Development of a pathology-supported genetic test for improved clinical management of patients diagnosed with multiple sclerosis

Jalali Sefid Dashti, Mahjoubeh (Stellenbosch : University of Stellenbosch, 2010-12)

Thesis (MScMedSc (Pathology. Chemical Pathology))--University of Stellenbosch, 2010.

Bibliography

Thesis

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.

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