RESEARCH ARTICLE
Molecular Etiology of Non-Dominant, Non-Syndromic, Mild-to-Moderate Childhood Hearing Impairment in Chinese Hans Yongchuan Chai,1,2 Xiuhong Pang,1,2 Dongye Chen,1,2 Lei Li,1,2 Ying Chen,1,2 Lianhua Sun,1,2 Xiaowen Wang,1,2 Hao Wu,1,2** and Tao Yang1,2* 1
Department of Otorhinolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 2
Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai, China
Manuscript Received: 10 February 2014; Manuscript Accepted: 21 August 2014
Childhood hearing impairment (HI) is genetically heterogeneous. Compared with the severe-to-profound hearing impairment, the molecular etiology of mild-to-moderate hearing impairment in children has been less well characterized, especially for those not inherited in the dominant mode. In this study, we recruited 114 probands with non-dominant, non-syndromic, mild-to-moderate childhood hearing impairment. Sequencing of GJB2, SLC26A4, and MTRNR1 identified causative mutations in 30.7% (35/114), 4.4% (5/114), and 4.4% (5/114) of subjects, respectively. A majority (62.9%) of bi-allelic GJB2 mutations have non-truncating mutations in at least one allele. In 10 multiplex probands with no GJB2, SLC26A4, and MTRNR1 mutations identified, targeted next-generation sequencing (NGS) of 79 known deafness genes did not identify any additional causes. Our data showed that the molecular etiology of mild-tomoderate childhood hearing impairment is considerably different from what reported for severe-to-profound hearing impairment and far from complete for those inherited in nondominant modes. Ó 2014 Wiley Periodicals, Inc.
Key words: deafness; non-syndromic; mild-to-moderate hearing loss; genetic etiology; target next-generation sequencing
INTRODUCTION Hearing impairment (HI) is the most common sensory disorder in children, occurring in 2–3.5 per 1,000 [Morton and Nance, 2006]. More than 50% of permanent childhood HI is attributable to genetic factors [Nance et al., 2006]. The molecular etiology of childhood HI is highly heterogeneous as more than 60 genes have been found to be associated with non-syndromic HI (Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org). A number of genetic etiology studies have been performed in large cohorts of hearing impaired subjects from various ethnic groups [Snoeckx et al., 2005; Dai et al., 2009]. Most of those studies, however, focused primarily on the severe-to-profound HI as subjects with severer degree of HI were generally more easily accessible (e.g., from deaf schools and auditory clinics). Though mutations in numerous
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Chai Y, Pang X, Chen D, Li L, Chen Y, Sun L, Wang X, Wu H, Yang T. 2014. Molecular etiology of non-dominant, nonsyndromic, mild-to-moderate childhood hearing impairment in Chinese Hans. Am J Med Genet Part A 164A:3115–3119.
deafness genes have been reported to be associated with dominant, mild-to-moderate HI, those mutations were relatively rare and limited to individual families. For majority of mild-to-moderate childhood HI that was inherited in non-dominant modes, the molecular etiology was less well characterized in large scale. Conflict of interest: None. Grant sponsor: National Science Foundation of China; Grant numbers: 81222010, 81371101, 81330023, 81170924; Grant sponsor: National Basic Research Program of China; Grant number: 2011CB504501; Grant sponsor: Minister of Science and Technology; Grant number: 2012BAI09B02; Grant sponsor: Specialized Research Fund for the Doctoral Program of Higher Education; Grant number: 20130073110011; Grant sponsor: Program for New Century Excellent Talents in University; Grant number: NCET-13-0376; Grant sponsor: Shanghai Municipal Education Commission; Grant sponsor: Shanghai Jiaotong University School of Medicine; Grant number: BXJ201221. Correspondence to: Tao Yang, Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai, China. E-mail: [email protected] Correspondence to: Hao Wu, Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 23 September 2014 DOI 10.1002/ajmg.a.36785
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3116 With the wide-spread implication of universal newborn hearing screening in recent years, childhood HI of milder degrees can be recognized more promptly and frequently [Halpin et al., 2010]. Those fast-accumulating cases of mild-to-moderate childhood HI demand a better understanding of the causes at the molecular level. In this study, we performed a molecular etiology study in a large cohort of Chinese Han probands with non-dominant, non-syndromic, mild-to-moderate childhood HI. We chose three deafness genes, GJB2, SLC26A4, and MTRNR1, for primary screening as they have been considered as the most common genetic causes of nonsyndromic HI in childhood [Morton and Nance, 2006] and some mutations in those genes have been previously linked to mild-tomoderate HI [Pollak et al., 2007; Suzuki et al., 2007; Berrettini et al., 2008]. Selected multiplex probands were also screened by targeted next-generation sequencing (NGS) of 79 known deafness genes in search for other possible causes.
