Genetic Association of PTPN22 Polymorphisms with Autoimmune Hepatitis and Primary Biliary Cholangitis in Japan

Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are liver-specific autoimmune conditions that are characterized by chronic hepatic damage and often lead to cirrhosis and hepatic failure. Specifically, the protein tyrosine phosphatase N22 (PTPN22) gene encodes the lymphoid protein tyrosine phosphatase, which acts as a negative regulator of T-cell receptor signaling. A missense single nucleotide polymorphism (SNP) (rs2476601) in PTPN22 has been linked to numerous autoimmune diseases in Caucasians. In the present series, nine SNPs in the PTPN22 gene were analyzed in 166 patients with AIH, 262 patients with PBC, and 322 healthy controls in the Japanese population using TaqMan assays. Although the functional rs3996649 and rs2476601 were non-polymorphic in all subject groups, the frequencies of the minor alleles at rs1217412, rs1217388, rs1217407, and rs2488458 were significantly decreased in AIH patients as compared with controls (all Pc < 0.05). There were no significant relationships with PTPN22 SNPs in PBC patients. Interestingly, the AAGTCCC haplotype was significantly associated with resistance to both AIH (odds ratio [OR] = 0.58, P = 0.0067) and PBC (OR = 0.58, P = 0.0048). SNPs in the PTPN22 gene may therefore play key roles in the genetic resistance to autoimmune liver disease in the Japanese.

PTPN22 Haplotypes in Patients and Controls. Pairwise linkage disequilibrium (LD) mapping confirmed that the seven tested alleles were in strong LD over a narrow range, with an LD index > 0.9 (Table 3). Strong LD was indicated in the same block for AIH and PBC patients and controls. Nine unique SNP haplotypes were found altogether, of which four had frequencies of > 5% (Table 4). Association analysis of haplotypes calculated by EM algorithms showed that haplotype 3, which was the only rs3765598 T, was significantly associated with resistance to both AIH (OR = 0.58, 95% CI 0.39-0.86, P = 0.0067) and PBC (OR = 0.58, 95% CI 0.40-0.85, P = 0.0048).
Association Between PTPN22 SNPs and Clinical Outcome. AIH and PBC patients were stratified according to disease progression. However, no SNP or haplotype in the PTPN22 gene was associated with either cirrhosis in AIH or a history of orthotopic transplantation and disease progression of PBC (data not shown).

