Atherosclerosis Related Gene Analysis

Objective (1) To study the clinical significance and mutation rate at point 3200,3206 of mtDNA 16srRNA and point 4136,4164,4216 of ND1 gene in patients with type 2 diabetes (T2DM) in Han people of HuBei province. (2)To investigate the pathogenesis of atherosclerosis in diabetes, and detect the expression of scavenger receptor CD36 in monocytes in patients Type 2 diabetes.Methods (1) Mitochondrial were detected using PCR restriction fragment length polymorphism (PCR-RFLP) analysis and Allele specific polymerase chain reaction (AS-PCR) and DNA sequencing. All mutations were analyzed by DNAstar,Primer ,mfold and Antherprot softwares. Five nucleotide substitutions of mtDNA (nt3200, nt3306, nt4136, nt4164, nt4216) were screened in 175 diabetics and 200 non-diabetic subjects.(2) In accordance with the criteria of the WHO ,diabetic patients were classified into two groups: well controlled diabetic patients (WCP)and poorly controlled diabetic patients(PCP)and compared with age and sex matched nondiabetic control group. The expression of CD36 protein and mRNA were evaluated by flow cytometry and RT-PCR.Results (l)In diabetic group, there were 2 carriers (1.14%) of 3200 T→C,11 (6.28%) of 3206 C→T, and 3 (1.71%) of 4216T→C(Tyr→His). Meanwhile, two novel mutations (7(4%)of 4164 (A→G) and4200 (A→T)were found. In control group, there were 8(4%) of 3206 (C→T) , 5(2.5%)of 4164( A→G). The point mutation 4136 (A→ G) was not found in two groups. The 3200 (T→C) mutation caused a great alteration in the minimal free energy secondary structure mode, while the 3206 mutation alter normal structure little. The protein secondary structure prediction revealed that there were difference between 4216 mutant and wild-type ND1 protein. (2) Flow cytometry and RT-PCR showed that the base intensity of monocyte CD36 protein expression (MFI) and CD36 mRNA were significantly higher in the diabetes group compared to controls(p<0.01). CD36 MFI and mRNA in the PCP were increased by 78% and 36%compared to the WCP. In the WCP and PCP group , CD36 MFI and mRNA were significantly higher in patients with diabetic atherosclerosis in comparison to patients with diabetic nonatherosclerosis(P<0.05). No significant difference was observed in CD14 expression between the different groups(P>0.05). The concentrations of plasma oxLDL were higher in the PCP group compared to WCP and control group(P<0.05), whereas oxLDL average values did not differ significantly between WCP and control group(P>0.05). In the WCP and PCP group, oxLDL levels were higher in patients with diabetic atherosclerosis than diabetic nonatherosclerosis(P<0.05). Conclusion (1) MtDNA 16SrRNA and ND1 gene mutations at nt3200 (T—C), and nt4216 (T-*C) might contribute to the pathogenesis of Type 2 DM with other genetic factors and environment factors; 3206(C-*T)^P 4164 (A-*G) might be gene polymorphism, and they might little contribute to the pathogenesis of Type 2 DM. (2) The increased expression of scavenger receptors CD36 may be one of the mchanism of accelerated atherosclerosis in diabetes. The poorly controlled diabetes patients are at higher risk for the vascular complications than those who well controlled.