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A novel early truncation mutation in OTOG causes prelingual mild hearing loss without vestibular dysfunction
European Journal of Medical Genetics xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: www.elsevier.com/locate/ejmg
A novel early truncation mutation in OTOG causes prelingual mild hearing loss without vestibular dysfunction Seyoung Yua,1, Hye Ji Choib,1, Joon Suk Leea, Hyun Jae Leeb, John Hoon Rima, Jae Young Choib, Heon Yung Geea,∗∗, Jinsei Jungb,∗ a b
Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea Department of Otorhinolaryngology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: OTOG Otogelin DFNB18 Prelingual Mild hearing loss
OTOG was identified as a nonsyndrmoic hearing loss gene in 2012 in two families with nonprogressive mild-tomoderate hearing loss. However, no further literature have this gene for nonsyndromic hearing loss. Furthermore, it is still unclear whether vestibular impairment is involved or not in patients with mutations in OTOG. This study presents a validated second report for homozygous causative mutations in OTOG of mild hearing loss. Whole exome sequencing (WES) was performed in a five-year-old male proband with mild hearing loss. The analysis of WES revealed a homozygous truncating mutation (c.330C > G; p.Tyr110*) in OTOG. The identified novel mutation, p.Tyr110*, leads to a null allele based on the fact that early truncated protein contains no functional domain of otogelin. While defects in otogelin previously reported to result in hearing loss and vestibular dysfunction, p.Tyr110* only caused nonsydromic and nonprogressive hearing loss without any vestibular impairment, indicating that vestibular phenotype would be variable. Given that mild hearing loss is not easy to be detected early, mutations of OTOG may be more prevalent than reported. Therefore, genetic evaluation for OTOG should be considered in children with mild hearing loss with/without vestibular dysfunction.
1. Introduction Congenital hearing loss is one of the most prevalent sensorial disorders, present in one of 500–1000 children. It is estimated that genetic factors contribute to about half cases of congenital hearing loss and about 50% of inherited cases are non-syndromic hearing loss (NSHL) (Lenz and Avraham, 2011). Of the more than 100 genes (> 140 loci) associated with NSHL, approximately 70% contribute to autosomal recessive pattern of inheritance (http://hereditaryhearingloss.org/) (Morton and Nance, 2006,Lenz and Avraham, 2011). Most of autosomal recessive non-syndromic hearing loss (AR-NSHL) is prelingual and severe-to-profound sensorineural hearing loss. However, several mutations on OTOG or OTOGL are associated with AR-NSHL with mild-tomoderate sensorineural hearing loss (Oonk et al., 2014). OTOG encodes otogelin protein, a noncollagenous component of the acellular gelatinous structures that cover the sensory epithelia of the inner ear; the tectorial membrane in cochlea, the otoconial membranes in the utricle and saccule, and the cupulae that cover the cristae
amullares of the semicircular canals in the vestibular organ (Simmler et al., 2000a). Otogelin is one of three major components in tectorial membrane besides alpha tectorin and beta tectorin (Schraders et al., 2012). Mutations in OTOG are known to cause DFNB18B (MIM 614945). To date, three causative mutations in OTOG have been found in two patients with DFNB18B: 1) a homozygous 1bp deletion, c.5508delC (p.Ala1838Profs*31) which leads to a frame shift and premature stop codon and 2) compound-heterozygous mutations: c.6347C > T (p.Pro2116Leu) and c.6559C > T (p.Arg2187*) (Schraders et al., 2012). Moderate hearing impairment with a U-shaped to flat audiogram is the most common type of hearing impairment associated with defects in components of the tectorial membrane and this was also observed in the affected individuals who had a homozygous OTOG mutation (Schraders et al., 2012). On the contrary, the affected individuals who have compound-heterozygous mutations had slightly down-sloping shaped audiograms (Schraders et al., 2012). In addition, most of affected individuals showed a delayed speech development which suggest a prelingual onset of hearing impairment (Schraders
∗
Corresponding author. Department of Otorhinolaryngology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, Republic of Korea. Corresponding author. Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, Republic of Korea. E-mail addresses: [email protected] (H.Y. Gee), [email protected] (J. Jung). 1 These authors contributed equally to this work. ∗∗
https://doi.org/10.1016/j.ejmg.2018.05.018 Received 12 November 2017; Received in revised form 9 May 2018; Accepted 21 May 2018 1769-7212/ © 2018 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Yu, S., European Journal of Medical Genetics (2018), https://doi.org/10.1016/j.ejmg.2018.05.018
European Journal of Medical Genetics xxx (xxxx) xxx–xxx
S. Yu et al.
Fig. 1. Pedigree and clnical phenotypes of YUHL62. (A) Family pedigree of YUHL62 with sporadic congenital hearing loss. (B) audiograms of subjects in YUHL62. (C) Ear drum finding and Temporal bone computed tomogram of YUHL62-21. (D) Vestibular function tests (upper: caloric test, lower: rotary chair test) were performed in YUHL62-21. Caloric test vis, visual; supp, suppression; enh, enhancement.