MATERIALS AND METHODS Patients A total of 114 probands with bilateral, permanent, non-dominant (as the probands or the affected family members had normal hearing parents), mild-to-moderate childhood sensorineural HI (mean PTA 54.1 dB HL, 95% CI 51.7–56.5 dB HL) were recruited through Xinhua Hospital, Shanghai, China. Clinical characteristics of the probands were summarized in Supplementary Table SI (see supporting information online). The age of the probands varied from a few months to 18 years old (mean age 8.4 years, 95% CI 7.5–9.3 years). Twenty-six probands had mild HI (mean PTA 33.3 dB HL, 95% CI 31.0–35.5 dB HL) and the remaining 88 probands had moderate HI (mean PTA 59.5 dB HL, 95% CI 58.0–61.0 dB HL). Dominant inheritance was excluded for the 31 multiplex probands. All subjects or their parents gave written, informed consent to participate in this study. This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine.
Auditory Evaluation Medical history and clinical questionnaire was obtained for all probands. A comprehensive auditory evaluation was performed including otoscopic examination, tympanometry, pure-tone audiometry (PTA) and/or auditory brainstem response (ABR, performed in all subjects under 3 years old and in some subjects between 3 and 6 years old). Degree of HI was calculated as the average of the hearing levels at 0.5, 1.0, 2.0, and 4.0 kHz for the better ear. The severity of HI was defined as mild (20–40 dB), moderate (41–70 dB), severe (71–95 dB), and profound (>95 dB).
Mutation Screening of GJB2, SLC26A4, and MTRNR1 Genomic DNA was extracted from the whole blood samples using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China). Mutation screening of GJB2, SLC26A4, and MTRNR1 was performed by PCR amplification and bi-directional sequencing as previously described [Ji et al., 2011]. Trans-configuration of compound
AMERICAN JOURNAL OF MEDICAL GENETICS PART A heterozygous GJB2 or SLC26A4 mutations was confirmed by parental genotyping. For multiplex probands, co-segregation of the detected mutations in GJB2, SLC26A4, and MTRNR1 was confirmed in their corresponding family members.
Targeted Next-Generation Sequencing of 79 Known Deafness Genes Targeted NGS of 79 known deafness genes (Supplementary Table SII) were performed as previously described [Yang et al., 2013]. Fragmented Genomic DNA was captured by a customized array designed to target all exons, splicing sites and flanking intronic sequences of 79 deafness genes (NimbleGen, Roche, Madison, WI). Captured DNA fragments were sequenced on Illumina HiSeq2000 Analyzers. Data analysis and bioinformatics processing were performed following standard Illumina procedure. Potentially pathogenic variants were defined as nonsense, missense, splice-site and indel variants that have allele frequencies under 0.01 (determined by NCBI dbSNP, 1000 Genomes and the in-house sequencing data of 200 Chinese Han normal hearing controls). Parental genotyping of the detected variants was performed by PCR amplification and Sanger sequencing for corresponding probands. For CNV detection, the NGS data generated from both the 10 multiplex probands and the 200 normal hearing controls were analyzed with the CNV calling tool CONTRA (Copy Number Analysis for Targeted Resequencing, http://contra-cnv.sourceforge.net/).
RESULTS Etiological Contribution of GJB2, SLC26A4, and MTRNR1 Mutations to Mild-to-Moderate HI Mutation screening of GJB2 identified bi-allelic mutations in 30.7% (35/114) of probands with mild-to-moderate HI (Table I). A majority of those probands (62.9%, 22/35) have non-truncating mutations (missense mutations) in at least one allele. On the contrary, only 37.1% (13/35) of probands have truncating mutations (frame-shifting indels) in both alleles. Two most prevalent GJB2 mutations identified in mild-to-moderate HI is p.V37I and c.235delC, accounting for 47.1% (33/70) and 40.0% (28/70) of all mutant alleles, respectively. Bi-allelic mutations of SLC26A4 were identified in 4.4% (5/114) of probands. c.919-2A>G was the predominant mutation for SLC26A4, accounting for 60% (6/10) of mutant alleles. The average age of the five probands carrying bi-allelic SLC26A4 mutations were 10 years old (95% CI 8.2–11.8 years), not significantly different (P ¼ 0.46) than the rest of the probands (mean age 8.4 years, 95% CI 7.4–9.3 years). It is worth noting that in this study we identified two novel missense variants, p.K61Q in GJB2 and p.V97E in SLC26A4. Though the pathogenicity of these two variants were not confirmed by functional studies, they were likely to be pathogenic because: (i) both variants occur at the evolutionary conserved amino acid positions (PhyloP scores of 3.34 and 2.38 for p.K61Q and p.V97E, respectively) and were predicted to be disease causing by the Mutation Taster Program (http://www.mutationtaster.org); (ii) they were not found in 200 ethnically-matched normal hearing
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TABLE I. Probands With Causative Mutations in GJB2, SLC26A4, and MTRNR1 Number (%) of probands Gene Genotype GJB2 p.V37I/p.V37I p.V37I/p.K61Q p.V37I/c.235delC c.235delC/c.299-300delAT c.235delC/c.235delC c.235delC/p.T86R c.235delC/c.507insAACG c.235delC/c.93delT SLC26A4 c.919-2A > G/c.919-2A > G c.919-2A > G/p.H723R p.R409H/p.H723R c.919-2A > G/p.V97E MTRNR1 m.1555G> A Total
Multiplex (n ¼ 31) 8 (25.8%) 3 (9.7%) — — 1 (3.2%) 2 (6.5%) 1 (3.2%) — 1 (3.2%) 2 (6.5%) 1 (3.2%) 1 (3.2%) — — 4 (12.9%) 4 (12.9%) 14 (45.2%)
Simplex (n ¼ 83) 27 (32.5%) 10 (15.7%) 1 (1.2%) 6 (7.2%) 3 (3.6%) 5 (6.0%) 1 (1.2%) 1 (1.2%) — 3 (3.6%) 1 (1.2%) — 1 (1.2%) 1 (1.2%) 1 (1.2%) 1 (1.2%) 31 (37.3%)
controls; and (iii) they were in compound heterozygosity with another known pathogenic GJB2 or SLC26A4 mutation in trans (confirmed by parental genotyping). The m.1555A>G mitochondrial mutation of MTRNR1 was identified in another 4.4% (5/114) of probands. All those m.1555A>G mutations were homoplasmic and maternally inherited with incomplete penetrance with family members. Exposure to aminoglycosides was recorded in three multiplex probands and not in the remaining two (one multiplex and one simplex proband).
