- Email: [email protected]
A novel mutation in the MYO7A gene is associated with Usher syndrome type 1 in a Chinese family
Accepted Manuscript A novel mutation in the MYO7A gene is associated with Usher syndrome type 1 in a Chinese family Xiaoguang He, Qi Peng, Siping Li, Pengyuan Zhu, Chunqiu Wu, Chunbao Rao, Jingqi Lin, Xiaomei Lu PII:
S0165-5876(17)30240-9
DOI:
10.1016/j.ijporl.2017.05.021
Reference:
PEDOT 8555
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 13 March 2017 Revised Date:
24 May 2017
Accepted Date: 25 May 2017
Please cite this article as: X. He, Q. Peng, S. Li, P. Zhu, C. Wu, C. Rao, J. Lin, X. Lu, A novel mutation in the MYO7A gene is associated with Usher syndrome type 1 in a Chinese family, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.05.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel mutation in the MYO7A gene is associated with Usher Syndrome type 1 in a Chinese family Running title: A novel mutation in MYO7A associated with USH1 2¶
, Siping Li2, Pengyuan Zhu3, Chunqiu Wu3, Chunbao
RI PT
Xiaoguang He1, 2¶, Qi Peng
Rao2, Jingqi Lin4, Xiaomei Lu1,2*
1. Department of neonates, Dongguan Children's Hospital, Dongguan, Guangdong,
SC
China
Dongguan, Guangdong, China
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2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
3. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China 4. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan,
¶
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Guangdong, China
These authors contributed equally to this work.
*Corresponding author: Department of Medical and Molecular Genetics, Dongguan
EP
Institute of Pediatrics, Dongguan, Guangdong, China Email：[email protected]
AC C
Tel: +86 86186505
ACCEPTED MANUSCRIPT A novel mutation in the MYO7A gene is associated with Usher Syndrome type 1 in a Chinese family Xiaoguang He1,2¶ , Qi Peng 2¶, Siping Li2 , Pengyuan Zhu3 , Chunqiu Wu3, Chunbao
RI PT
Rao2, Jingqi Lin4, Xiaomei Lu1,2* 1. Department of neonates, Dongguan Children's Hospital, Dongguan, Guangdong, China
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2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
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Dongguan, Guangdong, China
3. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China 4. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan, Guangdong, China
These authors contributed equally to this work.
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¶
*Corresponding author: Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, Guangdong, China Email：[email protected]
EP
Tel: +86 86186505
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Running title: A novel mutation in MYO7A associated with USH1 Funding sources: Supported by the Dongguan Bureau of Science and Technology for the City Key Program of Science and Technology (Project Number: 2013108101018) and the Science and Technology Planning Project of Guangdong Province (Project Number: 2014A020213001).
ACCEPTED MANUSCRIPT Abstract Objectives: We aimed to investigate the genetic causes of hearing loss in a Chinese proband with autosomal recessive congenital deafness. Methods: The targeted
RI PT
capture of 159 known deafness genes and next-generation sequencing were performed to study the genetic causes of hearing loss in the Chinese family. Sanger sequencing was employed to verify the variant mutations in members of this family. Results: The
SC
proband harbored two mutations in the MYO7A gene in the form of compound
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heterozygosity. She was found to be heterozygous for a novel insertion mutation c.3847_3848 ins TCTG (p.N1285LfsX24) in exon 30 and for the known mutation c.2239_2240delAG (p.R747S fsX16）in exon 19. The novel mutation was absent in the 1000 Genomes Project. These variants were carried in the heterozygous state by
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the parents and were therefore co-segregated with the genetic disease. Clinical re-assessment, including detailed audiologic and ocular examinations, revealed congenital deafness and retinitis pigmentosa in the proband. Collectively, the
EP
combination of audiometric, ophthalmologic and genetic examinations successfully
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confirmed the phenotype of Usher syndrome type 1 (USH1). Conclusion: This study demonstrates that the novel mutation c.3847_3848insTCTG (p. N1285LfsX24) in compound heterozygosity with c.2239_2240delAG in the MYO7A gene is the main cause of USH1 in the proband. Our study expands the mutational spectrum of MYO7A and provides a foundation for further investigations elucidating the MYO7A-related mechanisms of USH1. Key words: Usher syndrome; hearing loss; MYO7A; novel mutation
ACCEPTED MANUSCRIPT 1.Introduction Hearing loss is the most common sensory impairment and severely affects patients’ daily quality of life. It is etiologically heterogeneous, and it is estimated that
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at least two thirds of all cases of childhood-onset hearing loss have genetic causes[1, 2]. To date, more than 150 genes have been reported to be asssociated with deafness (http://deafnessvariationdatabase.org/). Some of those genes might result in distinct
SC
phenotypes other than hearing loss due to the genetic diversity, which can result in
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syndromic hearing loss[3]. The myosin VIIA (MYO7A) gene is one such gene and is associated with both syndromic and nonsyndromic sensorineural hearing loss. The MYO7A gene is located on chromosome 11q13.5 and encodes the actin-binding motor protein myosin VIIa[4]. The biggest transcript of MYO7A consists of 49 exons
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and encodes a 2,215-amino acid unconventional myosin named myosin VIIA. It plays an important role in intracellular transport, endocytosis, and intercellular adhesion[5]. Mutations in the MYO7A gene have been reported to be associated with a wide
EP
phenotypic spectrum, including congenital hearing impairment[6]. It is commonly
AC C
noted that MYO7A mutations are associated with Usher syndrome type 1 (USH1), an autosomal recessive group of symptoms characterized by pre-pubertal onset retinitis pigmentosa (RP), sensorineural hearing impairment and vestibular dysfunction[7,8]. Moreover, variants in the MYO7A gene have been linked to autosomal dominant non-syndromic hearing
loss
(DFNA11)
and
autosomal
recessive
deafness
(DFNB2)[9,10]. In this study, we performed gene capture and targeted next generation sequencing (NGS) for 159 deafness associated genes based on deafness
ACCEPTED MANUSCRIPT variation database （http://deafnessvariationdatabase.org/）in a Chinese proband who was diagnosed with non-syndromic hearing loss but was negative in a panel of 20 common deafness-associated mutations. Interestingly, we identified two variants in
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MYO7A in this family. Clinical re-assessment revealed congenital deafness and retinitis pigmentosa in the proband. Collectively, the combination of audiometric,
USH1, but not NSHL, in this family.
