Newborn hearing concurrent genetic screening for hearing impairment—A clinical practice in 58,397 neonates in Tianjin, China

International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

Contents lists available at ScienceDirect

International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl

Newborn hearing concurrent genetic screening for hearing impairment—A clinical practice in 58,397 neonates in Tianjin, China Junqing Zhang a,f,1, Peng Wang b,1, Bing Han e,1, Yibing Ding b, Lei Pan b, Jing Zou c, Haisheng Liu a, Xinzhi Pang a, Enqing Liu b, Hongyue Wang b, Hongyan Liu b, Xudong Zhang a, Xiu Cheng a, Dafei Feng c, Qian Li e, Dayong Wang e, Liang Zong e, Yuting Yi a, Ning Tian a, Feng Mu d, Geng Tian a, Yaqiu Chen b, Gongshu Liu b, Fuxia Zhang b, Xin Yi a,c,d, Ling Yang a,c,f,**, Qiuju Wang e,* a

BGI-Tianjin, Tianjin, China Tianjin Women and Children Healthcare Center, Tianjin, China c BGI-Shenzhen, Shenzhen, China d BGI-Beijing, Beijing, China e Department of Otolaryngology-Head and Neck Surgery, and Chinese PLA Institute of Otolaryngology, Chinese People’s Liberation Army General Hospital, Beijing, China f Tianjin Medical Genomics Technology Engineering Center, Tianjin, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 24 April 2013 Received in revised form 25 August 2013 Accepted 29 August 2013 Available online 8 September 2013

Objective: Newborn hearing screening (NHS) is used worldwide due to its feasibility and cost-efficiency. However, neonates with late-onset and progressive hearing impairment will be missed by NHS. Genetic factors account for an estimated 60% of congenital profound hearing loss. Our previous cohort studies were carried out in an innovative mode, i.e. hearing concurrent genetic screening, in newborns to improve the abilities or early diagnosis and intervention for the hearing defects. In this study, we performed the first clinical practice of this mode in Tianjin city. Methods: A large cohort of 58,397 neonates, born between December 2011 and December 2012, in 44 hospitals in Tianjin, were screened for 20 hot spot hearing loss associated mutations from GJB2, GJB3, SLC26A4 and MTRNR1(12S rRNA). The data of genetic screening results was comprehensively analyzed with newborn hearing screening (NHS) results. Results: We developed an accurate, high throughput genetic screening method and applied it to a total of 58,397 newborns in Tianjin. 3225 (5.52%) infants were detected to carry at least one mutation allele in GJB2, GJB3, SLC26A4 or MTRNR1. 34 (0.58%) infants were positive for hearing loss caused by GJB2 or SLC26A4 mutations (homozygote or compound heterozygote). 54(0.93%) infants are heterozygous of various genes. 109(1.87%) infants had the pathological mitochondrial DNA mutation. Conclusion: Accurate, comprehensive hearing loss associated genetic screening can facilitate genetic counseling and provides valuable prognostic information to affected infants. This united screening mode of this study was a promising clinical practice. ß 2013 Published by Elsevier Ireland Ltd.

Keywords: GJB2 GJB3 SLC26A4 MTRNR1 Newborn hearing screening Genetic screening

1. Introduction

* Corresponding author at: Department of Otolaryngology-Head and Neck Surgery, Chinese PLA Institute of Otolaryngology, Chinese People’s Liberation Army General Hospital, 28 Fuxing Road, Beijing 100853, China. Tel.: +86 10 68172228; fax: +86 10 68172228. ** Corresponding author at: BGI-Tianjin, E3 Building, Business Park-east, Airport Economic Area, Tianjin 300308, China. Tel.: +86 22 58838880 8097; fax: +86 22 58838880 8097. E-mail addresses: [email protected] (L. Yang), [email protected] (Q. Wang). 1 These authors contributed equally to this work. 0165-5876/$ – see front matter ß 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.ijporl.2013.08.038

Hearing loss is the most common congenital neurosensory disorder and affecting about 1–3 newborns in every 1000 live births [1–4]. In China, there are about 0.8 million children (<7 years old) with hearing impairment, with an annual increase of 30,000 children. The infants with profound hearing loss (90 dB HL), will suffer permanent hearing impairment with significant and irreversible deficits in linguistic, cognitive and educational development, if not detected and treated within the first postnatal year. But their condition can be improved if identified and positively intervened before 6 months of age [5].