Targeted NGS Identified No Additional Causes for Mild-to-Moderate HI Bi-allelic mutations in GJB2 and SLC26A4, and mitochondrial mutations in MTRNR1 were not identified in 17 multiplex and 52 simplex probands with mild-to-moderate HI. Among them, 10 multiplex probands had good DNA quality and quantity and were selected for further mutation screening by targeted NGS of 79 known deafness genes. The average depth for the targeted regions was 507.1-fold and 98.4% of the targeted regions were covered by 20 or more reads, demonstrating the high quality of the sequencing. All candidate variants detected by the targeted NGS (Supplementary Table SIII), however, were heterozygous and not consistent with the nondominant inheritance (Supplementary Fig. S1 in supporting information online). None of the candidate variants was reported to be associated with HI in the clinvar database. Parental genotyping showed no evidence of de novo variants. No potential pathogenic CNVs were identified by the NGS analysis.
DISCUSSION Our study presented a preliminary overview of the molecular etiology of non-dominant mild-to-moderate childhood HI in
Clinical information Total 35 13 1 6 4 7 2 1 1 5 2 1 1 1 5 5 45
(n ¼ 114) (30.7%) (11.4%) (0.9%) (5.3%) (3.5%) (6.1%) (1.8%) (0.9%) (0.9%) (4.4%) (1.8%) (0.9%) (0.9%) (0.9%) (4.4%) (4.4%) (40.4%)
Mean PTA (dB) 51.8 46.5 29.0 54.7 52.5 57.4 62.5 55.0 60.0 61.8 65.0 55.0 59.0 65.0 43.8 43.8 52.0
Mean age (years) 7.6 6.5 8.0 9.3 8.1 9.7 6.5 0.4 4.0 10.0 10.0 8.0 11.3 10.7 8.7 8.7 8.0
Chinese Hans. In comparison with our previous studies of severe-to-profound HI [Yang et al., 2013], it was considerably different in that: (i) bi-allelic GJB2 mutations accounted for a higher percentage of causes (30.7% vs. 19.7%, Fisher’s exact test, P ¼ 0.036, Fig. 1); (ii) the percentage of bi-allelic GJB2 mutations associated with non-truncating mutations was much higher (62.9% vs. 12.5%, Fisher’s exact test, P ¼ 8.1 106, Fig. 2); (iii) bi-allelic SLC26A4 mutations accounted for a lower percentage of causes (4.4% vs. 12.0%); and (iv) less causative mutations was identified in other known deafness genes (None vs. 15.8%). The high percentage of non-truncating mutations of GJB2 observed in our study of mild-to-moderate HI was in strong agreement with a genotype-phenotype correlation between nontruncating mutations of GJB2 and milder degree of HI. Consistent with our results, previous functional studies also showed that mutant GJB2 protein with truncating mutations has abolished plasma membrane expression and is void of gap junction plaque formation, while mutant GJB2 protein with non-truncating mutations, though functionally impaired in intercellular channel activity, still retains normal formation of gap junction plaques [Oguchi et al., 2005; Jara et al., 2012]. On the other side, the fact that 37.1% of bi-allelic GJB2 mutations associated with mild-to-moderate HI was indeed truncating in both alleles (Fig. 2), which suggested that additional genetic modifiers and environmental factors might also influence the degree of HI. Our study showed a significant etiological contribution of biallelic GJB2 mutations to mild-to-moderate HI (30.7%, Fig. 1) in Chinese Hans. Interestingly, however, in a study of 48 Australian elementary school children with slight/mild HI, no causative mutations of GJB2 were identified in 40 children of non-Asian ethnicity while homozygous p.V37I was identified in four of the eight children of Asian ethnicity [Dahl et al., 2006]. Apparently, the molecular etiology of mild-to-moderate HI can be variable among different ethnical groups. Based on our results, the p.V37I
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FIG. 1. Molecular etiology of non-dominant mild-to-moderate HI in Chinese Hans in comparison with that of severe-to-profound HI.
