Identification Of Gene Mutations For Patients Of 46,XY Disorders Of Sex Development And In Vitro Functional Studies Of NR5A1 Gene Rare Variants

Context46,XY disorders of sex development(DSD)is characterized by incomplete masculinization genitalia,with gonadal dysplasia,and with/without the presence of Mullerian structures.At least 30 genes related to 46,XY DSD have been found.However,the clinical phenotypes of patients with different gene mutations overlap,and accurate diagnosis relies on gene sequencing technology.The Sanger sequencing is time-consuming and high cost,and the gene detection rate of 46,XY DSD patients was about 20%.It is especially suitable for diseases in which multiple gene are involved.and the pathogenic mechanism of most patients is still unclear.Therefore,it is important to select appropriate gene sequencing methods to improve the gene detection rate of such patients.NR5A1(nuclear receptor subfamily 5 group A member 1)gene mutation is one of the most common pathogenic mechanism of 46,XY DSD,which are detected in about 10%-20%of 46,XY DSD patients.NR5A1 is a key transcription factor that regulates the expression of various steroid hormone synthesis and sexual development related genes.Thus,it plays an important role in the development of hypothalamic-pituitary-adrenal glands and hypothalamic-pituitary-gonads.Studying the clinical features of the NR5A1 gene mutations and the pathogenicity of the mutations can improve the understanding of this disease and the function of NR5A1 protein.ObjectiveThis study aims to explore the pathogenic genes in 46,XY DSD patients and determine the distribution and incidence of disease-causing genes in these patients;and to analyze the clinical phenotype of patients with NR5A1 gene rare variants and verify the pathogenicity of NR5A1 gene variants in vitro.MethodsPatients with 46,XY DSD admitted to Peking Union Medical College Hospital were recruited between 2016.11 and 2018.04.Targeted next-generation and Sanger sequencing were performed to investigate pathogenic gene variants and validate gene variants,respectively.The clinical and endocrinological characteristics of patients with NR5A1 variants were retrospectively analyzed.In vitro functional studies were used to analyze the pathogenicity of novel rare variants.Results(1)A total of 18 patients with 46,XY DSD were recruited in this study,ten of which were detected disease-causing mutations and the incidence was about 56%.Three patients with AR or SRD5A2 gene mutations were detected respectively,and the mutation rate was 16.7%.One patient with NR5A1,LHCGR,CYP17A1 or MAP3K1 gene mutation were detected,respectively,and the incidence rate was 5.6%.(2)A total of twelve patients with NR5A1 gene mutations were recruited in this study.Two of the patients were assigned as female gender.All patients with NR5A1 rare variants had normal adrenal function and showed genital defects.Physical examination showed that 5 patients had female external genitalia,3 patients showed clitoris hypertrophy,3 patients were ambiguous genitalia,and 1 patient was hypospadias,respectively.All patients had bilateral testis and 45%of patients had Mullerian structures.Hormone tests showed that except for 3 patients in prepuberty and 1 patient in mini-puberty,the other patients showed high gonadotropin level.Two patients showed the normal testosterone level.(3)A total of 41.7%NR5A1 gene mutations were recurrent,including p.Ser32Asn,p.Gly35Asp,p.Asn44del,p.Arg84Cys and p.Leu233_Glu234insLeuGlnLeu.And 58.3%were rare novel variants,including p.Ser21Phe,p.Lys45Glu,p.Ala82Ser,p.Arg89Ser,р.Gly91Asp,p.Arg255Profs*44 and p.Gln362Serfs*20.The results of the dual luciferase reporter system showed that only one novel variant Gly91Asp significantly reduced the effect of NR5A1 on CYP11A1 transcriptional activity;the Ser21Phe mutant slightly reduced the transcriptional activity,and the Ala82Ser and Arg89Ser mutants did not impact protein function.The results of the minigene reporter system showed thatс.244G>A(Arg82Ser)led to the deletion of 19 bases at the 3’ end of exon 2 or the entire exon 2 of the NR5A1 gene RNA.ConclusionTargeted next-generation sequencing is an effective method to identify disease-causing gene mutation in 46,XY DSD patients,and 56%patients were detected harboring rare variants of related genes.AR and SRD5A2 genes are the most common pathogenic genes,accounting for more than half of the 46,XY DSD patients.Patients with NR5A1 mutations are mainly characterized by genital defects,and roughly normal adrenal function.Only one novel missense variant affected the transcriptional activity of NR5A1 on CYP11A1 and one nominal missense variant leading to abnormal splicing of NR5A1 mRNA were verified by in vitro study,suggesting that the further confirmatory research is required for elucidating the pathogenicity of rare variants.