HSD3B2 gen mutasyonunun fonksiyonel analizi

HSD3B3 deficiency is rare form of the congenital adrenal hyperplasia arised from the inactivating mutation of HSD3B2 gene (3β-hydroxysteroid dehydrogenase). Even though it is not frequently seen in the world, the prevalence of the HSD3B2 deficiency is higher specifically in the Turkey. This disease is defined as defects in adrenal steroid synthesis in adrenal gland and gonads. Therefore, understanding and characterisation of the mutational area is very crucial. Also, the possible influence of the mutation can be changed depending on the position of the mutation in HSD3B2 gene. The aim of this study to determine in vitro characterisation of the HSD3B2 enzyme with p.L304P mutant and try to understand the relationship the molecular structure and clinical symptoms. Also, influence of the p.L304P mutation on HSD3B2 enzymatic activity and rate of 17 OH pregnenolone conversion was investigated. In the experiment, pCMV3-HSD3B2 vector was used and target p.L304P mutation was created with site directed mutagenesis. All plasmids were transformed into the DH5 alpha. The presence of the p.L304P mutation and wild type HSD3B2 gene were controlled by sanger sequencing. Later, the plasmids with wild type and mutant were transfected into the HEK293T cell line. After the transfection, the cells were treated with 17 hydroxypregnenolone that is dissolved in the DMEM. The treatment was done at different time interval and different substrate concentrations. At last step, the DMEM was removed and the amount of 17 hydroxyprogesterone was measured with LCMS/MS. In conclusion, our experimental finding showed that the mutation dramatically cause decline in 17 hydroxyprogesterone. Also, the amount of the 17 hydroxypregnenolone was detected higher in wildtype as compared with mutant cell lines.