Identification And Pathogenicity Analysis Of DHX37 Variants In Patients With Disorders Of Sex Development

Background: Disorders of sex development（DSD）are a group of congenital diseases with inconsistent chromosomal,genetic and gonadal phenotype structures,with a prevalence of about 1/4500 to 1/5500 in the population.Due to the complexity of phenotype and pathogenesis of DSD,only 35-45% of patients with 46,XY DSD and10% with 46,XX DSD have a clear genetic etology.DHX37 is a new candidate causative gene for 46,XY DSD disease,and the molecular mechanism of DHX37 gene mutation leading to 46,XY DSD disease is still unknown.Subjects and methods: In the thesis,40 patients with sporadic 46,XY DSD were chosen as research subjects.（1）Whole-exome sequencing（WES）was performed on the patients` DNA.Sanger sequencing was used to confirm candidate pathogenic variants of DHX37 among family members in comparison to the Gnom AD and an internal whole-exome sequencing database of 2,114 Han Chinese data.（2）Poly Phen-2,PROVEAN,Mutation Taster,Homolo Gene,Alphafold and other tools were used to predict the protein structure,function and conservation of DHX37 variants.（3）To investigate the pathogenicity of DHX37 variants,an in vitro functional assay was used:the ICC experiment was used to determine the subcellular localization of the DHX37 protein.In addition,the protein expression of DHX37 was investigated using a Western Blot experiment.（4）q PCR and Western Blot experiments were used to confirm the interference efficiency of DHX37 after the RNAi experiment.Following transcriptome sequencing and analysis,q PCR experiment was carried out to confirm the differentially expressed genes,in order to investigate the molecular mechanism of 46,XY DSD caused by DHX37 variant.Results:（1）In 40 patients with sporadic 46,XY DSD,two candidate pathogenic DHX37 variants were identified: c.319G>A（p.Glu107Lys）and c.2012G>C（p.Arg671Thr）.（2）Bioinformatics analysis revealed that the protein function of two variants were damaged.The number of hydrogen bonds and interacting amino acids of the variant protein were altered to varying degrees when compared with the structure of the wild-type DHX37 protein.（3）The ICC array revealed that both wild-type and variant protein were primarily found in the nucleolus.The expression levels of the variant proteins were significantly lower than the wild-type protein,according to a Western Blot array.（4）Following transcriptome sequencing analysis,63 DSD-related differentially expressed genes were identified,including 24 up-regulated genes and 39down-regulated genes.After verification by q PCR experiments,it was found that the m RNA expression trends of each gene were consistent with the transcriptome sequencing results.Conclusion:（1）In 40 patients with sporadic 46,XY DSD,two candidate pathogenic DHX37 variants were identified,neither of which had been reported previously.（2）In vitro assays revealed that two DHX37 variant proteins didn`t change the intracellular localization but significantly reducing protein expression levels,implying that the pathogenesis of DHX37 variation may be haploinsufficiency.（3）the verification of the differentially expressed genes by q PCR suggested that the DHX37 variant may affect GATA4-AR pathway of male gonad development or LHX9-NR5A1-RSPO1 pathway of female gonad development,resulting in 46,XY DSD.