U.S. patent application number 13/049612 was filed with the patent office on 2011-07-28 for methods of analysis of polymorphisms and uses thereof.
This patent application is currently assigned to SYNERGENZ BIOSCIENCE LIMITED. Invention is credited to Robert Peter Young.
Application Number | 20110182872 13/049612 |
Document ID | / |
Family ID | 37431471 |
Filed Date | 2011-07-28 |
United States Patent
Application |
20110182872 |
Kind Code |
A1 |
Young; Robert Peter |
July 28, 2011 |
METHODS OF ANALYSIS OF POLYMORPHISMS AND USES THEREOF
Abstract
The present invention provides methods for the assessment of
diseases that result from the combined or interactive effects of
two or more genetic variants, and in particular for diagnosing risk
of developing such diseases in subjects using an analysis of
genetic polymorphisms. Methods for the derivation of a net score
indicative of a subject's risk of developing a disease are
provided.
Inventors: |
Young; Robert Peter;
(Auckland, NZ) |
Assignee: |
SYNERGENZ BIOSCIENCE
LIMITED
HONG KONG
CN
|
Family ID: |
37431471 |
Appl. No.: |
13/049612 |
Filed: |
March 16, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11432770 |
May 10, 2006 |
7933722 |
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13049612 |
|
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Current U.S.
Class: |
424/94.1 ;
702/19 |
Current CPC
Class: |
C12Q 2600/172 20130101;
Y02A 90/24 20180101; Y02A 90/10 20180101; C12Q 1/6883 20130101;
C12Q 2600/156 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/94.1 ;
702/19 |
International
Class: |
A61K 38/43 20060101
A61K038/43; A61P 35/00 20060101 A61P035/00; G06F 19/00 20110101
G06F019/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2005 |
NZ |
540249 |
Aug 15, 2005 |
NZ |
541842 |
Claims
1. A method of assessing a subject's risk of developing a disease
which comprises: analysing a biological sample from said subject
for the presence or absence of a protective polymorphism and for
the presence or absence of a susceptibility polymorphism, wherein
said protective polymorphism and said susceptibility polymorphism
are associated with said disease; assigning a positive score for
each protective polymorphism and a negative score for each
susceptibility polymorphism or vice versa; calculating a net score
for said subject, said net score representing the balance between
the combined value of the protective polymorphism and the combined
value of the susceptibility polymorphism present in the subject
sample; wherein a net protective score is predictive of a reduced
risk of developing said disease and a net susceptibility score is
predictive of an increased risk of developing said disease.
2. A method according to claim 1 wherein the value assigned to each
protective polymorphism is the same.
3.-25. (canceled)
26. A method of determining a subject's risk of developing a
disease, said method comprising: obtaining the result of one or
more analyses of a sample from said subject to determine a presence
or absence of a protective polymorphism and a presence or absence
of a susceptibility polymorphism, and wherein said protective and
susceptibility polymorphisms are associated with said disease;
assigning a positive score for each protective polymorphism and a
negative score for each susceptibility polymorphism or vice versa;
and calculating a net score for said subject, said net score
representing a balance between a combined value of the protective
polymorphism and a combined value of the susceptibility
polymorphism present in the subject sample, wherein a net
protective score is predictive of a reduced risk of developing said
disease and a net susceptibility score is predictive of an
increased risk of developing said disease.
27. (canceled)
28. A method of treatment of a subject to decrease a risk of
developing a disease through alteration of the net score for said
subject as determined by a method as defined above, wherein said
method of treatment comprises: reversing, genotypically or
phenotypically, the presence and/or functional effect of one or
more susceptibility polymorphisms associated with said disease;
and/or replicating and/or mimicking, genotypically or
phenotypically, the presence and/or functional effect of one or
more protective polymorphisms associated with said disease 3) a)
reversing, genotypically or phenotypically, the presence,
functional effect, or presence and functional effect of one or more
susceptibility polymorphisms associated with said disease and b)
replicating, mimicking, or replicating and mimicking, genotypically
or phenotypically, the presence, functional effect, or presence and
functional effect of one or more protective polymorphisms
associated with said disease.
29. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to New Zealand Application
Nos. 540249, filed May 20, 2005 and 541842, filed Aug. 15, 2005,
both of which are incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention is concerned with methods for the
assessment of diseases that result from the combined or interactive
effects of two or more genetic variants, and in particular for
diagnosing risk of developing such diseases in subjects using an
analysis of genetic polymorphisms.
BACKGROUND OF THE INVENTION
[0003] Diseases that result from the combined or interactive
effects of two or more genetic variants, with or without
environmental factors, are called complex diseases and include
cancer, coronary artery disease, diabetes, stroke, and chronic
obstructive pulmonary disease (COPD). Although combining
non-genetic risk factors to determine a risk level of outcome has
been in applied to coronary artery disease, (by combining
individual factors such as blood pressure, gender, fasting
cholesterol, and smoking status), there are no such methods in
combining the effects of multiple genetic factors with non-genetic
factors. There is a growing realization that the complex diseases,
for which examples are given above, may result from the combined
effects of common genetic variants or polymorphisms rather than
mutations which are rare (believed to be present in less than 1% of
the general population). Moreover, these relatively common
polymorphisms can confer either susceptibility and/or protective
effects on the development of these diseases. In addition, the
likelihood that these polymorphisms are actually expressed (termed
penetrance) as a disease or clinical manifestation requires a
quantum of environmental exposure before such a genetic tendency
can be clinically detected.
SUMMARY OF THE INVENTION
[0004] Recent studies have identified a number of genetic variants
or polymorphisms that confer susceptibility to protection from
COPD, occupational COPD (OCOPD), and lung cancer. The biological
basis of just how these polymorphisms interact or combine to
determine risk remains unclear.
[0005] Surprisingly, it has now been found that an assessment
approach which determines a subject's net score following the
balancing of the number of polymorphisms associated with protection
from a disease against the number of polymorphisms associated with
susceptibility to that disease present in the subject is indicative
of that subject's risk quotient. Furthermore, it has presently been
determined that this approach is widely applicable, on a
disease-by-disease basis.
[0006] It is broadly to this approach to risk assessment that the
present invention is directed.
[0007] Accordingly, in a first aspect, the present invention
provides a method of assessing a subject's risk of developing a
disease which includes: [0008] analysing a biological sample from
said subject for the presence or absence of protective
polymorphisms and for the presence or absence of susceptibility
polymorphisms, wherein said protective and susceptibility
polymorphisms are associated with said disease; [0009] assigning a
positive score for each protective polymorphism and a negative
score for each susceptibility polymorphism or vice versa; [0010]
calculating a net score for said subject, said net score
representing the balance between the combined value of the
protective polymorphisms and the combined value of the
susceptibility polymorphisms present in the subject sample; [0011]
wherein a net protective score is predictive of a reduced risk of
developing said disease and a net susceptibility score is
predictive of an increased risk of developing said disease.
[0012] The value assigned to each protective polymorphism can be
the same or can be different. The value assigned to each
susceptibility polymorphism can be the same or can be different,
with either each protective polymorphism having a negative value
and each susceptibility polymorphism having a positive value, or
vice versa. When the disease is a lung disease, the protective
polymorphisms analysed can be selected from one or more of the
group consisting of: +760GG or +760CG within the gene encoding
superoxide dismutase 3 (SOD3); -1296TT within the promoter of the
gene encoding tissue inhibitor of metalloproteinase 3 (TIMP3); CC
(homozygous P allele) within codon 10 of the gene encoding
transforming growth factor beta (TGF.beta.); 2G2G within the
promoter of the gene encoding metalloproteinase 1 (MMP1); or one or
more polymorphisms in linkage disequilibrium with one or more of
these polymorphisms.
[0013] Linkage disequilibrium is a phenomenon in genetics whereby
two or more mutations or polymorphisms are in such close genetic
proximity that they are co-inherited. This means that in
genotyping, detection of one polymorphism as present implies the
presence of the other. (Reich, D. E. et al. Linkage disequilibrium
in the human genome. Nature 411:199-204 (2001), herein incorporated
by reference in its entirety).
[0014] Preferably, all polymorphisms of the group are analysed.
[0015] Preferably, the susceptibility polymorphisms analysed are
selected from one or more of the group consisting of: -82AA within
the promoter of the gene encoding human macrophage elastase
(MMP12); -1562CT or -1562TT within the promoter of the gene
encoding metalloproteinase 9 (MMP9); 1237AG or 1237AA (Tt or tt
allele genotypes) within the 3' region of the gene encoding
a1-antitrypsin (a1AT); or one or more polymorphisms in linkage
disequilibrium with one or more of these polymorphisms.
[0016] Preferably, all polymorphisms of the group are analysed.
[0017] In one embodiment each protective polymorphism is assigned a
value of -1 and each susceptibility polymorphism is assigned a
value of +1.
[0018] In another embodiment each protective polymorphism is
assigned a value of +1 and each susceptibility polymorphism is
assigned a value of -1.
[0019] When the disease is COPD, the protective polymorphisms
analysed can be selected from one or more of the group consisting
of: -765 CC or CG in the promoter of the gene encoding
cyclooxygenase 2 (COX2); Arg 130 Gln AA in the gene encoding
Interleukin-13 (IL-13); Asp 298 Glu TT in the gene encoding nitric
oxide synthase 3 (NOS3); Lys 420 Thr AA or AC in the gene encoding
vitamin binding protein (VDBP); Glu 416 Asp TT or TG in the gene
encoding VDBP; Ile 105 Val AA in the gene encoding glutathione
S-transferase (GSTP1); MS in the gene encoding a 1-antitrypsin (a 1
AT); the +489 GG geneotype in the gene encoding Tissue Necrosis
factor a (TNFa); the -308 GG geneotype in the gene encoding TNFa;
the C89Y AA or AG geneotype in the gene encodoing SMAD3; the 161 GG
genotype in the gene encodoing Mannose binding lectin 2 (MBL2); the
-1903 AA genotype in the gene encoding Chymase 1 (CMA1); the Arg
197 Gln AA genotype in the gene encoding N-Acetyl transferase 2
(NAT2); the His 139 Arg GG genotype in the gene encoding Microsomal
epoxide hydrolase (MEH); the -366 AA or AG genotype in the gene
encoding 5 Lipo-oxygenase (ALOX5); the HOM T2437C TT genotype in
the gene encoding Heat Shock Protein 70 (HSP 70); the exon 1 + 49
CT or TT genotype in the gene encoding Elafin; the Gln 27 Glu GG
genotype in the gene encoding 132 Adrenergic receptor (ADBR); the
-1607 1G1G or 1G2G genotype in the promoter of the gene encoding
Matrix Metalloproteinase 1 (MMP1); or one or more polymorphisms in
linkage disequilibrium with one or more of these polymorphisms.
Preferably, all polymorphisms of the group are analysed.
[0020] Preferably, the susceptibility polymorphisms analysed are
selected from one or more of the group consisting of: Arg 16 Gly GG
in the gene encoding .beta.2-adrenoreceptor (ADRB2); 105 AA in the
gene encoding Interleukin-18 (IL-18); -133 CC in the promoter of
the gene encoding IL-18; -675 5G5G in the promoter of the gene
encoding plasminogen activator inhibitor 1 (PAI-1); -1055 TT in the
promoter of the gene encoding IL-13; 874 TT in the gene encoding
interferon gamma (IFN?); the +489 AA or AG genotype in the gene
encoding TNFa; the -308 AA or AG genotype in the gene encoding
TNFa; the C89Y GG genotype in the gene encoding SMAD3; the E469K GG
genotype in the gene encoding Intracellular Adhesion molecule 1
(ICAM1); the Gly 881 Arg GC or CC genotype in the gene encoding
Caspase (NOD2); the -511 GG genotype in the gene encoding IL1B; the
Tyr 113 His TT genotype in the gene encoding MEH; the -366 GG
genotype in the gene encoding ALOX5; the HOM T2437C CC or CT
genotype in the gene encoding HSP 70; the +13924 AA genotype in the
gene encoding Chloride Channel Calcium-activated 1 (CLCA1); the
-159 CC genotype in the gene encoding Monocyte differentiation
antigen CD-14 (CD-14); or one or more polymorphisms in linkage
disequilibrium with one or more of these polymorphisms.
[0021] Preferably, all polymorphisms of the group are analysed.
[0022] In one embodiment each protective polymorphism is assigned a
value of -1 and each susceptibility polymorphism is assigned a
value of +1.
[0023] In one embodiment each protective polymorphism is assigned a
value of +1 and each susceptibility polymorphism is assigned a
value of -1.
[0024] When the disease is OCOPD, the protective polymorphisms
analysed can be selected from one or more of the group consisting
of: -765 CC or CG in the promoter of the gene encoding COX2; -251
AA In the promoter of the gene encoding interleukin-8 (IL-8); Lys
420 Thr AA in the gene encoding VDBP; Glu 416 Asp TT or TG in the
gene encoding VDBP; exon 3 T/C RR in the gene encoding microsomal
epoxide hydrolase (MEH); Arg 312 Gln AG or GG in the gene encoding
SOD3; MS or SS in the gene encoding a1AT; Asp 299 Gly AG or GG in
the gene encoding toll-like receptor 4 (TLR4); Gln 27 Glu CC in the
gene encoding ADRB2; -518 AA in the gene encoding IL-11; Asp 298
Glu TT in the gene encoding NOS3; or one or more polymorphisms in
linkage disequilibrium with one or more of these polymorphisms.
[0025] Preferably, all polymorphisms of the group are analysed.
[0026] Preferably, the susceptibility polymorphisms analysed are
selected from one or more of the group consisting of: -765 GG in
the promoter of the gene encoding COX2; 105 AA in the gene encoding
IL-18; -133 CC in the promoter of the gene encoding IL-18; -675
5G5G in the promoter of the gene encoding PAI-1; Lys 420 Thr CC in
the gene encoding VDBP; Glu 416 Asp GG in the gene encoding VDBP;
Ile 105 Val GG in the gene encoding GSTP1; Arg 312 Gln AA in the
gene encoding SOD3; -1055 TT in the promoter of the gene encoding
IL-13; 3' 1237 Tt or tt in the gene encoding a 1AT; -1607 2G2G in
the promoter of the gene encoding MMP1; or one or more
polymorphisms in linkage disequilibrium with one or more of these
polymorphisms.
[0027] Preferably, all polymorphisms of the group are analysed.
[0028] In one embodiment each protective polymorphism is assigned a
value of -1 and each susceptibility polymorphism is assigned a
value of +1.
[0029] In one embodiment each protective polymorphism is assigned a
value of +1 and each susceptibility polymorphism is assigned a
value of -1.
[0030] When the disease is lung cancer, the protective
polymorphisms analysed can be selected from one or more of the
group consisting of: the Asp 298 Glu TT genotype in the gene
encoding NOS3; the Arg 312 Gln CG or GG genotype in the gene
encoding SOD3; the Asn 357 Ser AG or GG genotype in the gene
encoding MMP12; the 105 AC or CC genotype in the gene encoding
IL-18; the -133 CG or GG genotype in the gene encoding IL-18; the
-765 CC or CG genotype in the promoter of the gene encoding COX2;
the -221 TT genotype in the gene encoding Mucin 5AC (MUC5AC); the
intron 1 C/T TT genotype in the gene encoding Arginase 1 (Arg1);
the Leu252Val GG genotype in the gene encoding Insulin-like growth
factor II receptor (IGF2R); the -1082 GG genotype in the gene
encoding Interleukin 10 (IL-10); the -251 AA genotype in the gene
encoding Interleukin 8 (IL-8); the Arg 399 Gln AA genotype in the
X-ray repair complementing defective in Chinese hamster 1 (XRCC1)
gene; the A870G GG genotype in the gene encoding cyclin D (CCND1);
the -751 GG genotype in the promoter of the xeroderma pigmentosum
complementation group D (XPD) gene; the Ile 462 Val AG or GG
genotype in the gene encoding cytochrome P450 1A1 (CYP1A1); the Ser
326 Cys GG genotype in the gene encoding 8-Oxoguanine DNA glycolase
(OGG1); the Phe 257 Ser CC genotype in the gene encoding REV1; or
one or more polymorphisms in linkage disequilibrium with any one or
more of these polymorphisms.
[0031] Preferably, all polymorphisms of the group are analysed.
[0032] Preferably, the susceptibility polymorphisms analysed are
selected from one or more of the group consisting of: the -786 TT
genotype in the promoter of the gene encoding NOS3; the Ala 15 Thr
GG genotype in the gene encoding anti-chymotrypsin (ACT); the 105
AA genotype in the gene encoding IL-18; the -133 CC genotype in the
promoter of the gene encoding IL-18; the 874 AA genotype in the
gene encoding IFN?; the -765 GG genotype in the promoter of the
gene encoding COX2; the -447 CC or GC genotype in the gene encoding
Connective tissue growth factor (CTGF); and the +161 AA or AG
genotype in the gene encoding MBL2; -511 GG genotype in the gene
encoding IL-1B; the A-670G AA genotype in the gene encoding FAS
(Apo-1/CD95); the Arg 197 Gln GG genotype in the gene encoding
N-acetyltransferase 2 (NAT2); the Ile462 Val AA genotype in the
gene encoding CYP1A1; the 1019 G/C Pst I CC or CG genotype in the
gene encoding cytochrome P450 2E1 (CYP2E1); the C/T Rsa I TT or TC
genotype in the gene encoding CYP2E1; the GSTM null genotype in the
gene encoding GSTM; the -1607 2G/2G genotype in the promoter of the
gene encoding MMP1; the Gln 185 Glu CC genotype in the gene
encoding Nibrin (NBS1); the Asp 148 Glu GG genotype in the gene
encoding Apex nuclease (APE1); or one or more polymorphisms in
linkage disequilibrium with any one or more of these
polymorphisms.
[0033] Preferably, all polymorphisms of the group are analysed.
[0034] In one embodiment each protective polymorphism is assigned a
value of -1 and each susceptibility polymorphism is assigned a
value of +1.
[0035] In one embodiment each protective polymorphism is assigned a
value of +1 and each susceptibility polymorphism is assigned a
value of -1.
[0036] In various embodiments the subject is or has been a
smoker.
[0037] Preferably, the methods of the invention are performed in
conjunction with an analysis of one or more risk factors, including
one or more epidemiological risk factors, associated with the risk
of developing a lung disease including COPD, emphysema, OCOPD, and
lung cancer. Such epidemiological risk factors include but are not
limited to smoking or exposure to tobacco smoke, age, sex, and
familial history.
[0038] In another aspect, the invention provides a method of
determining a subject's risk of developing a disease, said method
comprising [0039] obtaining the result of one or more analyses of a
sample from said subject to determine the presence or absence of
protective polymorphisms and the presence or absence of
susceptibility polymorphisms, and wherein said protective and
susceptibility polymorphisms are associated with said disease;
[0040] assigning a positive score for each protective polymorphism
and a negative score for each susceptibility polymorphism or vice
versa; [0041] calculating a net score for said subject, said net
score representing the balance between the combined value of the
protective polymorphisms and the combined value of the
susceptibility polymorphisms present in the subject sample; [0042]
wherein a net protective score is predictive of a reduced risk of
developing said disease and a net susceptibility score is
predictive of an increased risk of developing said disease.
[0043] In a further aspect the present invention provides a method
for assessing the risk of a subject developing a disease which
includes [0044] determining a net score for said subject in
accordance with the methods of the invention described above, in
combination with a score based on the presence or absence of one or
more epidemiological risk factors, [0045] wherein a net protective
score is predictive of a reduced risk of developing said disease
and a net susceptibility score is predictive of an increased
predisposition and/or susceptibility to said disease.
[0046] In another aspect, the present invention provides a kit for
assessing a subject's risk of developing a disease, said kit
comprising a means of analysing a sample from said subject for the
presence or absence of one or more protective polymorphisms and one
or more susceptibility polymorphisms as described herein.
[0047] In yet a further aspect, the present invention provides a
method of prophylactic or therapeutic intervention in relation to a
subject having a net susceptibility score for a disease as
determined by a method as defined above which includes the steps of
communicating to said subject said net susceptibility score, and
advising on changes to the subject's lifestyle that could reduce
the risk of developing said disease.
[0048] In still a further aspect, the present invention provides a
method of treatment of a subject to decrease to the risk of
developing a disease through alteration of the net score for said
subject as determined by a method as defined above, wherein said
method of treatment includes reversing, genotypically or
phenotypically, the presence and/or functional effect of one or
more susceptibility polymorphisms associated with said disease;
and/or replicating and/or mimicking, genotypically or
phenotypically, the presence and/or functional effect of one or
more protective polymorphisms associated with said disease.
BRIEF DESCRIPTION OF FIGURES
[0049] FIG. 1: depicts a graph showing combined frequencies of the
presence or absence of selected protective genotypes in the COPD
subjects and in resistant smokers.
[0050] FIG. 2: depicts a graph showing net scores for protective
and susceptibility polymorphisms in COPD subjects.
[0051] FIG. 3: depicts a graph showing net scores for protective
and susceptibility polymorphisms in OCOPD subjects.
[0052] FIG. 4: depicts a graph showing net scores for protective
and susceptibility polymorphisms in subjects with lung cancer.
[0053] FIG. 5: depicts a graph showing net scores for protective
and susceptibility polymorphisms in subjects with lung cancer.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0054] There is a need for a method for assessing a subject's risk
of developing a disease using genetic (and optionally non-genetic)
risk factors. In some embodiments, it is an object of the present
invention to go some way towards meeting this need and/or to
provide the public with a useful choice.
[0055] The present invention is directed to methods for the
assessment of the genetic risk quotient of a particular subject
with respect to a particular disease. The methods rely upon the
recognition that for many (if not all) diseases there exist genetic
polymorphisms which fall into two categories--namely those
indicative of a reduced risk of developing a particular disease
(which can be termed "protective polymorphisms" or "protective
SNPs") and those indicative of an increased risk of developing a
particular disease (which can be termed "susceptibility
polymorphisms" or "susceptibility SNPs").
[0056] As used herein, the phrase "risk of developing [a] disease"
means the likelihood that a subject to whom the risk applies will
develop the disease, and includes predisposition to, and potential
onset of the disease. Accordingly, the phrase "increased risk of
developing [a] disease" means that a subject having such an
increased risk possesses an hereditary inclination or tendency to
develop the disease. This does not mean that such a person will
actually develop the disease at any time, merely that he or she has
a greater likelihood of developing the disease compared to the
general population of individuals that either does not possess a
polymorphism associated with increased disease risk, or does
possess a polymorphism associated with decreased disease risk.
Subjects with an increased risk of developing the disease include
those with a predisposition to the disease, for example in the case
of COPD, a tendency or prediliction regardless of their lung
function at the time of assessment, for example, a subject who is
genetically inclined to COPD but who has normal lung function,
those at potential risk, for example in the case of COPD, subjects
with a tendency to mildly reduced lung function who are likely to
go on to suffer COPD if they keep smoking, and subjects with
potential onset of the disease, for example in the case of COPD,
subjects who have a tendency to poor lung function on spirometry
etc., consistent with COPD at the time of assessment.
[0057] Similarly, the phrase "decreased risk of developing [a]
disease" means that a subject having such a decreased risk
possesses an hereditary disinclination or reduced tendency to
develop the disease. This does not mean that such a person will not
develop the disease at any time, merely that he or she has a
decreased likelihood of developing the disease compared to the
general population of individuals that either does possess one or
more polymorphisms associated with increased disease risk, or does
not possess a polymorphism associated with decreased disease
risk.
[0058] It will be understood that in the context of the present
invention the term "polymorphism" means the occurrence together in
the same population at a rate greater than that attributable to
random mutation (usually greater than 1%) of two or more alternate
forms (such as alleles or genetic markers) of a chromosomal locus
that differ in nucleotide sequence or have variable numbers of
repeated nucleotide units. See
www.ornl.gov/sci/techresources/Human_Genome/publicat/97pr/09gloss.html#p.
Accordingly, the term "polymorphisms" is used herein contemplates
genetic variations, including single nucleotide substitutions,
insertions and deletions of nucleotides, repetitive sequences (such
as microsatellites), and the total or partial absence of genes (eg.
null mutations). As used herein, the term "polymorphisms" also
includes genotypes and haplotypes. A genotype is the genetic
composition at a specific locus or set of loci. A haplotype is a
set of closely linked genetic markers present on one chromosome
which are not easily separable by recombination, tend to be
inherited together, and can be in linkage disequilibrium. A
haplotype can be identified by patterns of polymorphisms such as
SNPs. Similarly, the term "single nucleotide polymorphism" or "SNP"
in the context of the present invention includes single base
nucleotide substitutions and short deletion and insertion
polymorphisms. It will further be understood that the term
"disease" is used herein in its widest possible sense, and includes
conditions which can be considered disorders and/or illnesses which
have a genetic basis or to which the genetic makeup of the subject
contributes.
[0059] Using case-control studies, the frequencies of several
genetic variants (polymorphisms) of candidate genes have been
compared in disease sufferers, for example, in chronic obstructive
pulmonary disease (COPD) sufferers, in occupational chronic
obstructive pulmonary disease (OCOPD) sufferers, and in lung cancer
sufferers, and in control subjects not suffering from the relevant
disease, for example smokers without lung cancer and with normal
lung function. The majority of these candidate genes have confirmed
(or likely) functional effects on gene expression or protein
function.
[0060] In various specific embodiments, the frequencies of
polymorphisms between blood donor controls, resistant subjects and
those with COPD, the frequencies of polymorphisms between blood
donor controls, resistant subjects and those with OCOPD, and the
frequencies of polymorphisms between blood donor controls,
resistant subjects and those with lung cancer, have been compared.
This has resulted in both protective and susceptibility
polymorphisms being identified for each disease.
[0061] The surprising finding relevant to this invention is that a
combined analysis of protective and susceptibility polymorphisms
discriminatory for a given disease yields a result that is
indicative of that subject's risk quotient for that disease. This
approach is widely applicable, on a disease-by-disease basis.
[0062] The present invention identifies methods of assessing the
risk of a subject developing a disease which includes determining
in said subject the presence or absence of protective and
susceptibility polymorphisms associated with said disease. A net
score for said subject is derived, said score representing the
balance between the combined value of the protective polymorphisms
present in said subject and the combined value of the
susceptibility polymorphisms present in said subject. A net
protective score is predictive of a reduced risk of developing said
disease, and a net susceptibility score is predictive of an
increased risk of developing said disease.
[0063] Within each category (protective polymorphisms,
susceptibility polymorphisms, respectively) the polymorphisms can
each be assigned the same value. For example, in the analyses
presented in the Examples herein, each protective polymorphism
associated with a given disease is assigned a value of +1, and each
susceptibility polymorphism is assigned a value of -1.
Alternatively, polymorphisms discriminatory for a disease within
the same category can each be assigned a different value to reflect
their discriminatory value for said disease. For example, a
polymorphism highly discriminatory of risk of developing a disease
can be assigned a high weighting, for example a polymorphism with a
high Odd's ratio can be considered highly discriminatory of
disease, and can be assigned a high weighting.
[0064] Accordingly, in a first aspect, the present invention
provides a method of assessing a subject's risk of developing a
disease which includes:
[0065] analysing a biological sample from said subject for the
presence or absence of protective polymorphisms and for the
presence or absence of susceptibility polymorphisms, wherein said
protective and susceptibility polymorphisms are associated with
said disease;
[0066] assigning a positive score for each protective polymorphism
and a negative score for each susceptibility polymorphism or vice
versa;
[0067] calculating a net score for said subject, said net score
representing the balance between the combined value of the
protective polymorphisms and the combined value of the
susceptibility polymorphisms present in the subject sample;
[0068] wherein a net protective score is predictive of a reduced
risk of developing said disease and a net susceptibility score is
predictive of an increased risk of developing said disease.
[0069] The subject sample can have already been analysed for the
presence or absence of one or more protective or susceptibility
polymorphisms, and the method includes the steps of [0070]
assigning a positive score for each protective polymorphism and a
negative score for each susceptibility polymorphism or vice versa;
[0071] calculating a net score for said subject, said net score
representing the balance between the combined value of the
protective polymorphisms and the combined value of the
susceptibility polymorphisms present in the subject sample; [0072]
wherein a net protective score is predictive of a reduced risk of
developing said disease and a net susceptibility score is
predictive of an increased risk of developing said disease.
[0073] In one embodiment described herein in Example 1, 17
susceptibility genetic polymorphisms and 19 protective genetic
polymorphisms identified as discriminatory for COPD were analysed
using methods of the invention. These analyses can be used to
determine the risk quotient of any subject for COPD, and in
particular to identify subjects at greater risk of developing lung
cancer.
[0074] In another embodiment described herein in Example 2, 11
susceptibility genetic polymorphisms and 11 protective genetic
polymorphisms identified as discriminatory for OCOPD are analysed
using methods of the invention. These analyses can be used to
determine the risk quotient of any subject for OCOPD, and in
particular to identify subjects at greater risk of developing
OCOPD.
[0075] In a further embodiment described herein in Example 3, 19
susceptibility genetic polymorphisms and 17 protective genetic
polymorphisms identified as discriminatory for lung cancer are
analysed using methods of the invention. These analyses can be used
to determine the risk quotient of any subject for lung cancer, and
in particular to identify subjects at greater risk of developing
lung cancer.
[0076] Susceptibility and protective polymorphisms can readily be
identified for other diseases using approaches similar to those
described in the Examples, as well as in PCT International
Application No. PCT/NZ02/00106 (published as WO 02/099134 and
herein incorporated by reference in its entirety) via which four
susceptibility and three protective polymorphisms discriminatory
for lung disease were identified.
[0077] The one or more polymorphisms can be detected directly or by
detection of one or more polymorphisms which are in linkage
disequilibrium with said one or more polymorphisms. As discussed
above, linkage disequilibrium is a phenomenon in genetics whereby
two or more mutations or polymorphisms are in such close genetic
proximity that they are co-inherited. This means that in
genotyping, detection of one polymorphism as present implies the
presence of the other. (Reich D E et al; Linkage disequilibrium in
the human genome, Nature 2001, 411:199-204.)
[0078] Examples of polymorphisms reported to be in linkage
disequilibrium are presented herein, and include the Interleukin-18
-133 C/G and 105 A/C polymorphisms, and the Vitamin D binding
protein Glu 416 Asp and Lys 420 Thr polymorphisms, as shown
below.
TABLE-US-00001 LD rs Alleles between Phenotype Gene SNPs numbers in
LD alleles in COPD Interleukin- IL18 -133 rs360721 C allele Strong
LD CC 18 C/G susceptible IL18 105 rs549908 A allele AA A/C
susceptible Vitamin D VDBP rs4588 A allele Strong LD AA/AC binding
Lys 420 protective protein Thr VDBP rs7041 T allele TT/TG Glu 416
protective Asp
[0079] It will be apparent that polymorphsisms in linkage
disequilibrium with one or more other polymorphism associated with
increased or decreased risk of developing COPD, emphysema, or both
COPD and emphysema will also provide utility as biomarkers for risk
of developing COPD, emphysema, or both COPD and emphysema. The data
presented herein shows that the frequency for SNPs in linkage
disequilibrium is very similar. Accordingly, these genetically
linked SNPs can be utilized in combined polymorphism analyses to
derive a level of risk comparable to that calculated from the
original SNP.
[0080] It will therefore be apparent that one or more polymorphisms
in linkage disequilibrium with the polymorphisms specified herein
can be identified, for example, using public data bases. Examples
of such polymorphisms reported to be in linkage disequilibrium with
the polymorphisms specified herein are presented herein in Table
21.
[0081] The methods of the invention are primarily reliant on
genetic information such as that derived from methods suitable to
the detection and identification of single nucleotide polymorphisms
(SNPs) associated with the specific disease for which a risk
assessment is desired. In some embodiments, a SNP is a single base
change or point mutation resulting in genetic variation between
individuals. SNPs occur in the human genome approximately once
every 100 to 300 bases, and can occur in coding or non-coding
regions. Due to the redundancy of the genetic code, a SNP in the
coding region may or may not change the amino acid sequence of a
protein product. A SNP in a non-coding region can, for example,
alter gene expression by, for example, modifying control regions
such as promoters, transcription factor binding sites, processing
sites, ribosomal binding sites, and affect gene transcription,
processing, and translation.
[0082] SNPs can facilitate large-scale association genetics
studies, and there has recently been great interest in SNP
discovery and detection. SNPs show great promise as markers for a
number of phenotypic traits (including latent traits), such as for
example, disease propensity and severity, wellness propensity, and
drug responsiveness including, for example, susceptibility to
adverse drug reactions. Knowledge of the association of a
particular SNP with a phenotypic trait, coupled with the knowledge
of whether an individual has said particular SNP, can enable the
targeting of diagnostic, preventative and therapeutic applications
to allow better disease management, to enhance understanding of
disease states and to ultimately facilitate the discovery of more
effective treatments, such as personalised treatment regimens.
[0083] Indeed, a number of databases have been constructed of known
SNPs, and for some such SNPs, the biological effect associated with
a SNP. For example, the NCBI SNP database "dbSNP" is incorporated
into NCBI's Entrez system and can be queried using the same
approach as the other Entrez databases such as PubMed and GenBank.
This database has records for over 1.5 million SNPs mapped onto the
human genome sequence. Each dbSNP entry includes the sequence
context of the polymorphism (i.e., the surrounding sequence), the
occurrence frequency of the polymorphism (by population or
individual), and the experimental method(s), protocols, and
conditions used to assay the variation, and can include information
associating a SNP with a particular phenotypic trait.
[0084] At least in part because of the potential impact on health
and wellness, there has been and continues to be a great deal of
effort to develop methods that reliably and rapidly identify SNPs.
This is no trivial task, at least in part because of the complexity
of human genomic DNA, with a haploid genome of 3.times.10.sup.9
base pairs, and the associated sensitivity and discriminatory
requirements.
[0085] Genotyping approaches to detect SNPs well-known in the art
include DNA sequencing, methods that require allele specific
hybridization of primers or probes, allele specific incorporation
of nucleotides to primers bound close to or adjacent to the
polymorphisms (often referred to as "single base extension", or
"minisequencing"), allele-specific ligation (joining) of
oligonucleotides (ligation chain reaction or ligation padlock
probes), allele-specific cleavage of oligonucleotides or PCR
products by restriction enzymes (restriction fragment length
polymorphisms analysis or RFLP) or chemical or other agents,
resolution of allele-dependent differences in electrophoretic or
chromatographic mobilities, by structure specific enzymes including
invasive structure specific enzymes, or mass spectrometry. Analysis
of amino acid variation is also possible where the SNP lies in a
coding region and results in an amino acid change.
[0086] DNA sequencing allows the direct determination and
identification of SNPs. The benefits in specificity and accuracy
are generally outweighed for screening purposes by the difficulties
inherent in whole genome, or even targeted subgenome,
sequencing.
[0087] Mini-sequencing involves allowing a primer to hybridize to
the DNA sequence adjacent to the SNP site on the test sample under
investigation. The primer is extended by one nucleotide using all
four differentially tagged fluorescent dideoxynucleotides (A,C,G,
or T), and a DNA polymerase. Only one of the four nucleotides
(homozygous case) or two of the four nucleotides (heterozygous
case) is incorporated. The base that is incorporated is
complementary to the nucleotide at the SNP position.
[0088] A number of methods currently used for SNP detection involve
site-specific and/or allele-specific hybridisation (Matsuzaki, H.
et al. Genome Res. 14:414-425 (2004); Matsuzaki, H. et al. Nat.
Methods 1:109-111 (2004); Sethi, A. A. et al. Clin. Chem.
50(2):443-446 (2004), each of the foregoing which is herein
incorporated by reference in its entirety). These methods are
largely reliant on the discriminatory binding of oligonucleotides
to target sequences containing the SNP of interest. The techniques
of Affymetrix (Santa Clara, Calif.) and Nanogen Inc. (San Diego,
Calif.) are particularly well-known, and utilize the fact that DNA
duplexes containing single base mismatches are much less stable
than duplexes that are perfectly base-paired. The presence of a
matched duplex is detected by fluorescence.
[0089] The majority of methods to detect or identify SNPs by
site-specific hybridisation require target amplification by methods
such as PCR to increase sensitivity and specificity (see, for
example U.S. Pat. No. 5,679,524, PCT publication WO 98/59066, PCT
publication WO 95/12607, each of the foregoing which is herein
incorporated by reference in its entirety). US Application
20050059030 (incorporated herein in its entirety) describes a
method for detecting a single nucleotide polymorphism in total
human DNA without prior amplification or complexity reduction to
selectively enrich for the target sequence, and without the aid of
any enzymatic reaction. The method utilises a single-step
hybridization involving two hybridization events: hybridization of
a first portion of the target sequence to a capture probe, and
hybridization of a second portion of said target sequence to a
detection probe. Both hybridization events happen in the same
reaction, and the order in which hybridisation occurs is not
critical.