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2. Patients and methods
SC
ophthalmologic and genetic examinations successfully confirmed the phenotype of
2.1 Patients
The proband, a 9-year-old girl with congenital deafness and her unaffected parents and brothers were recruited to participate in this study. All of them came from
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Guangdong Province and are part of the southern Han Chinese population. A clinical evaluation of the family was conducted, including a description of family history and detailed medical history and a physical examination. Audiometric
EP
tests covering otoscope examination, pure-tone audiometry (PTA), tympanometry
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audiometry, acoustic stapedial reflex, auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAEs) were performed. The audiological data were evaluated based on hearing impairment criteria established by the United States Joint Committee on Infant Hearing (JCIH).The degree of hearing loss was classified as mild (30–40 dB HL), moderate (41–70 dB HL), severe (71–90 dB HL) or profound (> 90 dB HL)[11].
After compound heterozygous mutations in the MYO7A
gene were found, detailed ophthalmologic examinations of the subjects, including
ACCEPTED MANUSCRIPT best-corrected visual acuities (BCVAs) measurements, slit-lamp biomicroscopy, intraocular pressure (IOP) tests, funduscopy, and visual field (VF) tests, were performed.
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This study was approved by and conducted in accordance with the protocol of the Institutional Medical and Ethics Committee of Dongguan Children's Hospital, and it conformed to the tenets of the Declaration of Helsinki. Written informed consent was
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2.2 Targeted deafness gene capturing and NGS
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obtained from the parents or legal guardians of the subjects.
Genomic DNA was extracted from peripheral blood samples of the subjects using the Blood DNA Kit (TIANGEN BIOTECH, Beijing, China) following the manufacturer's protocol. DNA yield and quality were determined using a NanoDrop
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8000 ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and 1% agarose gel electrophoresis was performed according to routine methods.
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Twenty hotspot mutations of four common deafness-associated genes (GJB2,
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GJB3, SLC26A4, and MTRNR1) were first screened by matrix-assisted laser desorption ionization time of flight-mass spectrometry (MALDI-TOF-MS). Once the screening results were negative, the genomic DNA was used to prepare a library with the SureSelectXT Reagent kit from Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA; Cat. no. G9611A). All exons and some adjacent flanking intronic sequencing of 159 known deafness genes were captured by a custom-designed array (Agilent Technologies Santa Clara, CA, USA). Sequencing
ACCEPTED MANUSCRIPT was performed using a BES 4000 sequencer with single-end 160-bp reads. After sequencing, TMAP and Torrent Variant Caller (TVC) were used to map to HG19 for variant call analysis.
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2.3 Sanger sequencing Potential mutations, which were detected by target gene capture and next generation sequencing, were verified by PCR amplification and Sanger sequencing.
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The following sequence-specific primers were synthesized: 5’-CAACTGAGTTC
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TTGACCTGTGCT-3’ and 5’-TGTGTGCACTT GTTTCATAGGTG-3’ for human MYO7A c.2239_2240delAG; 5’-CAGAGAGCCAAAGTCC AGAGG-3’ and 5’-CG AGGTGCTCAAGGGTGAATAG-3’ for human MYO7A c.3847_3848 insTCTG (p. L1283LfsX27). PCR was performed in a 50 µl reaction mixture; the PCR products
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were purified with a PCR purification kit (Takara, Japan) and sequenced using an ABI 3100 DNA sequencing machine (ABI, Foster City, CA, USA) and the ABI 3100 Analysis Software (ver. 3.7 NT).