1930

J. Zhang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

Hearing loss is etiologically heterogeneous, it is estimated that at least two thirds of cases of childhood-onset hearing loss have a genetic causes. The majority (around 70%) are non-syndromic hearing loss (NSHL), involving mutations in many genes [6,7]. GJB2 (Gap junction protein, beta-2) is the most prevalent causative gene for NSHL [8,9]. To date, more than 90 GJB2 mutations have been reported, most of which are found in patients with moderate to profound hearing loss [10]. GJB3 is the first deafness gene cloned in Chinese population in 1998 [11]. Mutations in GJB3 are associated with progressive hearing loss. Phenotype of c.538C > T and c.547G > A mutation of GJB3 was variable, with adult male patients presented progressive or late-onset profound deafness, while female carriers either subclinical or not detectable [12]. SLC26A4 gene mutations are major cause for Pendred syndrome(PDS) or enlarged vestibular aqueduct (EVA), presenting specific temporal bone malformation and congenital or earlyonset deafness [13]. The MTRNR1(12S rRNA) mutation m.1555A > G had been found to responsible for aminoglycosideinduced hearing loss worldwide, and m.1494 C > T mutation was another prevalent mutation in the Chinese population [14]. Some other researchers suggested these mutations may cause NHSL or mitochondrial associated SHL [15], and exhibited a considerable phenotypic variation with respect to severity, age-of-onset and penetrance of hearing loss without aminoglycoside exposure [16,17]. Newborn hearing screening (NHS) is used worldwide due to its feasibility and cost-efficiency. The referral rates among those screened range from 0.3% to 14% in various testing centers when using different methods [18–20]. However, neonates with lateonset or progressive hearing impairment will be missed by NHS, because studies revealed that the prevalence of permanent sensorineural hearing loss continues to increase about 50 percent during childhood, and doubles during adolescence [4,21]. Our previous cohort studies were carried out in an innovative mode of hearing concurrent gene screening in newborns, which can improve the abilities for early diagnosis and intervention for the hearing defects [22,23]. In order to verify and improve the clinical significance of the mode, we performed this procedure for 20 hot spot mutations from GJB2, GJB3, SLC26A4 and MTRNR1(12S rRNA), in 58,397 neonates hearing concurrent in Tianjin, China.

a repeated test of OAE plus AABR (automated auditory brainstem response) at the age of 42 days. The compound hearing test result was recorded as newborn hearing screening (NHS) ‘‘pass’’ or NHS ‘‘refer’’. Those who failed to pass the two-step test were referred to further comprehensive audiological assessment at 3 months. 2.3. Newborn genetic screening This genetic screening involved 20 hot spot mutations from 4 primary NSHL genes: GJB2, GJB3, SLC26A4 and MTRNR1 (12S rRNA). The multiple-PCR-based MALDI-TOF MS assay was used for genotyping. The reaction assay, which including primers and unextended probes, was designed specially (Table S1). Every mutation allele has an allele-specific extending probes with different molecular weight in the reaction, and could be separated by MALDI-TOF MS. All the reaction could be done in a single well or tube. The methods were validated with wild and mutant type DNA samples, from those who had been confirmed by Sanger sequencing previously. Samples with positive results were further confirmed by sequencing. The sensitivity and specificity was >99.9%. Chelating resin Chelex-100 (Biorad, USA) was used for DNA extraction [24]. The PCR was performed in a total volume of 5 mL consisting 1 mL of DNA template (10–25 ng/mL), 1 PCR buffer (including 2 mmol/L magnesium chloride), 2 mmol/L magnesium chloride, 500 mmol/L deoxy-nucleoside triphosphate (dNTP) mix, 0.1 pmol/mL of each preprimer, and 0.5 U of HotstarTaq. The PCR condition was as follow: denaturation at 94 8C for 15 minutes, followed by 45 cycles of 20 s at 94 8C, 30 s at 56 8C, 1 min at 72 8C, and a final extension of 3 min at 72 8C. Then the final primary PCR reaction mix was treated with shrimp alkaline phosphatase(SAP) to dephosphorylate remaining dNTPs. The iPLEX primers extension reaction was performed, which followed the iPLEX kit standard protocol (Sequenom, USA). To desalt the iPLEX extension products before mass spectrometric analysis, 6 mg clean resin was added into the 384-well PCR plate. After purification, 3–10 nL products were dispensed into a 384-element SpectroCHIP bioarray (Sequenom,USA). TYPE 4.0 software (Sequenom, USA) was used to process and analyze iPLEX Spectro CHIP bioarrays. 2.4. Description and interpretation of gene screening results

