Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Familial hypercholesterolaemia (FH) contributes significantly to the high death rate from cardiovascular disease worldwide. FH is a common autosomal co-dominant disease characterised by raised cholesterol levels and premature coronary heart disease (CHD). Whilst these features usually are very prominent in homozygotes the clinical diagnosis of heterozygotes is complicated by variable phenotypic expression. Specific founder genes in the low-density lipoprotein receptor (LDLR) gene have increased the prevalence of FH in South African Afrikaners, Indians, Jews and Coloureds, and screening for these known mutations allows unequivocal diagnosis of FH-affected individuals. The systematic molecular analysis of FH resulted in the identification of at least ten founder-type LDLR gene mutations among the 56 different gene defects described to date in the diverse South African population. DNA screening of 792 at-risk family members for the FH-related mutations identified in 379 index cases, allowed accurate disease diagnosis in an additional 340 relatives and exclusion of the relevant mutation in 452 individuals. This effort forms part of the MED PED FH initiative, a collaborative project to "Make Early Diagnosis and Prevent Early Deaths in MEDical PEDigrees with FH". Evaluation of clinical criteria versus DNA diagnosis of three founder-related mutations (D154N, D206E and V408M) in the South African population demonstrated that the sensitivity and specificity of diagnoses, based on total cholesterol values measured in family members of index cases recruited for this study, were 88% and 77%, respectively. A population-directed DNA diagnosis of FH is therefore justified in South Africa on a routine basis, since expression of the defective gene measured in biochemical tests does not allow accurate diagnosis of FH in all cases. The application of mutation detection was illustrated by prenatal diagnosis of FH performed for a couple who are both heterozygous for the most common Afrikaner mutation, D206E. The mutation was absent in the foetus and a normocholesterolaemic infant was born. Prenatal diagnosis of FH, aimed at the detection of homozygous cases, is particularly applicable in populations and families with molecularly defined LDLR gene mutations. The MED-PED approach resulted in accurate diagnosis and subsequent treatment of FH in more patients, and referral to lipid clinics where they could receive the intensive care their condition justifies. Molecularly diagnosed FH patients will be the first to benefit from future treatment approaches based on mutation type. / AFRIKAANSE OPSOMMING: Familiële hiprcholesterolemie dra grootliks by tot die wêreldwye hoë sterftesyfer van kardiovaskulêre siekte. FH is 'n algemene outosomale ko-dominante siekte wat gekenmerk word deur verhoogde cholesterolvlakke en vroeë koronêre hartsiekte. Terwyl hierdie kenmerke prominent is in homosigote, word die kliniese diagnose van heterosigote bemoeilik deur variasie in fenotipiese uitdrukking. Spesifieke stigtergene in die lae-digtheids lipoproteien reseptor (LDLR) geen het die voorkomssyfer van FH verhoog in Suid Afrikaanse Afrikaners, Indiërs, Jode en Kleurlinge. Sifting vir hierdie bekende mutasies maak akkurate diagnose van FH geaffekteerde individue moontlik. Die sistematiese molekulêre analise van FH het aangetoon dat ten minste tien van die 56 verskillende geen defekte wat tot dusver beskryf is in die Suid-Afrikaanse populasie stigtertipe LDLR geen mutasies is. DNA sifting van 792 familielede vir die FH-verwante mutasie in 379 indeksgevalle geïdentifiseer is, het akkurate diagnose moontlik gemaak in 340 addisionele familielede, en uitsluiting daarvan in 452 individue. Hierdie poging vorm deel van die MED-PED FH ("Make Early Diagnosis and Prevent Early Deaths in MEDical PEDigrees with FH) inisiatief. Evaluering van kliniese kriteria teenoor DNA diagnose van drie stigter verwante mutasies (D154N, D206E en V408M) in die Suid Afrikaanse populasie het getoon dat die sensitiwiteit en spesifisiteit van die diagnose, wat gebasseer is op totale cholesterol waardes in familielede van indeksgevalle, onderskeidelik 88% en 77% was. 'n Populasie gerigte DNA diagnose van FH is dus geregverdig in Suid-Afrika op "n roetine basis, omdat die defektiewe geen nie altyd in biochemiese toetse uitgedruk word nie. Die waarde van mutasie opsporing is geillustreer deur 'n voorgeboortelike diagnose van FH wat aangevra is vir ouers wat beide heterosigoties is vir die mees algemene Afrikaner mutasie, D206E. Die mutasie was afwesig in die fetus en 'n normocholesterolemiese baba is gebore. Voorgeboortelike diagnose van FH, wat gemik is op die opsporing van homosigotiese gevalle, is veral van toepassing in populasies en families met bekende LDLR geen mutasies. Die MED-PED benadering het gelei tot akkurate diagnose en daaropvolgende behandeling van FH in meer pasiënte, en verwysings na lipiedklinieke waar hulle intensiewe aandag kan geniet. Molekulêre gediagnoseerde FH pasiënte sal die eerste wees om baat te vind by toekomstige behandeling wat moontlik gebasseer sal word op mutasie status.

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.