[0090] US Application 20050042608 (herein incorporated by reference
in its entirety) describes a modification of the method of
electrochemical detection of nucleic acid hybridization of Thorp et
al. (U.S. Pat. No. 5,871,918, herein incorporated by reference in
its entirety). Briefly, capture probes are designed, each of which
has a different SNP base and a sequence of probe bases on each side
of the SNP base. The probe bases are complementary to the
corresponding target sequence adjacent to the SNP site. Each
capture probe is immobilized on a different electrode having a
non-conductive outer layer on a conductive working surface of a
substrate. The extent of hybridization between each capture probe
and the nucleic acid target is detected by detecting the
oxidation-reduction reaction at each electrode, utilizing a
transition metal complex. These differences in the oxidation rates
at the different electrodes are used to determine whether the
selected nucleic acid target has a single nucleotide polymorphism
at the selected SNP site.
[0091] The technique of Lynx Therapeutics (Hayward, Calif.) using
MEGATYPE.TM. technology can genotype very large numbers of SNPs
simultaneously from small or large pools of genomic material. This
technology uses fluorescently labeled probes and compares the
collected genomes of two populations, enabling detection and
recovery of DNA fragments spanning SNPs that distinguish the two
populations, without requiring prior SNP mapping or knowledge.
[0092] A number of other methods for detecting and identifying SNPs
exist. These include the use of mass spectrometry, for example, to
measure probes that hybridize to the SNP (Ross, P. L. et al.
Discrimination of single-nucleotide polymorphisms in human DNA
using peptide nucleic acid probes detected by MALDI-TOF mass
spectrometry. Anal. Chem. 69, 4197-4202 (1997), herein incorporated
by reference in its entirety). This technique varies in how rapidly
it can be performed, from a few samples per day to a high
throughput of 40,000 SNPs per day, using mass code tags. A
preferred example is the use of mass spectrometric determination of
a nucleic acid sequence which includes the polymorphisms of the
invention, for example, which includes the promoter of the COX2
gene or a complementary sequence. Such mass spectrometric methods
are known to those skilled in the art, and the genotyping methods
of the invention are amenable to adaptation for the mass
spectrometric detection of the polymorphisms of the invention, for
example, the COX2 promoter polymorphisms of the invention.
[0093] SNPs can also be determined by ligation-bit analysis. This
analysis requires two primers that hybridize to a target with a one
nucleotide gap between the primers. Each of the four nucleotides is
added to a separate reaction mixture containing DNA polymerase,
ligase, target DNA and the primers. The polymerase adds a
nucleotide to the 3' end of the first primer that is complementary
to the SNP, and the ligase then ligates the two adjacent primers
together. Upon heating of the sample, if ligation has occurred, the
now larger primer will remain hybridized and a signal, for example,
fluorescence, can be detected. A further discussion of these
methods can be found in U.S. Pat. Nos. 5,919,626; 5,945,283;
5,242,794; and 5,952,174 (each of the foregoing which is herein
incorporated by reference in its entirety).
[0094] U.S. Pat. No. 6,821,733 (herein incorporated by reference in
its entirety) describes methods to detect differences in the
sequence of two nucleic acid molecules that includes the steps of:
contacting two nucleic acids under conditions that allow the
formation of a four-way complex and branch migration; contacting
the four-way complex with a tracer molecule and a detection
molecule under conditions in which the detection molecule is
capable of binding the tracer molecule or the four-way complex; and
determining binding of the tracer molecule to the detection
molecule before and after exposure to the four-way complex.
Competition of the four-way complex with the tracer molecule for
binding to the detection molecule indicates a difference between
the two nucleic acids.
[0095] Protein- and proteomics-based approaches are also suitable
for polymorphism detection and analysis. Polymorphisms which result
in or are associated with variation in expressed proteins can be
detected directly by analysing said proteins. This typically
requires separation of the various proteins within a sample, by,
for example, gel electrophoresis or HPLC, and identification of
said proteins or peptides derived therefrom, for example by NMR or
protein sequencing such as chemical sequencing or more prevalently
mass spectrometry. Proteomic methodologies are well known in the
art, and have great potential for automation. For example,
integrated systems, such as the ProteomIQ.TM. system from Proteome
Systems, provide high throughput platforms for proteome analysis
combining sample preparation, protein separation, image acquisition
and analysis, protein processing, mass spectrometry and
bioinformatics technologies.
[0096] The majority of proteomic methods of protein identification
utilise mass spectrometry, including ion trap mass spectrometry,
liquid chromatography (LC) and LC/MSn mass spectrometry, gas
chromatography (GC) mass spectroscopy, Fourier transform-ion
cyclotron resonance-mass spectrometer (FT-MS), MALDI-TOF mass
spectrometry, and ESI mass spectrometry, and their derivatives.
Mass spectrometric methods are also useful in the determination of
post-translational modification of proteins, such as
phosphorylation or glycosylation, and thus have utility in
determining polymorphisms that result in or are associated with
variation in post-translational modifications of proteins.
[0097] Associated technologies are also well known, and include,
for example, protein processing devices such as the "Chemical
Inkjet Printer" comprising piezoelectric printing technology that
allows in situ enzymatic or chemical digestion of protein samples
electroblotted from 2-D PAGE gels to membranes by jetting the
enzyme or chemical directly onto the selected protein spots
(Sloane, A. J. et al. High throughput peptide mass fingerprinting
and protein macroarray analysis using chemical printing strategies.
Mol Cell Proteomics 1(7):490-9 (2002), herein incorporated by
reference in its entirety). After in-situ digestion and incubation
of the proteins, the membrane can be placed directly into the mass
spectrometer for peptide analysis.
[0098] A large number of methods reliant on the conformational
variability of nucleic acids have been developed to detect
SNPs.
[0099] For example, Single Strand Conformational Polymorphism
(SSCP, Orita et al., PNAS 86:2766-2770 (1989), herein incorporated
by reference in its entirety) is a method reliant on the ability of
single-stranded nucleic acids to form secondary structure in
solution under certain conditions. The secondary structure depends
on the base composition and can be altered by a single nucleotide
substitution, causing differences in electrophoretic mobility under
nondenaturing conditions. The various polymorphs are typically
detected by autoradiography when radioactively labelled, by silver
staining of bands, by hybridisation with detectably labelled probe
fragments or the use of fluorescent PCR primers which are
subsequently detected, for example by an automated DNA
sequencer.
[0100] Modifications of SSCP are well known in the art, and include
the use of differing gel running conditions, such as for example
differing temperature, or the addition of additives, and different
gel matrices. Other variations on SSCP are well known to the
skilled artisan, including, RNA-SSCP (Gasparini, P. et al. Scanning
the first part of the neurofibromatosis type 1 gene by RNA-SSCP:
identification of three novel mutations and of two new
polymorphisms. Hum Genet. 97(4):492-5 (1996), herein incorporated
by reference in its entirety), restriction endonuclease
fingerprinting-SSCP (Liu, Q. et al. Restriction endonuclease
fingerprinting (REF): a sensitive method for screening mutations in
long, contiguous segments of DNA. Biotechniques 18(3):470-7 (1995),
herein incorporated by reference in its entirety), dideoxy
fingerprinting (a hybrid between dideoxy sequencing and SSCP)
(Sarkar, G. et al. Dideoxy fingerprinting (ddF): a rapid and
efficient screen for the presence of mutations. Genomics 13:441-443
(1992), herein incorporated by reference in its entirety),
bi-directional dideoxy fingerprinting (in which the dideoxy
termination reaction is performed simultaneously with two opposing
primers) (Liu, Q. et al. Bi-directional dideoxy fingerprinting
(Bi-ddF): a rapid method for quantitative detection of mutations in
genomic regions of 300-600 bp. Hum Mol. Genet. 5(1):107-14 (1996),
herein incorporated by reference in its entirety), and Fluorescent
PCR-SSCP (in which PCR products are internally labelled with
multiple fluorescent dyes, can be digested with restriction
enzymes, followed by SSCP, and analysed on an automated DNA
sequencer able to detect the fluorescent dyes) (Makino, R. et al.
F-SSCP: fluorescence-based polymerase chain reaction-single-strand
conformation polymorphism (PCR-SSCP) analysis. PCR Methods Appl.
2(1):10-13 (1992), herein incorporated by reference in its
entirety).
[0101] Other methods which utilise the varying mobility of
different nucleic acid structures include Denaturing Gradient Gel
Electrophoresis (DGGE) (Cariello, N. F. et al. Resolution of a
missense mutant in human genomic DNA by denaturing gradient gel
electrophoresis and direct sequencing using in vitro DNA
amplification: HPRT Munich. Am J Hum Genet. 42(5):726-34 (1988),
herein incorporated by reference in its entirety), Temperature
Gradient Gel Electrophoresis (TGGE) (Riesner, D. et al.
Temperature-gradient gel electrophoresis for the detection of
polymorphic DNA and for quantitative polymerase chain reaction.
Electrophoresis. 13:632-6 (1992), herein incorporated by reference
in its entirety), and Heteroduplex Analysis (HET) (Keen, J. et al.
Rapid detection of single base mismatches as heteroduplexes on
Hydrolink gels. Trends Genet. 7(1):5 (1991), herein, incorporated
by reference in its entirety). Here, variation in the dissociation
of double stranded DNA (for example, due to base-pair mismatches)
results in a change in electrophoretic mobility. These mobility
shifts are used to detect nucleotide variations.
[0102] Denaturing High Pressure Liquid Chromatography (HPLC) is yet
a further method utilised to detect SNPs, using HPLC methods
well-known in the art as an alternative to the separation methods
described above (such as gel electophoresis) to detect, for
example, homoduplexes and heteroduplexes which elute from the HPLC
column at different rates, thereby enabling detection of mismatch
nucleotides and thus SNPs (Giordano, M. et al. Identification by
denaturing high-performance liquid chromatography of numerous
polymorphisms in a candidate region for multiple sclerosis
susceptibility. Genomics 56(3):247-53 (1999), herein incorporated
by reference in its entirety).
[0103] Yet further methods to detect SNPs rely on the differing
susceptibility of single stranded and double stranded nucleic acids
to cleavage by various agents, including chemical cleavage agents
and nucleolytic enzymes. For example, cleavage of mismatches within
RNA:DNA heteroduplexes by RNase A, of heteroduplexes by, for
example bacteriophage T4 endonuclease YII or T7 endonuclease I, of
the 5' end of the hairpin loops at the junction between single
stranded and double stranded DNA by cleavase I, and the
modification of mispaired nucleotides within heteroduplexes by
chemical agents commonly used in Maxam-Gilbert sequencing
chemistry, are all well known in the art.
[0104] Further examples include the Protein Translation Test (PTT),
used to resolve stop codons generated by variations which lead to a
premature termination of translation and to protein products of
reduced size, and the use of mismatch binding proteins (Moore, W.
et al. Mutation detection in the breast cancer gene BRCA1 using the
protein truncation test. Mol. Biotechnol. 14(2):89-97 (2000),
herein incorporated by reference in its entirety). Variations are
detected by binding of, for example, the MutS protein, a component
of Escherichia coli DNA mismatch repair system, or the human hMSH2
and GTBP proteins, to double stranded DNA heteroduplexes containing
mismatched bases. DNA duplexes are then incubated with the mismatch
binding protein, and variations are detected by mobility shift
assay. For example, a simple assay is based on the fact that the
binding of the mismatch binding protein to the heteroduplex
protects the heteroduplex from exonuclease degradation.
[0105] Those skilled in the art will know that a particular SNP,
particularly when it occurs in a regulatory region of a gene such
as a promoter, can be associated with altered expression of a gene.
Altered expression of a gene can also result when the SNP is
located in the coding region of a protein-encoding gene, for
example where the SNP is associated with codons of varying usage
and thus with tRNAs of differing abundance. Such altered expression
can be determined by methods well known in the art, and can thereby
be employed to detect such SNPs. Similarly, where a SNP occurs in
the coding region of a gene and results in a non-synonomous amino
acid substitution, such substitution can result in a change in the
function of the gene product. Similarly, in cases where the gene
product is an RNA, such SNPs can result in a change of function in
the RNA gene product. Any such change in function, for example as
assessed in an activity or functionality assay, can be employed to
detect such SNPs.
[0106] The above methods of detecting and identifying SNPs are
amenable to use in the methods of the invention.
[0107] In practicing the present invention to assess the risk a
particular subject faces with respect to a particular disease, that
subject will be assessed to determine the presence or absence of
polymorphisms (preferably SNPs) which are either associated with
protection from the disease or susceptibility to the disease.
[0108] In order to detect and identify SNPs in accordance with the
invention, a sample containing material to be tested is obtained
from the subject. The sample can be any sample potentially
containing the target SNPs (or target polypeptides, as the case may
be) and obtained from any bodily fluid (blood, urine, saliva, etc)
biopsies or other tissue preparations.
[0109] DNA or RNA can be isolated from the sample according to any
of a number of methods well known in the art. For example, methods
of purification of nucleic acids are described in Tijssen;
Laboratory Techniques in Biochemistry and Molecular Biology
Hybridization with nucleic acid probes Part 1: Theory and Nucleic
acid preparation, Elsevier, New York, N.Y. 1993, as well as in
Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning
Manual 1989 (each of the foregoing which is herein incorporated by
reference in its entirety).
[0110] Upon detection of the presence or absence of the
polymorphisms tested for, the critical step is to determine a net
susceptibility score for the subject. This score will represent the
balance between the combined value of the protective polymorphisms
present and the total value of the susceptibility polymorphisms
present, with a net protective score (i.e., a greater weight of
protective polymorphisms present than susceptibility polymorphisms)
being predictive of a reduced risk of developing the disease in
question. The reverse is true where there is a net susceptibility
score. To calculate where the balance lies, the individual
polymorphisms are assigned a value. In the simplest embodiment,
each polymorphisms within a category (i.e. protective or
susceptibility) is assigned an equal value, with each protective
polymorphism being -1 and each susceptibility polymorphism being +1
(or vice versa). It is however contemplated that the values
assigned to individual polymorphisms within a category can differ,
with some polymorphisms being assigned a value that reflects their
predictive or discriminatory value. For example, one particularly
strong protective polymorphism can have a value of -2, whereas
another more weakly protective polymorphism can have a value of
-0.75.
[0111] The net score, and the associated predictive outcome in
terms of the risk of the subject developing a particular disease,
can be represented in a number of ways. One example is as a graph
as more particularly exemplified herein.
[0112] Another example is a simple numerical score (eg +2 to
represent a subject with a net susceptibility score or -2 to
represent a subject with a net protective score). In each case, the
result is communicated to the subject with an explanation of what
that result means to that subject. Preferably, advice on ways the
subject can change their lifestyle so as to reduce the risk of
developing the disease is also communicated to the subject.
[0113] It will be appreciated that the methods of the invention can
be performed in conjunction with an analysis of other risk factors
known to be associated with a disease, such as COPD, emphysema,
OCOPD, or lung cancer. Such risk factors include epidemiological
risk factors associated with an increased risk of developing the
disease. Such risk factors include, but are not limited to smoking
and/or exposure to tobacco smoke, age, sex and familial history.
These risk factors can be used to augment an analysis of one or
more polymorphisms as herein described when assessing a subject's
risk of developing a disease such as COPD, emphysema, OCOPD, or
lung cancer.
[0114] The predictive methods of the invention allow a number of
therapeutic interventions and/or treatment regimens to be assessed
for suitability and implemented for a given subject, depending upon
the disease and the overall risk quotient. The simplest of these
can be the provision to a subject with a net susceptibility score
of motivation to implement a lifestyle change, for example, in the
case of OCOPD, to reduce exposure to aero-pollutants, for example,
by an occupational change or by the use of safety equipment in the
work place. Similarly where the subject is a current smoker, the
methods of the invention can provide motivation to quit smoking. In
this latter case, a `quit smoking` program can be followed, which
can include the use of anti-smoking medicaments (such as nicotine
patches and the like) as well as anti-addiction medicaments.
[0115] Other therapeutic interventions can involve altering the
balance between protective and susceptibility polymorphisms towards
a protective state (such as by neutralizing or reversing a
susceptibility polymorphism). The manner of therapeutic
intervention or treatment will be predicated by the nature of the
polymorphism(s) and the biological effect of said polymorphism(s).
For example, where a susceptibility polymorphism is associated with
a change in the expression of a gene, intervention or treatment is
preferably directed to the restoration of normal expression of said
gene, by, for example, administration of an agent capable of
modulating the expression of said gene. Where a polymorphism, such
as a SNP allele or genotype, is associated with decreased
expression of a gene, therapy can involve administration of an
agent capable of increasing the expression of said gene, and
conversely, where a polymorphism is associated with increased
expression of a gene, therapy can involve administration of an
agent capable of decreasing the expression of said gene. Methods
useful for the modulation of gene expression are well known in the
art. For example, in situations were a polymorphism is associated
with upregulated expression of a gene, therapy utilising, for
example, RNAi or antisense methodologies can be implemented to
decrease the abundance of mRNA and so decrease the expression of
said gene. Alternatively, therapy can involve methods directed to,
for example, modulating the activity of the product of said gene,
thereby compensating for the abnormal expression of said gene.
[0116] Where a susceptibility polymorphism is associated with
decreased gene product function or decreased levels of expression
of a gene product, therapeutic intervention or treatment can
involve augmenting or replacing of said function, or supplementing
the amount of gene product within the subject for example, by
administration of said gene product or a functional analogue
thereof. For example, where a polymorphism is associated with
decreased enzyme function, therapy can involve administration of
active enzyme or an enzyme analogue to the subject. Similarly,
where a polymorphism is associated with increased gene product
function, therapeutic intervention or treatment can involve
reduction of said function, for example, by administration of an
inhibitor of said gene product or an agent capable of decreasing
the level of said gene product in the subject. For example, where a
polymorphism is associated with increased enzyme function, therapy
can involve administration of an enzyme inhibitor to the
subject.
[0117] Likewise, when a protective polymorphism is associated with
upregulation of a particular gene or expression of an enzyme or
other protein, therapies can be directed to mimic such upregulation
or expression in an individual lacking the resistive genotype,
and/or delivery of such enzyme or other protein to such individual
Further, when a protective polymorphism is associated with
downregulation of a particular gene, or with diminished or
eliminated expression of an enzyme or other protein, desirable
therapies can be directed to mimicking such conditions in an
individual that lacks the protective genotype.
EXAMPLES
[0118] The invention will now be described in more detail, with
reference to non-limiting examples.
Example 1
Case Association Study--COPD Methods
Subject Recruitment
[0119] Subjects of European descent who had smoked a minimum of
fifteen pack years and diagnosed by a physician with chronic
obstructive pulmonary disease (COPD) were recruited. Subjects met
the following criteria: were over 50 years old and had developed
symptoms of breathlessness after 40 years of age, had a Forced
expiratory volume in one second (FEV1) as a percentage of predicted
<70% and a FEV1/FVC ratio (Forced expiratory volume in one
second/Forced vital capacity) of <79% (measured using American
Thoracic Society criteria). Two hundred and ninety-four subjects
were recruited, of these 58% were male, the mean FEV1/FVC (.+-.95%
confidence limits) was 51% (49-53), mean FEV1 as a percentage of
predicted was 43 (41-45). Mean age, cigarettes per day and pack
year history was 65 yrs (64-66), 24 cigarettes/day (22-25) and 50
pack years (41-55) respectively. Two hundred and seventeen European
subjects who had smoked a minimum of twenty pack years and who had
never suffered breathlessness and had not been diagnosed with an
obstructive lung disease in the past, in particular childhood
asthma or chronic obstructive lung disease, were also studied. This
control group was recruited through clubs for the elderly and
consisted of 63% male, the mean FEV1/FVC (95% CI) was 82% (81-83),
mean FEV1 as a percentage of predicted was 96 (95-97). Mean age,
cigarettes per day and pack year history was 59 yrs (57-61), 24
cigarettes/day (22-26) and 42 pack years (39-45) respectively.
Using a PCR based method [1, incorporated herein in its entirety by
reference], all subjects were genotyped for the
.alpha.1-antitrypsin mutations (S and Z alleles) and those with the
ZZ allele were excluded. The COPD and resistant smoker cohorts were
matched for subjects with the MZ genotype (5% in each cohort). 190
European blood donors (smoking status unknown) were recruited
consecutively through local blood donor services. Sixty-three
percent were men and their mean age was 50 years. On regression
analysis, the age difference and pack years difference observed
between COPD sufferers and resistant smokers was found not to
determine FEV or COPD.
[0120] This study shows that polymorphisms found in greater
frequency in COPD patients compared to controls (and/or resistant
smokers) can reflect an increased susceptibility to the development
of impaired lung function and COPD. Similarly, polymorphisms found
in greater frequency in resistant smokers compared to susceptible
smokers (COPD patients and/or controls) can reflect a protective
role.
Summary of Characteristics for the COPD, Resistant Smoker and
Healthy Blood Donors
TABLE-US-00002 [0121] Parameter COPD Resistant smokers Median (IQR)
N = 294 N = 217 Differences % male 58% 63% ns Age (yrs) 65 (64-66)
59 (57-61) P < 0.05 Pack years 50 (46-53) 42 (39-45) P < 0.05
Cigarettes/day 24 (22-25) 24 (22-26) ns FEV1 (L) 1.6 (0.7-2.5) 2.9
(2.8-3.0) P < 0.05 FEV1 % predict 43 (41-45) 96% (95-97) P <
0.05 FEV1/FVC 51 (49-53) 82 (81-83) P < 0.05
Means and 95% confidence limits Cyclo-oxygenase 2 (COX2) -765 G/C
Promoter Polymorphism and a1-antitrypsin Genotyping
[0122] Genomic DNA was extracted from whole blood samples [2,
herein incorporated by reference in its entirety]. The
Cyclo-oxygenase 2 -765 polymorphism was determined by minor
modifications of a previously published method [3, herein
incorporated by reference in its entirety]. The PCR reaction was
carried out in a total volume of 25 ul and contained 20 ng genomic
DNA, 500 pmol forward and reverse primers, 0.2 mM dNTPs, 10 mM
Tris-HCL (pH 8.4), 150 mM KCl, 1.0 mM MgCl.sub.2 and 1 unit of
polymerase (Life Technologies). Cycling times were incubations for
3 min at 95.degree. C. followed by 33 cycles of 50 s at 94.degree.
C., 60 s at 66.degree. C. and 60 s at 72.degree. C. A final
elongation of 10 min at 72.degree. C. then followed. 4 ul of PCR
products were visualised by ultraviolet trans-illumination of a 3%
agarose gel stained with ethidium bromide. An aliquot of 3 ul of
amplification product was digested for 1 hr with 4 units of AciI
(Roche Diagnostics, New Zealand) at 37.degree. C. Digested products
were separated on a 2.5% agarose gel run for 2.0 hours at 80 mV
with TBE buffer. The products were visualised against a 123 bp
ladder using ultraviolet transillumination after ethidium bromide
staining. Using a PCR based method referenced above [1, herein
incorporated by reference in its entirety], all COPD and resistant
smoker subjects were genotyped for the .alpha.1-antitrypsin S and Z
alleles.
[0123] Other Polymorphism Genotyping
[0124] Genomic DNA was extracted from whole blood samples [2].
Purified genomic DNA was aliquoted (10 ng/ul concentration) into 96
well plates and genotyped on a Sequenom.TM. system (Sequenom.TM.
Autoflex Mass Spectrometer and Samsung 24 pin nanodispenser) using
the following sequences, amplification conditions and methods.
[0125] The following conditions were used for the PCR multiplex
reaction: final concentrations were for 10.times. Buffer 15 mM
MgCl.sub.2 1.25.times., 25 mM MgCl.sub.2 1.625 mM, dNTP mix 25 mM
500 uM, primers 4 uM 100 nM, Taq polymerase (Qiagen hot start) 0.15
U/reaction, Genomic DNA 10 ng/ul. Cycling times were 95.degree. C.
for 15 mM, (5.degree. C. for 15 s, 56.degree. C. 30 s, 72.degree.
C. 30 s for 45 cycles with a prolonged extension time of 3 min to
finish. Shrimp alkaline phosphotase (SAP) treatment was used (2 ul
to 5 ul per PCR reaction) incubated at 35.degree. C. for 30 min and
extension reaction (add 2 ul to 7 ul after SAP treatment) with the
following volumes per reaction of: water, 0.76 ul; hME 10.times.
termination buffer, 0.2 ul; hME primer (10 uM), 1 ul; MassEXTEND
enzyme, 0.04 ul.
Sequenom Conditions for the Polymorphisms Genotyping--1
TABLE-US-00003 [0126] SNP_ID TERM WELL 2nd-PCRP 1st-PCRP Vitamin
ACT W1 ACGTTGGATGGCTTGTTAACCAGCTTTGCC
ACGTTGGATGTTTTTCAGACTGGCAGAGCG [SEQ. ID. NO. 2] DBP-420 [SEQ. ID.
NO. 1] Vitamin ACT W1 ACGTTGGATGTTTTTCAGACTGGCAGAGCG
ACGTTGGATGGCTTGTTAACCAGCTTTGCC [SEQ. ID. NO. 4] DBP-416 [SEQ. ID.
NO. 3] IL13 C- ACT W2 ACGTTGGATGCATGTCGCCTTTTCCTGCTC
ACGTTGGATGCAACACCCAACAGGCAAATG [SEQ. ID. NO. 6] 1055T [SEQ. ID. NO.
5] GSTP1- ACT W2 ACGTTGGATGTGGTGGACATGGTGAATGAC
ACGTTGGATGTGGTGCAGATGCTCACATAG [SEQ. ID. NO. 8] 105 [SEQ. ID. NO.
7] PAI1 G- ACT W2 ACGTTGGATGCACAGAGAGAGTCTGGACAC
ACGTTGGATGCTCTTGGTCTTTCCCTCATC [SEQ. ID. NO. 10] 675G [SEQ. ID. NO.
9] NOS3- ACT W3 ACGTTGGATGACAGCTCTGCATTCAGCACG
ACGTTGGATGAGTCAATCCCTTTGGTGCTC [SEQ. ID. NO. 12] 298 [SEQ. ID. NO.
11] IL13- ACT W3 ACGTTGGATGGTTTTCCAGCTTGCATGTCC
ACGTTGGATGCAATAGTCAGGTCCTGTCTC [SEQ. ID. NO. 14] Arg130Gln [SEQ.
ID. NO. 13] ADRB2- ACT W3 ACGTTGGATGGAACGGCAGCGCCTTCTTG
ACGTTGGATGACTTGGCAATGGCTGTGATG [SEQ. ID. NO. 16] Arg16Gly [SEQ. ID.
NO. 15] IFNG- CGT W5 ACGTTGGATGCAGACATTCACAATTGATTT
ACGTTGGATGGATAGTTCCAAACATGTGCG [SEQ. ID. NO. 18] A874T [SEQ. ID.
NO. 17] IL18-C- ACT W6 ACGTTGGATGGGGTATTCATAAGCTGAAAC
ACGTTGGATGCCTTCAAGTTCAGTGGTCAG [SEQ. ID. NO. 20] 133G [SEQ. ID. NO.
19] IL18- ACT W8 ACGTTGGATGGGTCAATGAAGAGAACTTGG
ACGTTGGATGAATGTTTATTGTAGAAAACC [SEQ. ID. NO. 22] A105C [SEQ. ID.
NO. 21]
Sequenom Conditions for the Polymorphisms Genotyping--2
TABLE-US-00004 [0127] SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC
PWARN UEP_DIR Vitamin DBP - 420 99 99.7 99.7 46.2 53.3 ML R Vitamin
DBP - 416 99 99.7 99.7 45.5 33.3 M F IL13 C-1055T 112 97.5 80 48.2
60 L R GSTP1 - 105 107 99.4 80 49.9 52.9 F PAI1 G-675G 109 97.9 80
59.3 66.7 g F NOS3 -298 186 98.1 65 61.2 63.2 F IL13-Arg130Gln 171
99.3 65 55.1 47.6 F ADRB2- Arg16Gly 187 88.2 65 65.1 58.3 F IFNG -
A874T 112 75.3 81.2 45.6 27.3 F IL18- C-133G 112 93.5 74.3 41.8
46.7 L F IL18- A105C 121 67.2 74.3 48.9 40 R
Sequenom Conditions for the Polymorphisms Genotyping--3
TABLE-US-00005 [0128] SNP_ID UEP_MASS UEP_SEQ EXT1_CALL EXT1_MASS
Vitamin DBP - 420 4518.9 AGCTTTGCCAGTTCC [SEQ. ID. NO. 23] A 4807.1
Vitamin DBP - 416 5524.6 AAAAGCAAAATTGCCTGA [SEQ. ID. NO. 24] T
5812.8 IL13 C-1055T 4405.9 TCCTGCTCTTCCCTC [SEQ. ID. NO. 25] T
4703.1 GSTP1 - 105 5099.3 ACCTCCGCTGCAAATAC [SEQ. ID. NO. 26] A
5396.5 PAI1 G-675G 5620.6 GAGTCTGGACACGTGGGG [SEQ. ID. NO. 27] DEL
5917.9 NOS3 -298 5813.8 TGCTGCAGGCCCCAGATGA [SEQ. ID. NO. 28] T
6102 IL13-Arg130Gln 6470.2 AGAAACTTTTTCGCGAGGGAC [SEQ. ID. NO. 29]
A 6767.4 ADRB2- Arg16Gly 7264.7 AGCGCCTTCTTGCTGGCACCCAAT [SEQ. ID.
NO. 30] A 7561.9 IFNG - A874T 6639.4 TCTTACAACACAAAATCAAATC [SEQ.
ID. NO. 31] T 6927.6 IL18- C-133G 4592 AGCTGAAACTTCTGG [SEQ. ID.
NO. 32] C 4865.2 IL18- A105C 6085 TCAAGCTTGCCAAAGTAATC [SEQ. ID.
NO. 33] A 6373.2
Sequenom Conditions for the Polymorphisms Genotyping--4
TABLE-US-00006 [0129] SNP_ID EXT1_SEQ EXT2_CALL EXT2_MASS EXT2_SEQ
1stPAUSE Vitamin AGCTTTGCCAGTTCCT [SEQ. ID. NO. 34] C 5136.4
AGCTTTGCCAGTTCCGT 4848.2 DBP - 420 [SEQ. ID. NO. 35] Vitamin
AAAAGCAAAATTGCCTGAT [SEQ. ID. NO. 36] G 6456.2
AAAAGCAAAATTGCCTGAGGC 5853.9 DBP-416 [SEQ. ID. NO. 37] IL13 C-
TCCTGCTCTTCCCTCA [SEQ. ID. NO. 38] C 5023.3 TCCTGCTCTTCCCTCGT
4735.1 1055T [SEQ. ID. NO. 39] GSTP1- ACCTCCGCTGCAAATACA [SEQ. ID.
NO. 40] G 5716.7 ACCTCCGCTGCAAATACGT 5428.5 105 [SEQ. ID. NO. 41]
PAI1 G- GAGTCTGGACACGTGGGGA [SEQ. ID. NO. 42] G 6247.1
GAGTCTGGACACGTGGGGGA 5949.9 675G [SEQ. ID. NO. 43] NOS3-
TGCTGCAGGCCCCAGATGAT [SEQ. ID. NO. G 6416.2 TGCTGCAGGCCCCAGATGAGC
6143 298 44] [SEQ. ID. NO. 45] IL13- AGAAACTTTTTCGCGAGGGACA G
7416.8 AGAAACTTTTTCGCGAGGGACGGT 6799.4 Arg130Gln [SEQ. ID. NO. 46]
[SEQ. ID. NO. 47] ADRB2- AGCGCCTTCTTGCTGGCACCCAATA G 8220.3
AGCGCCTTCTTGCTGGCACCCAATGGA 7593.9 Arg16Gly [SEQ. ID. NO. 48] [SEQ.
ID. NO. 49] IFNG- TCTTACAACACAAAATCAAATCT A 7225.8
TCTTACAACACAAAATCAAATCAC 6952.6 A874T [SEQ. ID. NO. 50] [SEQ. ID.
NO. 51] IL18- C- AGCTGAAACTTCTGGC [SEQ. ID. NO. 52] G 5218.4
AGCTGAAACTTCTGGGA 4921.2 133G [SEQ. ID. NO. 53] IL18-
TCAAGCTTGCCAAAGTAATCT C 7040.6 TCAAGCTTGCCAAAGTAATCGGA 6414.2 A105C
[SEQ. ID. NO. 54] [SEQ. ID. NO. 55]
Sequenom Conditions for the Polymorphisms Genotyping--5
TABLE-US-00007 [0130] SNP_ID 2nd-PCRP 1st-PCRP Lipoxygenase5-
ACGTTGGATGGAAGTCAGAGATGATGGCAG ACGTTGGATGATGAATCCTGGACCCAAGAC
366G/A [SEQ. ID. NO. 56] [SEQ. ID. NO. 57] TNFalpha+489G/A
ACGTTGGATGGAAAGATGTGCGCTGATAGG ACGTTGGATGGCCACATCTCTTTCTGCATC [SEQ.
ID. NO. 58] [SEQ. ID. NO. 59] SMAD3C89Y
ACGTTGGATGTTGCAGGTGTCCCATCGGAA [SEQ. ID. NO. 60]
ACGTTGGATGTAGCTCGTGGTGGCTGTGCA [SEQ. ID. NO. 61]
CaspaseGly881ArgG/C ACGTTGGATGGTGATCACCCAAGGCTTCAG [SEQ. ID. NO.
62] ACGTTGGATGGTCTGTTGACTCTTTTGGCC [SEQ. ID. NO. 63] MBL2+161G/A
ACGTTGGATGGTAGCTCTCCAGGCATCAAC [SEQ. ID. NO. 64]
ACGTTGGATGGTACCTGGTTCCCCCTTTTC [SEQ. ID. NO. 65] HSP70-HOM2437T/C
ACGTTGGATGTGATCTTGTTCACCTTGCCG [SEQ. ID. NO. 66]
ACGTTGGATGAGATCGAGGTGACGTTTGAC [SEQ. ID. NO. 67] CD14-159C/T
ACGTTGGATGAGACACAGAACCCTAGATGC [SEQ. ID. NO. 68]
ACGTTGGATGGCAATGAAGGATGTTTCAGG [SEQ. ID. NO. 69] Chymase1-1903G/A
ACGTTGGATGTAAGACAGCTCCACAGCATC [SEQ. ID. NO. 70]
ACGTTGGATGTTCCATTTCCTCACCCTCAG [SEQ. ID. NO. 71] TNFalpha-308G/A
ACGTTGGATGGATTTGTGTGTAGGACCCTG [SEQ. ID. NO. 72]
ACGTTGGATGGGTCCCCAAAAGAAATGGAG [SEQ. ID. NO. 73] CLCA1+13924T/A
ACGTTGGATGGGATTGGAGAACAAACTCAC [SEQ. ID. NO. 74]
ACGTTGGATGGGCAGCTGTTACACCAAAAG [SEQ. ID. NO. 75] MEHTyr113HisT/C
ACGTTGGATGCTGGCGTTTTGCAAACATAC [SEQ. ID. NO. 76]
ACGTTGGATGTTGACTGGAAGAAGCAGGTG [SEQ. ID. NO. 77] NAT2Arg197GlnG/A
ACGTTGGATGCCTGCCAAAGAAGAAACACC [SEQ. ID. NO. 78]
ACGTTGGATGACGTCTGCAGGTATGTATTC [SEQ. ID. NO. 79] MEHHis139ArgG/A
ACGTTGGATGACTTCATCCACGTGAAGCCC [SEQ. ID. NO. 80]
ACGTTGGATGAAACTCGTAGAAAGAGCCGG [SEQ. ID. NO. 81] IL-1B-511A/G
ACGTTGGATGATTTTCTCCTCAGAGGCTCC [SEQ. ID. NO. 82]
ACGTTGGATGTGTCTGTATTGAGGGTGTGG [SEQ. ID. NO. 83] ADRB2Gln27GluC/G
ACGTTGGATGTTGCTGGCACCCAATGGAAG [SEQ. ID. NO. 84]
ACGTTGGATGATGAGAGACATGACGATGCC [SEQ. ID. NO. 85] ICAM1E469KA/G
ACGTTGGATGACTCACAGAGCACATTCACG [SEQ. ID. NO. 86]
ACGTTGGATGTGTCACTCGAGATCTTGAGG [SEQ. ID. NO. 87]
Sequenom Conditions for the Polymorphisms Genotyping--6
TABLE-US-00008 [0131] SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC
UEP_DIR Lipoxygenase5-366G/A 104 99.6 73.4 59 70.6 F TNFalpha +
489G/A 96 99.6 73.4 45.5 38.9 F SMAD3C89Y 107 87.3 71.7 45.7 47.1 F
CaspaseGly881ArgG/C 111 97.2 81 52.9 58.8 R MBL2 + 161G/A 99 96.8
81 50.3 52.9 F HSP70-HOM2437T/C 107 99.3 81 62.2 65 R CD14-159C/T
92 98 76.7 53.3 50 F Chymase1-1903G/A 105 99.6 76.7 53.6 39.1 R
TNFalpha-308G/A 100 99.7 81.6 59.9 70.6 R CLCA1 + 13924T/A 101 98
98 45.3 36.8 R MEHTyr113HisT/C 103 97.7 82.2 48.7 42.1 R
NAT2Arg197GlnG/A 115 97.4 70 48.5 36.4 F MEHHis139ArgG/A 115 96.7
77.8 66 82.4 F IL-1B-511A/G 111 99.2 83 46 47.1 R ADRB2Gln27GluC/G
118 96.6 80 52.2 66.7 F ICAM1E469KA/G 115 98.8 95.8 51.5 52.9 R
Sequenom Conditions for the Polymorphisms Genotyping--7
TABLE-US-00009 [0132] SNP_ID UEP_MASS UEP_SEQ EXT1_CALL EXT1_MASS
Lipoxygenase5-366G/A 5209.4 GTGCCTGTGCTGGGCTC [SEQ. ID. NO. 88] A
5506.6 TNFalpha+489G/A 5638.7 GGATGGAGAGAAAAAAAC [SEQ. ID. NO. 89]
A 5935.9 SMAD3C89Y 5056.3 CCCTCATGTCATCTACT [SEQ. ID. NO. 90] A
5353.5 CaspaseGly881ArgG/C 5097.3 GTCACCCACTCTGTTGC [SEQ. ID. NO.
91] G 5370.5 MBL2+161G/A 5299.5 CAAAGATGGGCGTGATG [SEQ. ID. NO. 92]
A 5596.7 HSP70-HOM2437T/C 6026.9 CCTTGCCGGTGCTCTTGTCC [SEQ. ID. NO.
