- Email: [email protected]
Prevalence of A1555G mitochondrial mutation in Chinese newborns and the correlation with neonatal hearing screening
International Journal of Pediatric Otorhinolaryngology 75 (2011) 532–534
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Prevalence of A1555G mitochondrial mutation in Chinese newborns and the correlation with neonatal hearing screening Guanming Chen a, Xiyin Wang b, Siqing Fu b,* a b
Department of Otolaryngology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Medical Genetics, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 September 2010 Received in revised form 4 January 2011 Accepted 11 January 2011 Available online 15 February 2011
Objective: To investigate the feasibility of genetic screening for deafness causative genes in the process of newborn hearing screening in China. Methods: Total 865 newborn babies between November 2009 and March 2010 were enrolled for the simultaneous hearing and deafness causative gene screening in Tongji Hospital, Wuhan, China. Hearing screening followed a two-stage strategy with transient evoked otoacoustic emissions. Infants referred after the second-stage screening were tested by diagnostic auditory brainstem response (ABR). Genomic DNA was extracted from heel blood of newborns, and the mitochondrial 12S rRNA A1555G mutation was detected by polymerase chain reaction (PCR) based restriction fragment length polymorphism and confirmed by DNA sequencing. Results: In hearing screening, 134 out of the 865 newborns (15.5%) were referred after the first-stage screening and 86.6% (116/134) of them returned for the second stage. After the second-stage screening, 15 who were still referred were tested by diagnostic ABR and 3 of them failed the test. On the other hand, gene screening identified 6 of the 865 newborns (0.7%) harbored homoplasmic 12S rRNA A1555G mutation although they passed the hearing screening. Conclusion: It might be practical and effective to complement routine hearing screening in newborns with gene screening for the purpose of early diagnosis and discovery of the late-onset hearing loss. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hearing screening Newborn Hearing loss Mitochondrial mutation Genetic screening
1. Introduction Hearing loss is the most common birth defect and occurs more frequently than other conditions for which newborns are routinely screened [1]. Universal newborn hearing screening is now implemented in China and most of other countries around the world. Newborn hearing screening aims to provide early identification of hearing loss, promote early intervention and, in turn, facilitate the development of communication and auditory skills in children with hearing loss [2]. In developed countries, about twothirds of prelingual-onset sensorineural hearing loss have a genetic etiology [3,4]. Specifically, in familial cases of ototoxic sensorineural hearing loss, the aminoglycoside hypersensitivity is often maternally transmitted, suggesting the involvement of mutations in mitochondrial DNA (mtDNA) [5], which are associated with both aminoglycoside-induced and non-syndromic deafness [5,6]. So far, the A1555G mitochondrial mutation has been found to be one of the major causes of hearing loss in Chinese [7–11]. Li et al. screened the mitochondrial 12S rRNA gene in 128 Chinese
* Corresponding author. Tel.: +86 27 8369 2629; fax: +86 27 8369 2608. E-mail address: [email protected] (S. Fu). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.01.013
pediatric subjects with sporadic aminoglycoside-induced and nonsyndromic hearing loss and found that the incidence of A1555G mutation is approximately 13% and 2.9% in this pediatric population with aminoglycoside-induced and nonsyndromic hearing loss, respectively [9]. In addition, Liu et al. investigated mtDNA mutation in 498 nonsyndromic sensorineural hearing loss (NSHL) patients (290 from China and 208 from the USA) with or without history of aminoglycoside exposure, mtDNA A1555G mutation was found in all Chinese probands (15.5%) with mtDNA mutation, but only in 4 probands (1.9%) from the USA. Indeed, the high incidence of mtDNA A1555G mutation in Chinese probands with NSHL helps explain the high prevalence of aminoglycosideinduced deafness in China [10]. It is important to note that although mutations causing deafness are present at the time of conception, hearing loss resulting from these mutations is not present at birth. Consequently, most children who develop delayed onset hearing loss will pass the newborn hearing screening [12,13]. The addition of genetic screening as a complement to audiologic newborn screening will help identify infants with late-onset prelingual hearing loss [14,15]. Therefore, in this study we performed genetic screening on the common mutation in Chinese populations when hearing screening was carried out
G. Chen et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 532–534
in newborns, with the aim to investigate the feasibility of genetic screening for deafness causative genes in the process of newborn hearing screening in China.