development. In vestibular function test, the proband (YUHL62-21) showed normoreflexia in caloric test as well as rotary chair test (with slow harmonic acceleration) (Fig. 1D). In bithermic caloric test, canal paresis was 3.5% at the left ear (normal range ≤ 25%) and directional predonderance was 5.3% at the right ear (normal range ≤ 30%), which were within normal limits. In slow harmonic acceleration test, the gains of vestibulo-ocular reflex were normal without phase lead or lag at the frequencies of stimuli from 0.08 to 1.28 Hz. According to the pedigree, the affected individual was only YUHL62-21 and sporadic or autosomal recessive pattern of hearing loss was suspicious.
et al., 2012). Here, we report a Korean individual who exhibit early-onset mild NSHL. We performed whole exome sequencing (WES) and identified a nonsense mutation (hg19; chr11:17,514,667A > G) in OTOG. Our study suggests that genetic analysis of early-onset mild NSHL should include OTOG as a possible causative gene. 2. Clinical report The affected proband YUHL62-21 was five-year-old male (Fig. 1A). He did not have any medical history or syndromic features. He failed the newborn hearing screening with automated auditory brainstem response. At three months of age, the threshold of auditory brainstem response was 40 dB nHL at both ears (Supplementary Fig. 1). However, since there was no delay in speech development, his parents (YUHL6211 and −12) did not have further evaluation about the hearing impairment. At 5 years of age, YUHL62-21 individual visited tertiary medical center for the reason of hearing difficulty. He had mild sensorineural hearing loss with decreased threshold in pure-tone audiometry (30 and 41 dB HL at the right and left ear, respectively). The audiogram was relatively flat pattern (Fig. 1B). However, there was no sign for middle ear anomaly or infection in otoscopic finding and temporal bone computed tomogram (Fig. 1C). Word recognition score at the most comfortable level was 94% at both ears. His parents (YUHL62-11; male/36 years-old, YUHL62-12; female/35 years-old, and) had no definite hearing impairment (Fig. 1B) and his younger brother (YUHL62-22; male/2 years-old) passed neonatal hearing screening with automated auditory brainstem response. The affected proband did not have any vertigo symptoms and showed normal motor
3. Methods 3.1. Subjects This study was approved by the institutional review board of the Severance Hospital, Yonsei University Health System (IRB#4-20150659). After obtaining written informed consent from subjects or their parents, individuals with hearing loss were enrolled in the Yonsei University Hearing Loss (YUHL) cohort, and their clinical and pedigree data were recorded. The proband (−21) of YUHL62 family exhibited no other syndromic features except sensorineural hearing loss. All the subjects in YUHL62 did not have any history of non-inherited risk factors for hearing loss, such as perinatal viral infection, neonatal intensive care unit treatment, and middle ear infection. 3.2. Audiological and vestibular analyses Audiological evaluations, including pure-tone audiometry and 2
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recessive pattern. Given that the full length of otogelin consists of 2925 amino acids, the early truncated mutant otogelin (p.Tyr110*) may have complete loss-of-function and a homozygous mutation of OTOG was previously shown to be associated with AR-NSHL (Schraders et al., 2012). To date, there has been only one report that mild-to-moderate hearing loss was attributable to the mutations of OTOG in human (Schraders et al., 2012). Like other genes such as TECTA, COLL11A2, and OTOGL coding for the proteins of tectorial membrane, the mutations in OTOG showed early onset and non-progressive AR-NSHL (Verhoeven et al., 1998,McGuirt et al., 1999,Zheng et al., 2011). The patients with mutations in OTOG did not have severe-to-profound hearing deterioration. This finding is consistent with the fact that the pathogenesis of defect in otogelin does not involve in anchoring the tectorial membrane to the hair cells but just increase the resistance of the tectorial membrane (Simmler et al., 2000b). It is likely that cases of mild hearing impairment are not detected. This fact suggests that hearing loss caused by mutations in OTOG might be more common than diagnosed. In the ExAC, there are 20 loss-of-function variants which includes nonsense, frameshift, and obligatory splice site mutations (Supplementary Table 2). None of them is found homozygously, their MAF range from 0.00006 to 0.00096, and the sum of their allele frequencies is 0.004446. If we assume that human population is in HardyWeinberg equilibrium and q is 0.004446, about 2 out of 100,000 (q2 = 1.98 x 10−5) will have two loss-of-function alleles, meaning that 2 out of 100,000 individuals may have mild hearing loss due to OTOG mutations. These loss-of-function variants are functionally less harmful because mutant protein resulting from these variant are longer than p.Tyr110*, which is identified in this study. With lack of human data, it has not been clear whether hearing loss caused by mutations in OTOG is congenital or not. Although the previous report presented that the subjects with mutations in OTOG were prelingual hearing loss and showed delayed speech development, only