93] T 6324.1 CD14-159C/T 6068 CAGAATCCTTCCTGTTACGG [SEQ. ID. NO.
94] C 6341.1 Chymase1-1903G/A 6973.6 TCCACCAAGACTTAAGTTTTGCT [SEQ.
ID. NO. 95] G 7246.7 TNFalpha-308G/A 5156.4 GAGGCTGAACCCCGTCC [SEQ.
ID. NO. 96] G 5429.5 CLCA1+13924T/A 5759.8 CTTTTTCATAGAGTCCTGT
[SEQ. ID. NO. 97] A 6048 MEHTyr113HisT/C 5913.9 TTAGTCTTGAAGTGAGGGT
[SEQ. ID. NO. 98] T 6211.1 NAT2Arg197GlnG/A 6635.3
TACTTATTTACGCTTGAACCTC [SEQ. ID. NO. 99] A 6932.5 MEHHis139ArgG/A
5117.3 CCAGCTGCCCGCAGGCC [SEQ. ID. NO. 100] A 5414.5 IL-1B-511A/G
5203.4 AATTGACAGAGAGCTCC [SEQ. ID. NO. 101] G 5476.6
ADRB2Gln27GluC/G 4547 CACGACGTCACGCAG [SEQ. ID. NO. 102] C 4820.2
ICAM1E469KA/G 5090.3 CACATTCACGGTCACCT [SEQ. ID. NO. 103] G
5363.5
Sequenom Conditions for the Polymorphisms Genotyping--8
TABLE-US-00010 [0133] EXT2 EXT2 1.sup.st SNP_ID EXT1_SEQ CALL MASS
EXT2_SEQ PAUSE Lipoxygenase5- GTGCCTGTGCTGGGCTCA G 5826.8
GTGCCTGTGCTGGGCTCGT [SEQ. ID. NO. 105] 5538.6 366G/A [SEQ. ID. NO.
104] TNFalpha+489G/A GGATGGAGAGAAAAAAACA G 6256.1
GGATGGAGAGAAAAAAACGT [SEQ. ID. NO. 107] 5967.9 [SEQ. ID. NO. 106]
SMAD3C89Y CCCTCATGTCATCTACTA G 5658.7 CCCTCATGTCATCTACTGC [SEQ. ID.
NO. 109] 5385.5 [SEQ. ID. NO. 108] CaspaseGly881ArgG/C
GTCACCCACTCTGTTGCC C 5699.7 GTCACCCACTCTGTTGCGC [SEQ. ID. NO. 111]
5426.5 [SEQ. ID. NO. 110] MBL2+161G/A CAAAGATGGGCGTGATGA G 5901.9
CAAAGATGGGCGTGATGGC [SEQ. ID. NO. 113] 5628.7 [SEQ. ID. NO. 112]
HSP70-HOM2437T/C CCTTGCCGGTGCTCTTGTCCA C 6644.3
CCTTGCCGGTGCTCTTGTCCGT 6356.1 [SEQ. ID. NO. 114] [SEQ. ID. NO. 115]
CD14-159C/T CAGAATCCTTCCTGTTACGGC T 6645.3 CAGAATCCTTCCTGTTACGGTC
[SEQ. ID. NO. 117] 6372.2 [SEQ. ID. NO. 116] Chymase1-1903G/A
TCCACCAAGACTTAAGTTTTGCTC A 7550.9 TCCACCAAGACTTAAGTTTTGCTTC 7277.8
[SEQ. ID. NO. 118] [SEQ. ID. NO. 119] TNFalpha-308G/A
GAGGCTGAACCCCGTCCC A 5733.7 GAGGCTGAACCCCGTCCTC [SEQ. ID. NO. 121]
5460.6 [SEQ. ID. NO. 120] CLCA1+13924T/A CTTTTTCATAGAGTCCTGTT T
6659.4 CTTTTTCATAGAGTCCTGTAAC [SEQ. ID. NO. 123] 6073 [SEQ. ID. NO.
122] MEHTyr113HisT/C TTAGTCTTGAAGTGAGGGTA C 6531.3
TTAGTCTTGAAGTGAGGGTGT [SEQ. ID. NO. 125] 6243.1 [SEQ. ID. NO. 124]
NAT2Arg197GlnG/A TACTTATTTACGCTTGAACCTCA G 7261.8
TACTTATTTACGCTTGAACCTCGA 6964.5 [SEQ. ID. NO. 126] [SEQ. ID. NO.
127] MEHHis139ArgG/A CCAGCTGCCCGCAGGCCA G 5734.7
CCAGCTGCCCGCAGGCCGT [SEQ. ID. NO. 129] 5446.5 [SEQ. ID. NO. 128]
IL-1B-511A/G AATTGACAGAGAGCTCCC A 5820.8 AATTGACAGAGAGCTCCTG [SEQ.
ID. NO. 131] 5507.6 [SEQ. ID. NO. 130] ADRB2Gln27GluC/G
CACGACGTCACGCAGC G 5173.4 CACGACGTCACGCAGGA [SEQ. ID. NO. 133]
4876.2 [SEQ. ID. NO. 132] ICAM1E469KA/G CACATTCACGGTCACCTC A 5707.7
CACATTCACGGTCACCTTG [SEQ. ID. NO. 135] 5394.5 [SEQ. ID. NO.
134]
[0134] Results
[0135] Frequencies of individual polymorphisms are as follows:
TABLE-US-00011 TABLE 1 Polymorphism allele and genotype frequencies
in the COPD patients and resistant smokers. Cyclo-oxygenase 2 -765
G/C Allele* Genotype Frequency C G CC CG GG Controls n = 94 (%) 27
(14%) 161 (86%) 3 (3%) 21 (22%) 70 (75%) COPD n = 202 (%) 59 (15%)
345 (85%) 6 (3%) 47 (23%) 149 (74%) Resistant n = 172 (%) 85.sup.2
(25%).sup. 259 (75%) 14.sup.1 (8%).sup. 57 (33%) 101 (59%)
Beta2-adrenoreceptor Arg 16 Gly Allele* Genotype Frequency A G AA
AG GG Controls n = 182 (%) 152 (42%) 212 (58%) 26 (14%) 100 (55%)
.sup. 56 (31%) COPD n = 236 (%) 164 (34%) 308 (66%) 34 (14%) 96
(41%) 106.sup.3 (45%) Resistant n = 190 (%) 135 (36%) 245 (64%) 34
(18%) 67 (35%) 89.sup.4 (47%) Interleukin 18 105 A/C Allele*
Genotype Frequency C A CC AC AA Controls n = 184 (%) 118 (32%) 250
(68%) 22 (12%) 74 (40%) .sup. 88 (48%) COPD n = 240 (%) 122 (25%)
377.sup.6 (75%).sup. 21 (9%) 80 (33%) 139.sup.5, 7 (58%) Resistant
n = 196 (%) 113 (29%) 277 (71%) 16 (8%) 81 (41%) .sup. 99 (50%)
Interleukin 18 -133 C/G Allele* Genotype Frequency G C GG GC CC
Controls n = 187 (%) 120 (32%) 254 (68%) 23 (12%) 74 (40%) 90 (48%)
COPD n = 238 123 (26%) 353.sup.9 (74%).sup. 21 (9%) 81 (34%)
136.sup.8 (57%).sup. Resistant n = 195 (%) 113 (29%) 277 (71%) 16
(8%) 81 (42%) 98 (50%) Plasminogen activator inhibitor 1 -675 4G/5G
Allele* Genotype Frequency 5G 4G 5G5G 5G4G 4G4G Controls n = 186
(%) .sup. 158 (42%) 214 (58%) .sup. 31 (17%) 96 (52%) .sup. 59
(32%) COPD n = 237 (%) 219.sup.12 (46%) 255 (54%) 54.sup.10,11
(23%) 111 (47%) .sup. 72 (30%) Resistant n = 194 (%) .sup. 152
(39%) 236 (61%) .sup. 31 (16%) 90 (46%) 73.sup.10,11 (38%) Nitric
oxide synthase 3 Asp 298 Glu (T/G) Allele* Genotype Frequency T G
TT TG GG Controls n = 183 (%) 108 (30%) 258 (70%) .sup. 13 (7%) 82
(45%) 88 (48%) COPD n = 238 (%) 159 (42%) 317 (58%) .sup. 25 (10%)
109 (47%) 104 (43%) Resistant n = 194 (%) 136 (35%) 252 (65%)
28.sup.13 (15%) 80 (41%) 86 (44%) Vitamin D Binding Protein Lys 420
Thr (A/C) Allele* Genotype Frequency A C AA AC CC Controls n = 189
(%) .sup. 113 (30%) 265 (70%) .sup. 17 (9%) .sup. 79 (42%) 93 (49%)
COPD n = 250 (%) .sup. 147 (29%) 353 (71%) .sup. 24 (10%) .sup. 99
(40%) 127 (50%) Resistant n = 195 (%) 140.sup.15 (36%) 250 (64%)
25.sup.14 (13%) 90.sup.14 (46%) 80 (41%) Vitamin D Binding Protein
Glu 416 Asp (T/G) Allele* Genotype Frequency T G TT TG GG Controls
n = 188 (%) .sup. 162 (43%) 214 (57%) .sup. 35 (19%) .sup. 92 (49%)
61 (32%) COPD n = 240 (%) .sup. 230 (48%) 250 (52%) .sup. 57 (24%)
.sup. 116 (48%) 67 (28%) Resistant n = 197 (%) 193.sup.17 (49%) 201
(51%) 43.sup.16 (22%) 107.sup.16 (54%) 47 (24%) Glutathione S
Transferase P1 Ile 105 Val (A/G) Allele* Genotype Frequency A G AA
AG GG Controls n = 185 (%) .sup. 232 (63%) 138 (37%) .sup. 70 (38%)
92 (50%) 23 (12%) COPD n = 238 (%) .sup. 310 (65%) 166 (35%) .sup.
96 (40%) 118 (50%) 24 (10%) Resistant n = 194 (%) 269.sup.19 (69%)
119 (31%) 91.sup.18 (47%) 87 (45%) 16 (8%) Interferon-gamma 874 A/T
Allele* Genotype Frequency A T AA AT TT Controls n = 186 (%) 183
(49%) 189 (51%) .sup. 37 (20%) 109 (58%) 40 (22%) COPD n = 235 (%)
244 (52%) 226 (48%) 64.sup.20 (27%) 116 (49%) 55 (24%) Resistant n
= 193 (%) 208 (54%) 178 (46%) .sup. 51 (27%) 106 (55%) 36 (18%)
Interleukin-13 Arg 130 Gln (G/A) Allele* Genotype Frequency A G AA
AG GG Controls n = 184 (%) 67 (18%) 301 (82%) .sup. 3 (2%) 61 (33%)
120 (65%) COPD n = 237 (%) 86 (18%) 388 (82%) .sup. 8 (3%) 70 (30%)
159 (67%) Resistant n = 194 (%) 74 (19%) 314 (81%) 9.sup.21 (5%) 56
(28%) 129 (67%) Interleukin-13 -1055 C/T Allele* Genotype Frequency
T C TT TC CC Controls n = 182 (%) 65 (18%) 299 (82%) .sup. 5 (3%)
55 (30%) 122 (67%) COPD n = 234 (%) 94 (20%) 374 (80%) 8.sup.22
(4%) 78 (33%) 148 (63%) Resistant n = 192 (%) 72 (19%) 312 (81%)
.sup. 2 (1%) 68 (35%) 122 (64%) a1-antitrypsin S Allele* Genotype
Frequency M S MM MS SS COPD n = 202 (%) 391 (97%) 13 (3%) 189 (94%)
.sup. 13 (6%) .sup. 0 (0%) Resistant n = 189 (%) 350 (93%) 28 (7%)
162 (85%) 26.sup.23 (14%) 1.sup.23 (1%) *number of chromosomes
(2n)Genotype
[0136] 1. Genotype. CC/CG vs GG for resistant vs COPD, Odds ratio
(OR)=1.98, 95% confidence limits 1.3-3.1, .chi..sup.2 (Yates
corrected)=8.82, p=0.003, CC/CG=protective for COPD [0137] 2.
Allele. C vs G for resistant vs COPD, Odds ratio (OR)=1.92, 95%
confidence limits 1.3-2.8, .chi..sup.2 (Yates corrected)=11.56,
p<0.001, C=protective for COPD [0138] 3. Genotype. GG vs AG/AA
for COPD vs controls, Odds ratio (OR)=1.83, 95% confidence limits
1.2-2.8, .chi.2 (Yates corrected)=8.1, p=0.004, GG=susceptible to
COPD (depending on the presence of other snps) [0139] 4. Genotype.
GG vs AG/AA for resistant vs controls, Odds ratio (OR)=1.98, 95%
confidence limits 1.3-3.1, .chi.2 (Yates corrected)=9.43, p=0.002
GG=resistance (depending on the presence of other snps) [0140] 5.
Genotype. AA vs AC/CC for COPD vs controls, Odds ratio (OR)=1.50,
95% confidence limits 1.0-2.3, .chi.2 (Yates uncorrected)=4.26,
p=0.04, AA=susceptible to COPD [0141] 6. Allele. A vs C for COPD vs
control, Odds ratio (OR)=1.46, 95% confidence limits 1.1-2.0,
.chi.2 (Yates corrected)=5.76, p=0.02 [0142] 7. Genotype. AA vs
AC/CC for COPD vs resistant, Odds ratio (OR)=1.35, 95% confidence
limits 0.9-2.0, .chi.2 (Yates uncorrected)=2.39, p=0.12 (trend),
[0143] AA=susceptible to COPD [0144] 8. Genotype. CC vs CG/GG for
COPD vs controls, Odds ratio (OR)=1.44, 95% confidence limits
1.0-2.2, .chi.2 (Yates corrected)=3.4, p=0.06, CC=susceptible to
COPD [0145] 9. Allele. C vs G for COPD vs control, Odds ratio
(OR)=1.36, 95% confidence limits 1.0-1.9, .chi.2 (Yates
corrected)=53.7, p=0.05, C=susceptible to COPD [0146] 10. Genotype.
5G5G vs rest for COPD vs resistant, Odds ratio (OR)=1.55, 95%
confidence limits 0.9-2.6, .chi.2 (Yates uncorrected)=3.12, p=0.08,
5G5G=susceptible to COPD [0147] 11. Genotype. 5G5G vs rest for COPD
vs control, Odds ratio (OR)=1.48, 95% confidence limits 0.9-2.5,
.chi.2 (Yates uncorrected)=2.43, p=0.12, 5G5G=susceptible to COPD
[0148] 12. Allele. 5G vs 4G for COPD vs resistant, Odds ratio
(OR)=1.33, 95% confidence limits 1.0-1.8, .chi.2 (Yates
corrected)=4.02, p=0.05, 5G=susceptible to COPD [0149] 13.
Genotype. TT vs TG/GG for resistant vs controls, Odds ratio
(OR)=2.2, 95% confidence limits 1.0-4.7, .chi.2 (Yates
corrected)=4.49, p=0.03, TT genotype=protective for COPD [0150] 14.
Genotype. AA/AC vs CC for resistant vs COPD, Odds ratio (OR)=1.39,
95% confidence limits 0.9-2.1, .chi.2 (Yates uncorrected)=2.59,
p=0.10, AA/AC genotype=protective for COPD [0151] 15. Allele. A vs
C for resistant vs COPD, Odds ratio (OR)=1.34, 95% confidence
limits 1.0-1.8, .chi.2 (Yates corrected)=3.94, p=0.05, A
allele=protective for COPD [0152] 16. Genotype. TT/TG vs GG for
resistant vs controls, Odds ratio (OR)=1.53, 95% confidence limits
1.0-2.5, .chi.2 (Yates uncorrected)=3.52, p=0.06, TT/TG
genotype=protective for COPD [0153] 17. Allele. T vs G for
resistant vs control, Odds ratio (OR)=1.27, 95% confidence limits
1.0-1.7, .chi.2 (Yates corrected)=2.69, p=0.1, T allele=protective
for COPD [0154] 18. Genotype. AA vs AG/GG for resistant vs
controls, Odds ratio (OR)=1.45, 95% confidence limits 0.9-2.2,
.chi.2 (Yates uncorrected)=3.19, p=0.07, AA genotype=protective for
COPD [0155] 19. Allele. A vs G for resistant vs control, Odds ratio
(OR)=1.34, 95% confidence limits 1.0-1.8, .chi.2 (Yates
uncorrected)=3.71, p=0.05, A allele=protective for COPD [0156] 20.
Genotype. AA vs AT/TT for COPD vs controls, Odds ratio (OR)=1.51,
95% confidence limits 0.9-2.5, .chi.2 (Yates uncorrected)=3.07,
p=0.08, AA genotype=susceptible to COPD [0157] 21. Genotype. AA vs
AG/GG for resistant vs controls, Odds ratio (OR)=2.94, 95%
confidence limits 0.7-14.0, .chi.2 (Yates uncorrected)=2.78,
p=0.09, AA genotype=protective for COPD [0158] 22. Genotype. TT vs
TC/CC for COPD vs resistant, Odds ratio (OR)=6.03, 95% confidence
limits 1.1-42, .chi.2 (Yates corrected)=4.9, p=0.03, TT=susceptible
to COPD [0159] 23. Genotype. MS/SS vs MM for Resistant vs COPD,
Odds ratio (OR)=2.42, 95% confidence limits 1.2-5.1, .chi.2 (Yates
corrected)=5.7, p=0.01, S=protective for COPD
Tissue Necrosis Factor .alpha. +489 G/A Polymorphism Allele and
Genotype Frequency in the COPD Patients and Resistant Smokers.
TABLE-US-00012 [0160] 1. Allele* 2. Genotype Frequency A G AA AG GG
COPD 54 (11%) 430 (89%) 5 (2%) 44 (18%) 193 (80%) n = 242 (%)
Resistant 27 (7%) 347 (93%) 1 (1%) 25 (13%) 161 (86%) n = 187 (%)
*number of chromosomes (2n)
[0161] 1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio
(OR)=1.57, 95% confidence limits 0.9-2.7, .chi..sup.2 (Yates
corrected)=2.52, p=0.11, [0162] AA/AG=susceptible (GG=protective)
[0163] 2. Allele. A vs G for COPD vs resistant, Odds ratio
(OR)=1.61, 95% confidence limits 1.0-2.7, .chi..sup.2 (Yates
corrected)=3.38, p=0.07, [0164] A=susceptible
Tissue Necrosis Factor .alpha. -308 G/A Polymorphism Allele and
Genotype Frequency in the COPD Patients and Resistant Smokers.
TABLE-US-00013 [0165] 3. Allele* 4. Genotype Frequency A G AA AG GG
COPD 90 (19%) 394 (81%) 6 (2%) 78 (32%) 158 (65%) n = 242 (%)
Resistant 58 (15%) 322 (85%) 3 (2%) 52 (27%) 135 (71%) n = 190 (%)
*number of chromosomes (2n)
[0166] 1. Genotype. GG vs AG/AA for COPD vs resistant, Odds ratio
(OR)=0.77, 95% confidence limits 0.5-1.2, .chi..sup.2 (Yates
uncorrected)=1.62, p=0.20, [0167] GG=protective (AA/AG=susceptible)
trend [0168] 2. Allele. A vs G for COPD vs resistant, Odds ratio
(OR)=1.3, 95% confidence limits 0.9-1.9, .chi..sup.2 (Yates
uncorrected)=1.7, p=0.20, [0169] A=susceptible trend
SMAD3 C89Y Polymorphism Allele and Genotype Frequency in the COPD
Patients and Resistant Smokers.
TABLE-US-00014 [0170] 5. Allele* 6. Genotype Frequency A G AA AG GG
COPD n = 250 (%) 2 (1%) 498 (99%) 0 (0%) 2 (1%) 248 (99%) Resistant
n = 196 6 (2%) 386 (98%) 0 (0%) 6 (3%) 190 (97%) (%) *number of
chromosomes (2n)
[0171] 1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio
(OR)=0.26, 95% confidence limits 0.04-1.4, .chi..sup.2 (Yates
uncorrected)=3.19, p=0.07, [0172] AA/AG=protective (GG susceptible)
Intracellular Adhesion Molecule 1 (ICAM1) A/G E469K (rs5498)
Polymorphism Allele and Genotype Frequency in COPD Patients and
Resistant Smokers.
TABLE-US-00015 [0172] 7. Allele* 8. Genotype Frequency A G AA AG GG
COPD 259 (54%) 225 (46%) 73 (30%) 113 (47%) 56 (23%) n = 242 (%)
Resistant 217 (60%) 147 (40%) 64 (35%) 89 (49%) 29 (16%) n = 182
(%) *number of chromosomes (2n)
[0173] 1. Genotype. GG vs AG/GG for COPD vs resistant, Odds ratio
(OR)=1.60, 95% confidence limits 0.9-2.7, .chi..sup.2 (Yates
corrected)=3.37, p=0.07, [0174] GG=susceptibility [0175] 2. Allele.
G vs A for COPD vs resistant, Odds ratio (OR)=1.3, 95% confidence
limits 1.0-1.7, .chi..sup.2 (Yates corrected)=2.90, p=0.09
Caspase (NOD2) Gly881Arg Polymorphism Allele and Genotype
Frequencies in the COPD Patients and Resistant Smokers.
TABLE-US-00016 [0176] 9. Allele* 10. Genotype Frequency G C GG GC
CC COPD n = 247 486 (98%) 8 (2%) 239 (97%) 8 (3%) 0 (0%) Resistant
n = 195 388 (99.5%) 2 0.5%) 193 (99%) 2 (1%) 0 (0%) (%) *number of
chromosomes (2n)
[0177] 1. Genotype. CC/CG vs GG for COPD vs resistant, Odds ratio
(OR)=3.2, 95% confidence limits 0.6-22, .chi..sup.2 (Yates
uncorrected)=2.41, p=0.11 (1-tailed), [0178] GC/CC=susceptibility
(trend)
Mannose Binding Lectin 2(MBL2) +161 G/A Polymorphism Allele and
Genotype Frequencies in the COPD Patients and Resistant
Smokers.
TABLE-US-00017 [0179] 11. Allele* 12. Genotype Frequency A G AA AG
GG COPD n = 218 110 (25%) 326 (75%) 6 (3%) 98 (45%) 114 (52%) (%)
Resistant 66 (18%) 300 (82%) 6 (3%) 54 (30%) 123 (67%) n = 183 (%)
*number of chromosomes (2n)
[0180] 1. Genotype. GG vs rest for COPD vs resistant, Odds ratio
(OR)=0.53, 95% confidence limits 0.4-0.80, .chi..sup.2 (Yates
uncorrected)=8.55, p=0.003, [0181] GG=protective
Chymase 1 (CMA1)-1903 G/A Promoter Polymorphism Allele and Genotype
Frequencies in the COPD Patients and Resistant Smokers.
TABLE-US-00018 [0182] 13. Allele* 14. Genotype Frequency A G AA AG
GG COPD 259 (54%) 219 (46%) 67 (28%) 125 (52%) 47 (20%) n = 239 (%)
Resistant 209 (58%) 153 (42%) 63 (35%) 83 (46%) 35 (19%) n = 181
(%) *number of chromosomes (2n)
[0183] 1. Genotype. AA vs AG/GG for COPD vs resistant, Odds ratio
(OR)=0.73, 95% confidence limits 0.5-1.1, .chi..sup.2 (Yates
corrected)=1.91, p=0.17, [0184] AA genotype=protective trend
N-Acetyltransferase 2 Arg 197 Gln G/A Polymorphism Allele and
Genotype Frequencies in COPD and Resistant Smokers.
TABLE-US-00019 [0185] 15. Allele* 16. Genotype Frequency A G AA AG
GG COPD 136 (28%) 358 (72%) 14 (6%) 108 (44%) 125 (50%) n = 247 (%)
Resistant 125 (32%) 267 (68%) 21 (11%) 83 (42%) 92 (47%) n = 196
(%) *number of chromosomes (2n)
[0186] 1. Genotype. AA vs AG/GG for COPD vs resistant, Odds ratio
(OR)=0.50, 95% confidence limits 0.2-1.0, .chi..sup.2 (Yates
uncorrected)=3.82, p=0.05, [0187] AA genotype=protective
Interleukin 1B (IL-1B) -511 A/G Polymorphism Allele and Genotype
Frequencies in COPD and Resistant Smokers.
TABLE-US-00020 [0188] 17. Allele* 18. Genotype Frequency A G AA AG
GG COPD 160 (32%) 336 (68%) 31 (13%) 98 (40%) 119 (48%) n = 248 (%)
Resistant 142 (36%) 248 (64%) 27 (14%) 88 (45%) 80 (41%) n = 195
(%) *number of chromosomes (2n)
[0189] 1. Genotype. GG vs AA/AG for COPD vs resistant, Odds ratio
(OR)=1.3, 95% confidence limits 0.9-2.0, .chi..sup.2 (Yates
corrected)=1.86, p=0.17, [0190] GG genotype=susceptible trend
Microsomal Epoxide Hydrolase (MEH) Tyr 113 His T/C (Exon 3)
Polymorphism Allele and Genotype Frequency in COPD and Resistant
Smokers.
TABLE-US-00021 [0191] 19. Allele* 20. Genotype Frequency C T CC CT
TT COPD 137 (28%) 361 (72%) 18 (7%) 101 (41%) 130 (52%) n = 249 (%)
Resistant 130 (34%) 258 (66%) 19 (10%) 92 (47%) 83 (43%) n = 194
(%) *number of chromosomes (2n)
[0192] 1. Genotype. TT vs CT/CC for COPD vs resistant, Odds ratio
(OR)=1.5, 95% confidence limits 1.0-2.2, .chi..sup.2 (Yates
corrected)=3.51, p=0.06, [0193] TT genotype=susceptible
Microsomal Epoxide Hydrolase (MEH) His 139 Arg A/G (Exon 4)
Polymorphism Allele and Genotype Frequency in COPD and Resistant
Smokers.
TABLE-US-00022 [0194] 21. Allele* 22. Genotype Frequency A G AA AG
GG COPD n = 238 372 (78%) 104 (22%) 148 (62%) 76 (32%) 14 (6%) (%)
Resistant 277 (77%) 81 (23%) 114 (64%) 49 (27%) 16 (9%) n = 179 (%)
*number of chromosomes (2n)
[0195] 1. Genotype. GG vs AA/AG for COPD vs resistant, Odds ratio
(OR)=0.64, 95% confidence limits 0.3-1.4, .chi..sup.2 (Yates
uncorrected)=1.43, p=0.23, [0196] GG genotype=protective
(trend)
Lipo-Oxygenase -366 G/A Polymorphism Allele and Genotype
Frequencies in the COPD Patients and Resistant Smokers.
TABLE-US-00023 [0197] 23. Allele* 24. Genotype Frequency A G AA AG
GG COPD n = 247 21 (4%) 473 (96%) 1 (0.5%) 19 (7.5%) 227 (92%) (%)
Resistant 25 (7%) 359 (93%) 0 (0%) 25 (13%) 167 (87%) n = 192 (%)
*number of chromosomes (2n)
[0198] 1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio
(OR)=0.60, 95% confidence limits 0.3-1.1, .chi..sup.2 (Yates
corrected)=2.34, p=0.12, [0199] AA/AG genotype=protective (GG
susceptible) trend
Heat Shock Protein 70 (HSP 70) HOM T2437C Polymorphism Allele and
Genotype Frequencies in the COPD Patients and Resistant
Smokers.
TABLE-US-00024 [0200] 25. Allele* 26. Genotype Frequency C T CC CT
TT COPD n = 199 (%) 127 271 5 (3%) 117 (59%) 77 (39%) (32%) (68%)
Resistant n = 166 78 254 4 (2%) 70 (42%) 92 (56%) (%) (23%) (77%)
*number of chromosomes (2n)
[0201] 1. Genotype. CC/CT vs TT for COPD vs resistant, Odds ratio
(OR)=2.0, 95% confidence limits 1.3-3.1, .chi..sup.2 (Yates
uncorrected)=9.52, p=0.002, [0202] CC/CT genotype=susceptible
(TT=protective)
Chloride Channel Calcium-Activated 1 (CLCA1) +13924 T/A
Polymorphism Allele and Genotype Frequencies in the COPD Patients
and Resistant Smokers.
TABLE-US-00025 [0203] 27. Allele* 28. Genotype Frequency A T AA AT
TT COPD n = 224 282 166 84 (38%) 114 (51%) 26 (12%) (%) (63%) (37%)
Resistant n = 158 178 138 42 (27%) 94 (59%) 22 (14%) (%) (56%)
(44%) *number of chromosomes (2n)
[0204] 1. Genotype. AA vs AT/TT for COPD vs resistant, Odds ratio
(OR)=1.7, 95% confidence limits 1.0-2.7, .chi..sup.2 (Yates
corrected)=4.51, p=0.03, [0205] AA=susceptible
Monocyte Differentiation Antigen CD-14 -159 Promoter Polymorphism
Allele and Genotype Frequencies in the COPD Patients and Resistant
Smokers.
TABLE-US-00026 [0206] 29. Allele* 30. Genotype Frequency C T CC CT
TT COPD n = 240 268 212 77 (32%) 114 (48%) 49 (20%) (%) (56%) (44%)
Resistant n = 180 182 178 46 (25%) 90 (50%) 44 (24%) (%) (51%)
(49%) *number of chromosomes (2n)
[0207] 1. Genotype CC vs CT/TT for COPD vs Resistant, Odds ratio
(OR)=1.4, 95% confidence limits 0.9-2.2, .chi..sub.2 (Yates
uncorrected)=2.12, p=0.15, [0208] CC=susceptible (trend)
Elafin +49 C/T Polymorphism Allele and Genotype Frequencies in the
COPD Patients, Resistant Smokers and Controls.
TABLE-US-00027 [0209] 31. Allele* 32. Genotype Frequency C T CC CT
TT COPD n = 144 (%) 247 41 105 (73%) 37 (26%) 2 (1%) (86%) (14%)
Resistant n = 75 121 29 49 (65%) 23 (31%) 3 (4%) (%) (81%) (19%)
*number of chromosomes (2n)
[0210] 1. Genotype. CT/TT vs CC for COPD vs resistant, Odds ratio
(OR)=0.70, 95% [0211] confidence limits=0.4-1.3, .chi..sup.2 (Yates
uncorrected)=1.36, p=0.24, [0212] CT/TT genotype=protective (trend
only) [0213] 2. Allele: T vs C for COPD vs resistant, Odds ratio
(OR)=0.69, 95% confidence limits=0.4-1.2, .chi..sup.2 (Yates
uncorrected)=1.91, p=0.17, [0214] T genotype=protective (trend
only)
Beta2-Adrenoreceptor Gln 27 Glu Polymorphism Allele and Genotype
Frequency in the COPD Patients, Resistant Smokers and Controls.
TABLE-US-00028 [0215] 33. Allele* 34. Genotype Frequency C G CC CG
GG Controls 204 168 57 (31%) 89 (48%) 39 (21%) n = 185 (%) (55%)
(45%) COPD n = 238 268 208 67 (28%) 134 (56%) 37 (16%) (%) (56%)
(44%) Resistant 220 170 64 (33%) 92 (47%) 39 (20%) n = 195 (%)
(56%) (44%) *number of chromosomes (2n)
[0216] 1. Genotype. GG vs CG/CC for COPD vs resistant, Odds ratio
(OR)=0.74, 95% confidence limits=0.4-1.2, .chi..sup.2 (Yates
uncorrected)=1.47, p=0.23, [0217] GG=protective (trend) [0218] 2.
Genotype. GG vs CG/CC for COPD vs controls, Odds ratio (OR)=0.69,
95% confidence limits=0.4-1.2, .chi..sup.2 (Yates
uncorrected)=2.16, p=0.14, [0219] GG=protective (trend)
Maxtrix Metalloproteinase 1 (MMP1) -1607 1G/2G Polymorphism Allele
and Genotype Frequencies in COPD Patients, Resistant Smokers and
Controls.
TABLE-US-00029 [0220] 35. Allele* 36. Genotype Frequency 1G 2G 1G1G
1G2G 2G2G Controls n = 174 214 134 68 (39%) 78 (45%) 28 (16%) (%)
(61%) (39%) COPD n = 217 182 252 47 (22%) 88 (41%) 82 (38%) (%)
(42%) (58%) Resistant n = 187 186 188 46 (25%) 94 (50%) 47 (25%)
(%) (50%) (50%) *number of chromosomes (2n)
[0221] 1. Genotype. 1G1G vs rest for COPD vs controls, Odds ratio
(OR)=0.43, 95% confidence limits 0.3-0.7, ?.sup.2 (Yates
uncorrected)=13.3, p=0.0003 [0222] 1G1G genotype=protective [0223]
2. Allele. 1G vs 2G for COPD vs controls, Odds ration (OR)=0.45,
95% confidence limits 0.3-0.6, ?2 (Yates corrected)=28.8,
p<0.0001, [0224] 1G=protective [0225] 3. Genotype. 1G1G/1G2G vs
rest for COPD vs resistant smokers, Odds ratio (OR)=0.55, 95%
confidence limits 0.4-0.9, ?.sup.2 (Yates uncorrected)=6.83,
p=0.009 [0226] 1G1G/162G genotypes=protective [0227] 4. Allele. 1G
vs 2G for COPD vs resistant smokers, Odds ratio (OR)=0.73, 95%
confidence limits 0.6-1.0, ?2 (Yates corrected)=4.61, p=0.03,
[0228] 1G=protective [0229] 5. Genotype. 2G2G vs 1G1G/1G2G for COPD
vs controls, Odds ratio (OR)=3.17, 95% confidence limits 1.9-5.3,
?2 (Yates uncorrected)=21.4, p<0.0001 [0230] 2G2G
genotype=susceptible [0231] 6. Allele. 2G vs 1G for COPD vs
controls, Odds ratio (OR)=2.2, 95% confidence limits 1.6-3.0, ?2
(Yates corrected)=28.8, p<0.00001, [0232] 2G=susceptible [0233]
7. Genotype. 2G2G vs 1G1G/1G2G for COPD vs resistant, Odds ratio
(OR)=1.81, 95% confidence limits 1.2-2.9, ?2 (Yates
uncorrected)=6.83, p=0.009 [0234] 2G2G genotype=susceptible [0235]
8. Allele. 2G vs 1G for COPD vs resistant, Odds ratio (OR)=1.4, 95%
confidence limits 1.0-1.8, ?2 (Yates corrected)=4.61, p=0.0.03,
[0236] 2G=susceptible
[0237] Table 2 below provides a summary of the protective and
susceptibility polymorphisms determined for COPD.
