Identification And Functional Analysis Of Causal Gene In A Chinese Pedigree With Autosomal Dominant Hereditary Conductive Hearing Loss

To date, only55loci have been mapped for autosomal dominant nonsyndromicdeafness (DFNA) and a total of30DFNA-related genes have been cloned, most ofwhich are involved in sensorineural or mixed deafness. Research into the molecularmechanism of causative genes for conductive deafness caused by factors such ascongenital malformation of the external and middle ear has made slow progress becauseof the influence of environmental factors and a lack of pedigrees with simple conductivedeafness. In this study, an advanced genetics research strategy combininghigh-throughput sequencing, comparative genomic hybridization, and massspectrometry analysis was used to explore the molecular genetic mechanism of inheritedconductive DFNA in a rare Chinese pedigree. An attempt was made to identify newpathogenic genes of conductive deafness in the Chinese population and to performfunctional analysis as well as sporadic verification.In Section I, clinical data and genetic resources of a pedigree with conductivedeafness (Pedigree028) were collected, and conductive deafness-related pathogenicgenes were investigated using next-generation sequencing technology and advancedbioinformatics analysis tools. Pedigree028had a clinical phenotype of relatively simplecongenital conductive deafness with bilateral ptosis, external auditory canal stenosis,and middle ear malformation. Surgical exploration confirmed that the cause of deafnesswas congenital middle ear malformation, involving fixation of the stapes and osseoushyperplasia with external fixation of the head of the malleus. No causative genes wereidentified by linkage analysis.Exome sequencing was performed on six individuals selected from the pedigree,and sequencing data were compared with the human genome database for the screeningof rare group-specific variations, prediction of candidate single nucleotide polymorphisms and insertions/deletions, and further verification. However, nopathogenic genes were identified. More comprehensive genome re-sequencing wastherefore carried out on three individuals (two patients and a normal control) selectedfrom the pedigree to detect mutations in the non-coding region or structural variations inthe genome. During data verification, array-comparative genomic hybridization wasused for the genomic DNA analysis of one patient, and a decrease in the number ofcopies of large DNA fragments was detected on chromosome2p21. Whole-genomere-sequencing data were analyzed and breakpoint PCR successfully identified acausative59,482-bp heterozygous deletion in the chromosome245199520–45259001region, which included the entirety of the C gene. The formation of this mutation maybe related to the chromosomal recombination induced by insertion of the Alu sequence.In Section II, the function of C gene was discussed in terms of developmentalbiology, and data of sporadic patients with similar phenotypes to those in Pedigree028were collected to further confirm that C is the causative gene of conductive deafness inthis pedigree.C gene contains two exons that encode a protein of291amino acids. It is locateddirectly downstream of Hoxa2, and is jointly involved in the development of the secondbranchial arch. A deletion of Hoxa2leads to increased expression of C gene, and aduplication of the malleus–incus complex at the position of the second branchial archthat mirrors the distribution of normal otosteon. The homozygous deletion of C in miceis lethal, resulting in only the partial development of a skeleton derived from the secondbranchial arch. A homolog of C, Six1, was confirmed to be part of the Eya–Six–Paxgene regulatory network that is important for the development of the ear and kidney.Mutations in Six1weaken the interaction between Eya1and Six1. A total of203sporadic patients with conductive deafness were selected to verify C as the causativegene using breakpoint PCR and genetic screening; however, no causative C mutationswere detected in these patients.Future work: To continue to collect sporadic cases with a phenotype similar topatients of Pedigree028; to improve the Taqman probe method and further verify copy number variation in sporadic samples using quantitative PCR; and to develop a Ctransgenic animal model.