Genetic Diagnosis Of Disorders Of Sex Development And Preliminary Molecular Pathogenesis Of Related Genes

Section Ⅰ.Identification and Analysis of the Genetic Variation in Patients with Disorders of Sex Development through Proband-Only Medical Exome SequencingObjective: Disorders of sex development（DSD）are a large group of heterogeneous diseases characterized by atypia of chromosomes,gonads,or anatomical structures,with the incidence of most specific types of DSD below 1/100,000.The pathogeneses are complex,and the clinical diagnosis rate is low.Next-generation sequencing（NGS）technology has greatly improved the molecular diagnostic efficiency of genetic diseases by virtue of its rapid,massively parallel detection.This part of the study aimed to identify the genetic variation of Chinese DSD patients with proband-only medical exome sequencing（POMES）and evaluate its clinical application value.Method:（1）According to the inclusion criteria of DSD,DNA samples from 88 patients and their family members were collected,and NGS was performed for proband.The procedures include library preparation,hybridization,enrichment,capture and onmachine sequencing etc.（2）The quality of data was evaluated according to parameters such as sequencing depth,and the variation was screened by Ingenuity software.（3）Candidate gene variants were verified by Sanger sequencing,and pathogenicity was evaluated according to the guidelines of the American College of Medical Genetics and Genomics（ACMG）with further classification and statistics.Result:（1）Eighty-eight probands were divided into 46,XY DSD（68/88,77.3%）and 46,XX DSD（20/88,22.7%）according to the karyotype.The most common age of the patients is between 9 to 14 years old.（2）After the sequencing data were filtered and verified,the candidate gene variants were identified in 47 patients（53.4%,47/88）,with the most common variants in SRD5A2 and AR gene.（3）We reported 27 novel variants.The types of variation included missense,nonsense,frameshift,splice site,in-frame insertion/deletion and copy number variation.The pathogenic and likely pathogenic variants accounted for 44.4%（24/54）and 25.9%（14/54）,respectively.（4）The diagnostic rates of 46,XY DSD and 46,XX DSD were 45.6%（31/68）and 20%（4/20）,respectively.Conclusion:（1）This section studies the condition of Chinese DSD patients,and enriches the gene spectrum,mutation spectrum and phenotypic spectrum of the rare types of DSD in Chinese population.（2）POMES technology,combined with standardized variants classification,could be used as clinical first-tier diagnostic strategies for 46,XY DSD.Section Ⅱ.Molecular Genetic Analysis of Pedigree with Leydig Cell Dysplasia Type 1Objective: Leydig cell hypoplasia（LCH）is a rare disease and one of the causes of 46,XY DSD.Inactivating variants in the luteinizing hormone/chorionic gonadotrophin receptor（LHCGR）gene account for the underlying LCH pathogenicity.This part of the study aimed to summarize and analyze the clinical data,family diagram and molecular genetic characteristics of LCH patients.Method:（1）Clinical data were collected and analyzed from the proband.Related serum hormone levels were detected and hormone stimulation tests were perfomed.（2）DNA of the proband was performed through POMES technology and the candidate variants were verified with Sanger sequencing.（3）The CRYP-SKIP and BDGP prediction software was used to assess the pathogenicity of the variation,in combination with the functional domains of the proteins.（4）Pathological analysis of the abnormal gonadal tissues,which were surgically resected from the proband,was performed.Result:（1）The clinical features of probands mainly manifested as the incompatibility between external genital appearance and chromosome karyotype,inguinal hernia,low levels of serum basal testosterone and dihydrotestosterone,which did not respond to the stimulation of human chorionic gonadotropin.（2）The genetic testing revealed novel compound heterozygous variants in the LHCGR gene,including a splice site mutation（c.681-1 G>A）and a frameshift variant（c.1582₁585del ATAT,p.Ile528*）,which were inherited from their parents respectively.（3）According to the prediction software and the position of variants in protein domains,the two variants may affect the function of the receptor by alternative splicing to form abnormally spliced products and introducing termination to form the truncated proteins,respectively.The two variants can be categorized as pathogenic.（4）The histopathological analysis of the resected gonads found abnormal development of the testis,which accords with the diagnosis of LCH.Conclusion: This section identified two novel compound heterozygous variants in the LHCGR gene,further to broaden the genotype-phenotype correlation spectrum of LHCGR variants,and analyzed the differences in pathogenic mechanisms between different functional domains of the receptor.Section Ⅲ.Pathogenesis of Primary Ovarian Insufficiency caused by HFM1 gene variantsObjective: Primary ovarian insufficiency（POI）is a common disease of reproductive system with complicated etiologies.The meiotic gene HFM1 is considered to be associated with POI.However,the mechanism has not been studied yet.This part of study intended to to explore the possible pathogenesis with the HFM1 missense varaints,which were identified in patients with POI.Method:（1）The mutant plasmid was constructed based on the wild-type plasmid by site-directed mutagenesis.The wild-type and mutant plasmids were transiently transfected into HEK 293 T cells.（2）The transfected cells were harvested and the total RNA was extracted.Reverse transcription real-time polymerase chain reaction was perfomed,as well as the statistical analysis.（3）We extracted the total protein from transfected cells.The western blotting experiments was then performed.（4）The three-dimensional structure of the protein structure was constructed based on the protein sequence combined with SWISS-MODEL and Py MOL software,etc.Result:（1）Sequencing verified that seven mutant expression plasmids（c.148、c.1241、c.2206、c.2325、c.2651、c.3367 and c.3580）were successfully constructed.（2）The m RNA expression of HFM1 mutant and wild-type was no significant difference T test showed that the expression difference between the mutant and wild type were statistically significant.（3）At the protein expression level,there was no significant difference between the mutant type and the wild type.（4）Based on the protein structure model,amino acids changes（p.His414 Pro,p.Phe775 Leu and p.Ile884Ser）are located in the key functional domain of the HFM1 protein,while p.Gly736 Ser and p.Ser1123 Pro are located in important motifs.Conclusion: This section found that the identified missense variation in HFM1 gene does not affect the protein expression in vitro cells.According to the mutant location of the protein structure and the changes in the properties of amino acids,it is presumed that the pathogenic mechanism may be related to the important domain and motif and result in enzyme dysfunction,which provides a clue for further research.