaaaggct gtc 2332614DNAHomo
sapiens 32ttggtagaga tggggtctcc taggctggtc ttgaactcct ggrctcaagc
aatcttcctg 60cctcagcctt ccaaagtact gggattactg gcgtgggcca ccatgcctgg
cttgaaattt 120ttctatggct ttattctttc tccaagtaca gagtctaccc
aaccttctga gatctttggt 180tttcttttcc taggtaacta tagtacatac
ttatttatgt taaacaacag caatcacaca 240tttctttttc tatacagtca
tgctttatag gcaaataaag cctccgtctt aggctttctg 300gattttttca
aaagatgcaa ttcctggagt atgtttttac ttagagcaaa gcagcctagt
360ctcctatacc ttctgcatct gcagaaaagt tggttaaaca gactttgtaa
tgatgcccct 420tacaattctg aagggacttg tgaaatagtt tcacagagtt
tcagtgttag gtatatttga 480tcaatgctaa cttttggaaa actttggtgc
ctgtatgatt cagagggtag ggcagaatat 540taaattaatc acaacttctt
gtattttaac cattctgggt aaattgggat tccgtgacgc 600ccaggcaaaa ttat
6143320DNAHomo sapiens 33ttggtagaga tggggtctcc 203420DNAHomo
sapiens 34ataattttgc ctgggcgtca 2035633DNAHomo sapiens 35tatcttatat
cccctccaag cattcattaa ctgatggatt agtgagttgg ccttgagaag 60cataaaggct
cgtctccatg tgcttctaag cattgtgtct aagttctgtt tggtttcctg
120agtgaaactg tcttaatgtt accaacagaa gttaaatgcc taagagwttc
ttatacatgg 180gctgagtacc tctgtgactg ggcaagccac ctcacctcat
tttaccttgt ctgcaaaatg 240aggaactggg tcaactcatc gttcaaatct
cactgaaagc taattgatcg cttttgacag 300aagtagctcc cttgggccgt
atatttattt cctagcttgg aggaaggtgg ggacagacag 360aattgatgta
cacctttatt tttatctcta tggtaaacct gtgcatacta aagcattcct
420ctggtctttt gagatgagtg tatacattgt gtctggccct gtgcattttt
taccaagaag 480taagttttgt tgagtaaact tgggttgtat gaagaactgc
atgctcaccg tactcaagta 540gcttttgcta cctaaaggac agctgctcat
atgtacttga cttcctttaa agtgaaggat 600gatgacattt gaaaaacgga
ggttgaaaag gag 6333625DNAHomo sapiens 36tatcttatat cccctccaag cattc
253721DNAHomo sapiens 37ctccttttca acctccgttt t 21381081DNAHomo
sapiens 38ttgagcatgt gttatttaat gagttatacc tctgtcatat gtgtgtgttt
atatcacaaa 60ataacttatt tttataaaac catattttga gtcatcattt gtgacaatgt
cttcttttct 120ctggtataaa tgaggcatgt agaaagaaga ttgacatttg
ctagaagctt cccctttcct 180ctaactccac aataaaatgg atgctcataa
ttacatctgc tcctataagg tcaagatttc 240agggctggaa gtgaccttag
atcatttagg cccaacttgc cctcaggaaa ggaaactgag 300gcccagagat
gccttaagtg aattgcccaa tgtcacacgc tgagtcagtg gccagagcaa
360ggcttggatc cagttctctg ctccctttcc agagccttgt gatgtcttct
ctcctacagg 420aggtgaaaat aactgctgtg gctggttctg ttttgctgac
tgtaaattgg gtcatggtca 480gggacagtgc ataggtgtaa agaagttgct
ggttgggggt tctaatgcag gtttctccaa 540aagtgaatgc cctgttaaaa
aaaaattctt aacaaatata cagagatttt tttttwaaaa 600aagtgtgaca
gttctagaca cctagagagt aaagtgaaga agcctgtttt caggtttccc
660gcctccctga atttcccagc atggtccagg ctttgaaatt tatttatctg
cttttggcaa 720tggttgatgg gaatttccca catttatttt ttagctacag
agaaaggaca ttatctttaa 780aatctcttcg ttgttctctc tctttgagtg
aggagagaag atgtgaatcc tggcagtggt 840tcagagtgga cacagcccct
gtgtttgtgg cataggctct gtgggcccca tgccagggag 900cagtaccccc
gtgtaaagga gtgggggttt gtccatttgg atagagcaaa gatcctccac
960ctcaaatccc acaagaacag ttgccacaac ctgggcccta agcatctcat
tttcctatgt 1020agaaattaat gatctggagg agatggcaaa acattccttc
cagagcctgt gtggattttg 1080g 10813926DNAHomo sapiens 39ttgagcatgt
gttatttaat gagtta 264020DNAHomo sapiens 40ccaaaatcca cacaggctct
2041599DNAHomo sapiens 41tagtgctcag tatttccaac gttctgttta
