Study On The Clone And Polymorphism Of Adiponectin Gene

Objective:To clone the cDNA of human adiponectin for nucleotide sequencing and to investigate the relationship of Glyl5Gly polymorphism in adiponectin gene exon-2 with obesity and Type-2 diabetes.Methods:We extract total RNA of human adipose tissue and amplify the cDNA of human adiponectin by RT-PCR. The amplified product is cloned into plasmid pMD18-T by TA-cloning. The recombinant plasmid is then verified by sequencing. We collected data for analysis from three groups of subjects: 186 non-obesity subjects (Non-ob), 122 overweight or obese subjects (Obesity) and 213 patients with Type-2 diabetes (T2D). Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) is used to genotype the Glyl5Gly polymorphism of adiponectin gene exon-2 and the frequencies of genotype and allele are compared among the groups.Results:1. The cloning of cDNA of human adiponectin is successful. The recombinant plasmid (pMD18-T/ADPN) sequence is verified to share 100% homology with that of Genebank (Accession NM₀04797).2. There exists Gly15Gly polymorphism in adiponectin gene exon-2 among Han ethnic group in Tianjin, China. The results show that, the TT genotype frequencies are 57.5%, 59.0% and 56.3% in Non-ob, Obesity and T2D groups respectively;the TG genotype frequencies are 40.3%, 33.6% and 35.7%;the GG genotype frequencies are 2.2%, 7.4% and 8.0%. The GG genotype frequency ofT2D group is significantly higher than that of Non-ob group (P<0.05). There is no significant difference in allelic frequency among the groups.3. In Non-ob group, compared to those of TT genotype subjects, the SBP, LDL-C and FINS levels of TG genotype subjects are significantly higher (P<0.05), while their HDL-C concentrations are significantly lower (P<0:05);the SBP, LDL-C, FINS and HOMA-IR levels of TG+GG genotype subjects are significantly higher (SBP, LDL-C, FINS, PO.01;HOMA-IR, P<0.05), while their HDL-C and IAI levels are significantly lower (HDL-C, PO.01;IAI, P<0.05).4. In T2D group, the FINS levels of the GG genotype subjects are significantly higher than those of TT and TG genotype subjects (PO.01);the HOMA-IR levels of GG and TG genotype subjects are significantly higher than those of TT genotype subjects (PO.05), while their IAI levels are significantly lower (PO.05);the HOMA-IR levels of TG+GG genotype subjects are significantly higher than those of TT genotype subjects (PO.01), while their IAI levels are significantly lower (PO.01).Conclusion:The constructed clone of human adiponectin cDNA is verified by sequencing to share 100% homology with that of Genebank and is ready for further cloning expression. The Gly5Gly polymorphism in adiponectin gene exon-2 is significantly associated with hyperinsulinemia and dyslipidaemia in normal and Type-2 diabetic patients.