Analysis of two mutations in the MTHFR gene associated with mild hyperhomocysteinaemia – heterogeneous distribution in the South African population

Objective, The frequencies of mutations 677C→T and 1298A→C in the methylenetetrahydrofolate reductase (MTHFR) gene, previously shown to be associated with decreased enzyme activity that may lead to hyperhomocysteinaemia and consequently increased risk of cardiovascular disease (CVD), were determined in the South African population.Methods, Hinfi (677C→T) and MbolI (1298A→C) restriction enzyme analyses were performed on amplified DNA samples of 76 white, 73 coloured and 60 black subjects.Results, The mutant alleles of mutations 677C→T and 1298A..-+C were more common in the white (allele frequencies 0.36 and 0.37, respectively) than in the black population (0.04 and 0.09), while intermediate frequencies were detected in the coloured population (0,18 and 0.30); Homozygosity for mutation 677C → T was not detected in the black cohort, while this genotype was detected in 1 coloured (1.4%) and 8 white (105%) subjects, In the black population, 5% of the 60 subjects analysed were homozygous for mutation 1298A→C, compared with approximately 12% in both the white and coloured populations,Conclusions. Since hyperhomocysteinaemia is a risk factor for premature CVD, the heterogeneous distribution of the 677C→T and 1298A→C mutations across ethnic groups may partly explain ethnic differences in heart disease risk through decreased enzyme activity and hence increased homocysteine levels

Molecular investigation into regulatory regions of the LDLR gene involved in lipoprotein metabolism

Thesis (PhD) -- University of Stellenbosch, 2001.ENGLISH ABSTRACT: The advent of the new millennium saw the complete sequencing of the entire human genome. Only approximately 30 000 genes, much less than was initially predicted, have been identified to be responsible for the genetic diversity in humans. This discovery has prompted a shift in the approach to disease research, since one gene can be involved in numerous diseases. This phenomenon seems to be especially true for the low-density lipoprotein receptor (LDLR) gene. Various substances beside sterols can induce transcription of the LDLR gene. Non-communicable diseases (e.g. hypertension) are common in the developing world and contribute significantly to mortality rates. The fmding that a promoter variant (-175 g~t) in the LDLR gene is associated with elevated diastolic blood pressure may explain the phenomenon of high LDL-cholesterollevels in hypertensive individuals. Studies have demonstrated that the lowering of cholesterol, especially LDL-cholesterol, can reduce the incidence of hypertension. The -175 g~t variant is located in a newly described cis-acting regulatory element which contains a putative binding site for Yin Yang (YY)-l and also demonstrates great homology to the cAMP response element (CRE) which bind the Ca2+- dependent transcription factor, CRE binding protein (CREB). The fact that Ca2+ can induce transcription of the LDLR gene may, at least in part, explain the association between the - 175g~t variant and elevated diastolic blood pressure. Cholesterol is important for various processes, such as apoptosis, maintenance of cellular membranes and immune function. The -59 c-ot mutation in repeat 2 of the LDLR gene abolishes binding of the sterol regulatory element binding protein(SREBP) to the SRE-l site. SREBP is proteolytically activated during apoptosis by two caspases (CPP32 and Mch3) to induce cholesterol levels. Our results imply that the -59C/T mutation, in repeat 2 of the LDLR gene promoter, may inhibit apoptosis under normal immunological conditions. Atherosclerosis can be considered an immunological disease, since various humoral and cellular immune processes can be detected throughout the course of the disease. The fmding that certain lipoproteins can protect against infection by binding and lysing of pathogens, or competing with pathogens for cellular receptors, prompted the investigation into the potential role of variation in the LDLR gene promoter in immune function. A significant difference in allelic distribution was detected between asymptomatic HIY -infected subjects and fast progressors for the -124 c-ot variant (P=O.006), shown to increase (~160%) transcriptional activity of the LDLR gene. Of relevance to this particular study is the fact that human herpesvirus (HHV) 6 can transactivate CD4 promoters through a partial CRE site. It has been shown that the CREB and YYl can regulate viral and cellular promoters, and these transcription factors can potentially bind to the LDLR promoter at the FP2 site. The mutation enrichment in the LDLR gene promoter seen in the South African Black and Coloured population groups can possibly provide insight into the pathogenesis of various diseases. This could also potentially, provide novel targets for treatment, since manipulation of cholesterol levels may affect the pathogenesis of various diseases.AFRIKAANSE OPSOMMING: Die volledige DNA volgorde bepaling van die mensgenoom is voltooi vroeg in die nuwe millennium. Slegs ongeveer 30 000 gene is geidentifiseer, heelwat minder as wat in die verlede voorspel is, wat verantwoordelik is vir die genetiese diversiteit in die mens. Hierdie ontdekking het gelei tot 'n verandering in die benadering van navorsing ten opsigte van siektes, aangesien een geen 'n rol by verskeie siektes kan speel. Hierdie gewaarwording blyk veral waar te wees vir die lae digtheids lipoproteien reseptor (LDLR) geen. Verskeie stimuli, buiten sterole, kan transkripie van die LDLR geen inisieer. Verskeie siektes soos hipertensie is algemeen in die ontwikkelende wereld, en dra by tot die hoe mortaliteit syfers. Die bevinding dat 'n promoter variant in die LDLR geen (-175g-H) geassosieer is met verhoogde diastoliese bloeddruk, kan moontlik verhoogde lipiedvlakke in hipertensiewe individue verklaar. Studies het aangetoon dat die verlaging van cholesterol, veral LDL-cholesterol, die voorkorns van hipertensie kan verlaag. Die -175 g~t variant is gelee in 'n cis-regulerende element wat na bewering 'n bindingsetel vir die Yin Yang (YY)-l transkripsie faktor bevat asook sterk homologie met die cAMP respons element (CRE) toon, wat bind aan die Ca2 +_ afhanklike transkripie faktor, CRE bindings proteiene (CREB). Die feit dat Ca2+ transkripsie van die LDLR geen kan inisieer, kan dalk tot 'n mate, 'n verklaring bied vir die assosiasie tussen die -175 (g~t) variant en verhoogde diastoliese bloeddruk. Cholesterol is noodsaaklik vir verskeie prosesse soos apoptose, die instandhouding van selmembrane sowel as immuun funksies. Die -59 c-ot mutasie in die sterol regulerende element 1 (SRE-l) van die LDLR geen vernietig binding van die sterol regulerende element bindingsprotei'en (SREBP) aan SRE-l. SREBP word proteolities geaktiveer tydens apoptose deur twee kaspases (CPP32 en Mch3) om cholesterolvlakke te induseer. Ons resultate impliseer dat die -59C/T mutasie, in herhaling-2 van die LDLR-geen promoter, apoptose kan inhibeer onder normale immunologiese toestande. Aterosklerose kan beskou word as 'n immunologiese siekte, aangesien verskeie humorale en sellulere immuun prosesse deur die verloop van die siekte waargeneem kan word. Die feit dat Iipoproteiene beskermend kan wees teen infeksies, deur binding en lisering van virusse of kompeteer met patogene vir sellulere reseptore, het aanleiding gegee tot 'n ondersoek na die potensiele rol van variasies in die promoter area van die LDLR geen in immuun funksie. Betekenisvolle verskille in alleel verspreiding vir die -124c~t variant (P=0.006) is waargeneem tussen asimptomatiese MIV -geinfekteerde pasiente en individue met vinnige siekte progressie. In vitro studies het voorheen getoon dat die -124c~t 'n verhoging in LDLR geen transkripsie (160%) tot gevolg het. Dit is noemenswaardig dat 'n vroee studie getoon het dat die mens like herpesvirus-6 (MHV6) transaktivering van die CD4 promoters deur 'n gedeeltelike CRE bindingsetel kan bewerkstellig. Beide CREB en YYl kan virus en sellulere promotors reguleer, en hierdie transkripsie faktore toon bindingshomologie met die FP2 element van die LDLR promotor Die mutasie verryking van die LDLR geen promoter soos waargeneem in Suid Afrikaanse Swart en Kleurling populasies, kan moontlik lig werp op die patogenese van verskeie siektetoestande. Hierdie bevindinge kan potensieel nuwe teikens vir behandeling identifiseer, aangesien manipulasie van cholesterolvlakke 'n effek mag he op die patogenese van verskeie siektes