audiologic evaluations at 3.5 and more years were provided, thus it is unclear whether an early progressive hearing loss precedes the later stable phase of the hearing loss (Schraders et al., 2012). In the present study, the affected subject YUHL63-21 had a threshold data of ABR at three months of age. According to the data, YUHL63-21 had 40 dB nHL at both ears implying that mild hearing impairment might have already existed before or right after birth. Therefore, hearing loss caused by mutations in OTOG could start during the embryonic stage. With high possibility that hearing loss occurs congenitally, a mutation analysis about OTOG should be recommended if newborns have mild threshold shift in ABR. Another interesting finding in clinical evaluation was normal vestibular function in the patient with bi-allelic mutation of p.Tyr110*. In the previous report, vestibular dysfunction and delayed motor development were identified in the affected subjects with mutations in OTOG (Schraders et al., 2012). However, the affected proband (YUHL62-21)
speech audiometry, were performed. The average values of hearing thresholds (in dB) at 0.5, 1, 2, and 4 kHz were then calculated (PTA4). Vestibular function test, including bithermal caloric test (Slmed, Seoul, Korea) and rotary chair test (Neurokinetics, Pittsburgh, USA), were performed using videonystagmography as previously described (Jung et al., 2016). 3.3. Whole exome sequencing Peripheral blood obtained from affected individuals and their parents was used to performing genomic DNA (gDNA) extraction using RBC Lysis Solution, Cell Lysis Solution, and Protein Precipitation Solution (iNtRon Biotechnology, Inc). Whole exome capture was performed with the Agilent SureSelect V5 enrichment capture kit (Agilent Technologies) and the enriched library was then sequenced on the Illumina HiSeq 2500 (101 bases paired end). Image analysis and base calling were generated by the Illumina pipeline using default parameters. Sequence reads were mapped to the human reference genome assembly (NCBI build 3/hg19) using CLC Genomic Workbench (version 9.5.3) software (Quiagen). All variants with a minimum coverage of 2 were used. The variants were called using Basic Variant Caller of CLC Workbench and then annotated. Variant filtering was done as described previously (Jung et al., 2017). Variant filtering process is described in Supplementary Table 1. 4. Results We performed WES for the affect individual, YUHL62-21. WES data had 99.2%, 97.1%, and 90.4% of mappable bases represented by coverage of at least 1, 10, and 20 reads, respectively. The mean exome coverage was 67. As the pedigree revealed the fact that both parents were not affected (Fig. 1A), we first assumed that the patient would have a recessive inheritance. Therefore, we looked for variants which are homozygous or compound heterozygous in the affected individual. After filtering and inspection of WES data, a homozygous variant in OTOG, which encodes otogelin, remained. Mutations in OTOG are known to cause deafness (MIM 614945). The identified variant NM_001277269.1: c.330C > G; p.(Tyr110*) segregates with the affected status in this family; his parents and younger brother are heterozygous carriers (Table 1 and Fig. 2A). This mutation is a nonsense one which results in an early premature truncation of otogelin, resulting most likely in a null allele (Fig. 2B). 5. Discussion In this study, we performed WES to find a causative mutation of NSHL in a patient with sporadic mild hearing loss at a young age. We identified a homozygous nonsense mutation of c.330C > G (p.Tyr110*) in OTOG, which was causative for mild NSHL in autosomal Table 1 Mutation in OTOG identified in YUHL62-21 by whole exome sequencing. Gene Symbol
Individual
Sex
Nucleotide changea
Amino acid change
Exon (zygosity, segregation)
dbSNPb
ESPc
gnomADd
NBKe
MTf
CADDg
OTOG
YUHL62-21
M
c.330C > G
p.Tyr110*
4 (HOM, Mo, Fa)
rs574007567 G = 0.0002/1
ND
ND
G:0.00126
DC (1)
29.6
Abbreviations are as follows: DC, disease causing; Fa, heterozygous mutation identified in father; HOM, homozygous in affected individual; M, male; Mo, heterozygous mutation identified in mother; ND, no data available. a cDNA mutations are numbered according to human cDNA reference sequence NM_001277269.1 (OTOG); +1 corresponds to the A of ATG translation initiation codon. b dbSNP database (http://www.ncbi.nlm.nih.gov/SNP). c NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/). d gnomAD browser (http://exac.broadinstitute.org/). e National Biobank of Korea, Centers for Disease Control and Prevention, whole genome sequencing data of 398 Korean individuals. f Mutation taster (http://www.mutationtaster.org/). g Phred-like scores (scaled C scores) on Combined Annotation Dependent Depletion (http://cadd.gs.washington.edu/home/). 3
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Fig. 2. A mutation in OTOG identified by whole exome sequencing (WES). (A) A nonsense mutation in OTOG in YUHL62-21 was identified by WES. Sanger sequencing traces of exon 4 of OTOG in YUHL62-21, −11, −12, and −22. The altered base is highlighted. (B) Domain structure of OTOGELIN. The reported mutations in OTOG are shown. The new mutation identified in this study is underlined in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
doi.org/10.1016/j.ejmg.2018.05.018.
