IFIH1 Gene Polymorphisms In Type 1 Diabetes: Genetic Association Analysis And Genotype-phenotype Correlation In Chinese Han Population

Background and objective:The evaluation of susceptibility loci is an important addition to the current predictive and screening models in type 1 diabetes (T1D). IFIH1 single nucleotide polymorphisms (SNP) rs3747517, rs1990760 have been proved to be associated with T1D, and its importance is different in different ethnic groups. Considering that IFIH1 protein is a cytoplasmic helicaase which mediates induction of interferon response to viral RNA, this may be part of the immunity mechinsm of T1D. The purpose of this study was not only to replicate the associatic finding beween IFIH1 SNP and T1D in an independent Chinese Han sample, but also to evaluate the interactions with auto-antibodies (Glutamic acid decarboxylase antibody, GADA; Insulin autoantibody, IAA; protein tyrosine phosphatase IA2 antibody, IA-2A and Zine transporter 8 antibodies, ZnT8A) linked risk and HLA classⅡlinked risk, and to determine the genotype-phenotype correlation. All the results above will improve the current underatanding of how IFIH1 SNP involved in the pathological mechanism of T1D in Chinese Han polulation, and make the current predictive and screening models in T1D better.Methods:1. Subjects Patients with T1D (n=464) were recruited in Jiangsu province between 2008 and 2010 as patients participating in the studies, The median age was (29.7±13.7) years, and the male and female patients were 240 and 224 respectively, All the patients were diagnosed by 1999 WHO diagnostic criteria. The 465 healthy control subjects whose median age was (31.1±7.4) years and the male were 221 had no family history of diabetes and other autoimmune diseases, and their fasting and 2h postprandial blood glucose were normal. This study was approved by the appropriate ethical committees and informed consent was obtained from all participants. The positive and negative quality control serum samples were taken from the United States Barbara Davis Center for Childhood Diabetes Laboratory.2, The polymorphism of IFIH1 gene rs3747517 Genomic DNA was extracted from heparinized blood from the above diabetes patients and healthy controls using standard procedures, and were genotyped for the IFIH1 gene rs3747517 single nucleotide polymorphism (SNP). the SNP rs3747517 was genotyped by the polymerase chain reaction and restriction fragment polymorphism (PCR-RFLP) technique. The PCR products were digested with a HinⅢ, resolved on a 2% agarose gel and stained with ethidium bromide, observed the digestion results with the UV light.3, The polymorphism of IFIH1 gene rs1990760 Genomic DNA was extracted from heparinized blood from the above diabetes patients and healthy controls using standard procedures, and were genotyped for the IFIH1 SNP rs1990760 by using the Custom TaqMan SNP genotyping assay. The data were then analyzed by SDS software.4. The detection of islet autoantibodies GADA, IA-2A, IAA and ZnT8A 82 patients were selected from the T1D group randomly. Blood samples were collected from patients in the morning and were centrifugated. Serum samples were collected and were then stored in -20℃ready for use. GADA,IA-2A,IAA and ZnT8A were detected by radio-immunoprecipitation assay, and the results was represent with the antibody index (sample–negative control) /( positive control–negative control). 5. The examination of HLA-DR,DQ 79 patients were selected from the T1D group randomly. Peripheral blood samples were collected and genomic DNA was extracted. The genetic polymorphisms of HLA-DR,DQ were examined with polymerase chain reaction restriction sequence specific oligonacleotide (PCR-SSO) method.Results:1. In the case-control cohort, for rs3747517, the allelic frequencies: A allele was significantly lower in cases compared with control subjects (52.8% vs 66.45%), while G allele (47.2% vs. 33.55%) was the opposite. At the genotypic level, the frequencies of the AA (24.57% vs. 45.16%, P<0.001), AG (56.47% vs. 42.58%, P=0.01), and GG(18.97% vs. 12.26%, P=0.02) genotypes in cases were significantly different from control subjects. For rs1990760, the frequencies of alleles and CC, CT, TT genotypes were similar in cases and control subjects respectively (P>0.05).2. 82 subjects were selected randomly from the whole T1D patients (P=0.53 at the allelic level, P=0.15 at the genotypic level), and were tested for the four antibodies. According to the number of individual positive antibodies, they were stratified into two subsets: the first subset with at least one positive antibody, the second subset with more than one positive antibody. However, the frequencies of SNP rs3747517 alleles and genotypes did not differ statistically after stratification (P=0.29 for alleles, P=0.55 for genotypes), so as showed between the GADA, ICA, IAA and ZnT8A positive subsets (P=0.86 for alleles, P=0.95 for genotypes).3. 79 subjects were selected randomly from the whole T1D patients (At the allelic level, P=0.5 for rs3747517, P=0.72 for rs1990760; At the genotypic level, P=0.69 for rs3747517, P=0.77 for rs1990760), and were classified into two subsets: one with the susceptible DRB1 and DQB1 alleles (DRB1*04, 0301, 0901 or DQB1*0201, 0302) and the second subset with low-risk, neutral or protective DRB1 and DQB1 alleles (all others). These analyses showed no significant association between either of the two SNPs and HLA classⅡlinked risk in T1D (At the allelic level, P=0.68 for rs3747517, P=0.1 for rs1990760; At the genotypic level, P=0.8 for rs3747517, P=0.26 for rs1990760).4. According to demographic (age at diagnosis, gender and family history of diabetes), clinical (BMI) and biologic (fasting C-peptide and post meal 2 hour C-peptide) features, the randomly selected patients were stratified and observed about the distribution of different alleles and genotypes. For SNPs rs3747517 and rs1990760, allelic and genotypic frequencies in each subset did not differ statistically from the whole T1D group (P>0.05). Also, in each subset, the demographic, clinical and biologic features did not show significant difference between genotypes (P>0.05).Conclusions:In Chinese Han population, we conclude that IFIH1 rs3747517, but not rs1990760, associates with T1D significantly. G allele of IFIH1 rs3747517 may implicate the susceptibility of T1D, but SNP rs3747517 has no effect on the positive rates of GADA,ICA,IAA and ZnT8A. Besides, both of the SNPs have no interaction with HLA-DRB1,HLA-DQB1 linked risk, and are not preferentially associated with a particular disease phenotype (age at diagnosis, gender, family history of diabetes, BMI, fasting C-peptide and post meal 2 hour C-peptide).