Analysis Of Clinical And Genetic Features Of PJVK And OTOF Gene In Chinese Patients With Auditory Neuropathy Spectrum Disorder

Auditory neuropathy (AN), also known as auditory neuropathy spectrum disorder(ANSD), is used to describe auditory disorders due to dysfunction of the synapse of the inner hair cells and auditory nerve, and/or the auditory nerve itself. Unlike common sensorineural hearing loss, patients with this disorder typically present impaired speech understanding, and show normal to severely impaired speech detection and pure tone thresholds. It is distinguished from other hearing loss by the otological characteristics of preservation of otoacoustic emissions (OAEs) and/or cochlear microfonics (CMs) and absent or abnormal auditory brainstem response (ABR).To date, the etiologies, the exact sites of lesions and the pathogenesis of AN are still not well understood, which results in the difficulties of clinical interventions for these patients. In2008, Starr and his colleagues proposed classifing the term auditory neuropathy into types by the site of this disorder. If the lesions of AN located at the level of auditory nerve,while the inner hair cells and synapses were normal, the disorder would be classified as "auditory nerve disorder." Similarly, if the inner hair cell synapses were disordered but the auditory nerve was normal, then the disorder would be classified as "auditory synaptic disorder." Site-specific classification would be helpful to define the disorder more specifically, besides it would be a good guideline for clinical interventions. But, there were no clinical measures to distinguish the sites of this disorder precisely. In recent years, a series of genes related to AN were found,and these genes revealed the pathological mechanism of AN at the molecular level.These discovers made it possible to classify the term auditory neuropathy by the site of lesions. It was more important in non-syndromic hearing loss. To date, two genes have been found to be responsible for autosomal recessive non-syndromic auditory neuropathy:PJVK and OTOF gene.In this study, we screened PJVK gene in150Chinese patients with AN collected from2003to2011and OTOF gene in17patients with AN in order to analysis the clinical and genetic features of PJVK and OTOF gene in Chinese patients with AN. Then, we analysised the relationship of the genotype and the phenotype. And with this method, we discussed the clinical and genetic features of3temperature sensitive auditory neuropathy patients. This study tried to describe pathogenesis of AN from the the viewpoint of genetics, and the purpose of this study is to identify the clinical and genetic features of different types of AN.The thesis is divided into three parts:PART1:Clinical and Genetic Features of PJVK Gene in Chinese Patients with Auditory Neuropathy Spectrum DisorderWe enrolled150unrelated AN patients from20different provinces of China collected from2003to2011. The sex ratio of these patients was1:1.05. We designed7pairs of primers to amplify7exons of the PJVK gene. Polymerase chain reaction (PCR) and sequencing were performed to identify the mutations in PCR products of PJVK coding sequence. Novel mutations were screened in100unrelated normal-hearing control individuals. Then we investigated the genetic features of PJVK gene in Chinese AN patients and analysised the clinical characters of patients with PJVK gene mutations.Five different pathogenic variants were detected, among which four are novel, including1nonsense mutation (c.886C>T),3missense mutations(c.437G>A, c.740G>T and c.887G>A). Besides, we also identified1reported missense mutation(c.548G>A). Among the150patients with AN, mutations were identified in5(3.3%).The variant, c.886C>T(p.R296X), was identified in a temperature-sensitive auditory neuropathy patient. This mutation, which substitutes a arginine with a stop codon at exon7, leading to a putative shorter protein, was considered as pathogenic variant. It was not existed in other patients and controls.The four missense mutations, c.437G>A, c.740G>T, c.548G>A and c.887G>A, caused amino acids changing, and were not existed in controls. The regions of the four missense mutations are highly conserved among vertebrate species.We considered that these mutations had relationships with AN. The variant, c.437G>A, was identified in an AN patient who presented mild to moderately-severe hearing loss at23years old with speech discrimination score of the left ear was68%and right64%. The variant, c.548G>A, was identified in an AN patient who presented normal to moderately-severe hearing loss at21years old with speech discrimination score of the left ear was32%and right18%. The patient carried c.740G>T variant presented fluctuant hearing loss at the level of mild to moderate with speech discrimination score of the left ear was88%and right86%. The patient carried c.887G>A variant presented hearing loss after birth, the hearing level was from mild to moderately-severe and the speech discrimination score of the left ear was80%and right76%.PART2:Clinical and Genetic Features of OTOF Gene in Chinese Patients with Auditory Neuropathy Spectrum DisorderWe screened OTOF gene in17patients with AN.44pairs of primers were designed to amplify47exons of the OTOF gene. Polymerase chain reaction (PCR) and sequencing were performed to identify the mutations in PCR products of OTOF coding sequence. Novel mutations were screened in100unrelated normal-hearing control individuals. Then we investigated the genetic features of OTOF gene in these patients and analysised the clinical characters of patients with OTOF gene mutation.In this study, we detected11kinds of pathogenic variants and18kinds of SNPs. Of the11kinds of pathogenic variants,8were novel, including4frameshift mutation (p.G558A fsX21, p.L795S fsX5, p.I1108H fsX69, and c.5833delG),1nonsense mutation (p.Y1133X) and3missense mutations (p. Q265L, p. G541S and p.R1928C). Besides, we also identified3reported pathogenic mutations(p.N727S, p.Q994V fsX6, c. ivs4023+1G>A). These11mutations were only found in Chinese patients.The mutations, p. G541S and p.L795S fsx5, were identified in a temperature sensitive auditory neuropathy patient, which didn’t existed in other patients and controls. We considered that these mutations were novel pathogenic mutations associated with TSAN. In one young AN patient with profound hearing loss, we identified2deletion mutation(p.G558A fsX21, p.Q994V fsX6) and1missense mutations(p. Q265L). In another young children AN patient with profound hearing loss, we identified c.4023+1G>A in homozygosis. The mutations c.5833delG and p.Y1133X, were also identified in a young AN patient with profound hearing loss. In addition, we also found3mutations in heterozygosis: p.I1108H fsX69, p.N727S and p.R1928C. The patient carried p.I1108H fsX69variant presented severe hearing loss, p.N727S presented moderately-severe hearing loss, and p.R1928C presented profound hearing loss.PART3:Clinical and Genetic Features of Temperature Sensitive Auditory Neuropathy Spectrum Disorder Patients. We screened OTOF and PJVK gene in3patients with temperature sensitive auditory neuropathy and analysised the clinical characters of these patients.The p.Q994V fsX6mutation of OTOF and p.R296X mutation of PJVK were identified in one TSAN patient.This patient presented fluctuating hearing with the change of temperature in one day. The patient complained of difficulties of hearing and communication when he had a higher temperature, while he can communicate well and response to sound quickly when the temperature was lower. In another TSAN patient, we identified p. G541S and p.L795S fsx5mutations of OTOF gene. This patient presented obvious difficulties of communication when he was febrile.