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2.3 Bioinformatics and validation of the variants
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Sequence data were analyzed by alignment with the National Center for Biotechnology Information (NCBI) reference sequence of MYO7A using the DNA Star 5.0 software. The 1000 Genomes Project database (http://www.1000genomes.org/) and the dbSNP database of the NCBI (http://www.ncbi.nlm.nih.gov/) were used as references to assess the novelty of mutations found in this study. The online tool MutationTaster was used to predict the possible pathogenicity. 3. Results
ACCEPTED MANUSCRIPT 3.1. Clinical features A hearing-impaired subject and her normal-hearing parents and two brothers were enrolled in our study (Figure 1 A). The affected girl was born after a normal pregnancy and delivery. Deafness was noted and confirmed in the first few months of
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her life. Hearing loss was identified as stable and congenital. PTA of the proband showed symmetrical bilateral and profound sensorineural hearing loss in all frequencies (Figure 1 B). The audiograms of her parents and two brothers were normal. Auditory brainstem response did not show any evoked response at or below
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100 dB on both sides of the proband. She received a cochlear implant (CI) at the age of six years old. She had balance problems before cochlear implants, such as she
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started to walk at the age of 20 months, which is later than the normal children. However, her language function was developed now because she wore hearing aids for
half
a
year
before
cochlear
implant
and
also
went
to
the
special educational institutions from 3 years old to improve her language ability.
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Her parents or medical staff did not notice the eye problem of the proband before mutations in MYO7A gene were found and detailed ophthalmologic examinations performed. Through inquiry, we learned that the proband had difficulty with night
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vision. Her current BCVA was 0.6 in the left eye and 0.3 in the right eye. Fundus
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photography revealed atrophy of the retinal pigment epithelia, attenuated retinal blood vessels and a characteristic bone spicule-like pigment. Her macular region was relatively preserved according to fundus appearance results, and there was concentric narrowing of her field of vision. The anterior segment and intraocular pressure were both normal. No abnormal results were identified in the unaffected parents and two brothers. 3.2. Genetic analysis
ACCEPTED MANUSCRIPT The sequence analysis of SLC26A4 indicated that the proband presented a novel insertion mutation c.3847_3848 insTCTG (p. N1285LfsX24) in exon 30 and a known mutation c.2239_ 2240delAG in exon 19. Additionally, her mother and big brother
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were heterozygous carriers of the c.2239_ 2240del AG mutation, and her father was a heterozygous carrier of the c.3847_3848 insTCTG (p. N1285LfsX24) mutation (Figure 2). The reported mutation c.2239_2240delAG, occurring within the tail
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domain (IQ1 region) of the MYO7A protein, results in a premature termination of
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translation at amino acid 762 (p. R747SfsX16). In addition, the c.3847_3848 insTCTG (p. N1285LfsX24) mutation, which has not been previously reported, leads to a truncated protein with termination at amino acid 1308 (Figure 3). This novel mutation was absent in the 1000 Genomes Project and was predicted to be “disease
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causing” by MutationTaster, which strongly suggests that this novel mutation is closely associated with USH1. 4. Discussion
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In this Chinese family, we revealed two deleterious compound heterozygous
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mutations in the MYO7A gene: a novel insertion mutation c.3847_3848 insTCTG (p. N1285LfsX24) and
a reported deletion
mutation c.2239_2240delAG. The
c.3847_3848 insTCTG (p. N1285LfsX24) mutation has not been previously described in hearing loss patients. Through gene mutation analysis, we further confirmed that the compound heterozygous mutations in the MYO7A gene might be pathogenic because the two detected variants both cause a frameshift mutation resulting in a premature stop-codon and in a predicted truncated protein that could be
ACCEPTED MANUSCRIPT non-functional. The compound heterozygous variants co-segregated with their clinical phenotypes. In brief, this proband had symmetrical bilateral and profound sensorineural hearing loss in all frequencies and retinitis pigmentosa, which were
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consistent with the phenotype reported for USH1. These results indicate that the compound heterozygous mutations are likely the genetic cause of USH1 in this family.
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The human myosin VIIA protein contains a N-terminal motor domain (amino
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acids 1–729), a five IQ motif neck region (amino acids 745–857), a predicted short coiled coil domain (amino acids 858–935), a MyTH4 domain (amino acids 1017–1253), a 4.1-Ezrin-radixin-moesin (FERM) domain (amino acids 1258–1602), an SH3 domain (amino acids 1603–1672), and a second C-terminal MyTH4-FERM
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tandem domain (amino acids 1747–2205)[12]. Expression of the myosin VIIa protein in the cochlear hair cells, retinal photoreceptors, and retinal pigment epithelium has been detected[13]. In the hair cells of the inner ear, myosin VIIA interacts with other
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proteins to participate in the formation of the mechanotransduction complex, which is
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critical for detecting sound[14]. In the retinal pigment epithelium cells, myosin VIIA participates in the light cycle-dependent movement of melanosome transportation[15] and the normal functioning of the visual retinoid cycle[16]. In the photoreceptor cells, myosin VIIA is present at the connecting cilium and regulates opsin transport[13]. The C-terminal tail of myosin VIIA, which contains the SH3, MyTH4, and FERM domains (1605–2215), interacts with harmonin (USH1C)[17]. Both of the truncated mutant myosin VIIA proteins in this study lack the C-terminal tail and may thus lose
ACCEPTED MANUSCRIPT the ability to bind harmonin, which acts as a scaffold protein linking the proteins in the cell membrane to the proteins in the cytoskeleton. The additive effects of the two identified compound heterozygous mutations could cause a complete dysfunction of
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myosin VIIA in the proband, leading to the observed phenotype. The proband did not display any vestibular dysfunction, but we cannot rule out the possibility of dysfunction developing later in her life.
