Mutation analysis of the promoter region of CYBRD1, HFE, LTF, HAMP and SLC40A1 in a Tuberculosis cohort

Sittmann, Margarete (2014-04)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Tuberculosis (TB) is an epidemic disease characterised by wet, persistent coughing, weight loss, fever, fatigue, and blood in the sputum. It has been reported that one in every three individuals is currently infected with Mycobacterium tuberculosis, the causative agent of TB, and that 10% of them will develop the active disease. The high prevalence and low penetrance of this disease has resulted in increased research performed to ascertain what factors play a role in susceptibility to M. tuberculosis infection. Some factors known to play a role in a minority of cases may include: HIV infection, diabetes, alcohol abuse and old age, but racial differences in susceptibility have also been observed. However, the influence of genetic factors is gaining popularity in current research. M. tuberculosis requires iron to proliferate, which it must appropriate from its host. For this reason the genes involved in the metabolism of iron in the human host are of particular interest when considering susceptibility to M. tuberculosis infection. In order to determine whether the expression of these genes may influence disease susceptibility, the promoter region of the CYBRD1, HAMP, HFE, LTF and SLC40A1 genes have been targeted for investigation. The aim of this study was to determine whether DNA variation in the promoter region of these genes involved in iron metabolism is associated with M. tuberculosis susceptibility. The study cohort consisted of 49 TB patients and 51 healthy, unrelated, population-matched controls, all of whom were Black, Xhosa-speaking individuals. Initially, 15 patient samples were randomly selected for exploratory screening, utilising semi-automated bi-directional sequencing analysis. In this manner, 40 variants were identified of which 30 were previously described. The novel variants included CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; and LTF: -1377 T/G, -457 T/C, -437 C/G. A number of loci demonstrated a statistically significant difference in allele and genotype frequencies, and in iron parameter levels when comparing the patient and control groups and for each variant separately. In silico analyses revealed the creation and/or abolishment of several transcription factor binding sites (TFBSs) due to the presence or absence of certain identified variants. The change in the composition of TFBSs in the promoter region may lead to differential expression of the gene. This study served as a pilot investigation to identify promoter region variants in the candidate genes involved in iron metabolism, in TB patients. The results presented here indicate that the identified variants (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) could possibly contribute to the increased absorption of iron in the TB patient group, which could subsequently increase the occurrence of pathogenic infection. The findings of this study could further aid in the elucidation of the exact mechanism(s) by which iron, its metabolism, and the genes involved affect disease susceptibility, more specifically, M. tuberculosis susceptibility.

AFRIKAANSE OPSOMMING: Tuberkulose (TB) is „n epidemiese siekte gekarakteriseer deur nat, aanhoudende hoes, gewigsverlies, moegheid, en bloed in die speeksel. Dit is gerapporteer dat een in elke drie individue tans geïnfekteer is met Mycobacterium tuberculosis, die veroorsakende agent van TB, en dat 10% van dié individue die aktiewe vorm van die siekte sal ontwikkel. Die hoë voorkoms en lae effek van hierdie siekte het daartoe gelei dat meer navorsing mettertyd gedoen is om die faktore wat „n rol mag speel in M. tuberculosis infeksie, te bepaal. Sommige faktore bekend vir hul rol in „n minderheid van gevalle sluit in: MIV infeksie, diabetes, alkoholmisbruik en bejaardheid, maar etniese verskille in vatbaarheid vir die siekte is ook al waargeneem. Die waarskynlikheid van genetiese invloed op die ontwikkeling van TB word ook meer deur navorsers ondersoek. M. tuberculosis benodig yster om te vermeerder, wat dit moet bekom vanaf die gasheer. Vir hierdie rede is die gene betrokke by yster metabolisme in die menslike gasheer veral van belang vir die oorweging van vatbaarheid vir M. tuberculosis. Om te bepaal of die uitdrukking van hierdie gene moontlik „n invloed het op vatbaarheid vir die siekte, was die promoter areas van die CYBRD1, HAMP, HFE, LTF en SLC40A1 gene geteiken. Die doel van hierdie studie was om te bepaal of DNS variasie in die promoter area van hierdie gene betrokke in yster metabolisme moontlik verband kan hou met vatbaarheid vir M. tuberculosis. Die studie kohort het uit 49 TB pasiënte en 51 gesonde, onverwante, populasie-gepaarde kontroles, waarvan almal Swart, Xhosa-sprekende individue was, bestaan. Aanvanklik was 15 pasiënt monsters lukraak gekies vir ondersoekende sifting, deur die gebruik van semi-outomatiese twee-rigting volgordebepalings. Op hierdie manier is 40 variante geïdentifiseer waarvan 30 voorheen beskryf is. Die nuwe variante sluit in CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; en LTF: -1377 T/G, -457 T/C, -437 C/G. „n Aantal loci het statisties betekenisvolle verskille getoon in alleel en genotipe frekwensies, en in yster parameter vlakke met die vergelyking van die pasiënt groep met die kontrole groep. In silico analise het verder die skepping en/of afskaffing van verskeie transkripsiefaktor bindingsetels (TFBSs), as gevolg van die teenwoordigheid of afwesigheid van sekere variante, getoon. Die verandering in die samestelling van TFBSs in die promoter area kan lei tot differensiële uitdrukking van die geen. Dié studie het gedien as „n voorlopige ondersoek om te bepaal of promoter area variante, geïdentifiseer in kandidaat gene betrokke by yster metabolisme, „n invloed het in die ontwikkeling van TB. Die resultate wat hier gewys word dui aan dat die geïdentifiseerde variante (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) moontlik die verhoogde opname van yster kan veroorsaak, wat later die toename van die patogeniese infeksie kan veroorsaak. Die bevindinge van hierdie studie kan moontlik bydra tot die toeligting van die presiese meganisme(s) waardeur yster, yster metabolisme, en die betrokke gene vatbaarheid vir siekte, meer spesifiek M. tuberculosis vatbaarheid, beïnvloed.

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