Thesis (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. Current knowledge suggests that MS is associated with autoimmunity and that infectious agents and hereditary factors may be involved. The demonstration of a higher recurrence risk of MS in families (4-5%) compared with the general population (0.1%) provides strong evidence for a genetic basis. Extensive analyses of the entire human genome to identify new genes that may underlie MS have indicated that several genes may contribute to disease susceptibility, but these remain largely unidentified. In this study candidate genes involved in iron metabolism and immunology have been analysed for the first time within the context of both autoimmune and infectious disease susceptibility, in order to investigate the role of genetic and viral factors implicated in the pathogenesis of MS. The Z-DNA forming repeat polymorphism in the promoter region of the solute carrier family 11 (proton-coupled divalent ion transporters) member 1 (SLC11A1) gene was found to be significantly associated with MS (P<0.01) in the genetically homogeneous Afrikaner population of South Africa, but not in the German and French populations using a case-control study and transmission linkage disequilibrium approach, respectively. However, significant differences were observed in genotype distribution between German MS patients with a primary- and secondary progressive disease course (P<0.05), and between the German patients with relapsing remitting and primary progressive MS (P<0.05). These findings provide further evidence that the SLC11A1 gene is associated with MS, most likely due to its role in iron homeostasis. In order to investigate the influence of viruses in the apparent multi-step aetiology of MS, serum and peripheral blood mononuclear cells (PBMCs) of MS patients, close relatives and unrelated controls were screened for the presence of MS-associated retrovirus (MSRV) and two herpes virus (HHV-6 and EBV) sequences. Detection of the pol gene expression of MSRV in the serum RNA of 69% of South African MS patients and in 70% of their unaffected close relatives, whilst absent in the serum of 39 unrelated healthy control individuals (P<0.001), indicated that virus infections affect the population risk but not the familial risk in MS. HHV-6 sequences were also present at a significantly lower frequency (P<0.04) in the PBMCs of unrelated controls (5%) compared to MS patients (22.5%). A point mutation (77C^G) in the gene encoding protein-tyrosine phosphatase, receptortype C (PTPRC), which is essential for activation of T and B cells, was found to be associated with MS in the German population. Analysis of the Afrikaner and German study populations included in our study did not indicate a causative role for the PTPRC gene in MS. However, it seems likely that this mutation may contribute to disease expression, since in one of the South African families with two MS affected sibs, the most severely affected sister was heterozygous for the 77C-»G mutation. The PTPRC mutation may therefore be of significance in disease prognosis. The multidisciplinary study approach has led to a stepwise accumulation of scientific information, which forever changed our understanding of the disease process underlying MS. / AFRIKAANSE OPSOMMING: Veelvoudige sklerose (VS) is ‘n kroniese inflammatoriese siekte van die sentrale senuweestelsel. Oor die algemeen word aanvaar dat VS geassosieerd is met outoimmuniteit en dat infektiewe agente en oorerflike faktore ’n rol speel. Die hoër herhalingsrisiko van VS in families (4-5%) in vergelyking met die voorkoms in die algemene populasie (0.1%) dui op 'n genetiese basis. Alhoewel volledige analise van die mensgenoom om gene onderliggend aan VS te identifiseer aangedui het dat verskeie gene waarskynlik bydra tot vatbaarheid vir die siekte, is die aard van die gene wat betrokke is grootliks onbekend. In hierdie studie is kandidaatgene betrokke by ystermetabolisme en immunulogie vir die eerste keer geanaliseer binne die konteks van beide outoimmuun en infektiewe siekte vatbaarheid, ten einde die rol van genetiese en virale faktore in die patogenese van VS te ondersoek. Die Z-DNS herhalingsvolgorde polimorfisme in die promotor area van die SLC11A1 geen was betekenisvol