Role of gene polymorphisms of proinflammantory cytokines IL-1 in primary knee and hip osteoarthritis

Cilj istraživanja. Osteoartritis (OA) je česta bolest zglobova sa višestrukim genetskim rizikom. Genetska predispozicija za nastanak kompleksnih nasljednih bolesti kao što je OA velikih zglobova (kuka i koljena) uključuje interleukin-1 obitelj gena (IL1) na kromosomu 2. Također, jedan od lokusa koji mogu pogodovati razvoju OA nalazi se na kromosomu 6 (6p12.3-q13). U blizini ovog lokusa je gen koji kodira interleukin-17A (IL-17A). Specifični ciljevi ovog istraživanja bili su: 1) utvrditi polimorfizme gena koji kodiraju interleukin-1 (IL-1) i IL-17 u ispitanika s uznapredovalim oblikom primarnog OA (POA) koljena i kuka kojima je ugrađena totalna endoproteza (TEP) kuka, odnosno parcijalna endoproteza (PEP) ili TEP koljena i usporediti s kontrolnom skupinom iz hrvatske populacije dobrovoljnih darivatelja krvi (DDK), 2) istražiti značaj povezanosti rizika za razvoj POA koljena i kuka i frekvencija dodatnih polimorfnih markera za IL1 regiju [IL1A (rs1800587, C-889T) i IL1B (rs1143634, C+3954T)] kao i za IL17 gen, odnosno IL17A (rs2275913, G-197A), 3) ispitati da li su frekvencije genskih polimorfizma IL1A (rs1800587, C-889T), IL1B (rs1143634, C+3954T) i IL17A (rs2275913, G-197A) povezane s povećanim rizikom za nastanak POA koljena i kuka te 4) proširiti istraživanje uvođenjem rezultata prethodnog istraživanja s dva polimorfna markera za IL1 regiju [IL1B (rs16944, G-511A) i IL1RN varijabilni broj tandemskih ponavljanja (od engl. Variable Number Tandem Repeat - VNTR)] kako bi se definirao prošireni haplotip (IL1A C-889T, IL1B C+3954T, IL1B G-511A i IL1RN VNTR) i usporedio s predispozicijom za nastanak bolest(i). Ispitanici i metode. Koristeći studiju slučajeva i kontrola s 500 bolesnika koji boluju od uznapredovalog oblika POA (260 bolesnika s OA kuka kojima je ugrađena TEP kuka i 240 bolesnika s OA koljena kojima je ugrađena PEP ili TEP koljena), analizirane su frekvencije polimorfizama IL1 obitelji gena u hrvatskoj populaciji. Kontrolnu skupinu čini 531 osoba od kojih su većina zdravi DDK. Genotipizirana su dva polimorfizma jednog nukletioda (od engl. Single Nucleotide Polymorphisms - SNPs) u IL1 obitelji gena na poziciji IL1A (-889, C>T, rs1800587) i IL1B (+3594, C>T, rs1143634) te su njihove frekvencije uspoređene između bolesnika i kontrola. Haplotipivi su predviđeni kombiniranjem dobivenih rezultata i postojećih rezultata iz prethodnih vlastitih istraživanja s dva polimorfna markera za IL1 regiju: IL1B (-511, G>A, rs16944) i IL1RN (VNTR), kako bi se definirao prošireni haplotip [IL1A(rs1800587) - IL1B(rs1143634) - IL1B(rs16944) - IL1RN(VNTR)] i usporedio s predispozicijom za nastanak bolesti u istoj populaciji. Nadalje, genotipiziran je SNP -197 (G>A, rs2275913) IL17A gena u ukupno 358 bolesnika s uznapredovalim oblikom POA (186 bolesnika s OA koljena kojima je ugrađena PEP ili TEP koljena i 172 bolesnika s OA kuka kojima je ugrađena TEP kuka) te 136 zdrava DDK kao kontrolne skupine. Frekvencije alela genskog polimorfizma na poziciji -197G>A IL17A gena su otkrivene Taqman metodom. Rezultati. Analiza haplotipova otkrila je spolne nejednakosti i pokazala da osobe ženskog spola koje su nosioci 1-2-1-1 haplotipa imaju 6 puta manji rizik za nastanak OA koljena, dok osobe istog spola koje su nosioci 1-1-1-2 haplotipa imaju preko 2 puta veću predispoziciju za nastanak OA kuka. Nadalje, genotipiziranjem SNP -197 (G>A, rs2275913) IL17A gena nisu pronađene razlike između alelnih i genotipskih frekvencija između bolesnika i kontrola. Zaključci. Upalni medijatori, kao što je IL-1, mogu imati ulogu u patogenezi POA velikih zglobova, kao i da još neidentificirani specifični spolni čimbenik postoji u hrvatskoj populaciji koji determinira predispoziciju za nastanak POA velikih zglobova. Ovo je prva studija koja je odvojila spolove u procjeni genskog rizika IL1 lokusa za nastanak POA. Ovim istraživanjem nije pronađena značajna povezanost frekvencije genskog polimorfizma IL17A (rs2275913, G-197A) s povećanim rizikom za nastanak POA koljena i kuka u hrvatskoj populaciji. Također, ovo