TABLE-US-00030 TABLE 2 Summary of protective and susceptibility
polymorphisms for COPD Gene Polymorphism Role Cyclo-oxygenase 2
(COX2) COX2 -765 G/C CC/CG protective .beta.2-adrenoreceptor (ADBR)
ADBR Arg16Gly GG susceptible Interleukin-18 (IL18) IL18 -133 C/G CC
susceptible Interleukin-18 (IL18) IL18 105 A/C AA susceptible
Plasminogen activator inhibitor 1 (PAI-1) PAI-1 -675 4G/5G 5G5G
susceptible Nitric Oxide synthase 3 (NOS3) NOS3 298 Asp/Glu TT
protective Vitamin D Binding Protein (VDBP) VDBP Lys 420 Thr AA/AC
protective Vitamin D Binding Protein (VDBP) VDBP Glu 416 Asp TT/TG
protective Glutathione S Transferase (GSTP-1) GSTP1 Ile105Val AA
protective Interferon ? (IFN-?) IFN-? 874 A/T AA susceptible
Interleukin-13 (IL13) IL13 Arg 130 Gln AA protective Interleukin-13
(IL13) Il13 -1055C/T TT susceptible a1-antitrypsin (a1-AT) a1-AT S
allele MS protective Tissue Necrosis Factor .alpha. TNFa TNFa +489
G/A AA/AG susceptible GG protective Tissue Necrosis Factor .alpha.
TNFa TNFa -308 G/A GG protective AA/AG susceptible SMAD3 SMAD3 C89Y
AG AA/AG protective GG susceptible Intracellular adhesion molecule
1 ICAM1 E469K GG susceptible (ICAM1) A/G Caspase (NOD2) NOD2 Gly
881Arg GC/CC susceptible G/C Mannose binding lectin 2 (MBL2) MBL2
161 G/A GG protective Chymase 1 (CMA1) CMA1 -1903 G/A AA protective
N-Acetyl transferase 2 (NAT2) NAT2 Arg 197 Gln AA protective G/A
Interleukin 1B (IL1B) (IL1B) -511 A/G GG susceptible Microsomal
epoxide hydrolase (MEH) MEH Tyr 113 His TT susceptible T/C
Microsomal epoxide hydrolase (MEH) MEH His 139 Arg GG protective
G/A 5 Lipo-oxygenase (ALOX5) ALOX5 -366 G/A AA/AG protective GG
susceptible Heat Shock Protein 70 (HSP 70) HSP 70 HOM CC/CT
susceptible T2437C TT protective Chloride Channel Calcium-activated
1 CLCA1 +13924 AA susceptible (CLCA1) T/A Monocyte differentiation
antigen CD-14 CD-14 -159 C/T CC susceptible Elafin Elafin Exon 1
+49 CT/TT protective C/T B2-adrenergic receptor (ADBR) ADBR Gln 27
Glu GG protective C/G Matrix metalloproteinase 1 (MMP1) MMP1 -1607
1G1G/1G2G 1G/2G protective
[0238] The combined frequencies of the presence or absence of the
selected protective genotypes COX2 (-765) CC/CG, .beta.2
adreno-receptor AA, Interleukin-13 AA, Nitic Oxide Synthase 3 TT,
and Vitamin D Binding Protein AA observed in the COPD subjects and
in resistant smokers is presented below in Table 3.
TABLE-US-00031 TABLE 3 Combined frequencies of the presence or
absence of selected protective genotypes in COPD subjects and in
resistant smokers. Number of protective polymorphisms Cohorts 0 1
=2 Total COPD 136 (54%) 100 (40%) 16 (7%) 252 Resistant smokers 79
(40%) 83 (42%) 34 (17%) 196 % of smokers with COPD 136/215 100/183
16/50 (63%) (55%) (32%) Comparison Odd's ratio 95% CI ?2 P value 0
vs 1 vs 2+, Resist vs COPD -- -- 16.43 0.0003 2+ vs 0-1, Resist vs
COPD 3.1 1.6-6.1 12.36 0.0004 1+ vs 0, Resist vs COPD 1.74 1.2-2.6
7.71 0.006
[0239] The combined frequencies of the presence or absence of the
selected susceptibility genotypes Interleukin-18 105 AA, PAI-1 -675
5G5G, Interleukin-13 -1055 TT, and Interferon-? -874 AA observed in
the COPD subjects and in resistant smokers is presented below in
Table 4.
TABLE-US-00032 TABLE 4 Combined frequencies of the presence or
absence of selected susceptibility genotypes in the COPD subjects
and in resistant smokers. Number of protective polymorphisms
Cohorts 0 1 =2 Total COPD 66 (26%) 113 (45%) 73 (29%) 252 Resistant
smokers 69 (35%) 92 (47%) 35 (18%) 196 % of smokers with 66/135
113/205 73/108 COPD (49%) (55%) (68%) Comparison Odd's ratio 95% CI
?2 P value 0 vs 1 vs 2+, COPD vs Resist -- -- 8.72 0.01 2+ vs 0-1,
COPD vs Resist 1.9 1.2-3.0 6.84 0.009 1+ vs 0, COPD vs Resist 1.5
1.0-3.5 3.84 0.05
[0240] The combined frequencies of the presence or absence of the
protective genotypes COX2 (-765) CC/CG, Interleukin-13 AA, Nitic
Oxide Synthase 3 TT, Vitamin D Binding Protein AA/AC, GSTP1 AA, and
a1-antitypsin MS/SS, observed in the COPD subjects and in resistant
smokers is presented below in Table 5 and in FIG. 1.
TABLE-US-00033 TABLE 5 Combined frequencies of the presence or
absence of selected protective genotypes in the COPD subjects and
in resistant smokers. Number of protective polymorphisms Cohorts 0
1 =2 Total COPD 51 (19%) 64 (24%) 150 (57%) 265 Resistant smokers
16 (8%) 56 (27%) 133 (65%) 205 % of smokers with COPD 51/76 64/120
150/283 (76%) (53%) (53%) Comparison Odd's ratio 95% CI ?2 P value
0 vs 1 vs 2+, Resist vs COPD -- -- 12.14 0.0005 1+ vs 0, Resist vs
COPD 2.82 1.5-5.3 11.46 0.0004
[0241] Protective polymorphisms were assigned a score of +1 while
susceptibility polymorphisms were assigned a score of -1. For each
subject, a net score was then calculated according to the presence
of susceptibility and protective genotypes. This produced a linear
spread of values. When assessed as a range between -3 to +3, a
linear relationship as depicted in FIG. 2 was observed. This
analysis indicates that for subjects with a net score of -2 or
less, there was a 70% or greater risk of having COPD. In contrast,
for subjects with a net score of 2+ or greater the risk was
approximately 40% (see FIG. 2).
[0242] In an analysis in which the value of a given polymorphism
was weighted based on the Odd's ratio for that polymorphism
(generated by comparing its frequency between resistant and COPD
subjects), a linear relationship was again observed. This analysis
allowed for the distinction of smokers at high or low risk of
having COPD.
I. Example 2
Case Association Study--OCOPD Methods
Subject Recruitment
[0243] Subjects of European decent who had been exposed to chronic
smoking (minimum 15 pack years) and aero-pollutants in the work
place (noxious dusts or fumes) were identified from respiratory
clinics. After spirometric testing those with occupational chronic
obstructive pulmonary disease (OCOPD) with forced expiratory volume
in one second (FEV1) as a percentage of predicted <70% and a
FEV1/FVC ratio (Forced expiratory volume in one second/Forced vital
capacity) of <79% (measured using American Thoracic Society
criteria) were recruited. One hundred and thirty-nine subjects were
recruited, of these 70% were male, the mean FEV1/FVC (.+-.Standard
Deviation) was 54% (SD 15), mean FEV1 as a percentage of predicted
was 46 (SD 19). Mean age, cigarettes per day, and pack year history
was 62 yrs (SD 9), 25 cigarettes/day (SD 16) and 53 pack years (SD
31), respectively. One hundred and twelve European subjects who had
smoked a minimum of fifteen pack years and similarly been exposed
in the work place to potentially noxious dusts or fumes were also
studied. This control group was recruited through community studies
of lung function and were 81% male; the mean FEV1/FVC (SD) was 81%
(SD 8), and mean FEV1 as a percentage of predicted was 96 (SD 10).
Mean age, cigarettes per day and pack year history was 58 yrs (SD
11), 26 cigarettes/day (SD 14) and 45 pack years (SD 28),
respectively. Using a PCR based method [1], all subjects were
genotyped for the .alpha.1-antitrypsin mutations (M, S and Z
alleles) and those with the ZZ allele were excluded. The OCOPD and
resistant smoker cohorts were matched for subjects with the MZ
genotype (6% in each cohort). They were also matched for age
started smoking (mean 16 yr) and aged stopped smoking (mid
fifties). 190 European blood donors (smoking and occupational
exposure status unknown) were recruited consecutively through local
blood donor services. Sixty-three percent were men and their mean
age was 50 years. On regression analysis, the age difference and
pack years difference observed between OCOPD sufferers and
resistant smokers was found not to determine FEV or OCOPD.
Summary of Characteristics for the OCOPD and Exposed Resistant
Smoker Cohorts.
TABLE-US-00034 [0244] Parameter OCOPD Exposed resistant Mean (SD)
(N = 139) smokers (N = 112) Differences % male 70% 81% P < 0.05
Age (yrs) 62 (9) 58 (11) ns Pack years 53 (31) 45 (28) P < 0.05
Cigarettes/day 25 (16) 26 (14) ns FEV1 (L) 1.3 (0.7) 3.0 (0.7) P
< 0.05 FEV1 % predict 46 (19) 96% (10) P < 0.05 FEV1/FVC 54
(15) 81 (8) P < 0.05
Means and 1SD
[0245] Cyclooxygenase 2 (COX2) -765 G/C Promoter Polymorphism and
a1-Antitrypsin Genotyping
[0246] Genomic DNA was extracted from whole blood samples [2]. The
COX2 -765 polymorphism was determined by minor modifications of a
previously published method [3]. The PCR reaction was carried out
in a total volume of 25 ul and contained 20 ng genomic DNA, 500
pmol forward and reverse primers, 0.2 mM dNTPs, 10 mM Tris-HCL (pH
8.4), 150 mM KCl, 1.0 mM MgCl.sub.2 and 1 unit of Taq polymerase
(Life Technologies). Cycling times were incubations for 3 min at
95.degree. C. followed by 33 cycles of 50 s at 94.degree. C., 60 s
at 66.degree. C. and 60 s at 72.degree. C. A final elongation of 10
min at 72.degree. C. then followed. 4 ul of PCR products were
visualised by ultraviolet trans-illumination of a 6% agarose gel
stained with ethidium bromide. An aliquot of 3 ul of amplification
product was digested for 1 hr with 4 units of AciI (Roche
Diagnostics, New Zealand) at 37.degree. C. Digested products were
separated on a 2.5% agarose gel run for 2.0 hrs at 80 mV with TBE
buffer and visualised using ultraviolet transillumination after
ethidium bromide staining against a 123 bp ladder. Using a PCR
based method discussed above [3], all smoking subjects were
genotyped for the .alpha.1-antitrypsin M, S and Z alleles.
Genotyping of the Superoxide Dismutase 3 Arg 312 Gln
Polymorphism
[0247] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described [4, herein incorporated by reference
in its entirety]. Genotyping was done using minor modifications of
the above protocol optimised for laboratory conditions. The PCR
reactions were amplified in MJ Research thermocyclers in a total
volume of 25 .mu.l and contained 80 ng genomic DNA, 10 pmol forward
and reverse primers, 0.1 mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM
KCl, 1.0 mM MgCl.sub.2 and 0.5 unit of Taq polymerase (Qiagen).
Aliquots of amplification product were digested for 4 hrs with 5 U
of the relevant restriction enzymes (Roche Diagnostics, New
Zealand) at designated temperatures and conditions. Digested
products were separated on 8% polyacrylamide gels (49:1, Sigma).
The products were visualised by ultraviolet transillumination
following ethidium bromide staining and migration compared against
a 1 Kb plus ladder standard (Invitrogen). Genotypes were recorded
in data spreadsheets and statistical analysis performed.
Genotyping of the Microsomal Epoxide Hydrolase Exon 3 TC
Polymorphism
[0248] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described [5, herein incorporated by reference
in its entirety]. Genotyping was done using minor modifications of
the above protocol optimised for laboratory conditions. The PCR
reactions were amplified in MJ Research thermocyclers in a total
volume of 25 .mu.l and contained 80 ng genomic DNA, 100 ng forward
and reverse primers, 0.2 mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM
KCl, 1.5 mM MgCl.sub.2 and 1.0 unit of Taq polymerase (Qiagen).
Cycling conditions consisted of 94.degree. C. 60 s, 56.degree. C.
20 s, 72.degree. C. 20 s for 38 cycles with an extended last
extension of 3 min. Aliquots of amplification product were digested
for 4 hrs with 5 U of the relevant restriction enzymes Eco RV
(Roche Diagnostics, New Zealand) at designated temperature
conditions. Digested products were separated on 8% polyacrylamide
gels (49:1, Sigma). The products were visualised by ultraviolet
transillumination following ethidium bromide staining and migration
compared against a 1 Kb plus ladder standard (Invitrogen).
Genotypes were recorded in data spreadsheets and statistical
analysis performed.
Genotyping of the 3' 1237 G/A (T/T) Polymorphism of the
a1-Antitrypsin Gene
[0249] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described [Sandford A J et al., [6], each of
which is herein incorporated by reference in its entirety].
Genotyping was done using minor modifications of the above protocol
optimised for laboratory conditions The PCR reactions were
amplified in MJ Research thermocyclers in a total volume of 25
.mu.l and contained 80 ng genomic DNA, 100 ng forward and reverse
primers, 0.2 mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.5 mM
MgCl.sub.2 and 1.0 unit of Taq polymerase (Qiagen). Forward and
reverse prime sequences were 5'-CTACCAGGAATGGCCTTGTCC-3'
[SEQ.ID.NO.136] and 5'-CTCTCAGGTCTGGTGTCATCC-3' [SEQ.ID.NO.137].
Cycling conditions consisted of 94 C 60 s, 56 C 20 s, 72 C 20 s for
38 cycles with an extended last extension of 3 min. Aliquots of
amplification product were digested for 4 hrs with 2 Units of the
restriction enzymes Taq 1 (Roche Diagnostics, New Zealand) at
designated temperature conditions. Digested products were separated
on 3% agarose. The products were visualised by ultraviolet
transillumination following ethidium bromide staining and migration
compared against a 1 Kb plus ladder standard (Invitrogen).
Genotypes were recorded in data spreadsheets and statistical
analysis performed.
Genotyping of the Asp 299 Gly Polymorphism of the Toll-Like
Receptor 4 Gene
[0250] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described [6]. Genotyping was done using minor
modifications of the above protocol optimised for laboratory
conditions The PCR reactions were amplified in MJ Research
thermocyclers in a total volume of 25 .mu.l and contained 80 ng
genomic DNA, 100 ng forward and reverse primers, 0.2 mM dNTPs, 10
mM Tris-HCL (pH 8.4), 150 mM KCl, 1.5 mM MgCl.sub.2 and 1.0 unit of
Taq polymerase (Qiagen). Forward and reverse prime sequences were
5'-GATTAGCATACTTAGACTACTACCTCCATG-3' [SEQ.ID.NO.138] and
5'-GATCAACTTCTGAAAAAGCATTCCCAC-3' [SEQ.ID.NO.139]. Cycling
conditions consisted of 94.degree. C. 30 s, 55.degree. C. 30 s,
72.degree. C. 30 s for 30 cycles with an extended last extension of
3 min. Aliquots of amplification product were digested for 4 hrs
with 2 U of the restriction enzyme Nco I (Roche Diagnostics, New
Zealand) at designated temperature conditions. Digested products
were separated on 3% agarose gel. The products were visualised by
ultraviolet transillumination following ethidium bromide staining
and migration compared against a 1 Kb plus ladder standard
(Invitrogen). Genotypes were recorded in data spreadsheets and
statistical analysis performed.
Genotyping of the -1607 1G2G Polymorphism of the Matrix
Metalloproteinase 1 Gene
[0251] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described [Dunleavey L, et al]. Genotyping was
done using minor modifications of the above protocol optimised for
laboratory conditions The PCR reactions were amplified in MJ
Research thermocyclers in a total volume of 25 .mu.l and contained
80 ng genomic DNA, 100 ng forward and reverse primers, 200 mM
dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 1.5 mM MgCl.sub.2 and
1.0 unit of Taq polymerase (Qiagen). Forward and reverse prime
sequences were 3' TCGTGAGAATGTCTTCCCATT-3' [SEQ.ID.NO.140] and
5'-TCTTGGATTGATTTGAGATAAGTGAAATC-3' [SEQ.ID.NO.141]. Cycling
conditions consisted of 94 C 60 s, 55 C 30 s, 72 C 30 s for 35
cycles with an extended last extension of 3 min. Aliquots of
amplification product were digested for 4 hrs with 6 Units of the
restriction enzymes XmnI (Roche Diagnostics, New Zealand) at
designated temperature conditions. Digested products were separated
on 6% polyacrylamide gel. The products were visualised by
ultraviolet transillumination following ethidium bromide staining
and migration compared against a 1 Kb plus ladder standard
(Invitrogen). Genotypes were recorded in data spreadsheets and
statistical analysis performed.
Other Polymorphism Genotyping
[0252] Genomic DNA was extracted from whole blood samples [4].
Purified genomic DNA was aliquoted (10 ng/ul concentration) into 96
well plates and genotyped on a Sequenom.TM. system (Sequenom.TM.
Autoflex Mass Spectrometer and Samsung 24 pin nanodispenser) using
the sequences, amplification conditions and methods described
below.
[0253] The following conditions were used for the PCR multiplex
reaction: final concentrations were for 10.times. Buffer 15 mM
MgCl2 1.25.times., 25 mM MgCl2 1.625 mM, dNTP mix 25 mM 500 uM,
primers 4 uM 100 nM, Taq polymerase (Quiagen hot start) 0.15
u/reaction, Genomic DNA 10 ng/ul. Cycling times were 95.degree. C.
for 15 min, (5.degree. C. for 15 s, 56.degree. C. 30 s, 72.degree.
C. 30 s for 45 cycles with a prolonged extension time of 3 min to
finish. We used shrimp alkaline phosphotase (SAP) treatment (2 ul
to 5 ul PCR reaction) incubated at 35.degree. C. for 30 min and
extension reaction (add 2 ul to 7 ul after SAP treatment) with the
following volumes per reaction of water 0.76 ul, hME 10.times.
termination buffer 0.2 ul, hME primer (10 uM) 1 ul, MassEXTEND
enzyme 0.04 ul.
Sequenom Conditions for the Polymorphisms Genotyping--1
TABLE-US-00035 [0254] SNP_ID TERM WELL 2nd-PCRP 1st-PCRP VDBP - 420
ACT W1 ACGTTGGATGGCTTGTTAACCAGCTT ACGTTGGATGTTTTTCAGACTGGCAG TGCC
[SEQ. ID. NO. 142] AGCG [SEQ. ID. NO. 143] VDBP - 416 ACT W1
ACGTTGGATGTTTTTCAGACTGGCAG ACGTTGGATGGCTTGTTAACCAGCTTT AGCG [SEQ.
ID. NO. 144] GCC [SEQ. ID. NO. 145] ADRB2- ACT W2
ACGTTGGATGTTGCTGGCACCCAATG ACGTTGGATGATGAGAGACATGACGA Gln27Glu GAAG
[SEQ. ID. NO. 146] TGCC [SEQ. ID. NO. 147] GSTP1 -105 ACT W2
ACGTTGGATGTGGTGGACATGGTGAA ACGTTGGATGTGGTGCAGATGCTCAC TGAC [SEQ.
ID. NO. 148] ATAG [SEQ. ID. NO. 149] PAI1 G-675G ACT W2
ACGTTGGATGCACAGAGAGAGTCTGG ACGTTGGATGCTCTTGGTCTTTCCCTC ACAC [SEQ.
ID. NO. 150] ATC [SEQ. ID. NO. 151] IL-11 G518A ACT W3
ACGTTGGATGCCTCTGATCCTCTTTGC ACGTTGGATGAAGAGGGAGTGGAAG TTC [SEQ. ID.
NO. 152] GGAAG [SEQ. ID. NO. 153] NOS3 - 298 ACT W3
ACGTTGGATGACAGCTCTGCATTCAG ACGTTGGATGAGTCAATCCCTTTGGT CACG [SEQ.
ID. NO. 154] GCTC [SEQ. ID. NO. 155] IL-8 A-251T CGT W5
ACGTTGGATGACTGAAGCTCCACAAT ACGTTGGATGGCCACTCTAGTACTAT TTGG [SEQ.
ID. NO. 156] ATCTG [SEQ. ID. NO. 157] IL-18 C-133G ACT W6
ACGTTGGATGGGGTATTCATAAGCTG ACGTTGGATGCCTTCAAGTTCAGTGG AAAC [SEQ.
ID. NO. 158] TCAG [SEQ. ID. NO. 159] IL-18 A105C ACT W8
ACGTTGGATGGGTCAATGAAGAGAA ACGTTGGATGAATGTTTATTGTAGAA CTTGG [SEQ.
ID. NO. 160] AACC [SEQ. ID. NO. 161]
Sequenom Conditions for the Polymorphisms Genotyping--2
TABLE-US-00036 [0255] SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC
PWARN UEP_DIR VDBP - 420 99 99.7 99.7 46.2 53.3 ML R VDBP - 416 99
99.7 99.7 45.5 33.3 M F ADRB2-Gln27Glu 118 96.6 80 52.2 66.7 L F
GSTP1 -105 107 99.4 80 49.9 52.9 F PAI1 G-675G 109 97.9 80 59.3
66.7 g F IL-11 G518A 169 97.5 65 52.9 52.6 s F NOS3 - 298 186 98.1
65 61.2 63.2 F IL-8 A-251T 119 92.6 81.2 45.9 28.6 R IL-18 C-133G
112 93.5 74.3 41.8 46.7 L F IL-18 A105C 121 67.2 74.3 48.9 40 R
Sequenom Conditions for the Polymorphisms Genotyping--3
TABLE-US-00037 [0256] SNP_ID UEP_MASS UEP_SEQ EXT1_CALL EXT1_MASS
VDBP - 420 4518.9 AGCTTTGCCAGTTCC[SEQ. ID. NO. 162] A 4807.1 VDBP -
416 5524.6 AAAAGCAAAATTGCCTGA[SEQ. ID. NO. T 5812.8 163] ADRB2-
4547 CACGACGTCACGCAG[SEQ. ID. NO. 164] C 4820.2 Gln27Glu GSTP1 -105
5099.3 ACCTCCGCTGCAAATAC[SEQ. ID. NO. 165] A 5396.5 PAI1 G-675G
5620.6 GAGTCTGGACACGTGGGG[SEQ. ID. NO. DEL 5917.9 166] IL-11 G518A
5705.7 TCCATCTCTGTGGATCTCC[SEQ. ID. NO. A 6002.9 167] NOS3 - 298
5813.8 TGCTGCAGGCCCCAGATGA[SEQ. ID. NO. T 6102 168] IL-8 A-251T
6428.2 CACAATTTGGTGAATTATCAA[SEQ. ID. A 6716.4 NO. 169] IL-18
C-133G 4592 AGCTGAAACTTCTGG[SEQ. ID. NO. 170] C 4865.2 IL-18 A105C
6085 TCAAGCTTGCCAAAGTAATC[SEQ. ID. A 6373.2 NO. 171]
Sequenom Conditions for the Polymorphisms Genotyping--4
TABLE-US-00038 [0257] SNP_ID EXT1_SEQ EXT2_CALL EXT2_MASS EXT2_SEQ
1stPAUSE VDBP - 420 AGCTTTGCCAGTTCCT C 5136.4 AGCTTTGCCAGTTCCGT
4848.2 [SEQ. ID. NO. 172] [SEQ. ID. NO. 173] VDBP - 416
AAAAGCAAAATTGCCTGA G 6456.2 AAAAGCAAAATTGCCTGAG 5853.9 T [SEQ. ID.
NO. 174] GC [SEQ. ID. NO. 175] ADRB2- CACGACGTCACGCAGC G 5173.4
CACGACGTCACGCAGGA 4876.2 Gln27Glu [SEQ. ID. NO. 176] [SEQ. ID. NO.
177] GSTP1 -105 ACCTCCGCTGCAAATACA G 5716.7 ACCTCCGCTGCAAATACGT
5428.5 [SEQ. ID. NO. 178] [SEQ. ID. NO. 179] PAI1 G-675G
GAGTCTGGACACGTGGGG G 6247.1 GAGTCTGGACACGTGGGGG 5949.9 A [SEQ. ID.
NO. 180] A [SEQ. ID. NO. 181] IL-11 G518A TCCATCTCTGTGGATCTCC G
6323.1 TCCATCTCTGTGGATCTCC 6034.9 A [SEQ. ID. NO. 182] GT [SEQ. ID.
NO. 183] NOS3 - 298 TGCTGCAGGCCCCAGATG G 6416.2 TGCTGCAGGCCCCAGATGA
6143 AT [SEQ. ID. NO. 184] GC [SEQ. ID. NO. 185] IL-8 A-251T
CACAATTTGGTGAATTAT T 7029.6 CACAATTTGGTGAATTATC 6741.4 CAAT [SEQ.
ID. NO. 186] AAAT [SEQ. ID. NO. 187] IL-18 C-133G AGCTGAAACTTCTGGC
G 5218.4 AGCTGAAACTTCTGGGA 4921.2 [SEQ. ID. NO. 188] [SEQ. ID. NO.
189] IL-18 A105C TCAAGCTTGCCAAAGTAA C 7040.6 TCAAGCTTGCCAAAGTAAT
6414.2 TCT [SEQ. ID. NO. 190] CGGA[SEQ. ID. NO. 191]
[0258] Results
[0259] Frequencies of individual polymorphisms are as follows:
TABLE-US-00039 TABLE 6 Polymorphism allele and genotype frequency
in the OCOPD patients, exposed resistant smokers and controls.
Cyclo-oxygenase 2 -765 G/C Allele* Genotype Frequency C G CC CG GG
Controls n = 95 (%) 27 (14%) 161 (86%) 3 (3%) 21 (22%) 70 (75%)
OCOPD n = 82 (%) 22 (13%) 142.sup.4 (87%).sup. 2 (2%) 18 (22%)
62.sup.3 (76%).sup. Resistant n = 87 (%) 42.sup.2 (24%).sup. 132
(76%) 6.sup.1 (7%).sup. 30.sup.1 (34%).sup. 51 (59%) Glutathione S
Transferase P1 Ile 105 Val (A/G) Allele* Genotype Frequency A G AA
AG GG Controls n = 186 (%) 234 (63%) 138 (37%) 71 (38%) 92 (50%) 23
(12%) OCOPD n = 123 (%) 159 (65%) 87 (36%) 52 (42%) 55 (45%)
16.sup.5 (13%).sup. Resistant n = 98 (%) 136 (69%) 60 (31%) 44
(45%) 48 (49%) 6 (6%) Interleukin 18 105 C/A Allele* Genotype
Frequency C A CC AC AA Controls n = 185 (%) 119 (32%) 251 (68%) 22
(12%) 75 (40%) .sup. 88 (48%) OCOPD n = 122 (%) 62 (25%) 182 (75%)
12 (10%) 38 (31%) 72.sup.6,7 (59%) Resistant n = 98 (%) 60 (31%)
136 (69%) 6 (6%) 48 (49%) .sup. 44 (45%) Interleukin 18 -133 G/C
Allele* Genotype Frequency G C GG GC CC Controls n = 188 (%) 121
(32%) 255 (68%) 23 (12%) 75 (40%) .sup. 90 (48%) OCOPD n = 122 62
(25%) 182 (75%) 12 (10%) 38 (31%) 72.sup.8,9 (59%) Resistant n = 97
(%) 60 (31%) 134 (69%) 6 (6%) 48 (50%) .sup. 43 (44%) Interleukin 8
-251 A/T Allele* Genotype Frequency A T AA AT TT Controls n = 188
(%) 175 (47%) 201 (53%) .sup. 39 (21%) 97 (52%) 52 (28%) OCOPD n =
116 101 (44%) 131 (56%) .sup. 21 (18%) 59 (51%) 36 (31%) Resistant
n = 93 (%) 94.sup.11 (50%) 92 (49%) 26.sup.10 (28%) 42 (45%) 25
(27%) Vitamin D Binding Protein Lys 420 Thr (A/C) Allele* Genotype
Frequency A C AA AC CC Controls n = 189 (%) .sup. 113 (30%) 265
(70%) .sup. 17 (9%) 79 (42%) .sup. 93 (49%) OCOPD n = 122 (%) .sup.
62 (25%) 182 (75%) .sup. 5 (4%) 52 (43%) 65.sup.14 (53%) Resistant
n = 99 (%) 73.sup.13 (37%) 125 (63%) 12.sup.12 (12%) 49 (50%) .sup.
38 (38%) Vitamin D Binding Protein Glu 416 Asp (T/G) Allele*
Genotype Frequency T G TT TG GG Controls n = 189 (%) .sup. 163
(43%) 215 (57%) .sup. 35 (19%) .sup. 93 (49%) .sup. 61 (32%) OCOPD
n = 122 (%) .sup. 109 (45%) 135 (55%) .sup. 25 (21%) .sup. 59 (48%)
38.sup.17 (31%) Resistant n = 99 (%) 103.sup.16 (52%) 95 (48%)
23.sup.15 (23%) 57.sup.15 (58%) .sup. 19 (19%) Microsomal epxoide
hydrolase R/r Exon 3 T/C Allele* Genotype Frequency r R rr Rr RR
Controls n = 184 (%) 228 (62%) 140 (38%) 77 (42%) 74 (40%) .sup. 33
(18%) OCOPD n = 98 (%) 144 (74%) 52 (26%) 55 (56%) 34 (35%) .sup. 9
(9%) Resistant n = 102 (%) 135 (66%) 69 (34%) 52 (51%) 31 (30%)
19.sup.18 (19%) Super oxide dismutase 3 Arg 312 Gln Allele*
Genotype Frequency A G AA AG GG Controls n = 190 (%) .sup. 371
(98%) .sup. 9 (2%) 183 (96%) .sup. 5 (3%) .sup. 2 (1%) OCOPD n =
100 (%) 199.sup.20 (99%) .sup. 1 (1%) 99 (99%) .sup. 1 (1%) .sup. 0
(0%) Resistant n = 102 (%) .sup. 193 (95%) 11.sup.20 (5%) 92 (90%)
9.sup.19 (9%) 1.sup.19 (1%) a1-antitrypsin S Allele* Genotype
Frequency M S MM MS SS OCOPD n = 88 (%) 171 (97%) .sup. 5 (3%) 83
(94%) .sup. 5 (6%) 0 (0%) Resistant n = 94 (%) 175 (93%) 13.sup.22
(7%) 81 (86%) 13.sup.21 (14%) 0 (0%) Toll-like receptor 4 Asp 299
Gly A/G Allele* Genotype Frequency A G AA AG GG OCOPD n = 60 (%)
117 (98%) 1 (2%) 58 (98%) .sup. 1 (2%) 0 (0%) Resistant n = 34 (%)
65 (96%) 3 (4%) 31 (91%) 3.sup.23 (9%) 0 (0%) Beta2-adrenoreceptor
Gln 27 Glu Allele* Genotype Frequency C G CC CG GG Controls n = 186
(%) 204 (55%) 168 (45%) .sup. 57 (31%) 90 (48%) 39 (21%) OCOPD n =
122 (%) 129 (53%) 115 (47%) .sup. 32 (26%) 65 (53%) 25 (21%)
Resistant n = 99 (%) 117 (59%) 81 (41%) 38.sup.24 (38%) 41 (41%) 20
(20%) Interleukin 11 (IL-11) -518 G/A Allele* Genotype Frequency A
G AA AG GG OCOPD n = 119 (%) 110 (46%) 128 (54%) .sup. 22 (19%) 66
(55%) 31 (26%) Resistant n = 98 (%) 103 (53%) 93 (47%) 26.sup.25
(27%) 51 (52%) 21 (21%) Interleukin-13 -1055 C/T Allele* Genotype
Frequency T C TT TC CC Controls n = 182 (%) 65 (18%) 299 (82%)
.sup. 5 (3%) 55 (30%) 122 (67%) OCOPD n = 121 (%) 53 (22%) 189
(78%) 5.sup.26 (4%) 43 (36%) 73 (60%) Resistant n = 97 (%) 31 (16%)
163 (84%) .sup. 1 (1%) 29 (30%) 67 (69%) Plasminogen activator
inhibitor 1 -675 4G/5G Allele* Genotype Frequency 5G 4G 5G5G 5G4G
4G4G Controls n = 186 (%) .sup. 158 (42%) 214 (58%) .sup. 31 (17%)
96 (52%) 59 (32%) OCOPD n = 122 (%) 115.sup.28 (47%) 129 (53%)
29.sup.27 (24%) 57 (47%) 36 (30%) Resistant n = 98 (%) .sup. 76
(39%) 120 (61%) .sup. 14 (14%) 48 (49%) 36 (37%) Nitric oxide
synthase 3 Asp 298 Glu (T/G) Allele* Genotype Frequency T G TT TG
GG Controls n = 183 (%) 108 (30%) 258 (70%) .sup. 13 (7%) 82 (45%)
88 (48%) OCOPD n = 120 (%) 71 (30%) 169 (70%) .sup. 10 (8%) 51
(43%) 59 (49%) Resistant n = 99 (%) 71 (36%) 127 (64%) 15.sup.29,30
(15%) 41 (41%) 43 (43%) a1-antitrypsin 3' 1237 G/A (T/t) Allele*
Genotype Frequency T t TT Tt tt Controls n = 178 (%) 345 (97%) 11
(3%) .sup. 167 (94%) 11 (6%) .sup. 0 (0%).sup. COPD n = 61 (%) 109
(89%) 13 (11%).sup.32 50 (82%) 9 (15%).sup.31 2 (3%).sup.31
Resistant n = 35 (%) 67 (96%) 3 (4%).sup. 32 (91%) 3 (9%).sup. 0
(0%).sup. Matrix metalloproteinase 1 -1607 1G/2G Allele* Genotype
Frequency 1G 2G 1G1G 1G2G 2G2G Controls n = 174 (%) 214 (61%) 134
(39%).sup. 68 (39%) 78 (45%) 28 (16%).sup. COPD n = 93 (%) 90 (48%)
96 (52%).sup.34 24 (26%) 42 (45%) 27 (29%).sup.33 Resistant n = 94
(%) 99 (53%) 89 (47%).sup. 25 (27%) 49 (52%) 20(21%).sup. *number
of chromosomes (2n)
[0260] 1. Genotype. CC/CG vs GG for resistant vs OCOPD, Odds ratio
(OR)=2.2, 95% confidence limits=1.1-4.8, .chi..sup.2 (Yates
corrected)=4.76, P=1.03, CC/CG=protective [0261] 2. Allele. C vs G
for resistant vs OCOPD, Odds ratio (OR)=2.1, 95% confidence limits
1.1-3.8, .chi..sup.2 (Yates corrected)=5.65, p=0.02. C=protective
[0262] 3. Genotype. GG vs CG/CC for OCOPD vs resistant, Odds ratio
(OR)=0.5, 95% confidence limits=0.2-0.9, .chi..sup.2 (Yates
corrected)=4.76, P=0.03. GG=susceptible [0263] 4. Allele. G vs C
for OCOPD vs resistant, Odds ratio (OR)=0.5, 95% confidence limits
0.3-0.9, .chi..sup.2 (Yates corrected)=5.65, p=0.02. G=susceptible
[0264] 5. Genotype. GG vs AG/AA for OCOPD vs resistant, Odds ratio
(OR)=2.3, 95% confidence limits=0.8-6.9, .chi..sup.2 (Yates
uncorrected)=2.88, p=0.09. GG genotype=susceptible [0265] 6.
