Evaluation of the association between rs2296533 and rs7984870 gene polymorphisms, and RANKL gene expression in bone tissue

Podatki kažejo, da sta najpogostejša presnovna bolezen kosti, osteoporoza, in najpogostejša degenerativna bolezen sklepov, osteoartroza, verjetno povezani z motnjami v regulatornem sistemu OPG/RANK/RANKL. RANKL, osrednja tema te magistrske naloge, je protein, odgovoren za aktivnost, diferenciacijo in preživetje osteoklastov in s tem posledično za proces kostne premene. Pri omenjenih boleznih namreč povečano izražanje gena RANKL pospeši osteoklastogenenzo in razgradnjo kostnine. V genu za RANKL so odkrili številne genetske variacije, ki vplivajo na sintezo in/ali aktivnost tega proteina. Namen te naloge je bil ugotoviti, ali polimorfizma rs2296533 (SNP 1) in rs7984870 (SNP 2) vplivata na izražanje gena RANKL v humanem kostnem tkivu bolnikov z osteoporozo in osteoartrozo. V raziskavo smo vključili 166 slovenskih preiskovancev, ki smo jih razdelili v 3 skupine: preiskovanci z osteoporozo (OP) (n = 75), preiskovanci z osteoartrozo (OA) (n = 54) in zdravi preiskovanci (n = 24). DNA smo izolirali iz vzorcev krvi ter izvedli analizo dveh enonukleotidnih polimorfizmov (SNP), in sicer SNP 1 in SNP 2. Za analizo obeh polimorfozmov smo uporabili genotipizacijo s PCR-TaqMan sondami. Določili smo alelne in genotipske frekvence SNP 1 in SNP 2 slovenskih preiskovancev in ovrednotili njun klinični pomen. Ugotovili smo, da so bile genotipske frekvence pri obeh polimorfizmih primerljive (TT : TC : CC = 22,3 : 57,2 : 20,5 za SNP 1 in GG : GC : CC = 22,3 : 57,8 : 19,9 za SNP 2) ter da so se pari genotipov pojavljali sočasno, in sicer v 99,4 %. Redkejši alel je bil alel C (za oba SNP) in se je pojavljal z alelno frekvenco 49,1 % v primeru SNP 1 in 48,8 % v primeru SNP 2. Frekvenci genotipov nakazujeta vezano dedovanje obeh SNP skupaj, zato nas je zanimalo, ali tudi enako vplivata na izražanje gena za RANKL. Statistična analiza Kruskal-Wallisovega testa je pokazala, da se izražanje gena RANKL statistično značilno ne razlikuje v odvisnosti od genotipa, se pa statistično značilno pričakovano razlikuje v odvisnosti od diagnoze v primeru obeh SNP, in sicer je pri OP in OA pričakovano značilno višje. Zaključimo lahko, da je povečano izražanje gena RANKL pri preiskovancih z osteoporozo signifikantno v primerjavi z zdravimi preiskovanci. Pri preiskovancih z osteoartrozo pa smo ugotovili le statistično značilno večjo frekvenco mutiranega alela v primerjavi z zdravimi preiskovanci. Oba zaključka nakazujeta, da proučevana genetska polimorfizma v genu za RANKL prispevata h genetski nagnjenosti k osteoartrozi in osteoporozi.Osteoporosis, one of the most common metabolic bone disorders, and osteoarthritis, the common degenerative joint disease, are conditions in which, according to available data, the OPG/RANK/RANKL system dysfunction is most likely involved. The focus of this master thesis is RANKL, the protein responsible for activity, differentiation and survival of osteoclasts, and consequently, for the process of bone turnover. In the aforementioned diseases, the increased RANKL gene expression increases osteoclastogenesis and bone deterioration. A number of genetic variations affecting the synthesis and/or activity of this protein have been identified in the RANKL gene. The aim of this thesis was to determine whether gene polymorphisms rs2296533 (SNP 1) and rs7984870 (SNP 2) have any impact on the expression of RANKL in human bone tissue of osteoporotic and osteoarthritic subjects. The study included 166 subjects, divided into three groups: subjects with osteoporosis (OP) (n = 75), subjects with osteoarthritis (OA) (n = 54) and healthy subjects (n = 24). We extracted DNA from blood samples and analyzed two single nucleotide polymorphisms SNP1 and SNP2, using genotyping with hydrolysis probes. Allelic and genotypic frequencies of SNP 1 and SNP 2 were determined for Slovenian subjects and their clinical significance was evaluated. We discovered that the frequency of genotypes for both polymorphisms in the Slovenian population is similar (TT : TC : CC = 22,3 : 57,2 : 20,5 for SNP1 and GG : GC : CC = 22,3 : 57,8 : 19,9 for SNP2) and that the genotype pairs occur together in 99,4 %. The minor allele was allele C for both SNPs and is occurring with an allele frequency of 49,1 % in the case of SNP1 and 48,8 % in the case of SNP2. Frequencies of genotypes indicate linkage equilibrium of both SNPs, so we wondered whether they have the same effect on RANKL gene expression. The statistical analysis with the Kruskal–Wallis test showed that the RANKL gene expression is not statistically significantly different between the genotypes. However, there is a statistically significant difference between the diagnoses, namely the expression is as expected higher in OP and OA. In conclusion, there is a significantly higher expression of RANKL in the subjects with OP when compared with healthy subjects. In the OA group, only the frequency of mutated allele is significantly higher when compared with healthy subjects. Both conclusions indicate that the studied polymorphisms in the RANKL gene contribute to the susceptibility to osteoporosis and osteoarthritis