2. Subjects and methods 2.1. Subjects The newborn hearing screening and deafness-causing gene screening protocols have been developed by the Huazhong University of Science and Technology (HUST), and approved by the Ethics Committee of HUST. The hearing screening was a part of routine medical services provided to all newborns delivered in Tongji hospital, HUST, from November 2009 to March 2010. All parents were informed about the testing process and gave their consent. 2.2. Newborn hearing screening The screening method consisted of a two-stage screening approach with transient evoked otoacoustic emission (TEOAE). The initial screening was performed 3–7 days after birth before discharge. The newborns were always accompanied by their mother or guardian and were either asleep or drowsy. Outpatient screening was conducted 4–6 weeks after birth for infants who failed in the first-stage screening. If a baby did not pass the secondstage screen, he was referred for diagnostic testing. 2.3. Transient evoked otoacoustic emissions TEOAEs were recorded by an ILO-88 cochlear emission analyzer (Otodynamics, London, UK), using insert ear phones in a soundproofed room. The TEOAEs were obtained from 1 to 4 kHz, with stimuli consisting of clicks of 80 ms duration. The stimulus level in the outer ear was set at 83 3 dB SPL. The click rate was 80 s1, and post-stimulus analysis was in the range of 2– 20 ms. An amplifier gain suppression period of 2.5 ms was used to remove the primary stimulus artifact. A total of 260 sweeps was averaged above the noise rejection level of 50 dB. The stimuli were presented in the nonlinear mode, in which every fourth click stimulus was inverted and three times greater in amplitude than the three preceding clicks. The TEOAE responses were classified as either ‘‘pass’’ or ‘‘fail’’ following each stimulus data set. The pass criterion was as follows: the TEOAE spectrum was recorded at least 3 dB above the noise floor (i.e., SNR 3 dB) for all three frequency bands at 2.4, 3.2 and 4.0 kHz. Reproducibility percentages of 70% and stimulus stability of 80% were taken into account as acceptable for analysis [16].
533
2.5. Mitochondrial 12S rRNA A1555G mutation screening The heel blood leukocytes were routinely taken from the newborns for the screening of genetic diseases. Genomic DNA was extracted from the blood using a routine desalting method. Mitochondrial DNA was amplified with the forward primer 1555F: 50 -GAG GAG CCT GTT CTG TAA TC-30 , and reverse primer 1555R: 50 CTT GGA CAA CCA GCT ATC AC30 (amplicion 832 bp). The amplification conditions were 95 8C for 5 min, then 32 cycles of 94 8C for 1 min, 56 8C for 45 s, and 72 8C for 1 min, with a final extension at 72 8C for 5 min. Restriction enzyme digestion was performed on PCR products in order to detect the 12S rRNA A1555G mutation. Upon digestion with Alw26I, PCR product of 832 bp from normal individuals would yield bands of 473 bp and 359 bp, whereas in affected individuals, the absence of the restriction site resulted in a band of 832 bp. The samples with the possible 12S rRNA A1555G mutation were further subjected to DNA sequencing by ABI 377 DNA Automatic Sequencer (Perking, USA) to verify the mutation. The result of genetic screening was designated as ‘‘pass’’ or ‘‘fail’’ with ‘‘Pass’’ meaning that an infant harbored no 12S rRNA A1555G mutation and ‘‘Fail’’ meaning that an infant harbored 12S rRNA A1555G homoplasmic mutation in the current testing method.
