Establishment And Application Of A Method For Gene Diagnosis Of 21-hydroxylase Deficiency

Objective:To establish a new method of 21-hydroxylase gene(CYP21A2) mutations for congenital adrenal hyperplasia(CAH) by using Fluorescence Polarization technology associate with direct sequencing,and meanwhile, testing 2 most common gene deletions and 8 point mutations of CYP21A2 gene in chinese patients to verifying the accuracy of the system and clinical exploitativeness,and then analysis the gene mutation which detected by the system, discussing the relationship between genotype and clinical phenotype.Methods:The peripheral blood samples of 40 normal people and 3 patients were collected and genomic DNA were extracted.To detect the gene deletion of CYP21A2,at frist,universal primers were designed to amplifiy CYP21A2 and CYP21A1 P allele fragments, and then used specific TAMRA fluorescence labeled probe to hybrid with amplification products, at last,used fluorescence polarization instrument to test FP value to determine whether the gene deletion was happened.We designed the sequence GAGACTAC on the primer whichwas specific to CYP21A2 to detect the Exon3 Del 8 bp(GAGACTAC) deletion.To detect point mutations,5 pairs highly specific primers for CYP21A2 gene were designed according to the sequence differences between CYP21A2 and its pseudogene.The whole CYP21A2 gene was amplified and sequenced to detect the most common type of 8 kinds of point mutations in Chinese people which including P30 L, i2 g, I172 N,E6Cluster, V281 L, 920-921 ins T, Q218 X and R356 W.Results:CYP21A2 gene deletion was not detected by Fluorescence polarization method, the results have no statistical differences compared with direct sequencing method, corresponding rate reach up to 100%. No point mutation of CYP21A2 gene was found in the 40 healthy people.The three patients,one case was detected with Exon1 G56 R, Exon7 c. 920-921 insert T heterozygous mutations, another two patients carried Exon4 I172 N, Exon8 Q318 X heterozygous mutations.Conclusion:This study established a new method of gene mutations for 21-OHD patients which based on the fluorescence polarization in combination with direct sequencing. The new method, with advantage of simple operation,lower cost and accurate results,was an effective way which can be widely used in clinical, not only can analyze the type of gene mutation, but also can provide basis for genetic counseling and which was helpful for prenatal diagnosis.