Study On Underlying Molecular Mechanisms Of Monoallelic Mutations Of GJB2Gene In Chinese Nonsyndromic Hearing-impaired Patients

Objective: To screen the nonsyndromic hearing impairment (NSHI) patientswith monoallelic mutations of the GJB2gene, and to summarize thecharacteristics of genotype and phenotype of monoallelic mutations of the GJB2gene and the genotype-phenotype correlation. To evaluate GJA1gene mutationsin NSHI patients with monoallelic GJB2mutations. To explore the underlyingpathogenic factors for the NSHI patients with monoallelic mutations of the GJB2gene, and to detect whether or not insertion or deletion of large fragmentnucleotides exists in the GJB2gene sequence.Material and Methods: In part one, we analyzed the results of genediagnosis for7514NSHI patients from the Deafness Molecular DiagnosticsCenter of the People’s Liberation Army General Hospital and chose217caseswith monoallelic mutations of the GJB2gene but without the other commondeafness gene mutations for further study, such as GJB3gene, GJB6gene,SLC26A4gene, mitochondria DNA, etc. We analyzed the genotype of the GJB2gene monoallelic mutations and the single nucleotide polymorphism(SNP) andsummarized the clinical characteristics of the patients with GJB2geneheterozygous mutations, including the general information of the patients, theaudiological results and the radiological imaging data. The appropriateaudiological tests were selected according to the patients’ condition, includingpure tone audiometry, children behavioural audiometry, acoustic impedanceaudiometry, auditory brainstem response (ABR),40Hz auditory event related potentials(40Hz AERP), auditory steady-state response (ASSR), otoacousticemissions(OAEs), etc. In part two, we sequenced the entire coding region of theGJA1gene in205nonsyndromic hearing-impaired patients carrying a pathogenicGJB2gene mutation and in111normal hearing controls. In part three, wedetected the full length of the GJB2gene sequence for201NSHI patients withGJB2gene monoallelic mutations and111adults with normal hearing by longchain two-step PCR method and0.8%agarose gel electrophoresis(AGE).Results: The monoallelic mutation rate of the GJB2gene was2.89%(217/7541) in the Chinese NSHI patients. Among the217NSHI patientswith monoallelic GJB2gene mutations, there are119males and98females. Therewere17types of monoallelic pathologic mutations of the GJB2gene, the mostcommon types of monoallelic mutations were235delC heterozygous,299delATheterozygous and176del16heterozygous, with152cases (70.05%),28cases(12.90%),6cases (2.76%), respectively. There were100cases(46.08%) withoutany SNP in the patients with monoallelic GJB2gene mutations,53cases (24.42%)only with heterozygous109G>A,32cases (14.75%) with heterozygous of79G>Aand341A>G,24cases（11.06%）only with heterozygous79G>A, and8cases(3.69%) with other types of heterozygous SNP. The results of pure toneaudiometry test for191cases were132cases (69.11%) with very severe hearingloss,25cases (13.09%) with severe hearing loss,25cases (13.09%) withmoderate hearing loss,7cases (3.66%) with mild hearing loss, and2cases (1.05%)with normal hearing; there were162cases (84.82%) with bilateral symmetricalhearing loss and29cases (15.18%) with binaural nonsymmetrical hearing loss.There were176patients (92.14%,176/191) with frameshift mutations,13patients(6.81%,13/191）with missense mutations and2patients (1.05%,2/191) withnonsense mutations, and123patients (69.89%,123/176）with very severe hearingloss and frameshift mutations. The results of acoustic impedance audiometry test for74cases were102ears (68.92%) with type A of tympanogram,27ears(18.24%) with type As,9ears (6.08%) with type C,6ears (4.05%) with type Ad,2ears (1.35%) with type B and2ears (1.35%) with type D; there were37ears(25.00%) with the acoustic reflex threshold less than or equal to95dB,18ears(12.16%) with the acoustic reflex threshold between96dB and110dB and93ears(62.84%) without acoustic reflex; there were48ears (32.88%) with recruitment.The results of ABR for66cases were63ears (52.50%) without response,15ears(12.50%) with the threshold of ABR between30dB nHL and60dB nHL,23ears(19.17%) with the threshold between61dB nHL and90dB nHL, and19ears(15.83%) with the threshold more than90dB nHL. The results of40Hz AERP for35cases were5ears (7.14%) with the threshold of40Hz AERP less than or equalto60dB nHL,14ears (20.00%) with the threshold between61dB nHL and90dBnHL,35ears (50.00%) with the threshold between91dB nHL and120dB nHL,and12ears (22.86%) without response more than120dB nHL. The threshold ofASSR was the average threshold at0.5、1、2、4kHz, and the results of ASSR for29cases were8ears (13.79%) with the threshold of ASSR less than or equal to80HLcg,38ears (65.52%) with the threshold between81HLcg and119HLcg,12ears (20.69%) with the threshold equal to or more than120HLcg. The results ofOAEs for47cases were87ears (92.55%) without OAEs response and only7earswith OAEs response. Three of95patients were found having malformations ininner ear by temporal bone HRCT. Five of the205patients were found havingmonoallelic GJA1gene mutations,3(1.45%) having IVS2+1insA mutations,1having456G>A mutation, and1having717G>A mutation. Four individuals fromthe control group were found having monoallelic GJA1gene mutations,3(2.70%)with IVS2+1insA mutations, and1with466A>G mutation. No significantdifference in the IVS2+1insA mutation rate was found between the two groups(continuity correction χ2=0.115, P=0.735＞0.05). Obvious increase or decrease of the sequence length of GJB2gene was not found in201NSHI patients withmonoallelic GJB2gene mutations and in the111cases from the control group.Conclusion: The common types of monoallelic mutations of the GJB2genemonoallelic mutations in Chinese NSHI patients were235delC heterozygous,299delAT heterozygous and176del16heterozygous. The main clinicalmanifestation of most NSHI patients with monoallelic mutations of the GJB2gene showed bilateral, symmetrical, prelingual and very severe hearing loss. Thegenotype-phenotype correlation of monoallelic mutations of the GJB2generevealed versatility and polymorphism. The most common tympanogram typeswere type A and type As, and the patients whose acoustic reflection could beelicited often had recruitment. Other audiological tests were important means forNSHI patients, such as ABR, OAEs,40Hz AERP and ASSR. Malformations ofthe inner ear could be found in NSHI patients with monoallelic GJB2genemutations. Pathologic mutations of GJA1gene do not appear in NSHI patientswith monoallelic GJB2gene mutations. Insertion or deletion of large fragmentnucleotides in the GJB2gene sequence was not found in NSHI patients withmonoallelic GJB2gene mutations.