2. Materials and methods 2.1. Enrollment and study design This study was a universal hearing screening concurrent genetic screening project in Tianjin, which is a municipality in the north of China adjacent to Beijing. This study was performed with the approval of the Institutional Review Boards of Tianjin women and children healthcare center. From December 2011 to December 2012, 58,397 newborns were recruited from 44 hospitals in Tianjin, the participation rate was 56.8% (58,397/102,830). Written informed consent was obtained from infant’s parents or guardians. All newborn information, including a list of risk factors for deafness was collected into a newborn screening database, with an additional blood spot obtained at the time of newborn metabolic screening. All the infants received both newborn hearing screening and genetic screening.

According to the newborn-hearing screening program, we use ‘‘pass’’, ‘‘refer’’, ‘‘carrier’’ to describe the gene screening results as our pervious study. The ‘pass’ was defined to mean that mutation(s) or genotypes associated with hearing loss were not found. Genetic testing ‘refer’ was defined to mean that a causative mutation(s) in the forms of either homozygote or compound heterozygote was found at GJB2, SLC26A or mtDNA 12S rRNA 1555A > G, 1494G > T [22]. The ‘‘carrier’’ means individual carried heterozygous mutation(s) and were described in the form like ‘‘GJB2 c.235delC carrier’’. Specially, for heterozygous for m.1555A > G or m.1494C > T mutation in mitochondrial DNA, we describe it heteroplasmy. 3. Result 3.1. Demographic characteristics and the results of newborn hearing screening

2.2. Newborn hearing screening A two-step hearing screening was performed in the infants. An otoacoustic emission(OAE) test was used as the initial screening at 48–72 h after delivery. If an infant failed to pass the OAE test, either bilateral or unilateral, it will be recorded as first step OAE ‘‘refer’’, and all the first step OAE ‘‘refer’’ infants were conducted to receive

A total of 58,397 newborns with a gender ratio of 1.00 (female):1.12 (male), were subjected to NHS in this study. Demography information of the infants and potential risk factors for hearing loss were shown in Table 1. Premature delivery (2378 cases, 4.07%) were the most frequent risk factor. Totally, 9.21% of the newborns were predisposed to one risk factor at least.

[(Fig._1)TD$IG]

J. Zhang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

1931

Table 1 Birth information of the newborns in the screening screening. Items

Samples

Newborns subjected in this study Gender Male Female Risk factors Craniofacial deformity PCHL family history NICU 5 days Low birth weight Premature delivery Hyperbilirubinemia Intrauterine infection Neonatal respiratory distress syndrome Asphyxia neonatorum Othersa

58,397

Frequency (%)

30,819 27,578

52.78 47.23

76 114 1102 103 2378 858 617 71 75 19

0.13 0.20 1.89 0.18 4.07 1.47 1.06 0.12 0.13 0.03

a Including drug and alcohol abuse during pregnancy, extra corporeal membrane oxygen (ECMO), mechanical ventilation2 days and Vira/bacterial meningitis.

3.2. Results and comprehensive analysis of concurrent genetic and hearing screening Genetic screening data of the 20 deafness-associated mutations in this study of 58,397 neonates, was shown in Table 2. If each allele was counted individually (one infant with two mutation alleles will be counted twice), total carrier frequency was 5.65% (3298/58,397). GJB2 c.235delC and SLC26A4 c.919-2A > G were the most frequency mutations detected in this study, with a carrier frequency of 1.96% and 1.56%, respectively, and the mutation spectrum was shown in Fig. 1. In this study, 5.52% (3225/58,397) infants carried at least one mutation allele or mitochondrial mutation in the genetic screening. 143(0.25%) infants were genetic ‘‘refer’’ and 3082 (5.28%) infants were genetic mutation carriers. For the genetic ‘‘refer’’ infants, 109 infants (0.19%) were screened to be MTRNR1 (12S rRNA) mutation carriers. 18 infants had two mutated alleles in

Fig. 1. Spectrum of 20 hot spot deafness-associated mutations.

