Mutation Screening Of Candidate Genes In A Chinese Pedigree With Autosomal Dominant Hereditary Non-syndromic Auditory Neuropathy Spectrum Disorder And Study Of Development Of NELL2in Postnatal Mouse Cochlea

Part one：Clinical Diagnosis and Mutation Screening of Candidate Genes in aChinese Pedigree with Autosomal Dominant Hereditary Non-syndromic AuditoryNeuropathy Spectrum Disorder（ANSD）Objective By performing Clinical audiological examination and mutationscreening of candidate genes in a Chinese pedigree with autosomal dominant hereditarynon-syndromic auditory neuropathy spectrum disorder（ANSD）, the research aims toconfirm whether there are such possible pathogenic gene mutations.Methods A pedigree of4generation,24people altogether is taken as the researchobject. First of all, they were examined by ABR、EOAE、Pure tone audiometry、CT andMRI，and then the patients’genomic DNA was extracted from peripheral vein blood, andprimers were designed by Internet free primer design software; Then, known auditoryneuropathy related genes were analyzed by direct sequencing and gene sequences wereanalyzed comparatively by DNAMan. Based on the previous experiments,we performedexome sequencing to the patients of this family to search for the possible candidate genes.Results In this pedigree，the characteristics of4members is in accordance with thediagnosis of non-syndromic auditory neuropathy spectrum disorder（ANSD）and themode of inheritance is consistent with the feature of autosomal dominant genetic disease. The exons of DIAPH3、OTOF、PJVK、GJB2、GJB3genes and mitochondrial12SrRNAgene of all patients and some of the other members with normal hearing were analyzedby direct sequencing. There is no possible susceptible mutation. Then we found a newpoint mutation in the NELL2gene in this pedigrees by exome sequencing technology.Conclusions All the known pathogenic mutations cannot be detected in thisChinese ANSD family, but the mutation of NELL2gene was found in all patients in thisfamily, which suggests that NELL2mutation may result in non-syndromic ANSD andNELL2might be a new disease-causing gene and should be further studied. Part two: Study of Development of NELL2in Postnatal Mouse CochleaObjective Based on the results of mutations in the gene screening for a Chinesepedigree with ANSD，this research aims to study the expression of gene NELL2inpostnatal mouse cochlea.Methods C57mouse are used as the experimental animals, and postnatal day0（P0）、P3、P7、P14、P28are chosen to be the time node. Immunofluorescence staining、Western Blot and RT-PCR are the main experimental techniques.The expression anddistribution of NELL2in different developmental stages of postnatal mouse in cochleawith the levels of NELL2protein and NELL2mRNA are observed.Results The research indicates that NELL2expresses in the mouse cochlea, andthe expression increases with the age. It expresses in cytoplasm of the inner hair cells,outer hair cells, spiral ganglion neurons, the inner pillar cells and outer pillar cell in thecochlea. The morphous of Corti organ is immature during P0and P3. The expression ofNELL2is not obvious in this period，and the expression showed a gradually increasingfrom P7to P28. In spiral ganglion neurons, the expression of NELL2increased graduallyduring P0to P28. The Western Blot analysis showed a consistent trend with immunofluorescence. Data analysis is statistically significant. However, RT-PCR showedthat the expression of NELL2mRNA peaks at P7.And compared to that, the expression inP14and P28decreased significantly.Conclusions. The study of our team showed that the expression of NELL2in themouse cochlea has an increment trend as the age gradually increased. And it is coincidingin time with the morphological and functional maturation of the Corti organ. Our presentresults suggest that NELL2may play an important role in the development of Cortiorgan as well as maintenance of auditory functions. The results of expression of RT-PCRand Western Blot did not show consistency.It is presumed that the expression of NELL2should be regulated by one or a variety of mechanisms, and the study should be continued.