doi: 10.1111/ahg.12084
Exome Sequencing Identifies a Novel Frameshift Mutation
of MYO6 as the Cause of Autosomal Dominant
Nonsyndromic Hearing Loss in a Chinese Family
Jing Cheng1,† , Xueya Zhou2,3,† , Yu Lu1 , Jing Chen1 , Bing Han1 , Yuhua Zhu1 , Liyang Liu2 ,
Kwong-Wai Choy4 , Dongyi Han1 , Pak C. Sham3 , Michael Q. Zhang2,5∗ , Xuegong Zhang2∗ and
Huijun Yuan1∗
1
Institute of Otolaryngology, Chinese PLA General Hospital, Beijing, China
2
MOE Key Laboratory of Bioinformatics, Bioinformatics Division and Center for Synthetic and Systems Biology, TNLIST/Department
of Automation, Tsinghua University, Beijing, China
3
Department of Psychiatry and Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Hong Kong SAR, China
4
Li Ka Shing Institute of Health Sciences, Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University
of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
5
MCB, Center for Systems Biology, The University of Texas at Dallas, Richardson, TX, USA
Summary
Autosomal dominant types of nonsyndromic hearing loss (ADNSHL) are typically postlingual in onset and progressive.
High genetic heterogeneity, late onset age, and possible confounding due to nongenetic factors hinder the timely
molecular diagnoses for most patients. In this study, exome sequencing was applied to investigate a large Chinese family
segregating ADNSHL in which we initially failed to find strong evidence of linkage to any locus by whole-genome linkage
analysis. Two affected family members were selected for sequencing. We identified two novel mutations disrupting known
ADNSHL genes and shared by the sequenced samples: c.328C>A in COCH (DFNA9) resulting in a p.Q110K substitution
and a deletion c. 2814_2815delAA in MYO6 (DFNA22) causing a frameshift alteration p.R939Tfs∗ 2. The pathogenicity
of novel coding variants in ADNSHL genes was carefully evaluated by analysis of co-segregation with phenotype in the
pedigree and in light of established genotype–phenotype correlations. The frameshift deletion in MYO6 was confirmed
as the causative variant for this pedigree, whereas the missense mutation in COCH had no clinical significance. The
results allowed us to retrospectively identify the phenocopy in one patient that contributed to the negative finding in the
linkage scan. Our clinical data also supported the emerging genotype–phenotype correlation for DFNA22.
Keywords: Hearing loss, exome sequencing, molecular diagnosis, mutation, MYO6, DFNA22
Introduction
∗
Corresponding authors: Dr. Michael Q. Zhang, MCB, Center for
Systems Biology, The University of Texas at Dallas, Richardson,
TX, 75080, USA. Tel.: +1-972-883-2523; Fax: +1-970-883-5710;
E-mail: [email protected]. Dr. Xuegong Zhang, Bioinformatics Division, FIT 1-107, Tsinghua University, Beijing
10084, China. Tel: +86-10-62794919; Fax: +86-10-62773552;
E-mail: [email protected]. Dr. Huijun Yuan, Laboratory
of Molecular Genetics, Institute of Otolaryngology, Chinese PLA
General Hospital, Beijing 1000853, China. Tel.: +86-10-68287822;
Fax: +86-10-68156974; E-mail: [email protected]
†
These authors contributed equally to this work.
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Hearing loss (HL) is one of the most common sensorineural
defects that contribute to the public health burden. Depending on the diagnostic criteria, about 12 newborns with HL
can be diagnosed in every 1000, with age-related increase in
prevalence (Morton & Nance, 2006). In developed countries,
more than two-thirds of the cases can be attributed to genetic
causes (Smith et al., 2005). In 70% of families, HL is inherited as a classic Mendelian trait without symptoms in other
parts of the body (nonsyndromic) (Van Camp et al., 1997).
Autosomal recessive types of nonsyndromic HL (ARNSHL)
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A New Chinese DFNA22 Family
Figure 1 The pedigree structure of the family.