did not have any vestibular dysfunction in caloric and rotary chair tests. There are two possible explanations about the discrepant phenotype. One is that the mutation in OTOG may involve the variable degree of vestibular impairment depending on the genotypes, some of which are less susceptible and result in residual vestibular function. The second explanation is the age of the affected subject in the present study (5 years), which was too early to have vestibular impairment to the degree detected in the tests. In the previous report, the vestibular function tests were performed at age of 13, 15, 17, and 19 years, which were much later than that in the present study (Schraders et al., 2012). In this study, we identified a homozygous nonsense mutation in OTOG that causes mild AR-NSHL in a Korean child. Because mild hearing loss is not easy to be early detected, mutation of OTOG may be more prevalent than reported. Therefore, genetic evaluation about OTOG should be considered in children with mild hearing loss.
References Jung, J., Seo, Y.W., Choi, J.Y., Kim, S.H., 2016. Vestibular function is associated with residual low-frequency hearing loss in patients with bi-allelic mutations in the SLC26A4 gene. Hear. Res. 335, 33–39. Jung, J., Lee, J.S., Cho, K.J., Yu, S., Yoon, J.H., Yung Gee, H., et al., 2017. Genetic predisposition to sporadic congenital hearing loss in a pediatric population. Sci. Rep. 7, 45973. Lenz, D.R., Avraham, K.B., 2011. Hereditary hearing loss: from human mutation to mechanism. Hear. Res. 281, 3–10. McGuirt, W.T., Prasad, S.D., Griffith, A.J., Kunst, H.P., Green, G.E., Shpargel, K.B., et al., 1999. Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13). Nat. Genet. 23, 413–419. Morton, C.C., Nance, W.E., 2006. Newborn hearing screening–a silent revolution. N. Engl. J. Med. 354, 2151–2164. Oonk, A.M., Leijendeckers, J.M., Huygen, P.L., Schraders, M., del Campo, M., del Castillo, I., et al., 2014. Similar phenotypes caused by mutations in OTOG and OTOGL. Ear Hear. 35, e84–91. Schraders, M., Ruiz-Palmero, L., Kalay, E., Oostrik, J., del Castillo Francisco, J., Sezgin, O., et al., 2012. Mutations of the gene encoding otogelin are a cause of autosomalrecessive nonsyndromic moderate hearing impairment. Am. J. Hum. Genet. 91, 883–889. Simmler, M.C., Cohen-Salmon, M., El-Amraoui, A., Guillaud, L., Benichou, J.C., Petit, C., et al., 2000a. Targeted disruption of otog results in deafness and severe imbalance. Nat. Genet. 24, 139–143. Simmler, M.C., Zwaenepoel II, , Verpy, E., Guillaud, L., Elbaz, C., Petit, C., et al., 2000b. Twister mutant mice are defective for otogelin, a component specific to inner ear acellular membranes. Mamm. Genome Off. J. Int. Mamm. Genome Soc. 11, 961–966. Verhoeven, K., Van Laer, L., Kirschhofer, K., Legan, P.K., Hughes, D.C., Schatteman, I., et al., 1998. Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat. Genet. 19, 60–62. Zheng, J., Miller, K.K., Yang, T., Hildebrand, M.S., Shearer, A.E., DeLuca, A.P., et al., 2011. Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4). Proc. Natl. Acad. Sci. U.S.A. 108, 4218–4223.
Disclosure statement The authors declare that they have no competing interests. Acknowledgement This study was provided with bioresources from the National Biobank of Korea, Centers for Disease Control and Prevention, Republic of Korea (4845-301, 4851-302 and -307). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2015R1D1A1A01056685 to H.Y.G.) and by the Korea government (MSIP) (2016R1A2B4007268 to C.J.Y.) and Ministry of Health & Welfare, Republic of Korea (HI16C0142 to J.J.). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.
