Here, we present the results of a two-stage GWAS involving 492 biopsy-confirmed IgAN cases and 893 healthy controls. Despite the small sample size, our study is valuable in that we aimed to overcome the limitations of earlier studies by using a customized DNA chip, which is predominantly composed of exon and promoter regions and contained not only well-established but also unknown SNPs. In addition, we selected patients with biopsy-confirmed IgAN to overcome the small sample size. With this approach, we identified new susceptible loci of IgAN in the Korean population. The primary contribution from our study are: 1) we designed a customized DNA chip containing 98,667 SNPs; 2) we genotyped two candidate SNPs selected in the discovery stage using a validation cohort; and 3) we identified one susceptible SNP; rs2296136 in ANKRD16.
Despite remarkable progress since IgAN was first described by Berger et al. in 1968 , its pathogenesis has not yet been clearly defined. Inter-individual variation of disease course, differences in incidence among different ethnicities, and familial aggregation of the disease have suggested a genetic predisposition for IgAN . In the last two decades, there have been many candidate-gene association studies and linkage analyses for IgAN [6, 7]. However, those studies were underpowered, and no specific causative mutations for IgAN have been identified. GWASs have recently emerged as an alternative approach, allowing for the identification of susceptibility loci that were previously unrecognized .
The first GWAS of IgAN was performed in subjects of European ancestry by Feehally et al. . This study provided evidence for an association between IgAN and genes at HLA loci, across HLA-B, DRB1, DQA, and DQB. In several subsequent GWASs, nearly 20 risk variants for IgAN were identified (CFHR1, CFHR3, HORMAD2, TNFSF13, DEFA,ITGAM-ITGAX, VAV3, and CARD9, among others) [9,10,11]. Those loci are associated with the complement system, mucosal IgA production, and innate and acquired immunity . However, these previously reported SNPs are GWASs that were fixed and had less coverage of SNPs in the exon and promoter regions.
Recently, several large-scale GWASs on the population of East Asia have been reported. Yu et al.  conducted a GWAS to identify susceptibility loci for IgAN in Han Chinese and showed that IgAN is associated with SNPs of near genes involved in innate immunity. This study group also performed the largest GWAS of IgAN in Han Chinese and identified new susceptibility loci (rs7634389 in ST6GAL1, rs2074038 in ACCS, and rs2033562 in ODF1-KLF10) . The results of these previous studies have helped clinicians understand the pathogenesis of IgAN. However, considering the genetic differences and prevalence between East Asian countries, it is necessary to conduct GWAS of IgAN in the Korean population.
In the present study, we used the Axiom™ Genome-Wide Human Assay and found two SNPs with suggestive evidence for association with IgAN in the first stage (p ≤ 5 × 10− 5). Among these, rs2296136 in ANKRD16 showed significant association with IgAN in the validation stage. ANKRD16 is located at 10p15.1 and encodes the ankyrin repeat domain 16. Its function is unclear because only a few studies have investigated ANKD16. One study showed that ANKRD16 is associated with subtype differences of breast cancer . No study has reported an association between genetic variation in ANKRD16 and IgAN. We explored the effect of the variant on protein structure, function in missense SNPs and transcriptional activity in promoter SNPs. The probability of damage (probability > 0.8) for rs2296136 of ANKRD16 was validated by polyphen2. Further functional studies are needed to elucidate whether ANKRD16 can affect IgAN.
Performing validation study of GWAS results is important for extending the effect estimation and providing acceptable statistical evidence [20, 21]. Although our study had small sample size in the discovery stage, we also validated our results using an independent samples consisting of 310 biopsy-confirmed IgAN cases and 438 healthy controls.
Our study has some potential limitations. First, this GWAS was conducted in a relatively small patient population. Because of the small sample size, statistically significant SNPs of p < 1 × 10− 8 were not found. However, we found SNPs that were presumed to be related to IgAN and proceeded to validation. In the existing GWAS study, the most significant SNPs were mostly rare SNPs, and these significant SNPs were not significant when tested in other groups. Genetic polymorphic markers based on DNA in precision medicine are very important. Race, sex, and other factors affect the significance of these SNPs for any given disease. Therefore, we cannot say that the SNPs found in this study are statistically highly significant, but the SNPs reported through these studies may help to find additional markers. As described, to compensate for the sample size, we selected patients with biopsy-confirmed IgAN. Second, we focused only on SNPs with a minor allele frequency greater than 0.05 and so might have missed rarer variations associated with IgAN. Third, the patients included in this study were predominantly Korean, so the results should be generalized with caution. Finally, we did not assay gene expression in vivo or examine functional effects according to genetic variants, relying instead on in silico functional detection software. To improve these weakness, we are planning a follow-up study using expression quantitative trait loci (eQTL) analyses. Interestingly, however, both promoter and missense functional assay programs showed that the rs2296136 variant of ANKRD16 has important functional effects.