[The establishment and application of testing methods of tetraspanin 7 autoantibody in type 1 diabetes].

Objective: To establish the luciferase immunoprecipitation system assay (LIPS) to test tetraspanin 7 autoantibody (TSPAN7A) and evaluate its value in Chinese type 1 diabetes (T1D) patients. Methods: Renilla luciferase-tagged TSPAN7 plasmids were transfected into 293T cells to obtain Renilla luciferase-tagged TSPAN7 fusion protein. The cell lysate was incubated with sera overnight, followed by addition of protein A-agarose and extensive wash. Finally, the substrate of Renilla luciferase was added and luminescence was detected. Sera from 100 T1D patients [64 males and 36 females,with a mean age of (28±16) years], 119 type 2 diabetes (T2D) patients [78 males and 41 females,with a mean age of (47±12) years] and 98 healthy volunteers [55 males and 43 females,with a mean age of (28±12) years] from the Department of Metabolism and Endocrinology of the Second Xiangya Hospital, Central South University from 2014 to 2017, were tested by LIPS to evaluate the frequency of TSPAN7A. Radioligand binding assay (RLA) was used to test glutamic acid decarboxylase autoantibodies (GADA), protein tyrosine phosphatase-2 autoantibodies (IA-2A) and zinc transporter 8 autoantibodies (ZnT8A). Results: The frequencies of GADA, IA-2A, ZnT8A and TSPAN7A in T1D patients were 72.0%, 40.0%, 29.0% and 25.0%, respectively. After Bonferroni correction, the positivity of TSPAN7A was lower than GADA (P<0.001), but similar with IA-2A (P=0.035) and ZnT8A (P=0.630). The positivity of TSPAN7A in T1D patients was significantly higher than that in T2D (0.84%, P<0.001) and in healthy controls (1.02%, P<0.001). In combination with TSPAN7A, the positivity of islet autoantibodies in T1D patients increased from 82% to 85%. There was no significant difference in clinical characteristics between TSPAN7A-positive T1D and the other three islet autoantibodies-positive patients. Conclusion: This study succeeded in establishing LIPS method to assay TSPAN7A. Moreover, TSPAN7A are valid islet autoantibodies for T1D patients in China.

[Identification of HLA class â ¡ susceptible alleles and genotypes in latent autoimmune diabetes in adults].

Objective: To identify human leukocyte antigen (HLA)-DRB1, DQA1 and DQB1 susceptible alleles and genotypes in latent autoimmune diabetes in adults (LADA) patients of Chinese Han nationality. Methods: All subjects including 652 LADA patients and 1 181 healthy controls from 1999 to 2015 in Han nationality region of Hunan province were genotyped with high resolution at HLA-DRB1, DQA1 and DQB1 locus by PCR-sequence based typing (PCR-SBT). Frequencies of genotypes between patients and controls were compared by chi square test. Results: The DQA1 susceptible allele was DQA1*03 (OR=1.23, P(c)=0.028); the DQB1 susceptible alleles were DQB1*0201 (OR=2.24, P(c)<0.001), DQB1*0303 (OR=1.30, P(c)=0.030), DQB1*0304 (OR=10.23, P(c)=0.004) and DQB1*0401(OR=1.94, P(c)<0.001); and the DRB1 susceptible alleles were DRB1*0301 (OR=2.10, P(c)<0.001), DRB1*0405(OR=1.89, P(c)<0.001) and DRB1*0901(OR=1.36, P(c)=0.008), respectively in Chinese Han nationality LADA patients. The HLA-Ⅱ susceptible genotypes were DQA1*03/05 (OR=1.81, P(c)=0.007), DQB1*0201/0201(OR=5.74, P(c)<0.001), DQB1*0201/0303 (OR=2.58, P(c)=0.010), DRB1*0301/0901(OR=3.43, P(c)=0.028) and DRB1*0901/0901 (OR=1.82, P(c)=0.021), respectively in LADA patients. DQB1*0201 and DRB1*0301 were shared susceptible alleles for Chinese Han and Caucasian LADA patients, while DQA1*03, DQB1*0303, DQB1*0304, DQB1*0401, DRB1*0405 and DRB1*0901 were specific susceptible alleles for Chinese Han LADA patients. Interestingly, the DQB1*0303 allele was susceptible in Chinese while protective in Caucasian (OR: 1.30 vs 0.29). Conclusion: Susceptible alleles are DQA1*03, DQB1*0201, DQB1*0303, DQB1*0304, DQB1*0401, DRB1*0301, DRB1*0405 and DRB1*0901, and susceptible genotypes are DQA1*03/05, DQB1*0201/0201, DQB1*0201/0303, DRB1*0301/0901 and DRB1*0901/0901 in Chinese LADA patients.