Disease Causal Gene Screening And Characterisation In A Family With 46,XX Disorder Of Sex Development

Sex can be classified by genes,chromosomes,gonads,genitalia,psychological and social behaviors.Disorders of sex development(DSD)are conditions that the gonads or genitalia of patients are inconsistent with the genders determined by their karyotypes.Sex development can be divided into two stages:sex determination,and sex differentiation.Sex determination is mainly determined by the SRY gene that interacts with multiple genes.SOX9 gene,coding a transcription factor,plays an important regulatory role in pathways for male gonad development.Mutations in its coding region and structural variations of its regulatory region can make gene regulatory network for sex development imbalanced,and reslut in DSD.In this study,we identified a family with 46,XX DSD.In order to find the genetic abnormality that causes the condition,we performed a step-by-step screening for variants among the normal and infected members in this pedigre and made a comprehensive comparison of the transmition of the disease in this pedigree with cases reported previously.A molecular diagnosis is only made in-20%of DSDs patients,which implies that many unknown causes of DSDs exist.To study the causes of DSDs is very important for understanding of sex development.First,related hormones and CYP21A2 gene were analyzed.Second,multiplex ligation-dependent probe amplification(MLPA)was used for detecting insertion/deletion in WNT4,NR5A1,SOX9,NROB1 and other genes.Third,for the candidate disease causal genomic region,we characterized the variation using next-genenration sequencing,mater-pair sequencing,sanger sequencing and long range PCR.Fourth,to verify if the result of the experiment is right,we applied fluorescence in situ hybridization to locate the variation site.Finally,to look for the causes of the apparent genetic incomplete expression of the condition and possible interaction of deferent genes,we screened several genes in which mutations had been found to be associated with DSD in previous reports.The main results from this study include:1.A structrual variation upstream of SOX9 gene that leads to 46,XX DSD was identified.2.The duplications in upstream regulatory region of the SOX9 gene was located in chrl7:69256687-69952415(hgl9).3.As the carriers of the variants varied from normal to complete 46,XX DSD,we screened additional genes in which mutations had been reported to be associated with DSD,the intronic mutations reported in literatures did not show disease causal roles in this family.However we identified a new insertion variation intron 4 of DMRT1 gene.Our study provides further evidence that SOX9 gene's upstream regulatory region duplication can cause 46,XX DSD.Anew region of duplication was identified.It lay the foundations for the study of cause of DSD and have a strong influence on learn about sex development.