GJB2. 16 infants had two mutated alleles in SLC26A4. We further conducted an analysis of the clinical and genetic profiles for the infants with causative forms of mutations in GJB2 and SLC26A4, Detail information and follow-up results were shown in Table 3. In this study, we also detected 54 infants were heterozygous in various genes of GJB2, GJB3, SLC26A4 and MTRNR1. Especially, one infant was detected to carrier three mutations allele of GJB2 c.235delC (het) & GJB2 c.299_300delAT (het) & SLC26A4 c.1226G > A (het). The detail information of the heterozygous in two different genes in GJB2, GJB3, SLC26A4 were shown in Table 4. Association of newborn hearing screening and genetic screening data were shown in Table 5. We used the chi-squared test to compare the difference of genetic refer and genetic carrier frequency, among three groups of ‘‘audio pass’’, ‘‘audio bilateral refer’’ and ‘‘audio unilateral refer’’. GJB2 refer and SLC26A4 refer frequency is correlated with ‘‘audio bilateral refer’’ group and significant higher than ‘‘unilateral refer’’ group or ‘‘audio pass’’

Table 2 Mutation spectrum of 20 hot spot deafness associated allele in 58,397 neonates. Genetic screening

Mode of inheritance

GJB2 35delG 167delT 176_191del16 235delC 299_300delAT GJB3 538C > T 547G > A SLC26A4 281C > T 589G > A 919-2A > G 1174A > T 1226G > A 1229C > T IVS15 + 5G > A 1975G > C 2027T > A 2162C > T 2168A > G 12S rRNA 1494C > T 1555A > G

AR

Total

AR/AD

AR

Maternal inheritance

Heterzygousa

Total

Carrier frequency (%)

95% confidence interval

9 0 0 0 8 1 0 0 0 8 0 0 7 0 0 0 0 0 1 0 0 92 8 84

1520 9 0 70 1135 306 249 216 33 1403 20 20 902 45 58 49 19 74 42 5 169 17 0 17

1529 9 0 70 1143 307 249 216 33 1411 20 20 909 45 58 49 19 74 43 5 169 109 8 101

2.62 0.02 0.00 0.12 1.96 0.53 0.43 0.37 0.06 2.42 0.03 0.03 1.56 0.08 0.10 0.08 0.03 0.13 0.07 0.01 0.29 0.19 0.01 0.17

2.49–2.75 0.01–0.03 0.00–0.01 0.10–0.15 1.85–2.07 0.47–0.59 0.38–0.48 0.32–0.42 0.04–0.08 2.29–2.54 0.02–0.05 0.02–0.05 1.46–1.66 0.06–0.10 0.08–0.13 0.06–0.11 0.02–0.05 0.10–0.16 0.06–0.10 0.00–0.02 0.25–0.34 0.16–0.23 0.01–0.03 0.14–0.21

109

3189

3298

5.65

5.46–5.84

Homozygous

AR: autosomal recessive, AD: autosomal dominant. a Heterozygous in different genes were counted separately.

J. Zhang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

1932

Table 3 Newborn hearing screening result of the genetic affect infants. ID

Gender

Genotype

NHS result

Follow-up diagnosis

Risk factor(s)

19,686 16,982 2213 49,566 47,375 54,845 3489 29,553 10,687 57,183 5086 6803 14,173 19,317 42,957 46,766 44,194 17,990 9805

M M M M F M M F F F M M M M M F M M M

GJB2 235delC/235delC GJB2 235delC/235delC GJB2 235delC/235delC GJB2 235delC/235delC GJB2 235delC/235delC GJB2 235delC/235delC GJB2 235delC/235delC GJB2 235delC/235delC GJB2 299_300delAT/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT GJB2 235delC/299_300delAT,SLC26A4 1226G > A GJB2 35delG/176_191del16 SLC26A4 919-2A > G/919-2A > G

Refer Refer Refer Refer Refer Refer Pass Pass Refer Refer Refer Refer Refer Refer Refer Pass Refer Pass(L)Refer(R) Refer

Severe/severe Mild/moderate Severe/profound Severe/severe – – – – – – Severe/severe Severe/moderate – Profound/profound – – – – Profound/profound

17,165 21,484 35,511 38,001 5901 18,615 6264 2490 24,294 31,800 46,264 42,945 47,805 23,915 35,210

M M F F M M M F M M F F M F M

SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4 SLC26A4

Refer Refer Refer Refer Pass Pass Refer Pass Pass Refer Refer Refer Refer Pass(L)Refer(R) Refer

Severe/moderate Profound/severe Profound/profound Profound/profound – – – – – Moderate/profound Moderate/moderate Moderate/severe Moderate/moderate Mild/moderate –

— — Family history — — — — — — — Family history — — — — — — — Suspected deafnessassociated syndrome — — — — — — NICU  5 days — — — — — — Family history —

919-2A > G/919-2A > G 919-2A > G/919-2A > G 919-2A > G/919-2A > G 919-2A > G/919-2A > G 919-2A > G/919-2A > G 919-2A > G/919-2A > G 2027T > A/2027T > A 919-2A > G/2168A > G 919-2A > G/1174A > T 919-2A > G/1226G > A 919-2A > G/1226G > A 1226G > A/2168A > G 1226G > A/2168A > G 1229C > T/2168A > G 2027T > A/2168A > G

‘‘–’’, means that these infants were underway for audiological assessment after discharge; ‘‘—’’,means no risk factor identified.

