Genetic variation in COPD pathogenesis
Chronic obstructive pulmonary disease (COPD) is a disease state characterised by airflow limitation that is not fully reversible, due to chronic bronchitis and emphysema. Given its substantial health impact, it is vitally important that a better understanding of pathogenesis, risk factors and treatment of COPD is achieved. Cigarette smoking is the major risk factor for COPD. However, variability in the risk of developing COPD suggests that other factors, such as genetic variation, act in addition to cigarette smoke. Natural variations, or polymorphisms, in the deoxyribonucleic acid (DNA) sequence of genes may critically alter the function or rate of synthesis of their proteins. The best known polymorphism associated with COPD is the ZZ genotype of the a,-antitrypsin gene. As this is uncommon, polymorphisms in other genes may potentially contribute to COPD. Thus, the aims of this thesis were to examine the influence of candidate genes, relevant to the pathogenesis of COPD, on susceptibility to COPD and its disease severity. The selection of candidate genes was based on postulated mechanisms of pathogenesis (Abstract Figures 1 and 2)
Methods: The genotypes of 17 candidate genes were determined by polymerase chain reaction (PCR) ± restriction fragment length polymorphism (RFLP) analysis, on DNA extracted from the peripheral blood lymphocytes of participants. The frequencies of the polymorphisms in an Australian cohort of 205 COPD patients were compared to two control groups: 104 smokers with normal lung function and 207 blood donors, using Pearson's X2 test or Fisher's exact test, where appropriate (SPSS for Windows Version 9.0.1). In the 205 COPD patients, the genotypes of candidate genes were compared to standardised clinical phenotyping for chronic bronchitis and airflow limitation (spirometry and lung volumes) and emphysema (gas transfer and, wherever possible, high-resolution computed tomography (CT) quantification). A cohort of stable patients also had measurements of plasma MBL and TNF-α production from peripheral blood mononuclear cells (PBMCs) cultured with and without stimulation by LPS.
Results: As summarised in Abstract Tables 1 and 2, PI ZZ was found more frequently in COPD patients than controls. The MZ genotype was also increased in those with low gas transfer whereas variants in MMP9 and MPO were less common. PI Z, EPHX1 Argl39 and TNF -308A alleles were associated with chronic bronchitis. The low-producing MBL2 230A/A genotype was more frequent in COPD patients without, than with, chronic bronchitis. There was no association of GSTM1, GSTT1, GSTP1, CYPlA1, IL6, IL1RN, TGFB1, FCGR2A or FCGR3B polymorphisms with risk of COPD. Airflow limitation and gas trapping: In COPD patients, more severe airflow limitation was present with the ILIRN variants, EPHX1 Arg/Argl39, MPO -463AlA and CYP1A1 Ile/Val462. Patients with MPO ^63A/A or the CYP1A1 variant had increased gas trapping. Emphysema severity: Patients with IL1RN variants had more severely impaired gas transfer, whereas the MMP9 variant and GSTT1-0/GSTP1-lleflle haplotype had higher gas transfer. Those with the CYP1A1 Val462 allele had a more severe CT emphysema index. There was no association of GSTM1, IL6, TNF, FCGR2A or MBL2 polymorphisms with disease severity. Cytokine and defence molecule production: Patients with the TNF -308A variant had larger increments in TNF-a production with LPS-stimulation. CD14 polymorphism did not influence TNF-α production. The MBL2 230G>A variant was strongly associated with low blood MBL levels, with lowest levels in the A/A genotype. In addition, the variant MBL2 230A/A or G/A genotypes were associated with increased risk of admission for exacerbation and respiratory infection. Pulmonary hypertension: No association between VEGF genotypes and right ventricular systolic pressure was detected.
Discussion: This is the first study to systematically examine polymorphisms in candidate genes that span all the mechanisms of disease pathogenesis in COPD. Novel associations were detected for polymorphisms with susceptibility and disease severity of COPD. This study also demonstrates for the first time in COPD that there exist significant differences in TNF-α and MBL levels due to inter-individual gene variation. Thus the biological plausibility of inter-individual differences in COPD features being due to genetic variations is strongly supported by this study. The correlation of relevant genotypes to the COPD phenotype has direct clinical application for prognosis of at-risk individuals, targeted prevention and intervention, and development of novel pharmacological therapies.