Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter

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dc.contributor.advisorEley, Brianen_ZA
dc.contributor.advisorAnderson, Suzanneen_ZA
dc.contributor.authorPienaar, Sandra Margareten_ZA
dc.date.accessioned2014-11-11T06:58:16Z
dc.date.available2014-11-11T06:58:16Z
dc.date.issued2014en_ZA
dc.descriptionIncludes bibliographical references.en_ZA
dc.description.abstractThe 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.en_ZA
dc.identifier.apacitationPienaar, S. M. (2014). <i>Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Department of Paediatrics and Child Health. Retrieved from http://hdl.handle.net/11427/9534en_ZA
dc.identifier.chicagocitationPienaar, Sandra Margaret. <i>"Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Department of Paediatrics and Child Health, 2014. http://hdl.handle.net/11427/9534en_ZA
dc.identifier.citationPienaar, S. 2014. Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Pienaar, Sandra Margaret AB - 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. DA - 2014 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2014 T1 - Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter TI - Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter UR - http://hdl.handle.net/11427/9534 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/9534
dc.identifier.vancouvercitationPienaar SM. Tuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoter. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Department of Paediatrics and Child Health, 2014 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/9534en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDepartment of Paediatrics and Child Healthen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.titleTuberculosis and genes of the IL12/IL23/IFNγ pathway: Exploring functional significance of novel mutations in the IL12p40 promoteren_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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