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

[摘要] 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 comparedto 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.

[发布机构] Stellenbosch University