| Objective: Hearing loss,as a major sensory disorder that endangers human health,has been a worldwide public health problem.It is worth noting that more than 60% of hearing loss is caused by genetic factors,most of which are inherited by a single gene.X-linked hereditary deafness is relatively rare compared with autosomal hereditary deafness,and the incidence proportion in hereditary deafness is about 1%.The purpose of this study was to identify the pathogenic mutation of an X-linked dominant person with nonsyndromic deafness,detect the expression profile of the smpx gene in the zebrafish model,construct the smpx gene knockout and knockdown model,and explore the deafness mechanism of suspected pathogenic gene SMPX.Methods: Part ONE: Mutation analysis of pathogenic genes in deaf families The clinical data of the family were collected,and 2 patients and 1 person with a normal phenotype were sequenced by whole exon sequencing and bioinformatic analysis.Candidate mutations were obtained after data comparison and mutation screening.PCR-Sanger sequencing was used to detect mutations in family members and verify the correlation between mutations and phenotypes.Multispecies conserved analysis of mutations was performed using Homolo Gene.X chromosome inactivation test was used to investigate the cause of phenotypic heterogeneity. Part TWO: Cloning and expression analysis of SMPX gene in zebrafish The spatial and temporal expression of smpx in zebrafish was detected at m RNA level by the whole embryo in situ hybridization,and its subcellular localization was detected at the protein level by immunofluorescence staining.Part THREE: Establishment and phenotypic analysis of SMPX gene knockout and knockdown zebrafish models The g RNA was designed and synthesized,the smpx gene knockout models were constructed by CRISPR/Cas9 system,and the genotype and protein expression of mutants were verified by tail cutting sequencing and Western Blot.At the same time,the smpx gene Morpholino was designed and synthesized to verify the effectiveness of smpx-MO in vitro,and the smpx gene knockdown models were established by embryo microinjection technology.To observe the effects of smpx gene knockout and knockdown on the development and behavior of the inner ear,hair cells,and stereocilia of zebrafish.SMPX m RNA and SMPX m RNA(p.Q88E)were synthesized in vitro and co-injected with SMPX-MO to observe the phenotypic rescue of the colliculus cells and verify the pathogenicity of the mutation sites of SMPX C.262 c >G(p.Gln88Glu).Part FOUR: The deafness mechanism of SMPX gene Finally,transcriptome analysis should be conducted to explore the deafness mechanism of the SMPX gene.At the same time,real-time quantitative fluorescence PCR and TUNEL were used to verify the transcriptome data,which laid the foundation for clarifying the deafness mechanism of SMPX gene.Results: In this study,we reported a Chinese deafness family of 10 people with x-linked dominant inheritance in four generations.A new missense mutation of the known deafness gene SMPX(c.262 C >G,p.Gln88Glu)was identified by whole exon sequencing.This variant was co-isolated with the postverbal hearing loss phenotype in the family,and was highly conserved in mammals and missing in 300 healthy controls.In addition,X chromosome inactivation analysis revealed moderate genetic tilt in probands,suggesting that X chromosome inactivation offset may be one of the causes of phenotypic heterogeneity.The results of the whole embryo in situ hybridization showed that smpx was widely expressed in zebrafish embryo development,and specifically expressed in the inner ear,muscle tissue around the eye,and back brain of zebrafish.Immunostaining showed specific expression of smpx in the otolithoid membrane and the cuticular plate.The smpx knockout and knockdown did not affect the overall development of zebrafish but interfered with the development of the inner ear to some extent.The smpx knockout strain smpxzm3/-showed the decrease of 2dpf utricle,saccule,and the change of the number of the otolith.The smpxzm2/-and smpx-MO showed a slight decrease in the vesicle.The smpx knockout and knockdown resulted in a decrease in the number of neuromast in the posterior lateral line system of the trunk and the anterior lateral line system of the head,especially in the anterior lateral line system.The number of hair cells decreased in the posterior lateral neuromast.The smpx knockout and knockdown interfered with the development of stereocilia in inner ear hair cells to some extent,resulting in the reduction of the number and length of stereocilia to varying degrees.The smpx knockout and knockdown can weaken zebrafish’s response to sound and reduce activity.Compared with smpx-MO mutants,the hair cell clusters and the number of single hair cell clusters were partially saved,but they were weak compared with overexpressed SMPX m RNA(p.Q88E).Transcriptomic data showed that there were 1438 differentially expressed genes in the smpx knockout models,including 845 up-regulated genes and 593 down-regulated genes.Top30 down-regulated differential genes(fosab,erbb3 b,hspb1,hsp70 I,hsp70.1,hsp70.2,hsp70.3)were more enriched in the MAPK signaling pathway.Conclusion: 1.A new missense mutation of the SMPX(c.262C>G,p.Gln88Glu)was identified, which is the first report of a missense mutation of SMPX.2.X chromosome inactivation and migration may be one of the reasons for phenotypic heterogeneity.3.The smpx gene is specifically expressed in the otolithoid membrane and the cuticular plate,which may be involved in the maintenance and stability of stereocilium.The smpx knockout and knockdown models both interfere with the development of the inner ear and lateral line to a certain extent,leading to abnormal development of otoliths,reduced number of hair cells,and abnormal development of stereocilium,resulting in a weakened response to sound and reduced activity of zebrafish.4.SMPX may regulate the proliferation and differentiation of hair cells and stereocilium through the MAPK signaling pathway. |
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