mutation of GJB2, which has an extremely high allele frequency (6.2%) in Chinese Hans [Li et al., 2012], was the leading mutant allele identified in children with mild-to-moderate HI and likely the reason for the high etiological contribution of GJB2 mutations in Chinese Hans. Mutations in SLC26A4 are the major cause of HI associated with enlarged vestibular aqueduct. A previous study showed that biallelic mutations in SLC26A4 can be identified in approximately 11% of Chinese Han probands with severe-to-profound HI [Yang et al., 2013]. In contrast, bi-allelic SLC26A4 mutations were detected only in 4.4% of probands with mild-to-moderate HI in this study (Fig. 1). This difference was in agreement with previous reports that a majority of bi-allelic SLC26A4 mutations were associated with severe-to-profound HI [Albert et al., 2006; Chai et al., 2013]. It is worth noting that a significant portion of patients
with bi-allelic SLC26A4 mutations has been reported to have progressive HI during childhood [Albert et al., 2006]. The percentage of bi-allelic SLC26A4 mutations in mild-to-moderate HI, therefore, can be higher in children with younger age. In our study, the age of the five probands with bi-allelic SLC26A4 mutations was not significantly different from the rest of the probands (P ¼ 0.46). The mitochondrial m.A1555G mutation in MTRNR1 is associated with both aminoglycoside-induced and non-aminoglycosideinduced HI. In this study, we detected the m.A1555G mutation in 4.4% of probands with mild-to-moderate HI, a percentage similar to the previous report of severe-to-profound HI in Chinese Hans (Fig. 1). The previous study of severe-to-profound HI also detected 15.8% of probands with causative mutations in the rare deafness genes such as MYO15A, GPR98, TMC1, USH2A, and PCDH15 [Yang et al., 2013]. In this study, however, targeted NGS of 79 known deafness genes identified no causative mutations in the 10 selected multiplex probands with mild-to-moderate HI. This difference was likely attributable to the fact that most known recessive deafness genes were associated with severe-to-profound HI. On the other hand, our screening results of the multiplex probands also suggested that the molecular etiology of mild-tomoderate HI is far from being complete at the current stage. Other genetic causes, such as dominant mutations with incomplete penetrance or variable expressivity, recessive mutations in novel deafness genes or non-coding regions of known deafness genes, and large deletions or complex chromosome rearrangement, which is difficult to be detected by targeted NGS in this study, remain to be disclosed.
ACKNOWLEDGMENTS FIG. 2. Percentages of truncating (T) and non-truncating (N) mutations of GJB2 in Chinese Hans with mild-to-moderate and severe-to-profound childhood HI.
This research was supported by grants from National Science Foundation of China (81222010 and 81371101 to T.Y.; 81330023 and 81170924 to H.W.), National Basic Research Program of China (2011CB504501 to H.W.), Minister of Science and Technology (2012BAI09B02 to H.W.), Specialized Research Fund
CHAI ET AL. for the Doctoral Program of Higher Education (20130073110011 to T.Y.), Program for New Century Excellent Talents in University (NCET-13-0376 to T.Y.), the Oriental Scholar project from Shanghai Municipal Education Commission (to T.Y.) and the Ph.D. Innovation Foundation from Shanghai Jiaotong University School of Medicine (BXJ201221 to Y.C.).
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3119 in sporadic outpatients with nonsyndromic sensorineural hearing loss in China. Acta Otolaryngol 131:124–129. Li L, Lu J, Tao Z, Huang Q, Chai Y, Li X, Huang Z, Li Y, Xiang M, Yang J, Yao G, Wang Y, Yang T, Wu H. 2012. The p.V37I exclusive genotype of GJB2: A genetic risk-indicator of postnatal permanent childhood hearing impairment. PLoS ONE 7:e36621. Morton CC, Nance WE. 2006. Newborn hearing screening–a silent revolution. N Engl J Med 354:2151–2164. Nance WE, Lim BG, Dodson KM. 2006. Importance of congenital cytomegalovirus infections as a cause for pre-lingual hearing loss. J Clin Virol 35:221–225. Oguchi T, Ohtsuka A, Hashimoto S, Oshima A, Abe S, Kobayashi Y, Nagai K, Matsunaga T, Iwasaki S, Nakagawa T, Usami S. 2005. Clinical features of patients with GJB2 (connexin 26) mutations: Severity of hearing loss is correlated with genotypes and protein expression patterns. J Hum Genet 50:76–83. Pollak A, Skorka