| Part 1 The Prospective Study of Thyroid Autoimmunity in Type 1 DiabetesObjective:To investigate the relationship between type 1 diabetes and thyroid autoimmunity.Design:Prospective and case-control study.Subjects and Methods:120 type 1A diabetes,77 type 1B diabetes, and 130 Latent autoimmune diabetes in adults (LADA) patients were recruited. Physical examination was performed and sera were sampled for the measurement of thyroid peroxidase antibody (TPO-Ab), thyroglobulin antibody (TG-Ab), glutamic acid decarboxylase antibody (GADA) and uTSH. All the patients accepted follow-up 4 years later and above indexs were reexamined.Results:1. The frequency of TPO-Ab in type 1A diabetes was higher than in type 1B diabetes (27.5% vs 14.3% P<0.05), but had no difference in LADA patients (18.5%) at the time of registration. After the 4 years followed-up period, the prevalence of TPO-Ab in type 1A diabetes increased to 30.8%,which was higher than in type 1B diabetes and LADA patients (15.6%,20.0% respectively P<0.05). 2. Compared with those without thyroid antibodies, thyroid-antibody positive type 1A diabetes were more likely to have goiter and thyroid autoimmune disease family history. The prevalence of the high titer GADA (GADA≥0.3) were increased in type 1A diabetes with thyroid autoimmunity than those without before or after the followed-up (69.8% vs40.2%,58.1% vs 33.8%, P<0.05).3. Thyroid-antibody positive type 1B diabetes were more likely to be female and have lower C-peptide level and BMI compared with those without thyroid antibodies. (P<0.05).4. Compared with those without thyroid antibodies, LADA patients with thyroid antibodies were more likely to be female and have lower C-peptide level (P<0.05).52.9% thyroid-antibody positive LADA patients had high titer of GADA, which was significantly higher than those thyroid-antibody negatives (25.0, P<0.001).5. The frequency of TG-Ab and TPO-Ab in LADA1(GADA≥0.3) patients were higher than in LADA2 (0.05≤GAD-Ab<0.3) patients and type 1B diabetes before or after the followed-up (P<0.05), but had no difference from type 1A diabetes.6. Multiple logistic regression revealed that thyroid antibody positivity in the initial study were significantly associated with thyroid dysfunction in type 1 A, type 1B diabetes and LADA patients (OR=8.272, 6.5,4.045 respectively). Conclusion:1. LADA1 patients, similar to type 1A diabetes, have higher risk for thyroid autoimmunity than LADA2 and type 1B diabetes. LADA1 may be one of the components in autoimmune polyendocrine syndrome (APS).2. T1DM patients and LADA patients, especially those with high titer-GADA or females, are at high risk for thyroid autoimmunity.3. The presence of thyroid antibody may predict high risk for thyroid dysfunction, so regular screening of thyroid antibody is recommended in LADA and T1DM patients. Part 2 Epitope Analysis of GAD65 Autoantibodies in Type 1 Diabetes with Thyroid AutoimmunityObjective:To compare the epitopes of GAD65 autoantibodies in T1DM and LADA patients with and without thyroid autoimmunity.Design:Cross-sectional and case-control study.Subjects and Methods:The GADA levels and their relative epitope reactivities to N-terminal (GAD65-N), Middle (GAD65-M) and C-terminal (GAD65-C) regions of human GAD65 were determined by radioligand immunoassay in 112 T1DM,107LADA patients and 100 controls. TPO-Ab and TG-Ab were measured by radioimmunoassay.Results:1. Antibodies reactivity to N-terminal, Middle, C-terminal epitope of GAD65 was 11.6%,31.2%,68.8% in T1DM patients and 22.4%,31.8%, 47.7% in LADA patients respectively.2 The N-terminal of GAD65 was recognized by 22.4% of LADA patients compared with 11.6% in T1DM patients (P=0.002). More T1DM patients recognized C-terminal of GAD65 than LADA patients (68.8% vs 47.7％,P=0.002)。