The Function Of Gene Mutation In1α Hydroxylase

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 mutation in the25-hydroxyvitamin D3la-hydroxylase (la-hydroxylase, CYP27B1) gene. To date, nearly40mutations in the la-hydroxylase gene of PDDR patients have been identified. We did identify4novel mutations in5Chinese patients before but did no function study. So we established an overexpression cell culture model of CYP27B1in vitro, expressed both the wild type and the mutant gene, detected their hydroxylation products (l,25(OH)2D3)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 activity and the clinical feature.Methods:1. In order to get the mutant gene G57V and L333F, we did the site direct mutagenesis to the cDNA (pCMVXL5-CYP27Bl), amplificated the plasmids in E. coli, then extracted the plasmids to get sufficient DNA for transfection.2. In order to establish an overexpression cell culture model, human CYP27B1expression plasmids (pCMVXL5-CYP27Bl) containing the wild type and the mutant CYP27Blgene were transfected into293T cell by lipofectamine.3. In order to observe the enzymatic catalysis reaction, we added different amount of25OHD3to each plate36h after transfection to make different concentration of substrate. Then we incubated the plates in37℃,5%CO>2for8h.4. In order to detect the concentration of1,25(OH)2D3,we collected the cell culture fluid,did the extraction and purification through the cartridge pack, and the radioimmunoassay for25OHD3and1,25(OH)2D in all the sample. 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.5. 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 level of expression of wild type and the mutant CYP27B1gene was proved similar. There was enzymatic activity in expression products of both wild type and mutant gene, because both of them could produce detectable1,25(OH)2D3. There was significant difference in enzymatic activity between the wild type and the mutant gene expression products.Conclusion:We have successfully established a CYP27B1overexpression cell culture model. Based on this model, we were the first group to analyze the function of two novel CYP27B1missense mutations in Chinese. The data proved that there were significant differences in enzymatic activity between the wild type and the mutant gene G57V (56.8%) and L333F (70.75%), and there was some relationship between the mutant gene activity and the clinical feature of the patients.