tttaagatga aaattgctgt agttaataag 60cacttcccca tgtcattaaa atgcttaagg
atttttaatg accacataac agtccataat 120atgattaaac cccaatttac
tgaatcaatg ccatattgtt gggtctttag attgtctcct 180tttgtttctg
ctactgtgaa tgatcctgtg atgatcatct ttgtgtgtaa atctttgtcc
240cctcgccccc tcccctttta ttattttctt gggatagacc ccaggacaaa
aggtagaaaa 300gaacaaagtg ttaaamaatt tcttgataca tagccacaga
ttattttcct gaaagttctc 360aacatttata actacgagca gtatgtaaga
gagttatggt tggaatgatt ttaatgtctc 420tggggaattt aacaacaaaa
aaactttagg cttctttgga gagagacatg cccttaactc 480caccccgccc
tagaacagag acccagccca tccaagtcag cctccccagg tcctccacct
540tcaaaacagg caaacgaaat catttcttga ataattggta ggcttcaagg tcagatgtt
5994225DNAHomo sapiens 42tagtgctcag tatttccaac gttct 254323DNAHomo
sapiens 43aacatctgac cttgaagcct acc 2344599DNAHomo sapiens
44tagtgctcag tatttccaac gttctgttta tttaagatga aaattgctgt agttaataag
60cacttcccca tgtcattaaa atgcttaagg atttttaatg accacataac agtccataat
120atgattaaac cccaatttac tgaatcaatg ccatattgtt gggtctttag
attgtctcct 180tttgtttctg ctactgtgaa tgatcctgtg atgatcatct
ttgtgtgtaa atctttgtcc 240cctcgccccc tcccctttta ttattttctt
gggatagacc ccaggacaaa aggtagaaaa 300gaacaaagtg ttaaaaaatt
tcttgataca tagccacaga ttattttcct gaaagttcts 360aacatttata
actacgagca gtatgtaaga gagttatggt tggaatgatt ttaatgtctc
420tggggaattt aacaacaaaa aaactttagg cttctttgga gagagacatg
cccttaactc 480caccccgccc tagaacagag acccagccca tccaagtcag
cctccccagg tcctccacct 540tcaaaacagg caaacgaaat catttcttga
ataattggta ggcttcaagg tcagatgtt 59945641DNAHomo sapiens
45tgctatgtcc agtttacaca taaggatgtg caaatccagc aggttagctg agctgcccag
60gaatatccag gcaagaatga ccatattctg ataattactc aggcctctgc ctcatctccg
120ctgscccccc gccccctgac tctcttctga gtgccagatt cagcctccat
ttgaatgcca 180aatagacagg aaattagcat gcccagaatc cacgtcttta
gtgcactctc tccccagctc 240caaacctgtt actgcttgtg ttcaacatct
cagtaaagct caacaacatc gacccattac 300ttaggcctca aaccttgggt
ggcatcgtcg attgctcttt tctttcatac cccacattca 360acccatcagc
ccatcccaca ggcccaagtg tgtcctctct accttcaaag cgtgtgtggc
420atccaccgct tatcaccacc tctgccatta ccactggagt ccagtgccat
catctctcac 480ttggatgtgg ccagagtgtc tttgctggtc tccttcttgc
ttcctacctt tgtaacagcc 540tatcatctat ctctggtctc catagctcac
tcccatactt tgagagggcc tttgaaagcc 600ttagacagat catatcacag
acctctatac tgaaagtcgg g 6414625DNAHomo sapiens 46tgctatgtcc
agtttacaca taagg 254724DNAHomo sapiens 47cccgactttc agtatagagg tctg
2448284DNAHomo sapiens 48ccatctgtgg agcagagtca ctgaaaggaa
atactggaaa tactggaagc cacttggtgt 60tttatcaagg atgtgaggtt tcctggcaac
tttgtcgcca tatcatcatc atcatcacca 120tcatcatcat catcatcatc
atcatcatca tcatcatcat catcatctgc cctttaagtt 180ttctgcttgt
ttagaaaaga aatttataca gagcccccag tagcagctgt aagggggcag
240gttcttggag cagcccatcc tcaacattct tgctgctgat ggaa 2844920DNAHomo
sapiens 49ccatctgtgg agcagagtca 205020DNAHomo sapiens 50ttccatcagc
agcaagaatg 2051145DNAHomo sapiens 51tccacgcaga gaggatctaa
atctggctct ttgcaattgc cttcatacat gtgcatacac 60accacacaca cacacacaca
cacacacaca cacacacaca cagacacata catatgcaca 120caccccgact
caatggagga ccctc 1455221DNAHomo sapiens 52tccacgcaga gaggatctaa a
215320DNAHomo sapiens 53gagggtcctc cattgagtcg 20
* * * * *