3.46.0% T1DM patients and 51.6% LADA patients with thyroid autoimmunity had antibodies directed to both GAD65-M and GAD65-C, wich was higher than those without thyroid autoimmunity (14.5%,18.4% respectively, P<0.001).4. LADA patients with M+C-predominant GAD65Ab reactivity have higher risk for thyroid autoimmunity, lower C-peptide level, higher titer of GAD A (GADA≥0.3), and a need for insulin therapy compared with non-M+C-reactive patients (P<0.05).5. Multiple logistic regression revealed that GAD65-M+CAb positivity were significantly associated with thyroid autoimmunity in T1DM and LADA patients (OR=5.016,4.724).Conclusion:1.The majority of T1DM and LADA patients had antibodies directed to Middle and C-terminal epitopes of GAD65. GAD65Ab reacting with the N-terminal epitope were more common in LADA patients than TIDM, GAD65Ab binding to the C-terminal epitope were more frequent in T1DM than in LADA patients. These results indicate that there are both similarities and differences in the humoral response to GAD65 in LADA and T1DM patients.2. LADA patients with M+C-predominant GAD65 reactivity have clinical features more similar to those of T1DM patient.3. T1DM and LADA patients with GAD65-M+C epitope specific reactivity show an increase risk for thyroid autoimmunity. Part 3 The Association of CTLA-4 and HLA-DQ Gene Polymorphisms with T1DM and Thyroid AutoimmunityObjective:To investigate the association of CTLA-4+ 49A/G,CTLA-4 CT60 and HLA-DQ gene polymorphisms with T1DM and thyroid autoimmunity.Design:Cross-sectional and case-control study.Subjects and Methods:402 T1DM,231 LADA patients and 476 controls were recruited in the study. The CTLA-4+49 A/G and CT60 gene polymorphisms were analyzed by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. The polymorphisms of HLA-DQ were typed by PCR sequencing-base method. TPO-Ab and TG-Ab were determined by radioimmunoassay. GADA was measured by radioligand immunoassay.Results:1. The CTLA-4 +49 A/G polymorphism was significantly associated with T1DM complicated with thyroid autoimmunity (OR=2.454,P<0.001) and with T1DM alone (OR=1.531, P<0.05).2. The CTLA-4 CT60 polymorphism was also significantly associated with T1DM complicated with thyroid autoimmunity (OR=2.503, P<0.001) and with T1DM alone (OR= 1.428, P=0.011). 3. The CTLA-4 +49 A/G polymorphism was significantly associated with LADA (OR=1.463, P=0.02).The frequency of G allel of CTLA-4+ 49A/G and CT60 gene has no difference in LADA patients with and without thyroid autoimmunity.4. T1DM with thyroid autoimmuntiy were characterized by a significantly higher frequency of CTLA-4+49A/G, CT60 GG genotype (57.0%vs38.8%,76.6%vs58.3%,P=0.001) and HLA DQA1*03-DQB1*0401 haplotypes (11.3% vs 6.3%,P=0.0 42) compared to the patients without thyroid autoimmuntiy.5. The frequencies of HLA DQA1*03-DQB1*0401 and DQA1*05-DQB1*0201 haplotypes in LADA patients with thyroid autoimmunity were higher than those without.Conclusion:1. CTLA-4+49 A/G and CT60 gene polymorphism confers genetic susceptibility to type 1 diabetes, particularly in patients with thyroid autoimmunity. HLA-DQA1*03-DQB 1*0401 is a predisposing genetic marks for the development of thyroid autoimmunity in T1DM patients.2. CTLA-4+49 A/G gene polymorphism may be associated with LADA patients. HLA-DQA1*03-DQB1*0401 and DQA1*05-DQB1 *0201 is associated with the development of thyroid autoimmunity in LADA patients.
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