Discussion
In recent studies on the PTPN22 gene, the rs2476601 missense substitution SNP has been associated with multiple autoimmune diseases in Caucasians, including RA, SLE, Graves' disease, and Addison's disease. rs2476601 was also proposed to be functionally involved in interactions between Lyp and Csk kinase 13,14,27 . Another functional SNP that is located in the catalytic domain of Lyp, rs33996649, leads to reduced phosphatase activity and has been highlighted as an important genetic risk factor for RA and SLE 26 . The present analysis showed that both of these SNPs were non-polymorphic (rs2476601: C, rs33996649: G) among all subject groups, in accordance with earlier studies from Asia [19][20][21][22] . Our data confirmed that the rs2476601 and rs33996649 SNPs were not associated with Japanese autoimmune liver disease.
This study revealed a striking association between SNPs in the PTPN22 gene and resistance to AIH. Although a genome-wide association study showed that PTPN22 was not related to AIH in patients of European descent 13 , there have been no data regarding PTPN22 SNPs in Japanese AIH. Hence, our results raise several possibilities on an association between PTPN22 loci and AIH protection in the Japanese population. First, among the five significantly associated SNPs in PTPN22, rs2488457 in the promoter region might be an important factor in Asian populations, as seen in case-control studies where it increased the risk of RA 28,29 and ankylosing spondylitis 30 . Second, other potentially functional variants may be engaged in susceptibility to AIH as there are nine non-synonymous substitutions in addition to rs2476601 in exon 14 of PTPN22. Lastly, it is possible that the PTPN22 locus contains another, undefined functional variant in LD with rs3996649 or rs2476601. To address these prospects, we sequenced exon 14 of PTPN22 in 12 patients with AIH and 12 healthy controls whose genotypes were GG, AG, and AA at rs1217388 adjacent to rs2476601. No missense substitutions were detected in any sample, and all alleles were wild type ( Fig. 1). However, the strong LD across this region as evidenced by pairwise D' values near 1 (Table 3) made it difficult to ascertain whether these associated SNPs were independent protective factors of AIH. We compared haplotype frequencies between patients and controls to address this problem. Haplotype 3 containing AAGTCCC was less frequent in AIH and significantly associated with disease resistance (P = 0.0067, OR = 0.58). This novel haplotype contained minor and protective alleles concerning AIH susceptibility.
Although various autoimmune disorders have been associated with rs2476601, negligible relationships were found for systemic sclerosis, celiac disease, ulcerative colitis, Crohn's disease, multiple sclerosis, and psoriasis in a meta-analysis 31 . The study defined two groups of diseases with regard to their targeted tissues, and showed that most autoimmune diseases possessing an insignificant association with rs2476601 manifested in the skin, gastrointestinal tract, bile duct, or immune privileged sites. Such results indicated that the relationship of individual PTPN22 SNPs with autoimmune diseases depended on the localization of the affected tissues and suggested a role of targeted organ variation in disease manifestations. Regarding autoimmune liver disease, only one association study on PBC has been conducted that showed no relationship with rs2476601 in Canada 32    association studies have demonstrated a link between PTPN22 and PBC susceptibility [10][11][12]33,34 , which was supported by our data. PBC is characterized by slow, progressive destruction of the small bile ducts within the liver. Primary sclerosing cholangitis is another autoimmune disease targeting the bile ducts that is complicated by ulcerative colitis. Primary sclerosing cholangitis was also not associated with SNPs in rs2476601 35 . Interestingly, we witnessed that haplotypes containing the rs3765598 T allele were significantly associated with a 0.6 times less likelihood to develop PBC, which suggested that this haplotype may play an important role in protection from PBC in Japan. The limitations of this study are a small number of cases and controls and a narrow focus on few SNPs in this era of genome-wide association studies. Further investigation is needed to validate this association in other Asian countries.
In conclusion, the present study revealed PTPN22 gene SNP and haplotype associations with protection against AIH or PBC in a Japanese population. This gene may therefore play a crucial role in the pathogenesis of Japanese autoimmune liver disease, and further studies are warranted to clarify its role in AIH and PBC.

Patients and Methods
Subjects. The clinical and biochemical features of the 166 patients with AIH and 262 patients with PBC enrolled in this study between January 2001 and August 2015 are summarized in Table 5. We also recruited 322 volunteer control subjects from hospital staff who had indicated the absence of any major illnesses in a standard questionnaire. The racial background of all individuals was Japanese. All AIH patients had been diagnosed rs1217412 rs1217388 rs1217407 rs3765598 rs2488458 rs3789612 rs2488457  according to the scoring system of the International Autoimmune Hepatitis Group 36 and were classified as having type 1 AIH based on antibody profiles. The diagnosis of PBC was made according to criteria from the American Association for the Study of Liver Diseases 37 . Anti-nuclear antibody titers were determined by immunofluorescence using HEp-2 cells, for which a titer of ≥ 1:80 was considered a positive result 38 . Anti-mitochondrial antibody-M2 was measured by the enzyme-linked immunosorbent assay as reported previously 8 . All patients were negative for the hepatitis B surface antigen and antibodies to the hepatitis B core antigen, hepatitis C virus, and human immunodeficiency virus. Overlap syndromes were excluded. Liver cirrhosis was diagnosed by histological examination and/or characteristic clinical signs of advanced liver disease 39 . This study was reviewed and approved by the Institutional Review Board of Shinshu University Hospital (Matsumoto, Japan), and written informed consent was obtained from all participating subjects. The investigation was conducted according to the principals of the Declaration of Helsinki. PTPN22 Genotyping. Genomic DNA from patients and controls was isolated from whole blood samples using QuickGene-800 assays (Fujifilm, Tokyo, Japan).