Single-cell Transcriptome Analysis Of The Epididymis Of A Patient With 46,XY Disorder Of Sex Development

Background Disorders of sex development(DSD)are a set of congenital conditions in which the sex defined by sex chromosomes does not match the anatomic sex.It is mainly characterized by gonadal hypoplasia and/or deformed external genitalia.The incidence rate of sexual development disorder with a46,XY karyotype(46,XY DSD)was about 1:20000.The main characteristics of 46,XY DSD are gonadal dysplasia,feminized or ambiguous genitalia.Single-cell transcriptome sequencing is an important tool to explore the pathological changes at the cellular level,but it has not been utilized in the study of disorders of sex development.Using single-cell sequencing technology to analyse the epididymal cell composition,functional changes and the molecular mechanism behind 46,XY DSD patients is expected to provide an important reference for the diagnosis,treatment,as well as the preservation of fertility of such patients.Objective To explore the changes,along with their possible mechanisms,in the cell composition and functions of the epididymis of a patient with an NR5A1 gene mutation using a variety of bioinformatics tools.Methods 1)Targeted next-generation sequencing and Sanger sequencing were used to screen and validate the causative gene and the location of the mutation in a patient with 46,XY disorders of sex development,several pathogenicity prediction tools were used to confirm the deleteriousness of the mutations,and USCF Chimera was applied to predict the spatial conformation of the mutant protein.2)Single-cell sequencing was performed on the epididymis resected from this patient during cryptorchidectomy,and multiple single-cell sequencing data and regulatory network analysis softwares(Seurat,Cell Chat,pyscenic,cluster Profiler,Cytoscape,etc.)were used to perform dimensionality reduction,clustering,sub-clustering analysis,intercellular communication analysis as well as single-cell transcription factor analysis.3)Cell-type specific regulatory modules of transcription factor(regulons)were identified using regulon specificity score(RSS).To mitigate the sheer disparity among the cell numbers of different cell populations in this patient’s epididymis,we proposed a novel stratified bootstrapping method to calculate RSS and compared it with the original method.Results 1)Both targeted next-generation sequencing and Sanger sequencing revealed a heterozygous mutation at position 124 of the NR5A1 gene(c.124C>G/p.Q42E)in this patient,which has not been reported in East Asian populations.The mutation is located in the DNA binding domain of the encoded SF-1 protein.It leads to the substitution of a neutral amino acid for a more hydrophilic acidic amino acid,which may affect the transcriptional regulatory activity of NR5A1.All five prediction algorithms,including PROVEAN and Poly Phen-2,suggested the mutation to be pathogenic.2)13,316 cells were retained after filtering out low-quality cells.After dimensionality reduction and clustering,12 cell populations were detected in the single-cell sequencing data of the patient’s epididymis.The proportion of fibroblasts(cluster C0)was46.48%,while epididymal epithelial cells(cluster C2)accounted for only 9.21% of total cells.Further subclustering of cluster C2 showed that only 27 cells could be assigned to principal cells,which is the most abundant cell type in normal epididymides.3)A total of 821 ligand-receptor pairs were enriched in intercellular communication analysis,which shows that fibroblasts and epididymal epithelial cells are closely interacted with each other.IGF and Wnt signal pathways,which are related to the epithelial-mesenchymal transition process,were included in the enriched pathways between these two populations.It is worth noting that epithelial cells specifically express receptor IGF1 R,forming an interaction pair with its ligand IGF1,which is highly expressed in fibroblasts and participates in the fibrosis of the patient’s epididymis.4)By analysing active regulatory modules of transcription factor(regulons)in each cell population,two sets of homeobox family genes were found to be specifically turned "on" in fibroblasts and epididymal epithelial cells,including fibroblast-specific regulons(represented by HOXA5,HOXA7,HOXA10,HOXC6,HOXC8),and epididymal epithelial cell-specific regulons(represented by HOXB7 and HOXB8).Both sets of genes are widely involved in the epithelial-mesenchymal transition process.This suggests that the NR5A1 mutation may contribute to the transition from epithelial cells to fibroblasts in the epididymis of the patient by affecting the balance between these two sets of transcription factors.5)Compared to the original method,RSSs calculated by the stratified bootstrapping method can better distinguish specific regulons in smaller cell populations,which boost the ability of RSS to recognise specific regulons in each cell populations.Conclusions The mutation in the NR5A1 gene in this patient leads to a shift in the main cell population from epithelial cells to fibroblasts.In terms of mechanisms,both the result of Cell Chat and pyscenic shows that EMT-related pathway,especially IGF and Wnt,and the activation of HOX family transcription factors play important roles.This study not only elucidates epididymal dysfunction caused by NR5A1 mutations,but also provides an important reference for the diagnosis and treatment of patients with46,XY DSD caused by mutations of this gene.