Frequency of Usher syndrome type 1 in deaf children by massively parallel DNA sequencing
Journal of Human Genetics
Frequency of Usher syndrome type 1 in deaf children by massively parallel DNA sequencing
Usher syndrome type 1 (USH1) is the most severe of the three USH subtypes due to its profound hearing loss, absent vestibular response and retinitis pigmentosa appearing at a prepubescent age. Six causative genes have been identified for USH1, making early diagnosis and therapy possible through DNA testing. Targeted exon sequencing of selected genes using massively parallel DNA sequencing (MPS) technology enables clinicians to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using MPS along with direct sequence analysis, we screened 227 unrelated non-syndromic deaf children and detected recessive mutations in USH1 causative genes in five patients (2.2%): three patients harbored MYO7A mutations and one each carried CDH23 or PCDH15 mutations. As indicated by an earlier genotype-phenotype correlation study of the CDH23 and PCDH15 genes, we considered the latter two patients to have USH1. Based on clinical findings, it was also highly likely that one patient with MYO7A mutations possessed USH1 due to a late onset age of walking. This first report describing the frequency (1.3-2.2%) of USH1 among non-syndromic deaf children highlights the importance of comprehensive genetic testing for early disease diagnosis. Journal of Human Genetics (2016) 61, 419-422; doi:10.1038/jhg.2015.168; published online 21 January 2016
Usher syndrome (USH) is a collection of three autosomal recessive
disorder subtypes that results in hearing loss (HL), retinitis pigmentosa
(RP) and/or vestibular dysfunction. Among these, USH type 1 (USH1)
is the most severe due to its profound hearing loss, absent vestibular
response and RP appearing at a prepubescent age. USH type 2 (USH2)
shows congenital moderate-to-severe with a high-frequency sloping
HL and normal vestibular functions. RP of USH2 appears in the first
or second decades of life. USH type 3 (USH3) is typified by the
variable onset of progressive HL and RP and a range of vestibular
function impairment, from normal to absent.1
To date, 10 causal genes have been identified for USH: MYO7A
(USH1B), USH1C (USH1C), CDH23 (USH1D), PCDH15 (USH1F),
USH1G (USH1G) and CIB2 (USH1J) for USH1; USH2A (USH2A),
GPR98 (USH2C) and DFNB31 (USH2D) for USH2; and CLRN1
(USH3A) for USH3 (Hereditary Hearing Loss Homepage; http://
hereditaryhearingloss.org). As these target genes are large with many
exons, considerable labor and cost are required for their analysis by
using conventional Sanger sequencing. However, recent advances in
targeted re-sequencing by massively parallel DNA sequencing (MPS)
have made it possible to analyze all known causative genes
simultaneously;2,3 we recently employed MPS to identify the frequency of
USH-related gene mutations in Japanese USH1 patients4 and
characterize USH2 and USH3 patients.5,6
The diagnosis of USH in childhood based on clinical phenotypes
can be challenging since patients often appear to have non-syndromic
HL only in their youth until RP develops in later years. However, early
diagnosis through genetic testing provides many immediate and
longterm advantages for patients and their families.7 We previously
described a case in which MYO7A and GPR98 mutation analysis
allowed the diagnosis of USH prior to the appearance of visual
symptoms, and subsequent DNA testing enabled appropriate genetic
In the present study, we performed genetic analysis using MPS
technology to simultaneously screen for four USH1 causative
genes (MYO7A, USH1C, CDH23 and PCDH15) in unrelated,
non-syndromic, severe-to-profound HL children.