3. Results Among the 865 neonates screened in this study, 6 of the 865 newborns (0.7%) harbored homoplasmic 12S rRNA A1555G mutation as detected by PCR-RFLP and confirmed by DNA sequencing. It is important to note that all of these 6 newborns passed the hearing screening. For the TEOAE test, 15.5% of the 865 neonates (134/865) failed in the 1st hearing screening test, and 86.6% (116/134) of them returned for the second stage screening. The failure rate for 2nd screen was 12.9% (15/116) and the failure rate for diagnostic stage was 20% (3/15) because 3 of them failed the test when tested by diagnostic ABR. The three neonates were subjected to audiometry, the results showed that two had severe deafness in both ears and one had moderate deafness in both ears. The two neonates with severe deafness already got treatment and the one with moderate deafness is still in follow-up. According to the hearing screening protocol, all parents of failed neonates were asked to bring their children to repeat the test within a month following discharge. From the 116 of the 134 newborns who repeated the test, 101 were found to be normal, and 15 failed TEOAE. The 15 babies were then further tested by ABR, and 12 passed and 3 failed.
2.4. Diagnostic hearing testing 4. Discussion This diagnostic evaluation was conducted by the diagnostic auditory brainstem response (ABR). An evoked potential tester (Nicolet Spirit, Nicolet Inc., Madison, WI, USA) was used with an alternatively inverted click stimulus, pulse width of 0.1 ms, initial intensity of sound stimulus of 80 dB nHL, stimulus repetition rate of 11.9 times/s, analysis time of 10 ms, band-pass filtering of 10– 3000 Hz, and 2 or 3 replications of 1000 sweeps. The box of electrodes consisted of four electrodes: a forehead electrode as the recording electrode, acoustic stimulation of the bilateral mastoids as the reference electrodes, and a glabella electrode as the ground electrode. The impedance values of the electrodes were below 5 kV. A V response threshold for the ABR wave equal to or less than 30 dB nHL served as an index of normal hearing in the range of 2– 4 kHz [17].
In the present study, we found that six newborns who passed hearing screening harbored homoplasmic mitochondrial 12S rRNA A1555G mutation, indicating that they will be highly sensitive to amimoglycosides. Thus they could avoid irreversible hearing loss if their exposure to aminoglycosides is minimized in future. Based on our present data that 0.7% newborns harbor the mitochondrial 12S rRNA A1555G mutation, 126,000 Chinese children will carry this mutation given that about 18,000,000 children are born every year in China. In our previous study, we screened a total of 504,348 students in the Hubei province of China and identified 813 deaf students. Among the 813 cases of deafness, aminoglycoside-antibiotic-induced deafness accounted for 33.95% [18]. The frequency of the 12S rRNA
534
G. Chen et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 532–534
A1555G mutation is 13–25% in Chinese pediatric subjects with aminoglycoside-induced hearing loss [9,10]. Therefore, there is really an urgent need to implement gene screening for newborns in China to prevent hearing loss and control the incidence of hearing loss from the newborns. This should result in a drastic reduction in the incidence of this iatrogenic form of deafness. The hospital-based newborn hearing screening has been implemented for nearly ten years in China. The overall referral rate after the first-stage screening has a wide variation from 9.84% to 31.3% in China as reported previously [19,20]. In the population we screened in this study the referral rate was 15.5% and decreased significantly to 1.7% (15/865) on retest. Of the 865 newborns undergoing routine hearing screening, only 3 newborns were diagnosed as sensorineural hearing loss, thus the prevalence is 3.46:1000. This prevalence is higher than that reported by Nie et al. (2.86:1000) [21], but lower than that reported by Huang et al. (0.5%) [22]. It is unclear what percentage of post-lingual hearing loss is due to mtDNA mutations. It has been estimated that around 20% of inherited post-lingual hearing loss may be caused by mutations in the mitochondrial genome, but large ethnic differences might be present [23]. In total, newborns who harbored mutation accounted for 0.7% (6/865) in the present study, which was 7 times higher than the incidence of hearing loss (0.1%) by hearing screening [24]. Because hearing loss is not always present at birth and most children who develop delayed onset hearing loss will pass the newborn hearing screening [12,13], it might be an effective measure to complement routine hearing screening with mitochondrial 12S rRNA A1555G mutation screening for the purpose of early diagnosis and identification of the late-onset hearing loss. It is important to point out that the use of molecular test alone to detect genetic hearing loss is not currently feasible because the validity of the identified deafness genes for molecular test need further investigation and many uncertainties would arise in the interpretation of test results. Nevertheless, as an adjunct to audiologic screening, such genetic tests would provide a powerful and unprecedented strategy for identifying newborns at risk for the development of late-onset hearing loss [15]. Moreover, a recent survey revealed that Chinese parents have a predominantly positive attitudes toward genetic testing for prelingual deafness [25], indicating that genetic testing for newborn will be easily accepted by Chinese parents. In conclusion, our study suggests that it might be practical and effective to complement routine hearing screening in newborns with genetic screening for deafness susceptibility genes. With the improvement in genetic screening techniques for hearing loss and training of technicians, the combination of genetic screening in routine newborn hearing screening is expected to effectively reduce the incidence of hearing loss in the next 20–30 years in China.