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In conclusion, we described a patient with USH1 carrying biallelic mutations
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(c.3847_3848 insTCTG (p. N1285LfsX24)/c.2239_2240delAG) in the MYO7A gene. This finding of a novel mutation further expands the spectrum of known MYO7A mutations in the Chinese population. In future studies, we will focus on the construction of a transgenic mouse model carrying these mutations to reveal the
Conflicts of interest
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mechanism of hearing impairment.
We declare that we have no conflicts of interest.
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Acknowledgments
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This work was supported by the Dongguan Bureau of Science and Technology for the City Key Program of Science and Technology (Project Number: 2013108101018) and the Science and Technology Planning Project of Guangdong Province (Project Number: 2014A020213001). We also thank all the children and their parents for their cooperation during this work.
ACCEPTED MANUSCRIPT References 1.Petit C. Genes responsible for human hereditary deafness: symphony of a thousand. Nat Genet 1996;14:385-91.
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2.Willems PJ. Genetic Causes of Hearing Loss. N Engl J Med 2000;342:1101-9. 3.Ma Y, Xiao Y, Zhang F, Han Y et al. Novel compound heterozygous mutations in MYO7A gene associated with autosomal recessive sensorineural hearing loss in a Chinese family. Int J Pediatr
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Otorhinolaryngol 2016;83:179-85.
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4.Gibbs D, Diemer T, Khanobdee K, Hu J, Bok D, Williams DS. Function of MYO7A in the human RPE and the validity of shaker1 mice as a model for Usher Syndrome 1B. Invest Ophthalmol Vis Sci. 2010;51:1130-5.
5.Wu L, Pan L, Wei Z, Zhang M. Structure of MyTH4-FERM domains in myosin VIIa tail bound to
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cargo. Science 2011;331:757-60.
6.Zong L, Chen K, Wu X, Liu M, Jiang H. Compound heterozygous MYO7A mutations segregating Usher syndrome type 2 in a Han family. Int J Pediatr Otorhinolaryngol 2016;90:150-155.
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2010;55:327-35.
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7.Yan D, Liu XZ. Genetics and pathological mechanisms of Usher syndrome. J Hum Genet
8.Yoshimura H, Iwasaki S, Nishio S-y et al. Massively parallel DNA sequencing facilitates diagnosis of patients with usher syndrome type 1. PLoS ONE 2014;9:e90688. 9.Liu XZ, Walsh J, Mburu P et al. Mutations in the myosin VIIA gene cause non-syndromic recessive deafness. Nat Genet 1997;16:188-90. 10.Liu XZ, Walsh J, Tamagawa Y et al. Autosomal dominant non-syndromic deafness caused by a mutation in the myosin VIIA gene. Nat Genet 1997;17:268-9.
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11. Joint Committee on Infant Hearing. Year 2007 Position Statement: Principles and guidelines for early hearing detection and intervention programs. Pediatrics 2007; 120: 898-921.12.Inoue A, Ikebe M.
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Characterization of the motor activity of mammalian myosin VIIA. J Biol Chem 2003;278:5478-87. 13.Liu X, Udovichenko IP, Brown SDM, Steel KP, Williams DS. Myosin VIIa participates in opsin transport through the photoreceptor cilium. J Neurosci 1999;19:6267.
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14.Grati Mh, Kachar B. Myosin VIIa and sans localization at stereocilia upper tip-link density
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implicates these Usher syndrome proteins in mechanotransduction. Proc Natl Acad Sci USA 2011;108:11476-81.
15.El-Amraoui A, Schonn JS, Küssel-Andermann P et al. Myrip, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes. EMBO Rep 2002;3:463-70.
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16.Lopes VS, Gibbs D, Libby RT et al. The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65. Hum Mol Genet 2011;20:2560-70.
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17.Boëda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, Blanchard S, et al. Myosin VIIa,
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harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. EMBO J 2002;21:6689-99.
ACCEPTED MANUSCRIPT Figure Legends Fig. 1.(A) Pedigree of the family. Arrow indicates the proband. (B) PTA results of the proband and her family members.
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Fig. 2. Mutational analysis in the Chinese family. The DNA sequencing profile shows the c.3847_3848 insTCTG (p. N1285LfsX24) and c.2239_2240delAG mutations in MYO7A. The compound heterozygous variants co-segregated with the clinical
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phenotype.
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Fig 3. The domain structure of myosin VIIa showing the mutations. The reported mutation c.2239_2240delAG, occurring within the tail domain (IQ1 region) of the MYO7A protein, results in a premature termination of translation at amino acid 762 (p. R747SfsX16). The insertion mutation c.3847_3848 insTCTG introduced a premature
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termination codon (p. N1285LfsX24), which was predicted to truncate the protein.
