Title

Frequency and association of cytokine gene polymorphisms in tuberculosis disease sussceptibility and severity

Date of Award

2012

Document Type

Thesis

Degree Name

Doctor of Philosophy in Health Science (PhD)

Department

Biological and Biomedical Sciences

Abstract

One third of the world's populations are suspected to be infected with Mycobacterium tuberculosis with an estimation of 8-10 million new cases diagnosed annually. Pakistan is among the 22 high TB burden countries despite a low contribution from HIV. Among the several factors that contribute towards susceptibility to active tuberculosis, evolution of cytokine and chemokine responses are crucial for the disease progression and establishment. Cytokines modulate the activities of target cells and initiate immune response while chemokines are important in the recruitment of immune cells towards the site of infection. Polymorphisms in genes coding for cytokines and their receptors can have a broad effect on killing intracellular mycobacteria which reside and multiply within macrophages. The role of interferon gamma (IFN-y) is to activate macrophages to kill intracellular organisms. Single nucleotide polymorphisms (SNPs) in key cytokine genes may affect the functionality of IFN-y and may result in high and low producer phenotypes. A number of SNPs have been identified in the IFN-y response and regulatory genes that may predispose to mycobacterial disease. However, the relevance of polymorphism within these genes to the common phenotype of TB remains unclear. The frequency distributions of SNPs in different populations have been shown to be highly variable and this may be due to evolutionary pressures in different populations. Therefore, aim of the current study was to investigate functional gene polymorphisms in IFN-y (+874T—*A), its receptor (-273 to -741) and in associated regulatory cytokines and chemokines such as IL-10 (- 1082A—>G), TNF-a (-308G--4A), IL-6 (-174G-0C) and CCL-2 (-2518A-4G) to establish baseline frequencies and to investigate their influence on tuberculosis disease susceptibility and severity in indigenous population. Moreover, chemokine CCL-2, protein levels were also assessed on a subset of samples. When cytokine genotype frequencies were analysed in healthy individuals, Pakistani population seem to have a higher proportion of alleles that are associated with high IFN-y (7), high IL-10 (A), low TIVF-a (G), high IL-6 (G) and low CCL-2 (A) phenotypes as reported from other Asian populations compared to Caucasian, European and African populations and this underlines the importance of a 'local' reference population when evaluating the clinical relevance of cytokine gene polymorphisms. In relation to TB, the IFN-y T allele was found significantly higher in pulmonary TB (PTB) patients which was restricted to minimal and moderate PTB, increasing the risk by 2-3 fold. This T allele was also found to be linked between first intronic CA11 repeats and moderate PTB while AA was found to be linked between CA13 and disseminated extra-pulmonary TB (DTB). Two novel SNPs in 1FN-y receptor] promoter region at positions -255 (C--+T) and -129 (G—A) were found in association with advance PTB (PAD) and DTB respectively which suggest the link of these SNPs with TB disease severity. The IFN-y response cytokine SNPs were also found in association with differing susceptibility and severity of TB such as IL-10 (-1082) AhIgh with protection from PAD and ETB, TNF-a (-308) Ahigh with susceptibility to ETB and IL-6 CI' allele with protection from PTB. Investigation of CCL-2 genotype-phenotype relation was the next target of this study which showed that CCL-2 GG genotype and higher CCL-2 levels may play a role in TB disease localization but there was no significant relationship between CCL-2 genotypes and phenotypes. In terms of mutiloci interactions, the combination of IFN-y TT high or IFN-y AA1' with IL-6 GGhIgh was the most significant in determining disease severity which is in line with the reported function of IL-6 as part of the Th2 network. These findings suggest that identification of genetic biomarkers which may be predictive of disease progression or severity can provide extremely useful tools for National TB programs for th management of patients with differing disease severity and combinations of key cytokine genotypes would be more meaningful in determining the relationship of polymorphisms with TB disease severity.

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