geassosieerd met VS (P<0.01) in die geneties homogene Afrikaner populasie van Suid-Afrika. ’n Soortgelyke assosiasie kon egter nie aangetoon word in die Duitse en Franse populasies deur gebruik te maak van onderskeidelik ‘n gevalle-kontrole studie en transmissie-koppelings-disekwilibrium benadering nie. Betekenisvolle verskille in die genotipe verspreiding is egter tussen Duitse VS pasiente met ‘n sekonder- en primer progressiewe verloop van die siekte (P<0.05), en tussen die Duitse pasiente met terugvallende en primere progressiewe VS aangetoon (P<0.05). Hierdie bevinding verskaf verdere bewyse dat die SLC11A1 geen geassosieerd is met VS, heel waarskynlik weens die rol van die geen in yster-homeostase. Ten einde die invloed van virusse in die etiologie van VS te ondersoek is serum en witbloedselle van VS pasiente, naby-verwante familielede en nie-verwante kontroles getoets vir die teenwoordigheid van die VS-geassosieerde retrovirus (MSRV) en twee herpesvirus (HHV-6 en EBV) geenvolgordes. Die pol geen uitdrukking van MSRV was teenwoordig in die serum RNA van 69% van die Suid-Afrikaanse VS pasiente en in 70% van hul ongeaffekteerde naby-verwante familielede, terwyl dit afwesig was in 39 nieverwante kontrole individue (P<0.001). Dit dui daarop dat virusse waarskynlik die risiko vir VS meer in die populasie verhoog as in families. HHV-6 was ook teenwoordig teen ‘n beduidende laer frekwensie (P<0.04) in nie-verwante kontroles (5%) in vergeleke met VS pasiente. ‘n Puntmutasie (77C-G) in die geen wat kodeer vir die proteien tirosien fosfatase reseptor tipe C (PTPRC), wat belangrik is vir aktivering van T- en B-helperselle, is vroeer gevind om geassosieerd te wees met VS in die Duitse populasie. Analise van die Afrikaner en Duitse populasies in ons studie het egter geen bewyse gelewer dat die PTPRC geen ‘n rol speel in VS nie. Dit egter is moontlik dat hierdie mutasie bydra tot die uitdrukking van VS, aangesien die mees geaffekteerde VS pasient in een van die Suid- Afrikaanse families met twee geaffekteerde susters positief getoets het vir die mutasie. Die mutasie mag dus van belang wees in die prognose van VS. Die multidissiplinere studie-benadering en stapsgewyse insameling van wetenskaplike inligting het gelei tot ’n nuwe perspektief ten opsigte van die siekteproses onderliggend aan VS.

Thesis (MSc (Genetics))--University of Stellenbosch, 2007. / Multiple sclerosis (MS) is a disease that causes neurological dysfunction. Studies attempting to elucidate the role of genes in MS development may aid efforts to control the damage caused by the disease that affects two million people worldwide, e.g. improved diagnosis and treatment. Although the association of MS and genes has not been fully characterized the proposed genetic etiology has been supported by the observed association of MS with the Major Histocompatibility Complex (MHC), haplotype HLA-DRB1*1501, DRB5*0101, DQA1*0102, DQB1*0602. Iron, or rather the dysregulation thereof, has also been implicated as a precipitating factor in MS development. Considering the factors of iron dysregulation and the genes involved in iron regulation, this study aims to identify variation within genes involved in iron metabolism namely the high iron gene (HFE), solute-carrier family 40 (iron regulated transporter) member 1 gene (SLC40A1), hepcidin anti-microbial peptide (HAMP), cytochrome b reductase 1 (CYBRD1) and hemojuvelin (HJV). Screening of 40 patients (33 female, seven male; 33 Caucasian, seven Coloured) for each of the five genes was achieved by the Heteroduplex Single-Stranded Conformation Polymorphism (HEX-SSCP) technique. Semi-automated DNA sequencing allowed for verification and characterization of the variants detected. Results included identification of four novel variants present in only the Caucasian patient group, characterized as IVS4-53G→A (HFE) (one of 33 patients; 3%), IVS2-65delA (CYBRD1) (two of 32 patients; 6.3%), 3’UTR+26delACGTCACGTTTCAAAACTA (CYBRD1) (one of 31 patients; 3.2%) and 219delG (HJV) (two of 33 patients; 6%). In addition, a total of 15 previously described variants were identified (seven intronic and eight exonic) of which three were also prevalent in only the Caucasian patient group. This study aimed to investigate the differences ...