4
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: www.elsevier.com/locate/ejmg
A novel early truncation mutation in OTOG causes prelingual mild hearing loss without vestibular dysfunction Seyoung Yua,1, Hye Ji Choib,1, Joon Suk Leea, Hyun Jae Leeb, John Hoon Rima, Jae Young Choib, Heon Yung Geea,∗∗, Jinsei Jungb,∗ a b
Department of Pharmacology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea Department of Otorhinolaryngology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, 03722, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: OTOG Otogelin DFNB18 Prelingual Mild hearing loss
OTOG was identified as a nonsyndrmoic hearing loss gene in 2012 in two families with nonprogressive mild-tomoderate hearing loss. However, no further literature have this gene for nonsyndromic hearing loss. Furthermore, it is still unclear whether vestibular impairment is involved or not in patients with mutations in OTOG. This study presents a validated second report for homozygous causative mutations in OTOG of mild hearing loss. Whole exome sequencing (WES) was performed in a five-year-old male proband with mild hearing loss. The analysis of WES revealed a homozygous truncating mutation (c.330C > G; p.Tyr110*) in OTOG. The identified novel mutation, p.Tyr110*, leads to a null allele based on the fact that early truncated protein contains no functional domain of otogelin. While defects in otogelin previously reported to result in hearing loss and vestibular dysfunction, p.Tyr110* only caused nonsydromic and nonprogressive hearing loss without any vestibular impairment, indicating that vestibular phenotype would be variable. Given that mild hearing loss is not easy to be detected early, mutations of OTOG may be more prevalent than reported. Therefore, genetic evaluation for OTOG should be considered in children with mild hearing loss with/without vestibular dysfunction.
1. Introduction Congenital hearing loss is one of the most prevalent sensorial disorders, present in one of 500–1000 children. It is estimated that genetic factors contribute to about half cases of congenital hearing loss and about 50% of inherited cases are non-syndromic hearing loss (NSHL) (Lenz and Avraham, 2011). Of the more than 100 genes (> 140 loci) associated with NSHL, approximately 70% contribute to autosomal recessive pattern of inheritance (http://hereditaryhearingloss.org/) (Morton and Nance, 2006,Lenz and Avraham, 2011). Most of autosomal recessive non-syndromic hearing loss (AR-NSHL) is prelingual and severe-to-profound sensorineural hearing loss. However, several mutations on OTOG or OTOGL are associated with AR-NSHL with mild-tomoderate sensorineural hearing loss (Oonk et al., 2014). OTOG encodes otogelin protein, a noncollagenous component of the acellular gelatinous structures that cover the sensory epithelia of the inner ear; the tectorial membrane in cochlea, the otoconial membranes in the utricle and saccule, and the cupulae that cover the cristae
amullares of the semicircular canals in the vestibular organ (Simmler et al., 2000a). Otogelin is one of three major components in tectorial membrane besides alpha tectorin and beta tectorin (Schraders et al., 2012). Mutations in OTOG are known to cause DFNB18B (MIM 614945). To date, three causative mutations in OTOG have been found in two patients with DFNB18B: 1) a homozygous 1bp deletion, c.5508delC (p.Ala1838Profs*31) which leads to a frame shift and premature stop codon and 2) compound-heterozygous mutations: c.6347C > T (p.Pro2116Leu) and c.6559C > T (p.Arg2187*) (Schraders et al., 2012). Moderate hearing impairment with a U-shaped to flat audiogram is the most common type of hearing impairment associated with defects in components of the tectorial membrane and this was also observed in the affected individuals who had a homozygous OTOG mutation (Schraders et al., 2012). On the contrary, the affected individuals who have compound-heterozygous mutations had slightly down-sloping shaped audiograms (Schraders et al., 2012). In addition, most of affected individuals showed a delayed speech development which suggest a prelingual onset of hearing impairment (Schraders
∗
Corresponding author. Department of Otorhinolaryngology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, Republic of Korea. Corresponding author. Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752, Republic of Korea. E-mail addresses: [email protected] (H.Y. Gee), [email protected] (J. Jung). 1 These authors contributed equally to this work. ∗∗
https://doi.org/10.1016/j.ejmg.2018.05.018 Received 12 November 2017; Received in revised form 9 May 2018; Accepted 21 May 2018 1769-7212/ © 2018 Elsevier Masson SAS. All rights reserved.