A, Mueller-Malesinska M, Kostrzewa G, Kisiel B, Waligora J, Krajewski P, Oldak M, Korniszewski L, Skarzynski H, Ploski R. 2007. M34T and V37I mutations in GJB2 associated hearing impairment: Evidence for pathogenicity and reduced penetrance. Am J Med Genet Part A 143A:2534–2543. Snoeckx RL, Huygen PL, Feldmann D, Marlin S, Denoyelle F, Waligora J, Mueller-Malesinska M, Pollak A, Ploski R, Murgia A, Orzan E, Castorina P, Ambrosetti U, Nowakowska-Szyrwinska E, Bal J, Wiszniewski W, Janecke AR, Nekahm-Heis D, Seeman P, Bendova O, Kenna MA, Frangulov A, Rehm HL, Tekin M, Incesulu A, Dahl HH, du Sart D, Jenkins L, Lucas D, Bitner-Glindzicz M, Avraham KB, Brownstein Z, del Castillo I, Moreno F, Blin N, Pfister M, Sziklai I, Toth T, Kelley PM, Cohn ES, Van Maldergem L, Hilbert P, Roux AF, Mondain M, Hoefsloot LH, Cremers CW, Lopponen T, Lopponen H, Parving A, Gronskov K, Schrijver I, Roberson J, Gualandi F, Martini A, LinaGranade G, Pallares-Ruiz N, Correia C, Fialho G, Cryns K, Hilgert N, Van de Heyning P, Nishimura CJ, Smith RJ, Van Camp G. 2005. GJB2 mutations and degree of hearing loss: A multicenter study. Am J Hum Genet 77:945–957. Suzuki H, Oshima A, Tsukamoto K, Abe S, Kumakawa K, Nagai K, Satoh H, Kanda Y, Iwasaki S, Usami S. 2007. Clinical characteristics and genotypephenotype correlation of hearing loss patients with SLC26A4 mutations. Acta Otolaryngol 127:1292–1297. Yang T, Wei X, Chai Y, Li L, Wu H. 2013. Genetic etiology study of the nonsyndromic deafness in Chinese Hans by targeted next-generation sequencing. Orphanet J Rare Dis 8:85.
SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.
Molecular Etiology of Non-Dominant, Non-Syndromic, Mild-to-Moderate Childhood Hearing Impairment in Chinese Hans Yongchuan Chai,1,2 Xiuhong Pang,1,2 Dongye Chen,1,2 Lei Li,1,2 Ying Chen,1,2 Lianhua Sun,1,2 Xiaowen Wang,1,2 Hao Wu,1,2** and Tao Yang1,2* 1
Department of Otorhinolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 2
Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai, China
Manuscript Received: 10 February 2014; Manuscript Accepted: 21 August 2014
Childhood hearing impairment (HI) is genetically heterogeneous. Compared with the severe-to-profound hearing impairment, the molecular etiology of mild-to-moderate hearing impairment in children has been less well characterized, especially for those not inherited in the dominant mode. In this study, we recruited 114 probands with non-dominant, non-syndromic, mild-to-moderate childhood hearing impairment. Sequencing of GJB2, SLC26A4, and MTRNR1 identified causative mutations in 30.7% (35/114), 4.4% (5/114), and 4.4% (5/114) of subjects, respectively. A majority (62.9%) of bi-allelic GJB2 mutations have non-truncating mutations in at least one allele. In 10 multiplex probands with no GJB2, SLC26A4, and MTRNR1 mutations identified, targeted next-generation sequencing (NGS) of 79 known deafness genes did not identify any additional causes. Our data showed that the molecular etiology of mild-tomoderate childhood hearing impairment is considerably different from what reported for severe-to-profound hearing impairment and far from complete for those inherited in nondominant modes. Ó 2014 Wiley Periodicals, Inc.
Key words: deafness; non-syndromic; mild-to-moderate hearing loss; genetic etiology; target next-generation sequencing
INTRODUCTION Hearing impairment (HI) is the most common sensory disorder in children, occurring in 2–3.5 per 1,000 [Morton and Nance, 2006]. More than 50% of permanent childhood HI is attributable to genetic factors [Nance et al., 2006]. The molecular etiology of childhood HI is highly heterogeneous as more than 60 genes have been found to be associated with non-syndromic HI (Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org). A number of genetic etiology studies have been performed in large cohorts of hearing impaired subjects from various ethnic groups [Snoeckx et al., 2005; Dai et al., 2009]. Most of those studies, however, focused primarily on the severe-to-profound HI as subjects with severer degree of HI were generally more easily accessible (e.g., from deaf schools and auditory clinics). Though mutations in numerous
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Chai Y, Pang X, Chen D, Li L, Chen Y, Sun L, Wang X, Wu H, Yang T. 2014. Molecular etiology of non-dominant, nonsyndromic, mild-to-moderate childhood hearing impairment in Chinese Hans. Am J Med Genet Part A 164A:3115–3119.