Thesis (MScMedSc (Pathology. Chemical Pathology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown, due to its multifactorial nature with environmental and genetic factors contributing to the risk. Several investigations highlighted the important role of the genetic component influencing disease susceptibility and progression. In the present study genetic variations in the MTHFR (1298 A>C and 677 C>T) and HFE (845 G>A) genes previously, shown to affect folate and iron metabolism respectively, were studied in the context of MS. The aim of the study was to contribute the laboratory component of a pathology supported genetic testing approach used to identify a subgroup of MS patients with altered nutritional requirements due to genetic susceptibilities. The study population included 90 patients with a clinical diagnosis of MS and 49 control individuals, without any signs or symptoms of the disease, drawn from the same age- and population group. Three mutation detection systems were compared in terms of accuracy, sensitivity, cost effectiveness and ease of operation in relation to the MTHFR and HFE gene mutations analysed. Analytical validity of the genetic assays was an important consideration; therefore the respective real-time polymerase chain reaction (RT-PCR) methods were compared with direct DNA sequencing as the gold standard. The methodology included use of the ABI™ 7900HT, the Roche LightCycler® 480 II system and the Corbett Rotor-Gene™ 6000 5-plex HRM. The same genotype results were obtained for the DNA samples tested with the three RT-PCR methods. In terms of cost effectiveness, ease of operation and optimization, the Corbett Rotor-Gene™ 6000 5-plex HRM thermal cycler, with use of the ABI™ TaqMan Genotyping assays was found to be the most efficient for mutation detection using relatively small sample batches. Following successful standardization of the RT-PCR assays, genotype-phenotype correlation studies was performed in a subset of 43 MS patients with available data. Biochemical tests were previously done on blood samples at the National Health Laboratory Service (NHLS) chemical pathology laboratory at Tygerberg Academic Hospital. A novel finding of this study was that heterozygotes and homozygotes for mutation 1298 A>C in the MTHFR gene presented with lower serum iron levels (12.37 ± 5.91 μmol/l) in comparison to subjects without the C-allele (18.64 ± 7.15 μmol/l; P = 0.02). Furthermore, C-reactive protein (CRP) levels were found to be marginally significantly higher (P = 0.07) in the MTHFR 1298 A>C mutation-positive heterozygotes compared to subjects without the C-allele (6.65 ± 4.96 mg/l vs 2.93 ± 2.31 mg/l), linking inflammation to the presence of the MTHFR 1298 A>C mutation. In comparison, the MTHFR 677 C>T as well as the HFE 845 G>A mutation showed no correlation with transferrin saturation, ferritin, haemoglobin or CRP levels. The absence of increased iron status in HFE mutation carriers was in accordance previous findings suggesting altered iron metabolism in MS patients with this mutation. For the first time, high-throughput assays for functional polymorphisms in the MTHFR and HFE genes can now be offered as a routine service at the Tygerberg Academic Hospital. This application is used in combination with blood biochemistry tests as part of a comprehensive gene-based, pathology supported screening and intervention program aimed at improved quality of life in patients diagnosed with MS. / AFRIKAANSE OPSOMMING: Die etiologie van meervoudige sklerose (MS) is nog grootendeels onbekend, as gevolg van die multifaktoriale aard van die siekte, met omgewings- en genetiese faktore wat bydra tot die risiko. 'n Aantal ondersoeke het reeds die belangrikheid van die genetiese komponent vir die vatbaarheid vir die siekte en die progressie daarvan beklemtoon. In die huidige studie was genetiese variasies in die MTHFR (1298 A>C en 677 C>T) en HFE (845 G>A) gene bestudeer wat voorheen getoon het dat dit foliensuur- enystermetabolisme respektiewelik in die konteks van MS affekteer. Die doel van die studie was om die laboratorium komponent van 'n patologie-ondersteunde genetiese toets daar te stel wat gebruik kan word om 'n subgroep van MS pasiënte te identifiseer wat veranderderde voedingsbehoeftes het as gevolg van genetiese vatbaarheid. Die studiepopulasie het bestaan uit 90 pasiënte met 'n kliniese diagnose van MS en 49 kontroles sonder enige tekens of simptome van die siekte, wat ingesluit is vanuit dieselfde ouderdoms- en populasiegroep . Drie mutasie analise sisteme was vergelyk in terme van akkuraatheid, sensitwiteit, kostedoeltreffendheid en gemak van gebruik met betrekking tot die MTHFR en HFE geen mutasies. Analitiese geldigheid van die genetiese toetse was 'n belangrike oorweging; daarom was die onderskeie rieëltyd polimerase kettingreaksie (RT-PKR) metodes vergelyk met direkte DNA volgordebepaling as die goue standaard. Die metodologie het die ABI™ 7900HT, die Roche LightCycler® 480 II sisteem en die Corbett Rotor-Gene™ 6000 5-plex HRM ingesluit. Dieselfde genotipe resultate was met die verskillende metodes verkry vir die DNA monsters wat getoets is met die drie RT-PKR metodes. Wat betref kostedoeltreffendheid, gemak van gebruik en optimisering, was die gebruik van die Corbett Rotor-Gene™ 6000 5-plex HRM Thermal Cycler, met die ABI™ TaqMan Genotyping essays die mees effektief vir mutasie opsporing van relatief klein getalle monsters. Nadat die RT-PKR toetse suksesvol gestandardiseer was, was genotipe-fenotipe korrelasies uitgevoer in 'n subgroep van 43 MS pasiënte met die beskikbare data. Biochemiese toetse was voorheen gedoen op die betrokke bloedmonsters by die Nationale Gesondheid Laboratorium Diens (NHLS) se chemiese patologie laboratorium by Tygerberg Akademiese Hospitaal. 'n Nuwe bevinding van hierdie studie was dat heterosigote en homosigote vir die MTHFR 1298 A>C mutasie gepresenteer het met laer serum yster vlakke (12.37 ± 5.91 μmol/l) in vergelyking met individue sonder die C-alleel (18.64 ± 7.15 μmol/l; P = 0.02). Verder was die C-reaktiewe proteien (CRP) marginaal betekenisvol hoër (P = 0.07) in die MTHFR 1298 A>C heterosigote in vergelyking met individue sonder die C alleel (6.65 ± 4.96 mg/l vs 2.93 ± 2.31 mg/l), wat aandui dat inflammasie verhoog mag wees in die teenwoordigheid van die MTHFR 1298 A>C mutasie. In vergelyking hiermee het die MTHFR 677 C>T sowel as die HFE 845 G>A mutasies geen korrelasie met transferrien versadiging, ferritien, hemoglobien of CRP-vlakke getoon nie. Die afwesigheid van verhoogde yster status in MS pasiënte met die HFE mutasie was in ooreenstemming met vorige bevindinge wat veranderde ystermetabolisme in MS pasiënte met hierdie mutasie aangedui het. Vir die eerste keer is hoë deurvoer genetiese toetse nou vir funksionele polimorfismes in die MTHFR en HFE gene beskikbaar as 'n roetiene diens by die Tygerberg Akademiese Hospitaal. Dit kan gebruik word saam met bloed biochemiese toetse as deel van 'n omvattende geen-gebaseerde, patologie ondersteunde intervensie program wat daarop gemik is om die kwaliteit van lewe van pasiënte gediagnoseer met MS te verbeter. / Medical Research Council

Thesis (MMed)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: There is growing interest in how genetic and environmental risk factors interact to confer risk for dysregulated iron homeostasis, which is considered a possible pathogenic mechanism in multiple sclerosis (MS). While iron deficiency has been associated with greater disability and disease progression, cerebral accumulation and overload of insoluble iron has also been reported in MS patients. Variation in the matriptase-2 (TMPRSS6) gene has recently been described that may lead to reduced iron levels, which raised the question of whether it may be involved in dysfunctional iron regulation as a pathogenic mechanism in MS. The aims of the study were as follows: 1)) comparison of the allele frequencies and genotype distribution for TMPRSS6 A736V (rs855791, c.2207C>T) and HFE C282Y (rs1800562, c.845G>A) between patients diagnosed with MS and unaffected controls; 2) determination of the effects of clinical characteristics, relevant lifestyle factors and genotype on serum iron parameters in MS patients compared to population matched controls; and 3) determination of clinical outcome in relation to age of onset and degree of disability in MS patients. The study population included 121 Caucasian MS patients and 286 population-matched controls. Serum iron, transferrin, ferritin and transferrin saturation levels were available from previous studies and lifestyle factors were subsequently documented in a subgroup of 68 MS patients and 143 controls using the study questionnaire. Genotyping of TMPRSS6 A736V and HFE C282Y were performed using allele-specific TaqMan technology. The genotype distribution and allele frequencies of TMPRSS6 A736V and HFE C282Y did not differ between MS patients and controls. MS patients homozygous for the iron-lowering minor T-allele of TMPRSS6 A736V had significantly lower serum iron levels (p=0.03) and transferrin saturation levels (p=0.03) compared to CC homozygotes. In MS patients the iron-loading minor A-allele of HFE C282Y was also associated with a paradoxical decrease in serum ferritin (p<0.01) compared to GG homozygotes. When considering the combined effect of the minor alleles of TMPRSS6 A736V and HFE C282Y with opposite effects on iron levels, we found a significant reduction in serum ferritin levels (p<0.05), independent of age, sex, body mass index (BMI) or dietary red meat intake in MS patients. A similar effect was not observed in the population- and age-matched controls. Higher dietary red meat intake correlated significantly with increased ferritin only in controls (p=0.01 vs. 0.21 for MS patients). In the presence of the minor allele of HFE C282Y, the TMPRSS6 A736V CT and TT genotypes were associated with a significantly earlier age of onset of MS when the post hoc test was applied (p=0.04). All the study aims were successfully accomplished. Our results support the possibility of an epistatic effect between TMPRSS6 A736V and HFE C282Y associated with reduced ferritin levels in MS patients. Pathology-supported genetic testing (PSGT) applied in this study as a new concept for analysis of complex diseases with a genetic component, is well placed to optimise clinical management in patients with MS. / AFRIKAANSE OPSOMMING: Daar heers toenemende belangstelling in hoe die wisselwerking tussen genetiese en omgewingsfaktore die risiko tot wanregulering van yster-homeostase beïnvloed. Laasgenoemde is ‘n moontlike patogeniese meganisme vir meervoudige sklerose (MS). Alhoewel verhoogde gestremdheid en siekteprogressie met ystertekort geassosieer is, is ysterophoping in die serebrum asook ‘n oormaat onoplosbare yster al by MS-pasiënte gevind. Variasie in die matriptase-2 (TMPRSS6) geen wat tot verlaging in ystervlakke kan lei, is onlangs beskryf en laat die vraag ontstaan of dit betrokke is by wanregulering van yster-homeostase as patogeniese meganisme in MS. Die doelwitte van die studie was as volg: 1) vergelyking van alleelfrekwensies en genotipeverspreiding vir TMPRSS6 A736V (rs855791, c.2207C>T) en HFE C282Y (rs1800562, c.845G>A) tussen MS-pasiënte en ongeaffekteerde kontroles; 3) bepaling van die effekte van kliniese indikators, relevante leefstylfaktore en genotipe op serum yster parameters in MS-pasiënte in vergelyking met populasie-ooreenstemmende kontroles; en 4) bepaling van kliniese uitkoms ten opsigte van aanvangsouderdom en graad van MS-aantasting. Die studiepopulasie het uit 121 kaukasiese MS-pasiënte en 286 kontroles van dieselfde populasie, wat nie die siekte het nie, bestaan. Serum yster, transferrin, ferritien en transferrien-versadigingsvlakke was beskikbaar vanaf vorige studies. Leefstylfaktore is in ‘n subgroep van 68 MS-pasiënte en 143 kontroles gedokumenteer met behulp van die studie-vraelys. TMPRSS6 A736V en HFE C282Y genotipering is met alleel-spesifieke TaqMan-tegnologie uitgevoer. Beide pasiënte en kontroles het dieselfde genotipeverspreiding en alleelfrekwensies getoon. Die A-alleel van HFE C282Y is met ‘n paradoksale verlaging in serum ferritien geassosieer (p<0.01) in MS-pasiënte met TMPRSS6 A736V, moontlik weens geen-geen interaksie wat nie deur ouderdom, liggaamsmassa-indeks of inname van rooivleis in die dieet beïnvloed is nie (p<0.05) en nie by kontroles gevind is nie. MS-pasiënte wat homosigoties is vir die T-alleel van TMPRSS6 A736V, het statisties betekenisvolle laer serum ystervlakke (p=0.03) en transferrienversadiging (p=0.03) getoon in vergelyking met CC-homosigote. In MS-pasiënte was die yster-oorlading A-alleel van HFE C282Y ook geassosieer met ‘n paradoksale afname in serum ferritien (p<0.01) in vergelyking met GG-homosigote. Wanneer die gekombineerde effek van die risiko-geassosieerde allele van TMPRSS6 A736V en HFE C282Y met teenoorgestelde effekte op ystervlakke geanaliseer word, is daar ‘n statisties beteknisvolle afname in serum ferritienvlakke (p<0.05), onafhanklik van ouderdom, geslag, liggaamsmassa-indeks of rooivleisinname in MS-pasiënte. ‘n Soortgelyke effek is nie waargeneem in populasie- en geslag-gelyke kontroles nie. Die inname van rooivleis in die dieet was betekenisvol minder by MS-pasiënte teenoor kontroles (p=0.03) en dit het slegs betekenisvol met verhoogde ferritien by kontroles gekorreleer (p=0.01 teenoor 0.21 by MS-pasiënte). In die teenwoordigheid van die risiko-geassosieerde alleel van HFE C282Y, is die TMPRSS6 A736V CT en TT genotipes geassosieer met ‘n statisties-betekenisvolle vroeër aanvangsouderdom van MS soos bepaal met die post hoc-toets (p=0.04). Al die doelwitte van die studie is suksesvol uitgevoer. Die resultate ondersteun die moontlikheid van ‘n epistatiese effek tussen TMPRSS6 A736V en HFE C282Y wat geassosieer is met ‘n verlaging in ferritienvlakke in MS-pasiënte. Patologie-gesteunde genetiese toetsing soos toegepas in hierdie studie as ‘n nuwe konsep vir analise van komplekse siektes met ‘n genetiese komponent, is goed geplaas om kliniese hantering van MS-pasiënte te optimaliseer.