AU2012203045A1 - Methods Of Analysis Of Polymorphisms And Uses Thereof - Google Patents

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.

Description

1 Regulation 3.2 AUSTRALIA PATENTS ACT, 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT DIVISIONAL Name of Applicant: SYNERGENZ BIOSCIENCE LIMITED Actual Inventors: YOUNG, Robert Peter Address for service in AJ PARK, Level 11, 60 Marcus Clarke Street, Canberra ACT 2601, Australia: Australia Invention Title: Methods Of Analysis Of Polymorphisms And Uses Thereof Original application: Australian application 2006248189 dated 10 May 2006 The following statement is a full description of this invention, including the best method of performing it known to us.

2 "METHODS OF ANALYSIS OF POLYMORPHISMS AND USES THEREOF" FIELD OF THE INVENTION The present invention is concerned with methods for the assessment of diseases 5 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 10 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 15 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 20 present in less than 1% of the general population). Moreover, these relatively common polymorphisms may 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 25 detected. There is thus a need for a method for assessing a subject's risk of developing a disease using genetic (and optionally non-genetic) risk factors. 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. 30 SUMMARY OF THE INVENTION The Applicant's recent studies have identified a number of genetic variants or polymorphisms that confer susceptibility to protection from COPD, occupational COPD 3 (OCOPD), and lung cancer. The biological basis of just how these polymorphisms interact or combine to determine risk remains unclear. The Applicants have now surprisingly found that an assessment approach which determines a subject's net score following the balancing of the number of 5 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, the applicants have determined that this approach is widely applicable, on a disease-by-disease basis. It is broadly to this approach to risk assessment that the present invention is 10 directed. Accordingly, in a first aspect, the present invention provides 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 protective polymorphisms and for the presence or absence of susceptibility 15 polymorphisms, 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; calculating a net score for said subject, said net score representing the balance 20 between the combined value of the protective polymorphisms and the combined value of the susceptibility polymorphisms 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 developingsaid disease. 25 The value assigned to each protective polymorphism may be the same or may be different.The value assigned to each susceptibility polymorphism may be the same or may be different, with either each protective polymorphism having a negative value and each susceptibility polymorphism having a positive value, or vice vcrsa.When the disease is a lung disease, the protective polymorphisms analysed may be selected from 30 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); 4 CC (homozygous P allele) within codon 10 of the gene encoding transforming growth factor beta (TGFB); 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 5 polymorphisms. 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 infers the presence of the other. (Reich DE et al; Linkage disequilibrium in the human 10 genome, Nature 2001, 411:199-204.). Preferably, all polymorphisms of the group are analysed. 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 15 (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 al-antitrypsin (alAT); or 20 one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms. Preferably, all polymorphisms of the group are analysed. In one embodiment each protective polymorphism is assigned a value of -1 and each susceptibility polymorphism is assigned a value of +1. 25 In another embodiment each protective polymorphism is assigned a value of +1 and each susceptibility polymorphism is assigned a value of -1. When the disease is COPD, the protective polymorphisms analysed may 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); 30 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); 5 MS in the gene encoding al-antitrypsin (a1AT); the +489 GG geneotype in the gene encoding Tumour Necrosis factor a (TNFa); the -308 GG geneotype in the gene encoding TNFa; the C89Y AA or AG geneotype in the gene encodoing SMAD3; 5 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-Acctyl transferase 2 (NAT2); the His 139 Arg GG genotype in the gene encoding Microsomal epoxide 10 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; 15 the Gln 27 Glu GG genotype in the gene encoding P2 Adrenergic receptor (ADBR); the -1607 1GIG 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 20 these polymorphisms.Preferably, all polymorphisms of the group are analysed. Preferably, the susceptibility polymorphisms analysed are selected from one or more of the group consisting of: Ag 16 Gly GG in the gene encoding p2-adrenoreceptor (ADRB2); 105 AA in the gene encoding Interleukin-1 8 (IL-18); 25 -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-i); -1055 TT in the promoter of the gene encoding IL-13; 874 TT in the gene encoding interferon gamma (IFNy); 30 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); 6 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; 5 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); 10 or one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms. Preferably, all polymorphisms of the group are analysed. In one embodiment each protective polymorphism is assigned a value of -1 and each susceptibility polymorphism is assigned a value of +1. 15 In one embodiment each protective polymorphism is assigned a value of +1 and each susceptibility polymorphism is assigned a value of -1. When the disease is OCOPD, the protective polymorphisms analysed may be selected from one or more of the group consisting of: -765 CC or CG in the promoter of the gene encoding COX2; 20 -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; 25 MS or SS in the gene encoding alAT; 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 30 one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms. Preferably, all polymorphisms of the group are analysed. Preferably, the susceptibility polymorphisms analysed are selected from one or more of the group consisting of: 7 -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; 5 Lys 420 Thr CC in the gene encoding VDBP; Glu 416 Asp GG in the gene encoding VDBP; le 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; 10 3' 1237 Tt or tt in the gene encoding a1AT; -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. Preferably, all polymorphisms of the group are analysed. 15 In one embodiment each protective polymorphism is assigned a value of -1 and each susceptibility polymorphism is assigned a value of +1. In one embodiment each protective polymorphism is assigned a value of +1 and each susceptibility polymorphism is assigned a value of -1. When the disease is lung cancer, the protective polymorphisms analysed may be 20 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 MMP 12; the 105 AC or CC genotype in the gene encoding IL-18; 25 the -133 CG or GG genotype in the gene encoding IL-18; the -765 CC or CO 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 (Argi); the Leu252Val GG genotype in the gene encoding Insulin-like growth factor II 30 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 (XRCC 1) gene; 8 the A870G GG genotype in the gene encoding cyclin D (CCNDI); 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 5 (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 REVI; or one or more polymorphisms in linkage disequilibrium with any one or more 10 of these polymorphisms. Preferably, all polymorphisms of the group are analysed. 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; 15 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 IFNy; the -765 GG genotype in the promoter of the gene encoding COX2; 20 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. the -511 GG genotype in the gene encoding IL-1B; the A-670G AA genotype in the gene encoding FAS (Apo-1/CD95); 25 the Arg 197 Gln GG genotype in the gene encoding N-acetyltransferase 2 (NAT2); the Ile462 Val AA genotype in the gene encoding CYP I Al; the 1019 G/C Pst I CC or CG genotype in the gene encoding cytochrome P450 2E1 (CYP2E1); 30 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 MMPI; the Gln 185 Glu CC genotype in the gene encoding Nibrin (NBS 1); the Asp 148 Glu GG genotype in the gene encoding Apex nuclease (APE1); 9 or one or more polymorphisms in linkage disequilibrium with any one or more of these polymorphisms. Preferably, all polymorphisms of the group are analysed. In one embodiment each protective polymorphism is assigned a value of -1 and 5 each susceptibility polymorphism is assigned a value of +1. In one embodiment each protective polymorphism is assigned a value of +1 and each susceptibility polymorphism is assigned a value of -1. In various embodiments the subject is or has been a smoker. Preferably, the methods of the invention are performed in conjunction with an 10 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. In another aspect, the invention provides a method of determining a subject's 15 risk of developing a disease, said method comprising 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; 20 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 polymorphisms and the combined value of the susceptibility polymorphisms present in the subject sample; 25 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 developingsaid disease. In a further aspect the present invention provides a method for assessing the risk of a subject developing a disease which comprises 30 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, 10 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. In another aspect, the present invention provides a kit for assessing a subject's 5 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. 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 10 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. 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 15 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 20 and/or functional effect of one or more protective polymorphisms associated with said disease. BRIEF DESCRIPTION OF FIGURES Figure 1: depicts a graph showing combined frequencies of the presence or 25 absence of selected protective genotypes in the COP) subjects and in resistant smokers. Figure 2: depicts a graph showing net scores for protective and susceptibility polymorphisms in COPD subjects. Figure 3: depicts a graph showing net scores for protective and susceptibility 30 polymorphisms in OCOP) subjects. Figure 4: depicts a graph showing net scores for protective and susceptibility polymorphisms in subjects with lung cancer.

11 Figure 5: depicts a graph showing net scores for protective and susceptibility polymorphisms in subjects with lung cancer. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is directed to methods for the assessment of the genetic 5 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 10 developing a particular disease (which can be termed "susceptibility polymorphisms" or "susceptibility SNPs"). 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 15 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 20 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 25 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. 30 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 12 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. It will be understood that in the context of the present invention the term 5 "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/HumanGenome/publicat/97pr/09gloss.html#p. 10 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 15 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 may 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 20 nucleotide subsitutions 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 may be considered disorders and/or illnesses which have a genetic basis or to which the genetic makeup of the subject contributes. Using case-control studies, the frequencies of several genetic variants 25 (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 30 genes have confirmed (or likely) functional effects on gene expression or protein function. 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 13 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. The surprising finding by the Applicant relevant to this invention is that a 5 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. The present invention identifies methods of assessing the risk of a subject developing a disease which comprises determining in said subject the presence or 10 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 15 susceptibility score is predictive of an increased risk of developing said disease. 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 20 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 may 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 25 assigned a high weighting. Accordingly, in a first aspect, the present invention provides 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 protective polymorphisms and for the presence or absence of susceptibility 30 polymorphisms, 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; 14 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; wherein a net protective score is predictive of a reduced risk of developing said 5 disease and a net susceptibility score is predictive of an increased risk of developingsaid disease. The subject sample may have already been analysed for the presence or absence of one or more protective or susceptibility polymorphisms, and the method comprises the steps of 10 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 polymorphisms and the combined value of the susceptibility polymorphisms present in the subject sample; 15 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 developingsaid disease. In one embodiment described herein in Example 1, 17 susceptibility genetic polymorphisms and 19 protective genetic polymorphisms identified as discriminatory 20 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. In another embodiment described herein in Example 2, 11 susceptibility genetic polymorphisms and 11 protective genetic polymorphisms identified as discriminatory 25 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. In a further embodiment described herein in Example 3, 19 susceptibility genetic polymorphisms and 17 protective genetic polymorphisms identified as discriminatory 30 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. 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 15 International Application No. PCT/NZ02/00106 (published as WO 02/099134 and incorporated by reference) via which four susceptibility and three protective polymorphisms discriminatory for lung disease were identified. The one or more polymorphisms can be detected directly or by detection of one 5 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 infers the presence of the other. (Reich DE et al; Linkage 10 disequilibrium in the human genome, Nature 2001, 411:199-204.) 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. Gene SNPs rs Alleles in LD between Phenotype in numbers LD alleles COPD Interleukin-18 IL18 -133 rs360721 C allele Strong LD CC susceptible C/G IL18 105 rs549908 A allele AA susceptible A/C Vitamin D VDBP Lys rs4588 A allele Strong LD AA/AC binding protein 420 Thr I I protective VDBP Glu rs7041 T allele TT/TG protective 416 Asp 15 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 20 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. It will therefore be apparent that one or more polymorphisms in linkage disequilibrium with the polymorphisms specified herein can be identified, for example, 25 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.

16 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. SNP is a single base change or point mutation resulting in 5 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 10 promoters, transcription factor binding sites, processing sites, ribosomal binding sites, and affect gene transcription, processing, and translation. 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 15 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 20 enhance understanding of disease states and to ultimately facilitate the discovery of more effective treatments, such as personalised treatment regimens. 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 25 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 30 used to assay the variation, and can include information associating a SNP with a particular phenotypic trait. 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 17 of human genomic DNA, with a haploid genome of 3 x 109 base pairs, and the associated sensitivity and discriminatory requirements. Genotyping approaches to detect SNPs well-known in the art include DNA sequencing, methods that require allele specific hybridization of primers or probes, 5 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 oligonuclcotides (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 10 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. DNA sequencing allows the direct determination and identification of SNPs. 15 The benefits in specificity and accuracy are generally outweighed for screening purposes by the difficulties inherent in whole genome, or even targeted subgenome, sequencing. 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 20 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. A number of methods currently used for SNP detection involve site-specific 25 and/or allele-specific hybridisation. 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 30 base-paired. The presence of a matched duplex is detected by fluorescence. 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). US Application 20050059030 (incorporated herein in 18 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 5 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. US Application 20050042608 (incorporated herein in its entirety) describes a modification of the method of electrochemical detection of nucleic acid hybridization of 10 Thorp et al. (U.S. Pat. No. 5,871,918). 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 15 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. 20 The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPEIM 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 25 prior SNP mapping or knowledge. 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. 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 30 example is the use of mass spectrometric determination of a nucleic acid sequence which comprises 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 19 polymorphisms of the invention, for example, the COX2 promoter polymorphisms of the invention. 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 5 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, 10 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. US Patent 6,821,733 (incorporated herein 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 15 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 20 the detection molecule indicates a difference between the two nucleic acids. 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 25 electrophoresis or IPLC, 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 ProteomIQTM system from Proteome Systems, provide high throughput platforms 30 for proteome analysis combining sample preparation, protein separation, image acquisition and analysis, protein processing, mass spectrometry and bioinformatics technologies. The majority of proteomic methods of protein identification utilise mass spectrometry, including ion trap mass spectrometry, liquid chromatography (LC) and 20 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 5 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. Associated technologies are also well known, and include, for example, protein processing devices such as the "Chemical Inkjet Printer" comprising piezoelectric 10 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. After in-situ digestion and incubation of the proteins, the membrane can be placed directly into the mass spectrometer for peptide analysis. 15 A large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs. For example, Single Strand Conformational Polymorphism (SSCP, Orita et al, PNAS 1989 86:2766-2770) is a method reliant on the ability of single-stranded nucleic acids to form secondary structure in solution under certain conditions. The secondary 20 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 25 detected, for example by an automated DNA sequencer. 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, restriction endonuclease 30 fingerprinting-SSCP, dideoxy fingerprinting (a hybrid between dideoxy sequencing and SSCP), bi-directional dideoxy fingerprinting (in which the dideoxy termination reaction is performed simultaneously with two opposing primers), and Fluorescent PCR-SSCP (in which PCR products are internally labelled with multiple fluorescent dyes, may be 21 digested with restriction enzymes, followed by SSCP, and analysed on an automated DNA sequencer able to detect the fluorescent dyes). Other methods which utilise the varying mobility of different nucleic acid structures include Denaturing Gradient Gel Electrophoresis (DGGE), Temperature 5 Gradient Gel Electrophoresis (TGGE), and Heteroduplex Analysis (HET). 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. Denaturing High Pressure Liquid Chromatography (HPLC) is yet a further 10 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. 15 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 20 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. Further examples include the Protein Translation Test (PTT), used to resolve 25 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. 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 30 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.