(p < 0.05). GJB2 carrier and SLC26A4 carrier frequencies were correlated with ‘‘audio bilateral refer’’ and ‘‘audio unilateral refer’’ group and significantly higher than ‘‘audio pass’’ group, but there was no significant difference between unilateral cases and bilateral cases. There is no significant difference among the three group for MTRNR1 refer or GJB3 carrier. The results of newborn hearing screening (NHS) and genetic screening was shown in Fig. 2. 4. Discussion This study combined physical newborn hearing screening and genetic screening in a certain area (covered 51.6% neonates in Tianjin) in one year. In this study, we developed a special multiplePCR-based MALDI-TOF MS assay for the genotyping, and all the 20 hot spot mutations genotyping reactions could be performed simply in a single well of 384-well PCR plate, the high-throughput genotyping method made the genetic screening of the large cohorts (as many as 58,397 neonates) in Tianjin possible. The data in this study confirms that GJB2 is the most common causative gene for nonsyndromic hereditary HL in Chinese [25]. The carrier frequency of common mutations was 1.957% for c.235delC, 0.526% for c.299_300delAT and 0.120% for c.176_191del16. GJB2 c.35delG mutation carrier is common in Caucasian populations [26], but in this study, we detected only 0.015% (9/58,397) infants carrying c.35delG mutation in the Chinese population. Although seldomly reported in the Chinese population, GJB2 c.167delT mutation is common in Ashkennazi Jew [27], it is unexpected that in all the 58,397 infants, there is no c.167delT mutation detected, c.167delT was not supposed to be detected in the later screening. For GJB3, we detected 216 infants

carrying c.538C > T mutation and 33 carrying c.547G > A in the population. Science there is no significant difference in the GJB3 carrier frequency between the audio pass or refer group, we may conjecture GJB3 mutations carrier were normal at birth, but should receive special attention in their hear development. Mutations in the SLC26A4 gene have been identified as a major cause of nonsyndromic hearing loss associated with EVA syndrome and Pendred syndrome, with hearing loss found at birth or in early childhood. Common mutations of c.281C > T, c.589G > A, c.9192A > G, c.1174A > T, c.1226G > A, c.1229C > T, c.1975G > C, c.2027T > A, c.2162C > T, c.2168A > G and IVS15 + 5G > A were included in the screening. Previous study found that 97.9% of Chinese EVA patients carried SLC26A4 mutation, and EVA was found in 100% patients with bi-allelic mutations in SLC26A4 by CT scan [28]. In this study, 11 in 16 infants who were homozygotes or compound heterozygotes in SLC26A4 were confirmed to have EVA, while the other 5 were still being followed up. Mitochondrial gene MTRNR1 (12S rRNA) is the hot spot for mutations associated with aminoglycoside ototoxicity. In this study, all the infants received no aminoglycoside before the screening, among the 109 Mitochondrial MTRNR1 (12S rRNA) mutation carrier infants, 103 infants passed the NHS, while the other 6 infants were OAE referred. One referred infant who had family history and suspected deafness-associated syndrome, was diagnosed to be severe/moderate deafness in the follow-up audiology assessment. Other 5 referred infants were subjected to further diagnosis and family analysis, to found whether there are other possible causative conditions that had not been screened, the follow-up work were still underway. The most important thing was that for all the 109 infants with MTRNR1(12S rRNA mutation in

J. Zhang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

1933

Table 4 Newborn hearing screening result of heterozygous in different genes. ID

Gender

GJB2

GJB3

SLC26A4

NHS result

Follow-up diagnosis

Risk factor(s)

49,796 50,350 51,190 5689 7680 8269 20,098 26,405 26,434 30,576 37,575 41,822 43,942 23,007 438 10,112 34,877 42,294 10,995 12,756 17,407 9887 10,212 10,213 37,966 55,546 59,056 56,853 21,705 22,074 26,942 46,147 56,400 51,164 14,042 15,270 27,580 33,758 42,772 17,388 19,788 41,097 46,370

