The Auditory Characters For Newborns & Infants And The Deaf Gene Mutations And Genotype-Phenotype Correlations In Shandong Province

Background and objectives:Hearing loss is the common birth defect in newborns.There are about 20.57 million people with hearing loss and 0.1368 of newborns have hearing loss annually in china,which means that there are 20～30 thousand hearing loss children increased every year.As we know,it is a very important stage for speech & languagr development before three-year old.The congenital hearing loss will affect the speech,recognition,emotion,education and work opportunities.Therefore,the early diagnosis and early intervention is a better way to avoid dumb and promote children's development.In universal newborn hearing screening programe,there are many chanlenges on the aspects of diagnosis,intervention and rehabilitation presently. In the past decades,auditory brainstem response(ABR) was widely used in clinic as the main diagnosed mathod for infants with hearing loss.But the evoked potentials were very different in different stimulus.The click ABR was highly correlated with the threshold between 2000 to 4000Hz frequency range,while it might cause false nagative result in middle-low frenquency hearing loss.Recently,the frequency specific hearing tests were widely used clinically,including tone-burst ABR,tone-pip ABR,auditory steady state response(ASSR) and behavior hearing test.They could test infants with hearing loss in each frequency and provide the detailed information for diagnosis.More importantly,the frequency specific tests could provide the reliable profile of hearing loss and then make the hearing aid fitting more individually.The Joint Committee on Infant Hearing (JCIH) Year 2007 Position Statement further proposed that the frequency-specific assessment of ABR testing is needed to determine the degree and configuration of hearing loss in each ear for fitting of amplification devices. It also suggested the frequency-specific assessment can be applicated in the diagnosis tests, such as ASSR, tone-burst ABR and so on.In the prsent study, the frequency specific hearing tests were still lack of the clinical criteria for newborns and infants. In addition, the present data of tone-pip ABR and ASSR are mostly from the adults and children, lacking of the reports on newborns and infants. So we know little about the auditory development in the early stage of life,especially on the aspect of the frequency change.The previous study of our team revealed that the threshold of ABR in newborns and infants changed with the increaseing of the month.In this experiment, we analyzed the features of development of tone-pip ABR and ASSR, and established the data for normal infants within the first 6 months of life. This work will provide the theory basis and the application reference for early diagnosis and intervention of congenital hearing loss.The aim of this study is to evaluate the gene mutational analysis of the GJB2, SLC26A4, mtDNA A1555G on children with SNHL, and to identify the cause of SNHL in Shandong province and to explore the relationships between the gene mutations and their adiological characters.Methods:1.80 infants with normal hearing participated in the present study. These subjects were assigned to four groups including 2-day newborns as group 1,6-week old as group 2,3-month old as group 3 and 6-month old infants as group 4 for analysis. Every group had 20 cases (10male and 10 female) with 40 ears. The criteria for normal hearing of infants was:full-term infants with 38 to 41gestational weeks, the birth weight was not less than 2500g, the Apgar score after birth was not less than 8 within one minute, no history of newborn intensive care unite(NICU), no family history of hearing loss and not using any ototoxic drugs. Their physical examinations were within normal range.From Jan 2008 to July 2009,peripheral blood was collected from 146 children with SNHL confirmed by otoacoustic emissions (OAE),typernormetry,auditory brainstem response(ABR),tone-pip ABR,auditory steady state response(ASSR) and clinical physical examination,who aged 6 wks to 68 months.The degree of hearing loss was divided into mild(31～50dB),moderate(51～70dB),sever(71～90dB) and profound (≥91 dB)n HL according to WHO (1997) criteria.2. Measurement Method:①Case History:Before the hearing tests,the information about the history of diliverary,the disease history of the infants,physical examination,the history of pregnant mother and the family history of hearing loss etc.②GSI 901 tympernometry was used to measure the middle ear function. The pressure was produced from+200 daPa to-400 daPa at the speed of 50 daPa per second. The probe tone was produced at 1000 Hz and 226 Hz for the infants less than 6 months old and just 6 months old respectively.③TEOAEs were measured for each subject, using the Danmark Medsen diagnostic equipment, which could show PASS or REFER automatically.④The ABR was performed by the auditory evoked potential system(Medsen, ICS). The recording electrode was positioned on the foread, the ground electrode was positioned at the nasal placement, and the reference electrodes were positioned at the mastoid bilaterally. The impendence was not more than 3kΩacross any two of them. The stimulus was delivered through an ER-3A insert earphone at the rate of 20.1 per second. The output limit was 98 dBnHL. The superposition was 2000 times at each intensity.The band-pass filter was from 100 to 3000 Hz. The test intensity was given at 70 dB nHL firstly and decreased by 20 dB nHL untill the minimal level of the response threshold of wave V appeared.The tone-pip stimuli was produced in 2-0-2 blackman envelope, and the responses were recorded at the minimal level in nHL at the octave frequencies from 250 to 8000 Hz. The stimuli was delivered through an ER-3A insert earphone at the rate of 21.1 per second. The band-pass filter was from 100 to 1500 Hz.