22 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 5 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 10 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. The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention. 15 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. In order to detect and identify SNPs in accordance with the invention, a sample 20 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. DNA or RNA can be isolated from the sample according to any of a number of 25 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. 30 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 23 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 5 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 10 polymorphism may have a value of -2, whereas another more weakly protective polymorphism may have a value of -0.75. 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. 15 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 may change their lifestyle so as to reduce the risk of developing the disease is also communicated to the subject. 20 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 25 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. The predictive methods of the invention allow a number of therapeutic interventions and/or treatment regimens to be assessed for suitability and implemented 30 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 24 smoker, the methods of the invention can provide motivation to quit smoking. In this latter case, a 'quit smoking' program can be followed, which may include the use of anti-smoking medicaments (such as nicotine patches and the like) as well as anti addiction medicaments. 5 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 10 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 15 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 20 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. 25 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 30 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 25 subject. For example, where a polymorphism is associated with increased enzyme function, therapy can involve administration of an enzyme inhibitor to the subject. 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 5 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 10 genotype. EXAMPLES The invention will now be described in more detail, with reference to non limiting examples. EXAMPLE 1. CASE ASSOCIATION STUDY - COPD 15 METHODS Subject recruitment 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 20 symptoms of breathlessness after 40 years of age, had a Forced expiratory volume in one second (FEVi) 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 25 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, 30 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 FEVl/FVC ( 95%CI) was 82% (81-83), mean FEVI as a percentage of 26 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 al-antitrypsin mutations (S and Z alleles) and those with the ZZ 5 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 10 sufferers and resistant smokers was found not to determine FEV or COPD. 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 15 smokers (COPD patients and/or controls) can reflect a protective role. Summary of characteristics for the COPD, resistant smoker and healthy blood donors Parameter COPD Resistant smokers Differences Median (IQR) N=294 N=217 % 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 FEVI (L) 1.6 (0.7-2.5) 2.9 (2.8-3.0) P<0.05 FEVI %predict 43 (41-45) 96% (95-97) P<0.05 FEVJ/FVC 51(49-53) 82 (81-83) P<0.05 Means and 95% confidence limits Cyclo-oxygenase 2 (COX2) -765 GIC promoter polymorphism and al-antitrypsin 20 genotyping Genomic DNA was extracted from whole blood samples [2, incorporated herein in its entirety by reference]. The Cyclo-oxygenase 2 -765 polymorphism was 27 determined by minor modifications of a previously published method [3, incorporated herein in its entirety by reference]. The PCR reaction was carried out in a total volume of 25ul and contained 20 ng genomic DNA, 500pmol forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.0 mM MgCl 2 and 1 unit of 5 polymerase (Life Technologies). Cycling times were incubations for 3 min at 950C followed by 33 cycles of 50s at 94"C, 60s at 66"C and 60s at 72*C. A final elongation of 10 min at 72"C then followed. 4ul of PCR products were visualised by ultraviolet trans illumination of a 3% agarose gel stained with ethidium bromide. An aliquot of 3ul of amplification product was digested for 1 hr with 4 units of Acil (Roche Diagnostics, 10 New Zealand) at 37"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 123bp ladder using ultraviolet transillumination after ethidium bromide staining. Using a PCR based method referenced above [1], all COPD and resistant smoker subjects were genotyped for the al-antitrypsin S and Z alleles. 15 Other polymorphism genotyping 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 T m Autoflex Mass Spectrometer and Samsung 24 pin nanodispenser) using the following sequences, amplification conditions and methods. 20 The following conditions were used for the PCR multiplex reaction: final concentrations were for 10xBuffer 15 mM MgCl 2 1.25x, 25mM MgCl 2 1.625mM, dNTP mix 25 mM 500uM, primers 4 uM 100nM, Taq polymerase (Qiagen hot start) 0.15U/reaction, Genomic DNA 10 ng/ul. Cycling times were 95 0 C for 15 min, (5"C for 15 s, 56 0 C 30s, 72"C 30s for 45 cycles with a prolonged extension time of 3min to 25 finish. 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0 0 oj c zO C -34 RESULTS Frequencies of individual polymorphisms are as follows: Table 1. Polymorphism allele and genotype frequencies in the COPD patients and resistant smokers. Cyclo-oxygenne 2 -765 C/C Frequency Allele* Genotype 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 (%) 852(25%) 259(75%) 14'(8%) 57(33%) 101(59%) Beta2-adrenoreceptor Arg 16 Gly Frequency Allele* Genotype A G AA AG GG Controls n=182 (%) 152 (42%) 212(58%) 26 (14%) 100 (55%) 56 (31%) COPD n=236 (%) 164 (34%) 308 (66%) 34 (14%) 96 (41%) 106' (45%) Resistant n-190 (%/) 135 (36%) 245 (64%) 34 (18%) 67 (35%) 89 4 (47%) Interleukin 18 105 A/C Frequency Allele* Genotype C A CC AC AA Controls n=184 (%) 118 (32%) 250(68%) 22 (12%) 74 (40%) 88 (48%) COPD n=240 (%) 122 (25%) 3776 (75%) 21(9%) 80 (33%) 1395 (58%) Resistant n=196 (%) 113(29%) 277(71%) 16(8%) 81 (41%) 99(50%) Interleukin 18 -133 C/G Frequency Allele* Genotype G C GG GC CC Controls nl187 (%) 120 (32%) 254 (68%) 23 (12%) 74 (40%) 90 (48%) COPD n=238 123 (26%) 3539 (74%) 21(9%) 81(34%) 136g (57%) RIsislantn=195 (%) 113(29%) 277(71%) 16(8%) 81(42%) 98(50%) Plasminogen activator inhibitor 1 -675 4G/5G Frequency Allele* Genotype -35 5G 4G 5G5G 5G4G 4G4G Controls n=186 (%) 158 (42%) 214(58%) 31 (17%) 96 (52%) 59 (32%) COPD n-237 (/.) 219' (46%) 255(54%) 5410'11 (23%) 111 (47%) 72(30%) Resistant n=194 (%) 152(39%) 236 (61%) 31(16%) 90(46%) 7310'(38%) Nitric oxide synthase 3 Asp 298 Glu (T/G) Frequency Allele* Genotype T G TI TG GG Controls n=183 (%6) 108 (30%) 258 (70%) 13 (7%) 82 (45%) 88 (48%) COPD n=238 (%/) 159(42%) 317 (58%) 25(10%) 109(47%) 104(43%) Resistant n=194 (%) 136 (35%) 252(65%) 2813(15%) 80 (41%) 86 (44%) Vitamin D Binding Protein Lys 420 Thr (A/C) Frequency Allele* Genotype A C AA AC CC Controls n-189 (%) 113 (30%) 265 (70%) 17 (9%) 79 (42%) 93 (49%) COPD n=250 (%/) 147 (29%) 353 (71%) 24 (10%) 99 (40%) 127 (50%) Resistant n-195 (%) 140'5 (36%) 250(64%) 2514 (13%) 9014 (46%) 80 (41%) Vitamin D Binding Protein Glu 416 Asp (T/G) Frequency Allele* Genotype T G TT TG GG Controls n=188 (%) 162(43%) 214(57%) 35(19%) 92(49%) 61(32%) COPD n=240 (%6) 230 (48%) 250(52%) 57 (24%) 116 (48%) 67 (28%) Resistant n=197 (%) 193" (49%) 201 (51%) 43 (22%) 107'6 (54%) 47 (24%) Glutathione S Transferase P1 He 105 Val (A/G) Frequency Allele* Genotype A G AA AG GG Controls n=185 (%) 232 (63%) 138 (37%) 70 (38%) 92 (50%) 23 (12%) COPD n-238 (%) 310(65%) 166(35%) 96 (40%) 118(50%) 24 (10%) Resistant r194 (6) 269'9 (69%) 119 (31%) 911 (47%) 87(45%) 16(8%) Interferon-gamma 874 A/T Frequency Allele* Genotype -36 A T AA AT TT Controls n=186 (%) 183 (49%) 189(51%) 37(20%) 109 (58%) 40(22%) COPD n-235 (%) 244 (52%) 226(48%) 6420 (27%) 116 (49%) 55 (24%) Resistant n=193 (%) 208 (54%) 178 (46%) 51(27%) 106 (55%) 36 (18%) Interleuldn-13 Arg 130 GIn (G/A) Frequency Allele* Genotype A G AA AG GG Controls n=184 (%) 67 (18%) 301 (82%) 3 (2%) 61(33%) 120 (65%) COPD n=237 (%) 86 (18%) 388 (82%) 8 (3%) 70 (30%) 159 (67%) Resistant n=194 (%) 74(19%) 314(81%) 921(5%) 56(28%) 129(67%) Interleukin-13 -1055 C/T Frequency Allele* Genotype T C TT TC CC Controls nl182 (%) 65 (18%) 299(82%) 5 (3%) 55 (30%) 122 (67%) COPD n=234 (%) 94 (20%) 374 (80%) 8 22(4%) 78 (33%) 148 (63%) Resistant n=192 (%) 72(19%) 312(81%) 2(1%) 68(33%) 122(64%) al-antitrypsin S Frequency Allele* Genotype M S MM MS SS COPD n=202 (6) 391 (97%) 13 (3%) 189 (94%) 13 (6%) 0 (0%) Resistant n-189 (%) 350 (93%) 28 (7%) 162 (85%) 2623 (14%) 1 (1%) * number of chromosomes (2n)Genotype 1. Genotype. CC/CG vs GG for resistant vs COPD, Odds ratio (OR) =1.98,95% confidence limits 1.3-3.1, x2 (Yates corrected)= 8.82, p=0.003, CC/CG = protective for COPD 2. Allele. C vs G for resistant vs COPD, Odds ratio (OR) =1.92, 95% confidence limits 1. 3-2.8, x 2 (Yates corrected)= 11.56, p<0.001, C = protective for COPD 3. Genotype. GG vs AU/AA for COPfl vs controls, Odds ratio (OR) =1.83, 95% confidence limits 1.2-2.8, x 2 (Yates corrected) 8.1, p=0.

0 0 4 , GG = susceptible to COPD (depending on the presence of other snps) 4. Genotype. GG vs AG/AA fir resistant vs controls, Odds ratio (OR) =1.98, 95% confidence limits 1.3-3.1, x 2 (Yates corrected)-9.43, p-0.002 GG - resistance (depending on the presence of other snps) -37 5. Genotype. AA vs AC/CC for COPD vs controls, Odds ratio (OR) =1.50, 95% confidence limits 1.0-2.3, X 2 (Yates uncorrected)= 4.26, p-0.04, AA = susceptible to COPD 6. Allele. A vs C for COPD vs control, Odds ratio (OR) =1.46, 95% confidence limits 1.1-2.0, X2 (Yates corrected)= 5.76, p=0.02 7. Genotype. AA vs AC/CC for COPD vs resistant, Odds ratio (OR) =1.35, 95% confidence limits 0.9-2.0, X2 (Yates uncorrected)=2.39, p=0.1 2 (trend), AA = susceptible to COPD 8. Genotype. CC vs CG/GG for COPD vs controls, Odds ratio (OR) =1.44,95% confidence limits 1.0-2.2, - 2 (Yates corrected)= 3.4, p=0.06, CC = susceptible to COPD 9. Allele. C vs G for COPD vs control, Odds ratio (OR) =1.36, 95% confidence limits 1.0-1.9, 72 (Yates corrected)= 53.7, p=0.05, C susceptible to COPD 10. Genotype. 5G5G vs rest fbr COPD vs resistant, Odds ratio (OR) =1.55, 95% confidence limits 0.9-2.6, X2 (Yates uncorrected)= 3.12, p-0.08, 5G5G = susceptible to COPD 11. Genotype. 5G5G vs rest for COPD vs control, Odds ratio (OR) =1.48, 95% confidence limits 0.9-2.5, X2 (Yates uncorrected)= 2.43, p=0.

12 , 5G5G = susceptible to COPD 12. Allele. 5G vs 4G for COPD vs resistant, Odds ratio (OR) =1.33, 95% confidence limits 1.0-1.8, 2 (Yates corrected)4.02, p=0.05, 5G = susceptible to COPD 13. Genutype. TT vs TG/GG for resistant vs controls, Odds ratiu (OR) -2.2, 95% conFidcmx limits 1.0-4.7, X2 (Yates corrected)= 4.49, p=0.03, TT genotype = protective for COPD 14. Genotype. AA/AC vs CC for resistant vs COPD, Odds ratio (OR) =1.39, 95% confidence limits 0.9-2.1, X2 (Yates uncorrected)= 2.59, p=0.10, AAIAC genotype = protective for COPD 15. Allele. A vs C for resistant vs COPD, Odds ratio (OR) =1.34, 95% confidence limits 1.0-1.8, X2 (Yates corrected)=3.94, p=0.

0 5 , A allele = protective for COPD 16. Genotype. Tf/TG vs GG for resistant vs controls, Odds ratio (OR) =1.53, 95% confidence limits 1.0-2.5, X2 (Yates uncorrected)= 3.52, p-0.06, TT/TG genotype = protective for COPD 17. Allele. T vs G for resistant vs control, Odds ratio (OR) =1.27, 95% confidence limits 1.0-1.7, X2 (Yates corrected)-2.69, p-0.1, T allele - protective for COPD 18. Genotype. AA vs AG/GG for resistant vs controls, Odds ratio (OR) = 1.45, 95% confidence limits 0.9-2.2, X2 (Yatcs miunrcuted)- 3.19, p-0.0 7 , AA genutypc - prutcutivc fur COPD 19. Allele. A vs G for resistant vs control, Odds ratio (OR) =1.34, 95% confidence limits 1.0-1.8, X2 (Yates uncorrceted)=3.71, p=0.05, A allele = protective for COPD -38 20. Genotype. AA vs AT/I for COPD vs controls, Odds ratio (OR) =1.51, 95% confidence limits 0.9-2.5, x 2 (Yates uncorrected)= 3.07, p-0.

08 , AA genotype = susceptible to COPD 21. Genotype. AA vs AG/GG for resistant vs controls, Odds ratio (OR) =2.94, 95% confidence limits 0.7-14.0, X2 (Yates uncorrected)= 2.78, p=0.

09 , AA genotype = protective for COPD 22. Genotype. Tf vs TC/CC for COPD vs resistant. Odds ratio (OR) =6.03. 95% confidence limits 1.1-42. v2 (Yates corrected)- 4.9, p-0.03, TT= susceptible to COPD 23. Genotype. MS/SS vs MM for Resistant vs COPD, Odds ratio (OR) =2.42, 95% confidence limits 1.2-5.1, t 2 (Yates corrected)- 5.7, p 0

.

0 1, S= protective for COPD Tumour Necrosis Factor a +489 G/A polymorphism allele and genotype frequency in the COPD patients and resistant smokers. Frequency Allele* Genotype A G AA AG GG COPD n=242 (%) 54 (11%) 430 (89%) 5 (2%) 44 (18%) 193 (80%) Resistant n=187 (%) 27 (7%) 347 (93%) 1 (1%) 25 (13%) 161 (86%) * number of chromosomes (2n) 1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio (OR) =1.57, 95% confidence limits 0.9 2.7, xC (rates correcrea)= 2.32, p=O.11, AA/AG =susceptible (GG=protective) 2. Allele. A vs G for COPD vs resistant, Odds ratio (OR) =1.61, 95% confidence limits 1. 0-2.7, X 2 (Yates corrected)= 3.38, p=0.07, A =susceptible Tumour Necrosis Factor a -308 G/A polymorphism allele and genotype frequency in the COPD patients and resistant smokers. Frequency AIIele* Genotype A G AA AG GG COPD n=242 (%) 90 (19%) 394 (81%) 6 (2%) 78 (32%) 158 (65%) Resistant n=190 (%) 58 (15%) 322 (85%) 3 (2%) 52 (27/) 135 (71%) * number of chromosomes (2n) 1. Genotype. GG vs AG/AA for COPD vs resistant, Odds ratio (OR) =0.77, 95% confidence limits 0.5 1.2, x 2 (Yates uncorrected)= 1.62, p=0.

