| Objective Deafness has high genetic heterogeneity,and there are many genes causing deafness.Molecular diagnosis is the key.Only by establishing a good correlation among pathogenic genes,pathological mechanisms and therapeutic methods,can personalized and effective intervention be realized for patients.Our team identified a new gene of autosomal dominant deafness causing deafness,IFNLR1,and a candidate pathogenic gene of autosomal recessive auditory neuropathy,SH3GLB1,by whole exome sequencing.Previous functional studies showed that mutation of IFNLR1 gene caused changes in JAK/STAT3 signaling pathway,but the mechanism of deafness induced by this gene was not clear.A SH3GLB1 gene knockout mouse model was constructed.There was no significant hearing loss in mice,defects in cognitive behavior were observed.In order to further study the effects of the above two genes and gene mutations on hearing and central nervous system function,further explore the pathogenesis.This study is divided into two parts.First,The Ifnlr1 gene inner ear hair cell conditional knockout(IFNLR1-CKO)mouse model was constructed to observe its hearing phenotype and inner ear morphology.The human IFNLR1 c.296G>A(P.r G99HIS)mutant eukaryotic expression vector was constructed and transfected into cells in vitro to observe the effect of the mutant on cell morphology and function.Secondly,a comprehensive behavioral evaluation was conducted on the Sh3glb1 gene knockout(Sh3glb1-/-)mouse model constructed by our research group in the early stage.To study the effect of the deletion of gene expression on the function and morphology of central nervous system in mice.MethodsIn this study,a variety of experimental methods were used to investigate the functions of IFNLR1 and SH3GLB1 in the auditory system and nervous system.First,we studied the subcellular localization of IFNLR1 by transient transfection and immunostaining in HEK 293 T cell lines expressing normal and point mutated IFNLR1 proteins.At the mice level,Ifnlr1 inner ear conditioned knockout(Ifnlr1-CKO)mice were obtained,by the Cre-Loxp method.The auditory function,hair cell number and morphology of CKO mice were studied by auditory brain stem response test and immunohistochemical staining.Secondly,our team previously performed Sh3glb1 gene knockout on wild-type C57 BL/6 mice using CRISPR/Cas9 technology,and obtained the Sh3glb1 gene knockout mice model.A systematic analysis of hearing function was conducted,but no hearing loss was found in transgenic mice,which may be a compensatory or repair mechanism.Meanwhile,we found behavioral abnormalities in the mice.On this basis,behavioral experiments and brain immunofluorescence were used to explore the central nervous system function of Sh3glb1.Behavioral experiments include: tail suspension experiment and forced swimming experiment to evaluate depression state;Open field experiment to evaluate anxiety state;New object recognition experiment,step-down test and water maze experiment to evaluate learning and cognitive ability.A variety of cognitive behavioral abnormalities were found in Sh3glb1 knockout mice.To further investigate the pathological changes underlying behavioral abnormalities,We designed two immunofluorescence experiments.In experiment 1,Nestin+ and PCNA+ were used to label proliferating neural stem cells,and in experiment 2,Nestin+ and Sox2+ were used to label neural progenitor cells.Results1.New autosomal dominant deafness gene-IFNLR11.1 The expression position of the mutant IFNLR1 protein did not change,and the mutant IFNLR1 protein did not affect the normal morphology of the cells.Plasmid transfection showed that when normal and mutant IFNLR1 sequences were overexpressed on HEK 293 T cells,the translated protein was expressed in the cytoplasm and cell membrane.The cells in both groups showed normal cell morphology.1.2 Ifnlr1-CKO mouse model was obtained by Cre-Loxp gene editing technique.The breeding,genotype and phenotype were identified,and the animals were raised for 15 months.1.3 The Auditory Brainstem Response(ABR)shows that the Ifnlr1-CKO mouse has delayed,progressive hearing loss.There was no difference in hearing threshold between Ifnlr1-CKO mice and control mice at at the age of 1month(P>0.05).The hearing of mice in mutant groups at 32 k Hz was worse than that in control group at the age of 2 month and 3 month(P<0.05).The hearing of mice at 16 k Hz,24 k Hz and 32 k Hz was worse than the control group at the age of 7 month(P<0.05).The knockout mice showed greater hearing loss at all frequencies at 9 month of age(P<0.05)..Both wild group and knockout group showed severe hearing loss at 12 month and 15 month.1.4 Loss of hair cells in the inner ear of Ifnlr1-CKO mice.Observation of basement membrane in aged(9 months)mice.In the basal membrane of9-month old mice,different degrees of loss of internal and