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ACCEPTED MANUSCRIPT
S0165-5876(17)30240-9
DOI:
10.1016/j.ijporl.2017.05.021
Reference:
PEDOT 8555
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 13 March 2017 Revised Date:
24 May 2017
Accepted Date: 25 May 2017
Please cite this article as: X. He, Q. Peng, S. Li, P. Zhu, C. Wu, C. Rao, J. Lin, X. Lu, A novel mutation in the MYO7A gene is associated with Usher syndrome type 1 in a Chinese family, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.05.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel mutation in the MYO7A gene is associated with Usher Syndrome type 1 in a Chinese family Running title: A novel mutation in MYO7A associated with USH1 2¶
, Siping Li2, Pengyuan Zhu3, Chunqiu Wu3, Chunbao
RI PT
Xiaoguang He1, 2¶, Qi Peng
Rao2, Jingqi Lin4, Xiaomei Lu1,2*
1. Department of neonates, Dongguan Children's Hospital, Dongguan, Guangdong,
SC
China
Dongguan, Guangdong, China
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2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
3. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China 4. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan,
¶
TE D
Guangdong, China
These authors contributed equally to this work.
*Corresponding author: Department of Medical and Molecular Genetics, Dongguan
EP
Institute of Pediatrics, Dongguan, Guangdong, China Email：[email protected]
AC C
Tel: +86 86186505
ACCEPTED MANUSCRIPT A novel mutation in the MYO7A gene is associated with Usher Syndrome type 1 in a Chinese family Xiaoguang He1,2¶ , Qi Peng 2¶, Siping Li2 , Pengyuan Zhu3 , Chunqiu Wu3, Chunbao
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Rao2, Jingqi Lin4, Xiaomei Lu1,2* 1. Department of neonates, Dongguan Children's Hospital, Dongguan, Guangdong, China
SC
2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
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Dongguan, Guangdong, China
3. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China 4. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan, Guangdong, China
These authors contributed equally to this work.
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¶
*Corresponding author: Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, Guangdong, China Email：[email protected]
EP
Tel: +86 86186505
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Running title: A novel mutation in MYO7A associated with USH1 Funding sources: Supported by the Dongguan Bureau of Science and Technology for the City Key Program of Science and Technology (Project Number: 2013108101018) and the Science and Technology Planning Project of Guangdong Province (Project Number: 2014A020213001).
ACCEPTED MANUSCRIPT Abstract Objectives: We aimed to investigate the genetic causes of hearing loss in a Chinese proband with autosomal recessive congenital deafness. Methods: The targeted
RI PT
capture of 159 known deafness genes and next-generation sequencing were performed to study the genetic causes of hearing loss in the Chinese family. Sanger sequencing was employed to verify the variant mutations in members of this family. Results: The
SC
proband harbored two mutations in the MYO7A gene in the form of compound
M AN U
heterozygosity. She was found to be heterozygous for a novel insertion mutation c.3847_3848 ins TCTG (p.N1285LfsX24) in exon 30 and for the known mutation c.2239_2240delAG (p.R747S fsX16）in exon 19. The novel mutation was absent in the 1000 Genomes Project. These variants were carried in the heterozygous state by
TE D
the parents and were therefore co-segregated with the genetic disease. Clinical re-assessment, including detailed audiologic and ocular examinations, revealed congenital deafness and retinitis pigmentosa in the proband. Collectively, the
EP
combination of audiometric, ophthalmologic and genetic examinations successfully
AC C
confirmed the phenotype of Usher syndrome type 1 (USH1). Conclusion: This study demonstrates that the novel mutation c.3847_3848insTCTG (p. N1285LfsX24) in compound heterozygosity with c.2239_2240delAG in the MYO7A gene is the main cause of USH1 in the proband. Our study expands the mutational spectrum of MYO7A and provides a foundation for further investigations elucidating the MYO7A-related mechanisms of USH1. Key words: Usher syndrome; hearing loss; MYO7A; novel mutation
ACCEPTED MANUSCRIPT 1.Introduction Hearing loss is the most common sensory impairment and severely affects patients’ daily quality of life. It is etiologically heterogeneous, and it is estimated that
RI PT
at least two thirds of all cases of childhood-onset hearing loss have genetic causes[1, 2]. To date, more than 150 genes have been reported to be asssociated with deafness (http://deafnessvariationdatabase.org/). Some of those genes might result in distinct
SC
phenotypes other than hearing loss due to the genetic diversity, which can result in
M AN U
syndromic hearing loss[3]. The myosin VIIA (MYO7A) gene is one such gene and is associated with both syndromic and nonsyndromic sensorineural hearing loss. The MYO7A gene is located on chromosome 11q13.5 and encodes the actin-binding motor protein myosin VIIa[4]. The biggest transcript of MYO7A consists of 49 exons
TE D
and encodes a 2,215-amino acid unconventional myosin named myosin VIIA. It plays an important role in intracellular transport, endocytosis, and intercellular adhesion[5]. Mutations in the MYO7A gene have been reported to be associated with a wide
EP
phenotypic spectrum, including congenital hearing impairment[6]. It is commonly
AC C
noted that MYO7A mutations are associated with Usher syndrome type 1 (USH1), an autosomal recessive group of symptoms characterized by pre-pubertal onset retinitis pigmentosa (RP), sensorineural hearing impairment and vestibular dysfunction[7,8]. Moreover, variants in the MYO7A gene have been linked to autosomal dominant non-syndromic hearing
loss
(DFNA11)
and
autosomal
recessive
deafness
(DFNB2)[9,10]. In this study, we performed gene capture and targeted next generation sequencing (NGS) for 159 deafness associated genes based on deafness
ACCEPTED MANUSCRIPT variation database （http://deafnessvariationdatabase.org/）in a Chinese proband who was diagnosed with non-syndromic hearing loss but was negative in a panel of 20 common deafness-associated mutations. Interestingly, we identified two variants in
RI PT
MYO7A in this family. Clinical re-assessment revealed congenital deafness and retinitis pigmentosa in the proband. Collectively, the combination of audiometric,
USH1, but not NSHL, in this family.
