The Relationship Between The Genotype And Clinical Phenotype With 21-hydroxylase Deficiency (21-OHD), And The Discussion Of The Three Unreported Mutations

Objective:To investigate the genotype and clinical phenotype of 130 children with 21-hydroxylase deficiency?21-OHD?and to understand the CYP21A2 mutation of the children in southwest China,and to analyze whether there is pathogenicity in three unreported mutations.Methods : Taking 130 patients with diagnosis of 21-OHD from November 2012 to March 2017 in Children's Hospital Affiliated to Chongqing Medical University as the research object,and extract 2-5ml peripheral blood from everyone of them under the principle of informed consent.PCR primers are designed by gene sequence during mutation detection,amplify on a real-time fluorescent quantitative PCR,then perform sanger sequencing,defect and repeat mutations.First,design PCR primers,then amplify and multiple PCR,and analyze by DHPLC.According to the severity in the loss of enzyme caused by gene mutations,21-OHD are divided into group A?I2g,loss of large fragments,Q318 X,Q319X,E6 Cluster?forming compound heterozygous mutations orhomozygous mutations,these mutations lead 21 hydroxylase activity to only 0% to 1%;Group B?I172N,I173N?forming homozygous and compound heterozygous mutations that contain I172 N or I173 N,the enzyme activity retains about 1% to 10%;group C?P30L,P31 L,282 l,V281L?forming homozygous or compound heterozygous containing P30 L,P31L,282 l,V281 L,the enzymes keep 20% to 50%.The pathogenicity of unreported mutations determined by whether it meet the conditions of the support of biology and genetics,and clinical features.Until the above conditions are met at the same time,the pathogenic mutation probability increases,then the gene function need to be further determined.Results : The first diagnosis age of group A is 0.07-0.17 years old,median age is 0.12 years old;and the first diagnosis age of group B is0.14-6.1years old,median age is 3.4 years old;and the first diagnosis age of group C is aged between 8 and 16 years old,and the median age is 12.6years old.Three groups are compared according to age P values < 0.01,reminding that genetic mutations affect enzyme activity more and diagnosis age is smaller.Diagnosis age can estimate severity of disease to a certain extent.There are 84 cases salt wasting,namely SW?93.3%?in 90 cases in group A,and 4 cases of SW in 34 cases in group B?11.7%?.6 cases in group C with no SW.After comparing the P values < 0.01 in three groups,we have hint that the lower the residual enzyme activity,the more obviousof SW,the more serous clinical phenotype.In this research,there are 30 different types of point mutation?containing 86.4%?among the 130 children,large fragment deletion and gene conversion?4.1%?.The most common point mutation is I2g?containing 36.50%?,then I172N?7.76%?,and I173N?6.80%?in the third place,followed by Q319X?5.90%?,R357W?5%?,R356W?4.10%?,Q318X?2.70%?,and single body type?1.8%?etc.The most common type of SW is I2 g,secondly Q319X?5.90%?,R357W?5%?,R356W?4.10%?,deletion of large fragments and gene conversion?4.1%?.The most common type of simple virilizing,namely SV,is I172 N,followed by I173 N,the highest detection rate of nonclassic is P30 L In the detection of 3 unreported mutations,c.879₈80inscctgatcgggact ?M1R is confirmed extremely high pathogenic mutation,and there is a large possibility that R149 H is pathogenic,which need to be further functional identified.Conclusion:Genotype is closely related to clinical phenotype,which provides prediction of the severity of disease and is of great significance for guiding clinical work and prenatal diagnosis.Among the 130 children,the most common mutation is I2g?36.50%?,followed by I172N?7.76%?and I173N?6.80%?.The detection in the research on the 3 unreported mutations,according to the criterion that meet the conditions of the support of biology and genetics and clinical features at the same time,c.879₈80inscctgatcgggact?M1R is confirmed extremely high pathogenicmutation.And there is a large possibility that R149 H is pathogenic,which need to be further functional identified.