Genotype. AA vs AC/CC for OCOPD vs resistant, Odds ratio (OR)=1.8,
95% confidence limits=1.0-3.1, .chi..sup.2 (Yates corrected)=3.8,
p=0.05. AA=susceptibility [0266] 7. Genotype. AA vs AC/CC for OCOPD
vs controls, Odds ratio (OR)=1.6, 95% confidence limits 1.0-2.6,
.chi..sup.2 (Yates uncorrected)=3.86, p=0.05. AA=susceptibility
[0267] 8. Genotype. CC vs CG/GG for OCOPD vs controls, Odds ratio
(OR)=1.6, 95% confidence limits=1.0-2.6, .chi..sup.2 (Yates
uncorrected)=3.68, p=0.05. CC=susceptibility [0268] 9. Genotype. CC
vs CG/GG for OCOPD vs resistant, Odds ratio (OR)=1.8, 95%
confidence limits 1.0-3.2, .chi..sup.2 (Yates corrected)=4.10,
p=0.04. CC=susceptibility [0269] 10. Genotype. AA vs AT/TT for
OCOPD vs resistant, Odds ratio (OR)=1.8, 95% confidence
limits=0.9-3.6, .chi..sup.2 (Yates uncorrected)=2.88, p=0.09.
AA=protective [0270] 11. Allele. A vs T for OCOPD vs resistant,
Odds ratio (OR)=1.3, 95% confidence limits=0.9-2.0, .chi..sup.2
(Yates uncorrected)=2.3, p=0.15. A=protective [0271] 12. Genotype.
AA vs AC/CC for resistant vs OCOPD, Odds ratio (OR)=3.2, 95%
confidence limits=1.0-11.0, .chi..sup.2 (Yates corrected)=3.89,
p=0.05. AA genotype=protective [0272] 13. Allele. A vs C for
resistant vs OCOPD, Odds ratio (OR)=1.7, 95% confidence limits
1.1-2.6, .chi..sup.2 (Yates corrected)=6.24, p=0.01. A
allele=protective [0273] 14. Genotype. CC vs AC/AA for OCOPD vs
resistant, Odds ratio (OR)=1.8, 95% confidence limits=1.0-3.3,
.chi..sup.2 (Yates corrected)=4.29, p=0.04. CC
genotype=susceptibility [0274] 15. Genotype. TT/TG vs GG for
resistant vs OCOPD, Odds ratio (OR)=1.9, 95% confidence
limits=1.0-38, .chi..sup.2 (Yates uncorrected)=4.08, p=0.04. TT/TG
genotype=protective [0275] 16. Allele. T vs G for resistant vs
OCOPD, Odds ratio (OR)=1.3, 95% confidence limits 0.9-2.0,
.chi..sup.2 (Yates uncorrected)=2.36, p=0.12. A allele=protective
[0276] 17. Genotype. GG vs TT/TG for OCOPD vs resistant, Odds ratio
(OR)=0.5, 95% confidence limits=0.3-1.0, .chi..sup.2 (Yates
uncorrected)=4.1, p=0.04. GG genotype=susceptible [0277] 18.
Genotype. RR vs Rr/rr for resistant vs OCOPD, Odds ratio (OR)=2.3,
95% confidence limits=0.9-5.8, .chi..sup.2 (Yates uncorrected)=3.7,
p=0.05, RR genotype=protective [0278] 19. Genotype. AG/GG vs AA for
resistant vs OCOPD, Odds ratio (OR)=10.8, 95% confidence
limits=1.4-229, .chi..sup.2 (Yates corrected)=5.99 p=0.01. AG/GG
genotype=protective, AA susceptible [0279] 20. Allele. G vs A for
resistant vs OCOPD, Odds ratio (OR)=11.3, 95% confidence limits
1.5-237, .chi..sup.2 (Yates corrected)=6.77, p=0.001. G
allele=protective, A susceptible [0280] 21. Genotype. MS vs MM for
Resistant vs OCOPD, Odds ratio (OR)=2.7, 95% confidence limits
0.8-9.0, .chi..sup.2 (Yates uncorrected)=3.4, p=0.07. [0281]
MS=protective [0282] 22. Allele: S vs M allele for resistant vs
OCOPD, Odds ratio (OR)=2.5, 95% confidence limits 0.8-8.4,
.chi..sup.2 (Yates uncorrected)=3.24, p=0.07. [0283] 23. Genotype
AG vs AA in resistant vs OCOPD, Odd's Ratio (OR)=5.61, 95%
confidence limits 0.5-146, .chi..sup.2 (Yates uncorrected)=2.66,
p=0.10. AG=protective [0284] 24. Genotype. CC vs CG/GG for
resistant vs OOCOPD, Odds ratio (OR)=1.75, 95% confidence
limits=1.0-3.2, .chi..sup.2 (Yates uncorrected)=3.73, p=0.05.
CC=protective [0285] 25. Genotype: AA vs AG/GG for resistant vs
OCOPD, Odd's Ratio (OR)=1.6, 95% confidence limits 0.8-32,
.chi..sup.2 (Yates uncorrected)=2.02, p=0.16. AA=protective [0286]
26. Genotype. TT vs TC/CC for OCOPD vs resistant, Odds ratio
(OR)=6.03, 95% confidence limits 1.1-42, .chi..sup.2 (Yates
corrected)=4.9, p=0.03. TT=susceptible [0287] 27. Genotype. 5G5G vs
rest for OCOPD vs resistant, Odds ratio (OR)=1.9, 95% confidence
limits 0.9-4.0, .chi..sup.2 (Yates uncorrected)=3.11, p=0.08.
5G5G=susceptible [0288] 28. Allele. 50 vs 4G for OCOPD vs
resistant, Odds ratio (OR)=1.4, 95% confidence limits 0.9-2.1,
.chi..sup.2 (Yates corrected)=3.1, p=0.08. 5G=susceptible [0289]
29. Genotype. TT vs TG/GG for resistant vs controls, Odds ratio
(OR)=2.3, 95% confidence limits 1.0-5.5, .chi..sup.2 (Yates
corrected)=3.80, p=0.05. TT genotype=protective [0290] 30.
Genotype. TT vs TG/GG for resistant vs OCOPD, Odds ratio (OR)=1.9,
95% confidence limits 0.8-5.0, .chi..sup.2 (Yates
uncorrected)=2.49, p=0.11. TT genotype=protective [0291] 31.
Genotype: Tt/tt vs TT for COPD vs controls, Odd's Ratio (OR)=3.34,
95% confidence limits 1.3-8.9, .chi..sup.2 (Yates corrected)=6.28,
p=0.01. Tt/tt susceptible to OCOPD [0292] 32. Allele: t vs T for
COPD vs controls, Odd's Ratio (OR)=2.5, 95% confidence limits
1.0-6.3, .chi..sup.2 (Yates corrected)=4.1, p=0.04. t=susceptible
to OCOPD [0293] 33. Genotype. 2G2G vs 1G1G/1G2G for COPD vs
controls, Odds ratio (OR)=2.1, 95% confidence limits 1.1-4.1,
.chi..sup.2 (Yates corrected)=5.44, p=0.02. 2G2G
genotype=susceptible for OCOPD [0294] 34. Allele. 2G vs 1G for COPD
vs controls, Odds ratio (OR)=1.7, 95% confidence limits 1.2-2.5,
.chi..sup.2 (Yates corrected)=7.97, p=0.005. 2G=susceptible for
OCOPD
[0295] Table 7 below provides a summary of the protective and
susceptibility polymorphisms determined for OCOPD.
TABLE-US-00040 TABLE 7 Summary of protective and susceptibility
polymorphisms for OCOPD Gene Polymorphism Role Cyclo-oxygenase
(Cox) 2 Cox 2 -765 G/C CC/CG protective GG susceptible
.beta.2-adrenoreceptor (ADRB2) ADRB2 Gln 27Glu CC protective
Interleukin-18 (IL-18) IL-18 -133 C/G CC susceptible Interleukin-18
(IL-18) IL-18 105 A/C AA susceptible Plasminogen activator PAI-1
-675 4G/5G 5G5G inhibitor 1 (PAI-1) susceptible Nitric Oxide
synthase 3 (NOS3) NOS3 298 Asp/Glu TT protective Vitamin D Binding
Protein VDBR Lys 420 Thr AA protective (VDBR) CC susceptible
Vitamin D Binding Protein VDBP Glu 416 Asp TT/TG protective (VDBR)
GG susceptible Glutathione S Transferase GSTP1 Ile105Val GG
susceptible (GSTP1) Superoxide dismutase 3 (SOD3) SOD3 Arg 312 Gln
AG/GG protective AA susceptible a1-antitrypsin (a1AT) a1AT 3' 1237
G/A Tt/tt susceptible (T/t) a1-antitrypsin (a1AT) a1AT S allele MS
protective Toll-like receptor 4 (TLR4) TLR4 Asp 299 Gly AG/GG A/G
protective Interleukin-8 (IL-8) IL-8 -251 A/T AA protective
Interleukin 11 (IL-11) IL-11 -518 G/A AA protective Microsomal
epoxide MEH Exon 3 T/C RR protective hydrolase (MEH) (r/R)
Interleukin 13 (IL-13) IL-13 -1055 C/T TT susceptible Matrix
Metalloproteinase 1 MMP1 -1607 2G2G susceptible (MMP1) 1G/2G
[0296] The combined frequencies of the presence or absence of the
selected protective genotypes COX2 -765 CC/CG, NOS3 298 TT, a 1AT
MS/SS, SOD3 AG/GG, MEH Exon 3 RR, and VDBP 420 AA observed in the
OCOPD subjects and in resistant smokers is presented below in Table
8.
TABLE-US-00041 TABLE 8 Combined frequencies of the presence or
absence of protective genotypes in OCOPD subjects and in resistant
smokers. Number of protective polymorphisms Cohorts 0 1 =2 Total
OCOPD 34 (27%) 51 (41%) 39 (32%) 124 Resistant 20 (19%) 31 (30%) 53
(51%) 104 smokers % of 34/54 (63%) 51/82 (62%) 39/92 (42%) smokers
with OCOPD Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+,
Resist vs OCOPD -- -- 16.2 0.003 2+ vs 0-1, Resist vs OCOPD 2.3
1.3-4.0 8.15 0.004 0 vs 2+, OCOPD vs Resist 2.3 1.1-4.9 4.97
0.03
[0297] The combined frequencies of the presence or absence of the
selected susceptibility genotypes MMP1-1607 2G2G, GSTP1 105 GG,
PAI-1-675 5G5G, IL-13 -1055 TT, and VDBP 416 GG, observed in the
OCOPD subjects and in resistant smokers is presented below in Table
9.
TABLE-US-00042 TABLE 9 Combined frequencies of the presence or
absence of selected susceptibility genotypes in OCOPD subjects and
in resistant smokers. Number of protective polymorphisms Cohorts 0
1 =2 Total OCOPD 45 (36%) 55 (44%) 24 (20%) 124 Resistant 55 (54%)
37 (37%) 9 (9%) 101 smokers % of 45/100 (45%) 55/92 (60%) 24/33
(73%) smokers with OCOPD Comparison Odd's ratio 95% CI ?2 P value 0
vs 1 vs 2+, OCOPD vs Resist -- -- 9.1 0.01 2+ vs 0-1, OCOPD vs
Resist 2.5 1.0-6.0 4.05 0.04 0+ vs 1-2+, Resist vs OCOPD 2.1
1.2-3.7 6.72 0.01
[0298] Protective polymorphisms were assigned a score of +1 while
susceptibility polymorphisms were assigned a score of -1. For each
subject, a net score was then calculated according to the presence
of susceptibility and protective genotypes. This produced a linear
spread of values, as shown in Table 10. When assessed as a range
between -2 to +3, a linear relationship as depicted in FIG. 3 was
observed. This analysis indicates that for subjects with a net
score of -1 or less, there was an approximately 70% or greater risk
of having OCOPD. In contrast, for subjects with a net score of 2+
or greater, the risk was approximately 25% (see FIG. 3). As a point
of clarification, it is noted that FIG. 3 depicts the sum of the
protective and susceptibility polymorphisms combined, rather than
simply the sum of the protective polymorphisms in one graph and the
sum of the susceptibility polymorphisms in another graph. Thus, the
SNP score can be negative if there are only susceptibility
polymorphisms, positive, if there are only protective
polymorphisms, or either positive or negative, depending upon the
relative numbers of protective to susceptibility polymorphisms.
TABLE-US-00043 TABLE 10 Combined presence or absence of protective
and susceptibility polymorphisms Score combining protective and
susceptibility polymorphisms -2 -1 0 1 2 3 OCOPD n = 124 8 28 33 39
11 5 Resistant n = 101 2 11 23 27 23 15 % OCOPD 80% 72% 59% 59% 32%
25%
II. Example 3
Case Association Study--Lung Cancer
Methods
Subject Recruitment
[0299] Subjects of European decent who had smoked a minimum of
fifteen pack years and diagnosed with lung cancer were recruited.
Subjects met the following criteria: diagnosed with lung cancer
based on radiological and histological grounds, including primary
lung cancers with histological types of small cell lung cancer,
squamous cell lung cancer, adenocarinoma of the lung, non-small
cell cancer (where histological markers can not distinguish the
subtype) and broncho-alveolar carcinoma. Subjects can be of any age
and at any stage of treatment after the diagnosis had been
confirmed. One hundred and nine subjects were recruited, of these
58% were male, the mean FEV1/FVC (.+-.95% confidence limits) was
51% (49-53), mean FEV1 as a percentage of predicted was 43 (41-45).
Mean age, cigarettes per day and pack year history was 65 yrs
(64-66), 24 cigarettes/day (22-25) and 50 pack years (41-55)
respectively. Two hundred and seventeen European subjects who had
smoked a minimum of twenty pack years and who had never suffered
breathlessness and had not been diagnosed with an obstructive lung
disease or lung cancer in the past were also studied. This control
group was recruited through clubs for the elderly and consisted of
63% male, the mean FEV1/FVC (95% CI) was 82% (81-83), mean FEV1 as
a percentage of predicted was 96 (95-97). Mean age, cigarettes per
day and pack year history was 59 yrs (57-61), 24 cigarettes/day
(22-26) and 42 pack years (39-45) respectively. Using a PCR based
method [1], all subjects were genotyped for the
.alpha.1-antitrypsin mutations (S and Z alleles) and those with the
ZZ allele were excluded. 190 European blood donors (smoking status
unknown) were recruited consecutively through local blood donor
services. Sixty-three percent were men and their mean age was 50
years. On regression analysis, the age difference and pack years
difference observed between lung cancer sufferers and resistant
smokers was found not to determine FEV or lung cancer.
[0300] This study shows that polymorphisms found in greater
frequency in lung cancer patients compared to resistant smokers can
reflect an increased susceptibility to the development of lung
cancer. Similarly, polymorphisms found in greater frequency in
resistant smokers compared to lung cancer can reflect a protective
role.
[0301] Summary of Characteristics.
TABLE-US-00044 Parameter Lung Cancer Resistant smokers Median (IQR)
N = 109 N = 200 Differences % male 52% 64% ns Age (yrs) 68 (11) 60
(12) P < 0.05 Pack years 40 (31) 43 (25) P < 0.05
Cigarettes/day 18 (11) 24 (12) ns FEV1 (L) 1.7 (0.6) 2.8 (0.7) P
< 0.05 FEV1 % predict 67 (22) 96% (10) P < 0.05 FEV1/FVC 59
(14) 82 (8) P < 0.05
[0302] Means and 95% confidence limits
Glutathione S-Transferase Null Polymorphisms Genotyping
[0303] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described [7, herein incorporated by reference
in its entirety]. Genotyping was done using minor modifications of
the above protocol optimised for our own laboratory conditions The
PCR reactions were amplified in MJ Research thermocyclers in a
total volume of 25 .mu.l and contained 80 ng genomic DNA, 100 ng
forward and reverse primers, 200 mM dNTPs, 20 mM Tris-HCL (pH 8.4),
50 mM KCl, 2.5 mM MgCl2 and 1.0 unit of Taq polymerase (Qiagen).
Forward, internal (GSTM4) and reverse prime sequences were 5'
CTGCCCTACTTGATTGATGG-3' [SEQ.ID.NO.192], 5' ATCTTCTCCTCTTCTGTCTC-3'
[SEQ.ID.NO.193] and 5'-TTCTGGATTGTAGCAGATCA-3' [SEQ.ID.NO.194].
Cycling conditions consisted of 94 C 60 s, 59 C 30 s, 72 C 30 s for
35 cycles with an extended last extension of 3 min. Digested
products were separated on 3% agarose gel. The products were
visualised by ultraviolet transillumination following ethidium
bromide staining and migration compared against a 1 Kb plus ladder
standard (Invitrogen). Genotypes were recorded in data spreadsheets
and statistical analysis performed.
Cyclooxygenase 2 Polymorphisms Genotyping
[0304] Genomic DNA was extracted from whole blood samples
(Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning
Manual. 1989). The Cyclo-oxygenase 2 -765 polymorphism was
determined by minor modifications of a previously published method
(Papafili A, et al., 2002, incorporated in its entirety herein by
reference)). The PCR reaction was carried out in a total volume of
25 ul and contained 20 ng genomic DNA, 500 pmol forward and reverse
primers, 0.2 mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.0 mM
MgCl.sub.2 and 1 unit of polymerase (Life Technologies). Cycling
times were incubations for 3 min at 95.degree. C. followed by 33
cycles of 50 s at 94.degree. C., 60 s at 66.degree. C. and 60 s at
72.degree. C. A final elongation of 10 min at 72.degree. C. then
followed. 4 ul of PCR products were visualised by ultraviolet
trans-illumination of a 3% agarose gel stained with ethidium
bromide. An aliquot of 3 ul of amplification product was digested
for 1 hr with 4 units of AciI (Roche Diagnostics, New Zealand) at
37.degree. C. Digested products were separated on a 2.5% agarose
gel run for 2.0 hours at 80 mV with TBE buffer. The products were
visualised against a 123 bp ladder using ultraviolet
transillumination after ethidium bromide staining.
Matrix Metalloproteinase 1 -1607 1G/2G Polymorphisms Genotyping
[0305] Genomic DNA was extracted using standard phenol and
chloroform methods. Cohorts of patients and controls were
configured in to 96-well PCR format containing strategic negative
controls. The assay primers, PCR conditions and RFLP assays details
have been previously described (Dunleavey, L. et al. Rapid genotype
analysis of the matrix metalloproteinase-1 gene 1G/2G polymorphism
that is associated with risk of cancer. Matrix Biol. 19(2):175-7
(2000), herein incorporated by reference in its entirety).
Genotyping was done using minor modifications of the above protocol
optimised for our own laboratory conditions The PCR reactions were
amplified in MJ Research thermocyclers in a total volume of 25
.mu.l and contained 80 ng genomic DNA, 100 ng forward and reverse
primers, 200 mM dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 1.5 mM
MgCl.sub.2 and 1.0 unit of Taq polymerase (Qiagen). Forward and
reverse prime sequences were 3' TCGTGAGAATGTCTTCCCATT-3'
[SEQ.ID.NO.195] and
5'-TCTTGGATTGATTTGAGATAAGTGAAATC-3'[SEQ.ID.NO.196]. Cycling
conditions consisted of 94 C 60 s, 55 C 30 s, 72 C 30 s for 35
cycles with an extended last extension of 3 min Aliquots of
amplification product were digested for 4 hrs with 6 Units of the
restriction enzymes XmnI (Roche Diagnostics, New Zealand) at
designated temperature conditions. Digested products were separated
on 6% polyacrylamide gel. The products were visualised by
ultraviolet transillumination following ethidium bromide staining
and migration compared against a 1 Kb plus ladder standard
(Invitrogen). Genotypes were recorded in data spreadsheets and
statistical analysis performed.
Polymorphism Genotyping Using the Sequenom Autoflex Mass
Spectrometer
[0306] Genomic DNA was extracted from whole blood samples [2].
Purified genomic DNA was aliquoted (10 ng/ul concentration) into 96
well plates and genotyped on a Sequenom.TM. system (Sequenom.TM.
Autoflex Mass Spectrometer and Samsung 24 pin nanodispenser) using
the following sequences, amplification conditions and methods. The
following conditions were used for the PCR multiplex reaction:
final concentrations were for 10.times. Buffer 15 mM MgCl2
1.25.times., 25 mM MgCl2 1.625 mM, dNTP mix 25 mM 500 uM, primers 4
uM 100 nM, Taq polymerase (Quiagen hot start) 0.15 U/reaction,
Genomic DNA 10 ng/ul. Cycling times were 95.degree. C. for 15 min,
(5.degree. C. for 15 s, 56.degree. C. 30 s, 72.degree. C. 30 s for
45 cycles with a prolonged extension time of 3 min to finish. We
used shrimp alkaline phosphotase (SAP) treatment (2 ul to 5 ul per
PCR reaction) incubated at 35.degree. C. for 30 min and extension
reaction (add 2 ul to 7 ul after SAP treatment) with the following
volumes per reaction of: water, 0.76 ul; hME 10.times. termination
buffer, 0.2 ul; hME primer (10 uM), 1 ul; MassEXTEND enzyme, 0.04
ul.
Sequenom Conditions for the Polymorphisms Genotyping--1
TABLE-US-00045 [0307] TERM SNP_ID 2nd-PCRP 1st-PCRP ACT
CYP2E1_1019G/CPst1 ACGTTGGATGAAACCAGAGGGAAGCAAAGG
ACGTTGGATGTCATTGGTTGTGCTGCACCT [SEQ. ID. NO. 197] [SEQ. ID. NO.
198] ACT XPD-751 G/T ACGTTGGATGCACCAGGAACCGTTTATGGC
ACGTTGGATGAGCAGCTAGAATCAGAGGAG [SEQ. ID. NO. 199] [SEQ. ID. NO.
200] ACT IL-18 105 A/C ACGTTGGATGGTCAATGAAGAGAACTTGGTC
ACGTTGGATGAATGTTTATTGTAGAAAACC [SEQ. ID. NO. 201] [SEQ. ID. NO.
202] ACT IL-18-133 G/C ACGTTGGATGGGGTATTCATAAGCTGAAAC
ACGTTGGATGCCTTCAAGTTCAGTGGTCAG [SEQ. ID. NO. 203] [SEQ. ID. NO.
204] ACT CYP 1A1 Ile462Val ACGTTGGATGGTGATTATCTTTGGCATGGG
ACGTTGGATGGGATAGCCAGGAAGAGAAAG [SEQ. ID. NO. 205] [SEQ. ID. NO.
206] ACT MMP12 Asn 357 Ser A/G ACGTTGGATGCCCTATTTCTTTGTCTTCAC
ACGTTGGATGCTTGGGATAATTTGGCTCTG [SEQ. ID. NO. 207] [SEQ. ID. NO.
208] ACT OGG1 Ser 326 Cys G/C ACGTTGGATGGGAACCCTTTCTGCGCTTTG
ACGTTGGATGCCTACAGGTGCTGTTCAGTG [SEQ. ID. NO. 209] [SEQ. ID. NO.
210] ACT NAT2 Arg 197 Gln A/G ACGTTGGATGCCTGCCAAAGAAGAAACACC
ACGTTGGATGACGTCTGCAGGTATGTATTC [SEQ. ID. NO. 211] [SEQ. ID. NO.
212] ACT CYP2E1_C/T Rsa1 ACGTTGGATGGTTCTTAATTCATAGGTTGC
ACGTTGGATGCTTCATTTCTCATCATATTTTC [SEQ. ID. NO. 213] [SEQ. ID. NO.
214] ACG CCND1 A870G ACGTTGGATGTAGGTGTCTCCCCCTGTAAG
ACGTTGGATGTCCTCTCCAGAGTGATCAAG [SEQ. ID. NO. 215] [SEQ. ID. NO.
216] ACG ILB1-511 A/G ACGTTGGATGATTTTCTCCTCAGAGGCTCC
ACGTTGGATGTGTCTGTATTGAGGGTGTGG [SEQ. ID. NO. 217] [SEQ. ID. NO.
218] ACG FAS_A-670G ACGTTGGATGTTGTGGCTGCAACATGAGAG
ACGTTGGATGCTATGGCGCAACATCTGTAC [SEQ. ID. NO. 219] [SEQ. ID. NO.
220] ACG NOS3-786 T/C ACGTTGGATGACTGTAGTTTCCCTAGTCCC
ACGTTGGATGAGTCAGCAGAGAGACTAGGG [SEQ. ID. NO. 221] [SEQ. ID. NO.
222] ACT ACT_Ala15Thr ACGTTGGATGGAGTTGAGAATGGAGAGAATG
ACGTTGGATGTCAAGTGGGCTGTTAGGGTG [SEQ. ID. NO. 223] [SEQ. ID. NO.
224] ACT SOD3 Arg 312 Gln ACGTTGGATGTGCTGCGTGGTGGGCGTGTG
ACGTTGGATGGGCCTTGCACTCGCTCTCG [SEQ. ID. NO. 225] [SEQ. ID. NO. 226]
ACT NOS3 Asp 298 Glu ACGTTGGATGAAACGGTCGCTTCGACGTGC
ACGTTGGATGACCTCAAGGACCAGCTCGG [SEQ. ID. NO. 227] [SEQ. ID. NO. 228]
CGT IL-8-251 A/T ACGTTGGATGACTGAAGCTCCACAATTTGG
ACGTTGGATGGCCACTCTAGTACTATATCTG [SEQ. ID. NO. 229] [SEQ. ID. NO.
230] CGT IFN gamma 874 A/T ACGTTGGATGCAGACATTCACAATTGATTT
ACGTTGGATGGATAGTTCCAAACATGTGCG [SEQ. ID. NO. 231] [SEQ. ID. NO.
232] ACT XRCC1 Arg 399 Gln G/A ACGTTGGATGTAAGGAGTGGGTGCTGGACT
ACGTTGGATGAGGATAAGGAGCAGGGTTGG [SEQ. ID. NO. 233] [SEQ. ID. NO.
234]
Sequenom Conditions for the Polymorphisms Genotyping--2
TABLE-US-00046 [0308] SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC
PWARN UEP_DIR UEP_MASS CYP2E1_1019G/CPst 1 119 95.2 71.3 46.7 47.1
F 5256.4 XPD -751 G/T 113 97.6 71.3 49.8 47.4 F 5689.7 IL-18 105
A/C 120 65.6 71.3 49.8 36.4 R 6702.4 IL-18 -133 G/C 112 93.5 81.3
47.1 42.1 F 5811.8 CYP 1A1 Ile462Val 102 98.2 81.3 55.6 55 F 6222.1
MMP12 Asn 357 Ser A/G 95 92.6 81.3 48 30.4 F 7070.6 OGG1 Ser 326
Cys G/C 99 96.5 82.2 58.9 70.6 R 5227.4 NAT2 Arg 197 Gln A/G 115
97.4 70 48.5 36.4 F 6635.3 CYP2E1_C/T Rsa1 105 62.8 77.8 46.4 26.1
R 7018.6 CCND1 A870G 106 98.1 83 45.8 47.1 R 5034.3 ILB1 -511 A/G
111 99.2 83 46 47.1 R 5203.4 FAS_A-670G 103 99.2 83 54.4 50 R 6166
NOS3 -786 T/C 114 97.5 83 61.8 61.9 F 6358.1 ACT_Ala15Thr 118 93.4
68.2 45.2 47.1 F 5136.4 SOD3 Arg 312 Gln 119 63.2 68.2 55.5 57.9 F
5855.8 NOS3 Asp 298 Glu 113 82.2 68.2 65.4 66.7 F 6432.2 IL-8 -251
A/T 119 92.6 75.8 45.9 28.6 R 6428.2 IFN gamma 874 A/T 112 75.3
75.8 46.4 26.1 F 6943.6 XRCC1 Arg 399 Gln G/A 109 93.6 93.6 66.8
82.4 F 5099.3
Sequenom Conditions for the Polymorphisms Genotyping--3
TABLE-US-00047 [0309] EXT1 EXT1 EXT2 SNP_ID UEP_SEQ CALL MASS
EXT1_SEQ CALL CYP2E1_1019G/CPst1 TTCTTGGTTCAGGAGAG C 5529.6
TTCTTGGTTCAGGAGAGC G [SEQ. ID. NO. 235] [SEQ. ID. NO. 236] XPD-751
G/T GCAATCTGCTCTATCCTCT T 5977.9 GCAATCTGCTCTATCCTCTT G [SEQ. ID.
NO. 237] [SEQ. ID. NO. 238] IL-18 105 A/C ATTCAAGCTTGCCAAAGTAATC A
6990.6 ATTCAAGCTTGCCAAAGTAAT C [SEQ. ID. NO. 239] CT [SEQ. ID. NO.
240] IL-18 -133 G/C CATAAGCTGAAACTTCTGG C 6085 CATAAGCTGAAACTTCTGGC
G [SEQ. ID. NO. 241] [SEQ. ID. NO. 242] CYP 1A1 Ile462Val
GGAAGTGTATCGGTGAGACC A 6519.3 GGAAGTGTATCGGTGAGACC G [SEQ. ID. NO.
243] A [SEQ. ID. NO. 244] MMP12 Asn 357 Ser TGACAAATACTGGTTAATTAGCA
A 7367.8 TGACAAATACTGGTTAATTAG G A/G [SEQ. ID. NO. 245] CAA [SEQ.
ID. NO. 246] OGG1 Ser 326 Cys GCTCCTGAGCATGGCGG G 5500.6
GCTCCTGAGCATGGCGGC C G/C [SEQ. ID. NO. 247] [SEQ. ID. NO. 248] NAT2
Arg 197 Gln TACTTATTTACGCTTGAACCTC A 6932.5 TACTTATTTACGCTTGAACCT G
A/G [SEQ. ID. NO. 249] CA [SEQ. ID. NO. 250] CYP2E1_C/T Rsa1
CTTAATTCATAGGTTGCAATTTT T 7315.8 CTTAATTCATAGGTTGCAATT C [SEQ. ID.
NO. 251] TTA [SEQ. ID. NO. 252] CCND1 A870G ACATCACCCTCACTTAC[SEQ.
ID. G 5307.5 ACATCACCCTCACTTACC A NO. 253] [SEQ. ID. NO. 254] ILB1
-511 A/G AATTGACAGAGAGCTCC G 5476.6 AATTGACAGAGAGCTCCC A [SEQ. ID.
NO. 255] [SEQ. ID. NO. 256] FAS_A-670G ATGAGAGGCTCACAGACGTT G
6439.2 ATGAGAGGCTCACAGACGTT A [SEQ. ID. NO. 257] C [SEQ. ID. NO.
258] NOS3-786 T/C GGCATCAAGCTCTTCCCTGGC C 6631.3
GGCATCAAGCTCTTCCCTGG T [SEQ. ID. NO. 259] CC [SEQ. ID. NO. 260]
ACT_Ala15Thr GAATGTTACCTCTCCTG[SEQ. ID. A 5433.6 GAATGTTACCTCTCCTGA
G NO. 261] [SEQ. ID. NO. 262] SOD3 Arg 312 Gln GCACTCAGAGCGCAAGAAG
C 6129 GCACTCAGAGCGCAAGAAGC G [SEQ. ID. NO. 263] [SEQ. ID. NO. 264]
NOS3 Asp 298 Glu GCTGCTGCAGGCCCCAGATGA T 6720.4
GCTGCTGCAGGCCCCAGATG G [SEQ. ID. NO. 265] AT [SEQ. ID. NO. 266]
IL-8 -251 A/T CACAATTTGGTGAATTATCAA A 6716.4 CACAATTTGGTGAATTATCAA
T [SEQ. ID. NO. 267] T [SEQ. ID. NO. 268] IFN gamma 874 A/T
TTCTTACAACACAAAATCAAATC T 7231.8 TTCTTACAACACAAAATCAAA A [SEQ. ID.
NO. 269] TCT [SEQ. ID. NO. 270] XRCC1 Arg 399 Gln TCGGCGGCTGCCCTCCC
A 5396.5 TCGGCGGCTGCCCTCCCA G G/A [SEQ. ID. NO. 271] [SEQ. ID. NO.
272]
Sequenom Conditions for the Polymorphisms Genotyping--4
TABLE-US-00048 [0310] SNP_ID EXT2_MASS EXT2_SEQ 1stPAUSE
CYP2E1_1019G/CPst1 5873.8 TTCTTGGTTCAGGAGAGGT[SEQ. ID. NO. 273]
5585.6 XPD -751 G/T 6292.1 GCAATCTGCTCTATCCTCTGC[SEQ. ID. NO. 274]
6018.9 IL-18 105 A/C 7658 ATTCAAGCTTGCCAAAGTAATCGGA[SEQ. ID. 7031.6
NO. 275] IL-18 -133 G/C 6438.2 CATAAGCTGAAACTTCTGGGA[SEQ. ID. NO.
6141 276] CYP 1A1 Ile462Val 6839.5 GGAAGTGTATCGGTGAGACCGT[SEQ. ID.
NO. 6551.3 277] MMP12 Asn 357 Ser A/G 7688
TGACAAATACTGGTTAATTAGCAGT[SEQ. ID. 7399.8 NO. 278] OGG1 Ser 326 Cys
G/C 5853.8 GCTCCTGAGCATGGCGGGA[SEQ. ID. NO. 279] 5556.6 NAT2 Arg
197 Gln A/G 7261.8 TACTTATTTACGCTTGAACCTCGA[SEQ. ID. 6964.5 NO.
280] CYP2E1_C/T Rsa1 7636 CTTAATTCATAGGTTGCAATTTTGT[SEQ. ID. 7347.8
NO. 281] CCND1 A870G 5651.7 ACATCACCCTCACTTACTG[SEQ. ID. NO. 282]
5338.5 ILB1 -511 A/G 5820.8 AATTGACAGAGAGCTCCTG[SEQ. ID. NO. 283]
5507.6 FAS_A-670G 6743.4 ATGAGAGGCTCACAGACGTTTC[SEQ. ID. NO. 6470.2
284] NOS3-786 T/C 6975.5 GGCATCAAGCTCTTCCCTGGCTG[SEQ. ID. 6662.3
NO. 285] ACT_Ala15Thr 5738.7 GAATGTTACCTCTCCTGGC[SEQ. ID. NO. 286]
5465.6 SOD3 Arg 312 Gln 7116.6 GCACTCAGAGCGCAAGAAGGGGC[SEQ. ID.
6185 NO. 287] NOS3 Asp 298 Glu 7034.6 GCTGCTGCAGGCCCCAGATGAGC[SEQ.
ID. 6761.4 NO. 288] IL-8 -251 A/T 7029.6
CACAATTTGGTGAATTATCAAAT[SEQ. ID. NO. 6741.4 289] IFN gamma 874 A/T
7530 TTCTTACAACACAAAATCAAATCAC[SEQ. ID. 7256.8 NO. 290] XRCC1 Arg
399 Gln G/A 6054.9 TCGGCGGCTGCCCTCCCGGA[SEQ. ID. NO. 5428.5
291]
Sequenom Conditions for the Polymorphisms Genotyping--5
TABLE-US-00049 [0311] TERM SNP_ID 2nd-PCRP 1st-PCRP ACT CTGF-447G/C
ACGTTGGATGAGGTAGCTGAAGAG ACGTTGGATGGCCTATAGCCTCTAA GCAAAC [SEQ. ID.
NO. 292] AACGC [SEQ. ID. NO. 293] ACT NBS1 Gln185Glu
ACGTTGGATGCTTTCAATTTGTGGA ACGTTGGATGTGTGCACTCATTTGT G/C GGCTG [SEQ.
ID. NO. 294] GGACG [SEQ. ID. NO. 295] ACT MBL2 161 G/A
ACGTTGGATGGTAGCTCTCCAGGCA ACGTTGGATGGTACCTGGTTCCCCC TCAAC [SEQ. ID.
NO. 296] TTTTC [SEQ. ID. NO. 297] ACT IGF2R Leu252Val
ACGTTGGATGACACCAGGCGTTTGA ACGTTGGATGAAAAACGCCAACAGC C/G TGTTG [SEQ.
ID. NO. 298] ATCGG [SEQ. ID. NO. 299] ACT MUC5AC -221 C/T
ACGTTGGATGAGGCGGAGATGGGT ACGTTGGATGAGTCTAGGGTGGGG GTGTC [SEQ. ID.
NO. 300] TATGTG [SEQ. ID. NO. 301] ACT Arg1 intron1 C/T
ACGTTGGATGATGTGTGGATTCACA ACGTTGGATGGGGTTGGCAACTCTA GCTCG [SEQ. ID.
NO. 302] AAAGG [SEQ. ID. NO. 303] ACT REV1 Phe257Ser
ACGTTGGATGCTCTGAAATCAGTGC ACGTTGGATGATGGTCAACAGTGTT C/T TGCTC [SEQ.
ID. NO. 304] GCCAG [SEQ. ID. NO. 305] ACT Apex1 Asp148Glu
ACGTTGGATGCACCTCTTGATTGCT ACGTTGGATGACCCGGCCTTCCTGA G/T TTCCC [SEQ.
ID. NO. 306] TCATG [SEQ. ID. NO. 307] ACG IL-10 -1082 A/G
ACGTTGGATGATTCCATGGAGGCTG ACGTTGGATGGACAACACTACTAAG GATAG [SEQ. ID.