M F M F F M F F F M F F M M M M M F F F F M F F F M F M F M F M M F M M F M F M F F M

235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 235delC 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT 299_300delAT

wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt 538C > T 538C > T 538C > T 538C > T 538C > T 538C > T wt wt wt wt wt wt wt wt wt wt wt

919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 281C > T 2027T > A 2027T > A 2027T > A 1975G > C 1975G > C 1975G > C 1229C > T 1229C > T 1229C > T 1229C > T 1174A > T wt wt wt wt wt wt 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 919-2A > G 2168A > G 2168A > G 2168A > G 2168A > G

Pass Pass Pass Pass Pass Pass Refer(L)Pass(R) Pass Pass(L)Refer(R) Pass Pass Refer Pass(L)Refer(R) Pass Pass Pass Pass Pass Pass Pass Pass Pass Refer(L)Pass(R) Refer Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass

– – – – – – – – Normal/moderate – – – – – – – – – – – – – Moderate/normal Moderate/moderate – – – – – – – – – – – – – – – – – – –

7175 33,177 23,556 37,142 57,194 31,905 2719 59,143 45,437 45,932 24,686

M M F M F M M F M M M

299_300delAT 299_300delAT 299_300delAT 299_300delAT 176_191del16 176_191del16 176_191del16 wt wt wt wt

wt wt 538C > T 538C > T wt wt 538C > T 538C > T 538C > T 538C > T 538C > T

1226G > A 1226G > A wt wt 919-2A > G 919-2A > G wt 919-2A > G 589G > A 1975G > C 1226G > A

Pass Pass Pass Pass Pass Refer(L)Pass(R) Pass Pass Pass Pass Refer

– – – – – Normal – – – – –

— — — — — — — — — Intrauterine infection — — Craniofacial deformity — — — — — — — — — — — — — — — — — — — — — Family history — — — — — — — Suspected deafnessassociated syndrome Family history — — — — — — — — — —

‘‘–’’, means that these infants were underway for audiological assessment after discharge; ‘‘—’’,means no risk factor identified.

the screening, should be kept away from aminoglycosides all through the whole life-time to avoid drug-inducing hearing loss. This screening mode provided direct clues about the pathogenesis of hearing loss for certain patients and could lead to proper management. Provided that 1–3% newborns have hearing defects and genetic factors account for 60% of profound hearing loss. In this study, we found 34 infants with two mutated alleles in GJB2 or SLC26A4, accounting for 0.58% of the 58,397 infants, among them 24 infants of these infants had been diagnosed to be bilateral sensorineural hearing loss, these infants should receive cochlear implantation as early as possible, the other infants carried high risk causative mutations with normal hearing at birth could obtain the information on how to optimize the listening environment to refrain from hearing impairment in their hearing development, e.g.

avoid head trauma in daily life, and treatment timely if their auditory threshold changed. For the 3118 hearing loss associated mutation carriers, especially 54 carriers for more than one allele, who might have a risk of progressive hearing loss, we advised them to pay attention of hearing health in their daily life. 109(1.87%) infants with m.1555A > G or m.1494C > T mutation could be aware of the hearing susceptibility very early in their life, and they were conduct to avoid drug-induced hearing loss by keeping away from aminoglycosides. Some limitations about the project deserve to be considered. Firstly, the screening panel covered 20 common mutations accounting only for about 20–30% hereditary hearing loss in Chinese population. Some important late-onset and progressive profound deafness genes or sites should be added in the screening.

J. Zhang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

1934

Table 5 Association between newborn hearing screening and genetic screening results. Audio pass (55,890)

GJB2 refer SLC26A4 refer MTRNR refer GJB2 carrier GJB3 carrier SLC26A4 carrier a

[(Fig._2)TD$IG]

Audio bilateral refer (348)

Audio unilateral refer (422)

Unavailable (1737)

Total (58,397)

n

%

n

%

n

%

n

%

n

%

2 4 103 1392 239 1316

0.04 0.07 1.84 24.91 4.28 23.55

14 10 1 26 3 21

40.23 28.74 2.87 74.71 8.62 60.34

1 1 0 32 2 23

2.37 2.37 0 75.83 4.74 54.50

1 1 5 70 5 43

0.58 0.58 2.88 40.30 2.88 24.76

18 16 109 1520a 239a 1403a

0.31 0.27 1.87 26.03 4.09 24.03

Heterozygous in different genes were counted separately.

