REPLICATION OF DNA POLYMORPHISMS ASSOCIATED WITH MULTIPLE SCLEROSIS IN GENOME WIDE ASSOCIATION STUDIES IN SLOVENIAN PATIENTS

Multipla skleroza (MS) je kompleksna avtoimunska vnetna bolezen centralnega živčnega sistema. V procesu razvoja bolezni propadajo ovojnice živčnih vlaken zaradi izgube mielina oz. demielinizacije. Tipični simptomi bolezni so optični nevtritis ali vnetje vidnega živca, težave s koordinacijo gibov in ravnotežjem, občutek težkih udov. Najpogosteje zbolijo osebe med 20. in 40. letom starosti, ženske so bolj dovzetne za obolenje. Vzrok za nastanek MS je neznan, najverjetneje pa bolezen nastane zaradi kombinacje okoljskih dejavnikov, okužb in genetskih dejavnikov. Velik napredek pri genetiki MS so prispevale asociacijske študije celotnega genoma (GWAs, ang. za genome wide association studies), s pomočjo katerih identificiramo polimorfizme posameznega nukleotida (SNP-je, ang. za single nucleotide polymorphism), ki povečajo tveganje za nastanek bolezni. Do danes so z MS povezali več kot 100 lokusov na genomu. Pri pregledu do sedaj objavljenih GWA študij za MS pri drugih populacijah smo ugotovili, da so 3 SNP-ji, rs2104286 v genu IL2RA, rs12927355 v genu CLEC16A in rs6677309 v genu CD58, pokazali najmočnejšo statistično povezavo z MS. V okviru diplomske naloge smo izbrane signale replicirali na skupini 180 slovenskih bolnikov z MS in 210 zdravih posameznikov. Izbrane SNP-je smo genotipizirali z metodo verižne reakcije s polimerazo (PCR), ki ji je nato sledila metoda polimorfizmov dolžin restrikcijskih encimov (RFLP) ali metoda talilne krivulje visoke ločljivosti (HRM). Za SNP rs2104286 na genu IL2RA smo ugotovili močno statistično značilno povezavo z MS. Frekvenca alela G je povišana v skupini bolnikov z MS (0,417) v primerjavi z zdravimi posamezniki (0,227, p = 3,23x10-8). Nadalje smo za SNP rs6677309 na genu CD58 ugotovili višjo frekvenco alela A v skupini bolnikov z MS (0,901) v primerjavi z zdravimi posamezniki (0,871) ter za SNP rs12927355 na genu CLE16A ugotovili povišano frekvenca alela G v skupini bolnikov z MS (0,684) v primerjavi s kontrolno skupino (0,639), vendar statistično značilne povezave nismo dosegli, najverjetneje zaradi premajhnega števila vzorcev. Rezultati nakazujejo na pomembno vlogo izbranih polimorfizmov pri nastanku MS v slovenski populaciji.Multiple sclerosis (MS) is complex autoimmune disease of central nervous system. During disease development sheath of nerve fibers are failing due to the loss of myelin. Typical symptoms include optical nevtritis or optic neuritis, difficulty with coordination and balance and a feeling of heavy limbs. MS most commonly affect people between 20 and 40 years of age, and is more common in women. The main cause of MS is unknown, it is believed that it is caused by a combination of genetic and environmental factors as well as infections. Genome-wide association (GWA) studies have made great progress in genetics of MS. The purpose of GWA studies is to identify single nucleotide polymorphisms (SNPs) which are associated with risk to develop a disease. To date, GWA studies have identified more than 100 loci in the human genome that contribute to the development of MS. Three SNPs, rs2104286 in IL2RA gene, rs12927355 in CLEC16A gene and rs6677309 in CD58 gene showed strong statistically significant association with MS in GWA studies from different populations. The aim of our study was to analyze those SNPs in 180 Slovenian MS patients and 210 Slovenian controls. Selected SNPs were genotyped using polymerase chain reaction (PCR), followed by restriction fragment length polymorphism (RFLP) and high resolution melting analysis (HRM). For SNP rs2104286 in IL2RA gene we found strong statistically significant association with MS. Frequency of allele G was higher in a group of MS patients (0,417) compared to healthy controls (0,227, p = 3,23 x10-8). Further, frequency of allele A of SNP rs6677309 in CD58 gene was higher in MS patients (0,901) compared to controls (0,871) and frequency of allele G of SNP rs12927355 in CLEC16A gene was higher in MS patients (0,684) compared to controls (0,639), however, statistical significance was not reached, probably due to small sample size. Results of our study indicate the important role of selected polymorphisms in the development of MS in the Slovenian population