| Objective Osteopetrosis is a group of heterogeneous diseases characterized by syndromic deafness,increased bone density,hepatosplenomegaly,pancytopenia,and mental retardation.According to clinical phenotype and genetic pattern,it is classified into three subtypes including autosomal recessive osteopetrosis(ARO),autosomal dominant osteopetrosis(ADO)and intermediate autosomal recessive osteopetrosis(IARO).According to previous paper,twelve genes are associated with osteopetrosis in humans.The research intends to explore the genetic etiology of sporadic deafness patient with osteopetrosis in a consanguineous family which is collected in the early stage and clarify the pathogenic mechanism,so as to provide genetic counseling,marriage and childbirth guidance,prenatal diagnosis,enrich the mutation spectrum as much as possible and provide further basis for clinical diagnosis.Methods The family was recruited by the Department of Otolaryngology-Head and Neck Surgery,Taizhou People’s Hospital.According to the medical history inquiry and examination results,the pedigree was drawn.After informed consent was signed,the peripheral venous blood of the family members was collected,and the whole genomic DNA was extracted with the kit.Primers were designed for all exons and flanking sequences of three common deafness genes(GJB2,SLC26A4 and mitochondrial MTRNR1).After the products were amplified by nested polymerase chain reaction(PCR)and verified by electrophoresis,they were screened by Sanger sequencing technology.The results were analyzed by Sequencher5.4 software.If no suspicious pathogenic mutation was found,406 known deafness genes were screened by Next-generation sequencing(NGS).The conserved amino acids of mutant sites and their effects on protein structure and function were analyzed by online tools software such as PROVEAN,Mutation tasting,Polyphen2,Exome Variant Server and db SNP.Verify by Sanger sequencing in the family.The suspected pathogenic mutations of genotype-phenotypic co-separation were screened in 400 normal hearing people of the same race to determine their occurrence frequency in normal people.Results 1)Phenotype: The proband was identified with bilateral mixed deafness,multiple fractures of the whole body,scoliosis,increased bone density,ossification of maxillary sinus,cleft palate,teeth disorder or absence,hepatosplenomegaly,anemia,thrombocytopenia,repeated frontal infection,short stature,mental retardation and macular degeneration of the eye.The other family members in the pedigree did not see the above clinical phenotype of the proband.2)Genotype: The new mutation c.175 dup A(p.M59Nfs*8)of CLCN7 gene showed genotype-phenotypic co-separation in the family.The proband was homozygous mutation but her normal parents,aunt and brother were all heterozygous mutation,and the screening was negative in 400 normal hearing control population.Conclusion According to the family inheritance mode and clinical phenotype,the proband was suspected of intermediate autosomal recessive osteopetrosis with syndromic deafness.The c.175 dup A(p.M59Nfs*8)mutation of CLCN7 gene is likely to be the hereditary pathogenic factor in this family.After the mutation inserted adenine nucleotide at the 175 th base,resulting in the change of the coding amino acid at the 59 th site from methionine to asparagine,the stop codon appeared after the next eight amino acids,which led to the termination of the encoding of CLCN7 at the 67 th site and loss of protein function.The probable pathogenic mechanism of the mutation was the deletion of CLCN7 protein,which caused the lack of chloride and hydrogen ions that needed by V-ATPase,making the failure of lacunar acidification of fold membrane in lysosomes and osteoclasts.Cleft palate may be a new phenotype of intermediate autosomal recessive osteosclerosis.This study enriched the spectrum of genotype and genotype of IARO,and further provided a theoretical basis for genetic counseling,marriage and birth guidance and prenatal diagnosis of syndromic deafness. |
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