Subjects.
Between 2002 and 2015, a total of 223 Japanese patients were diagnosed as having IgG4-RD at Shinshu University Hospital or its affiliated institutions. Among them, 99 patients were excluded for the reason of no stored DNA samples (n = 55) or (2) no contrast-enhanced computed tomography (CT) analysis due to renal impairment or allergy (n = 44). The remaining 124 patients with IgG4-RD (median age: 67 years, 75% male) were included in this retrospective study along with 344 previously reported volunteer healthy subjects [11]. IgG4-RD was diagnosed based on the Japanese Comprehensive Diagnostic Criteria for IgG4-RD [12]. Contrast-enhanced CT findings were reviewed by an experienced radiologist (Y.F.). Periaortitis and periarteritis were defined as vessel wall thickness and wall enhancement either partially or circumferentially on CT images, as reported previously [4]. The protocol of this study was reviewed and approved by the Institutional Review Board of Shinshu University School of Medicine in Matsumoto, Japan (approval number: 571). Written informed consent was obtained from all participating patients. The investigation was conducted according to the principals of the Declaration of Helsinki.
GWAS and SNP genotyping.
Genomic DNA was isolated from whole-blood samples using Quick Gene-800 assays (Kurabo, Osaka, Japan). Genotyping was done with the GeneChip Human Mapping 500K Array Set (Affymetrix, CA, USA) according to the manufacturer’s protocol in the first stage of this analysis. Samples with a < 93% genotype call rate were excluded from the study, as were SNPs with a call rate of < 95% or a minor allele frequency of < 5% overall. From a prior genome-wide association analysis between patients with IgG4-related periaortitis/periarteritis (n = 28) and those without (n = 47), we extracted 3 candidate genes (interleukin 1 receptor type 1 [IL1R1]; 2q11.2-q12.1, calcium/calmodulin-dependent protein kinase II alpha [CAMK2A]; 5q32, and vacuolar protein sorting 13 homolog B [VPS13B]; 8q22.2) containing highly significant SNPs (i.e., P < 10− 4). SNPs in the IL1R1 gene were examined to identify possible susceptibility loci to IgG4-related periaortitis/periarteritis since this gene has been established as an important mediator involved in many cytokine-induced immune and inflammatory responses [13]. Three SNPs that showed a significant association with IgG4-related periaortitis/periarteritis by a GWAS were selected along with 5 additional SNPs. In total, 8 SNPs (rs3917225, rs2287049, rs2287048, rs3917273, rs2160227, rs951192, rs3917318, and rs7582198) having minor allele frequencies of > 5% in the ILR1 gene were evaluated. The genotyping of all SNPs was performed using the ABI TaqMan allelic discrimination kit and the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, MA, USA) following the manufacturer’s instructions.
Measurement of serum IL-1β levels.
Serum samples were obtained and stored below − 20 °C until measurements. Serum IL-1β levels were examined by a commercially available ELISA kit (Proteintech, IL, USA) following the manufacturer’s instructions.
Statistical analysis.
We assessed the significance of allele distribution among patients using the chi-square test by means of 2 × 2 comparisons (df = 1). Differences between groups were examined by Mann–Whitney U-test. P-values were subjected to Bonferroni’s correction by multiplication by the number of different alleles observed in each locus (Pc). Association strength was estimated by calculating the odds ratio (OR) and 95% confidence interval (CI). A P < 0.05 was considered statistically significant. Statistical analyses were performed using IBM SPSS statistics software version 24 (IBM, IL, USA). Genetic power was calculated using G*Power version 3.1.9.6 [14].