Identification of novel candidate genes for susceptibility to tuberculosis by identifying disease-causing mutations in individuals with Primary Immunodeficiency disorders

Schlechter, Nikola (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Mendelian susceptibility to mycobacterial disease (MSMD) is a rare primary immunodeficiency disorder believed to affect less than 1:1 000 000 individuals globally. It is characterized by increased susceptibility to weakly virulent mycobacterial infections, such as Bacille Calmette Guerin, and in some cases more virulent agents, such as Mycobacterium tuberculosis, possibly implying a monogenic predisposition to tuberculosis (TB). TB claimed the lives of ~1.5 million people worldwide in 2014, making it the leading cause of death due to a single infectious agent and a very high-priority health problem. Ten genes have been associated with MSMD, five of which are also involved in TB susceptibility. In half of all MSMD patients, however, no mutations are found in these genes, highlighting the need to identify yet undiscovered MSMD-causing genes. The present study aimed to identify novel MSMD-causing mutations by using whole exome sequencing (WES), and to determine whether the genes containing these variants are associated with increased TB susceptibility. It also investigated a potential link between MSMD and tuberculosis meningitis (TBM) by investigating variants identified in known MSMD-causing genes in TBM cases. Three MSMD patients and five of their healthy parents, as well as 10 TBM patients and 10 controls, were recruited. WES was performed on the Illumina HiSeq and produced ~60 000 - 75 000 variants per individual. Numerous filtering and variant prioritization tools were used to identify three plausible MSMD-causing variants in the MSMD patients based on in silico predictions of their potential to be disease-causing and the function of the genes they are situated in. These include two variants in transporter associated with antigen processing 1 (TAP1) and one in mitogen-activated protein kinase kinase kinase 14 (MAP3K14). In vitro functional studies verified their involvement in disease. Case-control association studies were performed using known single nucleotide polymorphisms in these two genes in a cohort of TB cases and controls. Four variants in three MSMD-causing genes were also identified in four of the TBM patients, which were absent from controls, and predicted to cause disease. One variant was identified in each MSMD patient. The heterozygous I296M variant in TAP1 was absent from ethnically matched controls and has not been previously identified according to ExAC Browser, the 1000 Genomes Project and ESP6500. The variant is predicted to be deleterious and may alter antigen presentation to molecules of the type I major histocompatibility complex. The homozygous V345M variant in MAP3K14 was absent from the above-mentioned databases, and predicted to be deleterious. It can potentially inhibit effective nuclear factor-kappa B signaling and thus lead to aberrant lymphoid immunity. The P67S variant in TAP1 has not previously been identified in homozygous form, although it has been recorded in the heterozygous state in three Asians and one European. This heterozygous variant is predicted to be a benign polymorphism. However, homozygous variants can have more severe effects on protein function, and could influence different ethnic populations dissimilarly. In summary, these results identify three novel putative variants involved in MSMD and thus increased TB susceptibility. It also hints at a potential link between MSMD and TBM, which should be investigated further. Identification of MSMD-causing mutations can inform treatment strategies by aiding in the implementation of patient-specific vaccine strategies and treatment regimes. It can lead to identification of at-risk relatives, and also provide novel candidate genes to be evaluated for increased TB susceptibility in the general population.

