Study On Auditory Function Of Ifnlr1 Variant Knock-In Mouse Model And Identification Of New Pathogenic Variant Of FOXC1 Gene

Part Ⅰ Identification and exploration of audiology and cochlea morphology of Ifnlr1 gene mutation knockin mouse modelObjectiveAutosomal dominant nonsyndromic deafness（ADNSHL）is mainly characterized by progressive sensorineural deafness.In this study,the p.Arg99 His mutation of IFNLR1 gene,which was constructed by the new deafness gene Ifnlr1 identified earlier,was knocked into the mouse model,and its hearing phenotypes were identified and analyzed,its physiological,biochemical and pathological manifestations were studied,and the function of IFNLR1 gene and the related mechanism of the gene mutation leading to ADNSHL were explored,which laid the foundation for the treatment and prevention of diseases.Methods（1）PCR-Sanger sequencing was applied to the experimental animals after breeding and cage expansion,and the genotype was identified according to the sequencing results;（2）Auditory function and pathological analysis: ABR audiometry,immunofluorescence staining and protein immunoblotting experiments were carried out on 1-month-old and 3-month-old mouse models to analyze the auditory function and possible pathological manifestations of the inner ear.Results（1）wild-type and homozygous mice were selected as control group(Ifnlr1^+/+)and mutant group(Ifnlr1^{Arg98His/Arg98His})respectively according to the sequencing peak map.（2）ABR audiometry showed that there was no difference between 1-month-old and 3-month-old mutant mice and control mice（P>0.05）;Immunofluorescence showed that there was no significant difference in the expression position of Ifnlr1 protein between mutant and wildtype mice.The fluorescence intensity of Ifnlr1 KI mice was lower than wildtype mice（P<0.05）.WB showed that expression of Ifnlr1 in mutant mice was lower than that in wildtype mice（P<0.05）.The morphology of hair cells was intact and there was no significant difference in the number.ConclusionsThe audiological study of Ifnlr1 gene mutation knock-in mouse model showed that there was no significant difference between homozygous and wild-type adult mice in hearing function.The inner ear of mice was dissected,and immunofluorescence and western blot experiments showed that the expression of Ifnlr1 protein in homozygous mice inner ear was decreased,but no abnormal morphology of inner ear was found.Part Ⅱ A novel variant in FOXC1 associated with atypical Axenfeld-Rieger syndromeObjectiveMutations in the Forkhead Box C1（FOXC1）are known to cause autosomal dominant hereditary Axenfeld-Rieger syndrome,which is a genetic disorder characterized by ocular and systemic features including glaucoma,variable dental defects,craniofacial dysmorphism and hearing loss.Due to late-onset of ocular disorders and lack of typical presentation,clinical diagnosis presents a huge challenge.In this study,we described a pathogenic in-frame variant in FOXC1 in one 5-year-old boy who is presented with hypertelorism,pupil deformation in both eyes,conductive hearing loss,and dental defects.MethodsWhole exome sequencing and clinical data analysis.ResultsBy whole exome sequencing,we identified a 3 bp deletion in FOXC1,c.516₅18del GCG（p.Arg173del）as the disease-causing variant,which was de novo and not detected in the parents,and could be classified as a “pathogenic variant” according to the American College of Medical Genetics and Genomics guidelines.After confirmation of this FOXC1 variant,clinical data on Axenfeld-Rieger syndromeassociated clinical features were collected and analyzed.Furthermore,Although the affected individual present hearing loss,however,the hearing loss is conductive and is reversible during the follow-up,which might not link to the FOXC1 variant and is coincidental.ConclusionsRoutine examination of FOXC1 is necessary for the genetic diagnosis of hypertelorism-associated syndrome.These findings may assist clinicians in reaching correct clinical and molecular diagnoses,and providing appropriate genetic counseling.