Genetic Etiology Analysis Of Five Deafness Families And Functional Identification Of A Novel Variant In CDH23 Gene

Background:Hearing loss(HL)is one of the most common sensory deficit diseases,which can occur in people of all ages,leading to severe speech communication disorders and affecting the quality of life of patients.The incidence of deafness is high and the etiology is complex.The incidence of deafness in infants worldwide is about 2‰～6‰.The etiology mainly includes genetic factors and environmental factors,of which the former account for about 50%～60%.With the continuous development of preventive medicine and the improvement of human health awareness,deafness caused by environmental factors has gradually decreased,and genetic factors have become more prominent.Hereditary hearing loss is mostly a monogenic disease with strong genetic heterogeneity.The rapid development of genetic testing technology has promoted the exploration of the etiology of hereditary deafness.More than 200 deafness related genes have been identified,and new genes are constantly being discovered related to deafness.However,there is no effective cure for hereditary deafness.The main treatment methods are hearing aid and cochlear implantation,but the cost is relatively expensive and the effect is not as good as expected.The prevention of hereditary deafness has become a hot topic in the world.Genetic testing of related members of deafness families can identify the pathogenic genes and reduce the risk of reproduction of children with deafness.Objective:Genetic testing of the five deafness families was performed to clarify the pathogenic causes and to provide basis for genetic counseling of the families and prenatal diagnosis of at-risk fetuses.At the same time,the function of a novel variant of CDH23 gene was identified in vitro to further explore the mechanism of deafness.Methods:1.The detailed clinical data of five deafness families were collected,including clinical manifestations,audiological examination and imaging examination.The genetic maps of the families were drawn and the possible modes of inheritance were judged.2.The DNA samples were extracted from the probands of five families.Whole exome sequencing was performed on the probands of families 1,2 and 3,and Sanger sequencing was performed on the related members.The deafness hot spot gene screening was performed for the probands of families 4 and 5 with autosomal recessive deafness.Afterwords,the probands of family 4 and 5,who had only single site mutation of SLC26A4 gene and negative screening results,were subjected to whole exome sequencing and Sanger sequencing of related family members.Finally,all relevant variants detected were analyzed for pathogenicity according to the ACMG guidelines.3.The deletion of SLC26A4 gene exon 5 in family 4 was verified by real-time fluorescence quantitative PCR.4.The RNA samples of the proband and his parents in family 5 were extracted for reverse transcription,and the c DNA products were sequenced.At the same time,the protein samples of the proband and his parents were extracted for Western Blotting.Results:The modes of inheritance in families 1-5 were X-linked dominant,autosomal dominant,X-linked recessive,autosomal recessive,and autosomal recessive,respectively.A total of 43 participants were involved,including 16 deaf patients.Among them,the temporal bone CT of the proband in family 3showed dilatation of the inner ear canal and abnormal communication with the cochlea.Besides,the proband of family 4 showed enlarged bilateral vestibular aqueduct on CT scan of temporal bone.PRPS1 gene c.46 T>C mutation、OSBPL2 gene c.564＿565 ins G mutation、POU3F4 gene c.520G>T mutation、SLC26A4 gene IVS7-2A>G mutation and heterozygous deletion in exon 5、CDH23 gene c.6688 del G mutation were separately detected in pedigrees 1-5.According to the ACMG guideline,all the above mutations are "pathogenic",and the diseases caused by the related gene variants are consistent with the family inheritance pattern.Among them,OSBPL2 gene c.564＿565 ins G mutation、POU3F4 gene c.520G>T mutation、SLC26A4 gene heterozygous deletion in exon 5、CDH23 gene c.6688 del G mutation were the first report mutations.The deletion of exon 5 of SLC26A4 gene in family 4 was verified by real-time fluorescence quantitative PCR,which was consistent with the results of next generation sequencing.In addition,the deletion variant of CDH23 gene in family 5 was identified in vitro,and the results of reverse transcription product sequencing were consistent with the results of whole exon sequencing.Western Blotting results showed that the proband had a truncated protein,and his parents expressed the truncated protein and normal protein.Conclusions:1.PRPS1 gene,OSBPL2 gene,POU3F4 gene,SLC26A4 gene,CDH23 gene pathogenic variants may be the cause of the five deafness families.Family 1 was diagnosed with syndromic hearing loss,the other four families had non-syndromic hearing loss;2.OSBPL2 gene c.564＿565 ins G,POU3F4 gene c.520G>T,SLC26A4 gene exon 5 deletion,CDH23 gene c.6688 del G mutation were all reported for the first time,which enriched the mutation spectrum of human deafness gene;3.The results of gene detection have provided a strong basis for genetic counseling and prenatal diagnosis of high-risk fetuses;4.In vitro functional identification experiments showed that the homozygous variant of CDH23 gene c.6688 del G did not affect m RNA transcription,but could affect protein translation,which further clarified the pathogenic mechanism of the mutation.