PROCEEDINGS OF THE LATVIAN ACADEMY OF SCIENCES. Section B, Vol. 63 (2009), No. 4/5 (663/664), pp. 214–221. DOI: 10.2478/v10046-009-0045-2 ASSOCIATION STUDY OF GENETIC VARIANTS IN THE 14q11 – 14q13 PROTEASOMAL GENES CLUSTER WITH JUVENILE IDIOPATHIC ARTHRITIS (JIA) IN LATVIAN POPULATION Ilva Trapiòa*,**, Ingrîda Rumba-Rozenfelde**, Nikolajs Sjakste**,***, Jeïizaveta Sokolovska*,**,***, Olga Sugoka*, and Tatjana Sjakste* * Institute of Biology, University of Latvia, Miera iela 3, Salaspils, LV-2169, LATVIA; e-mail: [email protected] ** Faculty of Medicine, University of Latvia, Ðarlotes iela 1a, Rîga, LV-1001, LATVIA *** Latvian Institute of Organic Synthesis, Aizkraukles iela 21, Rîga, LV-1006, LATVIA Communicated by Andrejs Çrglis The possible role of proteasomes in the development of autoimmune diseases was hypothesised after discovery of the involvement of proteasomal LMP2 and LMP7 subunits in antigene process- ing. The objective of this study was to determine the association between allelic variants of the genes encoding proteasomal proteins PSME1, PSME2 and PSMA6 and juvenile idiopathic arthri- tis (JIA) in the Latvian population. One Indel G-4543CA-4544®GA and four SNPs related to the PSMA6 gene (A-2486®G and C-1910®T, upstream promoter, C-110®A of promoter, and C-8®G of 5’UTR), of two cSNP in PSME1 (G1457®A:Val104, exon 6 and C2536®A: Lys244®Thr, exon 11) and in PSME2 (C1153®G:Arg61®Gly, exon 4 and A1440®C:His89®Pro, exon 6) were geno- typed by means of primer-specific PCR, CAPS assay and/or sequencing in case/control study composed from the 156 JIA patients and 214 healthy individuals. Allele frequency and genotype distribution was similar in cases and controls for Indel, and SNPs A-2486®G, C-1910®T and C-8®GofPSMA6, as well as for all studied cSNPs in PSME1 and PSME2 genes. Differences in A-110 allele and CG genotype frequencies were close to the statistically significant P level in JIA patients and healthy individuals, however, when an additive model was applied, the difference in the C-110®A locus turned out to be statistically significant. The results support the hypothesis of the possible association of PSMA6 gene allelic variants with JIA in the Latvian population. Key words: PSMA6, chromosome 14, SNP polymorphism, juvenile idiopathic arthritis, associa- tion study. INTRODUCTION most individual “JIA risk alleles” suggest that gene–gene interactions, which are poorly described or understood at Juvenile idiopathic arthritis (juvenile rheumatoid arthritis, present, likely influence whether pathology develops (Glass JIA) is the most prevalent paediatric rheumatic diagnosis and Giannini, 1999). This consideration encourages exten- among children in many countries (Glass and Giannini, sion of the search for JIA candidate genes. 1999). JIA is a clinically heterogeneous group of pheno- types that have in common chronic inflammatory synovitis The chromosome 14q region appears to be prospective for in children under the age of 16 (Prahalad et al., 2001). the above goal, as it harbours several genes of interest. Seven subtypes of JIA, based on the clinical characteristics Linkage of the locus to autoimmune diseases has been al- at onset are distinguished; including the more frequent ready reported (John and Worthington, 2001). Possible can- oligo, poly, psoriatic and systemic JIA variants (Petty at al., didate genes include the genes encoding the nuclear factor 2004). The genetic contributions to disease risk have been kappa B inhibitor (NFKBI) on 14q13 (le Beau et al., 1992), observed in many case/control studies (Glass and Giannini, the transcription factor FOS on 14q23, TGF-beta3 on 1999; Prahalad et al., 2001). A genome scan revealed link- 14q24, and immunoglobulin heavy chain locus at 14q32.33 age of JIA to the HLA region, additional evidence support- (Kamnasaran and Cox, 2002). Several proteasomal genes ing linkage of JIA was observed at 1p36, 19p13, and 20q13 are located at 14q including proteasomal activator subunits (Thompson et al., 2004). However, the low odds ratios for 1 and 2 (PSME1 and PSME2, respectively) and proteasome 214 Proc. Latvian Acad. Sci., Section B, Vol. 63 (2009), No. 4/5. subunit of beta type 5 (PSMB5) at 14q11.2, proteasome DNA extraction was performed using a kit for genomic subunits of alpha type 6 (PSMA6) at 14q13, and alpha type DNA extraction from nucleated blood cells (Fermentas, 3(PSMA3) at 14q23 (Chistyakov et al., 2000). Vilnius, Lithuania). DNA quantity and quality was deter- mined by electrophoresis in agarose gel. The proteasome is a multisubunit enzyme complex which 1457 2536 plays a central role in the degradation of proteins control- Genotyping of 6 SNP including G ®A and C ®A 1153 1440 ling transcription rate, cell-cycle progression and apoptosis; of PSME1;C ®GandA ®CofPSME2;and -2486 -8 therefore, an important role in pathogenesis of human dis- A ®GandC ®GofPSMA6 was conducted