Conflict of interest None to declare.
Acknowledgments We thank all the babies and their parents for their kind participation in the study. We greatly appreciate the editorial assistance of Dr. Yingqun Wang. References [1] J.S. Oghalai, L. Chen, M.L. Brennan, R. Tonini, S. Manolidis, Neonatal hearing loss in the indigent, Laryngoscope 112 (2002) 281–286. [2] Y.S. Sininger, A. Martinez, L. Eisenberg, E. Christensen, A. Grimes, J. Hu, Newborn hearing screening speeds diagnosis and access to intervention by 20–25 months, J. Am. Acad. Audiol. 20 (2009) 49–57. [3] C. Petit, J. Levilliers, J.P. Hardelin, Molecular genetics of hearing loss, Annu. Rev. Genet. 35 (2001) 589–646. [4] R.J. Smith, J.F. Bale Jr., K.R. White, Sensorineural hearing loss in children, Lancet 365 (2005) 879–890. [5] N. Fischel-Ghodsian, Mitochondrial deafness mutations reviewed, Hum. Mutat. 13 (1999) 261–270. [6] G. Van Camp, R.J. Smith, Maternally inherited hearing impairment, Clin. Genet. 57 (2000) 409–414. [7] D.N. Hu, W.Q. Qiu, B.T. Wu, L.Z. Fang, F. Zhou, Y.P. Gu, et al., Genetic aspects of antibiotic induced deafness: mitochondrial inheritance, J. Med. Genet. 28 (1991) 79–83. [8] X. Bu, G. Xing, M. Yan, Audiological and molecular findings in a large family with maternally inherited sensorineural hearing loss, J. Audiol. Med. 9 (2000) 61–69. [9] Z. Li, R. Li, J. Chen, Z. Liao, Y. Zhu, Y. Qian, et al., Mutational analysis of the mitochondrial 12S rRNA gene in Chinese pediatric subjects with aminoglycosideinduced and non-syndromic hearing loss, Hum. Genet. 117 (2005) 9–15. [10] X.Z. Liu, S. Angeli, X.M. Ouyang, W. Liu, X.M. Ke, Y.H. Liu, et al., Audiological and genetic features of the mtDNA mutations, Acta Otolaryngol. 128 (2008) 732–738. [11] X.M. Ouyang, D. Yan, H.J. Yuan, D. Pu, L.L. Du, D.Y. Han, et al., The genetic bases for non-syndromic hearing loss among Chinese, J. Hum. Genet. 54 (2009) 131–140. [12] G.E. Green, R.J. Smith, J.P. Bent, E.S. Cohn, Genetic testing to identify deaf newborns, JAMA 284 (2000) 1245. [13] V. Norris, K.S. Arnos, W.D. Hanks, X.J. Xia, W.E. Nance, A. Pandya, Does universal newborn hearing screening identify all children with GJB2 (Connexin 26) deafness? Penetrance of GJB2 deafness, Ear Hear. 27 (2006) 732–741. [14] S.K. Burton, K. Withrow, K.S. Arnos, A.L. Kalfoglou, A. Pandya, A focus group study of consumer attitudes toward genetic testing and newborn screening for deafness, Genet. Med. 8 (2006) 779–783. [15] C.C. Morton, W.E. Nance, Newborn hearing screening-a silent revolution, N. Engl. J. Med. 354 (2006) 2151–2164. [16] B. Gunduz, Y.A. Bayazit, F. Celenk, C. Saridog˘an, A.G. Guclu, E. Orcan, et al., Absence of contralateral suppression of transiently evoked otoacoustic emissions in fibromyalgia syndrome, J. Laryngol. Otol. 122 (2008) 1047–1051. [17] S.J. Norton, M.P. Gorga, J.E. Widen, R.C. Folsom, Y. Sininger, B.C. -W, et al., Identification of neonatal hearing impairment: summary and recommendations, Ear Hear. 21 (2000) 529–535. [18] S. Fu, G. Chen, J. Dong, L. Zhang, Prevalence and etiology of hearing loss in primary and middle school students in the Hubei province of China, Audiol. Neurootol. 