Masters Degrees (Medical Microbiology)

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Browsing Masters Degrees (Medical Microbiology) by browse.metadata.advisor "Glashoff, Richard"

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    Development of functional immune readouts for the confirmation of mendelian susceptibility to mycobacterial disease and related primary immunodeficiencies
    (Stellenbosch : Stellenbosch University, 2019-04) Van Coller, Ansia; Glashoff, Richard; Esser, Monika; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Microbiology.
    Background: Mendelian Susceptibility to Mycobacterial Disease (MSMD) is a primary immunodeficiency (PID) characterised by a predisposition to infection by weakly-pathogenic mycobacteria. In countries with a high prevalence of tuberculosis, individuals with MSMD are also prone to severe, persistent, unusual or recurrent infections by pathogenic Mycobacterium tuberculosis. Several MSMD-associated genes have been described, including IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1, NEMO, ISG15, IRF8, TYK2, and CYBB, many resulting in a disruption of IL-12 and IFN-γ cytokine axis, which is essential for control of mycobacterial infections. This genetic heterogeneity results in many distinct disorders, which vary in their mode of inheritance and clinical presentation. An accurate molecular diagnosis, confirmed by immune functional studies, is essential to ensure that the patient receives optimal treatment and prophylaxis for infections. The aim of this study was to implement and optimise a set of immune phenotyping and functional validation tests for the key pathway, the IFN-γ and IL-12 cytokine axis, involved in MSMD, and to use these assays to assess immune function in a cohort of suspected MSMD patients. Methodology: Blood was collected from 17 participants with MSMD-like clinical phenotypes. DNA was extracted and PBMCs were isolated from the patients’ blood. Whole exome sequencing (WES) was performed and the resulting data was processed using an in-house bioinformatics pipeline, TAPER™. A set of flow cytometry and ELISA-based functional assays were implemented and optimised to assess the integrity of the IL-12-IFN-γ pathway. IFN-γR1 and IL-12Rβ1 expression were assessed by means of standard surface flow cytometry, and IFN-γ and IL-12 signalling was assessed by the detection of pSTAT1 and pSTAT4 respectively through intracellular phospho-specific flow cytometry. IFN-γ-induced IL-12 production as well as IL-12-induced IFN-γ production was also assessed by ELISA after 48-hour in vitro stimulation. The functional and genetic data were then reconciled in order to confirm the extent of functional impairment associated with each genetic variant. Results: Plausible disease-causing variants were identified through genetic investigations for 11 of the 17 participants. Variants in MSMD-associated genes were found in 8 of these patients, although only one of the identified variants, IFNGR1 (c.818del4), has been described before. Variants in genes not previously associated with MSMD were also found, including variants in IKZF1, NOD2, IRAK1, IKBKB, and NFKB2. All the functional assays were optimised and the combination of the three assays for the assessment of the integrity of the IL-12-IFN-γ pathway was successful in identifying immune deficits in essentially all of the participants included in this study. Conclusions: The current study led to the implementation of functional immune readouts that allowed for the evaluation of the functional impact of both novel and previously described genetic variants on the IL-12-IFN-γ pathway. The results generated from the functional assays were highly variable and often defects within the same gene lead to different phenotypes, which emphasises the importance of in vitro functional confirmation of all PIDs. Hence it would be beneficial to apply these assays routinely for patients with suspected PID relating to mycobacterial susceptibility. A molecular diagnosis with confirmed functional impairment paves the way for targeted treatment and improved disease management options for these patients.