Development of functional immune readouts for the confirmation of mendelian susceptibility to mycobacterial disease and related primary immunodeficiencies

Thesis (MSc)--Stellenbosch University, 2019.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.Agtergrond: Mendeliese vatbaarheid vir mikobakteriële siektes (MSMD) is ‘n primêre immuundefek (PID) wat deur vatbaarheid tot infeksie deur minder-patogeniese mikobakterieë gekenmerk word. In lande met ‘n hoë voorkoms van tuberkulose, het hierdie individue ‘n hoër waarskynlikheid om ernstige, ongewone, aanhoudende of herhalende infeksies met die patogeniese Mycobacterium tuberculosis te kry. Verskeie gene wat met MSMD geassosieer word is reeds beskryf, onder andere IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1, NEMO, ISG15, IRF8, TYK2 en CYBB. Hierdie gene lei almal tot die ontwrigting van die IL-12-IFN-γ sitokien padweë wat noodsaaklik is vir die beheer van mikobakteriële infeksies. Hierdie genetiese diversiteit lei tot verskeie unieke versteurings wat verskil in die manier van oorerflikheid en kliniese voorkoms. ‘n Akkurate molekulêre diagnose, wat bevestig is deur funksionele studies, is daarom belangrik om te verseker dat die pasiënt optimale behandeling en profilakse vir infeksies ontvang. Die doel van hierdie studie was om ‘n stel immuun-fenotipering en funksionele validering toetse te implementeer en optimaliseer vir die MSMD kenmerkende sitokien padweë van IL-12 en IFN-γ. Metodes: Bloedmonsters van 17 pasiënte met ‘n kenmerkende MSMD kliniese fenotipe is vir DNS ekstraksie en PBMC isolasie geneem. Volledige-eksoom-volgordebepaling (WES) is op pasiënt DNS uitgevoer en die data is deur ‘n binne-huis bioinformatika pyplyn, TAPER™, verwerk. ‘n Stel vloeisitometrie en ELISA-gebaseerde funksionele toetse was geïmplementeer en geöptimaliseer om the integriteit van die IL-12 en IFN-γ padweë te ondersoek. IFN-γR1 en IL-12Rβ1 uitdrukking is deur middel van standard vloeisitometrie bepaal, en IFN-γ en IL-12 sein-oordrag is bepaal deur ondersoek van pSTAT1 en pSTAT4 deur middel van fosfo-spesifieke vloeisitometrie. IFN-γ-geïnduseerde-IL-12-produksie, asook IL-12-geïnduseerde-IFN-γ-produksie, is deur ELISA bepaal na 48 uur in vitro stimulasie met die onderskeie sitokiene. Funksionele en genetiese data was versoen om MSMD te bevestig of uit te sluit as die betrokke PID. Resultate: Moontlike patogeniese genetiese variante is geïdentifiseer in 11 van die 17 pasiënte. Variante in MSMD-veroorsakende gene is in 8 van hierdie pasiënte gevind, maar slegs een van hierde variante, IFNGR1 (c.818del4), is al voorheen beskryf. Variante is ook in gene gevind wat nog nie voorheen met MSMD geassosieer is nie, insluitend IKZF1, NOD2, IRAK1, IKBKB en NFKB2. Die funksionele toetse was suksesvol geöptimaliseer en die kombinasie van hierdie toetse vir die integriteit van die IL-12 en IFN-γ padweë het immuundefekte in die meerderheid van die pasiënte geïdentifiseer. Gevolgtrekkings: Hierdie studie het gelei tot die implementering van funksionele immuunassesseringstoetse wat dit moonlik maak om die impak van beide nuwe en vooraf-beskryfde genetiese variante op die IL-12 en IFN-γ padweë te bepaal. Die resultate van die funksionele toetse het gewissel en dikwels het variante in dieselfde geen tot verskillende fenotipes gelei. Dít beklemtoon die noodsaaklikheid van in vitro funksionele bevestiging van alle PIDs. Dit sal voordelig wees om hierdie toetse op ‘n roetine wyse aan te wend vir pasiënte wat vermoedelik ‘n PID het wat verwant is aan tuberkulose-vatbaarheid. ‘n Molekulêre diagnose sal lei tot beter behandelingopsies vir hierdie pasiënte, omdat die molekulêre meganisme van die individu se siekte toegelig is