15 (2010) 394–398. [19] J. Pan, Risk factors for hearing loss in 16567 neonates, Chin. J. Perinat. Med. 9 (2006) 275–276 (Chinese). [20] Z. Cai, L. Huang, H. En, S. Peng, Y. Zhen, B. Qi, et al., Infants hearing screening in thecoasted villages of Shandong province of China, Chin. J. Otorhinolaryngol. Head Neck Surg. 41 (2006) 104–406 (Chinese). [21] W. Nie, L. Gong, Y. Liu, L. Xiang, Q. Lin, Y. Qi, et al., Hearing screening of 10501 newborns, Natl. Med. J. China 83 (2003) 274–277 (Chinese). [22] L. Huang, Z. Cai, H. Zhang, S. Peng, D. Wu, L. Wang, et al., Study on multi-area universal newborn hearing screening in countryside of China, Lin Chung Er Bi Yan Hou Tou Jing Wai Ke Za Zhi 23 (2009) 737–742 (Chinese). [23] X. Estivill, N. Govea, A. Barcelo, E. Perello, C. Badenas, E. Romero, et al., Familial progressive sensorineural deafness is mainly due to the mtDNA A1555G mutation and is enhanced by treatment with aminoglycosides, Am. J. Hum. Genet. 62 (1998) 27–35. [24] X. Sun, C. Qu, L. Yang, J. Yan, J. Xia, Y. Chen, et al., Study on the disabilities in aged 0–7 years children in Shenzhen, China, Chin. J. Epidemiol. 24 (2003) 1016–1019 (Chinese). [25] S. Fu, J. Dong, C. Wang, G. Chen, Parental attitudes toward genetic testing for prelingual deafness in China, Int. J. Pediatr. Otorhinolaryngol. 74 (2010) 1122–1125.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Prevalence of A1555G mitochondrial mutation in Chinese newborns and the correlation with neonatal hearing screening Guanming Chen a, Xiyin Wang b, Siqing Fu b,* a b
Department of Otolaryngology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China Department of Medical Genetics, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 20 September 2010 Received in revised form 4 January 2011 Accepted 11 January 2011 Available online 15 February 2011
Objective: To investigate the feasibility of genetic screening for deafness causative genes in the process of newborn hearing screening in China. Methods: Total 865 newborn babies between November 2009 and March 2010 were enrolled for the simultaneous hearing and deafness causative gene screening in Tongji Hospital, Wuhan, China. Hearing screening followed a two-stage strategy with transient evoked otoacoustic emissions. Infants referred after the second-stage screening were tested by diagnostic auditory brainstem response (ABR). Genomic DNA was extracted from heel blood of newborns, and the mitochondrial 12S rRNA A1555G mutation was detected by polymerase chain reaction (PCR) based restriction fragment length polymorphism and confirmed by DNA sequencing. Results: In hearing screening, 134 out of the 865 newborns (15.5%) were referred after the first-stage screening and 86.6% (116/134) of them returned for the second stage. After the second-stage screening, 15 who were still referred were tested by diagnostic ABR and 3 of them failed the test. On the other hand, gene screening identified 6 of the 865 newborns (0.7%) harbored homoplasmic 12S rRNA A1555G mutation although they passed the hearing screening. Conclusion: It might be practical and effective to complement routine hearing screening in newborns with gene screening for the purpose of early diagnosis and discovery of the late-onset hearing loss. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hearing screening Newborn Hearing loss Mitochondrial mutation Genetic screening