AFRIKAANSE OPSOMMING: Mendeliese vatbaarheid vir mikobakteriële siekte (MVMS) is ‘n baie seldsame primêre immuniteitsgebrek wat minder as 1 uit elke 1 000 000 individue wêreldwyd affekteer. Dit word gekenmerk deur verhoogde vatbaarheid vir mikobakteriële infeksies van lae virulensie, soos Bacille Calmette Guerin, en in sommige gevalle meer virulente agense soos Mycobacterium tuberculosis, wat moontlik op ‘n monogeniese vatbaarheid vir tuberkulose (TB) kan dui. TB was die oorsaak van ongeveer 1.5 miljoen sterftes wêreldwyd in 2014. Dit is dus die leidende oorsaak van dood as gevolg van ‘n enkele aansteeklike agens en moet gevolglik as ‘n baie hoë-prioriteit gesondheidsprobleem beskou word. Tot op hede is tien gene geassosieer met MVMS, waarvan vyf ook betrokke is by TB-vatbaarheid. In soveel as die helfte van alle MVMS-pasiënte is daar egter geen mutasies in enige van hierdie gene gevind nie. Dit beklemtoon die belang daarvan om steeds onbekende gene, wat MVMS veroorsaak, te identifiseer. Die doelwit van die huidige studie was om nuwe mutasies wat MVMS veroorsaak te identifiseer, deur gebruik te maak van volledige eksoomvolgordebepaling (VEV) met gevorderde tegnieke, en om vas te stel of die gene, wat hierdie variante bevat, geassosieer kan word met verhoogde vatbaarheid vir TB. Dit het ook ‘n potensiële skakel tussen MVMS en tuberkulose meningitis (TBM) ondersoek, deur die teenwoordigheid van variante in die gene wat voorheen met MVMS geassosiëer is in TBM pasiënte te ondersoek. Drie MVMS-pasiënte en vyf van hulle gesonde ouers, asook 10 TBM pasiënte en 10 kontroles, is gewerf. VEV is uitgevoer op die Illumina HiSeq en het ~60 000 - 75 000 variante per pasiënt opgelewer. Verskeie filter- en variantprioritiseringsmetodes is gebruik drie potensiële variante wat moontlik MVMS in hierdie pasiënte kan veroorsaak, te identifiseer gebaseer op in silico voorspellings van hul potensiaal om siekte te veroorsaak, asook die funksie van die gene waarin hulle voorkom. Dit sluit in twee variante in TAP1 en een in MAP3K14. In vitro funksionele studies is uitgevoer om die betrokkenheid van hierdie twee gene in MVMS te bewys. Gevallekontrole assosiasiestudies is uitgevoer deur gebruik te maak van bekende enkelaminosuur-polimorfismes in die twee nuut-ontdekte MVMS gene in ‘n studiegroep van TB-gevalle en kontroles. Vier variante teenwoordig in drie van die gene wat MVMS veroorsaak is ook in vier van die TBM pasiënte ontdek, en was afwesig in die kontrolegroep. Hulle is voorspel om siekte te veroorsaak. Een variant is in elke MVMS pasiënt identifiseer. Die heterosigotiese I296M-variant in TAP1 was afwesig in kontroles van dieselfde etniese groep en is nog nooit tevore beskryf volgens “ExAC Browser”, die “1000 Genomes Project” en “ESP6500” nie. Die variant is voorspel om nadelig te wees en mag die voorstelling van antigeen aan molekule van die Klas I Major Histoverenigbaarheidskompleks affekteer. Die homosigotiese V345M-variant in MAP3K14 is afwesig van die voorgenoemde databasisse en word voorspel om nadelig te wees. Dit kan moontlik effektiewe seintransduksie deur kernfaktor-kappa B verhoed en dus lei tot afwykende limfoïede immuniteit. Die P67S-variant in TAP1 is nog nooit tevore in homosigotiese vorm beskryf nie, alhoewel dit in heterosigotiese vorm in drie Asiate een Europeër opgeteken is. Hierdie heterosigotiese variant word voorspel om ‘n benigne polimorfisme te wees. Homosigotiese variante kan egter meer ernstige uitwerkings op proteïenfunksie hê en kan verskillende etniese populasies op verskillende maniere beïnvloed. Ter opsomming identifiseer hierdie resultate drie nuwe variante wat vermoedelik betrokke mag wees by MVMS en dus verhoogde vatbaarheid vir TB. Dit identifiseer ook ‘n potensiële skakel tussen MVMS en TBM, wat verder ondersoek moet word. Die identifisering van mutasies wat MVMS veroorsaak kan behandelingstrategieë beïnvloed deur by te dra tot die implementering van pasiënt-spesifieke inentingstrategiëe en behandelingsregimes. Dit kan lei tot die identifisering van hӧe-risiko familielede, en ook nuwe kandidaatgene identifiseer wat evalueer kan word vir hul betrokkenheid in verhoogde TB-vatbaarheid onder die algemene bevolking.

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