Screening of the human tumor necrosis factor (TNF) gene and its receptor 1 (TNFR1) gene for DNA alterations and the subsequent investigation of these and an IL12p40 polymorphism for an association with paediatric tuberculosis

Abstract

Infection with Mycobacterium tuberculosis is characterised by diverse outcomes; the majority of infected individuals remain well and yet others develop disease ranging from limited pulmonary tuberculosis to severe disseminated disease. The reasons for this diverse outcome are poorly understood, but host factors are thought to play an important role. In particular, a genetic component to susceptibility to tuberculosis has been proposed. An important clue was the description of a group of Maltese children with an unusual susceptibility to progressive non-tuberculous mycobacterial infections. These patients showed defective tumor necrosis factor (TNF) production in response to endotoxin and a failure to upregulate TNF production in response to interferon gamma as well as diminished interferon gamma production during T-cell proliferation. They were found to lack expression of the interferon gamma receptor ligand-binding chain (IFN-yR1) on their cell surfaces due to a single point substitution resulting in a truncated protein. Since then other defects in the type 1 cytokine pathway leading to susceptibility to non-tuberculous mycobacteria, as well as to tuberculosis, have been described in rare isolated cases. From these findings, the hypothesis arose that less severe mutations in such pathways might individually, or in combination, lead to increased susceptibility to tuberculosis in the general population. The following study forms part of a larger multi-centre collaboration, which aims to better understand the genetic basis of susceptibility to mycobacterial infection by addressing this hypothesis. The approach taken has been the recruitment and immuno-phenotyping of a large group of children with tuberculosis as well as control subjects. Candidate genes, of the type 1 cytokine pathways being investigated, include interferon gamma, interleukin­12 and their receptors and TNF. The focus of the study described in this thesis has been the screening of a sub-cohort of patients and control subjects for DNA sequence alterations in the TNF and TNFR1 genes. The individuals in this cohort were selected on the basis of their whole blood stimulation assays, where either high or low levels of TNF in response to non-specific stimulatory factors, were the determining criteria. It was assumed that these two phenotypic groupings would be enriched for gene variants contributing to the TNF responses recorded in the stimulation assays. Once identified, these polymorphisms would be screened for frequencies in the broader patient and control groupings and assessed for any association with susceptibility to tuberculosis. This study was considered important in attempting to explain which genes and their polymorphisms are involved in determining the high prevalence of tuberculosis in African populations.