1. Introduction Hearing loss is the most common birth defect and occurs more frequently than other conditions for which newborns are routinely screened [1]. Universal newborn hearing screening is now implemented in China and most of other countries around the world. Newborn hearing screening aims to provide early identification of hearing loss, promote early intervention and, in turn, facilitate the development of communication and auditory skills in children with hearing loss [2]. In developed countries, about twothirds of prelingual-onset sensorineural hearing loss have a genetic etiology [3,4]. Specifically, in familial cases of ototoxic sensorineural hearing loss, the aminoglycoside hypersensitivity is often maternally transmitted, suggesting the involvement of mutations in mitochondrial DNA (mtDNA) [5], which are associated with both aminoglycoside-induced and non-syndromic deafness [5,6]. So far, the A1555G mitochondrial mutation has been found to be one of the major causes of hearing loss in Chinese [7–11]. Li et al. screened the mitochondrial 12S rRNA gene in 128 Chinese
* Corresponding author. Tel.: +86 27 8369 2629; fax: +86 27 8369 2608. E-mail address: [email protected] (S. Fu). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.01.013
pediatric subjects with sporadic aminoglycoside-induced and nonsyndromic hearing loss and found that the incidence of A1555G mutation is approximately 13% and 2.9% in this pediatric population with aminoglycoside-induced and nonsyndromic hearing loss, respectively [9]. In addition, Liu et al. investigated mtDNA mutation in 498 nonsyndromic sensorineural hearing loss (NSHL) patients (290 from China and 208 from the USA) with or without history of aminoglycoside exposure, mtDNA A1555G mutation was found in all Chinese probands (15.5%) with mtDNA mutation, but only in 4 probands (1.9%) from the USA. Indeed, the high incidence of mtDNA A1555G mutation in Chinese probands with NSHL helps explain the high prevalence of aminoglycosideinduced deafness in China [10]. It is important to note that although mutations causing deafness are present at the time of conception, hearing loss resulting from these mutations is not present at birth. Consequently, most children who develop delayed onset hearing loss will pass the newborn hearing screening [12,13]. The addition of genetic screening as a complement to audiologic newborn screening will help identify infants with late-onset prelingual hearing loss [14,15]. Therefore, in this study we performed genetic screening on the common mutation in Chinese populations when hearing screening was carried out
G. Chen et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 532–534
in newborns, with the aim to investigate the feasibility of genetic screening for deafness causative genes in the process of newborn hearing screening in China.