deafness genes have been reported to be associated with dominant, mild-to-moderate HI, those mutations were relatively rare and limited to individual families. For majority of mild-to-moderate childhood HI that was inherited in non-dominant modes, the molecular etiology was less well characterized in large scale. Conflict of interest: None. Grant sponsor: National Science Foundation of China; Grant numbers: 81222010, 81371101, 81330023, 81170924; Grant sponsor: National Basic Research Program of China; Grant number: 2011CB504501; Grant sponsor: Minister of Science and Technology; Grant number: 2012BAI09B02; Grant sponsor: Specialized Research Fund for the Doctoral Program of Higher Education; Grant number: 20130073110011; Grant sponsor: Program for New Century Excellent Talents in University; Grant number: NCET-13-0376; Grant sponsor: Shanghai Municipal Education Commission; Grant sponsor: Shanghai Jiaotong University School of Medicine; Grant number: BXJ201221. Correspondence to: Tao Yang, Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai, China. E-mail: [email protected] Correspondence to: Hao Wu, Department of Otolaryngology-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 23 September 2014 DOI 10.1002/ajmg.a.36785
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3116 With the wide-spread implication of universal newborn hearing screening in recent years, childhood HI of milder degrees can be recognized more promptly and frequently [Halpin et al., 2010]. Those fast-accumulating cases of mild-to-moderate childhood HI demand a better understanding of the causes at the molecular level. In this study, we performed a molecular etiology study in a large cohort of Chinese Han probands with non-dominant, non-syndromic, mild-to-moderate childhood HI. We chose three deafness genes, GJB2, SLC26A4, and MTRNR1, for primary screening as they have been considered as the most common genetic causes of nonsyndromic HI in childhood [Morton and Nance, 2006] and some mutations in those genes have been previously linked to mild-tomoderate HI [Pollak et al., 2007; Suzuki et al., 2007; Berrettini et al., 2008]. Selected multiplex probands were also screened by targeted next-generation sequencing (NGS) of 79 known deafness genes in search for other possible causes.
MATERIALS AND METHODS Patients A total of 114 probands with bilateral, permanent, non-dominant (as the probands or the affected family members had normal hearing parents), mild-to-moderate childhood sensorineural HI (mean PTA 54.1 dB HL, 95% CI 51.7–56.5 dB HL) were recruited through Xinhua Hospital, Shanghai, China. Clinical characteristics of the probands were summarized in Supplementary Table SI (see supporting information online). The age of the probands varied from a few months to 18 years old (mean age 8.4 years, 95% CI 7.5–9.3 years). Twenty-six probands had mild HI (mean PTA 33.3 dB HL, 95% CI 31.0–35.5 dB HL) and the remaining 88 probands had moderate HI (mean PTA 59.5 dB HL, 95% CI 58.0–61.0 dB HL). Dominant inheritance was excluded for the 31 multiplex probands. All subjects or their parents gave written, informed consent to participate in this study. This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiaotong University School of Medicine.
Auditory Evaluation Medical history and clinical questionnaire was obtained for all probands. A comprehensive auditory evaluation was performed including otoscopic examination, tympanometry, pure-tone audiometry (PTA) and/or auditory brainstem response (ABR, performed in all subjects under 3 years old and in some subjects between 3 and 6 years old). Degree of HI was calculated as the average of the hearing levels at 0.5, 1.0, 2.0, and 4.0 kHz for the better ear. The severity of HI was defined as mild (20–40 dB), moderate (41–70 dB), severe (71–95 dB), and profound (>95 dB).
Mutation Screening of GJB2, SLC26A4, and MTRNR1 Genomic DNA was extracted from the whole blood samples using the Blood DNA kit (TIANGEN BIOTECH, Beijing, China). Mutation screening of GJB2, SLC26A4, and MTRNR1 was performed by PCR amplification and bi-directional sequencing as previously described [Ji et al., 2011]. Trans-configuration of compound
AMERICAN JOURNAL OF MEDICAL GENETICS PART A heterozygous GJB2 or SLC26A4 mutations was confirmed by parental genotyping. For multiplex probands, co-segregation of the detected mutations in GJB2, SLC26A4, and MTRNR1 was confirmed in their corresponding family members.
Targeted Next-Generation Sequencing of 79 Known Deafness Genes Targeted NGS of 79 known deafness genes (Supplementary Table SII) were performed as previously described [Yang et al., 2013]. Fragmented Genomic DNA was captured by a customized array designed to target all exons, splicing sites and flanking intronic sequences of 79 deafness genes (NimbleGen, Roche, Madison, WI). Captured DNA fragments were sequenced on Illumina HiSeq2000 Analyzers. Data analysis and bioinformatics processing were performed following standard Illumina procedure. Potentially pathogenic variants were defined as nonsense, missense, splice-site and indel variants that have allele frequencies under 0.01 (determined by NCBI dbSNP, 1000 Genomes and the in-house sequencing data of 200 Chinese Han normal hearing controls). Parental genotyping of the detected variants was performed by PCR amplification and Sanger sequencing for corresponding probands. For CNV detection, the NGS data generated from both the 10 multiplex probands and the 200 normal hearing controls were analyzed with the CNV calling tool CONTRA (Copy Number Analysis for Targeted Resequencing, http://contra-cnv.sourceforge.net/).