M AN U
2. Patients and methods
SC
ophthalmologic and genetic examinations successfully confirmed the phenotype of
2.1 Patients
The proband, a 9-year-old girl with congenital deafness and her unaffected parents and brothers were recruited to participate in this study. All of them came from
TE D
Guangdong Province and are part of the southern Han Chinese population. A clinical evaluation of the family was conducted, including a description of family history and detailed medical history and a physical examination. Audiometric
EP
tests covering otoscope examination, pure-tone audiometry (PTA), tympanometry
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audiometry, acoustic stapedial reflex, auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAEs) were performed. The audiological data were evaluated based on hearing impairment criteria established by the United States Joint Committee on Infant Hearing (JCIH).The degree of hearing loss was classified as mild (30–40 dB HL), moderate (41–70 dB HL), severe (71–90 dB HL) or profound (> 90 dB HL)[11].
After compound heterozygous mutations in the MYO7A
gene were found, detailed ophthalmologic examinations of the subjects, including
ACCEPTED MANUSCRIPT best-corrected visual acuities (BCVAs) measurements, slit-lamp biomicroscopy, intraocular pressure (IOP) tests, funduscopy, and visual field (VF) tests, were performed.
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This study was approved by and conducted in accordance with the protocol of the Institutional Medical and Ethics Committee of Dongguan Children's Hospital, and it conformed to the tenets of the Declaration of Helsinki. Written informed consent was
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2.2 Targeted deafness gene capturing and NGS
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obtained from the parents or legal guardians of the subjects.
Genomic DNA was extracted from peripheral blood samples of the subjects using the Blood DNA Kit (TIANGEN BIOTECH, Beijing, China) following the manufacturer's protocol. DNA yield and quality were determined using a NanoDrop
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8000 ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and 1% agarose gel electrophoresis was performed according to routine methods.
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Twenty hotspot mutations of four common deafness-associated genes (GJB2,
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GJB3, SLC26A4, and MTRNR1) were first screened by matrix-assisted laser desorption ionization time of flight-mass spectrometry (MALDI-TOF-MS). Once the screening results were negative, the genomic DNA was used to prepare a library with the SureSelectXT Reagent kit from Agilent Technologies (Agilent Technologies, Santa Clara, CA, USA; Cat. no. G9611A). All exons and some adjacent flanking intronic sequencing of 159 known deafness genes were captured by a custom-designed array (Agilent Technologies Santa Clara, CA, USA). Sequencing
ACCEPTED MANUSCRIPT was performed using a BES 4000 sequencer with single-end 160-bp reads. After sequencing, TMAP and Torrent Variant Caller (TVC) were used to map to HG19 for variant call analysis.
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2.3 Sanger sequencing Potential mutations, which were detected by target gene capture and next generation sequencing, were verified by PCR amplification and Sanger sequencing.
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The following sequence-specific primers were synthesized: 5’-CAACTGAGTTC
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TTGACCTGTGCT-3’ and 5’-TGTGTGCACTT GTTTCATAGGTG-3’ for human MYO7A c.2239_2240delAG; 5’-CAGAGAGCCAAAGTCC AGAGG-3’ and 5’-CG AGGTGCTCAAGGGTGAATAG-3’ for human MYO7A c.3847_3848 insTCTG (p. L1283LfsX27). PCR was performed in a 50 µl reaction mixture; the PCR products
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were purified with a PCR purification kit (Takara, Japan) and sequenced using an ABI 3100 DNA sequencing machine (ABI, Foster City, CA, USA) and the ABI 3100 Analysis Software (ver. 3.7 NT).