je prva studija koja pokazuje i nedostatak povezanosti rs2275913 SNP IL17A gena s OA koljena.Objectives. Osteoarthritis (OA) is a frequent joint disease with complex genetic risk. Genetic predisposition to the complex hereditary disease like OA of the large joints (hip and knee) includes the interleukin-1 gene (IL1) cluster on chromosome 2. Also, one of the loci that could predispose the development of OA is located on chromosome 6 (6p12.3-q13). Near this locus is a gene, which encodes the interleukin-17A (IL-17A). The specific objectives of this study were: 1) to identify the interleukin-1 (IL-1) and IL-17 gene polymorphisms in patients with advanced primary knee OA (KOA) and hip OA (HOA) treated because of their illness with total hip replacement (THR) or partial/total knee replacement (PKR/TKR), and compared to a control group of voluntary blood donors from Croatian population, 2) explore the importance of association risk for developing KOA and HOA and frequency of additional polymorphic markers for IL1 gene locus [IL1A (rs1800587, C-889T) and IL1B (rs1143634, C +3954 T )] as well as IL17 gene, i.e. IL17A (rs2275913, G-197A), 3) to examine whether the frequency of polymorphisms IL1A (rs1800587, C-889T), IL1B (rs1143634, C +3954 T), and IL17A (rs2275913, G-197A) are associated with an increased risk of primary KOA and HOA, and 4) to extend this research by introducing the results of previous studies with two polymorphic markers for the IL1 region [IL1B (rs16944, G-511A) and IL1RN Variable Number Tandem Repeat (VNTR)] in order to search for a putative extended risk haplotype (consisting of 1: C-889T IL1A, 2: IL1B C+3954T, 3: IL1B G-511A, and 4: IL1RN VNTR markers) during association analyses with the disease(s). Patients and Methods. Using a case - control study with 500 OA patients (240 KOA and 260 HOA patients, all with joint replacement), we analyzed frequencies of IL1 gene cluster polymorphisms in Croatian Caucasian population. The control samples came from 531 healthy persons with a majority of voluntary blood donors. Two single nucleotide polymorphisms (SNPs) were genotyped in the IL1 gene locus at IL1A (-889, C>T, rs1800587) and IL1B (+3594, C>T, rs1143634), and their frequencies were compared between patients and controls. Haplotypes prediction was done by combining current data with previous results on gene polymorphisms (IL1B, rs16944, and the IL-1 receptor antagonist gene [IL1RN] Variable Number Tandem Repeat [VNTR]) for the same population. Furthermore, IL17A SNP of HOA patients were analyzed in our population, and analysis was extended on KOA patients. A total of 358 OA patients, of which 186 KOA patients with PKR or TKR and 172 HOA patients with THR, and 136 healthy voluntary blood donors as controls were genotyped for the IL17A gene SNP -197(G>A, rs2275913). The frequencies of alleles of the IL17A gene SNP at -197G>A were detected by Taqman method. Results. Haplotype analyses revealed gender disparities and showed that women carriers of the 1-2-1-1 haplotype [IL1A(rs1800587) - IL1B(rs1143634) - IL1B(rs16944) - IL1RN(VNTR)] had six-fold lower risk to develop KOA, whereas carriers of the 1-1-1-2 haplotype had over two-fold higher predisposition to HOA. Furthermore, genotyping of the SNP -197 (G> A, rs2275913) IL17A gene did not found differences in the allelic and genotypic frequencies between patients and controls. Conclusions. Our results differ from earlier studies in Caucasian subpopulations which may be due to the fact that this is the first study to separate genders in assessing the IL1- locus genetic risk of OA. The results suggest that both inflammatory mediators like IL-1 might be implicated in the pathogenesis of primary OA in large joints and that as yet unidentified gender specific factors exist in a Croatian Caucasian population which determines predisposition for development of primary OA of the large joints. Furthermore, no significant association was found for the IL17A gene SNP rs2275913 with KOA and HOA in Croatian population. This is the first study that shows lack of association of the rs 2275913 SNP in the IL17A gene with KOA