Genetic risk factors in cardiovascular disease in the South African population

Thesis (M. Sc. Med. Sc.) -- University of Stellenbosch, 1998.Full text to be digitised and attached to bibliographic record

Lipoprotein(a) determination and risk of cardiovascular disease in South African patients with familial hypercholesterolaemia

CITATION: Scholtz, C. L. et al 2000. Lipoprotein(a) determination and risk of cardiovascular disease in South African patients with familial hypercholesterolaemia. South African Medical Journal, 90(4):374-378.The original publication is available at http://www.samj.org.zaObjective. A raised plasma level of lipoprotein(a) (Lp(a)) is an established genetic risk factor for coronary heart disease (CHD), particularly in patients with concomitant elevation of low-density lipoprotein (LDL) cholesterol. The current study focused on the comparison of two commercially available Lp(a) assay kits to determine whether differences observed in measured Lp(a) levels could be deemed negligible in CHD risk assessment in familial hypercholesterolaemic (FH) patients. Design. To compare results obtained on duplicate plasma samples using two commercially available Lp(a) measuring kits, the immunoradiometric assay (RIA) and the enzyme-linked immunoabsorbent assay (ELISA). Setting. Division of Human Genetics, Department of Obstetrics and Gynaecology, University of Stellenbosch, Tygerberg, South Africa and the Institute for Medical Biology and Human Genetics, University of Innsbruck, Austria. Subjects. Plasma samples were obtained from 146 family members of 65 molecularly characterised South African FH families for comparative analysis. Results. Using the RIA method, 34 samples (23%) considered to be in the normal range by the ELISA technique, were placed in the high-risk group (> 30 mg/dl). Only one sample, considered to have a normal Lp(a) level with the RIA method, was categorised by the ELISA technique as high risk. Conclusion. Our data demonstrate that measurements of Lp(a) using the RIA method (the only assay available in South Africa at the time of this study) differ significantly from those obtained by the reference ELISA technique, suggesting that misclassification could lead to inaccurate CHD risk assessment. This is an important consideration in Afrikaner FH families, where plasma levels of Lp(a) have been shown to be elevated significantly in FH patients compared with non-FH individuals.Publisher’s versio