The Clinical And Laboratory Study Of Differential Diagnosis Of Young And Middle-aged Non-obese Diabetes Patients

Objective Diabetes mellitus has now reached epidemic proportions globally. Patients that are not easily classified as either type 1 or type 2 diabetes (T1DM or T2DM) are increasing wordwide, especially in Asian countries. However, the early diagnosis of autoimmune diabetes and early initiating of insulin treatment will have great benefits on the effective control of blood glucose and prevention of the development of long-term diabetes complications. This study will analyze and compare the characteristics of clinical parameters, islet autoantibodies, T lymphocytes and cytokines of the young and middle-aged non-obese diabetes patients; try to explore the valuable immune biomarkers for early diagnosis of autoimmune diabetes; and in the meanwhile, to further clarify the possible role of T lymphocytes and cytokines in the pathogenesis of type 1 diabetes. In addition, a patient with mitochondrial diabetes (mtDM) was included in our study subjects. This study will furthermore explore the clinical and gene features of the patient and her pedigree members, intending to improve our understanding of mtDM.Methods The recent onset diabetes patients aged 18-45 years old, with body mass index (BMI) <23kg/m² and no past history of obese in the department of Endocrinology, Peking Union Medical College Hospital from 2007 to 2009 will be recruited in this study. Clinical informations will be collected and analyzed. Radioimmunoassay methods will be used to detect islet autoantibodies. T lymphocytes will be analyzed by flow cytometry. Cytokines such as IFN-γ, IL-2, IL-4 and IL-17 will be measured by flow cytometry, and ELISA methods will be implemented to measure the level of TGF-β. Genomic DNA was isolated from the peripheral blood leucocytes of the pedigree members. A certain gene fragment was amplified by PCR methods and then the PCR products were sequenced in an automated DNA sequencer.ResultsThe proportion of various types of diabetes of the study population and their general characteristicsExcluding one mtDM patient, there were 102 diabetic patients enrolled in this study. The percentage of T1DM was 68.6%, of T2DM was 20.6%, and 11 (10.8%) patients met the diagnosis criteria of latent autoimmune diabetes in adults (LADA). The mean age of onset of T1DM patients was (24.8+6.6 years) younger than that of T2DM (34.8±7.5 years) and LADA (42.2±5.1 years) patients (P<0.01). Diabetes family history was more common in T2DM (70.6%) than that in T1DM (27.4%) and LADA (28.6%) (P<0.01). The mean blood pressure of T2DM patients (130/80mmHg) was higher than that of T1DM (110/70mmHg) and LADA (110/70mmHg) patients (P<0.05).Clinical manifestations of T1DM, T2DM and LADA patients at diabetes onsetThe percentage of patients having obvious symptoms such as thirst, polydipsia, polyuria and weight loss in T1DM group (91.9%) was significantly higher than that of T2DM (12.5%) and LADA (37.5%) group (P<0.01). Diabetic ketoacidosis (DKA) was more common in T1DM patients (71.0%) than that in T2DM (6.3%) and LADA (30.0%) patients (P<0.01), and LADA patients were more prone to DKA than T2DM patients (P<0.01). Fasting blood glucose of T2DM patients (11.5±4.5mmol/L) was lower than that of T1DM (20.3±8.8mmol/L) and LADA (16.3±4.9mmol/L) patients (P<0.01). Glycated hemoglobin (HbA_1c) of T2DM patients (8.7+2.8%) was also obviously lower than that of T1DM (11.1±1.4%) and LADA (9.6±1.1%) patients (P<0.01).Biochemical indices of T1DM, T2DM and LADA patientsTriglyceride level of T2DM patients (1.6mmol/L) was higher than that of T1DM (0.9mmol/L) and LADA (0.8mmol/L) patients (P<0.05). High density lipoprotein-cholesterol level of T2DM patients (1.0mmol/L) was lower than that of T1DM (1.5mmol/L) and LADA (1.5mmol/L) patients (P<0.01). Total cholesterol and low density lipoprotein-cholesterol levels of the three groups were similar (P>0.05). Fasting C peptide level of T2DM patients (1.4±0.7ng/ml) was significantly higher than that of T1DM (0.4±0.3ng/ml) and LADA (0.4±0.2ng/ml) patients (P<0.01). Prevalence of GAD₆₅,ICA,IAA and IA-2A in T1DM,LADA and