Virulence Genes Of Two Chinese Han Hereditary Deafness Pedigrees

Part 1 A study associated with a maternally inherited deafness family in ChinaObjectiveTo clarify the genetic etiology and pathogenesis of maternally transmitted hearing loss in a large Chinese family, for the prevention and treatment of hereditary hearing loss to provide a theoretical basis.MethodsOne Han Chinese family was ascertained through the Otology Clinic. Clinical data was obtained from this deafness family. According to the mode of inheritance through mothers, we extract the whole genome DNA in leukocyte from all family members available. PCR and sequencing the whole mitochondrion DNA and GJB2 gene to detect the possible mutation(s) associated with deafness. Pyrosequencing was used to quantitate the level of heteroplasmic mutation, which is associated with the severity and age-at-onset of hearing loss. The mutation of evolutionary conservation is calculated by comparing the human mtDNA variants with other species. Steady-state level of mitochondrial RNAs was examined by use lymphobastoid cell lines from affected matrilineal relatives and controls. These cell lines were further assessed for the effects of the mitochondrial protein synthesis and endogenous respiration.ResultsThe maternal family members had symmetrical, late-onset deafness, which began with tinnitus and high-frequency hearing loss, and the deafness was independence with use of aminoglycoside antibiotics. A CT scan of temporal bones of proband is normal. There is no other significant abnormal in this family. The mitochondrial DNA in this family has 43 mutations, belongs to haplotype Z. A novel heteroplasmic mutation T12201C in tRNA His of mitochondrial DNA had been found, which disrupts a highly conservative base-pairing (5A-68U) on the acceptor stem of tRNA His. The level of heteroplasmy in T12201C was roughly correlated with the age of onset and degree of deafness. No other mutations had been found important in mitochondrial DNA. Heterotic 235 delC in GJB2 gene had been found in seven maternal members including both deafness and normal hearing persons. Mitochondrial tRNA Northern analysis revealed that the T12201C mutation caused 75% reduction in steady-state levels of tRNA His,47% reduction in the rate of mitochondrial translation, and 37% reduction in the rate of total O2 consumption in the lymphoblastoid cell lines compared to the controls. The difference was statistically significant.ConclusionsA novel heteroplasmic mutation T12201C in tRNA His of mitochondrion DNA is mostly possible mutation induced the maternally inherited deafness in this family. A failure in tRNA metabolism causes a significant decrease in mitochondrial protein synthesis and respiratory phenotype, which results in a decline in ATP production and increasing production of oxygen reactive species, and eventually hearing loss. The level of heteroplasmic T12201C is associated with the age of onset and the degree of deafness. Further more, the tissue-specificity of this pathogenic mtDNA mutation is likely duie to the involvement of nuclear modifier genes or tissue-specific differences in tRNA metabolism.Part 2The deafness of Waardenburg Syndrome typeâ…¡in a Chinese familyObjectiveTo investigate clinical and molecular features, relationship between phenotype and genotype in a large Chinese Waardenburg Syndrome family. MethodsA family we obtained was identified with Waardenburg Syndrome in China. The proband with WS2 was discovered in clinic service, and then intimate clinic information and venous blood were collected in each member available by family investigation in their hometown. The whole genome DNA was extracted, and all 9 exons of MITF gene and the frequent hereditary deafness genes GJB2 and mitochondrial DNA (mtDNA) 12S rRNA were detected simultaneously by polymerase chain reaction (PCR). The mutations were analyzed and compared with the reference sequence to definite the genetic reason of WS2 in this family. We determined whether the gene mutations lead to changes of amino acids and evolution conservatism analysis of amino acids in MITF protein was made across many species. Screening the mutation discovered in one hundred controls with the same genetic background.ResultsAccording to the clinical features, it is consistent with WS2 diagnostic criteria previously described. The main abnormalities are sensorineural deafness, brown freckles on the skin, premature graying, and heterochromia irides in this family, but no dystopia canthorum in any person. The pedigree chart demonstrates autosomal dominant inheritance, but family members show varying degrees of clinical manifestation. An interesting novel heterozygous mutation c. [742-743delAAinsT; 746-747delCA] has been found in MITF gene exon 8. Twelve members have the mutation, which is co-segregate with phenotype of WS2 in the family. This mutation leads to an advanced termination codon TAG, which makes a truncated protein with only 247 amino acids, compared with the 419 amino acids in normal protein. The mutation makes MITF protein lost the complete functional structural domain bHLH-Zip, and leads to haploinsufficiency. This amino acid K (Lys) is highly conserved among many species. Intriguingly, the deaf proband had MITF gene heterozygous mutation as well as GJB2 gene c. [109G>A]+[235delC] compound heterozygous pathogenic mutations, which also lead to hereditary deafness. Nobody except the proband has double recessive pathogenic mutations in GJB2 gene and no pathogenic variants in mtDNA 12S rRNA gene are identified in all members of this pedigree. Meanwhile, no any mutation or polymorphism was observed in exon 8 of MITF gene in one hundred normal hearing controls with the same genetic background and c. [109G>A]+[235delC] in GJB2 gene was also not found in one hundred controls.ConclusionsWS2 has multivariate clinical phenotype, the novel rare pattern of inconsecutive heterozygous mutation, c. [742-743delAAinsT; 746-747delCA], in MITF gene exon 8 was the main genetic etiology of this WS2 family. The proband has both GJB2 gene and MITF gene mutations. Therefore, a digenic effect may happen in the deafness of proband with MITF and GJB2 genes. This new miscellaneous frameshift mutation in MITF gene expands mutation databases of WS2A in chinese patients. Relationship between phenotype and genotype of this family needs to be studied further in the future.