Mutation Analysis Of The Pathogenic Gene Of A Chinese Deaf Family And The Function Of The Deafness Gene ILDR1 In Zebrafish

The MY07A gene encodes a protein classified as an unconventional myosin. Mutations of myosin ⅦA have been implicated in Usher syndrome type 1B, atypical Usher syndrome, non-syndromic autosomal recessive hearing impairment (DFNB2) and autosomal dominant hearing impairment (DFNA11), different mutations in this gene result in various phenotypes. We collected a family with non-syndromic autosomal dominant progressive hearing impairment from Jiangmen. Affected members of the family present with an ascending audiogram affecting low and middle frequencies at young ages and then affecting all frequencies with increasing age. To identify the gene responsible for the hearing impairment in this family, Illumina Cyto-12 Chip was used for genome-wide linkage analysis. Finally, we mapped the disease loci to the 11q13.4-11q14.1 interval that resembles the previously published DFNA11 family. One new variant, a c.2003G-A (p.R668H) mutation of the myosin ⅦA gene, is heterozygous in all affected family members and absent in 100 healthy individuals. This mutation is predicted to result in a p.R668H amino acid substitution. R668 is located in a region of the myosin ⅦA motor domain that is highly conserved among different species. Molecular modeling predicts that the conserved R668 residue plays important structural roles in linking different lobes of motor domain together. This novel mutation is expected to disrupt or seriously disturb the function of the motor domain. In the actin-activated ATPase activity assay, the rate of NADH oxidation was higher in the wild-type myosin ⅦA, indicating that the ATPase activity in the p.R668H mutant myosin ⅦA was significantly destroyed. Conclusion: Deafness in this family is resulted from a c.2003G-A mutation which seriously reduced the ATPase activity.ILDR1 (Immunoglobulin-like domain containing receptor 1) was a less characterized gene which was first identified in lymphoma cells. It has three splice variants. Recently, ILDR1 was found to be causative for a familial deafness, DFNB42. However, the etiology and mechanism of DFNB42 caused by dysfunction of ILDR1 was not well elucidated. In order to reveal the possible etiology of deafness resulted from ILDRl dysfunction, we characterized the function of ILDR1 in zebrafish using morpholino technique. As a result, ILDRl morphant zebrafish had reduced number of lateral line neuromasts. The development of semicircular canals were also delayed which resulted in the fusion of otolith. Injection of human ILDR1 mRNA to ILDR1 morphant zebrafish can rescue the phenotype of abnormal inner ear and reduced number of lateral line neuromasts. The subsequent zebrafish genome-wide gene expression profile in 48h and 72h revealed that compared with control, ILDR1 morphant zebrafish have 130 genes decreased and 102 genes increased with at least two fold changes. Among changed genes, the expression of atplb2b was reduced by three fold in ILDR1 morphant fish compared with the controls. In addition, injection of zebrafish ILDR1 mRNA to ILDR1 morphant fish can enhance atplb2b expression. Furthermore, injection of atp1b2b mRNA can partly rescue the phenotype resulted from the knockdown of ILDR1. Finally, we concluded that ILDR1 regulation of atplb2b is crucial for the development of semicircular canals and otolith and for the migration of lateral line neuromasts in zebrafish.