T2DM patientsThe prevalence of GAD₆₅ (64.3%) in T1DM patients was higher than that of IAA (17.1%) (P<0.05). GAD₆₅ in LADA patients was also more common than IAA (P<0.05). The prevalence of GAD₆₅ and ICA in T2DM patients (9.5% and 4.8%) was significantly lower than that in T1DM and LADA patients (P<0.05).Frequency distributions of GAD₆₅,ICA,IAA and IA-2A according to age at diagnosis in T1DM patientsIn T1DM patients, the prevalence of GAD₆₅ was similar in different age groups (P>0.05). Patients in the youngest age group tested positive more often for ICA than those in the older age groups (P<0.05). The youngest age group also had a higher prevalence of positive IAA and IA-2A than the older age groups, although the difference was not statistically significant.Combinations of the four autoantibodies in T1DM patientsThe patients had the highest prevalence of positive GAD₆₅ (14.3%) when considering only one antibody positive. Combining the determinations of two antibodies, GAD₆₅ and/or ICA was positive in 75.7% of patients, being the most frequent combination; the positive prevalence of GAD₆₅ and/or IA-2A combination was next to that of GAD₆₅ and/or ICA, being positive in 74.3% of patients.T lymphocyte in T1DM, T2DM patients and normal control subjectsThe frequency of CD4+CD25+ T-cell in T1DM, T2DM patients and normal control subjects was 9.08 + 3.93%, 8.50+4.16% and 8.83 + 3.87% respectively, the difference was not statistically significant (P>0.05). The frequencies of CD8+ T-cell and CD4+CD25+ -Foxp3+ T-cell were also similar in the three groups.Intracellular production of IFN-γ, IL-2, IL-4 and IL-17 in T1DM, T2DM patients and normal control subjectsIntracellular IFN-γand IL-4 expression of CD4+ T-cell in T1DM patients (1.73 + 0.79% and 2.18±1.97%) was lower than that in T2DM patients (5.45±2.39% and 4.81±2.37%) and normal control subjects (5.81±2.69% and 5.30±2.66%) (P<0.05). IL-2 and IL-17 producting CD4±T-cell was similar in the three groups (P>0.05).TGF-βlevel in serum of TIDM, T2DM patients and normal control subjectsThe mean TGF-βlevel in serum of TIDM, T2DM patients and normal control subjects was 252.92±52.80, 242.96±50.75, 247.05±63.46 and 250.75±50.89 pg/ml respectively, and the difference was not statistically significant (P>0.05).Mutational analysis of the mtDM pedigree membersDNA analysis showed that members of the maternal side all harboured the tRNA⁽Leu(UUR) A3243G mutation. The paternal members did not have the mutation. The heteroplasmy levels of the maternal relatives were variable.Conclusion In the diabetic population that have no evidences of obesity, T1DM accounts for the majority of them, and the incidence of T2DM is relatively low. Compared to T2DM, T1DM patients have a younger age of onset, more obvious symptoms, more ketoacidosis, higher level of blood glucose, poorer islet cell function and less genetic predisposition. The characteristic of LADA is between that of T1DM and T2DM. The prevalence of GAD₆₅ is not influenced by age, and it is the most sensitive autoantibody marker for adult-onset T1DM and LADA. The prevalence of IAA in adult-onset T1DM and LADA patients is very low. GAD₆₅ and/or ICA is the best cost/benefit test combination for adult-onset autoimmune diabetes. Imbalance between CD8+Teff and Treg and breakdown of peripheral tolerance may be one of the important pathogenesis of T1DM. However, the frequencies of these two types of T lymphocytes do not alter in peripheral blood of T1DM patients. Cytokines are important mediators of immune response, and their roles in the pathogenesis of T1DM are very complicated. There exists great controversy on the change of cytokines level in peripheral blood of T1DM patients. Teff, Treg and IFN-γ, IL-2, IL-4, IL-17, TGF-βmay not be used as reliable markers for autoimmune diabetes. The main cause of diabetes in this pedigree is the tRNA⁽Leu(UUR) A3243G mutation and the heteroplasmy level of this mutation is positively associated with earlier age-of-onset and increasing severity of diabetes. Specific types of diabetes should be in mind when diabetes presents itself with special transmission mode or with other extra-pancreatic manifestations.