RESULTS Etiological Contribution of GJB2, SLC26A4, and MTRNR1 Mutations to Mild-to-Moderate HI Mutation screening of GJB2 identified bi-allelic mutations in 30.7% (35/114) of probands with mild-to-moderate HI (Table I). A majority of those probands (62.9%, 22/35) have non-truncating mutations (missense mutations) in at least one allele. On the contrary, only 37.1% (13/35) of probands have truncating mutations (frame-shifting indels) in both alleles. Two most prevalent GJB2 mutations identified in mild-to-moderate HI is p.V37I and c.235delC, accounting for 47.1% (33/70) and 40.0% (28/70) of all mutant alleles, respectively. Bi-allelic mutations of SLC26A4 were identified in 4.4% (5/114) of probands. c.919-2A>G was the predominant mutation for SLC26A4, accounting for 60% (6/10) of mutant alleles. The average age of the five probands carrying bi-allelic SLC26A4 mutations were 10 years old (95% CI 8.2–11.8 years), not significantly different (P ¼ 0.46) than the rest of the probands (mean age 8.4 years, 95% CI 7.4–9.3 years). It is worth noting that in this study we identified two novel missense variants, p.K61Q in GJB2 and p.V97E in SLC26A4. Though the pathogenicity of these two variants were not confirmed by functional studies, they were likely to be pathogenic because: (i) both variants occur at the evolutionary conserved amino acid positions (PhyloP scores of 3.34 and 2.38 for p.K61Q and p.V97E, respectively) and were predicted to be disease causing by the Mutation Taster Program (http://www.mutationtaster.org); (ii) they were not found in 200 ethnically-matched normal hearing
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TABLE I. Probands With Causative Mutations in GJB2, SLC26A4, and MTRNR1 Number (%) of probands Gene Genotype GJB2 p.V37I/p.V37I p.V37I/p.K61Q p.V37I/c.235delC c.235delC/c.299-300delAT c.235delC/c.235delC c.235delC/p.T86R c.235delC/c.507insAACG c.235delC/c.93delT SLC26A4 c.919-2A > G/c.919-2A > G c.919-2A > G/p.H723R p.R409H/p.H723R c.919-2A > G/p.V97E MTRNR1 m.1555G> A Total
Multiplex (n ¼ 31) 8 (25.8%) 3 (9.7%) — — 1 (3.2%) 2 (6.5%) 1 (3.2%) — 1 (3.2%) 2 (6.5%) 1 (3.2%) 1 (3.2%) — — 4 (12.9%) 4 (12.9%) 14 (45.2%)
Simplex (n ¼ 83) 27 (32.5%) 10 (15.7%) 1 (1.2%) 6 (7.2%) 3 (3.6%) 5 (6.0%) 1 (1.2%) 1 (1.2%) — 3 (3.6%) 1 (1.2%) — 1 (1.2%) 1 (1.2%) 1 (1.2%) 1 (1.2%) 31 (37.3%)
controls; and (iii) they were in compound heterozygosity with another known pathogenic GJB2 or SLC26A4 mutation in trans (confirmed by parental genotyping). The m.1555A>G mitochondrial mutation of MTRNR1 was identified in another 4.4% (5/114) of probands. All those m.1555A>G mutations were homoplasmic and maternally inherited with incomplete penetrance with family members. Exposure to aminoglycosides was recorded in three multiplex probands and not in the remaining two (one multiplex and one simplex proband).
Targeted NGS Identified No Additional Causes for Mild-to-Moderate HI Bi-allelic mutations in GJB2 and SLC26A4, and mitochondrial mutations in MTRNR1 were not identified in 17 multiplex and 52 simplex probands with mild-to-moderate HI. Among them, 10 multiplex probands had good DNA quality and quantity and were selected for further mutation screening by targeted NGS of 79 known deafness genes. The average depth for the targeted regions was 507.1-fold and 98.4% of the targeted regions were covered by 20 or more reads, demonstrating the high quality of the sequencing. All candidate variants detected by the targeted NGS (Supplementary Table SIII), however, were heterozygous and not consistent with the nondominant inheritance (Supplementary Fig. S1 in supporting information online). None of the candidate variants was reported to be associated with HI in the clinvar database. Parental genotyping showed no evidence of de novo variants. No potential pathogenic CNVs were identified by the NGS analysis.
