Study On The Correlativity Between G-protein B3 Subunit Gene C825T Genotype And Obesity

Obesity has become a big problem for modern people. The diseases associated with obesity mainly include : hypertention, coronary heart disease, hyperlipemia, erbral infarction, diabetes,etc. Thus the study on obesity has drawn more and more attention. G-protein b3 Subunit Gene has polymorphic situs of C825T. The study by Gutersohn on allele gene analysis among 1153 subjects of five groups,indicates that polymorphism of this situs is related to the occurrence of obesity. This study also indicates that polymorphism of C825T situs on G-protein b3 Subunit Gene has something to do with the restoration of the pregnant women's normal postnatal weight. The polymorphism of C825T situs on G-protein b3 Subunit Gene is referred to the C(?)T change at nucleotide 825 of theGNB3 gene. This change activates an arval splice site, whichactivates a cryptic splice site that results in alternative splicing of exon 9.The alternative splicing of exon 9 causes the deletion of 498-620bp in mRNA nucleotide sequence transcripted from extron 9. This leads to the decrease of codogenic amino acids 41 and the deletion of helico-region GNB3 structure which results in the structural change inβ3 subunit . The structural change inβ3 subunit will enhance the activity of G protein and increase the gene expression of Giα2 subunit. The increase of the gene expression of Gia2 subunit will cause the hyperplasy of adipocytes and the lipid accumulation within cells. Thus, the final result is the occurrence of obesity.In this study, we applied the technology of real time fluorescence PCR combined with MGB to make quick screening of the polymorphism of C825T situs on G-protein b3 Subunit Gene. With the analysis on the polymorphism of C825T situs on G-protein b3 Subunit Gene among the subjects of different regions, the distribution frequency of polymorphism of C825T situs on G-protein b3 Subunit Gene can be measured.Meanwhile we measured the height , weight, body fat content and the BMI of the subject to analyze the dependablity between the obesity-related trait and polymorphism of C825T situs on G-protein b3 Subunit Gene, which can provide scientific basis for preventive intervention on obesity.SAMPLE AND METHODSample dateEDTA anticoagulated blood of 20 subjects over 18 years old from 21 provinces ,serve as the sample for domestic distribution of polymorphism of C825T situs on G-protein b3, and EDTA anticoagulated blood of 207 individuals having health examination in the Sir Run Run Shao Hospital, serve as the sample for the dependablity betwween the obesity-related trait and polymorphism of C825T situs on G-proteinb3Subunit Gene.Of the 207 individuals, 138 are males, 69 females. The average age is 42.3±10.3. Mononuclearcell was separated from the sample within 3 hours out of the body and kept in frig -20℃.MethodPeripheral blood mononuclear cell separationWe took suction of 5 ml anticoagulated blood (EDTA), separated PBMCs with FICOLL, and then scoured it with PBS buffer solution 3 times.Genome DNA extractionQiagen genome nucleic acid extraction kit was applied according to the manipulation procedures on the instruction.PRC reactionPRC reaction was performed with 2μl extracted genome DNA in 20μl reaction volume. The compositions of reaction volume include the following sustances: 10×Buffer 2μl dNTP 10mM/L (0.4μl),MgCl₂ 25mmol/L (1.4μl), 2.5U TaqDNA pclymerase (0.5μl), formwork 2μl, upper and downstream idioprimer 20pmol (0.5μl,respectively) , deionized water 12.7μl.The primer sequence is5'-AGGTCTG ATCCCTGACCC AC-3 and 5'-TGCTTAGAGCAACACTGAGGG-3'. 3-min apomorphosis at 94℃ and then circulation for 30times under the following conditions: apomorphosis 94℃20s, renaturation 56℃ 20, elongation 72℃ 30s, elongation for 10 min at 72℃,cooling at 25℃ for 5 min. Lastly, perform agarose electrophoresis.Nucleotide sequencing sequencing with end termination We detected C825T gene polymorphism with fluorescence PCR and MGB and conducted real-time PCR reaction with 4μl genome DNA in 40μl reaction volume.The components of reaction volume include : 10× Buffer 4μl,dNTP 10mM/L (0.8μl),MgCl₂ 25mmol/L (2μl_,2U TaqDNA pclymerase, formwork 4μl,upper and downstream idioprimer 1μl,specific exploring needle 0.3μl, deionized water 26.1μl The primer sequence is 5' -TCTCCCACGAGAGCATCA -3'and5'-GTCG ICGTAGCCAGCGAA-3.' The sequence of exploring needle is5 '-FAM-ATCACGTCCGTGGC—MGBNFQ and5'-VIC-AAGGCCACAGACGT—MGBNFQ. 3-min apomorphosis at94℃ and then circulation for 40 times under the following conditions: 94℃ 10s, 60℃ 50s.Collecting light source at 60℃. Obesity-related trait determinationThe determing traits include height,weight, waist girth, hip girth, and fat content. The fat content was measured with body reconstituent analysator Inbody 3.0.Statistical AnalysisStatistical package SPSS 11.0 was used to make statistical analysis on the data.RESULTSThe comparison between real time fluorescence PCR and nucleotide sequencingPolymorphism of C825T situs on G-protein b3 Subunit Gene of 20 samples was determined with nueleotide sequencing with the result of 5 cases of C/C homozygotic type, 6 cases of T/T, 9 cases of C/T. This result coincide with the result determined with fluorescence PCR. They are in corresponding relation.Frequency analysis of polymorphism of C825T situs on G-protein b3 Subunit GeneThe result indi cates that among the 420 subjects from 21 provinces , the distribution frequency of 825T monoploid and 825C monoploid was 46.90% and 53.10% respectively.The distribution frequency of genetype 825T/T ,825T/C and 825C/C was 22.38% ,51.42% and 28.10%,respectively.Other genetypes were not found, which is in accordance with Hardy-Weinberg Law.The relationship between polymorphism of C825T situs on G-protein b3 Subunit Gene and obesity-related traitsThe weight of the 825T/T genotype class was significantly higher than that of the 825C/C class. The differences among other classes are statistically insignificant. BMI of the 825T/T genotype was 25.8±2.9, much higher than that of 25T/C(23.7±3.3 )and 825C/C (24.0±4.1). There was no significant difference between BMI of 825T/C and BMI of 825C/C.The fat content of 825T/T genotype was 30.3±5.9, much higherthan that of 825T/C (27.19±6.0) and 825C7C (27.6±5.9) .Combining the data of 825T/C class and 825C/C class, we compared it with that of 825T/T class and found th25T/T class.CONCLUTIONSat weight was of no significant difference,while BMI and fat content were significantly higher in 8With the establishment of quick screening methord for polymorphism of C825T situs on G-protein b3 Subunit Gene by using real time fluorescence PCR, both the sensitiveness and the specificness of the methord were 100%.The obtained domestic distribution frequency of gene polymorphism and genotype of GNB3 T835C is in accordance with the Hardy-Weinberg law.GNB3 genotype is associated with body fat content.The body fat content of 825T/T genotype is significantly higher than that of 825T/C and 825C/C.