Clinical Genetic Mechanisms For Auditory Neuropathy

Since the 1980's of the last century,audiologists,ear phsiologists,clinical doctors in otorhinolaryngology and molecular geneticists have started to attend a clinically distinct type of heating disorder-Auditory Neuropathy(AN).AN is a disorder with an abnormal hearing function in which sound signals can be delievered from outer ear to middle ear to inner ear but they fail to transmit to the brain.Therefore,its main otological characteristics are normal evoked otoacoustic emission(EOAE)readings,but obviously abnormal auditory brainstem responses (ABR).The etiology for AN remains less clear,authough the reports up to date implicate causes in genetics,immunology,virus infection,toxic agents and metabolism.In recent years increasing studies concerned the genomic basis for AN.Nowadays the studies of the underlying genes for sporadic or familial AN cases using molecular genetics has become one of hot spots in the domain of otological studies worldwide,which is of great importance in further understanding and exploring the etiological underpinns for AN.The present study was to analyse the clinical data for the sporadic and familial cases collected through the national network for collecting genetic resources of hearing loss maintained by the Institute of Otolaryngology of the Chinese People's Liberation Army(PLA)General Hospital.To determine the genetic causes,two candidate genes was also screened.The thesis describing the study is divided into three parts:PART 1:clinical characteristic of Auditory neuropathyOne hundred and forty six sporadic or familial AN cases was selected from the patients who visited our clinic during 2003-2006.Their features was characterised by analysing the clinical and audiological testing data,including identification of the AN subtypes based on their distinct clinical features,ages of onset,accompaning symptoms,and molecular epidemiological characters.For the 146 cases,gender ratio was 1:1.35(male:female)and the onset ages for 78.1%patients were less than 20 years.Audigrams showed that low frequency hearing loss(with low frequency ascending or flipped U-shape curve)taked up 77.3%.Other characteristics include(1)speech recognition rate was inproporanately lower than the pure tone threshold;(2).acoustic stapedial reflex was not induced or the threshold increased.Among 268 ears,auditory brainstem response(ABR)TypeⅠcurve was not present;13 ears(bilateral in six cases and singular in one case)induced TypeⅤcurve;five ears(bilateral in one case and singular in three cases)induced TypeⅠcurve.Distortion product otoacoustic emissions(DPOAE)tests reveal responses at all frequencies,and opposite side inhibition reaction disappeared.Based on auditory steady state response(ASSR) average over a range of frequencies,light heating loss accounted for 0.7%; moderate 22.5%;severe 59%;and profound 17.8%,indicating that ASSR was useful for judging the speech recognition ability of the AN patients.Based on 40 Hz auditory event related potential(40 Hz AERP),the four levels of hearing loss were 6.4%,18%,45.3%and 30.3%,respectively,indicating that this test could provide an objective evaluation for low to medium frequency hearing loss,and jointly used with ABR localize the lesion site in the brain-stem.According to electrocohleogram(ECochG),64.5%patients had-SP/AP＞1 or unrecognizable AP,and only 4.3%showed-SP/AP＜0.4.Twenty patients received an imaging exam on their skulls,revealing 19 normal cases and one case,identified by CT scan of the temporal bone,had bilateral middle ear mastoiditis and bilateral siagantritis.Overall,normal OAEs,absent ABR,decreased speech recognition unproporanately with the pure tone hearing,absent acoustic stapedial reflex were the standard for auditory neuropathy.In our study,auditory neuropathy can be distinguished different types according to whether the patient had simultaneous phenomenons.There were 23 patiants who had simultaneous phenomenons.According to the age of onset,there were 41 patients blow 12 years old,and we called them pedo-AN.There were 6 patients affected only one ear.And there were 8 patiants comes from 4 families among the whole objects.PART 2:screening the mutation of OTOF gene in auditory neuropathy.We designed 45 pairs of primers to amplify 48 exons of the OTOF gene. Polymerase chain reaction(PCR)and sequencing were performed to identify the mutations in PCR products of OTOF coding sequence in the members of groups that involved pedigree and isolated 76 patients suffered from auditory neuropathy and 92 persons as control.34 mutations located on different exons and introns of OTOF.Among the total,15 mutations located on exons,including 7 missense mutations,2 deletion mutations,and 6 SNPs which didn't change amino acids.19 mutations located on introns.1 deletion mutation(2975-2978delAG)and 2 missense mutations(A53T and R1607W)only existed in a temperature-sensitive auditory neuropathy.We had tested the parents of the patient who had normal acoustic sensibility.Mother carried the same deletion mutation,and father carried the same missense mutations.All of the three mutations caused amino acids changing,and were not existed in other patients and controls.We considered that these mutations had relationsheip with AN.5 mutations on exons(A35V,R82C,R49W,372A/G and 4677G/A)were SNPs which verified by statistics.4 mutations on exons(76C/A,129C/T, 4767C/T and 5418C/T)only showed heterozygosis form,they existed both in the patients and the controls.19 mutations on introns were all considered SNPs, because all of them located far away from coding region,and existed both in the patients and the controls.3 mutations(1740delC,D398E and E594K)only existed in 1 patiant respectively,but the 3 patiants didn't display special phaenotype.Mutation screening for OTOF allowed further characterization of this disease. The patients with OTOF mutation presented typeⅡ,and they were suitable for cochlea implant.PART 3:screening the mutation of GJB1 gene in auditory neurophathy.In the study we designed 2 pairs of primers to the GJB1 gene.The subjects were 41 sporadic male cases with auditory neuropathy and 1 X-linked familial auditory neuropathy including 3 patiants.We also choose 52 male as control.Only one point mutation was found in 2 sporadic cases,which were 512C/T located on the unique exon,and didn't cause amino acid changing.ConclusionIn the study,we analyzed the clinical audiology and genetics characteristic of auditory neurophathy respectively.The candidate genes of OTOF and GJB1 were screened in the cases with auditory neuropathy as well as in the controls.We found 34 point mutations on OTOF including the mutations which caused the amino acid changed in a temperature-sensitive auditory neuropathy.The results suggest that OTOF gene may contribute to the mechanism of auditory neuropathy, while GJB1 gene may be ruled out.Our study may help us for further understanding the auditory neuropathy.