DISCUSSION Our study presented a preliminary overview of the molecular etiology of non-dominant mild-to-moderate childhood HI in
Clinical information Total 35 13 1 6 4 7 2 1 1 5 2 1 1 1 5 5 45
(n ¼ 114) (30.7%) (11.4%) (0.9%) (5.3%) (3.5%) (6.1%) (1.8%) (0.9%) (0.9%) (4.4%) (1.8%) (0.9%) (0.9%) (0.9%) (4.4%) (4.4%) (40.4%)
Mean PTA (dB) 51.8 46.5 29.0 54.7 52.5 57.4 62.5 55.0 60.0 61.8 65.0 55.0 59.0 65.0 43.8 43.8 52.0
Mean age (years) 7.6 6.5 8.0 9.3 8.1 9.7 6.5 0.4 4.0 10.0 10.0 8.0 11.3 10.7 8.7 8.7 8.0
Chinese Hans. In comparison with our previous studies of severe-to-profound HI [Yang et al., 2013], it was considerably different in that: (i) bi-allelic GJB2 mutations accounted for a higher percentage of causes (30.7% vs. 19.7%, Fisher’s exact test, P ¼ 0.036, Fig. 1); (ii) the percentage of bi-allelic GJB2 mutations associated with non-truncating mutations was much higher (62.9% vs. 12.5%, Fisher’s exact test, P ¼ 8.1 106, Fig. 2); (iii) bi-allelic SLC26A4 mutations accounted for a lower percentage of causes (4.4% vs. 12.0%); and (iv) less causative mutations was identified in other known deafness genes (None vs. 15.8%). The high percentage of non-truncating mutations of GJB2 observed in our study of mild-to-moderate HI was in strong agreement with a genotype-phenotype correlation between nontruncating mutations of GJB2 and milder degree of HI. Consistent with our results, previous functional studies also showed that mutant GJB2 protein with truncating mutations has abolished plasma membrane expression and is void of gap junction plaque formation, while mutant GJB2 protein with non-truncating mutations, though functionally impaired in intercellular channel activity, still retains normal formation of gap junction plaques [Oguchi et al., 2005; Jara et al., 2012]. On the other side, the fact that 37.1% of bi-allelic GJB2 mutations associated with mild-to-moderate HI was indeed truncating in both alleles (Fig. 2), which suggested that additional genetic modifiers and environmental factors might also influence the degree of HI. Our study showed a significant etiological contribution of biallelic GJB2 mutations to mild-to-moderate HI (30.7%, Fig. 1) in Chinese Hans. Interestingly, however, in a study of 48 Australian elementary school children with slight/mild HI, no causative mutations of GJB2 were identified in 40 children of non-Asian ethnicity while homozygous p.V37I was identified in four of the eight children of Asian ethnicity [Dahl et al., 2006]. Apparently, the molecular etiology of mild-to-moderate HI can be variable among different ethnical groups. Based on our results, the p.V37I
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FIG. 1. Molecular etiology of non-dominant mild-to-moderate HI in Chinese Hans in comparison with that of severe-to-profound HI.
mutation of GJB2, which has an extremely high allele frequency (6.2%) in Chinese Hans [Li et al., 2012], was the leading mutant allele identified in children with mild-to-moderate HI and likely the reason for the high etiological contribution of GJB2 mutations in Chinese Hans. Mutations in SLC26A4 are the major cause of HI associated with enlarged vestibular aqueduct. A previous study showed that biallelic mutations in SLC26A4 can be identified in approximately 11% of Chinese Han probands with severe-to-profound HI [Yang et al., 2013]. In contrast, bi-allelic SLC26A4 mutations were detected only in 4.4% of probands with mild-to-moderate HI in this study (Fig. 1). This difference was in agreement with previous reports that a majority of bi-allelic SLC26A4 mutations were associated with severe-to-profound HI [Albert et al., 2006; Chai et al., 2013]. It is worth noting that a significant portion of patients
with bi-allelic SLC26A4 mutations has been reported to have progressive HI during childhood [Albert et al., 2006]. The percentage of bi-allelic SLC26A4 mutations in mild-to-moderate HI, therefore, can be higher in children with younger age. In our study, the age of the five probands with bi-allelic SLC26A4 mutations was not significantly different from the rest of the probands (P ¼ 0.46). The mitochondrial m.A1555G mutation in MTRNR1 is associated with both aminoglycoside-induced and non-aminoglycosideinduced HI. In this study, we detected the m.A1555G mutation in 4.4% of probands with mild-to-moderate HI, a percentage similar to the previous report of severe-to-profound HI in Chinese Hans (Fig. 1). The previous study of severe-to-profound HI also detected 15.8% of probands with causative mutations in the rare deafness genes such as MYO15A, GPR98, TMC1, USH2A, and PCDH15 [Yang et al., 2013]. In this study, however, targeted NGS of 79 known deafness genes identified no causative mutations in the 10 selected multiplex probands with mild-to-moderate HI. This difference was likely attributable to the fact that most known recessive deafness genes were associated with severe-to-profound HI. On the other hand, our screening results of the multiplex probands also suggested that the molecular etiology of mild-tomoderate HI is far from being complete at the current stage. Other genetic causes, such as dominant mutations with incomplete penetrance or variable expressivity, recessive mutations in novel deafness genes or non-coding regions of known deafness genes, and large deletions or complex chromosome rearrangement, which is difficult to be detected by targeted NGS in this study, remain to be disclosed.
ACKNOWLEDGMENTS FIG. 2. Percentages of truncating (T) and non-truncating (N) mutations of GJB2 in Chinese Hans with mild-to-moderate and severe-to-profound childhood HI.
This research was supported by grants from National Science Foundation of China (81222010 and 81371101 to T.Y.; 81330023 and 81170924 to H.W.), National Basic Research Program of China (2011CB504501 to H.W.), Minister of Science and Technology (2012BAI09B02 to H.W.), Specialized Research Fund
CHAI ET AL. for the Doctoral Program of Higher Education (20130073110011 to T.Y.), Program for New Century Excellent Talents in University (NCET-13-0376 to T.Y.), the Oriental Scholar project from Shanghai Municipal Education Commission (to T.Y.) and the Ph.D. Innovation Foundation from Shanghai Jiaotong University School of Medicine (BXJ201221 to Y.C.).
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