The Cyp17a1 Gene Sequence Analysis And Function Prediction Of Two Sisters And Their Parents With 17a-hydroxylase Deficiency

Background and ObjectiveMutations in the CYP17A1gene(cytochrome P450,family 17,subfamily A, polypeptide 1) can impair steroid biosynthesis in the adrenals and gonads,resulting in 17a-hydroxylase/17,20-lyase(P450c17)deficiency,leading to hypertension, hypokalemia sexual infantilism and primary amenorrhea in females(46,XX), pseudohermaphroditism in males(46,XY);at present,about 56 mutations in the CYP17 gene associated with 17a-hydroxylase/17,20-lyase deficiency have been described.Now,the reserch of 17a-hydroxylase deficiency mainly focused on two aspects. First,sequenced the CYP17A1 gene directly to identify the mutations and constructed a recombinant containing the mutations,then,the function of the mutant proteins was identified at the cell level;Second,A three-dimensional computer modeling for P450c17 enzyme was built to analysis and predict how these mutations affected the space structure and function of the proteins by use of bioinformatics software and dabase information.To date,there were more than 300 clinical reports associated with 17a-hydroxylase deficiency which have been described,including above 50 cases in our country,however,the methods of molecular biology was limited to several hospitals.Thus,we analyzed clinical characteristics of two sisters with P450c17 deficiency,and sequenced CYP17A1 gene of two sisters and their parents,the aim was to understand the molecular mechanism in P450c17 deficiency with the computer modeling for P450c17 enzyme.Materials and MethodsProband,patient I,a 15 yr-old female with karyotype of 46,XX;patientⅡ,the younger sister of the former,but her karyotype is 46,XY,the two sisters were diagnosed as P450c17 deficiency according to their clinical features and basal hormone assays in the first affiliated hospital of ZhengZhou university;First genomic DNA was extracted from peripheral blood leukocytes of two sisters and their parents, then all eight extons and intron/exton boundaries of CYP17A1 gene were amplified by PCR using eight pairs of primers,the amplified PCR products were purified by agarose gel and then directly sequenced.In order to confirm the DNA sequences of different alleles,some fragments were inserted into pGEM-T Easy vector and then subclone sequenced.At the end,sequencing results were compared with the established human CYP17A1 sequence(GeneBand M63871) and the computer model of the human P450c17 was used to futher understand how these mutations of CYP17A1 gene affected P450c17 enzymatic activity.Result1.Missense mutation in extron 6,the substitution of 6535 C＞T cause 362 amion acid from Arg to Cys,the two sisters and their father were heterozgous mutations,but there was normal sequence in their mother's.2.A 9bp deletion there existsed 9bp deletion in two sisters and their mother, which was D487 F489del in extron 8,and their father was noraml.3.Synonymous mutation①codon 46 from CAT to CAC,H46H;codon 65 from TCT to TCG,S65S in extron 1;②codon 283 from GAT to GAC,D283D in extron 5.4.CYP17 gene polymorphisms a C base inserted into the upstream of 5'-flanking in four individuals,that is:IVS4-2insC.5.Computer modeling for P450c17 enzyme R362C in extron 6 destroyed the function of P450c17 enzyme through subtly disrupting the eletron transfer from POR to P450c17 and the deletion of D487_S488_F489 in extron 8 changed the structure ofβ-folding.ConclusionWe analyzed the CYP17A1 gene features in two sisiters with P450c17 deficiency and therir parents,a novol compound heterozyous mutation were identified:missense mutation R362C in extron 6,which is common in Brazil,and D487 S488 F489del in extron 8,which is prevalent in Southeast Asia.and described its molecular mechanism by the computer model of the human P450c17.