2. Subjects and methods 2.1. Subjects The newborn hearing screening and deafness-causing gene screening protocols have been developed by the Huazhong University of Science and Technology (HUST), and approved by the Ethics Committee of HUST. The hearing screening was a part of routine medical services provided to all newborns delivered in Tongji hospital, HUST, from November 2009 to March 2010. All parents were informed about the testing process and gave their consent. 2.2. Newborn hearing screening The screening method consisted of a two-stage screening approach with transient evoked otoacoustic emission (TEOAE). The initial screening was performed 3–7 days after birth before discharge. The newborns were always accompanied by their mother or guardian and were either asleep or drowsy. Outpatient screening was conducted 4–6 weeks after birth for infants who failed in the first-stage screening. If a baby did not pass the secondstage screen, he was referred for diagnostic testing. 2.3. Transient evoked otoacoustic emissions TEOAEs were recorded by an ILO-88 cochlear emission analyzer (Otodynamics, London, UK), using insert ear phones in a soundproofed room. The TEOAEs were obtained from 1 to 4 kHz, with stimuli consisting of clicks of 80 ms duration. The stimulus level in the outer ear was set at 83 3 dB SPL. The click rate was 80 s1, and post-stimulus analysis was in the range of 2– 20 ms. An amplifier gain suppression period of 2.5 ms was used to remove the primary stimulus artifact. A total of 260 sweeps was averaged above the noise rejection level of 50 dB. The stimuli were presented in the nonlinear mode, in which every fourth click stimulus was inverted and three times greater in amplitude than the three preceding clicks. The TEOAE responses were classified as either ‘‘pass’’ or ‘‘fail’’ following each stimulus data set. The pass criterion was as follows: the TEOAE spectrum was recorded at least 3 dB above the noise floor (i.e., SNR 3 dB) for all three frequency bands at 2.4, 3.2 and 4.0 kHz. Reproducibility percentages of 70% and stimulus stability of 80% were taken into account as acceptable for analysis [16].
533
2.5. Mitochondrial 12S rRNA A1555G mutation screening The heel blood leukocytes were routinely taken from the newborns for the screening of genetic diseases. Genomic DNA was extracted from the blood using a routine desalting method. Mitochondrial DNA was amplified with the forward primer 1555F: 50 -GAG GAG CCT GTT CTG TAA TC-30 , and reverse primer 1555R: 50 CTT GGA CAA CCA GCT ATC AC30 (amplicion 832 bp). The amplification conditions were 95 8C for 5 min, then 32 cycles of 94 8C for 1 min, 56 8C for 45 s, and 72 8C for 1 min, with a final extension at 72 8C for 5 min. Restriction enzyme digestion was performed on PCR products in order to detect the 12S rRNA A1555G mutation. Upon digestion with Alw26I, PCR product of 832 bp from normal individuals would yield bands of 473 bp and 359 bp, whereas in affected individuals, the absence of the restriction site resulted in a band of 832 bp. The samples with the possible 12S rRNA A1555G mutation were further subjected to DNA sequencing by ABI 377 DNA Automatic Sequencer (Perking, USA) to verify the mutation. The result of genetic screening was designated as ‘‘pass’’ or ‘‘fail’’ with ‘‘Pass’’ meaning that an infant harbored no 12S rRNA A1555G mutation and ‘‘Fail’’ meaning that an infant harbored 12S rRNA A1555G homoplasmic mutation in the current testing method.
3. Results Among the 865 neonates screened in this study, 6 of the 865 newborns (0.7%) harbored homoplasmic 12S rRNA A1555G mutation as detected by PCR-RFLP and confirmed by DNA sequencing. It is important to note that all of these 6 newborns passed the hearing screening. For the TEOAE test, 15.5% of the 865 neonates (134/865) failed in the 1st hearing screening test, and 86.6% (116/134) of them returned for the second stage screening. The failure rate for 2nd screen was 12.9% (15/116) and the failure rate for diagnostic stage was 20% (3/15) because 3 of them failed the test when tested by diagnostic ABR. The three neonates were subjected to audiometry, the results showed that two had severe deafness in both ears and one had moderate deafness in both ears. The two neonates with severe deafness already got treatment and the one with moderate deafness is still in follow-up. According to the hearing screening protocol, all parents of failed neonates were asked to bring their children to repeat the test within a month following discharge. From the 116 of the 134 newborns who repeated the test, 101 were found to be normal, and 15 failed TEOAE. The 15 babies were then further tested by ABR, and 12 passed and 3 failed.
