Research Of Clinical Characteristics And Genetic Features Of DIAPH3Gene In Chinese Patients With Auditory Neuropathy Spectrum Disorder

Auditory neuropathy spectrum disorder (ANSD), also named auditoryneuropathy (AN), is used to describe auditory disorders due to a dysfunction ininner hair cells (IHC) and auditory nerve synapses and/or auditory nerve only,while function of outer hair cells (OHC) is normal. Unlike common sensorineuralhearing loss, patients with this disorder show special clinical characteristics, andtypically present absent or grossly abnormal auditory brainstem responses (ABRs)with normal otoacoustic emissions (OAEs) and/or cochlear microphonics (CMs).Pure tone audiometry of auditory neuropathy spectrum disorder has differentclinical manifestation. The shape of the audiometric curves tends to vary, fallingwithin the low frequencies ascending curve, flat curve, high frequency slopingcurve, et al. Pure-tone audiometry thresholds vary significantly ranging fromnormal to profound levels.The cause of ANSD is complex, mainly including genetic and environmentalfactors, and the genetic factors is very important. With the rapid development ofmolecular genetics, more and more focus is performed on the genetic factors, anda variety of genes associated with ANSD have been identified. The DIAPH3geneis the first found gene related to nonsyndromic autosomal dominant inheritedauditory neuropathy spectrum disorder, and is also considered to have possibleassociation with recessive inheritance of nonsyndromic auditory neuropathy.Our study mainly includes two parts: the first one is the research of Clinicalcharacteristics of auditory neuropathy spectrum disorder, while the other is the genetic features analysis of DIAPH3gene in patients with auditory neuropathyspectrum disorder. This research is intended to undertake an in-depth study ofclinical features and genetic characteristics in patients with auditory neuropathyspectrum disorder. The research of Clinical characteristics of Chinese auditoryneuropathy spectrum disorder focus on the hearing loss features of auditoryneuropathy spectrum disorder with different onset ages and the imagingcharacteristics of cochlear nerve in patients with ANSD in China.In the research of genetic features of DIAPH3gene in Chinese patients withauditory neuropathy spectrum disorder, we screened the mutations of DIAPH3gene in125patients with auditory neuropathy spectrum disorder and gave a studyof genetics manifestations. Combining the clinical research and the genetic study,we focused on the study in multiple perspective from etiology, pathologicalpositions and pathological mechanisms to guide the intervention and treatment ofauditory neuropathy spectrum disorder.PART1: Research of Clinical characteristics of auditory neuropathyspectrum disorderⅠ. Hearing loss features of ANSD with different onset ages88subjects (174ears) with auditory neuropathy spectrum disorder (ANSD)were enrolled in this study who had been screened for the mutation of OTOF,PJVK and SLC17A8gene and had good records of pure tone audiometry. The sexratio of the above subjects was0.7:1, while the onset age was between2and33,with a average age of14.4years old. The characteristics of pure tone audiogramswere analyzed in11subjects (20ears) with auditory neuropathy spectrum disorderwith onset age<6years old (Group A),12subjects (24ears) with onset age rangingfrom6years to12years (Group B),42subjects (84ears) with onset age rangingfrom12years to18years (Group C), and23subjects (46ears) with onset age>18 years (Group D). This study analyzed the degree and configuration of hearing lossof patients in the above4groups with auditory neuropathy spectrum disorder.Besides, the relationship between hearing loss degree and onset age was analyzedstatistically.This study found that the shape of the audiometric curves tended to vary,according to the onset age of auditory neuropathy spectrum disorder. Subjects inGroup A mainly presented flat curve with hearing loss at all frequencies, whilesubjects in Group B, C and D display low frequency ascending pattern in themajority of cases. Besides, the results indicated that the degree of hearing loss wasremarkably different in the groups of ANSD patients with different onset ages.Subjects in Group A showed severe to profound sensorineural hearing loss, andsubjects in Group B presented moderate to severe hearing loss, while the hearingloss of subjects in Group C and D displayed mild to moderate hearing loss.In this study, we also investigated the correlation between the hearing lossdegree and the onset age, and we finally found that the configuration and degreeof subjects’ hearing loss tended to vary, according to the onset age of auditoryneuropathy spectrum disorder. Characteristics of hearing loss were remarkablydifferent in the groups of ANSD patients with different onset ages. This study alsoprovided important clinical data for the further research in mechanisms ofauditory neuropathy spectrum disorder.