Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Non-Alcoholic Fatty Liver Disease (NAFLD) is the most prevalent chronic liver disease in Western countries and is considered the hepatic manifestation of the Metabolic Syndrome (MetS). Its heterogeneous nature ranges from hepatic steatosis through steatohepatitis to advanced fibrosis and cirrhosis where the ingestion of significant amounts of alcohol has been excluded. The disease profile of NAFLD and its necro-inflammatory subset Nonalcoholic Steatohepatitis (NASH) were described in the parent study, which provided a clinically well-characterised patient cohort for the present investigation. South African patients with NASH had significantly higher mean serum cholesterol and triglyceride levels than those with fatty liver only. The objective of this study was to implement a high-throughput real-time polymerase chain reaction (PCR) method in our laboratory to enable the assessment of cardiovascular genetic risk factors in NAFLD patients. The specific aims were to determine the clinical utility and perform analytical validation of each mutation included in the multi-gene cardiovascular disease (CVD) screening assay. The Pathology Supported Genetic Testing (PSGT) concept developed at our department provides a practical approach to personalized medicine. The CVD multi-gene screen analyses key metabolic pathways relating to atherogenic dyslipidaemia, chronic inflammation, hypercoagulation and iron dysregulation implicated in insulin resistance, which is known to be a universal factor in the pathogenesis of NAFLD. Deleterious low-penetrance mutations in the APOE (APOE2 and E4 alleles), MTHFR (677C>T and 1298A>C), F2 (20210G>A), FV (1691G>A, Leiden) and HFE (C282Y and H63D) genes were included for analysis due to their important role as genetic contributors to these biological processes. A total of 178 patients diagnosed with NAFLD and 75 controls were studied using direct DNA sequencing and a RT-PCR system for mutation detection. In addition, two patients with high ferritin levels were included as case studies. A significant association was found between HFE mutations and elevated Alanine Transaminase (ALT) levels in the NAFLD population (p = 0.04). This discovery is interpreted as the identification of a subset of patients at greater risk of developing progressive liver damage who would benefit most from genetic testing to direct more aggressive therapy at an earlier stage. The necessity of an integrative, systems-based network approach was demonstrated to more accurately distinguish between Hereditary Haemochromatosis (HH) and Insulin Resistance-associated Hepatic Iron Overload (IR-HIO) syndrome in obese patients. The PSGT approach to personalized medicine facilitates diagnosis of CVD subtypes, prevention of cumulative risk and the formulation of gene-based intervention programs tailored to the needs of the patient. These findings support the clinical utility of the CVD multi-gene test to guide chronic disease risk management in patients with NAFLD. The HFE mutation detection component of this test is of particular relevance in directing an effective treatment strategy in patients with a medical history of CVD and/or high iron stores. / AFRIKAANSE OPSOMMING: Nie-Alkoholiese Vettige Lewer Siekte (NAFLD) is die mees algemene kroniese lewer siekte in Westerse lande en word bestempel as die hepatiese manifestasie van die Metaboliese Sindroom (MetS). Die heterogene natuur van NAFLD strek van hepatiese steatose deur steatohepatietis tot gevorderde fibrose en sirrose waar grootskaalse alkohol inname uitgesluit is. Die siekte-profiel van NAFLD en sy nekro-inflammatoriese subtipe Nie-Alkoholiese Steatohepatietis (NASH) is reeds beskryf in die ouer studie, wat ‗n klinies goed-gekarakteriseerde pasiënt groep vir die huidige ondersoek daar gestel het. Suid-Afrikaanse pasiënte met NASH het beduidend hoër gemiddelde serum cholesterol en trigliseried vlakke in vergelyking met slegs vettige lewer. Die doel van hierdie studie was om ‗n hoë deurvoer rieëltyd polimerase kettingreaksie (RT-PCR) metode in ons laboratorium te implimenteer om kardiovaskulêre genetiese risiko faktore in NAFLD pasiënte te ondersoek. Die spesifieke mikpunte was om die kliniese nut en analitiese geldigheid van elke mutasie wat ingesluit is in die multi-geen kardiovaskulêre siekte (KVS) siftings toets vas te stel. Die Patologie Ondersteunde Genetiese Toetsing (PSGT) konsep wat by ons departement ontwikkel is, verskaf ‗n praktiese benadering tot persoonlike medisyne. Die KVS multi-geen toets analiseer belangrike metaboliese weë verwant aan atherogene dyslipidemie, kroniese inflammasie, oormatige bloedstolling en yster disregulering wat betrokke is by insulien weerstand wat bekend is as ‗n universele factor in the patogenese van NAFLD. Nadelige lae-penetrasie mutasies in die APOE (APOE2 en E4 allele), MTHFR (677C>T en 1298A>C) F2 (20210G>A), FV (1691G>A, Leiden) en HFE (C282Y en H63D) gene was ingesluit vir analise as gevolg van hul belangrike rol as genetiese bydraers tot die bogenoemde biologiese prosesse. ‗n Totaal van 178 pasiënte gediagnoseer met NAFLD en 75 kontroles is bestudeer deur gebruik te maak van direkte DNA volgordebepaling en ‗n RT-PCR metode vir mutasie opsporing. Twee pasiënte met verhoogde ferritien vlakke is ook as gevalle studies ingesluit. ‗n Beduidende assosiasie is gevind tussen HFE mutasies en verhoogde Alanien Transaminase (ALT) vlakke in die NAFLD studiepopulasie (p = 0.04) wat aanduidend is van ‗n subgroup van pasiënte wat die meeste baat sal vind uit genetiese toetsing om meer aggressiewe behandeling te rig op' n vroeër stadium. Die noodsaaklikheid van 'n geïntegreerde, stelsels-gebaseerde netwerk benadering is gewys om meer akkuraat te onderskei tussen Oorerflike Hemochromatose (HH) en Insulien Weerstand-geassosieerde Hepatiese Yster Oorlading (IR-HIO) sindroom in vetsugtige pasiënte. Die PSGT benadering tot persoonlike medisyne formuleer geen-gebaseerde intervensie programme aangepas tot die behoeftes van die pasiënt ek maak diagnose van KVS-subtipes en voorkoming van kumulatiewe risiko moontlik. Hierdie bevindinge ondersteun die kliniese nut van die KVS multi-geen toets om riglyne vir die risikobestuur van kroniese siektes soos NAFLD daar te stel. Die HFE mutasie opsporings komponent van hierdie toets is van besondere belang om 'n effektiewe strategie vir die behandeling van pasiënte met 'n mediese geskiedenis van KVS en/of hoë yster vlakke daar te stel.

Thesis (MSCMedSc)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world. The disease spectrum of NAFLD extends from steatosis (types 1,2) to non-alcoholic steatohepatitis (NASH) with inflammation (types 3,4). The aims of the study were 1) to analytically validate high-throughput real time polymerase chain reaction (RT-PCR) assays for three selected single nucleotide polymorphisms (SNPs), FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) and PPARγ rs1801282 (Pro12Ala, 34 C>G), and 2) to perform genotype-phenotype association studies in relation to biochemical abnormalities, disease severity and age of onset. A total of 119 patients with fatty liver identified on ultrasound, including 88 histologically confirmed NAFLD patients, and 166 control individuals were genotyped for the three selected SNPs. RT-PCR validated against direct sequencing as the gold standard was used for detection of genetic variation. All three SNPs were in Hardy Weinberg equilibrium in the study population, except for a deviation in genotype distribution detected for PPARγ rs1801282 in the NAFLD patient subgroup (p<0.001). After adjustment for age and gender, the risk-associated FTO rs9939609 A-allele was detected at a significantly higher frequency in the Caucasian compared with Coloured patients (p=0.005). The opposite was detected for the risk-associated TNF-α rs1800629 A-allele, which occurred at a significantly higher frequency in the Coloured compared with Caucasian NAFLD patients (p=0.034). The onset of fatty liver disease symptoms was on average 5 years younger in the presence of each risk-associated TNF-α rs1800629 A-allele (p=0.028). When considered in the context of an inferred genotype risk score ranging from 0-6, disease onset occurred on average 3 years earlier (p=0.008) in the presence of each risk-associated FTO A-allele, TNF-α A-allele or PPARγ C-allele. After adjustment for age, gender and race, no differences in genotype distribution or allele frequencies were observed between histologically confirmed NAFLD (types 1,2) and NASH (types 3,4) patients, while the minor allele frequency for the TNF-α rs1800629 was significantly higher in the total NAFLD (types 1-4) (p=0.047) as well as