Please cite this article as: Yu, S., European Journal of Medical Genetics (2018), https://doi.org/10.1016/j.ejmg.2018.05.018
European Journal of Medical Genetics xxx (xxxx) xxx–xxx
S. Yu et al.
Fig. 1. Pedigree and clnical phenotypes of YUHL62. (A) Family pedigree of YUHL62 with sporadic congenital hearing loss. (B) audiograms of subjects in YUHL62. (C) Ear drum finding and Temporal bone computed tomogram of YUHL62-21. (D) Vestibular function tests (upper: caloric test, lower: rotary chair test) were performed in YUHL62-21. Caloric test vis, visual; supp, suppression; enh, enhancement.
development. In vestibular function test, the proband (YUHL62-21) showed normoreflexia in caloric test as well as rotary chair test (with slow harmonic acceleration) (Fig. 1D). In bithermic caloric test, canal paresis was 3.5% at the left ear (normal range ≤ 25%) and directional predonderance was 5.3% at the right ear (normal range ≤ 30%), which were within normal limits. In slow harmonic acceleration test, the gains of vestibulo-ocular reflex were normal without phase lead or lag at the frequencies of stimuli from 0.08 to 1.28 Hz. According to the pedigree, the affected individual was only YUHL62-21 and sporadic or autosomal recessive pattern of hearing loss was suspicious.
et al., 2012). Here, we report a Korean individual who exhibit early-onset mild NSHL. We performed whole exome sequencing (WES) and identified a nonsense mutation (hg19; chr11:17,514,667A > G) in OTOG. Our study suggests that genetic analysis of early-onset mild NSHL should include OTOG as a possible causative gene. 2. Clinical report The affected proband YUHL62-21 was five-year-old male (Fig. 1A). He did not have any medical history or syndromic features. He failed the newborn hearing screening with automated auditory brainstem response. At three months of age, the threshold of auditory brainstem response was 40 dB nHL at both ears (Supplementary Fig. 1). However, since there was no delay in speech development, his parents (YUHL6211 and −12) did not have further evaluation about the hearing impairment. At 5 years of age, YUHL62-21 individual visited tertiary medical center for the reason of hearing difficulty. He had mild sensorineural hearing loss with decreased threshold in pure-tone audiometry (30 and 41 dB HL at the right and left ear, respectively). The audiogram was relatively flat pattern (Fig. 1B). However, there was no sign for middle ear anomaly or infection in otoscopic finding and temporal bone computed tomogram (Fig. 1C). Word recognition score at the most comfortable level was 94% at both ears. His parents (YUHL62-11; male/36 years-old, YUHL62-12; female/35 years-old, and) had no definite hearing impairment (Fig. 1B) and his younger brother (YUHL62-22; male/2 years-old) passed neonatal hearing screening with automated auditory brainstem response. The affected proband did not have any vertigo symptoms and showed normal motor
3. Methods 3.1. Subjects This study was approved by the institutional review board of the Severance Hospital, Yonsei University Health System (IRB#4-20150659). After obtaining written informed consent from subjects or their parents, individuals with hearing loss were enrolled in the Yonsei University Hearing Loss (YUHL) cohort, and their clinical and pedigree data were recorded. The proband (−21) of YUHL62 family exhibited no other syndromic features except sensorineural hearing loss. All the subjects in YUHL62 did not have any history of non-inherited risk factors for hearing loss, such as perinatal viral infection, neonatal intensive care unit treatment, and middle ear infection. 3.2. Audiological and vestibular analyses Audiological evaluations, including pure-tone audiometry and 2
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recessive pattern. Given that the full length of otogelin consists of 2925 amino acids, the early truncated mutant otogelin (p.Tyr110*) may have complete loss-of-function and a homozygous mutation of OTOG was previously shown to be associated with AR-NSHL (Schraders et al., 2012). To date, there has been only one report that mild-to-moderate hearing loss was attributable to the mutations of OTOG in human (Schraders et al., 2012). Like other genes such as TECTA, COLL11A2, and OTOGL coding for the proteins of tectorial membrane, the mutations in OTOG showed early onset and non-progressive AR-NSHL (Verhoeven et al., 1998,McGuirt et al., 1999,Zheng et al., 2011). The patients with mutations in OTOG did not have severe-to-profound hearing deterioration. This finding is consistent with the fact that the pathogenesis of defect in otogelin does not involve in anchoring the tectorial membrane to the hair cells but just increase the resistance of the tectorial membrane (Simmler et al., 2000b). It is likely that cases of mild hearing impairment are not detected. This fact suggests that hearing loss caused by mutations in OTOG might be more common than diagnosed. In the ExAC, there are 20 loss-of-function variants which includes nonsense, frameshift, and obligatory splice site mutations (Supplementary Table 2). None of them is found homozygously, their MAF range from 0.00006 to 0.00096, and the sum of their allele frequencies is 0.004446. If we assume that human population is in HardyWeinberg equilibrium and q is 0.004446, about 2 out of 100,000 (q2 = 1.98 x 10−5) will have two loss-of-function alleles, meaning that 2 out of 100,000 individuals may have mild hearing loss due to OTOG mutations. These loss-of-function variants are functionally less harmful because mutant protein resulting from these variant are longer than p.Tyr110*, which