⑤The ASSR measurements were presented by the ASSR system(Medsen, ICS) with the carrier frequencies from 250 to 8000Hz and the modulation rates from 77 to 102 Hz. The recording was lasting 8 minutes in the 100%amplitude-modulated (AM) and 25%frequency-modulated (FM) through an ER-3A insert earphone. The amplification gain was 200k, the band-pass filter was from 65 to 105 Hz. The stimulus was produced synchronously at 12 frequencies bilaterally with 5 dB steps. The output limit was 120 dB nHL.The minimal level of responses were detected in nHL automatically. Every electro-physilogial hearing assements above were conducted in the sound-proof room, and the ambient noise was not more than 17dB(A).⑥The high resolution CT scan was used for infants with hearing loss.⑦The polymerase chain reaction(PCR) and sequencing technique were used to analyze the coding region of GJB2,SLC26A4,mtDNA A1555G gene.The sequences were analysed with DNAStar.3.Statistical AnalysisAll data were analyzed by using SPSS 10.0 software packages, T test and one-way analysis were employed. The differences between groups were recognized statistically significant as the p value not more than 0.05. If there were statistical differences amongs the groups, the SNK (Student-Newman-Keuls) test were used for comparing every two groups. The difference between two groups was recognized statistically significant as the q value not more than 0.05.Results:1. For click ABR at 70dB nHL, the absolute and interpeak wave latencies ofⅠ,Ⅲ,Ⅴ,Ⅰ-Ⅲ,Ⅲ-ⅤandⅠ-Ⅴdecreased as the age increased (p<0.05). The developmental changes were obvious in waveⅠandⅢbefore 6 wk and 3-month respectively (p<0.05).2.The tone-pip ABR had similar waveforms as the click ABR, its wave latencies decreased and the waveforms got better with the age and frequency increased.3.The thresholds of tone-pip ABR and ASSR had different thresholds at 250 to 8000 Hz in 0～6-month infants, but no physiological differences of them. The tone-pip ABR thresholds were significantly lower than those of ASSR from 0.5 to 8 kHz (p<0.05).4. Both ASSR and ABR had satable and similar audiograms between different age groups in infants.5. 44 of 146 (30.14%)cases with SNHL were found to carry GJB2 mutations,18 cases were identified as 235delC/235delC homozygotes,4 of 44 were identified as 235delC/176-191dell6 compound heterozygotes,3 were identified as 235delC/299～300delAT compound heterozygotes,19 were identified as 235delC heterozygotes.6. 23 of 146 (15.75%) cases were found to carry SLA26A4 mutations,including 10 IVA7-2 A>G homozygotes and 13 heterozygotes.The incidence of hearing loss caused by SLA26A4 gene mutations was 6.85%.7. Only 1 of 146(0.68%) was found to carry mtDNA A1555G mutations, who was heterozygotes.8. Totally,128 of 146 (87.67%) cases were received high resilution CT scan and 27 cases of them had positive results.56.52%of cases with SLC26A4 mutations had inner ear malformation, which was higher than GJB2 and no-mutation groups.4 cases with GJB2 mutations were diagnosed with enlarged vestibular aqueduct (EVA), a novel reported in Shandong Province for the first time.9. The audiological characters of GJB2 and SLA26A4 mutations were diversity. The degree of the hearing loss were mostly sever to profound for GJB2 mutations while moderate to sever for SLC26A4 mutations.Conclusions:1. The wave latency ofⅠ,Ⅲ,Ⅴand inter-wave latency ofⅠ-Ⅲ,Ⅲ-ⅤandⅠ-Ⅴof tone-pip ABR were decreased as the age increase, while the thresholds had no physiology changes within 0～6 month after birth.2.Both tone-pip ABR and ASSR had stable frequency specificity. Compared with the ASSR, tone-pip ABR had lower response threshold and maybe nearer to the hearing level of the infant.3. The incidence of GJB2, SLC26A4 and mtDNA A1555G mutations were 30.14%,15.75%and 0.68%respectively for children with NSHI. The main mutations of GJB2 gene were 235delC/235delC,235delC/176-191del16 and 235delC/299delAT. 23.97%(35/146) of all with NSHI were diagnosed with heredity though the conbine tests of Molecular genetic method. Screening and diagnosis for these three mutations are nesseory and effective to the children with SNHL found by universal newborn hearing screening programe.4. The audiological characteristics of GJB2 and SLA26A4 mutations were diversity.5. Molecular genetic screening and diagnosis for GJB2, SLA26A4 and mtDNA A1555G mutations were nesseory and effective on the basis of universal newborn hearing screening programe.Innovations and Meanings:1.This study first established the normal data of tone-pip ABR and ASSR, and investigated the frequency specificity between them in infants aged 0～6 months,which provided the referenced evidence for the, development of human auditory system and effective policy of frequency specific hearing tests in clinical practice.2. This study first reported the incidence of GJB2, SLA26A4 and mtDNA A1555G mutations in a large sample in Shandong province of China, on the basis of universal newborn hearing screening program. This study also investigated the relationship between gene mutations and clinical denonstrations,and reported that 4 cases with inner ear malformation carried GJB2 but no SLA26A4 mutations.