AR And CAPN10 Gene Polymorphisms And Their Association With The Metabolic Syndrome In Elderly Men

BackgroundThe metabolic syndrome (MS) is a group of complicated metabolic disorders, including dysglycemia, dyslipidemia, hypertension and obesity. With the development of the social economy, the incidence of MS is accompanied with age increase. It is estimated that about 1/4 of the adults in the whole world could have MS. The risk of death from cardiovascular diseases or apoplexy in subjects with MS is twice of those without MS. The risk for T2DM in patients with MS has approximately 5 times.Recently, the relationship between androgen and the metabolic syndrome in elderly men has been paid much attention. Studies revealed that there were low-levels of DHEAS, TT, SHBG, FT and AR in elderly men with MS. Most epidemiology data show that low-level testosterone is a risk factor of MS and diabetes mellitus in elderly men. It has been documented that the low-levels of TT, FT, SHBG are correlated intensity with MS. Androgens act through the androgen receptor (AR). The expression and function of AR may controlled by the CAG repeat polymorphism in AR gene. This study intended to evaluate the relationship between the AR gene polymorphism and MS in old men from the aspects of AR gene polymorphism -AR expression -metabolic syndrome and to provide the theoretical evidences for the prevention and treatment of MS.Calpain10 is the first positional cloned predisposing gene of diabetes. Studies indicated that the subjects with the Calpain10-SNP43G/G genotype had a higher risk of diabetes, higher systolic blood pressure and triglyceride level than those with a G/A or A/A genotype. It was reported that SNP43 G/G genotype was related with central obesity or insulin resistance. Calpain 10 is possibly one of the predisposing genes of MS. Objectives(1) To construct the expression vector with 0, 21, 33 CAG repeats in exon 1 of human androgen receptor gene, which contains the full length AR coding sequence, full-length 5'-UTR and the partial 3'-UTR, the identified transcription and post-transcriptional regulatory regions. To observe the effects of different CAG repeats on AR gene transcription and expression by transfecting of the AR expression vector series into HEK293 cells.(2) To evaluate the association of the AR gene CAG repeat polymorphism with MS.(3) To investigate the distribution of the CAPN 10 gene SNP43 G/A polymorphism in elderly Han nationality population and its relation to MS.Objects312 men age 60 years or older were randomly selected from the epidemic survey data-bank. Among these old men, 119 (age 60-83, 68.97±5.39) suffered from MS; 193 subjects without MS were divided into three groups: group 1 with 1 component of metabolic disorder included 89 subjects (age 60-82, 68.06±4.82); group 2 with 2 components of metabolic disorders included 51 subjects (age 60-81, 68.29±5.35); group 3 with no metabolic disorder included 53 subjects (age 60-85, 68.43±5.21). The participants were all Han people living in the Wanshoulu Community of Haidian District in Beijing .All the MS subjects met the criteria for metabolic syndrome established by international diabetes federation IDF in 2005. Methods(1) Construction of expression vector with 0, 21, and 33 CAG repeats in exon 1 of human androgen receptor geneThe Genomic DNA with 0, 21, 33 CAG repeats were used as the templates in overlap extention PCR through which the desired gene was amplified. The spliced fragments were cloned into eukaryotic vector pAR-IRES2-EGFP.(2) Expression of AR in HEK293 cells The mutant recombinants with 0, 21, and 33 CAG repeats were transfected into HEK293 cells. Expression of AR was detected by RT-PCR and Western blot.(3) Detection of Calpain 10 gene polymorphismThe genomic DNA was extracted from the outer vein blood leukocytes by Phenol/Chloroform DNA extraction method. The SNP43 G/A polymorphism was determined by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and viewed by PCR product gel electrophorogram.Results(1) The expression vectors with correct 0, 21, 33 CAG repeats were confirmed by restriction enzymes and DNA sequencing, respectively.(2) The relationship between the expression of AR mRNA and AR protein and the CAG repeats: At AR mRNA level, CAG₀ is the highest, CAG33 is the second, CAG21 is the lowest, but the difference is not significant; At AR protein level, the difference is significant. CAG₀ is the highest, CAG21 and CAG33 has little difference.(3) Clinical feature of four groups: Weight, BMI, waist, Hip, SBP, DBP, TG, FBG rose gradually along with the increase of the number of the features of MS. However, the HDL level was inversely correlated with the number of clinical features. The variability of MS group is more significant than other three groups.(4) The mean CAG repeat length in the AR gene was 22.82±3.58 (range 10-33) in MS, 22.72±4.11 (range 8-34) in the group with no component of metabolic disorders, 22.25±3.24 (range 11-30) in group 1, and 22.41±3.54 (range 12-32) in group 2, respectivly. There was no significant difference in four groups.(5) According to the length of CAG, the objects were divided into two groups: the short CAG group (n<22) and the long CAG group (n>22). There was no significant difference of CAG constituent ratio in four groups. Weight, BMI, waist and Hip in the long CAG group were higher than those in the short CAG group. (6) Chi square test indicated that the frequencies of SNP-43 genotype did not differ in four groups (P>0.05). There was no difference of the allele gene frequency in four groups (P>0.05). Most genotype and allele gene were GG and G.(7) Multiple stepwise regression analysis showed BMI, Hip, weight, age, TC and FBG were positively correlated with central obesity. HDL was negatively correlated with it. Logistic multiple regression analysis showed TG, FBG, waist, SBP were associated with MS. CAG repeats and genotype of SNP-43 did not contribute to MS.Conclusions(1) There is no linear inverse relationship between the AR expression level and normal range of CAG repeat lengths, but the expression of CAGO is the highest.(2) The people of the long CAG group (n>22) have higher weight, BMI, waist and hip. BMI, Hip, weight, age, TC, FBG and HDL are related to central obesity.(3) The frequencies of Calpain 10 gene SNP43 G/A polymorphism genotype and allele gene in four groups are not different (P>0.05) . The most populated genotype and allele gene were GG and G.(4) TG, FBG, waist, SBP were risk factors of MS. CAG repeats and genotype of SNP-43 are not related to MS.