| Objective: Type 2 diabetes mellitus(T2DM) is the most common form of diabetes mellitus(DM) and a heterogeneous disease characterized by insulin resistance and impaired β-cell function, at the moment we can not provide a complete explanation for the pathogenesis of T2DM yet. Human leukocyte antigen(HLA) system is known as one of the most complex antigen system, which carries genetic information that approaches 1‰, and possesses polymorphisms. It is also one of the major gene in determining disease susceptibility. Previous studies suggested that many diseases including T2DM had a common HLA predisposition , it was essential to study HLA-associated susceptibility to the genetic characteristies of disease. With the developing of molecular biology typing techniques, especially the presence of PCR, there appeared to be many gene typing methods such as PCR-RFLP, PCR-SSP and PCR-SSO, many studies about HLA-associated susceptibility to T2DM have been doing. Rich et al found that HLA-DR4 genotypes were associated with T2DM in 1991, a DR4 haplotype was transmitted from the T2DM parent to the type 1 diabetes mellitus(T1DM) offspring more often than expected and this phenomenon might represent a homogeneous subset of diabetes susceptibility, thereafter Wu Songhua et al had also reported associations between T2DM and HLA-DQA1 loci, Tuomi and Fukui et al observed respectively the HLA distribution in patients with antibodyes to glutamic acid decarboxylase in T2DM who tested positive. However, the study was undertaken to investigate the association of familial T2DM with HLA- DRB1*, DQB1 genes, especially these genes were either itself or others in a haplotype or their interaction, so far there was no report about it. In this study, six previously identified familial T2DM study subjects were selected from a random samples in Tang shan Hans to analyze the association of familial T2DM with HLA-DRB1*-DQB1* haplotype.Methods: 1. genomic DNA: According to standard criteria, patients with T2DM and their available family members had been recruited from six families, 22 patients satisfied the WHO criteria for T2DM. Briefly, genomic DNA was extracted from the peripheral blood samples using saturation phenol/chloroform from six families consisting of parents and affected and unaffected children. 2. PCR amplification: DRB and DQB primers were provided by the Biotest company, Germany. 100μl PCR reaction mixture for DRB low resolution typing contained: 60.5μl H2O, 10.0μl 10×PCR buffer, 16.0μl dNTP(2mM), 8.0μl primer Mix C, 0.5μl Taq(5U/μl) and 5μl DNA(50ng/μl). The genericamplification should give a product of 277bp, in presence of a DR2 positive DNA an additional amplification product of 399bp would be seen. 50μl PCR reaction mixture for DQB low resolution typing contained: 32.5μl H2O, 5.0μl 10×PCR buffer, 8.0μl dNTP(2mM), 2.0μl primer Mix A or B, 0.5μl Taq(5U/μl) and 2μl DNA(50ng/μl). The A amplification should give a product of 246bp, and the B amplification a product of 231bp, the control band should be seen at 422bp in the PCR samples without specific PCR product. Programed the thermocycler as follows: 95oC pre- denaturing 5min, and then 95 oC denaturing 15sec, 55 oC annealing 15sec, 72 oC extending 15sec, total 30 cycles, final extending 72oC 6min. 3. determination of HLA-DRB1*, DQB1* alleles: Using the polymerase chain reaction- sequence specific oligonucleotide (PCR-SSO) probe and enzyme linked probe hybridization assay (ELPHA). 24 DRB1-SSO, 23 DQB1-SSO probes were coated wells of microtest plate respectively. According to the procedure , 3 different strips were required for each DRB or DQB low resolution typing. After transfered of PCR product in a 2 ml reaction tube, denaturation solution were added, the double stranded PCR products were denaturated to single stranded products, added neutralizing solution and H2O, distributed diluted PCR product into each well for the hybridization , washed microwells and unbound probes were removed in this washing step, distributed conjugate dilut...
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