Development of a high-throughput genotyping assay for detection of functional polymorphisms involved in homocysteine metabolism and the methylation process implicated in multiple sclerosis

[摘要] ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown due to themultifactorial nature of disease susceptibility determined by both environmental andgenetic factors. Progress has been made in identifying the genetic component of MS,as well as the possible interactions with the environment. In this study singlenucleotide polymorphisms (SNPs) in theFTO(rs9939609, Intron 1 T>A),MTR(rs1805087, 2756 A>G),MTRR(rs1801394, 66 A>G),MTHFR(rs1801133, 677 C>Tandrs1801131, 1298 A>C) andCOMT(rs4680, 472 G>A) genes involved in themethylation metabolic pathway were studied in the context of MS.The overall objective of this study was to elucidate the mechanism underlying raisedhomocysteine levels in MS patients. The specific aims were 1) to analytically validatehigh throughput real-time polymerase chain reaction (RT-PCR) genotyping assaysfor the 6 selected SNPs against direct sequencing as the gold standard for 2)possible integration into a pathology-supported genetic testing strategy aimed atimproved clinical management of MS. The study population included a total of 114unrelated Caucasian MS patients (98 females and 16 males) and 195 unrelatedCaucasian control individuals without a diagnosis of neurological disease (128females and 67 males).A novel finding of this study was that the risk-associated FTO rs9939609 A-allele wasassociated with raised homocysteine levels (p=0.003) in patients diagnosed with MS,but not in controls. Furthermore, homocysteine levels correlated significantly withbody mass index (BMI) (p=0.046) and total cholesterol levels (p=0.048). Bothhomocysteine (p=0.011) and BMI (p=0.017) were significantly reduced withincreasing intake of folate in the diet, while high saturated/trans fat intake correlatedsignificantly with increased BMI (p<0.001). High physical activity correlated withreduced BMI (p<0.006) in the study population, adjusted for age, gender and diseasestatus. Daily intake of at least five fruit and vegetable portions and theCOMTrs4680(472 G>A) AA genotype had a favourable lowering effect on MS disability asassessed by the expanded disability status scale (EDSS) (p=0.035), while smokingincreased MS disability significantly (p<0.001). All SNPs studied were found to be inHardy-Weinberg equilibrium (HWE), with no significant differences detected betweenpatients and control individuals in genotype distribution or allele frequencies. This study has shown for the first time that the underlying disease process of MSmoderates the effect of the FTO rs9939609 polymorphism on homocysteine levels,which is consistent with the role of FTO in demethylation and epigenetic changes.Identification of FTO rs9939609 reinforces the importance of adequate folate intakein the diet that can be assessed accurately with use of the Medical History andLifestyle Questionnaire applied in this study.Finally, the finding that raised homocysteine levels and BMI are significantlyinfluenced by lifestyle factors such as diet and physical activity in our study cohort,offers a solution to counteract the detrimental effects of genetic risk factorscontributing to the development of these established vascular risk factors for MS.Combining this information withFTOrs9939609 andCOMTrs4680 genotyping mayin future translate into a comprehensive pathology supported genetic testing strategyaimed at improved risk management and quality of life in MS patients.

[发布机构] Stellenbosch University