VITAMIN D RECEPTOR POLYMORPHISMS AMONG THE TURKISH POPULATION ARE ASSOCIATED WITH MULTIPLE SCLEROSIS
Bulan B1, Hoscan AY1, Keskin SN1, Cavus A1, Culcu EA1, Isik N2, List EO2, Arman A*4
*Corresponding Author: Dr. Ahmet Arman, The Department of Medical Genetics, Marmara Teaching and Research Hospital, Marmara University, 34899, Pendik, Istanbul/TURKEY. Tel #: +90-216- 657 0606, Fax #: +90-216-414 47 31, e-mail: ahmetarman@marmara.edu.tr, ORCID Id: https://orcid.org/0000-0001-5547-0024
page: 10

MATERIAL AND METHODS

Patients and controls A total of 474 ethnically matched participants from the Turkish population were enrolled in the study. Of the 474 participants, 271 were diagnosed with MS. Within the MS patients 2 of them had Primary Progressive Multiple Sclerosis (PPMS), 184 of them had Relapsing Remitting Multiple Sclerosis (RRMS) and 85 of them had Secondary Progressive Multiple Sclerosis (SPMS). 203 individuals served as healthy controls. All patients were referred to Goztepe Training and Research Hospital and were clinically diagnosed with MS, according to the McDonalds criteria [27]. A blood sample was collected from each person in order to obtain genomic DNA. The study protocol and consent were approved by Marmara University Medical School Clinical Research Ethic Committee. Written informed consent was obtained from all of the participants and there was no patient or control younger than the age of 16 in the study. Genotyping of polymorphisms Genomic DNA was extracted by using the salting out method, as previously described [28]. Polymorphism regions Fok-I (rs2225870), Bsm-I (rs1544410) and Taq-I (rs731236) were amplified by a polymerase chain reaction (PCR) (Techne Tc312) using specific primers, visible in Table 1. PCR was carried by a total volume of 25 μl reaction containing 0.5 μg of genomic DNA, 2.5 μl 10x buffer, 1.5 mM MgCl2, 0.5 μM forward primer, 0.5 μM reverse primer, 0.2 mM dNTP, and 0.5 U Taq polymerase. The PCR sample were denatured at 94C for 3 min (1x) for initial denaturation and the main PCR cycle for denaturation is at 94C for 30 secs, annealing at 69C for 30 secs, extension at 72C for 45 secs (all cycles 40x), and final extension was done at 72C for 10 min (1x) (annealing 69C for Fok-I, 66C for Bsm-I and 68C for Taq-I). The PCR products were digested by Fok-I, Bsm-I, and Taq-I restriction enzymes (CutSmart, New England Biolabs inc.). 10 μl of PCR product was mixed with 5 U restriction enzyme, 3 μl 10X reaction buffer and incubated overnight at 37C for Fok-I, at 65C for 3 hours for Bsm-I, at 65C for 3 hours for Taq-I. The digested PCR products were run on 1.5% agarose gel electrophoreses and genotyping was determined based on fragment size of digested PCR products. Digestion of Fok-I gives C/C (343 bp for homozygote mutant), T/C (343 bp, 267 bp, 76 bp for heterozygote) and T/T (267 bp, 76 bp for homozygote wild type). The digestion of Bsm-I gives A/A (531 bp for homozygote mutant), G/A (531 bp, 329 bp, 202 bp for heterozygote) and G/G (329 bp, 202 bp for homozygote wild type). The digestion of Taq-I gives T/T (479 bp for homozygote wild type), C/T (479 bp, 294 bp, 185 bp for heterozygote) and C/C (294 bp, 185 bp for homozygote mutant). Statistical analysis Comparison of genotype or allele between MS and control or MS subtypes were determined by using Pearsons chi-square test. The odds ratio and a 95% confidence interval were also used. Values of p<0.05 were considered significant. Data was analyzed with the SPSS 21.0 program. The statistical power of this study was calculated by using the G*Power program version of 3.1.9.6.



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