is identified in this study. With lack of human data, it has not been clear whether hearing loss caused by mutations in OTOG is congenital or not. Although the previous report presented that the subjects with mutations in OTOG were prelingual hearing loss and showed delayed speech development, only audiologic evaluations at 3.5 and more years were provided, thus it is unclear whether an early progressive hearing loss precedes the later stable phase of the hearing loss (Schraders et al., 2012). In the present study, the affected subject YUHL63-21 had a threshold data of ABR at three months of age. According to the data, YUHL63-21 had 40 dB nHL at both ears implying that mild hearing impairment might have already existed before or right after birth. Therefore, hearing loss caused by mutations in OTOG could start during the embryonic stage. With high possibility that hearing loss occurs congenitally, a mutation analysis about OTOG should be recommended if newborns have mild threshold shift in ABR. Another interesting finding in clinical evaluation was normal vestibular function in the patient with bi-allelic mutation of p.Tyr110*. In the previous report, vestibular dysfunction and delayed motor development were identified in the affected subjects with mutations in OTOG (Schraders et al., 2012). However, the affected proband (YUHL62-21)
speech audiometry, were performed. The average values of hearing thresholds (in dB) at 0.5, 1, 2, and 4 kHz were then calculated (PTA4). Vestibular function test, including bithermal caloric test (Slmed, Seoul, Korea) and rotary chair test (Neurokinetics, Pittsburgh, USA), were performed using videonystagmography as previously described (Jung et al., 2016). 3.3. Whole exome sequencing Peripheral blood obtained from affected individuals and their parents was used to performing genomic DNA (gDNA) extraction using RBC Lysis Solution, Cell Lysis Solution, and Protein Precipitation Solution (iNtRon Biotechnology, Inc). Whole exome capture was performed with the Agilent SureSelect V5 enrichment capture kit (Agilent Technologies) and the enriched library was then sequenced on the Illumina HiSeq 2500 (101 bases paired end). Image analysis and base calling were generated by the Illumina pipeline using default parameters. Sequence reads were mapped to the human reference genome assembly (NCBI build 3/hg19) using CLC Genomic Workbench (version 9.5.3) software (Quiagen). All variants with a minimum coverage of 2 were used. The variants were called using Basic Variant Caller of CLC Workbench and then annotated. Variant filtering was done as described previously (Jung et al., 2017). Variant filtering process is described in Supplementary Table 1. 4. Results We performed WES for the affect individual, YUHL62-21. WES data had 99.2%, 97.1%, and 90.4% of mappable bases represented by coverage of at least 1, 10, and 20 reads, respectively. The mean exome coverage was 67. As the pedigree revealed the fact that both parents were not affected (Fig. 1A), we first assumed that the patient would have a recessive inheritance. Therefore, we looked for variants which are homozygous or compound heterozygous in the affected individual. After filtering and inspection of WES data, a homozygous variant in OTOG, which encodes otogelin, remained. Mutations in OTOG are known to cause deafness (MIM 614945). The identified variant NM_001277269.1: c.330C > G; p.(Tyr110*) segregates with the affected status in this family; his parents and younger brother are heterozygous carriers (Table 1 and Fig. 2A). This mutation is a nonsense one which results in an early premature truncation of otogelin, resulting most likely in a null allele (Fig. 2B). 5. Discussion In this study, we performed WES to find a causative mutation of NSHL in a patient with sporadic mild hearing loss at a young age. We identified a homozygous nonsense mutation of c.330C > G (p.Tyr110*) in OTOG, which was causative for mild NSHL in autosomal Table 1 Mutation in OTOG identified in YUHL62-21 by whole exome sequencing. Gene Symbol
Individual
Sex
Nucleotide changea
Amino acid change
Exon (zygosity, segregation)
dbSNPb
ESPc
gnomADd
NBKe
MTf
CADDg
OTOG
YUHL62-21
M
c.330C > G
p.Tyr110*
4 (HOM, Mo, Fa)
rs574007567 G = 0.0002/1
ND
ND
G:0.00126
DC (1)
29.6
Abbreviations are as follows: DC, disease causing; Fa, heterozygous mutation identified in father; HOM, homozygous in affected individual; M, male; Mo, heterozygous mutation identified in mother; ND, no data available. a cDNA mutations are numbered according to human cDNA reference sequence NM_001277269.1 (OTOG); +1 corresponds to the A of ATG translation initiation codon. b dbSNP database (http://www.ncbi.nlm.nih.gov/SNP). c NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/). d gnomAD browser (http://exac.broadinstitute.org/). e National Biobank of Korea, Centers for Disease Control and Prevention, whole genome sequencing data of 398 Korean individuals. f Mutation taster (http://www.mutationtaster.org/). g Phred-like scores (scaled C scores) on Combined Annotation Dependent Depletion (http://cadd.gs.washington.edu/home/). 3
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Fig. 2. A mutation in OTOG identified by whole exome sequencing (WES). (A) A nonsense mutation in OTOG in YUHL62-21 was identified by WES. Sanger sequencing traces of exon 4 of OTOG in YUHL62-21, −11, −12, and −22. The altered base is highlighted. (B) Domain structure of OTOGELIN. The reported mutations in OTOG are shown. The new mutation identified in this study is underlined in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
doi.org/10.1016/j.ejmg.2018.05.018.