Fig. 2. Comprehensive scan of newborn hearing screening (NHS) and genetic (A), including heterozygous in different genes; (B), 13 infants were OAE pass but AABR refer; (C), gene screening data of 20 hot spot mutations in 4 genes.

To improve the screening panel, large-scale retrospective research on Chinese population with NSHL need to be conducted. The good news was that the whole exome sequencing or targeted-region sequencing and massively parallel sequencing (MPS), had been proved to be efficient means for genetic diagnosis of hearing loss [29,30], and could be effective complements to the hot spot mutations screening. Secondly, genetic heterogeneity of hearing loss makes it hard to unscramble precisely and accordingly lag in clinical application. Besides, the genetic results of late-onset and progressive hearing loss, which need long-term follow-up to conform, might lead to unjustified parental anxiety about their infant’s health. Theoretically, all the participants should be further followed up, but for such a large cohort, it is really a challenging work to timely and

accurately diagnostic of all the children. Efforts of ongoing care and management of their audiologic and related clinical needs and genetic counseling, were being given to children with HL their families. We will further discuss about the follow-up study in another research paper. In conclusion, accurate, comprehensive genetic testing can facilitate genetic counseling and provides valuable prognostic information to affected infants. The early predict can eventually to improve the quality of life by providing early treatment and avoid the hazard to hearing loss and early treatment, especially for individuals who were at risk for aminoglycoside ototoxicuty. This study had developed a promising clinical practice of the united screening mold of newborn hearing concurrent genetic defects screening.

J. Zhang et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1929–1935

Conflicts of interest We declare that we have no conflicts of interest. Acknowledgements This work was supported by grants of the National Major Science Research Project (No. 2014CB943000) and Tianjin Binhai New Area Science and Technology Commission (No. 2011BK120011), Beijing Post Doc Innovation and Practice Workstation Project (No. 2012-QQ-34) and the National Natural Science Foundation of China, Major Project (No. 81120108009). We also thank all the neonates and their parents for their cooperation during this work.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijporl.2013. 08.038. References [1] L.A. Schimmenti, B. Warman, M.R. Schleiss, K.A. Daly, J.A. Ross, M. McCann, et al., Evaluation of newborn screening bloodspot-based genetic testing as second tier screen for bedside newborn hearing screening, Genet. Med. 13 (2011) 1006–1010. [2] A. Kral, G.M. O’Donoghue, Profound deafness in childhood, N. Engl. J. Med. 363 (2010) 1438–1450. [3] C. Kennedy, D. McCann, M.J. Campbell, L. Kimm, R. Thornton, Universal newborn screening for permanent childhood hearing impairment: an 8-year follow-up of a controlled trial, Lancet 366 (2005) 660–662. [4] C.C. Morton, W.E. Nance, Newborn hearing screening – a silent revolution, N. Engl. J. Med. 354 (2006) 2151–2164. [5] C. Yoshinaga-Itano, A.L. Sedey, D.K. Coulter, A.L. Mehl, Language of early- and later-identified children with hearing loss, Pediatrics 102 (1998) 1161–1171. [6] C. Petit, Genes responsible for human hereditary deafness: symphony of a thousand, Nat. Genet. 14 (1996) 385–391. [7] P.J. Willems, Genetic causes of hearing loss, N. Engl. J. Med. 342 (2000) 1101–1109. [8] P.M. Kelley, D.J. Harris, B.C. Comer, J.W. Askew, T. Fowler, S.D. Smith, et al., Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss, Am. J. Hum. Genet. 62 (1998) 792–799. [9] V.M. Ballana E, R. Rabionet, P. Gasparini, X. Estivill, Connexins and Deafness Homepage, 2013 World wide web URL: http://www.crg.es/deafness. [10] K. Tsukada, S. Nishio, S. Usami, A large cohort study of GJB2 mutations in Japanese hearing loss patients, Clin. Genet. 78 (2010) 464–470. [11] J.H. Xia, C.Y. Liu, B.S. Tang, Q. Pan, L. Huang, H.P. Dai, et al., Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment, Nat. Genet. 20 (1998) 370–373.

