Pathogenesis Of Digenic Variants In Nonsyndromic Hearing Loss

Objective:A pedigree of hearing loss(HL)with PCDH15 and CDH23 digenic heterozygous variants was found through pedigree investigation and pathogenic gene screening.In this experiment,the functions of the variant sites were studied and the pathogenicity of digenic variants were discussed preliminarily.Methods:We collected peripheral blood samples from all members in the family.The whole-exome sequencing and Sanger sequencing were performed to identify the pathogenic genes;the pathogenicity of variants were predicting by bioinformatics analysis and protein conservation analysis;PCDH15 and CDH23 wild-type(WT)and mutant-type(MT)eukaryotic expressions vectors were constructed.The western blot and immunofluorescence were used to detect expression and subcellular localization of the proteins;Real-time quantitative PCR(q PCR)was used to detect expression of Harmonin,PIST,PDZD7 and Whirlin;Meanwhile,PCDH15 and CDH23 wild-type and mutant-type prokaryotic expression vectors were constructed to further study the pathogenic mechanism of the digenic variants in the future.Results:1.Sequencing results showed that three affected individuals(Ⅱ-2,Ⅱ-3 and Ⅱ-4)carried PCDH15 c.4765 del C /(p.Leu1589Serfs*13)and CDH23 c.9617G>A(p.Arg3206His)digenic heterozygous variants,and Ⅰ-1 carried PCDH15 c.4765 del C/(p.Leu1589Serfs*13),Ⅰ-2 and Ⅱ-1 carried CDH23 c.9617G>A(p.Arg3206His).2.The Leu1589 and Arg3206 are highly conserved throughout species and two variants are more pathogenic by bioinformatics analysis.3.The variant in PCDH15 would cause truncated protein and reduced protein expression;The protein expression of CDH23-WT and CDH23-MT did not change significantly.4.Variants in PCDH15 and CDH23 did not change localization.However,protein aggregations were observed in the cytoplasm of the mutant type cells.5.It was showed that the expression of Whirlin and PIST was no significant change in PCDH15-MT transfected cells,while the expression of Harmonin and PDZD7 decreased.It shows that PCDH15 variant will reduce the expression of Harmonin and PDZD7;The expression of Whirlin,PIST,Harmonin and PDZD7 in CDH23-MT transfected cells did not change significantly,indicating that the variant in CDH23 will not affect the expression of interacting genes.6.PCDH15 was not expressed in BL21(DE3 pLys S),while CDH23 can be expressed in prokaryotic cells and the variant has no effect on protein expression according to prokaryotic expression.In addition,we intend to obtain the proteins by prokaryotic expression for further study.Conclusion:It was showed that PCDH15 and CDH23 digenic heterozygous variants probably cause HL for the pedigree through molecular biology technology and cytology experiments.