MATERIALS AND METHODS
Among the 1373 Japanese HL patients registered in our DNA sample bank
from 53 otorhinolaryngology departments across Japan, we selected 227
patients who met the criteria of: (i) congenital HL (i.e., HL onset was
prelingual/early at o6 years of age), (ii) severe-to-profound HL (above 71
dB on average over 500, 1000, 2000 and 4000 Hz in the better hearing ear) and
(iii) DNA sampling prior to 10 years of age due to the prepubertal nature
Of the 227 non-syndromic deaf children screened, 21 were from autosomal
recessive families, 22 from autosomal dominant families and 184 from sporadic
onset families. There were 127 boys and 100 girls. All subjects (or guardians)
gave prior written informed consent for participation in the study. This study
was approved by the Ethics Committee of Shinshu University School of
Massively Parallel Sequencing
Targeted genes. We screened for mutations in MYO7A [NM_000260], USH1C
[NM_153676], CDH23 [NM_022124] and PCDH15 [NM_033056].
Amplicon library preparation. Amplicon libraries for MPS analysis were
prepared according to the manufacturer?s instructions with an Ion AmpliSeq
Custom Panel (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) for
63 standard genes that reportedly cause non-syndromic HL (including MYO7A,
USH1C, CDH23 and PCDH15) as described elsewhere.9 The amplicon libraries
were diluted to 20 pM, and equal amounts of six libraries from six patients were
pooled for one sequence reaction.
Emulsion polymerase chain reaction and sequencing. Emulsion polymerase
chain reaction and sequencing were performed according to the manufacturer?s
instructions and the protocol of an earlier report.9 MPS analysis was performed
with an Ion Torrent PGM using an Ion PGM 200 Sequencing Kit and Ion 318
Chip (Life Technologies).
Base call and data analysis. Sequence results were mapped against the human
genome sequence (build GRCh37/hg19) with the Torrent Mapping Alignment
Program. After sequence mapping, variant regions were compiled with Torrent
Variant Caller plug-in software. Each variant effect was then analyzed using
ANNOVAR software.10,11 Identified missense, nonsense, insertion/deletion and
splicing variants were further selected if their incidence was less than 1% of the
1000 Genome database, the 6500 exome variants in the Exome Variant Server,
the data set of 1208 Japanese exome variants in the Human Genetic Variation
Database and 269 in-house Japanese normally hearing controls. We excluded all
pathogenic mutations of CDH23-caused HL (DFNB12), on which we have
To predict the pathogenicity of missense variants, the following functional
prediction software included in ANNOVAR was used: Sorting Intolerant from
Tolerant (SIFT; http://sift.jcvi.org/), Polymorphism Phenotyping (PolyPhen2;
http://genetics.bwh.harvard.edu/pph2/), LRT (http://www.genetics.wustl.edu/
jflab/lrt_query.html) and MutationTaster (http://www.mutationtaster.org/).
Candidate mutations were confirmed using Sanger sequencing, and segregation
analysis was also performed using samples from the patients? family members.
The sequencing data are available in the DDBJ databank of Japan (Accession
Mutation analysis of 4 selected USH1-associated genes in 227
nonsyndromic deaf children revealed 9 different probable pathogenic
variants, among which 7 were novel. We observed one frameshift
mutation, four nonsense mutations, one splice site mutation and three
missense mutations (Table 1).
Whereas the nonsense, frameshift and splice site mutations were all
considered pathogenic, the missense mutations were presumed to be
probable pathogenic variants based on the results of prediction
software evaluation of pathogenicity (Table 1). These residues were
well conserved among several species. Functional prediction software
(Polyphen2, SIFT, MutationTaster and LRT) indicated mutations to
be damaging at scores of 1.0, 1.0, 1.0 and 1.0, respectively.
In the cohort, five patients had recessive mutations in a USH1
causative gene (2.2%). Of them, three were in MYO7A, one was in
CDH23 and one was in PCDH15 (Table 2).
The family histories of the five patients identified in this study were
compatible with autosomal recessive inheritance (Figure 1). Although
all patients entered this study before 12 months of age, no common
responsible genes, such as GJB2 or mitochondrial 1555AG
mutations, were found at the time. Genetic testing using MPS was
later carried out in 2013?2014.