Ⅱ.Imaging characteristics of cochlear nerve in patients with ANSD in China19patients with auditory neuropathy spectrum disorder were enrolled in thisstudy who had undergone cochlear nerve high-resolution magnetic resonanceimaging (HRMRI). The sex ratio of the above subjects was1.38:1, while the agewas between1and44, with a average age of17.3years old. There is one auditoryneuropathy spectrum disorder patient with movement disorders, one patient with optic nerve atrophy, two patients with family history of hearing loss. All the abovesubjects were studied by history collection, physical examination and audiologicalexamination, including pure tone audiometry, acoustic impedance, ABR, DPOAE,speech perception testing, et al. This study analyzed the imaging characteristics ofcochlear nerve in patients with ANSD, and also analyzed the audiometric featuresof cochlear nerve deficiency patients with ANSD.In this study, the19ANSD patients showed a high occurrence of cochlearnerve deficiency which was21.05%(4/19). Within the4patients (8ears）havingsmall cochlear nerves, one patient had movement disorders, one patient had opticnerve atrophy, and two patients had family histories of hearing loss. These4patients presented different configuration and degree of hearing loss. Two patientspresented flat hearing curve with moderate hearing loss at all frequencies, anotherone showed low frequency ascending pattern with moderate to severe hearingloss，while and the other displayed high frequency sloping curve with moderate tosevere hearing loss.This study also found that patients with cochlear nerve deficiency had badspeech discrimination scores, with7out of8ears showed speech discriminationscores less than40%.PART2:The genetic features analysis of DIAPH3gene in patients with ANSDWe enrolled125patients with auditory neuropathy spectrum disorderdiagnosed in our hospital from2003to2013. These patients came from24provinces and cities in China with the male to female ratio of1:1.02. We designed30pairs of PCR primers according to the29exons of the DIAPH3gene.Polymerase chain reaction (PCR) and direct sequencing was performed to identifymutations in the DIAPH3gene. We screened the mutations of DIAPH3gene in the125ANSD patients, and also screened the novel mutations in100unrelatedindividuals with normal hearing. Finally, we analyzed the clinical features andgenetic characteristics of the ANSD patients with DIAPH3gene mutations.In this study,46variations in DIAPH3gene were totally found in the125ANSD patients. Out of the46variations,22variations were located on the exonsof the DIAPH3gene, including7possible pathogenic mutation(c.1425G>A,c.1749T>C，c.2605A>C，c.-44C>G，c.-179G>A，c.-27C>T和c.*196A>T),10single nucleotide polymorphisms (SNP) in the coding region, and5SNP in thenon-coding region. The other24variations were located on the introns of theDIAPH3gene, of which11variations were first reported in the research.The variations of c.1425G>A and c.2605A>C were missense mutation. Thevariation of c.1749T>C was synonymous mutation, while c.-44C>G，c.-179G>A，c.-27C>T和c.*196A>T were mutations in the non-coding region. These7variations didn’t exist in other ANSD patients or100healthy members in thecontrols, so we considered that these seven mutations may have relationships withauditory neuropathy spectrum disorder. Among the125Chinese patients withANSD，mutations were found in7(5.6%).The patient carried c.1425G>A mutation had an onset of ANSD at12yearsold, and her clinical manifestation was an all-frequency, moderate sensorineuralhearing loss. The speech discrimination scores were48%(left ear) and40%(rightear). The variation, c.1749T>C, was identified in a patient who presented hearingloss after birth, and the hearing loss was profound with speech discriminationscores of96%(left ear) and37%(right ear). The patient with c.2605A>Cmutation had an onset of ANSD at18years old, and manifested an low-frequency,mild sensorineural hearing loss with speech discrimination scores of90%(left ear)and88%(right ear). The mutation, c.-44C>G, was identified in a patient whopresented hearing loss at21years old, and the hearing loss was moderate mainly at low frequencies with speech discrimination scores of88%(left ear) and76%(right ear). The mutation, c.-179G>A, was identified in a patient who presentedhearing loss at22years old, and the hearing loss was moderate mainly at lowfrequencies with speech discrimination scores of32%(left ear) and18%(rightear). The mutation, c.-27C>T, was identified in a patient who presented hearingloss at15years old, and the hearing loss was moderate at low frequencies withspeech discrimination scores of56%(left ear) and68%(right ear). The variation,c.1425G>A, was identified in a patient who presented hearing loss at3years old,and the hearing loss was moderate to severe at all frequencies.