DNA microarray analysis of rheumatoid arthritis susceptibility genes identified by genome-wide association studies

Vyse described the genetics of rheumatic disease [1]. In the past several years, genome-wide association studies (GWAS) have led to the identifi cation of six high-risk rheumatoid arthritis (RA) susceptibility genes – namely, CD244, PADI4, SLC22A2, PTPN22, CTLA4, and STAT4 (summarized in [2]). In vitro studies using mutant alleles and cultured cells have revealed the individual up-regulation of CD244, PADI4, SLC22A2, and PTPN22 [2-6]; however, studies on the expression of RA susceptibility genes in RA patients are rare. We therefore investigated the expression of the above-mentioned six RA susceptibility genes in 112 RA patients using DNA microarray analysis. Th is study aims to clarify whether DNA microarray analysis and GWAS produce comparable results with respect to RA susceptibility genes. Total RNA extracted from total peripheral blood cells obtained from 112 RA patients and 45 healthy individuals was used to prepare aminoallyl RNA. As a reference, mixed RNA from 45 healthy individuals was used. Th e aminoallyl RNA of each individual and the reference was subjected to Cy3 and Cy5 labeling, respectively, and was hybridized with an oligonucleotide-based DNA micro-array. Th e data obtained were analyzed by nonparametric statistical group comparison. Th e intensities of the no-probe spots were used as the background. Th e median and standard deviation of the background intensity were calculated. Th e genes with an intensity value that was less than the median plus 2 standard deviation of the background intensity were identifi ed as null. Th e Cy3/ Cy5 ratios of all spots on the DNA micro array were normalized using the global ratio median. Only gene expression data that were collected from at least 80% of samples from each group were selected for further analysis. Th e unpaired Mann–Whitney test was used to determine statistically signifi cant diff erences in the mRNA expression levels between the RA and healthy groups. Statistical signifi cance was set at P <0.05. Th e results of our DNA microarray analysis showed that the expressions of four out of the six RA susceptibility genes were signifi cantly higher in RA patients than in healthy individuals (1.0 x 10 –16 to 2.32 x 10 –5) (Table 1). As described above, the upregulation of these four genes (CD244, PADI4, SLC22A2, and PTPN22) has been previously confi rmed in in vitro studies. We found, however, that CTLA4 expression levels were similar between the RA and control groups, whereas STAT4 expression was signifi …