The onset of walking in two patients (#3840 and #4627) was normal
(12 and 17 months, respectively), while that in three patients
(JHLB1637, JHLB624 and #4859) was delayed (24, 24 and 31,
respectively; Table 2).
At the time of MPS testing, the identified patients were between 2
and 10 years old. Three had received a unilateral cochlear implant (CI)
and two had received bilateral CIs (Table 2). One patient (#3840)
had not experienced night blindness by the age of 10 years and
ophthalmologic data were therefore not available.
In this report, we identified nine mutations among three USH1
causative genes (MYO7A, CDH23 and PCDH15) in five patients.
However, since mutations among these genes could have resulted in
non-syndromic HL as well as USH1 (Hereditary Hearing Loss
Homepage; http://hereditaryhearingloss.org), a careful differential
diagnosis was crucial. As suggested by an earlier genotype?phenotype
correlation study, USH1D (CDH23) and USH1F (PCDH15) were
typically associated with truncating mutations, while DFNB12
(CDH23) and DFNB23 (PCDH15), which had a milder phenotype,
were associated with non-truncating mutations.13,14. Accordingly, we
considered the diagnosis in 2 patients (JHLB624 and #4859) to be
USH1 based on genetic findings.
No obvious correlations have been reported between mutations in
the MYO7A gene and the resulting phenotype.1 However, clinical
confirmation of hallmark symptoms may enable the differential
diagnosis of non-syndromic HL and USH1. The most frequent clinical
sign of USH1 in a cohort of prelinguistically deaf children was a
delayed onset of walking (420 months) due to bilateral vestibular
dysfunction.15 Therefore, it was highly likely that the clinical subtype
in one patient with
mutations (JHLB1637) was USH1B
because he began walking at 24 months of age. Astuto et al. evaluated
the published clinical data for non-syndromic HL patients with
mutations (DFNB2). They concluded that there was no
convincing evidence supporting a DFNB2 phenotype in patients with
recessive MYO7A mutations and that such deaf individuals most likely
had USH. In the present study, we considered the remaining two
patients with recessive mutations in MYO7A (#3840 and #4627) to
possess non-syndromic HL (DFNB2) because their onset time of
walking was normal. However, careful monitoring for ophthalmic
symptoms is needed.
Based on the above findings, we calculated that the frequency of
USH1 patients in 227 deaf children was 1.3?2.2% (3?5/227) on the
basis of MPS. We have performed mutation screening of four major
USH1-causing genes. It is known that the majority of cases of USH1
are caused by four genes (MYO7A, USH1C, CDH23 and PCDH15).
We have not included USH1G (USH1G) and USH1 J (CIB2) in this
study, because these USH1-causing genes have been reported to be
very rare.16?19 Based on microarray analysis, Kimberling et al. showed
that in 155 deaf children receiving CIs, 1.9% (3/155) carried recessive
USH1 mutations. Of them, however, two patients had non-truncating
recessive mutations in CDH23. We considered these to be cases of
non-syndromic HL, resulting in an adjusted frequency of USH1 in
deaf children of 0.6% (1/155). This difference (1.3?2.2% vs 0.6%) may
be attributed to the method of genetic testing (MPS vs microarray
analysis) and/or the mutation spectrum between Japanese (and by
association other Asian populations) and populations with European
with our findings.
According to a conservative estimate of the frequency of childhood
deafness of approximately 1/1000,20 we can calculate the incidence of
USH1 in the Japanese population to be 1.3?2.2 per 100 000
individuals. Previous studies have reported the prevalence of USH1
based on clinical data as 1.1?2.0 per 100 000,21?24 which is compatible
With regard to treatment, deaf children identified as harboring
USH1 causative mutations should be offered unilateral or bilateral CIs.
There is a strong need to provide USH children with the best hearing
amplification available, with a preference for CIs, accompanied by
intensive training and habilitation before the onset of RP.25 In fact, all
patients highlighted in this study have received a CI, two bilaterally,
with another (JHLB1637) about to receive a second implant. Careful
ongoing surveillance for visual symptoms is also needed for deaf
children with identified USH1 causative mutations.
