Study On The Role Of Zinc Transporter 8 In The Diagnosis Of Type 1 Diabetes Mellitus And Its Related Mechanism

Part IAssociation of SLC30A8Gene rs13266634C/T polymorphism with type1diabetes:case-control study and meta-analysisObjectives:To evaluate the relationship between SLC30A8gene rs13266634C/T polymorphism and type1diabetes mellitus(T1DM). Methods:A total of351T1DM patients and429healthy subjects were enrolled in this case-control study from September2008to November2010. The T1DM group included183males and168females, aged (20±11)years; and the control group included201males and228females, aged (23±4)years. All the participants underwent genotyping of the SLC30A8gene rs13266634polymorphism. In addition, systematic review and meta-analysis of the publishing literature on genetic association of SLC30A8rs13266634polymorphism and T1DM were performed in Pubmed and CNKI. Results:Our case-control study showed that rs13266634C/T polymorphism was not associated with T1DM(odds ratio=0.96,95%confidence interval (95%CI)=0.79to1.18, P=0.71). The literature search identified5studies that analyzed the association of the SLC30A8gene rs13266634polymorphism with T1DM, including10646T1DM patients and10207healthy controls. Since I2=0%and Pheterogeneity=0.595, all the studies were combined with fixed effects model. Odds ratios of C allele to T allele was1.02(95%CI:0.98to1.06, P=0.38), and Begg’s test revealed no significant publication bias. Conclusion:Our results indicated that SLC30A8gene rs13266634polymorphism is not associated with increased risk for T1DM. Part IIDiagnostic Value of Zinc Transporter eight autoantibody (ZnT8A) in Type1Diabetes MellitusObjective:To explore the prevalence of zinc transporter8autoantibody (ZnT8A) and the diagnistoc value of ZnT8A in the Chinese patients with type1diabetes mellitus (T1DM). Methods:Peripheral blood samples were collected from873type1diabetic patients and102healthy controls to detected the islet autoantibodies, zinc transporter8antibody (ZnT8A), glutamic acid decarboxyase antibody (GADA) and protein-tyrosine-phosphatase-2autoantibody (IA2A). The ZnT8A, IAA, GADA, and IA2A were detected by radioligand assay. Results:(1) The prevalence of islet autoantibodies(GADA48.7%, ZnT8A43.2%, IAA35.5%, IA-2A27.4%) were markedly higher than healthy controls (all P<0.01).(2) On the base of testing GADA, IAA and IA-2A, the positive rate with one antibody will increase by11.6%, and with two positive antibodies will increase by16.6%when ZnT8A is further tested (all P<0.01).(3) The prevalence of ZnT8A was11.6%in patients previously classified as autoantibodies-negitive.(4) ZnT8A were more often found in patients who already had autobodies against GAD or IA-2than those negtive for these.(5) The prevalence of ZnT8A in the patients with lower C-peptide(<200pmol/l) higher than that of C-peptide≥200pmol/l (P<0.05). However, The prevalence of ZnT8A was also related to duration of disease. Conclusions:The positive rate of antibody increase by11.6%in T1DM patients when ZnT8A is further tested. ZnT8A can improve the diagnostic sensitivity. However, ZnT8A are more often found in patients with other islet autoantibodies. In addition, the positive rate of ZnT8are also related to C-peptide level and disease duration. PartⅢIdentification of HLA-A*0201Restricted T Cell Epitopes for ZnT8Islet Cell AutoantigensObjective:To identify a panel of peptides derived from zinc transporter eight, which is one of the most important antigens of type1diabetes mellitus, through online programs prediction and peptide binding assay. Methods:Firstly, putative HLA-A*0201CD8+T-cell epitopes were predicted with the online programs SYFPEITHI and BIMAS. Then, the ability of peptides to bind HLA-A*0201and the stability of peptide/HLA-A*0201complexes were confirmed by peptide binding assays using the HLA-A2molecule in TAP-deficient174Xcem.T2cells in vitro. Results:(1)Tweny-one peptides who has high-affinity to HLA-A2*0201were identified by SYFPEITHI and BIMAS systems.(2) Peptides ZnT8107-115, ZnT8107-116, ZnT8153-161, ZnT8165-173, ZnT8221-229, ZnT8245-254, ZnT8273-282and ZnT8314-322stabilized HLA-A2expression at levels≥80%.However, only two peptide/HLA-A2complexes formed with ZnT8107-115and ZnT8107-116were stable at physiological temperature.(3) The adhesion of ZnT816-25, ZnT8115-123, ZnT8200-208, ZnT8292-300, ZnT8346-355and ZnT8291-300with HLA-A2in vitro is between40%and80%, but these peptides/HLA-A2complexes all unstable at physiological temperatures. Conclusions:(1) ZnT8autoantigens may be one of the initiate factors inducing autoimmune attack.(2) There existed discordant results between high-affinity epitopes derived from online programs and peptide binding assays in vitro.(3) ZnT8107-115and ZnT8107-116may be the pathogentic peptides of T1DM because of the high affinity to HLA-A2and high stability of the peptides/HLA-A2complexes.