2 0, -39 GG-protective (AA/AG =susceptible) trend 2. Allele. A vs G for COPD vs resistant, Odds ratio (OR) =1.3, 95% confidence limits 0.9-1.9, X 2 (Yates uncorrected)= 1.7, p=0.20, A =susceptible trend SMAD3 C89Y polymorphism allele and genotype frequency in the COPD patients and resistant smokers. Frequency AIleIe* Genotype 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) 1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio (OR) =0.26, 95% confidence limits 0.04-1.4, X 2 (Yates uncorrected)= 3.19, p=0.07, 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. Frequency AIIeIe* Genotype A G AA AG GG COPD n=242 (/6) 259(54%) 225 (46%) 73 (30%) 113 (47%) 56 (23%) Resistant n=182 (%) 217(60%) 147 (40%) 64 (35%) 89 (49%) 29 (16%) * number of chromosomes (2n) 1. Genotype. GG vs AG/GG for COPD vs resistant, Odds ratio (OR) =1.60, 95% confidence limits 0.9 2.7, x 2 (Yates corrected)= 3.37, p=0.07, GG =susceptibility 2. Allele. Gvs A for COPD vs resistant, Odds ratio (OR) =1.3, 95% confidence limits 1.0-1.7, X 2 (Yates corrected)= 2.90, p-0.09 Caspase (NOD2) Gly881Arg polymorphism allele and genotype frequencies in the COPD patients and resistant smokers. Frequency Alele* Genotype G C GG GC CC -40 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) 1. Genotype. CC/CG vs GG fir COPD vs resistant, Odds ratio (OR) =3.2, 95% confidence limits 0.6 22, X2 (Yates uncorrected)= 2.41, p-0.11 (1-tailed), GC/CC=susceptibility (trend) Mannose binding lectin 2(MBL2) +161 G/A polymorphism allele and genotype frequencies in the COPD patients and resistant smokers. Frequency AIIeIe* Genotype A G AA AG GG COPD n=218 (%) 110 (25%) 326 (75%) 6 (3%) 98(45%) 114 (52%) Resistant n=183 (%) 66 (18%) 300 (82%) 6 (3%) 54 (30%) 123 (67%) * number of chromosomes (2n) 1. Genotype. GG vs rest for COPD vs resistant, Odds ratio (OR) =0.53, 95% confidence limits 0.4 0.80, X 2 (Yates uncorrected)= 8.55, p=0.003, GG -protective Chymase 1 (CMA1) -1903 G/A promoter polymorphism allele and genotype frequencies in the COPD patients and resistant smokers. Frequency AIIeIe* Genotype A G AA AG GG COPD n=239 (%) 259 (54%) 219 (46%) 67 (28%) 125 (52%) 47 (20%) Resistant n=181 (%) 209(58%) 153 (42%) 63 (35%) 83 (46%) 35 (19%) * number of chromosomes (2n) 1. Genotype. AA vs AG/GG for COPD vs resistant, Odds ratio (OR) =0.73, 95% confidence limits 0.5 1.1, x 2 (Yates corrected)= 1.91, p=0.17, AA genotype -- protective trend N-Acetyltransferase 2 Arg 197 Gin G/A polymorphism allele and genotype frequencies in COPD and resistant smokers. Frequency AIIeIe* Genotype A G AA AG GG -41 COPD n=247 (%) 136(28%) 358 (72%) 14 (6%) 108 (44%) 125 (50%) Resistant n=196 (%/) 125 (32%) 267 (68%) 21 (11%) 83 (42%) 92 (47%) * number of chromosomes (2n) 1. Genotype. AA vs AG/GG for COPD vs resistant, Odds ratio (OR) =0.50, 95% confidence limits 0.2 1.0, X 2 (Yates uncorrected)= 3.82, p=0.05, AA genotype = protective Interleukin 1B (IL-1b) -511 A/G polymorphism allele and genotype frequencies in COPD and resistant smokers. Frequency AIIeIe* Genotype A G AA AG GG COPD n=248 (%) 160 (32%) 336 (68%) 31 (13%) 98(40%) 119 (48%) Resistant n=195 (%) 142(36%) 248 (64%) 27 (14%) 88 (45%) 80 (41%) * number of chromosomes (2n) 1. Genotype. GG vs AA/AG for COPD vs resistant, Odds ratio (OR) =1.3, 95% confidence limits 0.9 2.0, x 2 (Yates corrected)= 1.86, p=0.17, 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. Frequency AIIeIe* Genotype C T CC CT TT COPD n=249 (%) 137 (28%) 361 (72%) 18(7%) 101 (41%) 130 (52%) Resistant r194 (%) 130 (34%) 258 (66%) 19 (10%) 92 (47%/a) 83 (43%) * number of chromosomes (2n) 1. Genotype. TT vs CT/CC for COPD vs resistant, Odds ratio (OR) =1.5, 95% confidence limits 1.0 2.2, x 2 (Yates corrected)= 3.51, p=0.06, TT genotype = susceptible Microsomal epoxide hydrolase (MEH) His 139 Arg A/G (exon 4) polymorphism allele and genotype frequency in COPD and resistant smokers. Frequency AIIeIe* Genotype A G AA AG GG COPD n=238 (%) 372 (78%) 104 (22%) 148 (62%) 76(32%) 14(6%) Resistant n=179 (%) 277(77%) 81(23%) 114 (64%) 49(27%) 16 (9%) * number of chromosomes (2n) 1. Genotype. GG vs AA/AG for COPD vs resistant, Odds ratio (OR) =0.64, 95% confidence limits 0.3 1.4, x 2 (Yates uncorrected)= 1.43, p=0.23, GG genotype = protective (trend) Lipo-oxygenase -366 G/A polymorphism allele and genotype frequencies in the COPD patients and resistant smokers. Frequency AIIeIe* Genotype A G AA AG GG COPD n=247 (%) 21(4%) 473 (96%) 1(0.5%) 19 (7.5%) 227 (92%) Resistant n=192 (%) 25 (7%) 359 (93%) 0 (0%) 25 (13%) 167 (87%) * mimher nf chrnmnsnms (2n) 1. Genotype. AA/AG vs GG for COPD vs resistant, Odds ratio (OR) =0.60, 95% confidence limits 0.3-1.1, X 2 (Yates corrected)= 2.34, p=0.1 2 , 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. Frequency AIIeIe* Genotype C T CC CT TT COPD n=199 (%) 127 (32%) 271 (68%) 5 (3%) 117 (59%) 77 (39%) Resistant n=166 (%/) 78 (23%) 254 (77%) 4 (2%) 70 (42%) 92 (56%) * number of chromosomes (2n) 1. Genotype. CC/CT vs TT for COPD vs resistant, Odds ratio (OR) =20, 95% confidence limits 1.3 3.1, x 2 (Yates uncorrected)= 9.52, p=0.00 2 , 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. Frequency AIIeIe* Genotype A T AA AT TT COPD n=224 (%) 282(63%) 166 (37%) 84 (38%) 114 (51%) 26 (12%) Resistant n=158 (94) 178 (56%) 138 (44%) 42 (27%) 94 (59%) 22 (14%) -43 * number of chromosomes (2n) 1. Genotype. AA vs AT/fT for COPD vs resistant, Odds ratio (OR) =1.7, 95% confidence limits 1.0 2.7, x 2 (Yates corrected)= 4.51, p=0.03, AA=susceptible Monocyte differentiation antigen CD-14 -159 promoter polymorphism allele and genotype frequencies in the COPD patients and resistant smokers. Frequency AIIeIe* Genotype C T CC CT TT COPD n=240 (%) 268 (56%) 212 (44%) 77 (32%) 114 (48%) 49 (20%) Resistant n=180 (%) 182 (51%) 178 (49%) 46 (25%) 90 (50%) 44 (24%) * number of chromosomes (2n) 1. Genotype.CC vs CTtTI for COPD vs Resistant, Odds ratio (OR) =1.4, 95% confidence limits 0.9 2.2, x 2 (Yates uncorrected)= 2.12, p=0.15, CC = susceptible (trend) Elafin +49 C/R polymorphism allele and genotype frequencies in the COPD patients, resistant smokers and controls. Frequency Allele* Genotype C T CC CT TT COPD n=144 (%) 247 (86%) 41 (14%) 105 (73%) 37(26%) 2(1%) Resistant n=75 (%) 121 (81%) 29 (19%) 49 (65%) 23 (31%) 3 (4%) * number of chromosomes (2n) 1. Genotype. CT/TT vs CC for COPD vs resistant, Odds ratio (OR) 0.70, 95% confidence limits= 0.4-1.3 , X2 (Yates uncorrected)= 1.36, p=0.

24 , CT/IT genntype = prntective (tend nnly) 2. Allele: T vs C for COPD vs resistant, Odds ratio (OR) = 0.69, 95% confidence limits= 0.4-1.2, X 2 (Yates uncorrected)- 1.91, p-0.

17 , T genotype = protective (trend only) Beta2-adrenoreceptor Gin 27 GIn polymorphism allele and genotype frequency in the COPD patients, resistant smokers and controls. Frequency AlIele* Genotype -44 C G CC CG GG Controls n=185 (%) 204(55%) 168 (45%) 57(31%) 89(48%) 39(21%) COPD n=238 (%) 268 (56%) 208 (44%) 67(28%) 134(56%) 37(16%) Resistant n=195 (%) 220 (56%) 170 (44%) 64 (33%) 92 (47%) 39 (20%) * number of chromosomes (2n) 1. Genotype. GGvs CG/CC for COPD vs resistant, Odds ratio (OR) = 0.74, 95% confidence limits = 0.4-1.2, X 2 (Yates uncorrected)= 1.47, p=0.23, GG =protective (trend) 2. Genotype. GG vs CG/CC for COPD vs controls, Odds ratio (OR)= 0.69, 95% confidence limits = 0.4-1.2, X 2 (Yates uncorrected)= 2.16 , p=0.14, GG =protective (trend) Maxtrix metalloproteinase 1 (MMP1) -1607 lG/2G polymorphism allele and genotype frequencies in COPD patients, resistant smokers and controls. Frequency Allele* Genotype IG 2G IGIGC 1Gc2Gj 2G2G Controls n-174 (%) 214 (61%) 134 (39%) 68 (39%) 78 (45%) 28 (16%) COPD n=217 (%) 182 (42%) 252(58%) 47(22%) 88(41%) 82(38%) Resistant n=187 (%) 186 (50%) 188 (50%) 46 (25%) 94 (50%) 47 (25%) * number of chromosomes (2n) 1. Genotype. IGIG vs rest for COPD vs controls, Odds ratio (OR) =0.43, 95% confidence limits 0.3-0.7, ; (Yates uncorrected)= 13.3, p=O.0003 IGIG genotype =protective 2. Allele. IG vs 2G for COPD vs controls, Odds ration (OR) =0.45, 95% confidence limits 0.3-0.6, X2 (Yates corrected)= 28.8, p<0.0001, I G = protective 3. Genotype. lG1G/1G2G vs rest for COPD vs resistant smokers, Odds ratio (OR) 0.55, 95% confidence limits 0.4-0.9, X 2 (Yates uncorrected)= 6.83, p=0.009 IGIG/162G genotypes =protective 4. Allele. iG vs 2G for COPD vs resistant smokers, Odds ratio (OR) =0.73, 95% confidence limits 0.6-1.0, X 2 (Yates corrected)= 4.61, p=0.03, I G = protective -45 5. Genotype. 2G2G vs 1G1G/1G2G for COPD vs controls, Odds ratio (OR) =3.17, 95% confidence limits 1.9-5.3, x 2 (Yates uncorrected)= 21.4, p<0.0001 2G2G genotype =susceptible 6. Allele. 2G vs 1G for COPD vs controls, Odds ratio (OR) =2.2, 95% confidence limits 1.6-3.0, X2 (Yates corrected)= 28.8, p<0.00001, 2G = susceptible 7. Genotype. 2G2G vs 1G1G/1G2G for COPD vs resistant, Odds ratio (OR) =1.81, 95% confidence limits 1.2-2.9, X 2 (Yates uncorrected)= 6.83, p=0.009 2G2G genotype =susceptible 8. Allele. 2G vs 1G for COPD vs resistant, Odds ratio (OR) =1.4, 95% confidence limits 1.0-1.8, X2 (Yates corrected)= 4.61, p=0.0.03, 2G = susceptible Table 2 below provides a summary of the protective and susceptibility polymorphisms determined for COPD. Table 2. Summary of protective and susceptibility polymorphisms for COPD Gene Polymorphism Role Cyclo-oxygenase 2 (COX2) COX2 -765 G/C CC/CG protective P2-adrenoreceptor (ADBR) ADBR Argl6Gly GG susceptible Interleukin -18 (IL18) [I18 -133 C/G CC susceptible Interleukin -18 (IL18) IL18 105 A/C AA susceptible Plasminogen activator inhibitor 1 (PAI) PAI-I -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 4 16 Asp TTITG protective Glutathione S Transferase (GSTP-1) GSTP1 IlO15Val AA protective Interferon y (IFN-y) IFN-y 874 A/T AA susceptible Interleukin-13 (L13) IL13 Arg 130 Gln AA protective Interleukin-13 (L13) 1113 -1055C 'IT susceptible al-antitrypsin (al-AT) al-AT S allele MS protective Tumour Necrosis Factor a TNFa TNFa +489 G/A AA/AG susceptible GG protective -46 Tumour Necrosis Factor a TNFa TNFa -308 G/A GG protective AA/AG susceptible SMAD3 SMAD3 C89Y AU AA/AU protective GG susceptible Intracellular adhesion molecule 1 (ICAMI) ICAM1 E469K A/G GG susceptible Caspase (NOD2) NOD2 Gly 881Arg G/C GC/CC susceptible Mannose binding lectin 2 (MBL2) MBL2 161 G/A GG protective Chymase 1 (CMAl) CMA1 -1903 G/A AA protective N- Acetyl transferase 2 (NAT2) NAT2 Arg 197 Gin AA protective G/A Interleukin 13 (ILIB) (IB) -511 A/G GG susceptible Microsomal epoxide hydrolase (MEH) MEH Tyr 113 His T/C TT susceptible Microsomal epoxide hydrolase (MEH) MEH His 139 Arg G/A GG protective 5 Lipo-oxygenase (ALOX5) ALOX5 -366 G/A AA/AG0 protective GG susceptible Heat Shock Protein 70 (HSP 70) HSP 70 HOM T2437C CC/CT susceptible TT protective Chloride Channel Calcium-activated 1 (CLCA1) CLCA1 +13924 T/A AA susceptible Monocyte differentiation antigen CD-14 CD-14 -159 C/T CC susceptible Elafin Elafin Exon 1 +49 C/T CT/TT protective B2-adrenergic receptor (ADBR) ADBR Gin 27 Glu C/G GG protective Matrix metalloproteinase 1 (MMP1) MMP1 -1607 1G/2G 1G1G/1G2G protective The combined frequencies of the presence or absence of the selected protective genotypes COX2 (-765) CC/CG, P2 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 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 -47 % of smokers with COPD 136/215 (63%) 100/183 16/50 (55%1_6) (32%) Comparison Odd's ratio 95% CI X2 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 The combined frequencies of the presence or absence of the selected susceptibility genotypes Interleukin-18 105 AA, PAI-i -675 5G5G, Interleukin-13 -1055 TT, and Interferon-y -874 AA observed in the COPD subjects and in resistant smokers is presented below in Table 4. 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 COPD 66/135 113/205 73/108 (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 The combined frequencies of the presence or absence of the protective genotypes COX2 (-765) CCICG, Interleukin-13 AA, Nitic Oxide Synthase 3 TT, Vitamin D Binding Protein AA/AC, GSTP1 AA, and al-antitypsin MS/SS, observed in the COPD subjects and in resistant smokers is presented below in Table 5 and in Figure 1.