In conclusion, based on MPS, this study showed the frequency of
USH1 among deaf children to be 1.3?2.2% and underscored the
importance of comprehensive genetic testing for diagnosing USH
among non-syndromic deaf children. Otolaryngologists and
audiologists should bear USH in mind when dealing with deaf children with
the aims of prompt therapy and habilitation.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
This study was supported by a Health and Labour Sciences Research Grant for
Research on Rare and Intractable Diseases and Comprehensive Research on
Disability Health and Welfare from the Ministry of Health, Labour, and
Welfare of Japan (S.U.) and by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science, and Culture of Japan (S.U.). The authors thank
Mr Trevor Ralph for his help in preparing the manuscript.
1 Yan, D. & Liu , X. Z. Genetics and pathological mechanisms of Usher syndrome . J. Hum. Genet . 55 , 327 - 335 ( 2010 ).
2 Miyagawa, M. , Naito , T. , Nishio , S. Y. , Kamatani , N. & Usami , S. Targeted exon sequencing successfully discovers rare causative genes and clarifies the molecular epidemiology of Japanese deafness patients . PLoS ONE 8 , e71381 ( 2013 ).
3 Shearer, A. E. , Black-Ziegelbein , E. A. , Hildebrand , M. S. , Eppsteiner , R. W. , Ravi , H. , Joshi , S. et al. Advancing genetic testing for deafness with genomic technology . J. Med. Genet . 50 , 627 - 634 ( 2013 ).
4 Yoshimura, H., Iwasaki , S. , Nishio , S. Y. , Kumakawa , K. , Tono , T. , Kobayashi , Y. et al. Massively parallel DNA sequencing facilitates diagnosis of patients with Usher syndrome type 1 . PLoS ONE 9 , e90688 ( 2014 ).
5 Moteki, H., Yoshimura , H. , Azaiez , H. , Booth , K. T. , Shearer , A. E. , Sloan , C. M. et al. USH2 caused by GPR98 mutation diagnosed by massively parallel sequencing in advance of the occurrence of visual symptoms . Ann. Otol. Rhinol. Laryngol. 124 , 123S - 128S ( 2015 ).
6 Yoshimura, H., Oshikawa , C. , Nakayama , J. , Moteki , H. & Usami , S. Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports . Ann. Otol. Rhinol. Laryngol . 124 , 94S - 99S ( 2015 ).
7 Kimberling, W. J., Hildebrand , M. S. , Shearer , A. E. , Jensen , M. L. , Halder , J. A. , Trzupek , K. et al. Frequency of Usher syndrome in two pediatric populations: implications for genetic screening of deaf and hard of hearing children . Genet. Med . 12 , 512 - 516 ( 2010 ).
8 Yoshimura, H., Iwasaki , S. , Kanda , Y. , Nakanishi , H. , Murata , T. , Iwasa , Y. et al. An Usher syndrome type 1 patient diagnosed before the appearance of visual symptoms by MYO7A mutation analysis . Int. J. Pediatr. Otorhinolaryngol . 77 , 298 - 302 ( 2013 ).
9 Miyagawa, M. , Nishio , S.Y. , Ikeda , T. , Fukushima , K. & Usami , S. Massively parallel DNA sequencing successfully identifies new causative mutations in deafness genes in patients with cochlear implantation and EAS . PLoS ONE 8 , e75793 ( 2013 ).
10 Chang, X. & Wang , K. wANNOVAR: annotating genetic variants for personal genomes via the web . J. Med. Genet . 49 , 433 - 436 ( 2012 ).
11 Wang, K. , Li , M. & Hakonarson , H. ANNOVAR : functional annotation of genetic variants from high-throughput sequencing data . Nucleic Acids Res . 38 , e164 ( 2010 ).