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2.3 Bioinformatics and validation of the variants
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Sequence data were analyzed by alignment with the National Center for Biotechnology Information (NCBI) reference sequence of MYO7A using the DNA Star 5.0 software. The 1000 Genomes Project database (http://www.1000genomes.org/) and the dbSNP database of the NCBI (http://www.ncbi.nlm.nih.gov/) were used as references to assess the novelty of mutations found in this study. The online tool MutationTaster was used to predict the possible pathogenicity. 3. Results
ACCEPTED MANUSCRIPT 3.1. Clinical features A hearing-impaired subject and her normal-hearing parents and two brothers were enrolled in our study (Figure 1 A). The affected girl was born after a normal pregnancy and delivery. Deafness was noted and confirmed in the first few months of
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her life. Hearing loss was identified as stable and congenital. PTA of the proband showed symmetrical bilateral and profound sensorineural hearing loss in all frequencies (Figure 1 B). The audiograms of her parents and two brothers were normal. Auditory brainstem response did not show any evoked response at or below
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100 dB on both sides of the proband. She received a cochlear implant (CI) at the age of six years old. She had balance problems before cochlear implants, such as she
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started to walk at the age of 20 months, which is later than the normal children. However, her language function was developed now because she wore hearing aids for
half
a
year
before
cochlear
implant
and
also
went
to
the
special educational institutions from 3 years old to improve her language ability.
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Her parents or medical staff did not notice the eye problem of the proband before mutations in MYO7A gene were found and detailed ophthalmologic examinations performed. Through inquiry, we learned that the proband had difficulty with night
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vision. Her current BCVA was 0.6 in the left eye and 0.3 in the right eye. Fundus
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photography revealed atrophy of the retinal pigment epithelia, attenuated retinal blood vessels and a characteristic bone spicule-like pigment. Her macular region was relatively preserved according to fundus appearance results, and there was concentric narrowing of her field of vision. The anterior segment and intraocular pressure were both normal. No abnormal results were identified in the unaffected parents and two brothers. 3.2. Genetic analysis
ACCEPTED MANUSCRIPT The sequence analysis of SLC26A4 indicated that the proband presented a novel insertion mutation c.3847_3848 insTCTG (p. N1285LfsX24) in exon 30 and a known mutation c.2239_ 2240delAG in exon 19. Additionally, her mother and big brother
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were heterozygous carriers of the c.2239_ 2240del AG mutation, and her father was a heterozygous carrier of the c.3847_3848 insTCTG (p. N1285LfsX24) mutation (Figure 2). The reported mutation c.2239_2240delAG, occurring within the tail
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domain (IQ1 region) of the MYO7A protein, results in a premature termination of
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translation at amino acid 762 (p. R747SfsX16). In addition, the c.3847_3848 insTCTG (p. N1285LfsX24) mutation, which has not been previously reported, leads to a truncated protein with termination at amino acid 1308 (Figure 3). This novel mutation was absent in the 1000 Genomes Project and was predicted to be “disease
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causing” by MutationTaster, which strongly suggests that this novel mutation is closely associated with USH1. 4. Discussion
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In this Chinese family, we revealed two deleterious compound heterozygous
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mutations in the MYO7A gene: a novel insertion mutation c.3847_3848 insTCTG (p. N1285LfsX24) and
a reported deletion
mutation c.2239_2240delAG. The
c.3847_3848 insTCTG (p. N1285LfsX24) mutation has not been previously described in hearing loss patients. Through gene mutation analysis, we further confirmed that the compound heterozygous mutations in the MYO7A gene might be pathogenic because the two detected variants both cause a frameshift mutation resulting in a premature stop-codon and in a predicted truncated protein that could be
ACCEPTED MANUSCRIPT non-functional. The compound heterozygous variants co-segregated with their clinical phenotypes. In brief, this proband had symmetrical bilateral and profound sensorineural hearing loss in all frequencies and retinitis pigmentosa, which were
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consistent with the phenotype reported for USH1. These results indicate that the compound heterozygous mutations are likely the genetic cause of USH1 in this family.
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The human myosin VIIA protein contains a N-terminal motor domain (amino
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acids 1–729), a five IQ motif neck region (amino acids 745–857), a predicted short coiled coil domain (amino acids 858–935), a MyTH4 domain (amino acids 1017–1253), a 4.1-Ezrin-radixin-moesin (FERM) domain (amino acids 1258–1602), an SH3 domain (amino acids 1603–1672), and a second C-terminal MyTH4-FERM
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tandem domain (amino acids 1747–2205)[12]. Expression of the myosin VIIa protein in the cochlear hair cells, retinal photoreceptors, and retinal pigment epithelium has been detected[13]. In the hair cells of the inner ear, myosin VIIA interacts with other
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proteins to participate in the formation of the mechanotransduction complex, which is
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critical for detecting sound[14]. In the retinal pigment epithelium cells, myosin VIIA participates in the light cycle-dependent movement of melanosome transportation[15] and the normal functioning of the visual retinoid cycle[16]. In the photoreceptor cells, myosin VIIA is present at the connecting cilium and regulates opsin transport[13]. The C-terminal tail of myosin VIIA, which contains the SH3, MyTH4, and FERM domains (1605–2215), interacts with harmonin (USH1C)[17]. Both of the truncated mutant myosin VIIA proteins in this study lack the C-terminal tail and may thus lose
ACCEPTED MANUSCRIPT the ability to bind harmonin, which acts as a scaffold protein linking the proteins in the cell membrane to the proteins in the cytoskeleton. The additive effects of the two identified compound heterozygous mutations could cause a complete dysfunction of
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myosin VIIA in the proband, leading to the observed phenotype. The proband did not display any vestibular dysfunction, but we cannot rule out the possibility of dysfunction developing later in her life.