external hair cells were observed in the knockout group compared with the wild control group,especially the bottom turn.2.A candidate gene for auditory neuropathy-SH3GLB12.1 Our team previously constructed a mice model with Sh3glb1 gene knockout.The aging Sh3glb1-/-mouse model was obtained through cage expansion and genotype identification.2.2 Behavioral experiments showed that the experimental group(Sh3glb1-/-mice)and the same cage wild type control group(Sh3glb1 +/+ mice)showed significant differences in the Morris water maze experiment(P<0.05),but there was no significant difference between homozygous and hybrid mice after gene editing(P>0.05).There was no significant difference in the resting time between the experimental group(Sh3glb1-/-mouse)and the control group(Sh3glb1+/+ mice)in the tail suspension experiment and the forced swimming experiment(P>0.05).There was no significant difference in the exploration time of central region(P>0.05).In the new object recognition experiment,there was no significant difference in the exploration time of new objects(P>0.05).There was no significant difference in the incubation period of the jump experiment(P>0.05).2.3 The staining results of brain tissue sections showed that there were differences in the fluorescence expression in the hippocampus of the experimental group,the control group and the young group.Both the young group and the old group had nerve regeneration,and the cells expressing Nestin and PCNA were expressed.The results of experiment 2 showed that Nestin+ and Sox2+ cells were present in the brain tissue of experimental group,control group and young group,but no colocalization was expressed.Conclusions1.New autosomal dominant deafness gene-IFNLR11.1 The mutation of IFNLR1 p.Arg99 His does not change the normal morphology of HEK 293 T cells.However,it can lead to the change of IFNLR1 protein expression localization..1.2 Conditional knockout of Ifnlr1 gene in the inner ear causes delayed and progressive hearing loss in mice.1.3 Conditional knockout of Ifnlr1 gene in the inner ear results in the loss of outer hair cells and the morphological changes of inner and outer hair cells in mice.2.A candidate gene for auditory neuropathy-SH3GLB12.1 Sh3glb1 gene knockout would not cause depression and anxiety,but would lead to the decline of learning and memory ability in mice.2.2 Sh3glb1 gene knockout may inhibit the regeneration of neurons in the hippocampus of the mice brain.This may be one of the reasons why Sh3glb1 knockout mice showed decreased learning and memory ability.Objective Myhre syndrome is a rare multisystem genetic disorder that is caused by de novo heterozygous gain-of-function variants in SMAD4.The main clinical phenotypes of patients with Myhre syndrome include small stature,autism,developmental delay,left-sided heart defects,hearing loss and often have a characteristic facial appearance.Early clinical diagnosis of Myhre syndrome remains a major challenge due to its delayed onset.In order to achieve early diagnosis and clinical intervention,we explore the high efficiency of identifying diseases through genetic testing and the necessary for early prevention of late-onset defects through genetic testing technology.Methods1.Data of a child with abnormal facial and finger development were collected,included clinical phenotype,family history,physical examination,and two years of clinical follow-up.2.Genetic diagnosis: Whole exome sequencing and bioinformatics analysis are used to detect pathogenic variants.3.Literature review of Myhre syndrome and SMAD4 gene.Results1.This Chinese newborn has facial features typical of Myhre syndrome,growth retardation,and complete syndactyly that has not been described before.2.A recurrent de novo missense variant c.1498A>G p.I500V(p.Ile500Val)in SMAD4 was detected confirming the clinical diagnosis of Myhre syndrome at the age of 38 days.The infant appears to be the youngest reported case of Myhre syndrome.3.Review the literatures noted several common features in Myhre syndrome patients including hearing loss(72.7%),cardiovascular problems(65.0%),characteristic facial features(26.0%-54.5%),finger and toe abnormalities(3.9%-48.1%),short stature(45.5%)and respiratory(30.0%).Conclusions1.Early diagnosis of Myhre syndrome is difficult.Whole exome sequencing in children with facial and finger development abnormalities is helpful for early diagnosis of Myhre syndrome,and for early intervention and rehabilitation of children.2.In this study,a retrospective analysis of deafness phenotypes and finger/toe abnormalities in patients with Myhre syndrome showed that although some patients with Myhre syndrome had normal hearing,most patients usually had early onset or progressive hearing loss in one or both sides,with significant phenotypic heterogeneity. |
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