NO. 308] GCTTC [SEQ. ID. NO. 309]
Sequenom Conditions for the Polymorphisms Genotyping--6
TABLE-US-00050 [0312] SNP_ID AMP_LEN UP_CONF MP_CONF Tm(NN) PcGC
PWARN UEP_DIR UEP_MASS CTGF-447G/C 119 98.2 65 52 52.9 R 5090.3
NBS1 Gln185Glu G/C 118 97 65 51 52.9 R 5192.4 MBL2 161 G/A 99 96.8
65 50.3 52.9 F 5299.5 IGF2R Leu252Val C/G 114 98.5 67.8 68.6 82.4 F
5206.4 MUC5AC -221 C/T 119 81.8 67.8 56.9 64.7 g R 5273.4 Arg1
intron1 C/T 102 99.6 67.8 53.3 52.6 R 5932.9 REV1 Phe257Ser C/T 105
99.6 67.8 57.5 55 R 6003.9 Apex1 Asp148Glu G/T 114 92.9 67.8 46.8
35 F 6113 IL-10 -1082 A/G 107 98 68.8 51.2 58.8 R 4977.2
Sequenom Conditions for the Polymorphisms Genotyping--7
TABLE-US-00051 [0313] SNP_ID UEP_SEQ EXT1_CALL EXT1_MASS EXT1_SEQ
CTGF-447G/C AAAAGGTTTCTCCCCCC G 5363.5 AAAAGGTTTCTCCCCCCC [SEQ. ID.
NO. 310] [SEQ. ID. NO. 311] NBS1 Gln185Glu AGGCTGCTTCTTGGACT G
5465.6 AGGCTGCTTCTTGGACTC G/C [SEQ. ID. NO. 312] [SEQ. ID. NO. 313]
MBL2 161 G/A CAAAGATGGGCGTGATG A 5596.7 CAAAGATGGGCGTGATGA [SEQ.
ID. NO. 314] [SEQ. ID. NO. 315] IGF2R Leu252Val GCCAGCCCCGGGACGGA C
5479.6 GCCAGCCCCGGGACGGA C/G [SEQ. ID. NO. 316] C [SEQ. ID. NO.
317] MUC5AC -221 ATGGGTGTGTCTGCCGG T 5570.6 ATGGGTGTGTCTGCCGGA C/T
[SEQ. ID. NO. 318] [SEQ. ID. NO. 319] Arg1 intron1 C/T
GGCTGTAAGGAAATCTGGG T 6230.1 GGCTGTAAGGAAATCTGG [SEQ. ID. NO. 320]
GA [SEQ. ID. NO. 321] REV1 Phe257Ser CCTTATCCTCCTCCTGGGAA T 6301.1
CCTTATCCTCCTCCTGGG C/T [SEQ. ID. NO. 322] AAA [SEQ. ID. NO. 323]
Apex1 Asp148Glu TGTTTCATTTCTATAGGCGA T 6401.2 TGTTTCATTTCTATAGGCG
G/T [SEQ. ID. NO. 324] AT [SEQ. ID. NO. 325] IL-10 -1082 A/G
CCTATCCCTACTTCCCC G 5250.4 CCTATCCCTACTTCCCCC [SEQ. ID. NO. 326]
[SEQ. ID. NO. 327]
Sequenom conditions for the polymorphisms Genotyping--8
TABLE-US-00052 SNP_ID EXT2_CALL EXT2_MASS EXT2_SEQ 1stPAUSE
CTGF-447G/C C 5716.7 AAAAGGTTTCTCCCCCCGA 5419.5 [SEQ. ID. NO. 328]
NBS1 Gln185Glu C 5818.8 AGGCTGCTTCTTGGACTGA 5521.6 G/C [SEQ. ID.
NO. 329] MBL2 161 G/A G 5901.9 CAAAGATGGGCGTGATGGC 5628.7 [SEQ. ID.
NO. 330] IGF2R Leu252Val G 5823.8 GCCAGCCCCGGGACGGAGT 5535.6 C/G
[SEQ. ID. NO. 331] MUC5AC -221 C/T C 5890.8 ATGGGTGTGTCTGCCGGGT
5602.6 [SEQ. ID. NO. 332] Arg1 intron1 C/T C 6879.5
GGCTGTAAGGAAATCTGGGGGT 6262.1 [SEQ. ID. NO. 333] REV1 Phe257Ser C
6630.3 CCTTATCCTCCTCCTGGGAAGA 6333.1 C/T [SEQ. ID. NO. 334] Apex1
Asp148Glu G 7068.6 TGTTTCATTTCTATAGGCGAGGA 6442.2 G/T [SEQ. ID. NO.
335] IL-10 -1082 A/G A 5858.8 CCTATCCCTACTTCCCCTTC 5281.4 [SEQ. ID.
NO. 336]
[0314] Results
[0315] Frequencies of individual polymorphisms are as follows:
TABLE-US-00053 TABLE 11 Polymorphism allele and genotype
frequencies in the Lung cancer patients, resistant smokers and
controls. Nitric oxide synthase 3 Asp 298 Glu (T/G) Allele*
Genotype Frequency T G TT TG GG Controls n = 183 (%) 108 (30%) 258
(70%) 13 (7%) 82 (45%) 88 (48%) Lung Cancer n = 107 (%) 71 (33%)
143 (67%) 9 (8%) 53 (50%) 45 (42%) Resistant n = 198 (%) 135 (34%)
261 (66%) 28.sup.1.2 (14%) 79 (40%) 91 (46%) Nitric oxide synthase
3 -786 T/C Allele* Genotype Frequency C T CC CT TT Controls n = 183
(%) Lung Cancer n = 107 (%) 82 (38%) 132 (62%) 16 (15%) 50 (47%)
41.sup.3 (38%) Resistant n = 198 (%) 166 (42%) 228 (58%) 31 (16%)
104 (53%) 62 (31%) Super oxide dismutase 3 Arg 312 Gln C/G Allele*
Genotype Frequency C G CC CG GG Controls n = 190 (%) 371 (98%) 9
(2%) 183 (96%) 5 (3%) 2 (1%) Lung Cancer n = 104 (%) 208 (100%) 0
(0%) 104 (100%) 0 (0%) 0 (0%) Resistant n = 182 (%) 390 (98%) 10
(3%) 191 (95%) 8.sup.4 (4%) 1.sup.4 (1%) XRCC1 Arg 399 Gln A/G
Allele* Genotype Frequency A G AA AG GG Controls n = 190 (%) Lung
Cancer n = 103 (%) 68 (33%) 138 (67%) 4 (4%) 60 (58%) 39 (38%)
Resistant n = 193 (%) 132 (34%) 254 (66%) 18.sup.5 (9%) 96 (50%) 79
(41%) Interleukin 8 -251 A/T Allele* Genotype Frequency A T AA AT
TT Controls n = 188 (%) 175 (47%) 201 (53%) 39 (21%) 97 (52%) 52
(28%) Lung Cancer n = 90 68 (38%) 112 (62%) 6 (7%) 56 (52%) 28
(31%) Resistant n = 199 (%) 192.sup.7 (48%) 206 (52%) 45.sup.6
(23%) 102 (51%) 52 (26%) Anti-chymotrypsin Ala -15 Thr Allele*
Genotype Frequency A G AA AG GG Lung Cancer n = 108 99 (46%)
117.sup.9 (54%) 24 (22%) 51 (47%) 33.sup.8 (31%) Resistant n = 196
(%) 207 (53%) 185 (47%) 52 (27%) 103 (53%) 41 (21%) Cyclin D1 A 870
G Lung Cancer n = 107 109 (51%) 105 (49%) 25.sup.11 (23%) 59 (55%)
23 (21%) Resistant n = 199 (%) 188 (47%) 210 (53%) 45 (23%) 98
(49%) 56.sup.10 (28%) Interleukin 1B -511 A/G Lung Cancer n = 107
64 (30%) 150 (70%) 12 (11%) 40 (37%) 55.sup.12 (51%) Resistant n =
198 (%) 143 (36%) 253 (64%) 23 (12%) 97 (49%) 78 (39%) FAS
(Apo-1/CD 95) A -670 G Lung Cancer n = 106 121.sup.14 (57%) 91
(43%) 32.sup.13 (30%) 57 (54%) 17 (16%) Resistant n = 198 (%) 202
(51%) 194 (49%) 45 (23%) 112 (57%) 41 (21%) XPD 751 T/G Allele*
Genotype Frequency G T GG TG TT Lung Cancer n = 108 72 (33%) 144
(66%) 11 (10%) 50 (46%) 47 (44%) Resistant n = 197 (%) 147 (37%)
247 (63%) 31.sup.15 (16%) 85 (43%) 81 (41%) Cytochrome P450 1A1 Ile
462 Val G/A Allele* Genotype Frequency G A GG AG AA Lung Cancer n =
109 5 (2%) 213 (98%) 0 (0%) 5 (5%) 104.sup.16 (95%) Resistant n =
199 (%) 20 (5%) 378 (95%) 13.sup.16 (1%) 18.sup.16 (9%) 180.sup.2
(90%) MMP12 Asn 357 Ser Lung Cancer n = 109 8 (4%) 210 (96%) 1 (1%)
6 (5%) 102 (94%) Resistant n = 199 (%) 21 (5%) 377 (95%) 0.sup.17
(0%) 21.sup.17 (11%) 178 (89%) 8-oxoguanine DNA glycosylase Ser 326
Cys C/G Allele* Genotype Frequency G C GG CG CC Lung Cancer n = 109
40 (18%) 178 (82%) 2 (2%) 36 (33%) 71 (65%) Resistant n = 199 (%)
100 (25%) 298 (75%) 14.sup.18 (7%) 72 (36%) 113 (57%)
N-Acetyltransferase 2 Arg 197 Gln G/A Allele* Genotype Frequency A
G AA AG GG Lung Cancer n = 106 55 (26%) 157 (74%) 9 (8%) 37 (35%)
60.sup.19 (57%) Resistant n = 195 (%) 122 (31%) 268 (69%) 17 (9%)
88 (45%) 90 (46%) Cytochrome P450 2E1 1019 G/C Pst1 Allele*
Genotype Frequency C G CC CG GG Lung Cancer n = 109 10 (5%) 208
(95%) 0 (0%) 10.sup.20 (9%) 99 (91%) Resistant n = 197 (%) 11 (3%)
383 (97%) 0 (0%) 11 (6%) 186 (94%) Cytochrome P450 2E1 C/T Rsa I
Allele* Genotype Frequency T C TT TC CC Lung Cancer n = 108 11 (5%)
205 (95%) 0 (0%) 11.sup.21 (10%) 97 (90%) Resistant n = 198 (%) 11
(3%) 385 (97%) 0 (0%) 11 (6%) 187 (94%) Interleukin 18 105 A/C
Allele* Genotype Frequency C A CC AC AA Lung Cancer n = 107 50
(23%) 164 (77%) 8 (8%) 34 (33%) 65.sup.22 (61%) Resistant n = 200
(%) 116 (29%) 284 (71%) 17.sup.22 (9%) 82.sup.22 (41%) 101 (50%)
Interleukin 18 -133 C/G Allele* Genotype Frequency G C GG CG CC
Lung Cancer n = 109 52 (24%) 166 (76%) 8 (7%) 36 (33%) 65.sup.23
(60%) Resistant n = 198 (%) 117 (30%) 279 (70%) 17.sup.23 (9%)
83.sup.23 (42%) 98 (49%) Glutathione S-Transferase M null Allele*
Frequency Null Wild Controls n = 178 75 (42%) 103 (58%) Lung Cancer
n = 107 67.sup.24 (58%) 48 (42%) Resistant n = 182 100 (55%) 82
(45%) Interferon-gamma 874 A/T Allele* Genotype Frequency A T AA AT
TT Controls n = 186 (%) 183 (49%) 189 (51%) 37 (20%) 109 (58%) 40
(22%) Lung cancer n = 106 (%) 116 (55%) 96 (45%) 34.sup.25,26 (32%)
48 (45%) 24 (23%) Resistant n = 196 (%) 209 (53%) 183 (47%) 50
(26%) 109 (56%) 37 (19%) Cyclooxygenase -765 C/G Allele* Genotype
Frequency C G CC CG GG Controls n = 95 (%) 27 (14%) 161 (86%) 3
(3%) 21 (22%) 70 (75%) Lung Cancer n = 109 (%) 34 (16%) 184
(84%).sup.30 5 (5%.sup.27) 24 (22%).sup.27 80 (73%).sup.29
Resistant n = 158 (%) 75 (24%).sup.28 241 (76%) 11 (7%) 53 (34%) 94
(59%) Matrix metalloproteinase 1 -1607 1G/2G Allele* Genotype
Frequency 1G 2G 1G1G 1G2G 2G2G Controls n = 174 214 (61%) 134 (39%)
68 (39%) 78 (45%) 28 (16%) (%) Lung Cancer n = 67 58 (43%) 76
(57%).sup.32 13 (19%) 32 (48%) 22 (33%).sup.31 (%) Resistant n =
171 167 (49%) 175 (51%) 41 (24%) 85 (50%) 45 (26%) (%) *number of
chromosomes (2n)
[0316] 1. Genotype. TT vs TG/GG for resistant vs lung cancer, Odds
ratio (OR)=1.8, 95% confidence limits 0.8-4.3, .chi..sup.2 (Yates
uncorrected)=2.14, p=0.14, TT genotype=protective [0317] 2.
Genotype. TT vs TG/GG for resistant vs controls, Odds ratio
(OR)=2.2, 95% confidence limits 1.0-4.6, .chi..sup.2 (Yates
corrected)=4.2, p=0.04, TT genotype=protective [0318] 3. Genotype.
TT vs CC/CT for Lung cancer vs resistant, Odds ratio (OR)=1.4, 95%
confidence limits 0.8-2.3, .chi..sup.2 (Yates uncorrected)=1.45,
p=0.23, TT genotype=susceptible [0319] 4. Genotype CG/GG vs CC for
resistant vs lung cancer, Yates uncorrected=3.38, P=0.07 and
Fisher's Two tailed test, P=0.03. CG/GG=protective [0320] 5.
Genotype. AA vs AG/GG for resistant vs lung cancer, Odds ratio
(OR)=2.6, 95% confidence limits 0.8-9.2, .chi..sup.2 (Yates
uncorrected)=2.89, p=0.09. AA genotype=protective [0321] 6.
Genotype. AA vs AT/TT for resistant vs lung cancer, Odds ratio
(OR)=4.1, 95% confidence limits=1.6=11.2, .chi..sup.2 (Yates
corrected)=9.8, p=0.002, AA=protective [0322] 7. Allele. A vs T for
resistant smokers vs lung cancer, Odds ratio (OR)=1.5, 95%
confidence limits 1.0-2.2, .chi..sup.2 (Yates corrected)=5.07,
p=0.02, A=protective [0323] 8. Genotype. GG vs AA/AG for Lung
cancer vs resistant, Odds ratio (OR)=1.7, 95% confidence
limits=0.9-2.9, .chi..sup.2 (Yates uncorrected)=3.51, p=0.06,
GG=susceptible [0324] 9. Allele. G vs A for lung cancer vs
resistant smokers, Odds ratio (OR)=1.3, 95% confidence limits
0.9-1.9, .chi..sup.2 (Yates uncorrected)=2.71, p=0.10, [0325]
G=susceptible [0326] 10. Genotype. GG vs AG/AA for Resistant vs
lung cancer, Odds ratio (OR)=1.4, 95% confidence limits=0.8-2.6,
.chi..sup.2 (Yates uncorrected)=1.6, p=0.20, GG=protective [0327]
11. Genotype. AG/AA vs GG for Lung cancer vs resistant, Odds ratio
(OR)=1.4, 95% confidence limits=0.8-2.6, .chi..sup.2 (Yates
uncorrected)=1.6, p=0.20, AA=susceptible [0328] 12. Genotype. GG vs
AA/AG for Lung cancer vs resistant, Odds ratio (OR)=1.6, 95%
confidence limits=1-2.7, .chi..sup.2 (Yates uncorrected)=4.07,
p=0.04, GG=susceptible [0329] 13. Genotype. AA vs AG/GG for Lung
cancer vs resistant, Odds ratio (OR)=1.5, 95% confidence
limits=0.8-2.6, .chi..sup.2 (Yates uncorrected)=2.03, p=0.15,
AA=susceptible [0330] 14. Allele. A vs G for Lung cancer vs
resistant, Odds ratio (OR)=1.3, 95% confidence limits 0.9-1.8,
.chi..sup.2 (Yates uncorrected)=2.04, p=0.15, A=susceptible [0331]
15. Genotype. GG vs TG/TT for Resistant vs lung cancer, Odds ratio
(OR)=1.7, 95% confidence limits=0.8-3.7, .chi..sup.2 (Yates
uncorrected)=1.81, p=0.18, GG=protective [0332] 16. Genotype. AG/GG
vs AA for Resistant vs lung cancer, Odds ratio (OR)=2.2, 95%
confidence limits=0.7-6.9, .chi..sup.2 (Yates uncorrected)=2.41,
p=0.12, GG/AG=protective, AA=susceptible [0333] 17. Genotype. GG/AG
vs AA for Resistant vs COPD, Odds ratio (OR)=1.7, 95% confidence
limits=0.7-4.6, .chi..sup.2 (Yates uncorrected)=1.45, p=0.23,
GG/AG=protective [0334] 18. Genotype. GG vs CG/CC for Resistant vs
lung cancer, Odds ratio (OR)=4.0, 95% confidence limits=0.9-26.3,
.chi..sup.2 (Yates uncorrected)=3.87, p=0.05, GG=protective [0335]
19. Genotype. GG vs AG/AA for Lung cancer vs resistant, Odds ratio
(OR)=1.5, 95% confidence limits=0.9-2.5, .chi..sup.2 (Yates
uncorrected)=3.0, p=0.08, GG=susceptible [0336] 20. Genotype. CG vs
GG for Lung cancer and resistant, Odds ratio (OR)=1.7, 95%
confidence limits=0.7-4.5, .chi..sup.2 (Yates uncorrected)=1.42,
p=0.23, CG=susceptible [0337] 21. Genotype. TC vs CC for Lung
cancer and resistant, Odds ratio (OR)=1.9, 95% confidence
limits=0.8-5.0, .chi..sup.2 (Yates uncorrected)=2.24, p=0.13,
TC=susceptible [0338] 22. Genotype. AA vs AC/CC for Lung cancer and
resistant, Odds ratio (OR)=1.6, 95% confidence limits=1.0-2.6,
.chi..sup.2 (Yates uncorrected)=3.51, p=0.06, AA=susceptible, AC/CC
protective [0339] 23. Genotype. CC vs CG/GG for Lung cancer and
resistant, Odds ratio (OR)=1.5, 95% confidence limits=0.9-2.5,
.chi..sup.2 (Yates uncorrected)=2.90, p=0.09, CC=susceptible, CG/GG
protective [0340] 24. Genotype. Null vs wild for Lung cancer and
controls, Odds ratio (OR)=1.92, 95% confidence limits=1.2-3.2,
.chi..sup.2 (Yates corrected)=6.64, p=0.01, Null=susceptible [0341]
25. Genotype. AA vs AT/TT for lung cancer vs resistant, Odds ratio
(OR)=1.4, 95% confidence limits 0.8-2.4, .chi..sup.2 (Yates
uncorrected)=1.48, p=0.22, AA genotype=susceptible [0342] 26.
Genotype. AA vs AT/TT for lung cancer vs controls, Odds ratio
(OR)=1.9, 95% confidence limits 1.1-3.4, .chi..sup.2 (Yates
corrected)=5.45, p=0.02, AA genotype=susceptible to lung cancer
[0343] 27. Genotype. CC/CG vs GG for Lung cancer vs resistant, Odds
ratio (OR)=0.53, 95% confidence limits=0.3-0.9, .chi..sup.2 (Yates
corrected)=4.9, P=0.03 CC/CG=protective [0344] 28. Allele. C vs G
for Lung cancer vs resistant, Odds ratio (OR)=0.59, 95% confidence
limits 0.4-0.9, .chi..sup.2 (Yates corrected)=4.8, p=0.03,
C=protective [0345] 29. Genotype. GG vs CG/CC for Lung cancer vs
resistant, Odds ratio (OR)=1.88, 95% confidence limits=1.1-3.3,
.chi..sup.2 (Yates corrected)=4.9, P=0.03 GG=susceptible (when
compared against resistant smokers but not controls) [0346] 30.
Allele. G vs C for Lung cancer vs resistant, Odds ratio (OR)=1.7,
95% confidence limits 1.1-2.7, .chi..sup.2 (Yates corrected)=4.8,
p=0.03, G=susceptible (when compared against resistant smokers but
not controls) [0347] 31. Genotype. 2G2G vs 1G1G/1G2G for Lung
cancer vs controls, Odds ratio (OR)=2.55, 95% confidence limits
1.3-5.1, .chi..sup.2 (Yates corrected)=7.3, p=0.007 2G2G
genotype=susceptible [0348] 32. Allele. 2G vs 1G for Lung cancer vs
controls, Odds ratio (OR)=2.1, 95% confidence limits 1.4-3.2,
.chi..sup.2 (Yates corrected)=12.3, p=0.0004, 2G=susceptible
Connective Tissue Growth Factor (CTGF) -447 G/C Polymorphism Allele
and Genotype Frequencies in the Lung Cancer and Resistant
Smokers.
TABLE-US-00054 [0349] 37. Allele* 38. Genotype Frequency G C GG GC
CC Lung cancer n = 109 201 17 92 17 0 (%) (92%) (8%) (84%) (16%)
(0%) Resistant n = 200 379 21 179 21 0 (%) (95%) (5%) (90%) (10%)
(0%) *number of chromosomes (2n)
[0350] 1. Genotype. GC/CC vs GG for lung cancer vs resistant, Odds
ratio (OR)=1.6, 95% confidence limits 0.8-3.3, .chi..sup.2 (Yates
uncorrected)=1.70, p=0.19, GC/CC genotype=susceptibility
(trend)
Mucin 5AC (Muc5AC)-221 C/T Polymorphism Allele and Genotype
Frequencies in the Lung Cancer and Resistant Smokers.
TABLE-US-00055 [0351] 39. Allele* 40. Genotype Frequency C T CC CT
TT Lung cancer n = 109 177 41 73 31 5 (%) (81%) (19%) (67%) (28%)
(5%) Resistant n = 195 296 94 119 58 18 (%) (76%) (24%) (61%) (30%)
(9%) *number of chromosomes (2n)
[0352] 1. Genotype. TT vs CC/CT for lung cancer vs resistant, Odds
ratio (OR)=0.47, 95% confidence limits 0.2-1.4, .chi..sup.2 (Yates
uncorrected)=2.16, p=0.14, TT genotype=protective (trend)
Mannose Binding Lectin (MBL2) 161 G/A Polymorphism Allele and
Genotype Frequencies in the Lung Cancer and Resistant Smokers.
TABLE-US-00056 [0353] 41. Allele* 42. Genotype Frequency G A GG AG
AA Lung cancer n = 105 173 37 71 31 3 (%) (82%) (18%) (67%) (30%)
(3%) Resistant n = 197 338 56 147 44 6 (%) (86%) (14%) (75%) (22%)
(3%) *number of chromosomes (2n)
[0354] 1. Genotype. AG/AA vs GG for lung cancer vs resistant, Odds
ratio (OR)=1.4, 95% confidence limits 0.8-2.4, .chi..sup.2 (Yates
uncorrected)=1.67, p=0.20, AG/AA genotype=susceptibility
(trend)
Nibrin (NBS1) Gln185Glu G/C Polymorphism Allele and Genotype
Frequencies in the Lung Cancer and Resistant Smokers.
TABLE-US-00057 [0355] 43. Allele* 44. Genotype Frequency G C GG GC
CC Lung cancer n = 109 150 68 54 42 13 (%) (69%) (31%) (50%) (39%)
(12%) Resistant n = 199 295 103 107 81 11 (%) (74%) (26%) (54%)
(41%) (6%) *number of chromosomes (2n)
[0356] 1. Genotype. CC vs CG/GG for lung cancer vs resistant, Odds
ratio (OR)=2.3, 95% confidence limits 0.9-5.8, .chi..sup.2 (Yates
uncorrected)=4.01, p=0.05, CC genotype=susceptibility
Arginase 1 (Arg1) Intron 1 C/T Polymorphism Allele and Genotype
Frequencies in the Lung Cancer and Resistant Smokers.
TABLE-US-00058 [0357] 45. Allele* 46. Genotype Frequency C T CC CT
TT Lung cancer n = 105 137 73 45 47 13 (%) (65%) (35%) (43%) (45%)
(12%) Resistant n = 180 203 157 65 73 42 (%) (56%) (44%) (36%)
(41%) (23%) *number of chromosomes (2n)
[0358] 1. Genotype. TT vs CC/CT for lung cancer vs resistant, Odds
ratio (OR)=0.46, 95% confidence limits 0.2-0.95, .chi..sup.2 (Yates
uncorrected)=5.11, p=0.02, TT genotype=protective [0359] 2. Allele.
T vs C for lung cancer vs resistant, Odds ratio (OR)=0.69, 95%
confidence limits 0.5-1.0, .chi..sup.2 (Yates corrected)=3.96,
p=0.05, T allele=protective
REV1 Phe257Ser C/T Polymorphism Allele and Genotype Frequencies in
the Lung Cancer and Resistant Smokers.
TABLE-US-00059 [0360] 47. Allele* 48. Genotype Frequency C T CC CT
TT Lung cancer n = 109 129 89 39 51 19 (%) (59%) (41%) (36%) (47%)
(17%) Resistant n = 192 242 142 83 76 33 (%) (63%) (37%) (43%)
(40%) (17%) *number of chromosomes (2n)
[0361] 1. Genotype. CC vs CT/TT for lung cancer vs resistant, Odds
ratio (OR)=0.73, 95% confidence limits 0.4-1.2, .chi..sup.2 (Yates
uncorrected)=1.6, p=0.20, CC genotype=protective (trend)
Insulin-Like Growth Factor II Receptor (IGF2R) Leu252Val C/G
Polymorphism Allele and Genotype Frequencies in the Lung Cancer and
Resistant Smokers.
TABLE-US-00060 [0362] 49. Allele* 50. Genotype Frequency C G CC CG
GG Lung cancer n = 109 190 28 82 26 1 (%) (87%) (13%) (75%) (24%)
(1%) Resistant n = 198 342 54 150 42 6 (%) (86%) (14%) (76%) (21%)
(3%) *number of chromosomes (2n)
[0363] 1. Genotype. GG vs CC/CG for lung cancer vs resistant, Odds
ratio (OR)=0.30, 95% confidence limits 0.01-2.5, .chi..sup.2 (Yates
uncorrected)=1.41, p=0.22 (1-tailed t-test), GG genotype=protective
(trend)
Apex Nuclease (APE1) Asp148Glu T/G Polymorphism Allele and Genotype
Frequencies in the Lung Cancer and Resistant Smokers.
TABLE-US-00061 [0364] 51. Allele* 52. Genotype Frequency T G TT TG
GG Lung cancer n = 109 124 94 39 46 24 (%) (57%) (43%) (36%) (42%)
(22%) Resistant n = 192 229 155 69 91 32 (%) (60%) (40%) (36%)
(47%) (17%) *number of chromosomes (2n)
[0365] 1. Genotype. GG vs TT/TG for lung cancer vs resistant, Odds
ratio (OR)=1.4, 95% confidence limits 0.8-2.7, .chi..sup.2 (Yates
uncorrected)=1.3, p=0.25, GG genotype=susceptibility (trend)
Interleukin 10 (IL-10)-1082 A/G polymorphism allele and genotype
frequencies in the lung cancer and resistant smokers.
TABLE-US-00062 [0365] 53. Allele* 54. Genotype Frequency G C GG GC
CC Lung cancer n = 98 91 105 16 59 23 (%) (46%) (54%) (16%) (60%)
(24%) Resistant n = 196 174 218 40 94 62 (%) (44%) (56%) (20%)
(48%) (32%) *number of chromosomes (2n)
[0366] 1. Genotype. GG vs GC/CC for lung cancer vs resistant, Odds
ratio (OR)=0.66, 95% confidence limits 0.4-1.2, .chi..sup.2 (Yates
uncorrected)=2.12, p=0.15, GG genotype=protective (trend)
[0367] Table 12 below provides a summary of the protective and
susceptibility polymorphisms determined for lung cancer.
TABLE-US-00063 TABLE 12 Summary of protective and susceptibility
polymorphisms in Lung Cancer patients relative to resistant smokers
(with normal lung function) Gene Polymorphism Role Nitric Oxide
synthase 3 (NOS3) NOS3 Asp 298 Glu TT protective Nitric Oxide
synthase 3 (NOS3) NOS3 -786 T/C TT susceptible Superoxide dismutase
3 (SOD3) SOD3 Arg 312 Gln CG/GG protective XRCC1 XRCC1 Arg 399 Gln
G/A AA protective Interleukin-8 (IL-8) IL-8 -251 A/T AA protective
Anti-chymotrypsin (ACT) ACT Ala 15 Thr GG susceptible Cyclin D
(CCND1) CCND1 A870G GG protective AA susceptible Interleukin 1B
(IL-1B) IL-1B -511 A/G GG susceptible FAS (Apo-1/CD95) FAS A-670G
AA susceptible XPD XPD -751 G/T GG protective CYP 1A1 CYP 1A1 Ile
462 Val GG/AG protective A/G AA susceptible Matrix
metalloproteinase 12 MMP12 Asn 357 Ser GG/AG protective (MMP12) A/G
8-Oxoguanine DNA glycolase OGG1 Ser 326 Cys G/C GG protective
(OGG1) N-acetyltransferase 2 (NAT2) NAT2 Arg 197 Gln A/G GG
susceptible CYP2E1 CYP2E1 1019 G/C Pst I CC/CG susceptible CYP2E1
CYP2E1 C/T Rsa I TT/TC susceptible Interleukin-18 (IL-18) IL-18 105
A/C AC/CC protective AA susceptible Interleukin-18 (IL-18) IL-18
-133 G/C CG/GG protective CC susceptible Glutathione S-transferase
M GSTM null Null susceptible Interferon gamma (IFN?) IFN? 874 A/T
AA susceptible Cyclo-oxygenase 2 (COX2) COX2 -765 G/C CC/CG
protective GG susceptible Matrix metalloproteinase 1 (MMP1) MMP
-1607 1G/2G 2G2G susceptible Connective tissue growth factor CTGF
-447 G/C GC/CC (CTGF) susceptible Mucin 5AC (MUC5AC) MUC5AC -221
C/T TT protective Mannose binding lectin 2 (MBL2) MBL2 +161 G/A
AG/AA susceptible Nibrin (NBS1) NBS1 Gln185Glu G/C CC susceptible
Arginase 1 (Arg1) Arg1 intron 1 C/T TT protective REV1 REV1
Phe257Ser C/T CC protective Insulin-like growth factor II receptor
IGF2R Leu252Val C/G GG protective (IGF2R) Apex nuclease (Apex or
APE1)) Apex Asp148Glu G/T GG susceptible Interleukin 10 (IL-10)
IL-10 -1082 A/G GG protective
[0368] The combined frequencies of the presence or absence of the
selected protective genotypes CYP1A1 GG/AG, OGG1 GG, CCND1 GG, NOS3
298 TT, IL-8 AA, and XRCC1 AA observed in the subjects with lung
cancer and in resistant smokers is presented below in Table 13.
TABLE-US-00064 TABLE 13 Combined frequencies of the presence or
absence of selected protective genotypes in subjects with lung
cancer and in resistant smokers. Number of protective polymorphisms
Cohorts 0 1 =2 Total Lung Cancer 66 (61%) 37 (34%) 6 (6%) 109
Resistant smokers 71 (36%) 86 (43%) 42 (21%) 199 % of smokers with
Lung 66/137 37/123 6/42 cancer (48%) (30%) (14%) Comparison Odd's
ratio 95% CI ?2 P value 0 vs 1 vs 2+, Resist vs Lung -- -- 22.3
<0.0001 cancer 2+ vs 0-1, Resist vs Lung cancer 4.6 1.8-12.5
11.87 0.0005 0 vs 2+, Lung cancer vs Resist 2.8 1.7-4.6 16.7
<0.0001
[0369] The combined frequencies of the presence or absence of the
selected susceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-1B
GG, and ACT 15 GG, observed in the subjects with lung cancer and in
resistant smokers is presented below in Table 14.
TABLE-US-00065 TABLE 14 Combined frequencies of the presence or
absence of selected susceptibility genotypes in subjects with lung
cancer and in resistant smokers. Number of susceptibility
polymorphisms Cohorts 0 1 =2 Total Lung Cancer 21 (19%) 52 (48%) 35
(33%) 108 Resistant smokers 71 (36%) 85 (43%) 42 (21%) 198 % of
smokers with 21/92 52/137 35/77 COPD (23%) (38%) (45%) Comparison
Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, -- -- 10.2 0.006 Lung
cancer vs Resist 2+ vs 0-1, Lung cancer vs Resist 1.8 1.0-3.1 4.1
0.04 0+ vs 1-2+, Resist vs COPD 2.3 1.3-4.2 8.2 0.004
[0370] The combined frequencies of the presence or absence of the
selected protective genotypes CYP1A1 GG/AG, OGG1 GG, CCND1 GG, NOS3
298 TT, SOD3 CG/GG, XPD GG, MMP12 GG/AG, and XRCC1 AA observed in
the subjects with lung cancer and in resistant smokers is presented
below in Table 15.
TABLE-US-00066 TABLE 15 Combined frequencies of the presence or
absence of selected protective genotypes in subjects with lung
cancer and in resistant smokers. Number of protective polymorphisms
n = 8 Cohorts 0 1 =2 Total Lung Cancer 54 (50%) 50 (46%) 5 (4%) 109
Resistant smokers 67 (34%) 83 (42%) 50 (25%) 199 % of smokers with
Lung 54/121 50/133 5/55 cancer (45%) (38%) (9%) Comparison Odd's
ratio 95% CI ?2 P value 0 vs 1 vs 2+, Resist vs Lung -- -- 21.5
<0.0001 cancer 2+ vs 0-1, Resist vs Lung cancer 6.9 2.5-20.5
18.7 <0.0001 0 vs 2+, Lung cancer vs Resist 2.0 1.2-3.2 6.96
0.008
[0371] The combined frequencies of the presence or absence of the
selected susceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-1B
GG, ACT 15 GG, NAT2 GG, IL-18 105 AA, and IFN? AA, observed in the
subjects with lung cancer and in resistant smokers is presented
below in Table 16.
TABLE-US-00067 TABLE 16 Combined frequencies of the presence or
absence of selected susceptibility genotypes in subjects with lung
cancer and in resistant smokers. Number of susceptibility
polymorphisms n = 7 Cohorts 1 2 =3 Total Lung Cancer 16 (15%) 35
(32%) 58 (53%) 109 Resistant smokers 65 (33%) 66 (33%) 69 (34%) 200
% of smokers with 16/81 35/101 58/127 COPD (20%) (35%) (46%)
Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, Lung cancer
-- -- 14.6 0.0007 vs Resist 3+ vs 1-2, Lung cancer vs Resist 2.2
1.3-5.6 9.4 0.002 1 vs 2-3+, Resist vs COPD 2.8 1.5-5.4 10.7
0.001
[0372] The combined frequencies of the presence or absence of the
selected protective genotypes CYP1A1 GG/AG, OGG1 GG, CCND1 GG, NOS3
298 TT, IL-8 AA, XRCC1 AA, and Cox 2 -765 CC/CG, observed in the
subjects with lung cancer and in resistant smokers is presented
below in Table 17.
TABLE-US-00068 TABLE 17 Combined frequencies of the presence or
absence of protective genotypes in the exposed smoking subjects
(Lung cancer subjects and resistant smokers). Number of protective
genotypes Cohorts 0 1 =2 Total Lung Cancer 45 (40%) 50 (43%) 19
(17%) 114 Resistant smokers 47 (23%) 79 (40%) 74 (37%) 200 % of
smokers with Lung 45/92 50/129 19/93 cancer (49%) (39%) (20%)
Comparison Odd's ratio 95% CI ?2 P value 0 vs 1 vs 2+, Resist vs
Lung -- -- 16.8 0.0002 cancer 2+ vs 0-1, Resist vs Lung cancer 2.94
1.6-5.4 13.44 0.0002 0 vs 2+, Lung cancer vs Resist 2.12 1.3-3.6
8.2 0.004
[0373] The combined frequencies of the presence or absence of the
selected susceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-B1
GG, ACT 15 GG, and MMP1 2G2G, observed in the subjects with lung
cancer and in resistant smokers is presented below in Table 18.
