Establishment Of Gene Diagnostic Methods For Three Common Disorders Of Sex Development Disease

Objective:Disorders of Sex Development(DSD)are a group of congenital diseases caused by the abnormal development of sex chromosomes,gonads,and accessory organs,which will affect the patient’s reproductive function to varying degrees.More importantly,it may induce relevant problems in patients’ sexual development,sexual psychology,sexual behavior,etc.,causing psychological and behavioral deviations.Improper handling may become a social problem.Therefore,early diagnosis and treatment of such patients are extremely important.The genetics diagnosis of DSD is the basis of treatment.Genetic diagnosis can improve the patient’s psychological state while assisting in clinical diagnosis,provide insight into the pathogenesis of the disease,and provide important references for clinicians to choose treatment options.This study intends to establish standardized genetic diagnosis methods for disease-causing genes in the three types of diseases with high incidence in DSD by collecting DSD cases to analyze the genetic causes of DSD patients and provide more theoretical basis for the treatment of DSD patients and prenatal diagnosis of patients.These three major pathogenic genes include Androgen Insensitivity Syndrome(AIS)pathogenic gene AR,5α-reductase Deficiency major disease-causing gene SRD5A2,congenital adrenal cortical hyperplasia(CAH)major pathogenic gene CYP21A2.Methods:Sanger sequencing combined with real time fluorescence quantitative PCR(Q-PCR)and multiplex ligase-dependent probe amplification(MLPA)molecular diagnostic techniques were used to establish diagnostic methods for AR gene,SRD5A2 gene,and CYP21A2 gene.And the constructed method was used to perform AR,SRD5A2,CYP21A2 gene analysis of 69 cases of clinical diagnosis of disorders of sex development,including 4 cases of congenital adrenal hyperplasia.Family analysis was performed on candidate pathogenic mutations.Results:1.Of the 69 cases with diagnosis of disorders of sex development,a total of 36 patients were found to have mutations.(1)Eight patients had mutations in the AR gene,with six different mutations,including two new mutations that were not reported: c.1792A>C and c.2606C>T.(2)Twenty-five patients had homozygous or complex heterozygous mutations in SRD5A2 gene,a total of sixteen different mutant alleles,of which three mutations have not been reported: c.269A> C,c.365A> G,c.662T> G.Twenty-one patients have SRD5A2 gene heterozygou mutation and five patients have homozygous mutations.The exon 1 and exon 4 of SRD5A2 gene are mutant hot zone.c.680G> A and c.16C>T are hot spot mutation.(3)Two patients had mutations in the CYP21A2 gene.One patient was a compound heterozygous mutation with a heterozygous deletion of CYP21A2 gene and a heterozygous mutation of c.518T>A.Another patient was a homozygous mutation of c.293-13C>G.2.Two families with fertility requirements were collected for prenatal genetic testing.The results showed that 1 fetus did not carry the same AR gene c.2248A>G mutation as the proband.The other fetus carries the same CYP21A2 c.293-13C>G homozygous mutation as the proband.Conclusion:1.A method for the diagnosis of AR and SRD5A2 genes based on the sequencing of specific primers for amplification with universal primers was constructed,and a genetic diagnosis system for CYP21A2 gene was established using nested PCR-specific amplification and sequencing technology,Q-PCR technology.2.The constructed method was uesed for detecting AR,SRD5A2 genes for 65 patients with 46,XY DSD.The genetic etiology of 34 patients with sexual development abnormalities was identified.8 patients had AR mutations and 26 patients had SRD5A2 mutations.Five different new mutations were detected in 5 patients.Among them,two new mutations were located in the AR gene and three new mutations were located in the SRD5A2 gene.These new mutation enriched the AR gene and SRD5A2 gene mutation spectrum.3.The established CYP21A2 gene diagnostic method was uesed for detecting CYP21A2 genes for 4 patients with CAH,and 2 cases has CYP21A2 gene mutations.4.Through this study,prenatal genetic diagnosis was successfully performed on two families with fertility requirements.