Investigating candidate genes identified by genome-wide studies of granulomatous diseases in susceptibility to tuberculosis: ANXA11 and the CADM family

Salie, Muneeb (2010-12)

Thesis (MScMedSc (Biomedical Sciences))--University of Stellenbosch, 2010.

Thesis presented in partial fulfilment of the requirements for the degree Master of Medical Science (Human Genetics) at the University of Stellenbosch.

Bibliography

Thesis

ENGLISH ABSTRACT: The infectious disease tuberculosis (TB) remains the leading cause of death worldwide by a single infectious agent, despite significant advances in biomedical sciences. The idea that host genetics plays a role in the development of disease was proposed by Haldane in 1949. The observation that only 10% of immunocompetent individuals develop disease while others are able to successfully contain it, further suggests that host genetics plays an important role. TB is thus a complex disease, with the causative bacterium, Mycobacterium tuberculosis, host genetic factors and environment all contributing to the development of disease. To date several genes have been implicated in TB susceptibility, albeit with small effect. Genome-wide association studies (GWAS) offer the means to identify novel susceptibility variants and pathways through their ability to interrogate polymorphisms throughout the genome without being limited by our understanding of the immune processes involved in TB infection and disease progression. TB and sarcoidosis are both granulomatous diseases, and we therefore hypothesized that the genes and their associated variants identified in recent GWAS conducted in West Africa for TB, and Germany for sarcoidosis, could alter susceptibility to TB in the South African Coloured (SAC) population. In the sarcoidosis GWAS, ANXA11 was shown to alter susceptibility to sarcoidosis; whereas in the TB GWAS, CADM1 was found to alter susceptibility to TB. This study tested the association with TB of 16 polymorphisms in 5 potential TB host susceptibility genes in the SAC population. A well designed case-control study was employed, using the TaqMan® genotyping system to type the various polymorphisms. Any polymorphism that was found to be significantly associated with susceptibility to TB was then subjected to further analysis to determine the functional effect of the polymorphism. Promoter methylation patterns were also investigated in ANXA11 as another mechanism to elucidate its role in TB susceptibility. A 3’ UTR ANXA11 polymorphism was found to be strongly associated with susceptibility to TB, including 3 haplotypes. The gene expression analysis identified differential transcriptional levels between individual with the different genotypes, with individuals homozygous for the A-allele exhibiting a 1.2-fold increase in gene expression relative to those homozygous for the G-allele. Methylation analysis however found no differences between cases and controls. In addition, 16 novel polymorphisms were also identified, 15 of which occurred in the 3’UTR of ANXA11. The mechanism of action of ANXA11 in TB susceptibility is hypothesised to be in the area of endocytosis, autophagy or apoptosis. A weak association was noted with one of the 5’ UTR polymorphisms of CADM3, which did not hold up to further analysis in the GWAS study, and no functional work was therefore done. This work facilitates our understanding of the role of host genetics in susceptibility to TB and adds to the growing amount of information available. Proper understanding of the role that host genetics plays in TB susceptibility could result in better treatment regimens and prediction of individuals who are at a greater risk of developing TB, a disease that still kills millions of individuals annually.

AFRIKAANSE OPSOMMING: Tuberkulose is verantwoordelik vir meer sterftes as enige ander aansteeklike siekte, ten spyte van die voortuitgang wat die Biomediese Wetenskappe tans beleef. In 1949 het Haldane voorgestel dat die genetiese samestelling van die gasheer ‘n rol speel in vatbaarheid vir aansteeklike siektes. Vir tuberkulose word hierdie aanname gesteun deur die feit dat slegs 10% van individue wat geïnfekteer word aktiewe simptome ontwikkel, terwyl 90% die siekte suksesvol sal afweer. Tuberkulose is dus ‘n komplekse siekte wat veroorsaak word deur Mycobacterium tuberculosis, maar wat beïnvloed word deur genetiese sowel as omgewingsfaktore. Verskeie gene is al geïdentifiseer wat ‘n rol speel in vatbaarheid vir tuberkulose, tog is hul invloed betreklik klein. Genoom-wye assosiasiestudies (GWAS) bied unieke geleenthede vir die identifisering van nuwe polimorfismes wat genetiese vatbaarheid kan beïnvloed. Hierdie tegniek kan die hele genoom fynkam, sonder dat enige vooropgestelde idees oor die immuunrespons teen tuberkulose ‘n invloed sal hê. Tuberkulose en sarkoïdose is albei siektes wat die vorming van granulomas veroorsaak. Verskeie gene met hul geassosieerde variante is geïdentifiseer in ‘n onlangse GWAS, wat gefokus het op populasies in Wes-Afrika en Duitsland. Ons hipotese was dat die polimorfismes wat in hierdie studie geïdentifiseer is, ‘n invloed kan hê op genetiese vatbaarheid vir TB in die Suid-Afrikaanse Kleurlingbevolking (SAK). Die sarkoïdose GWAS het bevind dat ANXA11 vatbaarheid vir die siekte beïnvloed, terwyl CADM1 in die tuberkulose GWAS geïdentifiseer is. Die studie het die assosiasie tussen 16 variante en tuberkulose vatbaarheid ondersoek in die SAK populasie. Die variante strek oor 5 potensiële tuberkulose vatbaarheidsgene. Goedbeplande pasiënt-kontrole assosiasiestudies is gedoen en die polimorfismes is gegenotipeer deur gebruik te maak van die TaqMan® genotiperingsisteem. Enige polimorfisme wat beduidend met tuberkulose geassosieer was, is verder geanaliseer om die moontlike funksionele invloed daarvan te bepaal. Promotormetileringspatrone van ANXA11 is ook geanaliseer, om ‘n addisionele meganisme in tuberkulose vatbaarheidheid te ondersoek. Na genotipering van die polimorfismes is ‘n 3’ UTR ANXA11 variant geïdentifiseer wat beduidend met tuberkulose vatbaarheid geassosieer was. Drie haplotipes is ook geïdentifiseer. Geenuitdrukkingsanalise het aangedui dat verskille in transkripsie vlakke voorkom in individue met verskillende genotipes. Individue wat homosigoties was vir die A-alleel het ‘n verhoging van 1.2 in geenuitdrukking gehad, relatief tot individue wat homosigoties was vir die G-alleel. Metileringsanalise het egter geen verskil aangedui tussen pasiënte en kontroles nie. Addisioneel, is 16 nuwe variante ontdek, waarvan 15 in die 3’UTR van ANXA11 geleë was. Die meganisme waarmee ANAX11 genetiese vatbaarheid vir tuberkulose beïnvloed, blyk in die area van endositose, apoptose of outofagie, te wees. ‘n Swak assosiasie is gevind vir ‘n 5’ UTR variant van CADM3 en is nie verder opgevolg in die GWAS nie. Gevolglik is geen funksionele studies op hierdie polimorfisme gedoen nie. Hierdie studie dra by tot ons kennis oor die rol wat die genetiese samestelling van die gasheer speel in vatbaarheid vir tuberkulose. Indien die rol van mensgenetika in tuberkulose vatbaarheid korrek verstaan word, kan behandeling van die siekte verbeter word en kan individue wat ‘n hoër risiko loop om tuberkulose te ontwikkel geïdentifiseer word.

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