Association Between Abdominal Aortic Calcification And Osteoporosis In Postmenopausal Women In Beijing The Functional Study Of Mutation In1α-hydroxylase Gene CYP27B1

Objective:To investigate whether abdominal aortic calcification (AAC) is independent1y associated with osteoporosis in postmenopausal women in Beijing, China. Two traits of osteoporosis-BMD of femoral neck and lumbar spine, and osteoporotic vertebra fractures were involved in the present study, and AAC-a surrogate marker of atherosclerosis, also an independent risk factor of cardiovascular morbidity and mortality, was semi-quantitatively assessed. Potential confounders like age, BMI, years since menopause, exercise, life style, cardiovascular risk fators were considered.Methods:A total of1606generally healthy postmenopausal women were randomly recruited based in communities in Beijing, China.Demographic information, osteoporosis risk factors and cardiovascular risk factor was acquired through standard qustionares. BMD of femoral neck and lumbar spine (L2-4)was measured by Dual Energy X-ray Absorptiometry.Vertebra fractures were diagnosed by lateral radiographs, and AAC on lateral radiographs was assessed by AAC-24score system established previously. Logistic regression models were applied to evaluate the association between AAC and BMD, vertebral fractures.Results:1) Lower BMD of femoral neck or lumbar spine was significantly associated with AAC sore. In a multinomal logistic regression model, after adjusted for age, years since menopause, education, exercise, smoking, drinking, serum lipid profiles, Cystatin-C, Hypertension, diabetes mellitus, BMI, Coronary heart disease, Cardiovascular risk score, BMD in femoral neck(OR:0.152,95%CI:0.033-0.702,P<0.05)was a independent indicator of AAC sore,but BMD in lumbar spine was not associated with AAC any more.2) On the other hand, higher AAC score was significantly associated with osteoporosis and osteopenia in femoral neck or lumbar spine. In a multinomal logistic regression model, after adjusted for age, BMI, years since menopause, education, Cystatin-C,P1NP, β-CTX, smoking, drinking, osteoporotic fracture family history, exercise, past occupational intensity, AAC score (OR:1.113,95%CI:1.014-1.222,P<0.01) was an independent indicator of femoral neck osteoporosis, but not of lumbar spine osteoporosis. However, advanced AAC(AAC score≥6)(OR:2.092,95%CI:1.185-3.69,P<0.05) was independently associated with lumbar spine osteoporosis.3) In a multinomal logistic regression model, after adjusted for age, years since menopause, education, past occupational intensity, exercise, Cystatin-C, P1NP, β-CTX, smoking, drinking, AAC score(OR:1.072,95%CI:1.010-1.137, P<0.05)was a independent predictor of osteoporotic vertebra fractures.Conclusion:BMD of femoral neck is independently associated with AAC score; AAC score is independently associated with femoral neck osteoporosis, however, the linkage between AAC score and lumbar spine osteoporosis is much weaker. AAC score is independently associated with osteoporotic vertebra fractures. Overall, there is an association between AAC, BMD and osteoporotic vertebra fractures, independent of potential confounders. Objective:Pseudo-Vitamin D-Deficiency Rickets (PDDR), is a rare autosomal recessive disorder characterized by the early onset of rickets with a severe syndrome of rickets, and the cause of the disease is mutations in the25-hydroxyvitamin D la-hydroxylase (1α-hydroxylase, CYP27B1) gene. To date, nearly40mutations in the CYP27B1gene of PDDR patients have been identified.5novel missense mutations were found in Chinese patients with PDDR in our previous study, but the functional changes of these mutant were unclear. So we established an overexpression cell culture model of CYP27B1in vitro, expressed both the wild type and the mutant gene, detected their hydroxylation products to analyze whether there were difference between the activity of wild type and the mutant gene and whether there were relationship between the mutant gene activities and the clinical features.Methods:In order to get the mutant gene G73W、L333F、R432C、R459C and R492W, we did the site direct mutagenesis to the cDNA(pCMVXL5-CYP27B1), amplificated the plasmids in E. coli, then extracted the plasmids to get sufficient DNA for transfectionIn order to establish an overexpression cell culture model, human CYP27B1expression plasmids (pCMVXL5-CYP27B1) containing the wild type and the mutant CYP27B1gene were transfected into293T cell by lipofectamine.In order to observe the enzymatic catalysis reaction, we added different amount of25OHD3to each plate36h after transfection to make overdose of substrate. Then we incubated the plates in37℃,5%CO2for8h.In order to detect the concentration of1,25(OH)2D, we collected the cell culture fluid,did the extraction and purification through the cartridge pack, and the radioimmunoassay for25OHD and1,25(OH)2D in all the samples. Then we did the split-plot design factor analysis using SPSS to see whether there were significant difference between the wild type and mutant gene.In order to prove the expression of CYP27B1, we extracted the total protein of the transfected293T cell, and then did the western blot for the CYP27B1protein. Results:We successfully established the293T overexpression cell culture model, and in293T overexpression cell culture model, the expression of wild type and the mutant CYP27B1gene were proved similar.There were enzymatic activity in expression product of both wild type and mutant gene, because both of them could produce detectable1,25(OH)2D.There were significant difference in enzymatic activity between the wild type and the mutant gene expression products. The enzymatic activity of mutant gene G73W、L333F、 R432C. R459C and R492W is6.8%,6.2%,6.9%,5.5%,12.1%of the wild type respectively.Conclusion:We have successfully established a CYP27B1overexpression cell culture model. Based on this model, we were the first group to analyze the function of four novel CYP27B1missense mutations in Chinese. The data proved that there were significant differences in enzymatic activity between the wild type and the mutant gene G73W、L333F、R432C、 R459C and R492W, and there was some relationship between the mutant gene activity and the clinical feature of the patients.