-48 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 X2 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 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 Figure 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 Figure 2). 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. EXAMPLE 2. CASE ASSOCIATION STUDY - OCOPD METHODS Subject recruitment 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 -49 (FEVI) 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 FEVI 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 FEV1I/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 al 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. Parameter OCOPD Exposed resistant Differences Mean (SD) (N=139) smokers (N=112) % 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 FE VI (L) 1.3(0.7) 3.0(0.7) P<0.05 FEV1 %predict 46(19) 96% (10) P<0.05 FEVI/FVC 54 (15) 81(8) P<0.05 Means and 1SD -50 Cyclooxygenase 2 (COX2) -765 6/C promoter polymorphism and al-antitrypsin genotyping 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 25ul and contained 20 ng genomic DNA, 500pmol forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCI, 1.0 mM MgCl2 and 1 unit of Taq polymerase (Life Technologies). Cycling times were incubations for 3 min at 95"C followed by 33 cycles of 50s at 94"C. 60s at 66 0 C and 60s at 72"C. A final elongation of 10 min at 72"C then followed. 4ul of PCR products were visualised by ultraviolet trans-illumination of a 6% agarose gel stained with ethidium bromide. An aliquot of 3ul of amplification product was digested for 1 hr with 4 units of AciI (Roche Diagnostics, New Zealand) at 37 0 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 123bp ladder. Using a PCR based method discussed above [3], all smoking subjects were genotyped for the aIi antitrypsin M, S and Z alleles. Genovyping of the superoxide dtsmuase 3 Arg 312 Gin polymorphism 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, incorporated in its entirety herein by reference]. 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 25p and contained 80ng genomic DNA, 10 pmol forward and reverse primers, 0.1mM dNIPs, 10 mM Iris-HCL (pH 8.4), 150 mM KC1, 1.0 mM MgC 2 and 0.5 unit of Taq polymerase (Qiagen). Aliquots of amplification product were digested for 4 hrs with 5U 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 -51 1Kb plus ladder standard (Invitrogen). Genotypes were recorded in data spreadsheets and statistical analysis performed. Genotyping of the Microsomal Epoxide Hydrolase Exon 3 TCpolymorphism 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, incorporated in its entirety herein by reference]. 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 25pl and contained 80ng genomic DNA, 100 ng forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.5 mM MgCl 2 and 1.0 unit of Taq polymerase (Qiagen). Cycling conditions consisted of 94*C 60s, 56*C 20s, 72 0 C 20s for 38 cycles with an extended last extension of 3min. Aliquots of amplification product were digested for 4 hrs with 5U 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 1Kb plus ladder standard (Invitrogen). Genotypes were recorded in data spreadsheets and statistical analysis performed. Genotyping of the 3' 1237 GIA (TIM) polymorphism of the al-antitrypsin gene 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 AJ et al.,[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 2 5[l and contained 80ng genomic DNA, 100 ng forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.5 mM MgCl 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 94C 60 s, 56C 20s, 72C 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 I (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 1Kb 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 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, incorporated in its entirety herein by reference]. 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 2 5 L' and contained 80ng genomic DNA, 100 ng forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.5 mM MgCl 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 0 C 30s, 55 0 C 30s, 72 0 C 30s for 30 cycles with an extended last extension of 3min. Aliquots of amplification product were digested for 4 hrs with 2U 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 1Kb plus ladder standard (Invitrogen). Genotypes were recorded in data spreadsheets and statistical analysis performed.

-53 Genotyping of the -1607 1G2G polymorphism of the matrix metalloproteinase 1 gene 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 l and contained 80ng genomic DNA, 100 ng forward and reverse primers, 200mM dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KCI, 1.5 mM MgCl 2 and 1.0 unit of Taq polymerase (Qiagen). Forward and reverse prime sequences were3' TCGTGAGAATGTCTTCCCATT-3' [SEQ.ID.NO.140] and 5'-TCTTGGATTGATTTGAGATAAGTGAAATC-3' [SEQ.ID.NO.141]. Cycling conditions consisted of 94C 60 s, 55C 30s, 72C 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 1Kb plus ladder standard (Invitrogen). Genotypes were recorded in data spreadsheets and statistical analysis performed. Other polymorphism genotyping 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 Sequenomm system (Sequenomtm Autoflex Mass Spectrometer and Samsung 24 pin nanodispenser) using the sequences, amplification conditions and methods described below. The following conditions were used for the PCR multiplex reaction: final concentrations were for l0xBuffer 15 mM MgCl2 1.25x, 25mM MgCl2 1.625mM, dNTP mix 25 mM 500uM, primers 4 uM 1OOnM, Taq polymerase (Quiagen hot start) 0.15u/reaction, Genomic DNA 10 ng/ul. Cycling times were 95"C for 15min, (5"C for 15s, 56 0 C 30s, 72 0 C 30s for 45 cycles with a prolonged extension time of 3min to finish. We used shrimp alkaline phosphotase (SAP) treatment (2ul to 5ul PCR reaction) incubated at -54 35 0 C for 30min and extension reaction (add 2ul to 7ul after SAP treatment) with the following volumes per reaction of water 0.76ul, hME lOx termination buffer 0.2ul, hME primer (1OuM) lul, MassEXTEND enzyme 0.04ul.

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~ 0 N 0 % C 0% N - CU Cc C @* C I - - - - - - a e - % N O - n o - - o o , < a - - - - g a sam -=2. e a se e =2.C 92o~ y a a 2820 y a n i N Ln cr Cn -f 'IT Nq N *n Mni ~ ~ '0 * . t,'i -U- n U 0 - - H-H 5 E4' N ~ N - N '0 '. U 0 CC UP 3 F- C U w whcy c> E 1 2 u PH ulU m Q N CLN< J L)ao U 0 Go 0 - !2 L. rn- 0 E0 - - - 58 RESULTS Frequencies of individual polymorphisms are as follows: Table 6. Polymorphism allele and genotype frequency in the OCOPD patients, exposed resistant smokers and controls. Cyclo-oxygenase 2 -765 G/C Frequency Allele* Genotype C G CC CG GG Controls n=95 (%) 27(14%) 161 (86%) 3 (3%) 21(22%) 70(75%) OCOPD n=82 (%) 22 (13%) 1424 (87%) 2 (2%) 18 (22%) 623 (76%) Resistant n=87 (%) 422 (24%) 132(76%) 6' (7%) 30' (34%) 51 (59%) Glutathione S Transferase P1 lie 105 Val (A/G) Frequency Allele* Genotype 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- (13%) Resistant n=98 (%) 136(69%) 60(31%) 44(45%) 48 (49%) 6(6%) Interleukin 18 105 C/A Frequency Allele* Genotype C A CC AC AA Controls n=185 (%) 119(32%) 251 (68%) 22(12%) 75 (40%) 88(48%) OCOPD n=122 (%) 62(25%) 182(75%) 12(10%) 38 (31%) 72 7(59%) Resistant n=98 (%) 60(31%) 136(69%) 6(6%) 48 (49%) 44 (45%) Interleukin 18 -133 G/C Frequency Allele* Genotype CT C GlfG G3C CC Controls n=188 (%) 121 (32%) 255 (68%) 23 (12%) 75 (40%) 90(48%) OCOPD n=122 62(25%) 182(75%) 12(10%) 38 (31%) 72" (59%) Resistant n=97 (%) 60(31%) 134(69%) 6(6%) 48(50%) 43 (44%) Interleukin 8 -251 A/T Frequency Allele* Genotype -59 A T AA AT TT Controls n=188 (%) 175(47%) 201 (53%) 39(21%) 97(52%) 52(28%) OCOPD n-116 101 (11%) 131 (56%) 21 (18%) 59(51%) 36(31%) Resistant n=93 (%) 94" (50%) 92 (49%) 2610 (28%) 42 (45%) 25 (27%) Vitamin D Binding Protein Lys 420 Thr (A/C) Frequency Allele* Genotype A C AA AC CC Controls n=189 (%) 113 (30%) 265 (70%) 17(9%) 79(42%) 93(49%) OCOPD n=122 (%) 62 (25%) 182 (75%) 5 (4%) 52 (43%) 6514 (53%) Resistant n=99 (%) 73" (37%) 125 (63%) 1212 (12%) 49 (50%) 38 (38%) Vitamin D Binding Protein Glu 416 Asp (TIG) Frequency Allele* Genotype T G TT TG GG Controls n=189 (%) 163 (43%) 215 (57%) 35 (19%) 93 (49/.) 61(32%) OCOPD n=122 (%) 109(45%) 135 (55%) 25(21%) 59(48%) 3817 (31%) Resistant n=99 (%) 10316 (52%) 95 (48%) 2315 (23%) 5715 (58%) 19(19%) Microsomal epxoide hydrolase R/r Exon 3 T/C Frequency Allele* Genotype r R rr Rr RR Controls r184 (%) 228(62%) 140(38%) 77(42%) 74 (40%) 33 (18%) OCOPD n-98 (%) 144 (74%) 52 (26%) 55 (56%) 34 (35%) 9 (9%) Resistant n=102 (%) 135 (66%) 69 (34%) 52 (51%) 31 (30%) 191 (19%) Super oxide dismutase 3 Arg 312 Gln Frequency Allele* Genotype A G AA AG GG Controls r190 (%) 371 (98%) 9(2%) 183 (96%) 5(3%) 2(1%) OCOPD n=100 (%) 19920 (99%) 1 (1%) 99 (99%) 1 (1%) 0 (0%) Resistant n=102 (%) 193 (95%) 1120(5%) 92(90%) 9 (9%) 1 N (1%) al-antitrypsin S Frequency Allele* Genotype M S MM MS SS - 60 OCOPD n=88 (%) 171 (97%) 5 (3%) 83 (94%) 5 (6%) 0 (0%) Resistant ir-94 (%) 175 (93%) 1 132 (7%) 81(86%) 132 (14%) 0 (0%) Toll-like receptor 4 Asp 299 Gly A/G Frequency Allele* Genotype A G AA AG GG OCOPD n=60 (%) 117 (98%) 1(2%) 58 (98%) 1(2%) 0 (0%) Resistant n=34 (%) 65 (96%) 3 (4%) 31(91%) 32 (9%) 0 (0%) Beta2-adrenoreceptor Gin 27 Glu Frequency Allelc* Genotype C G CC CG GG Controls n=186 (%) 204 (55%) 168 (45%) 57(31%) 90(48%) 39(21%) OCOPD n=122 (%) 129(53%) 115(47%) 32(26%) 65(53%) 25(21%) Resistant n=99 (%) 117(59%) 181(41%) 3824 (38%) 41(41%) 20(20%) Interleukin 11 (IL-11) -518 G/A Frequency Allele* Genotype A G AA AG GG OCOPD n-119 (%) 110(46%) 128(54%) 22(19%) 66(55%) 31 (26%) Resistant n-98 (%) 103 (53%) 93 (47%) 26 25(27%) 51(52%) 21(21%) Interleukin-13 -1055 C/T Frequency Allele* Genotype T C TT TC CC Controls n=182 (%) 65(18%) 2999(82%) 5 (3%) 55 (30%) 122 (67%) OCOPD n=121 (%) 53 (22%) 189 (78%) 526 (4%) 43 (36%) 73(60%) Resistant n=97 (%) 31(16%) 163 (84%) 1(1%) 29 (30%) 67 (69%) Plasminogen activator inhibitor 1 -675 4G/5G Frequency Allele* Genotype 5G 4G 5G5G 5G4G 4G4G Controls n=186 (%) 158(42%) 214 (58%) 31(17%) 96(52%) 59(32%) OCOPD n=122 (%) 115 2(47%) 129(53%) 2927 (24%) 57(47%) 36(30%) Resistant n=98 (%) 76(39%) 120(61%) 14(14%) 48 (49%) 36 (37%W) - 61 Nitric oxide synthase 3 Asp 298 Glu (T/G) Frequency AlleIe* Genotype T G TT TG GG Controls n-183 (%) 108 (30%) 258 (70%) 13 (7%) 82 (45%) 88 (48%) OCOPD n-120 (%) 71(30%) 169(70%) 10(8%) 51(43%) 59 (49%) Resistant n=99 (%) 71(36%) 127 (64%) 1529" (15%) 41(41%) 43 (43%) al-antitrypsin 3' 1237 G/A (lit) Frequency AlleIe* Genotype T t TT Tt It Contmls n1 7R (%) 345 (97%) 11 (3%) 167 (94%) 11 (6%) 0 (0%) COPD n=61 (%) 109 (89%) 13 (11%)" 50 (82%) 9 (15%)" 2 (3%)" Resistant n=35 (%) 67 (96%) 3 (4%) 32 (91%) 3 (9%) 0 (0%) Matrix metalloproteinase 1 -1607 1G/2G Frequency Allele* Genotype IG 2G IGIG 1G2G 2G2G Controls n=174 (%) 214 (61%) 134 (39%) 68 (39%) 78 (45%) 28 (16%) COPD n=93 (%) 90 (48%) 96 (52%)"4 24 (26%) 42 (45%) 27 (29%)" Resistant n=94 (%) 99 (53%) 89 (47%) 25 (27%) 49 (52%) 20(21%) * number of chromosomes (2n) 1. Genotype. CC/CG vs GG for resistant vs OCOPD, Odds ratio (OR) =2.2, 95% confidence limits=1.1-4.8, X 2 (Yates corrected)- 4.76, P=0.03, CC/CG -protective 2. Allele. C vs G for resistant vs OCOPD, Odds ratio (OR) -2.1, 95% confidence limits 1.1- 3.8, x 2 (Yates corrected)= 5.65, p=0.02. C =protective 3. Genotype. GG vs CG/CC for OCOPD vs resistant, Odds ratio (OR) =0.5, 95% confidence limits=0.2-0.9, X 2 (Yates corrected)= 4.76, P=0.03. GG =susceptible 4. Allele. G vs C for OCOPD vs resistant, Odds ratio (OR) =0.5, 95% confidence limits 0.3- 0.9, X 2 (Yates corrected)= 5.65, p=0.02. G =susceptible 5. Genotype. GG vs AG/AA for OCOPD vs resistant, Odds ratio (OR) = 2.3, 95% confidence limits= 0.8-6.9, X 2 (Yates uncorrected)= 2.88, p-0.09. GG genotype = susceptible 6. Genotype. AA vs AC/CC for OCOPD vs resistant, Odds ratio (OR) =1.8, 95% confidence limits=1.0-3.1, x 2 (Yates corrected)=3.8, p=0.05. AA =susceptibility 7. Genotype. AA vs AC/CC for OCOPD vs controls, Odds ratio (OR) =1.6, 95% confidence limits 1.0-2.6, X 2 (Yates uncorrected)=3.86, p-0,05. AA - susceptibility - 62 8. Genotype. CC vs CG/GG for OCOPD vs controls, Odds ratio (OR) =1.6, 95% confidence limits=1.0-2.6, X 2 (Yates uncorrected)=3.68, p=0.05. CC =susceptibility 9. Genotype. CC vs CG/GG for OCOPD vs resistant, Odds ratio (OR) =1.8, 95% confidence limits 1.0-3.2, X 2 (Yates corrected)= 4.10, p=0.04. CC =susceptibility 10. Genotype. AA vs AT/TT for OCOPD vs resistant, Odds ratio (OR) =1.8, 95% confidence limits= 0.9-3.6, X' (Yates uncorrected)=2.88, p=0.0 9 . AA = protective 11. Allele. A vs T for OCOPD vs resistant, Odds ratio (OR) =1.3, 95% confidence limits= 0.9-2.0, x 2 (Yates uncorrected)= 2.3, pO.l15. A = protective 12. Genotype. AA vs AC/CC for resistant vs OCOPD, Odds ratio (OR) =3.2, 95% confidence limits = 1.0-11.0, X2 (Yates corrected)= 3.89, p=0.05. AA genotype =protective 13. Allele. A vs C for resistant vs OCOPD, Odds ratio (OR) =1.7, 95% confidence limits 1.1-2.6, X 2 (Yates corrected)=6.24, p=0.01. A allele = protective 14. Genotype. CC vs AC/AA for OCOPD vs resistant, Odds ratio (OR) =1.8, 95% confidence limits = 1.0-3.3, x 2 (Yates corrected)= 4.29, p=0.04. CC genotype = susceptibility 15. Genotype. TT/TG vs GG for resistant vs OCOPD, Odds ratio (OR) = 1.9, 95% confidence limits= 1.0-38, X 2 (Yates uncorrected)- 4.08, p-0.04. TTITG genotype - protective 16. Allele. T vs G for resistant vs OCOPD, Odds ratio (OR) =1.3, 95% confidence limits 0.9-2.0, X 2 (Yates uncorrected)=-2.36, p=0.12. A allele = protective 17. Genotype. GG vs TT/TG for OCOPD vs resistant, Odds ratio (OR) =0.5, 95% confidence limits= 0.3-1.0, X 2 (Yates uncorrected)= 4.1, p=0.04. GG genotype = susceptible 18. Genotype. RR vs Rr/rr for resistant vs OCOPD, Odds ratio (OR) = 2.3, 95% confidence limits= 0.9-5.8, x 2 (Yates uncorrected)= 3.7, p=0.05, RR genotype = protective 19. Genotype. AG/GG vs AA for resistant vs OCOPD, Odds ratio (OR) = 10.8, 95% confidence limits= 1.4-229, x 2 (Yates corrected)= 5.99 p=0.01. AG/GG genotype = protective, AA susceptible 20. Allele. G vs A for resistant vs OCOPD, Odds ratio (OR) =11.3, 95% confidence limits 1.5-237, ) 2 (Yates corrected)=6.77, p=0.001. G allele = protective, A susceptible 21. Genotype. MS vs MM for Resistant vs OCOPD, Odds ratio (OR) =-2.7, 95% confidence limits 0.8-9.0, x 2 (Yates uncorrected)= 3.4, p=0.07. MS-protective 22. Allele: S vs M allele for resistant vs OCOPD, Odds ratio (OR) =2.5, 95% confidence limits 0.8 8.4, X 2 (Yates uncorrected)= 3.24, p=0.07. 23. Genotype AG vs AA in resistant vs OCOPD, Odd's Ratio (OR)= 5.61, 95% confidence limits 0.5 146, X 2 (Yates uncorrected)= 2.66, p=0.10. AG = protective 24. Genotype. CC vs CG/GG for resistant vs OOCOPD, Odds ratio (OR) = 1.75, 95% confidence limits = 1.0-3.2, x 2 (Yates uncorrected)= 3.73, p=0.05. CC =protective 25. Genotype: AA vs AG/GG for resistant vs OCOPD, Odd's Ratio (OR)= 1.6, 95% confidence limits 0.8-32, X2 (Yates uncorrected)= 2.02, p=0.16. AA = protective 26. Genotype. TT vs TC/CC for OCOPD vs resistant, Odds ratio (OR) =6.03, 95% confidence limits 1.1-42, X2 (Yates corrected)= 4.9, p=0.03. TT=susceptible - 63 27. Genotype. 5G5G vs rest for OCOPD vs resistant, Odds ratio (OR) = 1.9, 95% confidence limits 0.9-4.0, x 2 (Yates uncorrected)= 3.11, p-0.

