Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter
The aim of this work was to screen the IL12p40 gene promoter for association with TB disease. Initially a subcohort of children (TB cases and healthy controls) from a TB-endemic area was screened for DNA changes by the WAVE method. Thereafter, the entire paediatric cohort and a cohort of healthy adult controls were screened by Amplification Refractory Mutation System PCR. Functional testing was done by reporter assay and immunological phenotype was investigated by measurement of cytokines levels and cytokine receptor expression. WAVE screening identified two heterozygous SNPs, -1523 A/G and -1564 C/T. Statistical analysis showed that -1523 A/G may be protective against TB disease (p=0.02). This possibility was supported by the location of -1523 A/G occurring within a GTATA sequence reported to bind nuclear proteins. Specific ARMS-PCR assays were then designed for screening of additional paediatric subjects and healthy adult controls for these SNPs. Analysis of the larger group, showed that -1564 C/T may contribute to susceptibility to TB disease (p=0.03) Exploring functional relevance, normal and mutant promoter fragments were PCR amplified, using uniquely adapted primers that included restriction sites corresponding to those in the multiple cloning site of an expression vector, facilitating cloning. A truncated promoter and one with essential regions deleted, were created as negative controls. These five promoter fragments were cloned into the expression vector and functional differences tested by reporter. No significant functional differences between variant and normal promoter fragments were observed. A predictive immune phenotype was investigated by measurement of IFNγ, TNFα and IL12p70 cytokine levels and IL12βR1 receptor expression. While distinct patterns of cytokine responses were seen, these did not predict genotype. These results show that the IL12p40 gene promoter is highly conserved and sequence variants may be just one of many factors contributing to TB susceptibility.
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Department of Paediatrics and Child Health