1935

[12] Q.Z. Li, Q.J. Wang, L.D. Zhao, H. Yuan, L.N. Li, D.Y. Han, et al., Mutation analysis of GJB3 in Chinese population with non-syndromic hearing impairment, J. Audiol. Speech Pathol. 13 (2005) 145–148. [13] S.P. Pryor, A.C. Madeo, J.C. Reynolds, N.J. Sarlis, K.S. Arnos, W.E. Nance, et al., SLC26A4/PDS genotype-phenotype correlation in hearing loss with enlargement of the vestibular aqueduct (EVA): evidence that Pendred syndrome and nonsyndromic EVA are distinct clinical and genetic entities, J. Med. Genet. 42 (2005) 159–165. [14] S. Kupka, T. Toth, M. Wrobel, U. Zeissler, W. Szyfter, K. Szyfter, et al., Mutation A1555G in the 12S rRNA gene and its epidemiological importance in German, Hungarian, and Polish patients, Hum. Mutat. 19 (2002) 308–309. [15] N. Mezghani, M. Mnif, E. Mkaouar-Rebai, N. Kallel, N. Charfi, M. Abid, et al., A maternally inherited diabetes and deafness patient with the 12S rRNA m.1555A > G and the ND1 m.3308T > C mutations associated with multiple mitochondrial deletions, Biochem. Biophys. Res. Commun. (2013). [16] S. Hakli, M. Luotonen, M. Sorri, K. Majamaa, Audiological Follow-Up of Children with the m.1555A > G mutation in Mitochondrial DNA, Audiol. Neurootol. 18 (2013) 23–30. [17] S.Y. Lu, S. Nishio, K. Tsukada, T. Oguchi, K. Kobayashi, S. Abe, et al., Factors that affect hearing level in individuals with the mitochondrial 1555A.G. mutation, Clin. Genet. 75 (2009) 480–484. [18] C.J. Clemens, S.A. Davis, Minimizing false-positives in universal newborn hearing screening: a simple solution, Pediatrics 107 (2001) E29. [19] C.C. Wu, C.C. Hung, S.Y. Lin, W.S. Hsieh, P.N. Tsao, C.N. Lee, et al., Newborn genetic screening for hearing impairment: a preliminary study at a tertiary center, PloS ONE 6 (2011) e42231. [20] P.M. Watkin, M. Baldwin, Identifying deafness in early childhood: requirements after the newborn hearing screen, Arch. Dis. Child. 96 (2011) 62–66. [21] H.M. Fortnum, A.Q. Summerfield, D.H. Marshall, A.C. Davis, J.M. Bamford, Prevalence of permanent childhood hearing impairment in the United Kingdom and implications for universal neonatal hearing screening: questionnaire based ascertainment study, BMJ 323 (2001) 536–540. [22] Q.J. Wang, Y.L. Zhao, S.Q. Rao, Y.F. Guo, Y. He, L. Lan, et al., Newborn hearing concurrent gene screening can improve care for hearing loss: a study on 14,913 Chinese newborns, Int. J. Pediatr. Otorhinolaryngol. 75 (2011) 535–542. [23] Z. Zhang, W. Ding, X. Liu, B. Xu, W. Du, S. Nan, et al., Auditory screening concurrent deafness predisposing genes screening in 10,043 neonates in Gansu province, China, Int. J. Pediatr. Otorhinolaryngol. 76 (2012) 984–988. [24] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotechniques 10 (1991) 506–513. [25] P. Dai, F. Yu, B. Han, X. Liu, G. Wang, Q. Li, et al., GJB2 mutation spectrum in 2063 Chinese patients with nonsyndromic hearing impairment, J. Transl. Med. 7 (2009) 26. [26] N. Hilgert, R.J. Smith, G. Van Camp, Forty-six genes causing nonsyndromic hearing impairment: which ones should be analyzed in DNA diagnostics? Mut. Res. 681 (2009) 189–196. [27] O. Dagan, H. Hochner, H. Levi, A. Raas-Rothschild, M. Sagi, Genetic testing for hearing loss: different motivations for the same outcome, Am. J. Med. Genet. 113 (2002) 137–143. [28] P. Dai, Y. Yuan, D. Huang, X. Zhu, F. Yu, D. Kang, et al., Molecular etiology of hearing impairment in Inner Mongolia: mutations in SLC26A4 gene and relevant phenotype analysis, J. Transl. Med. 6 (2008) 74. [29] A.E. Shearer, A.P. DeLuca, M.S. Hildebrand, K.R. Taylor, J. Gurrola 2nd, T.E. Scherer, et al., Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing, Proc Natl Acad Sci U S A 107 (2010) 21104–21109. [30] X. Lin, W. Tang, S. Ahmad, J. Lu, C.C. Colby, J. Zhu, et al., Applications of targeted gene capture and next-generation sequencing technologies in studies of human deafness and other genetic disabilities, Hear. Res. 288 (2012) 67–76.