NASH subgroup (NAFLD types 3,4) (p=0.030) compared with obese patients without a histologically confirmed NAFLD diagnosis. A significant correlation was furthermore observed between the number of TNF-α rs1800629 A-alleles and increasing CRP levels (p=0.029), with a favourable reduced effect in the presence of low- to moderate alcohol intake. The average waist circumference of physically active NAFLD patients was 12% lower than in physically inactive patients (p=0.004). In view of the results presented in this study, the inclusion of the selected SNPs, and in particular the pro-inflammatory TNF-α rs1800629 polymorphism, may be considered as part of a comprehensive cardiovascular risk evaluation of NAFLD patients. Ultimately, early detection of patients with fatty liver disease symptoms and effective intervention based on the underlying disease mechanism may prevent progression from NAFLD to NASH, shown to be an independent risk factor for cardiovascular diseases. / AFRIKAANSE OPSOMMING: Nie-alkoholiese lewervervetting (NALV) is die mees algemene kroniese lewersiekte in die wêreld. Die siektespektrum van NALV strek van steatose (vervette lewer tipes 1,2) tot steatohepatitis met inflammasie (NASH tipes 3,4). Die doel van die studie was 1) om analities die hoë omset polimerase kettingreaksie (RT-PKR) metode te valideer vir die geselekteerde enkel nukleotied polimorfismes (ENPs) FTO rs9939609 (intron 1 T>A), TNF-α rs1800629 (-308 G>A) en PPARγ rs1801282 (Pro12Ala, 34 C>G), en 2) om genotipe-fenotipe assosiasie studies uit te voer ten opsigte van relevante biochemiese abnormaliteite, graad van die siekte en aanvangsouderdom. ’n Totaal van 119 pasiënte met vervette lewers is geïdentifiseer met behulp van ultraklank, insluited 88 histologies-bevestigde NALV pasiënte, en 166 kontrole individue. Hierdie pasiënte is gegenotipeer vir die 3 geselekteerde ENP’s. RT-PKR gevalideer met direkte DNA volgorde bepaling as die goue standaard, is gebruik vir opsporing van genetiese variasie. Al die ENP’s was in Hardy Weinberg ekwilibrium in die studie populasie, behalwe vir ’n afwyking in genotipe verspreiding waargeneem vir PPARγ in die NALV subgroep (p<0.001). Nadat aanpassings gemaak is vir ouderdom en geslag, is die risiko-geassosieerde FTO rs9939609 A-alleel waargeneem teen ’n betekenisvol hoër frekwensie in die Kaukasiese pasiënte in vergelyking met Kleurling pasiënte (p=0.005). Die teenoorgestelde is waargeneem vir die risiko-geassosieerde TNF-α rs1800629 A-alleel wat voorgekom het teen ’n betekenisvol hoër frekwensie in die Kleurling NALV pasiënte, in vergelyking met Kaukasiese NALV pasiënte (p=0.034). Die aanvang van NALV was gemiddeld 5 jaar vroeër in die teenwoordigheid van elke risiko-geassosieerde TNF-α rs1800629 A-alleel (p=0.028). Met inagneming van ’n genotipe risiko telling tussen 0–6, het aanvang van siekte gemiddeld 3 jaar vroeër voorgekom (p=0.008) in die teenwoordigheid van elke toenemende risiko-geassosieerde FTO A-alleel, TNF-α A-alleel en PPARγ C-alleel. Nadat aanpassings gemaak is vir ouderdom, geslag en ras, is geen verskille waargeneem in genotipe verspreiding of alleel frekwensies tussen histologies bevestigde NALV (tipes 1,2) en NASH (tipes 3,4) pasiënte nie, terwyl die minor alleel telling vir die TNF-α rs1800629 betekenisvol hoër was in die totale NALV (tipes 1–4) (p=0.047) asook die NASH subgroep (NALV tipes 3,4) (p=0.03) in vergelyking met vetsugtige pasiënte sonder ’n histologies bevestigde diagnose. ‘n Statisties beteknisvolle korrelasie is verder waargeneem tussen die aantal TNF-α rs1800629 A-allele en toenemende CRP vlakke (p=0.029), met n gunstige verlaagde effek in die teenwoordigheid van lae alcohol gebruik. Die gemiddelde middellyf-omtrek van fisies aktiewe NALV pasiënte was 12% minder as fisies onaktiewe pasiente (p=0.004). Na aanleiding van die resultate van hierdie studie behoort insluiting van geselekteerde ENP’s, en in besonder die pro-inflammatoriese TNF-α rs1800629 polimorfisme, as deel van ’n omvattende kardiovaskulere risiko evaluasie oorweeg te word. Aan die einde van die dag mag vroeë identifikasie van NALV pasiente en effektieve intervensie gebasseer op die onderliggende siekte meganisme, vordering tot NASH verhoed wat getoon is om ’n onafhanklike risiko faktor vir kardiovaskulêre siekte te wees. / Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology