The notion that insulin binds to a specific site on the cell membrane was first proposed many years ago. However, experimental proof of a membrane bound insulin receptor did not come until the early 1970s when biologically active radiolabelled insulin was used in direct binding studies (Cuatrecasas, 1971). Recent advances in understanding the mechanism of insulin action are the result of studies on the structure and function of the insulin receptor. The membrane receptor would appear to have two functions: firstly, it must bind insulin and secondly, it must couple insulin binding to insulin action. Defects in either of these receptor functions will result in an impaired response to insulin, or insulin resistance (Taylor, 1985). Insulin resistance is a common disorder in a number of disease states in man. For example, non-insulin-dependent diabetes mellitus and obesity are associated with mild insulin resistance (Bar et aZ., 1976). There are also a number of relatively rare syndromes of extreme insulin resistance in which there is either impaired receptor function, or an immunological defect resulting in the development of auto-antibodies against the insulin receptor (Taylor et aZ., 1985).Studies on insulin receptor defects associated with these disease states have led to progress in understanding the molecular mechanisms of insulin action. Ideally when investigating these disease states one should study insulin action on classical target cells such as adipocytes, hepatocytes or muscle. However, it is now well established that the kinetics of insulin binding to its membrane receptor is similar in all human tissue whether or not it is a target for insulin action. This has led to a great deal of research on the more accessible human tissues such as monocytes, erythrocytes, cultured fibroblasts and Epstein-Barr virus (EBV) transformed B-Iymphocytes. The most convenient tissue to study is EBV transformed B-Iymphocytes, as these cells can be taken from individual patients and grown in culture in large quantities, which facilitates biochemical studies. Despite these advantages, it is important to establish that this virus-induced receptor is a true insulin receptor and not an artifact of viral transformation. Studies on B-Iymphocyte proliferation have shown that the insulin receptor appears on the cell membrane during the proliferative phase of B-cell activation. However , this is a transient event and once the cell reaches maturation the insulin receptor is no longer evident (Marchalonis & Galbraith, 1987). The insulin receptor has also been demonstrated in a number of cultured human lymphoblastoid cell lines (Gavin et aL, 1983; Maegawa et aL, 1983). It seems, therefore, that the insulin receptor is normally expressed by blast cells. The purpose of this study was to investigate insulin binding characteristics on a human lymphoblastoid cell line with B-cell characteristics which was originally derived from a patient with Burkitt's lymphoma. These cells, which are known as Raji cells, are unusual in that they carry multiple copies of the EBV genome in their DNA. For this reason they provide a useful model system for studying the insulin receptor in EBV transformed lymphocytes. In addition, studies on the mechanism of insulin action in these cells should give some insight into the function of the insulin receptor during B-cell proliferation. In this study four major characteristics of insulin binding to insulin receptors on Raji cells are described. Firstly, on the basis of kinetic studies a model for insulin-receptor interaction was established. Secondly, processing of insulin and the receptor was investigated to determine whether the receptor is functional. A third aspect was elucidation of the receptor structure and the insulin binding site. Finally, the cross-reaction between insulin and type I IGF receptors was studied, and the cellular response mediated by the insulin receptor and growth factor receptor was determined. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1988.

No abstract available.

No abstract available. / Thesis (Ph.D)-University of Natal, 1986.

Thesis (MMedSc)-University of KwaZulu-Natal, Durban, 2006.

Almost a century has elapsed since the antigen-antibody interaction was first recognised as the basis of an immune response (Ehrlich, 1897). However, it was only in the 1930s, with the development of improved technologies that this concept was better understood, and led to the discovery of the amazing diversity and specificity of antibody molecules (Landsteiner, 1933). Theoretically, it is possible to make antibodies to a variety of biological substances and other chemicals, and therefore they are ideally suited as specific recognition elements to be used for analytical, cytological, functional, therapeutic and biochemical purposes. The development of the radioimmunoassay (RIA) thirty five years ago, revolutionised research in many areas of clinical and scientific investigation. This technique evolved rapidly from the discovery made by Berson et al. in 1956 that antibodies to insulin could be detected in patients treated with this hormone, by measuring the binding of radiolabeled insulin to these antibodies. Although in the past RIAs have been the most important assay system employing antibody and labelled tracer, the limitation was that reliance had to be placed on the chance development of a good polyclonal antibody. These shortcomings stimulated the search for monospecific antibodies of reproducible quality and sufficient quantity. The development and introduction of monoclonal antibody technology brought about a revolution in immune serology (Kohler and Milstein, 1975). Establishment of immortal cell lines which contained the genetic elements of antibody-producing cells was achieved by fusion between a myeloma cell line and spleen cells from an immunised donor. The resulting hybrids had the essential properties of both parents, namely, permanent growth and a high capacity for the synthesis and secretion of immunoglobulins, normally characteristics of plasmacytomas, together with the genetic elements defining a specific antibody. Gestational trophoblastic disease (GTD) is a neoplastic condition of the trophoblast and occurs as molar pregnancy in a benign or invasive form, or as choriocarcinoma in a malignant form. Effective therapy has been developed for the treatment of both choriocarcinoma and molar pregnancy, but the key to successful management of these patients lies in their prompt diagnosis and careful monitoring of response to treatment (Green-Thompson, 1986). Fortuitously, these tumours elaborate the human chorionic gonadotrophin hormone (hCG) and its free alpha (a) and beta (B) subunits and hence a ready marker for the tumour exists. Human chorionic gonadotrophin is one of a group of glycoprotein hormones, which includes luteinising hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH). These hormones are composed of two dissimilar subunits designated a and B, which are bound non-covalently in the intact molecule. The B-subunit of each glycoprotein hormone is unique and is responsible for the respective biological and immunological properties of the glycoproteins. In contrast, all four hormones possess an identical a-subunit which is coded for by a single gene (Fiddes and Goodman, 1979). The measurement of hCG and its free B-subunit, as so-called BhCG, for the diagnosis and monitoring of therapy in patients with GTD is now routinely practised throughout the world (Vaitukaitis et al., 1972). However it has been demonstrated by Bagshawe (1975) that when serum BhCG can no longer be measured by current RIA methods, up to 10" tumour cells may remain undetected. In addition, there have been isolated reports of two patients with choriocarcinoma in whom BhCG was undetectable in the serum but who appeared to be secreting only the a-subunit (Dawood et al, 1977). Furthermore, it has been suggested that measurement of free a-subunit rather than intact hCG or the free B-subunit is a more effective means of detecting persistent trophoblastic disease as well as tumour recurrence following treatment (Quigley et al, 1980a and b). Radioimmunoassays which measure the free a-subunit of hCG have been developed, but in general lack the specificity and sensitivity required (Gaspard et al, 1980; Kohorn et al, 1981). These assays employ polyclonal antisera which also detect epitopes common to the pituitary gonadotrophins. Thus there is a need to produce monoclonal antibodies which recognise regions of the free a-subunit which are hidden in the intact gonadotrophins. Such antibodies would provide the required specificity for use in RIAs but are limited in their use by their inherent lack of high affinity for the antigen. Fortunately, this drawback may be overcome by using monoclonal antibodies as labelled reagents in an alternative assay system, the immunoradiometric assay (IRMA), described by Miles and Hales (1968). The IRMA, particularly the two-site sandwich version of the assay, has been shown to provide greater sensitivity in addition to allowing enhanced specificity. This is a consequence of the use of two antibodies in excess to detect the analyte, each directed at a different epitope on the target molecule. The first antibody, referred to as the capture antibody, is usually linked to a solid-phase to facilitate easy separation and is added in excess relative to the target hormone to enhance antibody-antigen interaction, thereby allowing increased sensitivity in the measurement of analyte. The second antibody, referred to as the detection antibody, is labelled with a radioactive isotope or an enzyme to detect antigen already bound to the capture antibody. The application of monoclonal antibodies specific for the free a-subunit to a highly sensitive IRMA format is an obvious need. Hence this study was undertaken firstly, to raise and characterise monoclonal antibodies to the free a-subunit, secondly to develop an IRMA using these antibodies and finally to establish whether measurement of free a-subunit has any clinical advantage. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1990.

Genetic polymorphism of apolipoprotein (apo) E has been shown to account for a significant amount of variance in plasma lipid and lipoprotein levels, thereby contributing to the incidence of cardiovascular disease across populations. In this cross-sectional study apo E genotypes were determined in a sample of 173 healthy, middle-aged Indian women using a restriction isotyping method, in which DNA was amplified by PCR and the Cfol restricted DNA fragments were separated on a polyacrylamide gel, allowing unambiguous typing of the common apo E genotypes. Considering the three common alleles, e2, e3 and e4, a reduced frequency of the e2 allele was observed in the study population in comparison to other populations around the world. This finding underlines the heterogeneity of apo E allele frequencies in different populations. This study also investigated possible effects of apo E genotype on lipoprotein changes in this sample of women spanning the menopause. Apo E polymorphism was associated with significant differences in plasma lipid levels. Notably, total and low density lipoprotein cholesterol and more especially plasma triglyceride concentrations were increased in carriers of the e3/4 genotype. Two-way analysis of variance of the effect of apo E genotype and menopausal status on the lipid profile showed no significant interaction effect, indicating that the effects of apo E genotype on the lipid profile do not differ significantly between premenopausal and postmenopausal women. Age and to a lesser extent the waist hip ratio also correlated with lipid concentrations, but menopausal status had no apparent effect in this sample. This study confirms the potentially deleterious effect of the e4 allele, in a vulnerable population. The reduced occurrence of the E2 isoform, which is considered to offer a measure of protection against cardiovascular disease, may contribute to the relatively high incidence of coronary heart disease in the South African Indian population. However, the relatively low incidence of the e2 allele may protect this population against the occurrence of type III hyperlipoproteinaemia precipitated by diabetes and obesity in e2/2 homozygotes. / Thesis (M.Med.)-University of Natal, Durban, 1993.

One of the earliest recorded references to polyuria is found in the Papyrus Ebers (1500 BC) and much later the occurrence of "honey urine" was noted by an ancient Hindu physician, Sushrutha, in old Indian Sanskrit (400 BC). However, the first good clinical description of the disease is ascribed to Celsus, although the name "diabetes" was introduced by Aretaeus of Cappadocia. The body of knowledge which has accumulated since these early recordings to the present state of the art reflects a most impressive sojourn, punctuated by many milestones, each adding impetus to future attempts in a relentless endeavour to unravel the aetiopathogenesis of this common malady. However, this "sweet evil" (diabetes) remains an enigma in many ways. There is little doubt today that there are 2 major types of diabetes: juvenile onset diabetes, presently known as insulin-dependent diabetes mellitus (IDDM) and maturity onset diabetes, referred to as non-insulin dependent diabetes mellitus (NIDDM). In NIDDM aggregation of HLA types, evidence of cell mediated immunity and the presence of circulating islet cell antibodies, which are characteristically associated with IDDM, are not found. There is also a vast difference in concordance of diabetes in the co-twins between the two types of diabetes suggesting that a different mixture of genetic and environmental factors is operative in the pathogenesis of these two types of diabetes. In I960, Fajans and Conn drew attention to the existence of a form of diabetes with an onset before the age of 35 years. Their patients showed a substantial improvement in glucose tolerance when treated with an oral hypoglycaemic agent, tolbutamide. Subsequent to this report numerous studies from various parts of the world confirmed this entity of non-insulin dependent diabetes in the young (NIDDY) in White Caucasians. There are, however, several different syndromes presenting as mild carbohydrate intolerance in the first two to three decades of life. The classical form of NIDDY is a mild non-insulin requiring form of diabetes in which the disorder is inherited as a dominant trait; there is little progression of glucose intolerance, if any, with time, and the diabetes is rarely accompanied by vascular complications. This subtype of diabetes is referred to as MODY (maturity onset diabetes in the young) and thus constitutes a subset under the broad umbrella of NIDDY. However, recently compelling evidence for heterogeneity within MODY has been presented. This evidence is based on the prevalence of certain HLA antigens, insulin responses to oral glucose, occurrence of vascular complications, progression of hyperglycaemia to the stage of insulin requirement and failure to demonstrate autosomal dominant inheritance in some families studied. In the South African Indian population which has a high prevalence of diabetes, Campbell was the first to draw attention to NIDDY in Indians more than two decades ago. Since this initial report, nobody has really studied NIDDY in any depth in South Africa and certainly not in the Indian population. NIDDY in the local Indian population is of particular interest for the obvious reason that diagnostic and management problems arise daily in a population with a high prevalence of non-insulin dependent diabetes. It is vital that the clinical features, endocrine and associated biochemical aberrations be known in detail if this condition is to be managed appropriately and adequately. A study of these aspects therefore became the primary task of this thesis. To pre-empt any challenge that patients were not really diabetic, the strict criteria of the W.H.O. for the diagnosis of diabetes were chosen. It should therefore be borne in mind throughout this study that a group of rather severe diabetics were selected by design. The patients studied represent the rather extreme end of the spectrum. But, in the event, this selection proved advantageous in that it covered an unstudied part of the spectrum and some light could be shed on the natural history of the disorder. In the long term the purpose was to prepare the ground for what must become the thrust of future studies, namely the biochemical pathogenesis of NIDDM. If it is true that some forms of NIDDY are inherited dominantly, existing techniques should make it possible to identify a gene(s) locus and if this is done the biochemical basis of this disorder must be identifiable. In the present study direct examination of these aspects were not undertaken, but an attempt was certainly made to pinpoint those biochemical abnormalities which are perhaps primary or central to the whole disorder. / Thesis (M.D.)- University of Natal, Durban. 1982.

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 (MMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Background: Serum free light chains (FLC) are associated with imbalances in heavy and light chain production. Abnormal FLC ratios have been associated with risk of progression in certain diseases. Automated assays are available for their determination and they are used in the followup and management of patients with monoclonal gammopathies. Acceptable imprecision, specificity, accuracy and reproducibility between reagent batches is required to prevent under- or overestimation. Method validation is a standard process in every good laboratory to judge the acceptability of a new method. Reference intervals have been established in an older population, but it was considered important to verify these in our population. HIV is associated with B-cell dysfunction. As B-cell abnormalities are associated with disorders leading to monoclonal gammopathies, we postulated that the FLC levels and FLC ratio would be abnormal in HIV infected individuals. Methods and materials: Controls and pooled patient samples were used for the method validation study which included imprecision studies, linearity, recovery and interference studies, and method comparison studies, the latter compared our method to the same method used in another laboratory. For the reference interval study, blood was obtained from 120 healthy subjects. The following blood tests were performed: total protein, IgG, IgA, IgM, creatinine, protein electrophoresis, kappa FLC and lambda FLC. Using the kappa and lambda FLC results, a FLC ratio was determined. Three hundred and sixty-nine HIV positive subjects were then studied. The same tests were performed, as well as CD4+ counts and viral loads on the majority of them. Results: For the method validation study, precision, linearity and recovery was acceptable. Minimal interference was observed with haemolysis, lipaemia, bilirubin and rheumatoid factor. Our method showed comparable performance with the established method. For the reference interval study, all the creatinine values were normal, as were serum protein values. The serum protein electrophoreses were independently reviewed by 3 pathologists. Most were normal, with a few polyclonal increases seen, but no definite monoclonal bands. The 95% reference intervals for FLC’s as well as the FLC ratio were not statistically significantly different to the manufacturer’s recommendations. When examining the HIV positive study population, we found that FLC and FLC ratio were influenced by markers of HIV disease severity, such as CD4+ count, IgG, viral load, use of antiretroviral treatment and abnormal serum protein electrophoreses. Conclusion: The validation study of FLC showed excellent precision, acceptable bias, good linearity, good recovery and minimal interference, allowing routine introduction of the test. The 95% reference intervals obtained for our population were slightly higher than those recommended by the manufacturer. However, as most of the values fell within the manufacturer’s limits, we could accept the manufacturer’s recommended cut-offs. We found that FLC levels were definitely influenced by markers of HIV disease severity in our population and we postulate that they may be of use for follow-up of patients with HIV. / AFRIKAANSE OPSOMMING: Agtergrond: Serum vry ligte kettings (VLK) word geassosieer met ‘n wanbalans van ligte en swaar ketting produksie. Abnormale VLK ratios is geassosieer met ‘n risiko van verloop in sekere siektes. Geoutomatiseerde laboratorium toetse vir VLK is beskikbaar vir hul bepaling en word gebruik om pasiënte met monoklonale gammopatieë op te volg en te behandel. Aanvaarbare impresisie, spesifisiteit, akkuraatheid en herhaalbaarheid tussen reagens besendings is belangrik om onder- of oorbepaling te verhoed. Metode validasie is ’n standaard proses in elke goeie laboratorium om die aanvaarbaarheid van ’n nuwe metode te bepaal. Verwysingswaardes is al bepaal in ’n ouer populasie. Ons het besluit om die verwysingswaardes in ons populasie te bepaal. Mens-immuungebrekvirus (MIV) word geassosieer met B-sel disfunksie. Omdat B-sel abnormaliteite geassosieer word met afwykings wat tot monoklonale gammopatieë lei, het ons gepostuleer dat die VLK vlakke en VLK ratio abnormaal sal wees in MIV geïnfekteerde persone. Metodes en Materiale: Kontroles en pasiënt monsters is gebruik vir die metode validasie studie wat impresisie studies, lineariteit, herwinning, inmenging en metode korrelasie studies ingesluit het. In laasgenoemde geval is ons metode met dieselfde metode van ’n ander laboratorium vergelyk. Vir die verwysingswaardes studie is 120 gesonde persone se bloed gebruik. Die volgende toetse is bepaal: totale proteïen, IgG, IgA, IgM, kreatinien, proteïen elektroferese, kappa en lambda VLK. Die VLK ratio is bepaal deur die kappa en lambda resultate te gebruik. Driehonderd nege en sestig MIV-positiewe pasiente is gebruik vir die studie. Dieselfde toetse was gedoen, asook CD4+ tellings en virale ladings op die meerderheid van pasiente. Resultate: Vir die metode validasie studie, was presisie, lineariteit en herwinning aanvaarbaar. Minimale inmenging van hemolise, lipemie, bilirubien en rumatoïede factor is waargeneem. Ons metode het goed gekorreleer met die bepaalde metode. Die serum kreatinien en serum totale proteïen waardes was normaal tydens die verwysingswaardes studie. Die serum proteïen elektroferese was onafhanklik beoordeel deur 3 patoloë. Die meeste was normaal met enkele poliklonale verhogings, maar geen definitiewe monoklonale bande nie. Die 95% verwysings intervalle vir VLK en VLK ratio het nie statisties betekenisvol verskil van die vervaardiger se aanbevelings nie. In die studie van die MIV-positiewe studie populasie, het ons gevind dat VLK en VLK ratio beïnvloed word deur merkers van ernstige MIV siekte, soos CD4+ telling, IgG, virale lading, die gebruik van antiretrovale medikasie en abnormale serum proteïen elektroferese. Gevolgtrekking: Die validasie studie van VLK het uitstekende presisie, aanvaarbare partydigheid, goeie lineariteit, goeie herwinning en minimale inmenging gewys, wat die roetine instelling van die toets toegelaat het. Die 95% verwysingsintervalle wat vir ons populasie bepaal is, was effens hoër as die vervaardiger se aanbeveling. Die meeste van die waardes het egter binne die vervaardiger se limiete geval, dus kon ons die vervaardiger se afsnypunte aanvaar. Ons het gevind dat VLK vlakke definitief beïnvloed word deur merkers van die ernstigheidsgraad van MIV siekte in ons populasie en ons postuleer dat VLK van waarde kan wees met die opvolg van MIV pasiente. / NHLS / Harry Crossley for funding obtained

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.