0 8 . 5G5G = susceptible 28. Allele. 5G vs 4G for OCOPD vs resistant, Odds ratio (OR) =1.4, 95% confidence limits 0.9-2.1,

X

2 (Yates corrected)=3.1, p=0.08. 5G = susceptible 29. Genotype. TT vs TG/GG for resistant vs controls, Odds ratio (OR) =2.3, 95% confidence limits 1.0-5.5, j2 (Yates corrected)= 3.80, p=0.05. TT genotype -protective 30. Genotype. TT vs TG/GG for resistant vs OCOPD, Odds ratio (OR) -1.9, 95% confidence limits 0.8-5.0, )2 (Yates uncorrected)= 2.49, p=O. 11. TT genotype =protective 31. Genotype: Tt/tt vs TT for COPD vs controls, Odd's Ratio (OR) =3.34, 95% confidence limits 1.3 8.9, X 2 (Yates corrected) = 6.28, p=0.01. Tt/tt = susceptible to OCOPD 32. Allele: t vs T for COPD vs controls, Odd's Ratio (OR)=2.5, 95% confidence limits 1.0-6.3, x 2 (Yates corrected)= 4.1, p=0.04. t = susceptible to OCOPD 33. Genotype. 2G2G vs 1G1G/1G2G for COPD vs controls, Odds ratio (OR) =2.1, 95% confidence limits 1.1-4.1, X 2 (Yates corrected)= 5.44, p=0.02. 2G2G genotype =susceptible for OCOPD 34. Allele. 2G vs 1G for COPD vs controls, Odds ratio (OR) =1.7, 95% confidence limits 1.2-2.5, x 2 (Yates corrected)= 7.97, p=0.005. 2G susceptible for OCOPD Table 7 below provides a summary of the protective and susceptibility polymorphisms determined for OCOPD. 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 p2-adrenoreceptor (ADRB2) ADRB2 Gln 27Glu CC protective Interleuldn -18 (LL-18) IL-18 -133 C/G CC susceptible Interleukin -18 (IL-18) IL-18 105 A/C AA susceptible Plasminogen activator inhibitor I (PAI-1) PAI-i -675 4G/5G 5G5G susceptible Nitric Oxide synthase 3 (NOS3) NOS3 298 Asp/Glu TT protective Vitamin D Binding Protein (VDBR) VDBR Lys 420 Thr AA protective CC susceptible Vitamin D Binding Protein (VDBR) VDBP Glu 416 Asp TT/TG protective GG susceptible Glutathione S Transferase (GSTP1) GSTP1 Ile105Val GG susceptible Superoxide dismutase 3 (SOD3) SOD3 Arg 312 Gln AG/GG protective - 64 AA susceptible al-antitrypsin (alAT) alAT 3' 1237 G/A (T/t) Tt/tt susceptible al-antitrypsin (aIAT) cOAT S allele MS protective Toll-like receptor 4 (TLR4) TLR4 Asp 299 Gly A/G AG/GG protective Interleukin-8 (IL-8) IL-S -251 A/T AA protective Interleukin 11 (IL-11) IL-11 -518 G/A AA protective Microsomal epoxide hydrolase (MEH) MEH Exon 3 T/C (r/R) RR protective Interleukin 13 (IL-13) IL-13 -1055 C/T TT susceptible Matrix Metalloproteinase 1 (MMPI) MMP1 -1607 1G/2G 2G2G susceptible The combined frequencies of the presence or absence of the selected protective genotypes COX2 -765 CC/CG, NOS3 298 T1T, alAT 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 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 smokers 20(19%) 31(30%) 53 (51%) 104 % of smokers with OCOPD 34/54 (63%) 51/82 (62%) 39/92 (42%) Comparison Odd's ratio 95% CI X2 P value 0 vs I 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 1.14.9 4.97 0.03 The combined frequencies of the presence or absence of the selected susceptibility genotypes MMPI -1607 2G2G, GSTP1 105 GG, PAI-I -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.

- 65 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 smokers 55 (54%) 37 (37%) 9 (9%) 101 % of smokers with OCOPD 45/100 (45%) 55/92(60%) 24/33 (73%) Comparison Odd's ratio 95% CI X2 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 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 Figure 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 Figure 3). Table 10. Combined presence or absence of protective and susceptibility polymorphisms Score combining protective and susceptibility polymorphisms -2 -1 0 1 2 3 OCOPD nl124 8 28 33 39 11 5 Resistantn=101 2 11 23 27 23 15 % OCOPD 80% 72% 59% 59% 32% 25% - 66 EXAMPLE 3. CASE ASSOCIATION STUDY - LUNG CANCER METHODS Subject recruitment 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 could 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 al 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. This study shows that polymorphisms found in greater frequency in lung cancer patients compared to resistant smokers may reflect an increased susceptibility to the development of lung cancer. Similarly, polymorphisms found in greater frequency in resistant smokers compared to lung cancer may reflect a protective role.

- 67 Summary of characteristics. Parameter Lung Cancer Resistant smokers Differences Median (IQR) N=109 N=200 % 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 FE VI (L) 1.7(0.6) 2.8(0.7) P<0.05 FEVI %predict 67(22) 96% (10) P<0.05 FEVI/FVC 59(14) 82(8) P<0.05 Means and 95% confidence limits Glutathione S-transferase nul polymorphisms genotyping 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, incorporated herein in its entirety by reference]. 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 25d and contained 80ng genomic DNA, 100 ng forward and reverse primers, 200mM dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KC, 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 94C 60 s, 59C 30s, 72C 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 1Kb plus ladder standard (Invitrogen). Genotypes were recorded in data spreadsheets and statistical analysis performed.

- 68 Cyclooxygenase 2 polymorphisms genotyping 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 25ul and contained 20 ng genomic DNA, 500pmol forward and reverse primers, 0.2mM dNTPs, 10 mM Tris-HCL (pH 8.4), 150 mM KCl, 1.0 mM MgCl2 and 1 unit of polymerase (Life Technologies). Cycling times were incubations for 3 min at 95 0 C followed by 33 cycles of 50s at 94 0 C, 60s at 66 0 C and 60s at 72 0 C. A final elongation of 10 min at 72"C then followed. 4ul of PCR products were visualised by ultraviolet trans-illumination of a 3% agarose gel stained with ethidium bromide. An aliquot of 3ul of amplification product was digested for 1 hr with 4 units of AciI (Roche Diagnostics, New Zealand) at 37"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 123bp ladder using ultraviolet transillumination after ethidium bromide staining. Matrix metalloproteinase 1 -16071 G/2G polymorphisms genotyping 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 our own laboratory conditions The PCR reactions were amplified in MJ Research thermocyclers in a total volume of 25 1 d and contained 80ng genomic DNA, 100 ng forward and reverse primers, 200mM dNTPs, 20 mM Tris-HCL (pH 8.4), 50 mM KCl, 1.5 mM MgCl 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 94C 60 s, 55C 30s, 72C 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 -69 bromide staining and migration compared against a 1Kb plus ladder standard (Invitrogen). Genotypes were recorded in data spreadsheets and statistical analysis performed. Polymorphism genotyping using the Sequenom Autoflex Mass Spectrometer 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 SequenomTM system (SequenomTM 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 l0xBuffer 15 mM MgCl2 1.25x, 25mM MgCl2 1.625mM, dNTP mix 25 mM 500uM, primers 4 uM 100nM, Taq polymerase (Quiagen hot start) 0.15U/reaction, Genomic DNA 10 ng/ul. Cycling times were 95"C for 15 min, (5 0 C for 15 s, 56"C 30s, 72 0 C 30s for 45 cycles with a prolonged extension time of 3min to finish. We used shrimp alkaline phosphotase (SAP) treatment (2ul to 5ul per PCR reaction) incubated at 35 0 C for 30 min and extension reaction (add 2ul to 7ul after SAP treatment) with the following volumes per reaction of: water, 0.76ul; hME I0x termination buffer, 0.2ul; hME primer (lOuM), lul; MassEXTEND enzyme, 0.04ul.

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CiH 0 0 H r 0 Ci C o <<000 G G to 0 Ct HN HN(mH- H ')zS) f . - 4. U- < I < F7T U< U< 6 E7 o. U< U oo a o m a o30C a CD0C30C o < < e o C L Ci 1-r1- o r-- 1- <-4r- n 0 CL S 10 1 - a o >0te oo e O O' m Ce 1~ 0 < CN '0 N in CN 0 c o) _j N CN 0 C) LL N CC .- > <can.<)-:9e -e - D 0. ,- N : 0 - ,- , O CN c. - o oNo > U' 02z 2 a < fl < a U oo Ga.a LL C 0 0s 0 0D 01 004 0 a um cr z - m Fn LL P e oww <<<<< < < u)L SL em oo o o oo o e z-om em, 00<< 1 0L < 0 N 0 0 ND N C) N 0 " 0 W < 0 m V C4 0 0 0 < C o U-) U) uO uD U) co Co Ct 0 -(. -L A ;7 r - om g" gg cl 0 1: . 0t 00000000002000 0 c O6~6 Cz e 0aoo ooadao awa~aga C) 0Cr'z a o - _Lw WOWW wooWOoWo vv E< ~ ~ ~ ~ 66 opEOEEO E-EUQo ;7 o- -, (rj (C0 S4 CC OZCr mn CO N 7O CO 0 - N 0 NCh Cr0 * D cD cc ra- Nm v 0 0 Lei O Zz 0~ ri Cl T L O N cnr) t N _ cy .j Lf) ~~~ 1- f) 0<o C D wCO U)w Cw w0C <O (- en 0