TABLE-US-00069 TABLE 18 Combined frequencies of the presence or
absence of susceptibility genotypes in the exposed smoking subjects
(Lung cancer subjects and resistant smokers). Number of
susceptibility genotypes Cohorts 0-1 2-3 4-6 Total Lung Cancer 13
(12%) 66 (61%) 30 (28%) 109 Resistant smokers 54 (27%) 113 (56%) 33
(17%) 200 % of smokers with 13/67 66/179 30/63 COPD (19%) (37%)
(48%) Comparison Odd's ratio 95% CI ?2 P value 0-1 vs 2-3 vs 4-6,
-- -- 11.8 0.003 Lung cancer vs Resist 4-6 vs rest, Lung cancer vs
1.9 1.0-3.5 4.6 0.03 Resist 0-1 vs rest, Resist vs COPD 2.7 1.4-5.6
8.6 0.003
[0374] Protective polymorphisms were assigned a score of -1 while
susceptibility polymorphisms were assigned a score of +1. For each
subject, a net score was then calculated according to the presence
of susceptibility and protective genotypes. This produced a linear
spread of values, as shown in Table 14. When assessed as a range
between -2 to +4, a linear relationship as depicted in FIG. 4 was
observed. This analysis indicates that for subjects with a net
score of -2 or less, there was a minimal risk of having lung
cancer. For subjects with a net score of -1, there was an
approximately one in ten risk of having lung cancer. In contrast,
for subjects with a net score of 4+ or greater, the risk was
markedly increased to over 70% (see Table 19 and FIG. 4). It is
noted that for FIG. 4, unlike the data presented in FIG. 3, the
protective polymorphisms are assigned a negative value while the
susceptibility polymorphisms are assigned a positive value. The
precise value or sign given to each one is not critical, as long as
it is consistent between the types of polymorphisms.
TABLE-US-00070 TABLE 19 Combined presence or absence of protective
and susceptibility polymorphisms Score combining protective (-1)
and susceptibility (+1) polymorphisms -2 -1 0 1 2 3 4+ Lung cancer
0 (0%) 2 (2%) 10 (9%) 21 (19%) 38 (35%) 23 (21%) 15 (14%) N = 109
(%) Resistant smokers 6 (3%) 21 (11%) 39 (20%) 51 (26%) 52 (26%) 25
(13%) 6 (3%) N = 200 (%) % Lung cancer 0% 9% 20% 29% 42% 48%
71%
[0375] A further combined analysis was performed using a greater
number of polymorphisms. Again, this produced a linear spread of
values (Table 20). When assessed as a range between -3 to +5, a
linear relationship as depicted in FIG. 5 was observed. This
analysis indicates that for subjects with a net score of -2 or
less, there was a minimal risk of having lung cancer. In contrast,
for subjects with a net score of 5+ or greater, the risk was
markedly increased to 80% (see Table 20 and FIG. 5).
TABLE-US-00071 TABLE 20 Combined presence or absence of protective
and susceptibility polymorphisms SNP score for Lung cancer
according to the presence of protective(-1) and susceptibility (+1)
genotypes for all smokers Cohorts <-3 -2 -1 0 1 2 3 4 5+ Lung
cancer 0 (0%) 1 (1%) 3 (3%) 10 (9%) 25 (23%) 32 (29%) 20 (18%) 14
(13%) 4 (4%) N = 109 Resistant smokers 3 (2%) 12 (6%) 16 (8%) 34
(17%) 58 29%) 48 (24%) 21 (11%) 7 (4%) 1 (0.5%) N = 200 % Lung
cancer 0% 7% 16% 23% 30% 40% 49% 67% 80%
DISCUSSION
[0376] The methods of the invention allow the determination of risk
of disease to be assessed. For example, a simple scoring system in
which each polymorphism in a category (i.e. protective or
susceptibility) is assigned the same value allows the combined
effects of all potentially relevant polymorphisms to be factored
into the analysis. In other embodiments, the methods of the
invention utilize a scoring system with adjustment (weighting) for
the magnitude of the effect of each individual polymorphism, and
again allow all polymorphisms to be simultaneously analyzed.
[0377] In other embodiments, analyses can utilise path analysis
and/or Monte-Carlo analysis where the non-genetic and genetic
factors can be analyzed.
[0378] Similar results were observed in comparing the presence or
absence of susceptibility and resistant polymorphisms in smokers
with OCOPD, and in smokers with lung cancer and resistant
smokers.
[0379] The benefit of a net susceptibility score, having been
determined for a subject is that it provides the opportunity for
early prophylactic and/or therapeutic intervention. Such
intervention can be as simple as communicating the net
susceptibility score to the subject together with an explanation of
the implications of that score. This alone can cause a lifestyle or
occupational change, with the resultant beneficial effect for the
subject.
[0380] Other, more direct approaches to prophylaxis or therapy can
also be followed. These can include pharmaceutical or other
medicaments being administered directed at favourably altering the
net score of the subject together with other such approaches as
discussed herein.
[0381] Table 21 below presents representative examples of
polymorphisms in linkage disequilibrium with the polymorphisms
specified herein. Examples of such polymorphisms can be located
using public databases, such as that available online at world wide
web dot hapmap dot org. Specified polymorphisms are indicated in
the columns marked SNP NAME. Unique identifiers are indicated in
the columns marked RS NUMBER.
TABLE-US-00072 TABLE 21 Polymorphisms reported to be in linkage
disequilibrium (unless stated) with examples of specified
polymorphism. SNP NAME RS NUMBER SNP NAME RS NUMBER SNP NAME RS
NUMBER COX2 SNPs rs6684912 rs5277 rs7527769 rs2745559 rs2066823
rs7550380 rs12042763 rs4648263 rs2206594 rs4648250 rs4987012
rs6687495 rs4648251 rs20428 rs6681231 rs2223626 rs20429 rs13376484
rs689462 rs4648264 rs12064238 rs4648253 rs4648265 rs10911911
rs689465 rs4648266 rs12743673 rs12027712 rs4648267 rs10911910
rs689466 rs11567824 rs12743516 rs2745558 rs4648268 rs10911909
rs3918304 rs4648269 rs1119066 rs20415 rs4648270 rs1119065 rs20416
rs12759220 rs1119064 rs4648254 rs20430 rs10798053 rs11567815
rs4648271 rs12409744 -765G > C rs20417 rs11567825 rs10911908
rs4648256 rs4648273 rs10911907 rs20419 rs16825748 rs7416022
rs2734779 rs4648274 rs2745561 rs20420 rs16825745 rs10911906 rs20422
rs20432 rs2734776 rs20423 rs20433 rs2734777 rs5270 rs3218622
rs12084433 rs20424 rs2066826 rs2734778 rs5271 rs5278 rs2745560
rs4648257 rs4648276 rs2223627 rs11567819 rs20434 rs2383517
rs3134591 rs3218623 rs4295848 rs3134592 rs3218624 rs4428839 rs20426
rs5279 rs4609389 rs4648258 rs4648278 rs4428838 rs11567820
rs13306034 rs12131210 rs2745557 rs2853803 rs2179555 rs11567821
rs4648279 rs2143417 rs4648259 rs4648281 rs2143416 rs4648260
rs4648282 rs11583191 rs4648261 rs11567826 rs2383516 rs4648262
rs4648283 rs2383515 rs11567822 rs4648284 rs10911905 rs11567823
rs4648285 rs10911904 rs2066824 rs11567827 rs20427 rs4648286
rs4648287 rs1042719 rs5744244 rs5272 rs3729944 rs360722 rs4648288
rs3730182 rs5023207 rs5273 rs1042720 rs5744246 rs5274 rs6879202
rs5744247 rs3218625 rs3777124 -133 C/G rs360721 rs4648289 rs1803051
rs4988359 rs4648290 rs8192451 rs12721559 rs1051896 rs4987255
rs5744248 rs5275 rs3177007 rs5744249 1ADRB SNPs rs1126871 rs5744250
rs2082382 rs6885272 rs5744251 rs2082394 rs6889528 rs100000356
rs2082395 rs4521458 rs1834481 rs9325119 rs10463409 rs17215057
rs9325120 rs7702861 rs5744253 rs12189018 IL-18 SNPs rs5744254
rs11168066 rs187238 rs5744255 rs11959615 rs5744228 rs5744256
rs11958940 rs360718 rs5744257 rs4705270 rs360717 rs360720
rs10079142 rs5744229 rs5744258 rs9325121 rs100000353 rs5744259
rs11746634 rs5744231 rs5744260 rs11168067 rs5744232 rs5744261
rs9325122 rs7106524 105 A/C rs549908 rs11957351 rs189667 PAI-1 SNPs
rs11948371 rs12290658 rs6465787 rs11960649 rs12271175 rs7788533
rs1432622 rs11606049 rs6975620 rs1432623 rs360716 rs6956010
rs11168068 rs360715 rs12534508 rs17778257 rs360714 rs4729664
rs2400706 rs2043055 rs2527316 rs2895795 rs5744233 rs2854235
rs2400707 rs795467 rs10228765 rs2053044 rs12270240 rs2854225
rs17108803 rs100000354 rs2854226 rs12654778 rs4937113 rs2227707
rs11168070 rs100000355 rs2227631 rs11959427 rs360723 -675 4G/5G No
rs rs1042711 rs5744237 NOS3 SNPs rs1801704 rs5744238 rs2373962
Arg16Gly rs1042713 rs5744239 rs2373961 rs1042714 rs7932965
rs6951150 rs1042717 rs11214103 rs13238512 rs1800888 rs5744241
rs10247107 rs1042718 rs5744242 rs10276930 rs3729943 rs5744243
rs10277237 rs12703107 rs9282804 rs2282679 rs6946340 Asp298Glu
rs1799983 rs2282680 rs6946091 VDBP SNPs rs705117 rs6946415 rs222035
rs2070741 rs10952296 rs222036 rs2070742 rs13309715 rs16846943
rs6821541 rs10952297 rs7668653 rs222048 rs7784943 rs1491720
rs432031 rs11771443 rs16845007 rs432035 rs2243310 rs17830803
rs222049 rs1800783 Glu416Asp rs7041 rs222050 rs3918155 Lys420Thr
rs4588 rs12510584 rs3918156 rs3737553 rs17467825 rs2566519 rs9016
GSTP1 SNPs rs3918157 rs1352846 rs656652 rs3918158 rs222039 rs625978
rs3918159 rs3775154 rs6591251 rs2566516 rs222040 rs12278098
rs3918225 rs843005 rs612020 rs3918160 rs222041 rs12284337 rs1800779
rs7672977 rs12574108 rs2243311 rs705121 rs6591252 rs3918161
rs11723621 rs597717 rs10952298 rs2298850 rs688489 rs2070744
rs705120 rs597297 rs3918226 rs2298851 rs6591253 rs3918162 rs844806
rs6591254 rs3918163 rs1491709 rs7927381 rs3918164 rs705119
rs7940813 rs3918165 rs6845925 rs593055 rs1800781 rs12640255
rs7927657 rs13310854 rs12644050 rs614080 rs13310763 rs6845869
rs7941395 rs2853797 rs12640179 rs7941648 rs13311166 rs222042
rs7945035 rs13310774 rs3187319 rs2370141 rs2853798 rs222043
rs2370142 rs11974098 rs842999 rs7949394 rs3918166 rs222044
rs7949587 rs3730001 rs222045 rs6591255 rs3918167 rs16846912
rs8191430 rs3918168 rs222046 rs6591256 rs3918169 rs705118 rs8191431
rs3918170 rs222047 rs8191432 rs3793342 rs13142062 rs7109914
rs3793341 rs843000 rs4147580 rs1549758 rs3755967 rs8191436
rs1007311 rs1491710 rs8191437 rs9282803 rs2282678 rs17593068
rs8191438 rs2069718 rs7145047 rs8191439 rs3087272 rs7141735
rs8191440 rs2069719 rs11558264 rs8191441 rs9282708 rs6647 rs1079719
rs2069720 rs8350 rs1871041 rs1042274 rs2230075 rs4147581 rs2069721
rs1049800 rs8191444 rs2069734 S allele rs17580 rs8191445 rs2069722
rs2854258 rs2370143 rs2234687 rs2753937 rs8191446 rs7957366
rs2749547 rs3891249 rs2069723 rs1243162 rs8191447 rs2069724
rs2753938 rs12796085 rs2069725 rs2070709 rs8191448 rs4394909
rs17090719 rs762803 rs2069726 rs11846959 rs8191449 rs2069727
rs1802962 Ile105Val rs947894 IL-13 SNPs rs2749521 rs4986948 -1055
C/T rs1800925 rs2753939 rs675554 rs11575055 rs1802959 rs749174
rs2069755 rs1802961 rs8191450 rs2069741 rs1050469 rs743679
rs2069742 Z allele no rs rs1799811 rs2069743 rs1050520 rs11553890
rs2069756 rs12077 rs4986949 rs3212142 rs12233 rs8191451 rs2066960
rs13170 rs1871042 rs1295687 rs1303 rs11553892 rs3212145 rs1802960
rs4891 rs2069744 rs1243163 rs6413486 rs2069745 rs2073333 rs5031031
rs2069746 rs1243164 rs947895 rs2069747 rs7144409 IFN-SNPs rs2069748
rs7142803 rs2069707 rs1295686 rs1243165 rs3814242 Arg130Gln rs20541
rs1051052 rs2069709 rs2069749 rs1243166 rs2069710 rs1295685
rs11628917 rs2069711 rs848 rs11832 rs2069712 rs2069750 rs9944155
874 A/T rs2430561 rs847 1237 G/A rs11568814 rs2069713
a1-antitrypsin rs877081 SNPs rs1861494 rs709932 rs877082 rs2234685
rs11558261 rs877083 rs1861493 rs20546 rs877084 rs2069714 rs11558263
rs875989 rs2069715 F1028580 rs9944117 rs2069716 rs7145770 rs1884546
rs2069717 rs2239652 rs1884547 rs1885065 rs2735442 rs8046608
rs1884548 rs2569693 rs5743264 rs1243167 rs281439 rs5743266
rs17751614 rs281440 rs2076752 rs1884549 rs2569694 rs5743267
rs1243168 rs11575073 rs8061316 rs17090693 rs2569695 rs8061636
rs17824597 rs2075741 rs16948754 TNFa SNPs rs11575074 rs7206340
rs1799964 rs2569696 rs2076753 rs1800630 rs2735439 rs2067085
rs1799724 rs2569697 rs16948755 +489 G/A rs1800610 rs2075742
rs2111235 rs3093662 rs2569698 rs2111234 rs3093664 rs11669397
rs7190413 -308 G/A rs1800629 (1) rs901886 rs7206582 SMAD3 SNPs
rs885742 rs8045009 C89Y C89Y no rs (2) rs2569699 rs6500328 ICAM1
rs1056538 rs7500036 rs1799969 rs11549918 rs8057341 rs5493 rs2569700
rs12918060 rs5030381 rs2228615 rs7204911 rs5494 rs2569701 rs7500826
rs3093033 rs2569702 rs4785449 rs5495 rs2735440 rs12922299 rs1801714
rs2569703 rs11649521 rs13306429 rs10418913 rs13339578 rs2071441
rs1056536 rs17221417 rs5496 rs2569704 rs13331327 rs5497 rs11673661
rs11642482 rs13306430 rs2569705 rs11642646 E469K rs5498 rs10402760
rs17312836 rs5030400 rs2569706 rs5743268 rs2071440 rs2569707
rs5743269 rs5499 rs2735441 rs5743270 rs3093032 rs2436545 rs12925051
rs1057981 rs2436546 rs12929565 rs5500 rs2916060 rs13380733 rs5501
rs2916059 rs13380741 rs5030383 rs2916058 rs11647841 rs281436
rs2569708 rs10451131 rs923366 rs12972990 rs2066842 rs281437
rs735747 rs5743271 rs3093030 rs885743 rs7498256 rs5030384 NOD2 SNPs
rs5743272 rs5030385 rs4785224 rs5743273 rs3810159 rs5743261
rs2076754 rs281438 rs5743262 rs2066843 rs3093029 rs5743263
rs1078327 rs5743274 rs11645386 rs1031101 rs1861759 rs7187857
rs10824795
rs5743275 rs8061960 rs10824794 rs5743276 rs5743294 rs920725
rs2066844 rs2357791 rs7916582 rs5743277 rs7359452 rs920724
rs5743278 rs7203344 rs16933335 rs6413461 rs5743295 rs11003125
rs3813758 rs5743296 rs7100749 rs5743279 rs3135499 rs11003124
rs5743280 rs5743297 rs7084554 rs5743281 rs5743298 rs7096206
rs4785225 rs5743299 rs11003123 rs16948773 rs3135500 rs11575988
rs9931711 rs5743300 rs11575989 rs17313265 rs8056611 rs7095891
rs11646168 rs2357792 rs4647963 rs9925315 rs12600253 rs8179079
rs5743284 rs12598306 rs5030737 rs5743285 rs7205423 161 G/A
rs1800450 rs751271 rs718226 rs1800451 rs748855 MBL2 SNPs rs12246310
rs1861758 rs7899547 rs12255312 rs13332952 rs10824797 rs11003122
rs7198979 rs11003131 rs1982267 rs1861757 rs930506 rs1982266
rs7203691 rs930505 rs4935047 rs5743286 rs11003130 rs4935046
rs5743287 rs2384044 rs10824793 rs10521209 rs2384045 rs1838066
Gly881Arg rs2066845 rs5027257 rs1838065 rs5743289 rs2384046
rs930509 rs8063130 rs12263867 rs930508 rs2076756 rs11003129
rs930507 rs12920425 rs12221393 CMA1 SNPs rs12920040 rs2165811
rs1956920 rs12920558 rs12782244 rs1956921 rs12919099 rs11003128
-1903 G/A rs1800875 rs12920721 rs17664818 rs1800876 rs2076755
rs7475766 rs3759635 rs5743290 rs10824796 rs1956922 rs5743291
rs16933417 rs1956923 rs11642651 rs2165810 NAT2 SNPs rs1861756
rs11003127 rs11780272 rs749910 rs3925313 rs2101857 rs4990643
rs7094151 rs13363820 rs1077861 rs7071882 rs6984200 rs5743292
rs12264958 rs13277605 rs9921146 rs11003126 rs9987109 rs7820330
rs7596849 -366 G/A rs9550373 rs7460995 rs4848306 rs11542984
rs2087852 rs3087257 rs4769055 rs2101684 rs7556811 rs17074937
rs7011792 rs7556903 rs9671065 rs1390358 rs6743438 rs9579645
rs923796 rs6743427 rs9579646 rs4546703 rs6761336 rs4075131
rs4634684 rs6761335 rs4075132 rs2410556 rs6743338 rs9315043
rs11996129 rs6761245 rs9315044 rs4621844 rs6761237 rs4597169
rs11785247 rs6743330 rs9578037 rs1115783 rs6743326 rs9578196
rs1115784 rs6743322 rs4293222 rs1961456 rs6761220 rs10507391
rs1112005 rs6761218 rs12429692 rs11782802 rs5021469 rs4769871
rs973874 rs6710598 rs4769872 rs1495744 rs1143623 rs4769873
rs7832071 rs1143624 rs12430051 rs1805158 rs2708920 rs9315045
rs1801279 rs1143625 rs9670278 rs1041983 rs2853545 rs4503649
rs1801280 rs2708921 rs9508832 rs4986996 rs1143626 rs9670460
rs12720065 rs3087258 rs3885907 rs4986997 C-511T rs16944 rs3922435
rs1799929 rs3917346 rs9551957 Arg197Gln rs1799930 rs4986962
rs12018461 rs1208 rs1143627 rs9551958 rs1799931 MEH SNPs rs10467440
rs2552 Tyr113His rs1051740 (2) rs12017304 rs4646247 His139Arg
rs2234922 (2) rs9551959 rs971473 ALOX5AP SNPs rs11617473 rs721398
rs4076128 rs11147438 IL-1B SNPs rs9508830 rs10162089 rs10169916
rs4073259 rs9551960 rs13009179 rs4073260 rs9285075 rs4849127
rs11616333 rs12431114 rs4849126 rs4073261 rs4254165 rs7558108
rs4075474 rs4360791 rs13032029 rs4075473 rs17612031 rs13013349
rs9670115 rs3803277 rs12623093 rs9315042 rs3803278 rs3087255
rs3809376 rs12429469 rs3087256 rs12877064 rs17612099 rs6721954
rs9508831 rs9550576 rs12621220 rs9670503 rs4356336 rs4584668
rs2075800 rs2734714 rs4238137 CLCA1 SNPs rs6661730 rs17612127
rs2791519 rs2753377 rs4147063 rs2791518 rs2753378 rs4147064
rs5744302 rs2145412 rs4147062 rs1321697 rs2180762 rs9315046
rs2753338 rs1005569 rs9506352 rs2791517 rs5744325 rs9670531
rs5744303 rs5744326 rs9671182 rs2734706 rs1985554 rs9315047
rs2753345 rs1985555 rs17690694 rs2753347 rs100000102 rs9652070
rs2753348 rs100000103 rs17074966 rs2753349 rs1969719 rs4387455
rs5744304 rs2390102 rs4254166 rs5744305 rs5744329 rs4075692
rs1358826 rs1407142 rs17690748 rs2753359 rs2753384 rs9671124
rs5744306 rs2753385 rs9671125 rs2734711 rs5744330 rs9741436
rs5744307 rs5744331 rs9578197 rs2734712 rs926064 rs4769056
rs2753361 rs926065 rs11147439 rs2753364 rs926066 rs12721459
rs1555389 rs926067 rs4769874 rs2753365 rs2753386 HSP70 HOM SNPs
rs100000100 rs2180764 rs1043618 rs100000101 rs2734689 rs11576009
rs5744310 rs5744332 rs11557922 rs5744311 rs5744333 rs11576010
rs5744312 rs11161837 rs1008438 rs4656114 rs5744335 rs11576011
rs5744313 rs2038485 rs4713489 rs2753367 rs3765989 rs16867582
rs4656115 rs2734690 rs12526722 rs2734713 rs5744336 rs6933097
rs5744314 rs2734691 rs12213612 rs5744315 rs2734692 rs481825
rs5744316 rs5744337 rs7757853 rs5744317 rs5744338 rs7757496
rs5744318 rs2734694 rs9469057 rs926063 rs5744339 rs12182397
rs5744319 rs100000104 rs16867580 rs5744320 rs2791515 rs2075799
rs5744321 rs4656116 rs482145 rs5744322 rs5744342 rs2227957
rs5744323 rs5744343 T2437C rs2227956 rs5744324 rs2180761 rs2227955
rs2791516 rs5744344 rs5744345 rs5744443 rs6032038 rs1358825
rs5744444 rs6032039 rs2145410 rs3138074 rs2267863 rs2734695
rs13166911 rs6124692 rs5744346 rs2563310 +49 C/T No rs rs5744347
rs2569193 rs17333103 rs100000105 rs2569192 rs17333180 rs5744349
rs5744446 rs1983649 rs4655913 rs5744447 rs16989785 rs1321696
rs5744448 rs17424356 rs5744352 rs3138076 rs6017500 rs11583355
rs12519656 rs6032040 rs100000106 rs5744449 rs6017501 rs1321695
rs2915863 rs2664581 +13924 T/A rs1321694 rs3138078 rs17424474
rs2791514 rs6875483 rs17333381 rs2734696 rs2569191 rs1053826
rs5744354 rs5744451 rs2664533 rs2791513 rs5744452 rs1053831
rs2753332 rs100000098 rs2664520 rs2791512 rs17118968 rs2267864
rs2791511 rs5744455 rs13038355 rs2734697 -159 C/T rs2569190
rs13043296 CD14 SNPs rs2569189 rs13039213 rs6877461 rs2563303
rs6104049 rs3822356 rs3138079 rs13043503 rs6877437 rs2228049
rs6104050 rs12153256 rs13763 rs17424578 rs11554680 rs11556179
rs17424613 rs12109040 rs4914 rs6017502 rs12517200 Elafin SNPs
rs6094101 rs5744430 rs2868237 rs6130778 rs5744431 rs4632412
rs6130779 rs100000092 rs7347427 rs6104051 rs5744433 rs6032032
rs6104052 rs100000093 rs10854230 ADBR2 SNPs rs4912717 rs7347426
rs2082382 rs100000094 rs8183548 rs2082394 rs100000095 rs6104047
rs2082395 rs100000096 rs6513967 rs9325119 rs6864930 rs13038813
rs9325120 rs100000097 rs8118673 rs12189018 rs6864583 rs7346463
rs11168066 rs6864580 rs7362841 rs11959615 rs6889418 rs13042694
rs11958940 rs6889416 rs13038342 rs4705270 rs5744440 rs7363327
rs10079142 rs5744441 rs6073668 rs9325121 rs5744442 rs13044826
rs11746634 rs11168067 rs1800468 rs542603 rs9325122 rs4987025
rs574939 rs11957351 rs1800469 rs573764 rs11948371 rs11466314
rs7102189 rs11960649 rs12977628 rs575727 rs1432622 rs12977601
rs552306 rs1432623 rs12985978 rs634607 rs11168068 rs11466315
rs12286876 rs17778257 rs11551223 rs12285331 rs2400706 rs11551226
rs519806 rs2895795 rs11466316 rs12283571 rs2400707 rs13306706
rs2839969 rs2053044 rs13306707 rs2000609 rs17108803 rs13306708
rs7125865 rs12654778 rs9282871 rs570662 rs11168070 Leu10Pro
rs1982073 rs11225427 rs11959427 rs1800471 rs484915 rs1042711
rs13447341 rs470307 rs1801704 rs11466318 rs2408490 rs1042713
rs12976890 rs12279710 Gln27Glu rs1042714 rs12978333 rs685265
rs1042717 rs10420084 rs7107224 rs1800888 rs10418010 rs1155764
rs1042718 rs12983775 rs534191 SOD3 SNPs rs12462166 rs509332
Arg213Gly rs1799895 (2) rs2241715 rs12283759 TGFB1 SNPs rs9749548
rs2105581 rs1529717 rs7258445 rs470206 rs1046909 rs11466320
rs533621 rs2241712 rs11466321 -1607 G/GG rs1799750 rs2241713
rs8108052 rs470211 rs2241714 rs6508976 rs470146 rs11673525
rs8108632 rs2075847 rs2873369 rs11466324 rs473509 rs11083617
rs2241716 rs498186 rs11083616 rs2241717 GSTM1 polymorphism
rs4803458 rs2288873 Null Null allele No rs (2) rs11670143
rs12973435 MMP9 SNPs rs1982072 rs2014015 rs11696804 rs11668109
rs1989457 rs6104416 rs13345981 rs10406816 rs3933239 rs11666933
rs8102918 rs3933240 rs11466310 rs4803455 rs6094237 rs11466311 MMP1
SNPs rs11697325 rs2317130 rs529381 rs6130988 rs4803457 rs1144396
rs6073983 rs3087453 rs504875 rs6130989 rs1800820 rs526215 rs6130990
rs1054797 rs12280880 rs10211842 rs6073984 rs8125587 TIMP3 SNPs
rs6073985 rs3918253 rs5754289 rs8121146 rs2274755 rs5754290
rs6032620 rs2664538 rs9606994 rs11698788 rs3918254 rs7285034
rs6032621 rs6130993 rs13433582 rs6065912 rs3918255 rs1962223
rs6104417 rs2236416 rs8137129 rs3848720 rs6130994 rs1807471
rs13040272 rs3918256 rs7290885 rs6104418 rs3918281 rs5749511
rs3848721 rs3787268 rs11703366 rs3848722 rs3918257 rs4990774
rs6104419 rs6017725 -1296 T/C rs9619311 rs4810482 rs6032623
rs2234921 rs3761157 rs3918258 rs2234920 rs3761158 rs2250889
rs16991235 rs3761159 rs3918259 rs4638893 rs8113877 rs3918260
rs12169569 rs6065913 rs13969 rs5998639 rs6104420 rs6104427
rs7284166 rs6104421 rs6104428 rs5749512 rs3918240 rs2274756
rs6104422 rs6017726 rs3918278 rs3918261 rs3918241 rs6032624 -1562
C/T rs3918242 rs3918262 rs3918243 rs3918263 rs3918279 rs3918264
rs3918280 rs6130995 rs4578914 rs6130996 rs6017724 rs3918265
rs3918244 rs3918266 rs3918245 rs3918267 rs6130992 rs6073987
rs3918247 rs6073988 rs3918248 rs3918282 rs3918249 rs1802909
rs6104423 rs13925 rs6104424 rs20544 rs6104425 rs1056628 rs6104426
rs1802908 rs3918250 rs2664517 rs1805089 rs9509 rs3918251 rs3918268
rs13040572 rs3918269 rs13040580 rs3918270 rs3918252 MMP12 SNPs
rs8125581 -82 A/G rs2276109 (2) (1 = no other SNPs reported to be
in LD, 2 = no other SNPS reported to be in LD)
INDUSTRIAL APPLICATION
[0382] The present invention is directed to methods for assessing a
subject's risk of developing a disease. The methods include the
analysis of polymorphisms herein shown to be associated with
increased or decreased risk of developing a disease, or the
analysis of results obtained from such an analysis, and the
determination of a net risk score. Methods of treating subjects at
risk of developing a disease herein described are also provided.
Additional information regarding the above material, or subparts
thereof, can be found in U.S. patent application Ser. No.
10/479,525, filed Jun. 16, 2004; and PCT Application No.
PCT/NZ02/00106, filed Jun. 5, 2002, which further designates New
Zealand Application No. 512169, filed Jun. 5, 2001; New Zealand
Application No. 513016, filed Jul. 17, 2001, and New Zealand
Application No. 514275, filed Sep. 18, 2001, all of which are
incorporated by reference in their entireties. Additional
information can also be found in PCT application Nos. ______ and
______, filed May 10, 2006, entitled "Methods and Compositions for
Assessment of Pulmonary Function and Disorders" and "Methods of
Analysis of Polymorphisms and Uses Thereof" respectively, having
Agent Reference Nos. 542813JBM and 542814JBM respectively, both of
which are incorporated in their entireties by reference. PCT
Application Agent Reference No. 542813JBM claims priority to: NZ
application No. 539934, filed May 10, 2005; NZ application No.
541935, filed Aug. 19, 2005; and JP application No. 2005-360523,
filed Dec. 14, 2005, all of which are incorporated by reference in
their entireties. PCT Application Agent Reference No. 542814JBM
claims priority to: NZ application No. 540249, filed May 20, 2005;
and NZ application No. 541842, filed Aug. 15, 2005, all of which
are incorporated in their entireties by reference. Additional
information can also be found in U.S. Pat. No. ______, filed
concurrently with the instant application, entitled "Methods and
Compositions for Assessment of Pulmonary Function and Disorders",
attorney docket No; SGENZ.013AUS, incorporated by reference in its
entirety.
PUBLICATIONS
[0383] 1. Sandford A J, et al., 1999. Z and S mutations of the
.alpha.1-antitrypsin gene and the risk of chronic obstructive
pulmonary disease. Am J Respir Cell Mol. Biol. 20; 287-291. [0384]
2. Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning
Manual. 1989. [0385] 3. Papafili A, et al., 2002. Common promoter
variant in cyclooxygenase-2 represses gene expression. Arterioscler
Thromb Vasc Biol. 20; 1631-1635. [0386] 4. Ukkola, O., Erkkila, P.
H., Savolainen, M. J. & Kesaniemi, Y. A. 2001. Lack of
association between polymorphisms of catalase, copper zinc
superoxide dismutase (SOD), extracellular SOD and endothelial
nitric oxide synthase genes and macroangiopathy in patients with
type 2 diabetes mellitus. J Int Med 249; 451-459. [0387] 5. Smith C
A D & Harrison D J, 1997. Association between polymorphism in
gene for microsomal epoxide hydrolase and susceptibility to
emphysema. Lancet. 350; 630-633. [0388] 6. Lorenz E, et al., 2001.
Determination of the TLR4 genotype using allele-specific PRC.
Biotechniques. 31; 22-24. [0389] 7. Cantlay A M, Smith C A, Wallace
W A, Yap P L, Lamb D, Harrison D J. Heterogeneous expression and
polymorphic genotype of glutathione S-transferases in human lung.
Thorax. 1994, 49(10):1010-4.
[0390] All patents, publications, scientific articles, and other
documents and materials referenced or mentioned herein are
indicative of the levels of skill of those skilled in the art to
which the invention pertains, and each such referenced document and
material is hereby incorporated by reference to the same extent as
if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Any and all
materials and information from any such patents, publications,
scientific articles, web sites, electronically available
information, and other referenced materials or documents can be
physically incorporated into this specification.
[0391] The specific methods and compositions described herein are
representative of various embodiments or preferred embodiments and
are exemplary only and not intended as limitations on the scope of
the invention. Other objects, aspects, examples and embodiments
will occur to those skilled in the art upon consideration of this
specification, and are encompassed within the spirit of the
invention as defined by the scope of the claims. It will be readily
apparent to one skilled in the art that varying substitutions and
modifications can be made to the invention disclosed herein without
departing from the scope and spirit of the invention. The invention
illustratively described herein suitably can be practiced in the
absence of any element or elements, or limitation or limitations,
which is not specifically disclosed herein as essential. Thus, for
example, in each instance herein, in embodiments or examples of the
present invention, any of the terms "comprising", "consisting
essentially of", and "consisting of" can be replaced with either of
the other two terms in the specification, thus indicating
additional examples, having different scope, of various alternative
embodiments of the invention. Also, the terms "comprising",
"including", containing", etc. are to be read expansively and
without limitation. The methods and processes illustratively
described herein suitably can be practiced in differing orders of
steps, and that they are not necessarily restricted to the orders
of steps indicated herein or in the claims. It is also that as used
herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly
dictates otherwise. Thus, for example, a reference to "a host cell"
includes a plurality (for example, a culture or population) of such
host cells, and so forth. Under no circumstances may the patent be
interpreted to be limited to the specific examples or embodiments
or methods specifically disclosed herein. Under no circumstances
may the patent be interpreted to be limited by any statement made
by any Examiner or any other official or employee of the Patent and
Trademark Office unless such statement is specifically and without
qualification or reservation expressly adopted in a responsive
writing by the Applicant.
[0392] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed can be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended indicative claims.