by eases has been ascribed to the structure (Glickman et al., MALDI-TOF Mass Spectrometry approach (Wenzel et al., 2001; Almond et al., 2002; Naujokat et al., 2002; Sjakste et 2003) on a MALDI TOF Autoflex Bruker Daltonics using al., 2002a; 2002b). To a known extent, proteasomes inter- genoSNIP specific primers (Table 1). One base pair inser- -4543 -4544 fere also with the atherogenesis process. Proteasomes de- tion/deletion polymorphism (InDel) G CA ®GA -1910 grade IF-kB factor with subsequent activation of the and SNP C ®T were genotyped by CAPS (cleaved am- atherogenic NF-kB (Wilson et al., 2001). Oxydised LDL plified polymorphism) assay using correspondingly BseMI causes activation of the proteasome activity followed by de- and EcoNI restriction sites in the intergenic region upstream -110 cline of this activity. HDL is an inhibitor of the proteasome PSMA6 gene. SNP C ®A(PSMA6 promoter) was ana- activity (Vieira et al., 2000; Robbesyn et al., 2003). lysed by allele specific PCR. Information on the primer se- quences, amplified and restricted fragment sizes, SNPs The latter gene group appears to be of special interest as it GenBank accession numbers, and function of the polymor- was shown that the proteasomal protein LMP2 gene poly- phism is given in Table 1. morphism affects the susceptibility to, and severity of HLA-B27 associated juvenile idiopathic arthritis (Pryhuber PCR was performed in a final volume of 30 ml containing et al., 1996). around 100 ng genomic DNA, 10× DreamTag polymerase buffer (final concentration 1×), 25 mM MgCl2 (from 1.5 We have previously addressed the possible association of mM to 3 mM), 10 mM dNTP Mix (0.5mM), 10 pM forward the PSMA6 gene in the genetic susceptibility to various dis- and reversal primers (0.33 pM each) and 0.75 units of eases. The PSMA6 gene is encoded by a single-copy gene DreamTaq polymerase (Fermentas, Vilnius, Lithuania). on chromosome 14q13.2 between markers D14S960 and PCR denaturation at 95 °C 5 min was followed by 35–40 D14S889 (Sjakste et al., 2001). In the initial studies we in- cycles each of 30 sec of denaturation at 95 °C , 30 s of an- vestigated a microsatellite polymorphism in intron 6 of the nealing (at 55–63°C depending on the polymorphism ana- gene in several human populations (Sjakste et al., 2002c). lysed) and 60 s of extension at 72 °C, with final extension Further studies showed that this marker is associated with step of 7 min at 72 °C. PCR products were tested by elec- Graves’ disease (Sjakste et al., 2004) and diabetes mellitus trophoresis in 1% agarose gel. Allele specific PCR was per- type 2 (DM2, Kalis et al., 2002; Sjakste et al., 2007a). formed using allele specific forward primers differing only Genotyping of the single nucleotide polymorphism (SNP) in the 3’-end polymorphic nucleotide and the reversal in the promoter and 5’-UTR region of the PSMA6 gene, re- primer common for both alleles (Table 1). Presence or ab- sulted in the discovery of the association of the genotype sence of the amplified product directly indicated the pres- -110 -8 -8C/G and haplotype C /G with a higher risk of DM2 ence or absence of the corresponding allele. PCR product (Sjakste et al., 2007b). was digested in case of CAPS assay by the corresponding restriction enzyme. Digestion was performed in final vol- In this study we investigated whether chromosome 14q re- ume of 25 ml containing about 1,000 ng of DNA in PCR gion SNP is associated with increased risk of juvenile idio- product, 10× Buffer R (final concentration 1×) and 0.5 units pathic arthritis. of the corresponding restriction enzyme (Fermentas, Vilnius, Lithuania) during 2 hours at 55 °C or 37 °C for MATERIALS AND METHODS BseMI or EcoNI, correspondingly. Results of the digestion were tested by gel electophoresis in 1% or 1.5% agarose Case/control study description. The polymorphisms were gel. Fragment sizes of the digested amplicons are indicated genotyped in a case/control study of 156 juvenile idiopathic in Table 1. Primer design was performed by the Primer 3.0 arthritis patients and 214 subjects without JIA from the Lat- programme using the 14q11.2–14q13.2 sequence informa- vian population. The case group (63% females) consisted of tion at the NCBI nucleotide service (GenBank accession patients attending the outpatient clinic at the Pauls Stradiòð NT_026437). Primers were synthesised in “Metabion Inter- Clinical University Hospital and the Children’s Hospital national AG” (Germany). “Gaiïezers”. The group represented about 60% of all regis- tered in Latvia JIA patients. Patients (57% females) to the SNP functionality was analysed in silico for the affinity to Rîga Biíernieki Hospital specialised in trauma medicine, transcription factors (TF) using Genomatix software were recruited to participate in the study control group (DiAlign TF, Release 3.1, and MatInspector, Release 7.4 (without autoimmune diseases).