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r r (9: 0 < 0 (DI- H F- (9H 0 aD Q a C' N a . ET 0 6 D 0 6o Z C D00 D00 Q 0 20&< w 9 o d wO2G j C aog U2OC srC 0 z 2 '0 z eC D < * o O o 0 O - 0 - - u LI U It) 1- cc -NU 00z 0 H 0 00 2 r 0< 9 600 k ; < 0_ 0 0 H 0 - oo & 9 m ,w wU W .~~C . . . . . . - . . s e s e 00 0 p- < 0 0 P C $"* E'w8 .0 o < oD 0 - L i- . 0k f2 0 Q o CM - 0 < 1 UJ00000000< Cld CD 0 HO L * a o 0 o o (D CD 0 0 0- 0 0 0 t ( ) Q cm 0 D CD 0 C z 'eW 2 0 z 0 e o z 0 00 C 7 00oz 0s S. IJ F--~ i ~ :~ 0 L~ N ~ LUM L.. 0) (D L uU0 D0C Z0 H CD CD F- W 4 2. >t n0 0 a 78 RESULTS Frequencies of individual polymorphisms are as follows: 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) Frequency Allele* Genotype 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' (14%) 79(40%) 91(46%) Nitric oxide synthase 3 -786 T/C Frequency Allele* Genotype C T cc CT TT Controls n=183 (C) Lung Cancer n=107 (%) 82(38%) 132(62%) 16(15%) 50(47%) 413 (38%) Resistant n=198 (%) 166(42%) 228 (58%) 31(16%) 104(53%) 62(31%) Super oxide dismutase 3 Arg 312 Gin C/G Frequency Allele* Genotype 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%) 84 (4%) 14 (1% XRCC1 Arg 399 Gin A/G Frequency Allele* Genotype 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%) 185 (9%) 96 (50%) 79 (41%) Interleukin 8 -251 A/T 79 Frequency Allele* Genotype 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 (a) 1927 (48%) 206(52%) 456 (23%) 102 (51%) 52 (26%) Anti-chymotrypsin Ala -15 Thr Frequency Allele* Genotype A G AA AG GG Lung Cancer n=108 99 (46%) 1179 (54%) 24(22%) 51(47%) 338 (31%) Resistant n=196 (%) 207(53%) 185(47%) 52(27%) 103 (53%) 41 (21%) Cyclin D1 A 870 G Frequency Allele* Genotype A G AA AG GG Lung Cancer n=107 109 (51%) 105(49%) 25" (23%) 59 (55%) 23 (21%) Resistant n=199 (%) 188 (47%) 210(53%) 45 (23%) 98 (49%) 5610 (28%) Interleukin 1B -511 A/G Frequency Allele* Genotype A G AA AG GG Lung Cancer n=107 64 (30%) 150 (70%) 12 (11%) 40 (37%) 5512(51%) Resistant n-198 (%) 143 (36%) 253 (64%) 23 (12%) 97 (49%) 78 (39%) FAS (Apo-1/CD 95) A -670 G Frequency Allele* Genotype A G AA AG GG Lung Cancer n=106 1211" (57%) 91(43%) 32"1 (30%) 57(54%) 17(16%) Resistant n=198 (%) 202 (51%) 194 (49%) 45(23%) 112(57%) 41(21%) XPD 751 T/G Frequency Allele* Genotype G T GG TG TT Lung Cancer ni108 72 (33%) 144(66%) 11(10%) 50 (46%) 47 (44%) Resistant n=197 (%) 147(37%) 247(63%) 3115 (16%) 85(43%) 81(41%) 80 Cytochrome P450 tA1 t1e 462 Val G/A Frequency Allele* Genotype O A GG AG AA Lung Cancer n=109 5 (2%) 213 (98%) 0 (0%) 5 (5%) 1041s (95%) Resistant n=199 (%) 20(5%) 378(95%) 131 (1%) 1816(9%) 1802 (90%) MMP12 Asn 357 Ser Frequency Allele* Genotype G A GG AG AA Lung Cancer nl109 8 (4%) 210 (96%) 1 (1%) 6(5%) 102 (94%) Resistant n=199 (%) 21 (5%) 377(95%) 017(0%) 211 (11%) 178(89%) 8-oxognanine DNA glycosylase Ser 326 Cys C/G Frequency Allele* Genotype G3 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" (7%) 72(36%) 113(57%) N-Acetyltransferase 2 Arg 197 Gln G/A Frequency Allele* Genotype A G AA AG GG Lung Cancer n=106 55 (26%) 157 (74%) 9(8%) 37 (35%) 60' (57%) Resistant n=195 (%) 122 (31%) 268 (69%) 17 (9%) 88 (45%) 90 (46%) Cytochrome P450 2E1 1019 G/C Pstt Frequency Allele* Genotype C G CC CG GG Lung Cancer n=109 10 (5%) 208 (95%) 0(0%) 102 (9%) 99 (91%) Resistant n=197 (%) 11(3%) 383(97%) 0 (0%) 11(6%) 186(94%) Cytochrome P450 2El C/T Rsa I Frequency Allele* Genotype T C TT TC cc Lung Cancer n=108 11(5%) 205(95%) 0(0%) 1121(10%)(90) 81 Resistant n=198 (%) 11(3%) 385(97%) 0(0%) 11(6%) 187(94%) Interleukin 18 105 A/C Frequency Allele* Genotype C A CC AC AA Lung Cancer ni107 50 (23%) 164(77%) 8 (8%) 34 (33%) 652 (61%) Resistant n-200 (%) 116 (29%) 284(71%) 17" (9%) 822 (41%) 101 (50%) Interleukin 18 -133 C/G Frequency Allele* Genotype c C GG CG CC Lung Cancer nl109 52 (24%) 166(76%) 8 (7%) 36 (33%) 65"1 (60%) Resistant n=198 (%) 117 (30%) 279(70%) 17" (9%) 833 (42%) 98 (49%) Glutathione S-Transferase M null Frequency Allele* Null Wild Controls n=178 75 (42%) 103 (58%) Lung Cancer n=107 6724 (58%) 48 (42%) Resistant n=182 100 (55%) 82 (45%) Interferon-gamma 874 A/T Frequency Allele* Genotype 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"- (32%) 48 (45%) 24 (23%) Resistant n=196 (%) 209 (53%) 183(47%) 50(26%) 109(56%) 37(19%) Cyclooxygenase -765 C/G Frequency Allele* Genotype 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%)O 5 (5%2) 24(22%)" 80 (73%)" Resistant n=158 (%) 75 (24%)" 241 (76%) 11(7%) 53 (34%) 94 (59%) 82 Matrix metalloproteinase 1 -1607 1G/2G Frequency AlIele* Genotype 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%) 2 13 (19%) 32 (48%) 22 (33%)31 Resistant n=171 (%) 167 (49%) 175 (51%) 41 (24%) 85 (50%) 45 (26%) * number of chromosomes (2n) 1. Genotype. TT vs TG/GG for resistant vs lung cancer, Odds ratio (OR) =1.8, 95% confidence limits 0.8 4.3, x 2 (Yates uncorrected)= 2.14, p=0. 14, TI genotype =protective 2. Genotype. TT vs TG/GG for resistant vs controls, Odds ratio (OR) =2.2, 95% confidence limits 1.0 4.6, X 2 (Yates corrected)= 4.2, p=0.04, TT genotype =protective 3. Genotype. TT vs CC/CT for Lung cancer vs resistant, Odds ratio (OR) =1.4,95% confidence limits 0.8-2.3, t 2 (Yates uncorrected)= 1.45, p=0.23, TT genotype =susceptible 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 5. Genotype. AA vs AG/GG for resistant vs lung cancer, Odds ratio (OR) = 2.6, 95% confidence limits 0.8-9.2, i 2 (Yates uncorrected)= 2.89, p=0.09. AA genotype = protective 6. Genotype. AA vs AT/TT for resistant vs lung cancer, Odds ratio (OR) -4.1, 95% confidence limits=l.6=1 1.2, X 2 (Yates corrected)=9.8, p=0.002, AA= protective 7. Allele. A vs T for resistant smokers vs lung cancer, Odds ratio (OR) =1.5, 95% confidence limits 1.0 2.2, Xt 2 (Yates corrected)= 5.07, p=0.0 2 , A=protective 8. Genotype. GG vs AA/AG for Lung cancer vs resistant, Odds ratio (OR) = 1.7, 95% confidence limits= 0.9-2.9, x 2 (Yates uncorrected)=3.51, p=0.06, GG =susceptible 9. Allele. G vs A for lung cancer vs resistant smokers, Odds ratio (OR) =1.3, 95% confidence limits 0.9 1.9, X 2 (Yates uncorrected)= 2.71, p=0.10, G=susceptible 10. Genotype. GG vs AG/AA for Resistant vs lung cancer, Odds ratio (OR) =1.4, 95% confidence limits=0.8-2.6, X 2 (Yates uncorrected)=l.6, p=0.20, GG -protective 83 11. Genotype. AG/AA vs GG for Lung cancer vs resistant, Odds ratio (OR) =1.4, 95% confidence limits=0.8-2.6, X 2 (Yates uncorrected)=1.6, p=0.20, AA =susceptible 12. Genotype. GG vs AA/AG for Lung cancer vs resistant, Odds ratio (OR) = 1.6, 95% confidence limits= 1-2.7, X 2 (Yates uncorrected)= 4.07, p=0.04, GG =susceptible 13. Genotype. AA vs AG/GG for Lung cancer vs resistant, Odds ratio (OR) =1.5, 95% confidence limits0.8-2.6, x 2 (Yates uncorrected)=2.03, p=0.15, AA susceptible 14. Allele. A vs G fbr Lung cancer vs resistant, Odds ratio (OR) =1.3, 95% confidence limits 0.9-1.8, x 2 (Yates uncorrected)= 2.04, p=O.15, A= susceptible 15. Genotype. GG vs TG/TT for Resistant vs lung cancer, Odds ratio (OR) -1.7, 95% confidence limits= 0.8-3.7, x2 (Yates uncorrected)= 1.81, p=0.18, GG =protective 16. Genotype. AG/GG vs AA for Resistant vs lung cancer, Odds ratio (OR) =2.2, 95% confidence limits= 0.7-6.9, 2 (Yates uncorrected)=2.41, p=0.12, GG/AG =protective, AA=susceptible 17. Genotype. GG/AG vs AA for Resistant vs COPD, Odds ratio (OR) =1.7, 95% confidence limits= 0.7 4.6, x 2 (Yates uncorrected)=1.45, p=0.23, GG/AG =protective 18. Genotype. GG vs CG/CC for Resistant vs lung cancer, Odds ratio (OR) -4.0, 95% confidence limits=0.9-26.3, xz (Yates uncorrected)=3.87, p=0.05, GG=protective 19. Genotype. GG vs AG/AA for Lung cancer vs resistant, Odds ratio (OR) =1.5, 95% confidence limits=0.9-2.5, x 2 (Yates uncorrected)=3.0, p=0.08, GG =susceptible 20. Genotype. CG vs GG for Lung cancer and resistant, Odds ratio (OR) =1.7, 95% confidence limits=0.7 4.5, x 2 (Yates uncorrected)=1.42, p=0.

23 , CG =susceptible 21. Genotype. TC vs CC for Lung cancer and resistant, Odds ratio (OR) =1.9, 95% confidence limits=0.8 5.0, X2 (Yates uncorrected)=2.24, p=O.1 3 , TC =susceptible 22. Genotype. AA vs AC/CC for Lung cancer and resistant, Odds ratio (OR) =1.6, 95% confidence limits=1.0-2.6, X 2 (Yates uncorrected)=3.51, p=0.06, AA =susceptible, AC/CC protective 23. Genotype. CC vs CG/GG for Lung cancer and resistant, Odds ratio (OR) =1.5, 95% confidence limits=0.9-2.5, X 2 (Yates uncorrected)=2.90, p=0.09, CC =susceptible, CG/GG protective 84 24. Genotype. Null vs wild for Lung cancer and controls, Odds ratio (OR) =1.92, 95% confidence Iimits=1.2-3.2, X2 (Yates corrected)=6.64, p=0.01, Null =susceptible 25. Genotype. AA vs AT/IT for lung cancer vs resistant, Odds ratio (OR) =1.4, 95% confidence limits 0.8-2.4, %2 (Yates uncorrected)= 1.48, p-0.22, AA genotype = susceptible 26. Genotype. AA vs AT/IT for lung cancer vs controls, Odds ratio (OR) =1.9, 95% confidence limits 1.1-3.4, x2 (Yates corrected)= 5.45, pO0.02, AA genotype = susceptible to lung cancer 27. Genotype. CC/CG vs GG for Lung cancer vs resistant, Odds ratio (OR) =0.53, 95% confidence limits=0.3-0.9, X2 (Yates corrected)= 4.9, P=0.03 CC/CG -protective 28. Allele. C vs G for Lung cancer vs resistant, Odds ratio (OR) =0.59, 95% confidence limits 0.4- 0. 9, x 2 (Yates corrected)= 4.8, p=0.03, C =protective 29. Genotype. GG vs CG/CC for Lung cancer vs resistant, Odds ratio (OR) =1.88, 95% confidence limits=1.1-3.3, x 2 (Yates corrected)= 4.9, P=0.03 GG =susceptible (when compared against resistant smokers but not controls) 30. Allele. G vs C for Lung cancer vs resistant, Odds ratio (OR) =1.7, 95% confidence limits 1.1- 2.7, X 2 (Yates corrected)= 4.8, p=0.03, G -susceptible (when compared against resistant smokers but not controls) 31. Genotype. 2G2G vs 1G1G/1G2G for Lung cancer vs controls, Odds ratio (OR) =2.55, 95% confidence limits 1.3-5.1, X2 (Yates corrected)= 7.3, p=0.007 2G2G genotype =susceptible 32. Allele. 2G vs 1G for Lung cancer vs controls, Odds ratio (OR) =2.1, 95% confidence limits 1.4-3.2, X 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. Frequency Allele* Genotype 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%) 85 * number of chromosomes (2n) 1. Genotype. GC/CC vs GG for lung cancer vs resistant, Odds ratio (OR) =1.6, 95% confidence limits 0.8-3.3, X2 (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. Frequency Allele* Genotype C T CC CT TT Lung cancer n=109 177 41 73 31 5 (%) (81%) (19%) (67%) (28%) (5%) Resistant n=195 296 94 (24%) 119 58 is (/o) (76%) (61%) (30%) (9%) * number of chromosomes (2n) 1. Genotype. TT vs CC/CT for lung cancer vs resistant, Odds ratio (OR) =0.47, 95% confidence limits 0.2-1.4, x2 (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. Frequency AlIeIe* Genotype 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) 1. Genotype. AG/AA vs GG for lung cancer vs resistant, Odds ratio (OR) =1.4, 95% confidence limits 0.8-2.4, X2 (Yates uncorrected)= 1.67, p=0.

2 0, AG/AA genotype = susceptibility (trend) 86 Nibrin (NBS1) Gln185Giu G/C polymorphism allele and genotype frequencies in the lung cancer and resistant smokers. Frequency AlIeIe* Genotype 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) 1. Genotype. CC vs CG/GG for lung cancer vs resistant, Odds ratio (OR) -2.3, 95% confidence limits 0.9-5.8, X2 (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. Frequency AIlele* Genotype C T CC CT TT Lung cancer nA05 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) 1. Genotype. TT vs CC/CT for lung cancer vs resistant, Odds ratio (OR) =0.46, 95% confidence limits 0.2-0.95, x 2 (Yates uncorrected)= 5.11, p=0.0 2 , TT genotype = protective 2. Allele. T vs C for lung cancer vs resistant Odds ratio (OR) =0.69, 95% confidence limits 0.5-1.0, X 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.

87 Frequency Allele* Genotype C T CC CT TT Lung cancer n=109 129 89(41%) 39 51 19 (%) (59%) (36%) (47%) (17%) Resistant n=192 242 142 83 76 33 (%) (63%) (37%) (43%) (40%) (17%) * number of chromosomes (2n) 1. Genotype. CC vs CT/IT for lung cancer vs resistant, Odds ratio (OR) =0.73, 95% confidence limits 0.4-1.2, X (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. Frequency Allele* Genotype 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) 1. Genotype. GG vs CC/CG for lung cancer vs resistant Odds ratio (OR) =0.30, 95% confidence limits 0.01-2.5, X 2 (Yates uncorrected)= 1.41, p-0.22 (1-tailed t-test), GG genotype = protective (trend) Apex nuclease (APEl) Asp148Glu T/G polymorphism allele and genotype frequencies in the lung cancer and resistant smokers. Frequency Allele* Genotype T G T 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%) 88 * number of chromosomes (2n) 1. Genotype. GG vs TT/TG for lung cancer vs resistant, Odds ratio (OR) =1.4, 95% confidence limits 0.8-2.7, X 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. Frequency Allele* Genotype 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) 1. Genotype. GG vs GC/CC for lung cancer vs resistant, Odds ratio (OR) =0.66, 95% confidence limits 0.4-1.2, x2 (Yates uncorrected)= 2.12, p=0.15, GG genotype = protective (trend) Table 12 below provides a summary of the protective and susceptibility polymorphisms determined for lung cancer. 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 XRCCI XRCCI 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) CCNDI A870G GG protective 89 AA susceptible Interleukin lB (IL-1B) IL-lB -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 A/G GG/AG protective AA susceptible Matrix metalloproteinase 12 (MMPl2) MMP12 Asn 357 Ser A/G GG/AG protective 8-Oxoguanine DNA glycolase (OGG1) OGG1 Ser 326 Cys G/C GG protective N-acetyltransferase 2 (NAT2) NAT2 Arg 197 Gln A/G GG susceptible CYP2E1 CYP2EI 1019 G/C Pst I CC/CG susceptible CYP2E1 CYP2EI C/T Rsa I TT/TC susceptible Interleukin -18 (IL-18) I-18 105 A/C AC/CC protective AA susceptible Interleukin -18 (IL-18) IL-i8 -133 G/C CG/GG protective CC susceptible Glutathione S-transfbrase M GSTM null Null susceptible Interferon gamma (IFNy) IFNy 874 AT AA susceptible Cyclo-oxygenase 2 (COX2) COX2 -765 G/C CC/CG protective GG susceptible Matrix metalloproteinase 1 (MMPl) MMP -1607 1G/20 2G2G susceptible Connective tissue growth factor (CTGF) CTGF -447 G/C GC/CC susceptible Mucin 5AC (MUC5AC) MUC5AC -221 C/T TT protective Mannose binding lectin 2 (MBL2) MBL2 +161 G/A AG/AA susceptible Nibrin (NBSl) NBS1 Gln185GIu G/C CC susceptible Arginase 1 (Arg1) Argl intron 1 C/T TT protective REVI REVI Phe257Ser C/T CC protective Insulin-like growth factor II receptor IGF2R Leu252Val C/G GG protective (IGF2R) Apex nuclease (Apex or APEl)) Apex Asp 148Glu G/T GG susceptible Interleukin 10 (IL-10) IL-10 -1082 A/G GG protective The combined frequencies of the presence or absence of the selected protective genotypes CYPlA1 GG/AG, OGG1 GG, CCND1 GG, NOS3 298 TT, IL-8 AA, and 90 XRCC 1 AA observed in the subjects with lung cancer and in resistant smokers is presented below in Table 13. 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 (14%) cancer (48%) (30%) Comparison Odd's ratio 95% CI x2 P value 0 vs 1 vs 2+, Resist vs Lung cancer - - 22.3 <0.0001 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 The combined frequencies of the presence or absence of the selected susceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-lB GG, and ACT 15 GG, observed in the subjects with lung cancer and in resistant smokers is presented below in Table 14. 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 COPD 21/92 52/137 35/77 (23%) (38%) (45%) 91 Comparison Odd's ratio 95% CI x 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 The combined frequencies of the presence or absence of the selected protective genotypes CYPlA1 GG/AG, OGGi 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 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 (9%) cancer (45%) (38%) Comparison Odd's ratio 95% CI X2 P value 0 vs 1 vs 2+, Resist vs Lung cancer - - 21.5 <0.0001 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 The combined frequencies of the presence or absence of the selected susceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-lB GG, ACT 15 GG, NAT2 GG, IL-18 105 AA, and IFNy AA, observed in the subjects with lung cancer and in resistant smokers is presented below in Table 16.