2.4. Diagnostic hearing testing 4. Discussion This diagnostic evaluation was conducted by the diagnostic auditory brainstem response (ABR). An evoked potential tester (Nicolet Spirit, Nicolet Inc., Madison, WI, USA) was used with an alternatively inverted click stimulus, pulse width of 0.1 ms, initial intensity of sound stimulus of 80 dB nHL, stimulus repetition rate of 11.9 times/s, analysis time of 10 ms, band-pass filtering of 10– 3000 Hz, and 2 or 3 replications of 1000 sweeps. The box of electrodes consisted of four electrodes: a forehead electrode as the recording electrode, acoustic stimulation of the bilateral mastoids as the reference electrodes, and a glabella electrode as the ground electrode. The impedance values of the electrodes were below 5 kV. A V response threshold for the ABR wave equal to or less than 30 dB nHL served as an index of normal hearing in the range of 2– 4 kHz [17].
In the present study, we found that six newborns who passed hearing screening harbored homoplasmic mitochondrial 12S rRNA A1555G mutation, indicating that they will be highly sensitive to amimoglycosides. Thus they could avoid irreversible hearing loss if their exposure to aminoglycosides is minimized in future. Based on our present data that 0.7% newborns harbor the mitochondrial 12S rRNA A1555G mutation, 126,000 Chinese children will carry this mutation given that about 18,000,000 children are born every year in China. In our previous study, we screened a total of 504,348 students in the Hubei province of China and identified 813 deaf students. Among the 813 cases of deafness, aminoglycoside-antibiotic-induced deafness accounted for 33.95% [18]. The frequency of the 12S rRNA
534
G. Chen et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 532–534
A1555G mutation is 13–25% in Chinese pediatric subjects with aminoglycoside-induced hearing loss [9,10]. Therefore, there is really an urgent need to implement gene screening for newborns in China to prevent hearing loss and control the incidence of hearing loss from the newborns. This should result in a drastic reduction in the incidence of this iatrogenic form of deafness. The hospital-based newborn hearing screening has been implemented for nearly ten years in China. The overall referral rate after the first-stage screening has a wide variation from 9.84% to 31.3% in China as reported previously [19,20]. In the population we screened in this study the referral rate was 15.5% and decreased significantly to 1.7% (15/865) on retest. Of the 865 newborns undergoing routine hearing screening, only 3 newborns were diagnosed as sensorineural hearing loss, thus the prevalence is 3.46:1000. This prevalence is higher than that reported by Nie et al. (2.86:1000) [21], but lower than that reported by Huang et al. (0.5%) [22]. It is unclear what percentage of post-lingual hearing loss is due to mtDNA mutations. It has been estimated that around 20% of inherited post-lingual hearing loss may be caused by mutations in the mitochondrial genome, but large ethnic differences might be present [23]. In total, newborns who harbored mutation accounted for 0.7% (6/865) in the present study, which was 7 times higher than the incidence of hearing loss (0.1%) by hearing screening [24]. Because hearing loss is not always present at birth and most children who develop delayed onset hearing loss will pass the newborn hearing screening [12,13], it might be an effective measure to complement routine hearing screening with mitochondrial 12S rRNA A1555G mutation screening for the purpose of early diagnosis and identification of the late-onset hearing loss. It is important to point out that the use of molecular test alone to detect genetic hearing loss is not currently feasible because the validity of the identified deafness genes for molecular test need further investigation and many uncertainties would arise in the interpretation of test results. Nevertheless, as an adjunct to audiologic screening, such genetic tests would provide a powerful and unprecedented strategy for identifying newborns at risk for the development of late-onset hearing loss [15]. Moreover, a recent survey revealed that Chinese parents have a predominantly positive attitudes toward genetic testing for prelingual deafness [25], indicating that genetic testing for newborn will be easily accepted by Chinese parents. In conclusion, our study suggests that it might be practical and effective to complement routine hearing screening in newborns with genetic screening for deafness susceptibility genes. With the improvement in genetic screening techniques for hearing loss and training of technicians, the combination of genetic screening in routine newborn hearing screening is expected to effectively reduce the incidence of hearing loss in the next 20–30 years in China.
Conflict of interest None to declare.
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