12 Miyagawa, M. , Nishio , S.Y. & Usami , S. Prevalence and clinical features of hearing loss patients with CDH23 mutations: a large cohort study . PLoS ONE 7 , e40366 ( 2012 ).
13 Schultz, J. M. , Bhatti , R. , Madeo , A. C. , Turriff , A. , Muskett , J. A. , Zalewski , C. K. et al. Allelic hierarchy of CDH23 mutations causing non-syndromic deafness DFNB12 or Usher syndrome USH1D in compound heterozygotes . J. Med. Genet . 48 , 767 - 775 ( 2011 ).
14 Doucette, L. , Merner , N. D. , Cooke , S. , Ives , E. , Galutira , D. , Walsh , V. et al. Profound, prelingual nonsyndromic deafness maps to chromosome 10q21 and is caused by a novel missense mutation in the Usher syndrome type IF gene PCDH15 . Eur. J. Hum. Genet . 17 , 554 - 564 ( 2009 ).
15 Liu, X. Z. , Angeli , S. I. , Rajput , K. , Yan , D. , Hodges , A. V. , Eshraghi , A. et al. Cochlear implantation in individuals with Usher type 1 syndrome . Int. J. Pediatr. Otorhinolaryngol . 72 , 841 - 847 ( 2008 ).
16 Le Quesne Stabej , P. , Saihan , Z. , Rangesh , N., Steele-Stallard , H. B. , Ambrose , J. , Coffey , A. et al. Comprehensive sequence analysis of nine Usher syndrome genes in the UK National Collaborative Usher Study . J. Med. Genet . 49 , 27 - 36 ( 2012 ).
17 Bonnet, C. , Grati , M. , Marlin , S. , Levilliers , J. , Hardelin , J.P. , Parodi , M. et al. Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis . Orphanet J. Rare Dis . 6 , 21 ( 2011 ).
18 Ouyang, X. M. , Yan , D. , Du , L. L. , Hejtmancik , J. F. , Jacobson , S. G. , Nance , W. E. et al. Characterization of Usher syndrome type I gene mutations in an Usher syndrome patient population . Hum. Genet . 116 , 292 - 299 ( 2005 ).
19 Riazuddin, S. , Belyantseva , I. A. , Giese , A. P. , Lee , K. , Indzhykulian , A. A. , Nandamuri , S. P. et al. Alterations of the CIB2 calcium- and integrin-binding protein cause Usher syndrome type 1J and nonsyndromic deafness DFNB48 . Nat. Genet . 44 , 1265 - 1271 ( 2012 ).
20 Morton, C. C. & Nance , W. E. Newborn hearing screening-a silent revolution . N. Engl. J. Med . 354 , 2151 - 2164 ( 2006 ).
21 Grondahl, J. Estimation of prognosis and prevalence of retinitis pigmentosa and Usher syndrome in Norway . Clin. Genet . 31 , 255 - 264 ( 1987 ).
22 Hope, C. I. , Bundey , S. , Proops , D. & Fielder , A. R. Usher syndrome in the city of Birmingham-prevalence and clinical classification . Br. J. Ophthalmol . 81 , 46 - 53 ( 1997 ).
23 Rosenberg, T. , Haim , M. , Hauch , A. M. & Parving , A. The prevalence of Usher syndrome and other retinal dystrophy-hearing impairment associations . Clin. Genet . 51 , 314 - 321 ( 1997 ).
24 Spandau, U. H. & Rohrschneider , K. Prevalence and geographical distribution of Usher syndrome in Germany . Graefes Arch. Clin. Exp. Ophthalmol . 240 , 495 - 498 ( 2002 ).
25 Brownstein, Z. , Ben-Yosef , T. , Dagan , O. , Frydman , M. , Abeliovich , D. , Sagi , M. et al. The R245X mutation of PCDH15 in Ashkenazi Jewish children diagnosed with nonsyndromic hearing loss foreshadows retinitis pigmentosa . Pediatr. Res . 55 , 995 - 1000 ( 2004 ).
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