did not have any vestibular dysfunction in caloric and rotary chair tests. There are two possible explanations about the discrepant phenotype. One is that the mutation in OTOG may involve the variable degree of vestibular impairment depending on the genotypes, some of which are less susceptible and result in residual vestibular function. The second explanation is the age of the affected subject in the present study (5 years), which was too early to have vestibular impairment to the degree detected in the tests. In the previous report, the vestibular function tests were performed at age of 13, 15, 17, and 19 years, which were much later than that in the present study (Schraders et al., 2012). In this study, we identified a homozygous nonsense mutation in OTOG that causes mild AR-NSHL in a Korean child. Because mild hearing loss is not easy to be early detected, mutation of OTOG may be more prevalent than reported. Therefore, genetic evaluation about OTOG should be considered in children with mild hearing loss.
References Jung, J., Seo, Y.W., Choi, J.Y., Kim, S.H., 2016. Vestibular function is associated with residual low-frequency hearing loss in patients with bi-allelic mutations in the SLC26A4 gene. Hear. Res. 335, 33–39. Jung, J., Lee, J.S., Cho, K.J., Yu, S., Yoon, J.H., Yung Gee, H., et al., 2017. Genetic predisposition to sporadic congenital hearing loss in a pediatric population. Sci. Rep. 7, 45973. Lenz, D.R., Avraham, K.B., 2011. Hereditary hearing loss: from human mutation to mechanism. Hear. Res. 281, 3–10. McGuirt, W.T., Prasad, S.D., Griffith, A.J., Kunst, H.P., Green, G.E., Shpargel, K.B., et al., 1999. Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13). Nat. Genet. 23, 413–419. Morton, C.C., Nance, W.E., 2006. Newborn hearing screening–a silent revolution. N. Engl. J. Med. 354, 2151–2164. Oonk, A.M., Leijendeckers, J.M., Huygen, P.L., Schraders, M., del Campo, M., del Castillo, I., et al., 2014. Similar phenotypes caused by mutations in OTOG and OTOGL. Ear Hear. 35, e84–91. Schraders, M., Ruiz-Palmero, L., Kalay, E., Oostrik, J., del Castillo Francisco, J., Sezgin, O., et al., 2012. Mutations of the gene encoding otogelin are a cause of autosomalrecessive nonsyndromic moderate hearing impairment. Am. J. Hum. Genet. 91, 883–889. Simmler, M.C., Cohen-Salmon, M., El-Amraoui, A., Guillaud, L., Benichou, J.C., Petit, C., et al., 2000a. Targeted disruption of otog results in deafness and severe imbalance. Nat. Genet. 24, 139–143. Simmler, M.C., Zwaenepoel II, , Verpy, E., Guillaud, L., Elbaz, C., Petit, C., et al., 2000b. Twister mutant mice are defective for otogelin, a component specific to inner ear acellular membranes. Mamm. Genome Off. J. Int. Mamm. Genome Soc. 11, 961–966. Verhoeven, K., Van Laer, L., Kirschhofer, K., Legan, P.K., Hughes, D.C., Schatteman, I., et al., 1998. Mutations in the human alpha-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nat. Genet. 19, 60–62. Zheng, J., Miller, K.K., Yang, T., Hildebrand, M.S., Shearer, A.E., DeLuca, A.P., et al., 2011. Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4). Proc. Natl. Acad. Sci. U.S.A. 108, 4218–4223.
Disclosure statement The authors declare that they have no competing interests. Acknowledgement This study was provided with bioresources from the National Biobank of Korea, Centers for Disease Control and Prevention, Republic of Korea (4845-301, 4851-302 and -307). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2015R1D1A1A01056685 to H.Y.G.) and by the Korea government (MSIP) (2016R1A2B4007268 to C.J.Y.) and Ministry of Health & Welfare, Republic of Korea (HI16C0142 to J.J.). Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.
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