The Role Of Zinc Transporter 8 And Its Antibodies In Type 1 Diabetes

Background and objective:Type 1 diabetes (T1D) is an autoimmune disease characterized by the selective destruction of B cells of the islets of Langerhans. A variety of antigens expressed in B cells are the key targets by this autoimmune damage, which lead to the destruction of islet B cells and finally the lack of insulin secretion. Zinc is an important component during the process of insulin storage and secretion. B cells need effective and specific transport proteins to accumulate sufficient amount of zinc. A cation effux transporter ZnT8 (SLC30A8), which is one of the major autoantigens of B cells, plays an important role in the synthesis and secretion of insulin by influencing the concentration of zinc ions. The polymorphism of aa325 in SLC30A8 gene determines its peptide specific of antibodies in T1D patients . ZnT8 autoantibodies (ZnT8A) is very important in diagnose and prediction autoimmune diabetes . Combined detection of ZnT8A , glutamic acid decarboxylase antibody (GADA), insulinoma associated protein -2 (IA-2) antibody and anti-insulin antibodies (IAA) can screen the pre-clinical T1D patients in the first realtionships and diagnose clinical patients with T1D.Methods:Subjects: Patients with type 1 diabetes(n=152) were recruited in Jiangsu province between 2008 and 2009 as patients participating in the studies, The median age was 20 years,with lower and upper quartiles at 2 and 56 years, respectively, The median duration of diabetes was 4.0 (range 0.0–16.0) years. A total of 126 type 2 diabetes had a median age at diabetes onset of 36 years,Type 1 and type 2 diabetes were diagnosed by 1999 WHO diagnostic criteria, The male-to-female sex ratio in both groups was 1.1. The 102 healthy control subjects (median age 23 years) had no family history of diabetes and other autoimmune diseases, 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.1, The detection of islet autoantibodies ZnT8A and GADA, IA-2A, IAA:Each islet autoantibody assay was run with 16 matched control samples and an eight-step doubling dilution of a high-titer T1D sera standard prepared from a pool of nine new-onset sera, which at a 1:10 dilution, Normalization was achieved by using the immunoprecipitation index (sample–negative control) /( positive control–negative control). we use the radio-immunoprecipitation assay to detect the four islet autoantibodies, the results was represent with the antibody index.2, The polymorphism of SLC30A8 gene rs13266634:Genomic DNA was extracted from heparinized blood from the above diabetes patients and healthy controls using standard procedures, and were genotyped for the rs13266634 single nucleotide polymorphism (SNP). the SLC30A8 gene rs13266634 polymorphism was genotyped by the polymerase chain reaction and restriction fragment polymorphism(PCR-RFLP) technique, The PCR products were digested with a PvuII, resolved on a 1.5% agarose gel and stained with ethidium bromide, observed the digestion results with the UV light.Results:1, Results showed that ZnT8A,GADA,IA-2A and IAA antibodies were detected in 46.7% , 42.8% , 36.2% ,.27.0% of type 1 diabetes (n=152), The positive rates of two or more autoantibodies in type 1 diabetes were 38.4%, and that of ZnT8A was 1.6%, GADA3.2%, IA-2A 1.6% in type 2 diabetes respectively, no positive detection by IAA(n=126). In healthy subjects ZnT8A was 1.0%, GADA 1.0%, without IA-2A and IAA positive(n=102). The positive rate of combined detection of ZnT8A, GADA, IA-2A and IAA was 91.4% in patients with type 1 diabetes, which was significantly higher than that in type 2 diabetes and negative control group (P <0.01). In type 1 diabetes the positive rate of the four antibodies were GADA> ZnT8A> IA-2A> IAA, the combined measurement of four antibodies positive rate was significantly higher than the positive rate of single autoantibody.(P <0.01), the detection of serum ZnT8A, GADA, IA-2A , IAA might be regarded as clinical significance for classification, treatment and predict prognosis of diabetes.2, The SLC30A8 rs13266634 polymorphism (Arg325Trp) in type 1 diabetes was325Arg (C/C) genotype 32.2% (49/152), 325Trp (T/T) genotype 21.7% (33/152), and 46.1% (70/152) were C/T heterozygotes. furthermore, C/C, T/T, C/T genotypes were found in 48(45.3%), 23(21.7%) and 35(33.0%) of 106 health subjects and in 58(46.0%), 16(12.7%) and 52(41.3%) of 126 type 2 diabetes, respectively. and the aa325Arg(C/C) genotype was 42.3% among ZnT8 autoantibody-positive patients. The SLC30A8 SNP allele frequency (61.3%C and 38.7%T) in type 1 diabetes patients varied little with presence of ZnT8 autoantibody, suggesting that the SLC30A8 rs13266634 polymorphism did not modify the risk of type 1 diabetes.Conclusions:We conclude that ZnT8 autoantibodies have important diagnostic and predictive value in the autoimmune diabetes , and the combined detection of four kinds of antibodies in patients contribute to the prediction, early diagnosis and classification of type 1 diabetes, which can guide clinical treatment. There was no difference in allele and genotype frequencies of the SLC30A8 SNP rs13266634 between patients with type 1 diabetes and control subjects, concluding that the SLC30A8 SNP polymorphism could not be associated with the development of type 1 diabetes in jiangsu region.