92 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 COPD 16/81 35/101 58/127 (20%) (35%) (46%) Comparison Odd's 95% CI X2 P value ratio 0 vs 1 vs 2+, Lung cancer vs Resist - - 14.6 0.0007 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 The combined frequencies of the presence or absence of the selected protective genotypes CYPIA1 GG/AG, OGG1 GG, CCND1 GG, NOS3 298 TT, IL-8 AA, XRCCl 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 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 cancer 45/92(49%) 50/129 (39%) 19/93 (20%) Comparison Odd's ratio 95% CI X2 P value 0 vs 1 vs 2+, Resist vs Lung cancer - - 16.8 0.0002 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 93 The combined frequencies of the presence or absence of the selected susceptibility genotypes CYP2E1 1019 CC/CG, FAS AA, IL-1 GG, ACT 15 GG, and MIP 2G2G, observed in the subjects with lung cancer and in resistant smokers is presented below in Table 18. 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 COPD 13/67 66/179 30/63 (19%) (37%) (48%) Comparison Odd's ratio 95% CI X2 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 Resist 1.9 1.0-3.5 4.6 0.03 0-1 vs rest, Resist vs COPD 2.7 1.4-5.6 8.6 0.003 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 Figure 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 Figure 4).

94 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 2 10 21 38 23 15 N=109 (%) (0%) (2%) (9%) (19%) (35%) (21%) (14%) Resistant smokers 6 21 39 51 52 25 6 N=200 (%) (3%) (11%) (20%) (26%) (26%) (13%) (3%) % Lung cancer 0% 9% 20% 29% 42% 48% 71% A further combined analysis was performed using a greater number of polymorphisms. Again, this produced a linear spread of values, as shown in Table ?. When assessed as a range between -3 to +5, a linear relationship as depicted in Figure 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 Figure 5). 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 0 1 3 10 25 32 20 14 4 cancer (0%) (1%) (3%) (9%) (23%) (29%) (18%) (13%) (4%) N=109 Resistant 3 12 16 34 58 48 21 7 1 smokers (2%) (6%) (8%) (17%) 29%) (24%) (11%) (4%) (0.5%) N=200 % Lung 0% 7% 16% 23% 30% 40% 49% 67% 80% cancer DISCUSSION 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 95 (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. In other embodiments, analyses may utilise path anlaysis and/or Monte-Carlo analysis where the non-genetic and genetic factors can be analyzed. 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. 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 may be as simple as communicating the net susceptibility score to the subject together with an explanation of the implications of that score. This alone may cause a lifestyle or occupational change, with the resultant beneficial effect for the subject. 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. 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 at---- r---a- ag. Specified polymorphisms are indicated in the columns marked SNP NAME. Unique identifiers are indicated in the columns marked RS NUMBER. 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 0X2__ $Nsra6684912 rs5277 rs7527769 r32745559 rs2066823 rs7550380 rs12042763 rs4648263 rs2206594 rs4648250 rs4987012 rs6687495 rs4648251 rs20428 96 ______ rs5681231 rs2223626 rs20429 msl 3376484 rs689462 r&4648264 rl 2064238 rs4648253 rs4648265 miOel 0191 rs689465 rs4648266 _______ l 2743673 _______ rs12027712 _______ ra4648267 rslO9ll9lO rs689466 ral11567B24 sl 2743516 rs2745558 ra4648268 rsi 0911909 rs3918B304 rs4648259 rsl119066 rs204l5 rs4648270 m1l119065 rs20416 ru12759220 rsl 119064 rs4648254 rs20430 ml10798053 rsI1567815 rs4648271 sl 2409744 -765G>C rs204l7 rsl 1567825 rslO9llOOB rs4648256 rs4648273 m1l0011907 rs20419 rsl6825748 ms74l6022 rs2734779 rs4648274 ms2745561 rs20420 ml 6825745 ml 0911906 rs20422 rs20432 rs2734776 rs20423 rs20433 ms2734777 rs5270 rs3218622 ml 2084433 rs20424 rs2066826 ms2734778 rsS2ll rs5278 ms2745560 rs4646257 rs4648276 rs2223627 rs11567819 rs20434 ms2383617 rs3l 34501 rs32l8623 rs4295848 ms3l34592 ms32l 8624 ms4428839 m20426 m5279 rs4609389 rs4648258 r&4648278 rs4428838 rm1567820 msl 3306034 ms1213l2l0 ms2745557 ms2853803 ms2179555 ms156782l rs4648279 rs2143417 rs4048259 rs4648281 rs2143416 ms4648260 ra4648282 rsl 1583191 rs4648261 sl 1567B26 ms2383516 ma4646262 rs4648283 rs238351 5 rsl______ m1567822 _______ rs4648284 Wl0911905 m1l1567823 rs4648285 mlO1l9O94 rs______ 2066824 _________ m1567827 ___________________ _____rs__ 20427 _______ rs4648286 ms4648287 rs1042719 0 5744244 rs5272 ms3729944 ms360722 rs4648288 rs3730182 rs5023207 ms5273 m1042720 ms5744246 ms5274 rs6879202 ms5744247 ms3218625 ms3777124 -133C10 ms360721 _______rs4648289 __________ 103051 _______ r.4988359 _______rs4648290 rs81_____ ml92451 rsl______ I2721 559 97 rs1051896 rs4987255 rs5744248 rs5275 m3177007 rs5744249 rs1126871 rs5744250 rs2082382 rs6885272 rs5744251 rs2082394 rs6889528 rsl 00000356 rs2082395 rs4521458 rl 8344B1 rs9325119 rs10463409 rs17215057 rs9325120 rs7702861 rs5744253 rs12189018 M8 _ 1 P rs5744254 rs11168066 rs187238 rs5744255 rs11959615 rs5744228 rs5744256 rs11958940 rs360718 rs5744257 rs4705270 rs360717 rs360720 rsl0079142 rs5744229 rs5744258 rs9325121 rs100000353 rs5744259 rsl 1746634 rs5744231 rs5744260 rs11168067 rs5744232 ra5744261 rs9325122 rs7106524 105 A/C rs549908 rs11957351 rs189667 A M rs11948371 rs12290658 rs6465787 rs11960649 rs12271175 rs7788533 rsl432622 rs11606049 rs6975620 rsl432623 rs360716 rs6956010 rs11168068 rs360715 rs12534508 rs17778257 rs360714 r&4729664 rs2400706 rs2043055 rs2527316 rs2895795 rs5744233 rs2854235 rs2400707 rs795467 rsl 0228765 rs2053044 rs12270240 rs2854225 rs17108803 rs100000354 rs2854226 m12654778 rs4937113 rs2227707 rs11168070 rs100000355 rs2227631 ml11959427 rs360723 -675 4G/5G No ra rsl042711 rs5744237 PM:N %% rs1801704 ra5744238 rs2373962 Arpl6GI rsl042713 rs5744239 rs2373961 rs1042714 rs7932965 rs6951150 rs1042717 rs11214103 rs13238512 rsl 800888 rs5744241 rsl 0247107 rs1042718 rs5744242 rs10276930 rs3729943 rs5744243 r.1 0277237 rsl2703107 rs9282804 rs2282679 rs6946340 Asp298Glu rs1799983 rs2282680 rs6946091 _Mzp _ ___9 rs705117 rs6946415 rs222035 ra2070741 _ m0952296 rs222036 rs2070742 rs13309715 rs16846943 rs6821541 98 rsl0952297 rs7668653 rs222048 rs7784943 rs1491720 r&432031 rsl1771443 rs16845007 rs432035 rs2243310 rW17830803 rs222049 sml 800783 Glu4l6Asp rm7041 rs222050 rs3918155 Lya420Thr rs4588 rs12510584 rs3918156 rs3737553 rsl 7467825 rs2566519 rs9O1 t1D6 AWA rs3918157 ra1352846 rs656652 rs3918158 rs222039 rs625978 rs3918159 rs3775154 rs6591251 rs2566516 rs222040 rs12278098 rs3918225 rs843005 rs612020 rs3918160 rs222041 rsl2284337 ml 800779 rs7672977 rsl2574108 rs2243311 rs705121 rs6591252 rs3918161 rs11723621 ra597717 rs10952298 rs2298850 rs688489 rs2070744 rs705120 rs597297 rs3918226 rs2298851 m6591253 rs3918162 rs844806 rs6591254 rs3918163 rs1491709 rs7927381 rs3918164 rs705119 rs7940813 rs3918165 rs6845925 r593055 m1800781 rs12640255 rs7927657 rsl3310854 rs12644050 rs614080 rsl3310763 rm6845869 m7941395 m2853797 rs12640179 rs7941648 rs13311166 rs222042 rs7945035 rs13310774 rs3187319 rs2370141 m2853798 rs222043 rs2370142 rsl 1974098 rs842999 rs7949394 m3918166 rs222044 rs7949587 rs3730001 rs222045 rs6591255 rs3918167 ra16846912 m8191430 rs3918168 rs222046 rs6591256 rs3918169 rs705118 rs8191431 m3918170 rs222047 rs8191432 rs3793342 rs13142062 rs7109914 rs3793341 rs843000 rs4147580 Sm549758 rs3755967 rs8191436 rsl007311 rs1491710 rs8191437 sm9282803 rs2282678 rsl 7593068 rs8191438 rs2069718 rs7145047 rs8191439 rs3087272 r7141735 Om8191440 rs2069719 rs1 1558264 rs8191441 rs9282708 rs6647 99 ______ rsl079719 rs2069720 rs8350 rsl87104l rs1042274 rs2230075 rs4147581 rs2089721 51l049800 me8191444 rs2069734 S allele rs17580 ________ s191445 rs_______ 2069722 _________ 2854258 ms2370l43 rs2234687 rs2753937 Wf191446 rs7957366 rs2749547 rs3891249 rs2069723 rs1243152 rs8191447 rs2089724 rs2753938 rsI 2796085 rs2069725 rs2070709 rs8191448 rs4394909 rs17090719 ms762803 rs2069726 nal 1846959 rsf191449 rs2069727 rsl 802962 liel O5VeI rs947894 Im ~lir2749521 rs4986948 -1055 C/T rs1800925 rs2753939 ms675554 rs11575055 m1I802959 ms749174 rs2069755 nal 802961 rs8191450 rs2069741 m10O50469 ms743679 rs2069742 Z allele no ms sl 799811 rs2089743 rs1050520 rs11553890 rs2069756 rs12077 rs4986949 rs3212142 rs12233 rsf191451 rs2066960 rs13170 m1l871042 m1295687 m1303 rs11553892 rs3212145 rs1802960 rs4891 rs2069744 rs1243163 m86413486 ms2069745 m2073333 _______rs5031031 rs2069746 rs1243164 5s947895 rs2069747 m7144409 rs2069748 rB7142803 rs2069707 rs1295686 rs1243165 ms3814242 Argl30Gln rs20541 rs105 1052 ________ 2069709 rs2069749 rs1243166 rs206971 0 rs1295685 rs11628917 rs2069711 ra848 m1I1832 rs2069712 rs2069750 rs9944156 874 AlT ms2430561 rs847 12370G/A W11568814 rs2069713 rsf77081 rl 861494 rs709932 rsf77082 rs2234685 ra11558261 rsf77083 mal 861493 rs20546 rsf77084 rs2069714 _________ s11558263 _________ 875989 ms2069715 F1028580 m9944117 rs2089716 rs7l45770 rs188454A6 ms2069717 rs2239652 m1 884547 msl885065 rs2735442 rs046608 100 rsl 884548 rs2569693 rs5743264 rl243167 rs281439 rs5743266 rsl7751614 rs281440 rs2076752 rl 884549 rs2569694 rs5743267 1rsl243168 rs11575073 rs8061316 rl7090693 rs2569695 rs8061636 rsl 7824597 rs2075741 rsl6948754 rs11575074 rs7206340 rl 799964 rs2569696 rs2076753 rsl 800630 rs2735439 rs2067085 rs1799724 rs2569697 rs16948755 +4M9 O/A rs 01OOO rs2075742 rs2111235 rs3093662 rs2569698 rs2111234 rs3093664 rs11669397 rs7190413 -308 G/A ra1800629 (1) rs901886 m7206582 .MD ... rs885742 rs8045009 C89Y C8DY no rs (2) rs2569699 rs6500328 ..A . rs1056538 rs7500036 rs1799969 rs11549918 rs8057341 rs5493 rs2569700 rs12918060 rs5030381 rs2228615 rs7204911 rs5494 rs2569701 rs7500826 rs3093033 rs2569702 rs4785449 rs5495 rs2735440 m12922299 rs1801714 rs2569703 rs11649521 rsl3306429 rs10418913 rs13339578 rs2071441 rs1056536 m17221417 rs5496 rs2569704 rsl3331327 rs5497 rs11673661 rsl1642482 rsl3306430 rs2569705 rsl1642646 E469K rm5498 rs10402760 rsl7312836 rs5030400 rs2569706 rs5743268 rs2071440 rs2569707 rs5743269 rs5499 rs2735441 rs5743270 rs3093032 ra2436545 rs12925051 rsl057981 rs2436546 rs12929565 rs5500 rs2916060 rs13380733 rs5501 rs2916059 rs13380741 rs5030383 rs2916058 rs11647841 rs281436 rs2569708 rs10451131 rs923366 rs12972990 rs2066842 rs281437 rs735747 rs5743271 rs3093030 rs885743 rs7498256 rs5030384 x rs5743272 rs5030385 rs4785224 rs5743273 rs3810159 rs5743261 rs2076754 m281438 rs5743262 m2066843 rs3093029 rs5743263 rsl 078327 101 rs5743274 rs11645386 rs1031101 rs1861759 rs7187857 rsl0824795 rs5743275 rs8061 960 rsl 0824794 rs5743276 rs5743294 rs920725 rs2066844 rs2357791 rs7916582 rs5743277 rs7359452 rs920724 rs5743278 rs7203344 rl6933335 rs6413461 rs5743295 rs11003125 rs3813758 rs5743296 rs7100749 m5743279 rs3135499 rsl1003124 rs5743280 rs5743297 rs7084554 rs5743281 rs5743298 re7096206 rs4785225 rs5743299 rs11003123 rsl6948773 rs3135500 rs11575988 rs9931711 rs5743300 rsl1575989 rsl7313265 rs8056611 rs7095891 rl1646168 rs2357792 rs4647963 rs9925315 rs12600253 rs8179079 rs5743284 rs12598306 rs5030737 rs5743285 rs7205423 161 G/A rW1800450 rs751271 rs718226 rsl800451 rs748855 r______ rs12246310 rs1861758 rs7899547 rsl2255312 rsl3332952 rs10824797 rsl1003122 rs7198979 rs11003131 rsl982267 rsl861757 rs930506 rsl 982266 rs7203691 rs930505 rs4935047 rs5743286 rsl1003130 rs4935046 rs5743287 rs2384044 rsl 0824793 rs10521209 rs2384045 rm1838066 GIOBArg rs2066845 rs5027257 rs1838065 rs5743289 rs2384046 rs930509 rs8063130 rs12263867 rs930508 rs2076756 rs11003129 rs930507 rs12920425 r812221393 rs12920040 rs2165811 rsl956920 rsl2920558 rs12782244 rs1956921 ral2919099 rs11003128 -1903 G/A rs1800875 rs12920721 rs17664818 rs1800876 rs2076755 rs7475766 rs3759635 rs5743290 rs10824796 rsl 956922 rs5743291 rs16933417 rsl 956923 ra11642651 rs2165810 NAY2 MN rs1861756 rs11003127 rsl1780272 rs749910 rs3925313 rs2101857 rs4990643 rs7094151 rsl 3363820 rsl 077861 rs7071882 rs6984200 rs5743292 rs12264958 rsl 3277605 rs9921146 rs11003126 rs9987109 102 rs7820330 rs7596849 -366 G/A rs9550373 rs7460995 rs4845306 rsl 1542954 rs2087852 rs3087257 r&4769055 rs2101584 rs755581 1 51l7074937 ms7011792 ms7656903 rB9671065 rsl 390358 rs6743438 5s9579545 rs923796 rs6743427 rs9579646 rs4546703 rs676l 336 r&40751 31 rs4634684 rs6761 335 rs40751 32 ms2410556 rs6743335 rs9315043 rs1 1996129 rs6761245 rs9315044 m4621844 m6761237 rs4597169 sl 1785247 ms6743330 ms9575037 rs1115783 rs6743326 rs9578196 rslll5784 m86743322 r&4293222 m1l951456 rs6761220 m1l0507391 rs1112005 rs6761218 rs12429592 ml11782802 rs502l469 rB4769871 rs973874 rs6710596 rs4769872 sl 495744 rs1143823 rs4769573 rs7832071 rs1143624 rs12430051 m1 805158 m2708920 s93l 5045 ml5801279 m1I143625 ms9670275 rsl 041983 rs2853545 rs4503649 ml 801280 ms2708921 ms9508832 rs4986996 rl 143626 rs9670460 sl 2720065 rs3087258 rs3685907 rs4986997 C-5I1IT rs16944 m3922435 rs1799929 rs3917346 rs9551957 Argj9Gln~ ml1799930 m4986962 m1 2018461 rsl208 rs1143627 rs9551958 rs1 799931 W-"U -r10467440 ms2552 Tyrll13His rs1051740 (2) rsl2017304 _______ 4646247 Hial 39Arg ms2234922 (2) ms9551 959 ms971473 m1Y F:,F a1617473 rs72l398 rs4076128 ma11147438 rs9508830 rs10162089 r5l0l69916 ra4073259 r59551960 _______rs1 3009179 rs4073250 rs9285075 rs4849127 rs11616333 rsl2431114 rs4849l26 rs4073261 r&4254165 rs75581 08 rs4075474 5s4360791 ml 3032029 rs4075473 rsl 7612031 ml13013349 rs96701l5 rs3803277 sl 2623093 rs9315042 rs3803278 ms3087255 ms3509376 sl 2429469 rs3087256 rs12877064 msl 7612099 103 rs6721954 r39508831 rS93550.576 ms12621220 ms9670503 rs4356336 rs4584668 rs2075800 rs2734714 rs4238137 .ASNa m6661730 r917612127 rs2791519 r92753377 rs4147063 rs2791518 rs2753378 r94147064 ra5744302 rs2145412 rs4147062 rs1321697 rs2180762 rs9315046 rs2753338 rs1005569 rs9506352 r2791517 rs5744325 rs9670531 rs5744303 rs5744326 rs9671182 rs2734706 r91985554 rs9315047 rs2753345 rs1985555 rs17690694 rs2753347 rslOO000102 rs9652070 rs2753348 rs1O0000103 rs17074966 rs2753349 rs1969719 rs4387455 rs5744304 rs2390102 rs4254166 rs5744305 rs5744329 rs4075692 rs1358826 rs1407142 rs17690748 m2753359 m2753384 rs9671124 rs5744306 rs2753385 rs9671125 rs273471 1 rs5744330 rs9741436 rs5744307 rs5744331 rs9578197 rs2734712 rs926064 rs4769056 rs2753361 rs926065 rs11147439 rs2753364 rs926066 rs12721459 rs1555389 rs926067 rs4769874 rs2753365 rs2753386 o irs1 o00100 rs2180764 rs1043618 rslOO000101 rs2734689 rs11576009 rs5744310 rs5744332 rs11557922 rs5744311 rs5744333 rs11576010 r5744312 rs11161837 rs1008438 rs4656114 rs5744335 rs11576011 rs5744313 rs2038485 rs4713489 rs2753367 rs3765989 rs16867582 rs4656115 rs2734690 rs12526722 rs2734713 rs5744336 rs6933097 rs5744314 rs2734691 r912213612 rs5744315 ra2734692 rs481825 rs5744316 rs5744337 rs7757853 rs5744317 rs5744338 rs7757496 r574431 8 rs2734694 rs9469057 rs926063 rs5744339 rs12182397 rs5744319 rs1O0000104 rsl6867580 rs5744320 rs2791515 rs2075799 rs5744321 rs4656116 104 rs482145 rs5744322 rs5744342 mr2227957 r5744323 mr5744343 T2437C rs2227956 rs5744324 rs2180761 rs2227955 rs2791516 rs5744344 rs5744345 rs5744443 rs6032038 rsl358825 rs5744444 rs6032039 rs2145410 rs3138074 rs2267863 rs2734695 rs13166911 rs6124592 rs5744346 rs2563310 +49 CIT No rs rs5744347 rm2569193 rsl7333103 rsl000105 rs2569192 rsl7333180 rs5744349 rs5744446 rsl 983649 rs4655913 rs5744447 rs16989785 rsl321696 rs5744448 m17424356 rs5744352 rs3138076 rs6017500 rs11583355 rs12519656 rs6032040 rsl00000106 rs5744449 rs6017501 rsl321695 rs2915863 rs2664581 i13924 TIA rs1321694 rs3138078 rs17424474 .2791514 rs6875483 rsl7333381 rs2734696 rs2569191 rs1053826 rs5744354 rs5744451 rs2664533 rs2791513 rs5744452 rs1053831 rs2753332 rs100000098 rs2664520 rs2791512 rs17118968 rs2267864 rs2791511 rs5744455 rs13038355 rs2734697 -159 CIT rs2569190 rs13043296 CD14:SNPs rs2569189 rs13039213 rs6877461 rs2563303 rs6104049 rs3822356 rs3138079 rsl 3043503 rs5877437 rs2228049 rs6104050 rs12153256 rs13763 m17424578 rsl1554680 rs1556179 rsl7424613 rs12109040 m4914 m6017502 rsl2517200 kh* rs6094101 rs5744430 rs2868237 rs6130778 rs5744431 rs4632412 rs6130779 rsl00000092 rs7347427 rs6104051 rs5744433 rs6032032 rs6104052 ml 00000093 rs10854230 AM W o rs4912717 rs7347426 rs2082382 rsl00000094 rs8183548 m2082394 ml 00000095 rs6104047 rs2082395 rs100000096 m6513967 rs9325119 rs6864930 rs13038813 rs9325120 0rs00000097 rs8118673 rs12189018 r96864583 rs7346463 rsl1168066 105 rs6864580 rs7362841 rs11959615 rs6889418 r13042694 l m11958940 rs8889416 rs13038342 rs4705270 rs5744440 rs7363327 rs10079142 rs5744441 rs6073668 rs9325121 rs5744442 rs13044826 rs11746634 ra11168067 ra1800468 rs542603 rs9325122 rs4987025 rs574939 rs11957351 rs1800469 rs573764 ra11948371 ra11466314 rs7102189 rs11960649 rs12977628 rs575727 rs1432622 rs12977601 rs552306 rs1432623 rs12985978 rs634607 ra11168068 rs11466315 rs12286876 rs17778257 rs 11551223 rs12285331 rs2400706 rs11551226 rs519806 ra2895795 rs11466316 rs12283571 rs2400707 rsl3306706 rs2839969 rs2053044 rsl3306707 rs2000609 rs17108803 rsl3306708 rs7125865 rsl2654778 rs9282871 rs570662 rs11168070 LeulOPro rs1982073 rs11225427 rs11959427 rs1800471 rs484915 ra1042711 rs13447341 rs470307 rW1801704 rs11466318 rs2408490 rs1042713 rs12976890 rs12279710 G1n27Glu rs1042714 rs12978333 rs685265 rs1042717 rsl0420084 rs7l 07224 ra1800888 rs10418010 rs1155764 rs1042718 rs12983775 rs534191 rs12462166 rs509332 Arg2l3GIy rs1799895 (2) rs2241715 rs12283759 TOW~B18N s rs9749548 rs2105581 rs1529717 rs7258445 rs470206 ral 046909 rs11466320 rs533621 rs2241712 rs11466321 -1607 G/G3 rs1799750 ra2241713 rs8108052 rs470211 rs2241714 rs6508976 rs470146 rs11673525 rs8108632 rs2075847 ra2873369 rs11466324 rs473509 rs11083617 rs2241716 rs498186 ral 1083616 rs2241717 r&4803458 rs2288873 Null Null allele No ra (2) ra11670143 rs12973435 ra1982072 rs2014015 r11696804 ra11668109 rs1989457 rs61D4416 106 rs13345981 rs10406816 rs3933239 ra11666933 rs8102918 ra3933240 rs11466310 rs4803455 rs6094237 ra11465311 ? S F%% rs11697325 rs2317130 rs529381 rs6130988 rs4803457 rs1144396 rs6073983 rs3087453 rs504875 rs6130989 rsl800820 rs526215 rs6130990 rs1054797 rs12280880 rs10211842 rs6073984 rs125587 rs6073985 rs3918253 rs5754289 rs8121146 rs2274755 r95754290 rs6032620 rs2664538 rs9606994 rs11698788 rs3918254 rs7285034 rs6032621 rs6130993 rs13433582 rs6065912 rs3918255 rs1962223 rs6104417 rs2236416 rs8137129 rs3848720 rs6130994 rs1807471 ral 3040272 rs3918256 rs7290885 ra104418 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 rs5104421 rs6104428 rs5749512 ras3918240 rs2274756 ras104422 rs6017726 rs3918278 rs3918261 ra3918241 rs6032624 1562 C/T rs3918242 rs3918262 ras3918243 rs3918263 rs3918279 rs3918264 rs3918280 rs6130995 ra4578914 rs6130996 rsO17724 rs3918265 ras3918244 rs3918266 ra3918245 rs3918267 rs6130992 rs6073987 ra3918247 rs6073988 rs3918248 rs3918282 ra3918249 rW1802909 107 rs6104423 rs13925 rs6104424 rs20544 rsG104425 rs105628 rs6104426 rW1802908 rs3918250 rs2664517 rsl805089 rs9509 rs3918251 rs3918268 rs13040572 rs3918269 rs13040580 rs3918270 rs3918252 MPW_ _ P 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 The present invention is directed to methods for assessing a subject's risk of developing a disease. The methods comprise 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. Publications 1. Sandford AJ, et al., 1999. Z and S mutations of the al-antitrypsin gene and the risk of chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 20; 287-291. 2. Maniatis,T., Fritsch, E. F. and Sambrook, J., Molecular Cloning Manual. 1989. 3. Papafili A, et al., 2002. Common promoter variant in cyclooxygenase-2 represses gene expression. Arterioscler Thromb Vasc Biol. 20; 1631-1635. 4. Ukkola, 0., 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. 5. Smith CAD & Harrison DJ, 1997. Association between polymorphism in gene for microsomal epoxide hydrolase and susceptibility to emphysema. Lancet. 350; 630 633. 6. Lorenz E, et al., 2001. Determination of the TLR4 genotype using allele-specific PRC. Biotechniques. 31; 22-24.

