| ObjectiveTo detect the HLA-DRB1 gene association with type 1 diabetes mellitus and find out susceptible HLA-DRB1 genes of type 1 diabetes mellitus in order to provide theoretical basis for studying etiopathogenisis of type 1 diabetes mellitus.Materials and Methods30 patients with type 1 diabetes mellitus and 30 healthy controls were choosed for the study.The HLA-DRB1 alleles were identified in patients with T1DM and healthy control subjects using polymerase chain reaction with sequence specific oligonucleotide probe (PCR-SSO), so as to understand the relationship of gene frequency between patients and healthy controls. First, HLA-DRB1 typing were detected in 30 patients and 30 controls by low-resolution typing.Then the alleles of low-resolution typing which they have significant difference were subtyped by high-resolution typing and the results of high-resolution typing are analysed between T1DM and healthy controls as well.ResultsFirstly,12 kinds. HLA-DRB1 alleles of HLA-DRB1 low-resolution typing in T1DM group were detected, which are DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*O9, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15 and DRB1*16. And the range of frequencies is from 1.67%-21.67%. The alleles of DRB1*03, DRB1*04, DRB1*09, DRB1*12 were higher than controls and their allelic frequencies are 16.67%, 23.33%,21.67%,13.33%, respectively. Whereas DRB1*01, DRB1*13, DRB1*16 in patients were lower in gene frequency and all of their allelic frequencies are 1.67%.13 kinds of HLA-DRB1 alleles were detected in healthy controls, which is DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15 and DRB1*16. The range of frequencies is from 1.67%-16.67%. And the alleles of DRB1*12, DRB1*13, DRB1*14, DRB1*15 were higher in gene frequency in patients, they mean that 15%,10%,10%,16.67%, respectively. A decreased frequency was observed in DRB1*03, DRB1*10, DRB1*16 and they are 3.33%,1.67%, and 1.67%.Owing to the gene frequencies of alleles DRB1*03, DRB1*04 and DRB1*09 have significant difference between patients and healthy controls subjects (P<0.05), and the patients were higher than controls.which are DRB1*03 (16.67% VS 3.33%, P=0.015); DRB1*04 (23.33% VS 8.33%, P=0.024) and DRB1*09 (21.67% VS 8.33%, P=0.041) Therefore DRB1*03, DRB1*04, DRB1*09 are susceptible genes in patient with T1DM and have a positive correlated with T1DM. However, DRB1*15 (5% VS 16.67%, P=0.040) also has a significant difference between patients and healthy controls and the patient group are lower than control group. Therefore DRB1* 15 is protective gene and have a negative correlation with T1DM.No significant differences were observed in the haplotype DR3/DR4, DR3/DR9 in patients with T1DM and controls.Secondly, the patients and controls with significant differences of alleles of DRB1 (DRB1*03, DRB1*04, DRB1*09, DRB1*15) were subtyped by high-resolution of PCR-SSO.Only DRB1*03:01 was detected out of 10 patients with DRB1*03 and four kinds of DRB1*04 high-resolution alleles were observed in 14 patients, they are:DRB1*04:05 (9 cases) with the alleles of gene frequency 15%; DRB1*04=04 (2 cases), with the alleles of gene frequency 3.33%; DRB1*04:03 (1 case) with the alleles of gene frequency 1.67% and DRB1*04:01 (2 cases) with the alleles of gene frequency 3.33%. Among of them, the DRB1*04:05 alleles are prevalent in patients and the DRB1*04:04, DRB1*04:03, DRB1*04:01 alleles are not common appear. DRB1*09:01 were detected in 13 patients with DRB1*09 and DRB1* 15:01 was observed in 3 patients with DRB1*15.In the group of control subjects, one kind of DRB1*03 high-resolution alleles DRB1*03:01 in 2 healthy controls were found out and two kinds of DRB1*04 high-resolution alleles in 5 healthy controls with DRB1*04 were observed:DRB1*04:06 (4 cases) with the alleles of gene frequency is 6.67%; DRB1*04:05 (1 cases), with the alleles of gene frequencies 1.67%. One kind of DRB1*09 high-resolution alleles in 5 healthy controls was detected, which is DRB1*09:01. One kind of DRB1*15 high-resolution alleles in 10 healthy controls was found, which is DRB1*15:01.DRB1*03:01, DRB1*04:05, DRB1*09:01 have significant difference between patients and healthy controls, and the patients were higher than controls, that is:DRB1*03 :01 (16.67% VS 3.33%, P=0.015),DRB1*04:05(15% VS 1.67%, P=0.008),DRB1*09 :01 (21.67% VS 8.33%, P=0.041). Hence DRB1*03:01, DRB1*04:05, DRB1*19 :01 are susceptive genes and have a positive correlation with T1DM.Conclusion:PCR-SSO higher resolution typing is the typical method we used for this study and found that DRB1*03:01,DRB1*04:05,DRB1*09:01 are susceptible in patients with TIDM and the critical founding, which we reported firstly, that DRB1*04:05 is susceptible in patients with TIDM so as to provide theoretical basis for studying etiopathogenisis of type 1 diabetes mellitus in the future.
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