Identification And Cloning Of A Novel Causative Gene Of X-linked Recessive Inherited Syndromic Hearing Loss

Background:Hearing loss?HL?is the most common sensory defect leading to human communication disorders.Nowadays,it is estimated that nearly 300 million people worldwide suffer from HL,and 60%?80%of those cases could be attributed to genetic factors and show a significant genetic heterogeneity.At present,a substantial amount of nuclear genes have been identified to be implicated in genetic HL.However,X-linked HL is relatively infrequent and rarely reported.Objective:To investigate the audiological and genetical features of a five-generation Chinese family with an X-linked recessive syndromic HL?SHL?,and identify the underlying genetic defect by targeting X-chromosome capture and exome sequencing?XES?.Methods:?1?A detailed medical history were obtained by interviewing the family members,and the affected family members were subjected to a comprehensive examination,including clinical examinations,otoscopy and audiological tests,and laboratory tests,etc.The peripheral blood of family members were collected and used to isolate genomic DNA.?2?Two affected individuals??-13,?-9?and one normal hearing family member??-4?were included in XES to identify the candidate genes in the family,which were then screened and verified by cosegregation analysis by direct PCR and Sanger sequencing.?3?In silico analysis was used to study the conservation of the identified gene and the structural changes of the mutant protein as well as its potential pathogenic impact.?4?The expression analysis of identified gene was performed,including the relative mRNA expression level in different mouse tissues by qRT-PCR and the spatial and temporal expression during early embryonic development of zebrafish by whole mount in situ hybridization?WISH?.Results:?1?All six affected individuals were male;four were alive and diagnosed with congenital bilateral mixed or conductive hearing loss and inner ear malformation,suggesting an X-linked recessive inheritance pattern.?2?Five variants?including 3 non-synonymous SNPs and 2 indels?were obtained by XES and predicted to potentially have a functional impact on the genes,and the subsequent cosegregation analysis identified and conformed that a 2-base pair missense mutation of c.1717-1718GC>AA in exon 5 of GPRASP2 could be the genetic cause responsible for the SHL of the family,which was absent in 300 controls.?3?In silico analysis showed that the mutation of c.1717₁718 GC>AA in GPRASP2 caused a change of amino acid residue?p.A573N?,which is highly conserved in different species,and the structural modeling revealed that the p.A573N mutation could result in the steric hindrance and polarity alternation of side chain,and lead to the structural change of protein,which might impair the function of GPRASP2.?4?The Gprasp2 gene was strongly expressed in the mouse brain and cochlea,and the immunohistochemical study on the mouse cochlea showed that Gprasp2 was localized in multiple structures of the mouse cochlea and intensively detected in the spiral ganglion,stria vascularis,spiral ligament,inner hair cells and outer hair cells.The WISH result showed that zebrafish armc10?an orthologous gene of human GPRASP2?was maternally expressed during early embryonic development,then gradually expressed in otic vesicle and the nervous system of head since 24 hours post-fertilization.Conclusions:A novel X-linked recessive SHL-causative gene,GPRASP2,was identified in this study,which would contribute to the complementation of mutation spectrum of SHL-causative genes.