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In conclusion, we described a patient with USH1 carrying biallelic mutations
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(c.3847_3848 insTCTG (p. N1285LfsX24)/c.2239_2240delAG) in the MYO7A gene. This finding of a novel mutation further expands the spectrum of known MYO7A mutations in the Chinese population. In future studies, we will focus on the construction of a transgenic mouse model carrying these mutations to reveal the
Conflicts of interest
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mechanism of hearing impairment.
We declare that we have no conflicts of interest.
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Acknowledgments
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This work was supported by the Dongguan Bureau of Science and Technology for the City Key Program of Science and Technology (Project Number: 2013108101018) and the Science and Technology Planning Project of Guangdong Province (Project Number: 2014A020213001). We also thank all the children and their parents for their cooperation during this work.
ACCEPTED MANUSCRIPT References 1.Petit C. Genes responsible for human hereditary deafness: symphony of a thousand. Nat Genet 1996;14:385-91.
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2.Willems PJ. Genetic Causes of Hearing Loss. N Engl J Med 2000;342:1101-9. 3.Ma Y, Xiao Y, Zhang F, Han Y et al. Novel compound heterozygous mutations in MYO7A gene associated with autosomal recessive sensorineural hearing loss in a Chinese family. Int J Pediatr
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Otorhinolaryngol 2016;83:179-85.
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4.Gibbs D, Diemer T, Khanobdee K, Hu J, Bok D, Williams DS. Function of MYO7A in the human RPE and the validity of shaker1 mice as a model for Usher Syndrome 1B. Invest Ophthalmol Vis Sci. 2010;51:1130-5.
5.Wu L, Pan L, Wei Z, Zhang M. Structure of MyTH4-FERM domains in myosin VIIa tail bound to
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cargo. Science 2011;331:757-60.
6.Zong L, Chen K, Wu X, Liu M, Jiang H. Compound heterozygous MYO7A mutations segregating Usher syndrome type 2 in a Han family. Int J Pediatr Otorhinolaryngol 2016;90:150-155.
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2010;55:327-35.
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7.Yan D, Liu XZ. Genetics and pathological mechanisms of Usher syndrome. J Hum Genet
8.Yoshimura H, Iwasaki S, Nishio S-y et al. Massively parallel DNA sequencing facilitates diagnosis of patients with usher syndrome type 1. PLoS ONE 2014;9:e90688. 9.Liu XZ, Walsh J, Mburu P et al. Mutations in the myosin VIIA gene cause non-syndromic recessive deafness. Nat Genet 1997;16:188-90. 10.Liu XZ, Walsh J, Tamagawa Y et al. Autosomal dominant non-syndromic deafness caused by a mutation in the myosin VIIA gene. Nat Genet 1997;17:268-9.
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11. Joint Committee on Infant Hearing. Year 2007 Position Statement: Principles and guidelines for early hearing detection and intervention programs. Pediatrics 2007; 120: 898-921.12.Inoue A, Ikebe M.
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Characterization of the motor activity of mammalian myosin VIIA. J Biol Chem 2003;278:5478-87. 13.Liu X, Udovichenko IP, Brown SDM, Steel KP, Williams DS. Myosin VIIa participates in opsin transport through the photoreceptor cilium. J Neurosci 1999;19:6267.
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14.Grati Mh, Kachar B. Myosin VIIa and sans localization at stereocilia upper tip-link density
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implicates these Usher syndrome proteins in mechanotransduction. Proc Natl Acad Sci USA 2011;108:11476-81.
15.El-Amraoui A, Schonn JS, Küssel-Andermann P et al. Myrip, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes. EMBO Rep 2002;3:463-70.
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16.Lopes VS, Gibbs D, Libby RT et al. The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65. Hum Mol Genet 2011;20:2560-70.
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17.Boëda B, El-Amraoui A, Bahloul A, Goodyear R, Daviet L, Blanchard S, et al. Myosin VIIa,
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harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle. EMBO J 2002;21:6689-99.
ACCEPTED MANUSCRIPT Figure Legends Fig. 1.(A) Pedigree of the family. Arrow indicates the proband. (B) PTA results of the proband and her family members.
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Fig. 2. Mutational analysis in the Chinese family. The DNA sequencing profile shows the c.3847_3848 insTCTG (p. N1285LfsX24) and c.2239_2240delAG mutations in MYO7A. The compound heterozygous variants co-segregated with the clinical
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phenotype.
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Fig 3. The domain structure of myosin VIIa showing the mutations. The reported mutation c.2239_2240delAG, occurring within the tail domain (IQ1 region) of the MYO7A protein, results in a premature termination of translation at amino acid 762 (p. R747SfsX16). The insertion mutation c.3847_3848 insTCTG introduced a premature
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termination codon (p. N1285LfsX24), which was predicted to truncate the protein.
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