[0393] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0394] Other embodiments are within the following indicative
claims. In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
Sequence CWU 1
1
336130DNAArtificial SequenceSynthetic nucleic acid sequence
1acgttggatg gcttgttaac cagctttgcc 30230DNAArtificial
SequenceSynthetic nucleic acid sequence 2acgttggatg tttttcagac
tggcagagcg 30330DNAArtificial SequenceSynthetic nucleic acid
sequence 3acgttggatg tttttcagac tggcagagcg 30430DNAArtificial
SequenceSynthetic nucleic acid sequence 4acgttggatg gcttgttaac
cagctttgcc 30530DNAArtificial SequenceSynthetic nucleic acid
sequence 5acgttggatg catgtcgcct tttcctgctc 30630DNAArtificial
SequenceSynthetic nucleic acid sequence 6acgttggatg caacacccaa
caggcaaatg 30730DNAArtificial SequenceSynthetic nucleic acid
sequence 7acgttggatg tggtggacat ggtgaatgac 30830DNAArtificial
SequenceSynthetic nucleic acid sequence 8acgttggatg tggtgcagat
gctcacatag 30930DNAArtificial SequenceSynthetic nucleic acid
sequence 9acgttggatg cacagagaga gtctggacac 301030DNAArtificial
SequenceSynthetic nucleic acid sequence 10acgttggatg ctcttggtct
ttccctcatc 301130DNAArtificial SequenceSynthetic nucleic acid
sequence 11acgttggatg acagctctgc attcagcacg 301230DNAArtificial
SequenceSynthetic nucleic acid sequence 12acgttggatg agtcaatccc
tttggtgctc 301330DNAArtificial SequenceSynthetic nucleic acid
sequence 13acgttggatg gttttccagc ttgcatgtcc 301430DNAArtificial
SequenceSynthetic nucleic acid sequence 14acgttggatg caatagtcag
gtcctgtctc 301529DNAArtificial SequenceSynthetic nucleic acid
sequence 15acgttggatg gaacggcagc gccttcttg 291630DNAArtificial
SequenceSynthetic nucleic acid sequence 16acgttggatg acttggcaat
ggctgtgatg 301730DNAArtificial SequenceSynthetic nucleic acid
sequence 17acgttggatg cagacattca caattgattt 301830DNAArtificial
SequenceSynthetic nucleic acid sequence 18acgttggatg gatagttcca
aacatgtgcg 301930DNAArtificial SequenceSynthetic nucleic acid
sequence 19acgttggatg gggtattcat aagctgaaac 302030DNAArtificial
SequenceSynthetic nucleic acid sequence 20acgttggatg ccttcaagtt
cagtggtcag 302130DNAArtificial SequenceSynthetic nucleic acid
sequence 21acgttggatg ggtcaatgaa gagaacttgg 302230DNAArtificial
SequenceSynthetic nucleic acid sequence 22acgttggatg aatgtttatt
gtagaaaacc 302315DNAArtificial SequenceSynthetic nucleic acid
sequence 23agctttgcca gttcc 152418DNAArtificial SequenceSynthetic
nucleic acid sequence 24aaaagcaaaa ttgcctga 182515DNAArtificial
SequenceSynthetic nucleic acid sequence 25tcctgctctt ccctc
152617DNAArtificial SequenceSynthetic nucleic acid sequence
26acctccgctg caaatac 172718DNAArtificial SequenceSynthetic nucleic
acid sequence 27gagtctggac acgtgggg 182819DNAArtificial
SequenceSynthetic nucleic acid sequence 28tgctgcaggc cccagatga
192921DNAArtificial SequenceSynthetic nucleic acid sequence
29agaaactttt tcgcgaggga c 213024DNAArtificial SequenceSynthetic
nucleic acid sequence 30agcgccttct tgctggcacc caat
243122DNAArtificial SequenceSynthetic nucleic acid sequence
31tcttacaaca caaaatcaaa tc 223215DNAArtificial SequenceSynthetic
nucleic acid sequence 32agctgaaact tctgg 153320DNAArtificial
SequenceSynthetic nucleic acid sequence 33tcaagcttgc caaagtaatc
203416DNAArtificial SequenceSynthetic nucleic acid sequence
34agctttgcca gttcct 163517DNAArtificial SequenceSynthetic nucleic
acid sequence 35agctttgcca gttccgt 173619DNAArtificial
SequenceSynthetic nucleic acid sequence 36aaaagcaaaa ttgcctgat
193721DNAArtificial SequenceSynthetic nucleic acid sequence
37aaaagcaaaa ttgcctgagg c 213816DNAArtificial SequenceSynthetic
nucleic acid sequence 38tcctgctctt ccctca 163917DNAArtificial
SequenceSynthetic nucleic acid sequence 39tcctgctctt ccctcgt
174018DNAArtificial SequenceSynthetic nucleic acid sequence
40acctccgctg caaataca 184119DNAArtificial SequenceSynthetic nucleic
acid sequence 41acctccgctg caaatacgt 194219DNAArtificial
SequenceSynthetic nucleic acid sequence 42gagtctggac acgtgggga
194320DNAArtificial SequenceSynthetic nucleic acid sequence
43gagtctggac acgtggggga 204420DNAArtificial SequenceSynthetic
nucleic acid sequence 44tgctgcaggc cccagatgat 204521DNAArtificial
SequenceSynthetic nucleic acid sequence 45tgctgcaggc cccagatgag c
214622DNAArtificial SequenceSynthetic nucleic acid sequence
46agaaactttt tcgcgaggga ca 224724DNAArtificial SequenceSynthetic
nucleic acid sequence 47agaaactttt tcgcgaggga cggt
244825DNAArtificial SequenceSynthetic nucleic acid sequence
48agcgccttct tgctggcacc caata 254927DNAArtificial SequenceSynthetic
nucleic acid sequence 49agcgccttct tgctggcacc caatgga
275023DNAArtificial SequenceSynthetic nucleic acid sequence
50tcttacaaca caaaatcaaa tct 235124DNAArtificial SequenceSynthetic
nucleic acid sequence 51tcttacaaca caaaatcaaa tcac
245216DNAArtificial SequenceSynthetic nucleic acid sequence
52agctgaaact tctggc 165317DNAArtificial SequenceSynthetic nucleic
acid sequence 53agctgaaact tctggga 175421DNAArtificial
SequenceSynthetic nucleic acid sequence 54tcaagcttgc caaagtaatc t
215523DNAArtificial SequenceSynthetic nucleic acid sequence
55tcaagcttgc caaagtaatc gga 235630DNAArtificial SequenceSynthetic
nucleic acid sequence 56acgttggatg gaagtcagag atgatggcag
305730DNAArtificial SequenceSynthetic nucleic acid sequence
57acgttggatg atgaatcctg gacccaagac 305830DNAArtificial
SequenceSynthetic nucleic acid sequence 58acgttggatg gaaagatgtg
cgctgatagg 305930DNAArtificial SequenceSynthetic nucleic acid
sequence 59acgttggatg gccacatctc tttctgcatc 306030DNAArtificial
SequenceSynthetic nucleic acid sequence 60acgttggatg ttgcaggtgt
cccatcggaa 306130DNAArtificial SequenceSynthetic nucleic acid
sequence 61acgttggatg tagctcgtgg tggctgtgca 306230DNAArtificial
SequenceSynthetic nucleic acid sequence 62acgttggatg gtgatcaccc
aaggcttcag 306330DNAArtificial SequenceSynthetic nucleic acid
sequence 63acgttggatg gtctgttgac tcttttggcc 306430DNAArtificial
SequenceSynthetic nucleic acid sequence 64acgttggatg gtagctctcc
aggcatcaac 306530DNAArtificial SequenceSynthetic nucleic acid
sequence 65acgttggatg gtacctggtt cccccttttc 306630DNAArtificial
SequenceSynthetic nucleic acid sequence 66acgttggatg tgatcttgtt
caccttgccg 306730DNAArtificial SequenceSynthetic nucleic acid
sequence 67acgttggatg agatcgaggt gacgtttgac 306830DNAArtificial
SequenceSynthetic nucleic acid sequence 68acgttggatg agacacagaa
ccctagatgc 306930DNAArtificial SequenceSynthetic nucleic acid
sequence 69acgttggatg gcaatgaagg atgtttcagg 307030DNAArtificial
SequenceSynthetic nucleic acid sequence 70acgttggatg taagacagct
ccacagcatc 307130DNAArtificial SequenceSynthetic nucleic acid
sequence 71acgttggatg ttccatttcc tcaccctcag 307230DNAArtificial
SequenceSynthetic nucleic acid sequence 72acgttggatg gatttgtgtg
taggaccctg 307330DNAArtificial SequenceSynthetic nucleic acid
sequence 73acgttggatg ggtccccaaa agaaatggag 307430DNAArtificial
SequenceSynthetic nucleic acid sequence 74acgttggatg ggattggaga
acaaactcac 307530DNAArtificial SequenceSynthetic nucleic acid
sequence 75acgttggatg ggcagctgtt acaccaaaag 307630DNAArtificial
SequenceSynthetic nucleic acid sequence 76acgttggatg ctggcgtttt
gcaaacatac 307730DNAArtificial SequenceSynthetic nucleic acid
sequence 77acgttggatg ttgactggaa gaagcaggtg 307830DNAArtificial
SequenceSynthetic nucleic acid sequence 78acgttggatg cctgccaaag
aagaaacacc 307930DNAArtificial SequenceSynthetic nucleic acid
sequence 79acgttggatg acgtctgcag gtatgtattc 308030DNAArtificial
SequenceSynthetic nucleic acid sequence 80acgttggatg acttcatcca
cgtgaagccc 308130DNAArtificial SequenceSynthetic nucleic acid
sequence 81acgttggatg aaactcgtag aaagagccgg 308230DNAArtificial
SequenceSynthetic nucleic acid sequence 82acgttggatg attttctcct
cagaggctcc 308330DNAArtificial SequenceSynthetic nucleic acid
sequence 83acgttggatg tgtctgtatt gagggtgtgg 308430DNAArtificial
SequenceSynthetic nucleic acid sequence 84acgttggatg ttgctggcac
ccaatggaag 308530DNAArtificial SequenceSynthetic nucleic acid
sequence 85acgttggatg atgagagaca tgacgatgcc 308630DNAArtificial
SequenceSynthetic nucleic acid sequence 86acgttggatg actcacagag
cacattcacg 308730DNAArtificial SequenceSynthetic nucleic acid
sequence 87acgttggatg tgtcactcga gatcttgagg 308817DNAArtificial
SequenceSynthetic nucleic acid sequence 88gtgcctgtgc tgggctc
178918DNAArtificial SequenceSynthetic nucleic acid sequence
89ggatggagag aaaaaaac 189017DNAArtificial SequenceSynthetic nucleic
acid sequence 90ccctcatgtc atctact 179117DNAArtificial
SequenceSynthetic nucleic acid sequence 91gtcacccact ctgttgc
179217DNAArtificial SequenceSynthetic nucleic acid sequence
92caaagatggg cgtgatg 179320DNAArtificial SequenceSynthetic nucleic
acid sequence 93ccttgccggt gctcttgtcc 209420DNAArtificial
SequenceSynthetic nucleic acid sequence 94cagaatcctt cctgttacgg
209523DNAArtificial SequenceSynthetic nucleic acid sequence
95tccaccaaga cttaagtttt gct 239617DNAArtificial SequenceSynthetic
nucleic acid sequence 96gaggctgaac cccgtcc 179719DNAArtificial
SequenceSynthetic nucleic acid sequence 97ctttttcata gagtcctgt
199819DNAArtificial SequenceSynthetic nucleic acid sequence
98ttagtcttga agtgagggt 199922DNAArtificial SequenceSynthetic
nucleic acid sequence 99tacttattta cgcttgaacc tc
2210017DNAArtificial SequenceSynthetic nucleic acid sequence
100ccagctgccc gcaggcc 1710117DNAArtificial SequenceSynthetic
nucleic acid sequence 101aattgacaga gagctcc 1710215DNAArtificial
SequenceSynthetic nucleic acid sequence 102cacgacgtca cgcag
1510317DNAArtificial SequenceSynthetic nucleic acid sequence
103cacattcacg gtcacct 1710418DNAArtificial SequenceSynthetic
nucleic acid sequence 104gtgcctgtgc tgggctca 1810519DNAArtificial
SequenceSynthetic nucleic acid sequence 105gtgcctgtgc tgggctcgt
1910619DNAArtificial SequenceSynthetic nucleic acid sequence
106ggatggagag aaaaaaaca 1910720DNAArtificial SequenceSynthetic
nucleic acid sequence 107ggatggagag aaaaaaacgt 2010818DNAArtificial
SequenceSynthetic nucleic acid sequence 108ccctcatgtc atctacta
1810919DNAArtificial SequenceSynthetic nucleic acid sequence
109ccctcatgtc atctactgc 1911018DNAArtificial SequenceSynthetic
nucleic acid sequence 110gtcacccact ctgttgcc 1811119DNAArtificial
SequenceSynthetic nucleic acid sequence 111gtcacccact ctgttgcgc
1911218DNAArtificial SequenceSynthetic nucleic acid sequence
112caaagatggg cgtgatga 1811319DNAArtificial SequenceSynthetic
nucleic acid sequence 113caaagatggg cgtgatggc 1911421DNAArtificial
SequenceSynthetic nucleic acid sequence 114ccttgccggt gctcttgtcc a
2111522DNAArtificial SequenceSynthetic nucleic acid sequence
115ccttgccggt gctcttgtcc gt 2211621DNAArtificial SequenceSynthetic
nucleic acid sequence 116cagaatcctt cctgttacgg c
2111722DNAArtificial SequenceSynthetic nucleic acid sequence
117cagaatcctt cctgttacgg tc 2211824DNAArtificial SequenceSynthetic
nucleic acid sequence 118tccaccaaga cttaagtttt gctc
2411925DNAArtificial SequenceSynthetic nucleic acid sequence
119tccaccaaga cttaagtttt gcttc 2512018DNAArtificial
SequenceSynthetic nucleic acid sequence 120gaggctgaac cccgtccc
1812119DNAArtificial SequenceSynthetic nucleic acid sequence
121gaggctgaac cccgtcctc 1912220DNAArtificial SequenceSynthetic
nucleic acid sequence 122ctttttcata gagtcctgtt 2012322DNAArtificial
SequenceSynthetic nucleic acid sequence 123ctttttcata gagtcctgta ac
2212420DNAArtificial SequenceSynthetic nucleic acid sequence
124ttagtcttga agtgagggta 2012521DNAArtificial SequenceSynthetic
nucleic acid sequence 125ttagtcttga agtgagggtg t
2112623DNAArtificial SequenceSynthetic nucleic acid sequence
126tacttattta cgcttgaacc tca 2312724DNAArtificial SequenceSynthetic
nucleic acid sequence 127tacttattta cgcttgaacc tcga
2412818DNAArtificial SequenceSynthetic nucleic acid sequence
128ccagctgccc gcaggcca 1812919DNAArtificial SequenceSynthetic
nucleic acid sequence 129ccagctgccc gcaggccgt 1913018DNAArtificial
SequenceSynthetic nucleic acid sequence 130aattgacaga gagctccc
1813119DNAArtificial SequenceSynthetic nucleic acid sequence
131aattgacaga gagctcctg 1913216DNAArtificial SequenceSynthetic
nucleic acid sequence 132cacgacgtca cgcagc 1613317DNAArtificial
SequenceSynthetic nucleic acid sequence 133cacgacgtca cgcagga
1713418DNAArtificial SequenceSynthetic nucleic acid sequence
134cacattcacg gtcacctc 1813519DNAArtificial SequenceSynthetic
nucleic acid sequence 135cacattcacg gtcaccttg 1913621DNAArtificial
SequenceSynthetic nucleic acid sequence 136ctaccaggaa tggccttgtc c
2113721DNAArtificial SequenceSynthetic nucleic acid sequence
137ctctcaggtc tggtgtcatc c 2113830DNAArtificial SequenceSynthetic
nucleic acid sequence 138gattagcata cttagactac tacctccatg
3013927DNAArtificial SequenceSynthetic nucleic acid sequence
139gatcaacttc tgaaaaagca ttcccac 2714021DNAArtificial
SequenceSynthetic nucleic acid sequence 140tcgtgagaat gtcttcccat t
2114129DNAArtificial SequenceSynthetic nucleic acid sequence
141tcttggattg atttgagata agtgaaatc 2914230DNAArtificial
SequenceSynthetic nucleic acid sequence 142acgttggatg gcttgttaac
cagctttgcc 3014330DNAArtificial SequenceSynthetic nucleic acid
sequence 143acgttggatg tttttcagac tggcagagcg 3014430DNAArtificial
SequenceSynthetic nucleic acid sequence 144acgttggatg tttttcagac
tggcagagcg 3014530DNAArtificial SequenceSynthetic nucleic acid
sequence 145acgttggatg gcttgttaac cagctttgcc 3014630DNAArtificial
SequenceSynthetic nucleic acid sequence 146acgttggatg ttgctggcac
ccaatggaag 3014730DNAArtificial SequenceSynthetic nucleic acid
sequence 147acgttggatg atgagagaca tgacgatgcc 3014830DNAArtificial
SequenceSynthetic nucleic acid sequence 148acgttggatg tggtggacat
ggtgaatgac 3014930DNAArtificial SequenceSynthetic nucleic acid
sequence 149acgttggatg tggtgcagat gctcacatag 3015030DNAArtificial
SequenceSynthetic nucleic acid sequence 150acgttggatg cacagagaga
gtctggacac 3015130DNAArtificial SequenceSynthetic nucleic acid
sequence 151acgttggatg ctcttggtct ttccctcatc 3015230DNAArtificial
SequenceSynthetic nucleic acid sequence 152acgttggatg cctctgatcc
tctttgcttc 3015330DNAArtificial SequenceSynthetic nucleic acid
sequence 153acgttggatg aagagggagt ggaagggaag 3015430DNAArtificial
SequenceSynthetic nucleic acid sequence 154acgttggatg acagctctgc
attcagcacg 3015530DNAArtificial SequenceSynthetic nucleic acid
sequence 155acgttggatg agtcaatccc tttggtgctc 3015630DNAArtificial
SequenceSynthetic nucleic acid sequence 156acgttggatg actgaagctc
cacaatttgg 3015731DNAArtificial SequenceSynthetic nucleic acid
sequence 157acgttggatg gccactctag tactatatct g 3115830DNAArtificial
SequenceSynthetic nucleic acid sequence 158acgttggatg gggtattcat
aagctgaaac 3015930DNAArtificial SequenceSynthetic nucleic acid
sequence 159acgttggatg ccttcaagtt cagtggtcag 3016030DNAArtificial
SequenceSynthetic nucleic acid sequence 160acgttggatg ggtcaatgaa
gagaacttgg 3016130DNAArtificial SequenceSynthetic nucleic acid
sequence 161acgttggatg aatgtttatt gtagaaaacc 3016215DNAArtificial
SequenceSynthetic nucleic acid sequence 162agctttgcca gttcc
1516318DNAArtificial SequenceSynthetic nucleic acid sequence
163aaaagcaaaa ttgcctga 1816415DNAArtificial SequenceSynthetic
nucleic acid sequence 164cacgacgtca cgcag 1516517DNAArtificial
SequenceSynthetic nucleic acid sequence 165acctccgctg caaatac
1716618DNAArtificial SequenceSynthetic nucleic acid sequence
166gagtctggac acgtgggg 1816719DNAArtificial SequenceSynthetic
nucleic acid sequence 167tccatctctg tggatctcc 1916819DNAArtificial
SequenceSynthetic nucleic acid sequence 168tgctgcaggc cccagatga
1916921DNAArtificial SequenceSynthetic nucleic acid sequence
169cacaatttgg tgaattatca a 2117015DNAArtificial SequenceSynthetic
nucleic acid sequence 170agctgaaact tctgg 1517120DNAArtificial
SequenceSynthetic nucleic acid sequence 171tcaagcttgc caaagtaatc
2017216DNAArtificial SequenceSynthetic nucleic acid sequence
172agctttgcca gttcct 1617317DNAArtificial SequenceSynthetic nucleic
acid sequence 173agctttgcca gttccgt 1717419DNAArtificial
SequenceSynthetic nucleic acid sequence 174aaaagcaaaa ttgcctgat
1917521DNAArtificial SequenceSynthetic nucleic acid sequence
175aaaagcaaaa ttgcctgagg c 2117616DNAArtificial SequenceSynthetic
nucleic acid sequence 176cacgacgtca cgcagc 1617717DNAArtificial
SequenceSynthetic nucleic acid sequence 177cacgacgtca cgcagga
1717818DNAArtificial SequenceSynthetic nucleic acid sequence
178acctccgctg caaataca 1817919DNAArtificial SequenceSynthetic
nucleic acid sequence 179acctccgctg caaatacgt 1918019DNAArtificial
SequenceSynthetic nucleic acid sequence 180gagtctggac acgtgggga
1918120DNAArtificial SequenceSynthetic nucleic acid sequence
181gagtctggac acgtggggga 2018220DNAArtificial SequenceSynthetic
nucleic acid sequence 182tccatctctg tggatctcca 2018321DNAArtificial
SequenceSynthetic nucleic acid sequence 183tccatctctg tggatctccg t
2118420DNAArtificial SequenceSynthetic nucleic acid sequence
184tgctgcaggc cccagatgat 2018521DNAArtificial SequenceSynthetic
nucleic acid sequence 185tgctgcaggc cccagatgag c
2118622DNAArtificial SequenceSynthetic nucleic acid sequence
186cacaatttgg tgaattatca at 2218723DNAArtificial SequenceSynthetic
nucleic acid sequence 187cacaatttgg tgaattatca aat
2318816DNAArtificial SequenceSynthetic nucleic acid sequence
188agctgaaact tctggc 1618917DNAArtificial SequenceSynthetic nucleic
acid sequence 189agctgaaact tctggga 1719021DNAArtificial
SequenceSynthetic nucleic acid sequence 190tcaagcttgc caaagtaatc t
2119123DNAArtificial SequenceSynthetic nucleic acid sequence
191tcaagcttgc caaagtaatc gga 2319220DNAArtificial SequenceSynthetic
nucleic acid sequence 192ctgccctact tgattgatgg 2019320DNAArtificial
SequenceSynthetic nucleic acid sequence 193atcttctcct cttctgtctc
2019420DNAArtificial SequenceSynthetic nucleic acid sequence
194ttctggattg tagcagatca 2019521DNAArtificial SequenceSynthetic
nucleic acid sequence 195tcgtgagaat gtcttcccat t
2119629DNAArtificial SequenceSynthetic nucleic acid sequence
196tcttggattg atttgagata agtgaaatc 2919730DNAArtificial
SequenceSynthetic nucleic acid sequence 197acgttggatg aaaccagagg
gaagcaaagg 3019830DNAArtificial SequenceSynthetic nucleic acid
sequence 198acgttggatg tcattggttg tgctgcacct 3019930DNAArtificial
SequenceSynthetic nucleic acid sequence 199acgttggatg caccaggaac
cgtttatggc 3020030DNAArtificial SequenceSynthetic nucleic acid
sequence 200acgttggatg agcagctaga atcagaggag 3020131DNAArtificial
SequenceSynthetic nucleic acid sequence 201acgttggatg gtcaatgaag
agaacttggt c 3120230DNAArtificial SequenceSynthetic nucleic acid
sequence 202acgttggatg aatgtttatt gtagaaaacc 3020330DNAArtificial
SequenceSynthetic nucleic acid sequence 203acgttggatg gggtattcat
aagctgaaac 3020430DNAArtificial SequenceSynthetic nucleic acid
sequence 204acgttggatg ccttcaagtt cagtggtcag 3020530DNAArtificial
SequenceSynthetic nucleic acid sequence 205acgttggatg gtgattatct
ttggcatggg 3020630DNAArtificial SequenceSynthetic nucleic acid
sequence 206acgttggatg ggatagccag gaagagaaag 3020730DNAArtificial
SequenceSynthetic nucleic acid sequence 207acgttggatg ccctatttct
ttgtcttcac 3020830DNAArtificial SequenceSynthetic nucleic acid
sequence 208acgttggatg cttgggataa tttggctctg 3020930DNAArtificial
SequenceSynthetic nucleic acid sequence 209acgttggatg ggaacccttt
ctgcgctttg 3021030DNAArtificial SequenceSynthetic nucleic acid
sequence 210acgttggatg cctacaggtg ctgttcagtg 3021130DNAArtificial
SequenceSynthetic nucleic acid sequence 211acgttggatg cctgccaaag
aagaaacacc 3021230DNAArtificial SequenceSynthetic nucleic acid
sequence 212acgttggatg acgtctgcag gtatgtattc 3021330DNAArtificial
SequenceSynthetic nucleic acid sequence 213acgttggatg gttcttaatt
cataggttgc 3021432DNAArtificial SequenceSynthetic nucleic acid
sequence 214acgttggatg cttcatttct catcatattt tc
3221530DNAArtificial SequenceSynthetic nucleic acid sequence
215acgttggatg taggtgtctc cccctgtaag 3021630DNAArtificial
SequenceSynthetic nucleic acid sequence 216acgttggatg tcctctccag
agtgatcaag 3021730DNAArtificial SequenceSynthetic nucleic acid
sequence 217acgttggatg attttctcct cagaggctcc 3021830DNAArtificial
SequenceSynthetic nucleic acid sequence 218acgttggatg tgtctgtatt
gagggtgtgg 3021930DNAArtificial SequenceSynthetic nucleic acid
sequence 219acgttggatg ttgtggctgc aacatgagag 3022030DNAArtificial
SequenceSynthetic nucleic acid sequence 220acgttggatg ctatggcgca
acatctgtac 3022130DNAArtificial SequenceSynthetic nucleic acid
sequence 221acgttggatg actgtagttt ccctagtccc 3022230DNAArtificial
SequenceSynthetic nucleic acid sequence 222acgttggatg agtcagcaga
gagactaggg 3022331DNAArtificial SequenceSynthetic nucleic acid
sequence 223acgttggatg gagttgagaa tggagagaat g 3122430DNAArtificial
SequenceSynthetic nucleic acid sequence 224acgttggatg tcaagtgggc
tgttagggtg 3022530DNAArtificial SequenceSynthetic nucleic acid
sequence 225acgttggatg tgctgcgtgg tgggcgtgtg 3022629DNAArtificial
SequenceSynthetic nucleic acid sequence 226acgttggatg ggccttgcac
tcgctctcg 2922730DNAArtificial SequenceSynthetic nucleic acid
sequence 227acgttggatg aaacggtcgc ttcgacgtgc 3022829DNAArtificial
SequenceSynthetic nucleic acid sequence 228acgttggatg acctcaagga
ccagctcgg 2922930DNAArtificial SequenceSynthetic nucleic acid
sequence 229acgttggatg actgaagctc cacaatttgg 3023031DNAArtificial
SequenceSynthetic nucleic acid sequence 230acgttggatg gccactctag
tactatatct g 3123130DNAArtificial SequenceSynthetic nucleic acid
sequence 231acgttggatg cagacattca caattgattt 3023230DNAArtificial
SequenceSynthetic nucleic acid sequence 232acgttggatg gatagttcca
aacatgtgcg 3023330DNAArtificial SequenceSynthetic nucleic acid
sequence 233acgttggatg taaggagtgg gtgctggact 3023430DNAArtificial
SequenceSynthetic nucleic acid sequence 234acgttggatg aggataagga
gcagggttgg 3023517DNAArtificial SequenceSynthetic nucleic acid
sequence 235ttcttggttc aggagag 1723618DNAArtificial
SequenceSynthetic nucleic acid sequence 236ttcttggttc aggagagc
1823719DNAArtificial SequenceSynthetic nucleic acid sequence
237gcaatctgct ctatcctct 1923820DNAArtificial SequenceSynthetic
nucleic acid sequence 238gcaatctgct ctatcctctt 2023922DNAArtificial
SequenceSynthetic nucleic acid sequence 239attcaagctt gccaaagtaa tc
2224023DNAArtificial SequenceSynthetic nucleic acid sequence
240attcaagctt gccaaagtaa tct 2324119DNAArtificial SequenceSynthetic
nucleic acid sequence 241cataagctga aacttctgg 1924220DNAArtificial
SequenceSynthetic nucleic acid sequence 242cataagctga aacttctggc
2024320DNAArtificial SequenceSynthetic nucleic acid sequence
243ggaagtgtat cggtgagacc 2024421DNAArtificial SequenceSynthetic
nucleic acid sequence 244ggaagtgtat cggtgagacc a
2124523DNAArtificial SequenceSynthetic nucleic acid sequence
245tgacaaatac tggttaatta gca 2324624DNAArtificial SequenceSynthetic
nucleic acid sequence 246tgacaaatac tggttaatta gcaa
2424717DNAArtificial SequenceSynthetic nucleic acid sequence
247gctcctgagc atggcgg 1724818DNAArtificial SequenceSynthetic
nucleic acid sequence 248gctcctgagc atggcggc 1824922DNAArtificial
SequenceSynthetic nucleic acid sequence 249tacttattta cgcttgaacc tc
2225023DNAArtificial SequenceSynthetic nucleic acid sequence
250tacttattta cgcttgaacc tca 2325123DNAArtificial SequenceSynthetic
nucleic acid sequence 251cttaattcat aggttgcaat ttt
2325224DNAArtificial SequenceSynthetic nucleic acid sequence
252cttaattcat aggttgcaat ttta 2425317DNAArtificial
SequenceSynthetic nucleic acid sequence 253acatcaccct cacttac
1725418DNAArtificial SequenceSynthetic nucleic acid sequence
254acatcaccct cacttacc 1825517DNAArtificial SequenceSynthetic
nucleic acid sequence 255aattgacaga gagctcc 1725618DNAArtificial
SequenceSynthetic nucleic acid sequence 256aattgacaga gagctccc
1825720DNAArtificial SequenceSynthetic nucleic acid sequence
257atgagaggct cacagacgtt 2025821DNAArtificial SequenceSynthetic
nucleic acid sequence 258atgagaggct cacagacgtt c
2125921DNAArtificial SequenceSynthetic nucleic acid sequence
259ggcatcaagc tcttccctgg c 2126022DNAArtificial SequenceSynthetic
nucleic acid sequence 260ggcatcaagc tcttccctgg cc
2226117DNAArtificial SequenceSynthetic nucleic acid sequence
261gaatgttacc tctcctg 1726218DNAArtificial SequenceSynthetic
nucleic acid sequence 262gaatgttacc tctcctga 1826319DNAArtificial
SequenceSynthetic nucleic acid sequence 263gcactcagag cgcaagaag
1926420DNAArtificial SequenceSynthetic nucleic acid sequence
264gcactcagag cgcaagaagc 2026521DNAArtificial SequenceSynthetic
nucleic acid sequence 265gctgctgcag gccccagatg a
2126622DNAArtificial SequenceSynthetic nucleic acid sequence
266gctgctgcag gccccagatg at 2226721DNAArtificial SequenceSynthetic
nucleic acid sequence 267cacaatttgg tgaattatca a
2126822DNAArtificial SequenceSynthetic nucleic acid sequence
268cacaatttgg tgaattatca at 2226923DNAArtificial SequenceSynthetic
nucleic acid sequence 269ttcttacaac acaaaatcaa atc
2327024DNAArtificial SequenceSynthetic nucleic acid sequence
270ttcttacaac acaaaatcaa atct 2427117DNAArtificial
SequenceSynthetic nucleic acid sequence 271tcggcggctg ccctccc
1727218DNAArtificial SequenceSynthetic nucleic acid sequence
272tcggcggctg ccctccca 1827319DNAArtificial SequenceSynthetic
nucleic acid sequence 273ttcttggttc aggagaggt 1927421DNAArtificial
SequenceSynthetic nucleic acid sequence 274gcaatctgct ctatcctctg c
2127525DNAArtificial SequenceSynthetic nucleic acid sequence
275attcaagctt gccaaagtaa tcgga 2527621DNAArtificial
SequenceSynthetic nucleic acid sequence 276cataagctga aacttctggg a
2127722DNAArtificial SequenceSynthetic nucleic acid sequence
277ggaagtgtat cggtgagacc gt 2227825DNAArtificial SequenceSynthetic
nucleic acid sequence 278tgacaaatac tggttaatta gcagt
2527919DNAArtificial SequenceSynthetic nucleic acid sequence
279gctcctgagc atggcggga 1928024DNAArtificial SequenceSynthetic
nucleic acid sequence 280tacttattta cgcttgaacc tcga
2428125DNAArtificial SequenceSynthetic nucleic acid sequence
281cttaattcat aggttgcaat tttgt 2528219DNAArtificial
SequenceSynthetic nucleic acid sequence 282acatcaccct cacttactg
1928319DNAArtificial SequenceSynthetic nucleic acid sequence
283aattgacaga gagctcctg 1928422DNAArtificial SequenceSynthetic
nucleic acid sequence 284atgagaggct cacagacgtt tc
2228523DNAArtificial SequenceSynthetic nucleic acid sequence
285ggcatcaagc tcttccctgg ctg 2328619DNAArtificial SequenceSynthetic
nucleic acid sequence 286gaatgttacc tctcctggc 1928723DNAArtificial
SequenceSynthetic nucleic acid sequence 287gcactcagag cgcaagaagg
ggc 2328823DNAArtificial SequenceSynthetic nucleic acid sequence
288gctgctgcag gccccagatg agc 2328923DNAArtificial SequenceSynthetic
nucleic acid sequence 289cacaatttgg tgaattatca aat
2329025DNAArtificial SequenceSynthetic nucleic acid sequence
290ttcttacaac acaaaatcaa atcac 2529120DNAArtificial
SequenceSynthetic nucleic acid sequence 291tcggcggctg ccctcccgga
2029230DNAArtificial SequenceSynthetic nucleic acid sequence
292acgttggatg aggtagctga agaggcaaac 3029330DNAArtificial
SequenceSynthetic nucleic acid sequence 293acgttggatg gcctatagcc
tctaaaacgc 3029430DNAArtificial SequenceSynthetic nucleic acid
sequence 294acgttggatg ctttcaattt gtggaggctg 3029530DNAArtificial
SequenceSynthetic nucleic acid sequence 295acgttggatg tgtgcactca
tttgtggacg 3029630DNAArtificial SequenceSynthetic nucleic acid
sequence 296acgttggatg gtagctctcc aggcatcaac 3029730DNAArtificial
SequenceSynthetic nucleic acid sequence 297acgttggatg gtacctggtt
cccccttttc 3029830DNAArtificial SequenceSynthetic nucleic acid
sequence 298acgttggatg acaccaggcg tttgatgttg 3029930DNAArtificial
SequenceSynthetic nucleic acid sequence 299acgttggatg aaaaacgcca
acagcatcgg 3030029DNAArtificial SequenceSynthetic nucleic acid
sequence 300acgttggatg aggcggagat gggtgtgtc 2930130DNAArtificial
SequenceSynthetic nucleic acid sequence 301acgttggatg agtctagggt
ggggtatgtg 3030230DNAArtificial SequenceSynthetic nucleic acid
sequence 302acgttggatg atgtgtggat tcacagctcg 3030330DNAArtificial
SequenceSynthetic nucleic acid sequence 303acgttggatg gggttggcaa
ctctaaaagg 3030430DNAArtificial SequenceSynthetic nucleic acid
sequence 304acgttggatg ctctgaaatc agtgctgctc 3030530DNAArtificial
SequenceSynthetic nucleic acid sequence 305acgttggatg atggtcaaca
gtgttgccag 3030630DNAArtificial SequenceSynthetic nucleic acid
sequence 306acgttggatg cacctcttga ttgctttccc 3030730DNAArtificial
SequenceSynthetic nucleic acid sequence 307acgttggatg acccggcctt
cctgatcatg 3030830DNAArtificial SequenceSynthetic nucleic acid
sequence 308acgttggatg attccatgga ggctggatag 3030930DNAArtificial
SequenceSynthetic nucleic acid sequence 309acgttggatg gacaacacta
ctaaggcttc 3031017DNAArtificial SequenceSynthetic nucleic acid
sequence 310aaaaggtttc tcccccc 1731118DNAArtificial
SequenceSynthetic nucleic acid sequence 311aaaaggtttc tccccccc
1831217DNAArtificial SequenceSynthetic nucleic acid sequence
312aggctgcttc ttggact 1731318DNAArtificial SequenceSynthetic
nucleic acid sequence 313aggctgcttc ttggactc 1831417DNAArtificial
SequenceSynthetic nucleic acid sequence 314caaagatggg cgtgatg
1731518DNAArtificial SequenceSynthetic nucleic acid sequence
315caaagatggg cgtgatga 1831617DNAArtificial SequenceSynthetic
nucleic acid sequence 316gccagccccg ggacgga 1731718DNAArtificial
SequenceSynthetic nucleic acid sequence 317gccagccccg ggacggac
1831817DNAArtificial SequenceSynthetic nucleic acid sequence
318atgggtgtgt ctgccgg 1731918DNAArtificial SequenceSynthetic
nucleic acid sequence 319atgggtgtgt ctgccgga 1832019DNAArtificial
SequenceSynthetic nucleic acid sequence 320ggctgtaagg aaatctggg
1932120DNAArtificial SequenceSynthetic nucleic acid sequence
321ggctgtaagg aaatctggga 2032220DNAArtificial SequenceSynthetic
nucleic acid sequence 322ccttatcctc ctcctgggaa 2032321DNAArtificial
SequenceSynthetic nucleic acid sequence 323ccttatcctc ctcctgggaa a
2132420DNAArtificial SequenceSynthetic nucleic acid sequence
324tgtttcattt ctataggcga 2032521DNAArtificial SequenceSynthetic
nucleic acid sequence 325tgtttcattt ctataggcga t
2132617DNAArtificial SequenceSynthetic nucleic acid sequence
326cctatcccta cttcccc 1732718DNAArtificial SequenceSynthetic
nucleic acid sequence 327cctatcccta cttccccc 1832819DNAArtificial
SequenceSynthetic nucleic acid sequence 328aaaaggtttc tccccccga
1932919DNAArtificial SequenceSynthetic nucleic acid sequence
329aggctgcttc ttggactga 1933019DNAArtificial SequenceSynthetic
nucleic acid sequence 330caaagatggg cgtgatggc 1933119DNAArtificial
SequenceSynthetic nucleic acid sequence 331gccagccccg ggacggagt
1933219DNAArtificial SequenceSynthetic nucleic acid sequence
332atgggtgtgt ctgccgggt 1933322DNAArtificial SequenceSynthetic
nucleic acid sequence 333ggctgtaagg aaatctgggg gt
2233422DNAArtificial SequenceSynthetic nucleic acid sequence
334ccttatcctc ctcctgggaa ga 2233523DNAArtificial SequenceSynthetic
nucleic acid sequence 335tgtttcattt ctataggcga gga
2333620DNAArtificial SequenceSynthetic nucleic acid sequence
336cctatcccta cttccccttc 20
* * * * *
References