108 7. Cantlay AM, Smith CA, Wallace WA, Yap PL, Lamb D, Harrison DJ.Heterogeneous expression and polymorphic genotype of glutathione S transferases in human lung. Thorax. 1994, 49(10):1010-4. 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. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, web sites, electronically available information, and other referenced materials or documents. 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" may 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 may 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 109 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 Applicants. 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 may 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. 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. 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.

Claims (29)

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 protective polymorphisms and for the presence or absence of susceptibility polymorphisms, 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; 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; 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. A method according to claim 1 or claim 2 wherein the value assigned to each susceptibility polymorphism is the same.

4. A method according to any one of claims 1 to 3 wherein each protective polymorphism has a negative value and each susceptibility polymorphism having a positive value.

5. A method according to any one of claims 1 to 3 wherein each protective polymorphism has a positive value and each susceptibility polymorphism has a negative vahle.

6. A method according to any one of claims 1 to 5 wherein when the disease is a lung disease, the protective polymorphisms analysed may 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); 111 CC (homozygous P allele) within codon 10 of the gene encoding transforming growth actor beta (T LF b); 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.

7. A method according to claim 6 wherein all polymorphisms of the group are analysed.

8. A method according to any one of claims 1 to 7 wherein when the disease is a lung disease, 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); -1 562CT 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 al-antitrypsin (alAT); or one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms.

9. A method according to claim 8 wherein all polymorphisms of the group are analysed.

10. A method according to any one of claims 1 to 5 wherein when the disease is COPD, the protective polymorphisms analysed may 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 TlT 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 al-antitrypsin (alAT); the +489 GG geneotype in the gene encoding Tumour Necrosis factor a (TNFa); the -308 GG geneotype in the gene encoding TNFa; the C89Y AA or AG geneotype in the gene encodoing SMAD3; 112 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 Gn 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 P2 Adrenergic receptor (ADBR); the -1607 IGiG or IG2G 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.

11. A method according to claim 10 wherein all polymorphisms of the group are analysed.

12. A method according to any one of claims I to 5, 10 or 11 wherein when the disease is COPD, the susceptibility polymorphisms analysed are selected from one or more of the group consisting of: Arg 16 Gly GG in the gene encoding p2-adrenoreceptor (ADRB2); 105 AA in the gene encoding Interleukin-18 (IL-18); -133 CC in the prinuutr of th gcnu caluding IL-18; -675 5G5G in the promoter of the gene encoding plasminogen activator inhibitor I (PAI-1); -1055 TT in the promoter of the gene encoding IL-13; 874 TT in the gene encoding interferon gamma (IFNy); 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 (ICAMI); 113 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 (CLCAI); 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.

13. A method according to claim 12 wherein all polymorphisms of the group are analysed.

14. A method according to any one of claims 1 to 5 wherein when the disease is OCOPD, the protective polymorphisms analysed may 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 Tf 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 alAT; 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 Tf in the gene encoding NOS3; or one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms.

15. A method according to claim 14 wherein all polymorphisms of the group are analysed. 114

16. A method according to any one of claims I to 5, 14, or 15 wherein when the disease is OCOPD, 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 GSTP 1; 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 alAT; -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.

17. A method according to claim 16 wherein all polymorphisms of the group are analysed.

18. A method according to any one of claims 1 to 5 wherein when the disease is lung cancer, the protective polymorphisms analysed may 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 MMP 12; 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 (Argi); the Leu252Val GG genotype in the gene encoding Insulin-like growth factor II receptor (IGF2R); 115 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 (XRCC 1) gene; the A870G GG genotype in the gene encoding cyclin D (CCND 1); the -751 GG genotype in the promoter of the xeroderma pigmentosum complementation group D (XPD) gene ; the lie 462 Val AG or GG genotype in the gene encoding cytochrome P450 IAl (CYP1A1); the Ser 326 Cys GG genotype in the gene encoding 8-Oxoguanine DNA glycolase (OGGI); the Phe 257 Ser CC genotype in the gene encoding REVi; or one or more polymorphisms in linkage disequilibrium with any one or more of these polymorphisms.

19. A method according to claim 18 wherein all polymorphisms of the group are analysed.

20. A method according to any one of claims I to 5, 18 or 19 wherein when the disease is lung cancer, 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 IFNy; 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. the -511 GG genotype in the gene encoding IL-IB; the A-670G AA genotype in the gene encoding FAS (Apo-l/CD95); the Arg 197 Gln GG genotype in the gene encoding N-acetyltransferase 2 (NAT2); 116 the Ie462 Val AA genotype in the gene encoding CYP 1A1; 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 CYP2E 1; the GSTM null genotype in the gene encoding GSTM; the -1607 2G/2G genotype in the promoter of the gene encoding MMPT; the Gln 185 Glu CC genotype in the gene encoding Nibrin (NBS 1); the Asp 148 Glu GG genotype in the gene encoding Apex nuclease (APEl); or one or more polymorphisms in linkage disequilibrium with any one or more of these polymorphisms.

21. A method according to claim 18 wherein all polymorphisms of the group are analysed.

22. A method according to any one of claims 1 to 21 wherein each protective polymorphism is assigned a value of -1 and each susceptibility polymorphism is assigned a value of+1.

23. A method according to any one of claims 1 to 21 wherein each protective polymorphism is assigned a value of +1 and each susceptibility polymorphism is assigned a value of -1.

24. A method according to any one of claims 1 to 23 wherein the subject is or has been a smoker.

25. A method according to any one of claims I to 24 wherein the method comprises an analysis of one or more risk factors, including one or more epidemiological risk factors, associated with the risk of developing said disease.

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 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; assigning a positive score for each protective polymorphism and a negative score for each susceptibility polymorphism or vice versa; 117 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; 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 developingsaid disease.

27. 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 according to any one of claims 1 to 26, wherein said method 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.

28. 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 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.

29. 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 in accordance with a method of any one of claims 1 to 28.