Thirty-two individuals whose whole-genome SNP genotypes were available are indicated by “+.” Those samples were included in
haplotype and linkage analyses. Two selected patients for exome sequencing are indicated by “#.” Phenotype classification criteria are
described in the main text. Family members with available DNA were genotyped for the two prioritized variants in the ADNSHL genes
shared by the two sequenced samples.
are typically prelingual and present in 80% of HL patients,
whereas the autosomal dominant types (ADNSHL) are mostly
postlingual onset and progressive and account for 20% cases
(Hilgert et al., 2009). Establishing an exact genetic diagnosis
for HL patients is crucially important as it allows for prognosis, enables genetic counseling, and even suggests customized
treatment options (Robin et al., 2005).
Nonsyndromic HL is extremely heterogeneous, with at
least 70 genes identified to date, including 27 genes for
ADNSHL (http://hereditaryhearingloss.org, Sep. 2013 accessed). Therefore, molecular diagnosis of inherited HL can
be challenging, especially for ADNSHL. Mutations in some
ARNSHL genes including GJB2 and SLC26A4 can explain
up to half of the ARNSHL patients in some populations, but
no single gene can account for the majority of ADNSHL cases
(e.g., Shearer et al., 2013; Yang et al., 2013). Although mutations in certain genes can result in distinct audiogram profiles
(e.g., low frequency HL in WFS1 and DIAPH1; middle frequency HL in TECTA) or accompanying recognizable symptoms (e.g., vestibular dysfunction associated with mutations in
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COCH and MYO7A) (Street et al., 2008), for most cases, no
such distinguishing phenotypic feature can be used to infer
the underlying gene with high confidence. The traditional
method for ADNSHL diagnosis relies on the establishment of
linkage to known DFNA loci in large pedigrees, followed by
Sanger sequencing of candidate genes. This approach is not
robust to phenotype misclassification, phenocopies caused by
nongenetic causes, or the presence of locus heterogeneity
within the same pedigree (e.g., Abdelfatah et al., 2013).
Whole-exome sequencing has become a useful tool for
both new gene discovery and molecular diagnosis in clinical genetics (Ku et al., 2012). A number of recent studies
have reported successful application of targeted next generation sequencing for comprehensive genetic testing in HL
(e.g., Shearer et al., 2010; Shearer et al., 2013; Yang et al.,
2013). With increasing power to discover more variants however, there is a greater challenge in variant interpretation
(Cooper & Shendure, 2011). In the present study, we applied
whole-exome sequencing to investigate the genetic causes
of ADNSHL in a large Chinese pedigree. Interestingly,
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Figure 2 Typical audiograms of DFNA22 patients.
(A) The progression of HL in the proband at the age of 36 and 41 years. The affected family members typically showed flat shaped (B) or
gentle down-sloping (C) audiograms. (D) Age-related typical audiograms (ATRA) for all MYO6 mutation carriers whose audiograms
were available. The estimated annual threshold deterioration is shown above each frequency band (∗ P < 0.05, ∗∗ P < 0.01).
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Annals of Human Genetics (2014) 78,410–423
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A New Chinese DFNA22 Family
we discovered two novel mutations disrupting two known
ADNSHL genes and shared by the sequenced samples.
Co-segregation analysis and careful examination of known
genotype–phenotype correlations confirmed the pathogenicity of the frameshift deletion in the MYO6 gene and led to
the discovery of a new DFNA22 family.
Materials and Methods
Family Ascertainment and Clinical Evaluation
Figure 1 shows the ascertained pedigree of the Chinese family.
The pedigree spans five generations, showing an autosomal
dominant inheritance of HL. A total of 46 voluntary family members (including six spouses) participated in the study.
All participants signed an informed consent and completed
a questionnaire regarding the following aspects: subjective
degree and symmetry of hearing impairment, age at onset
and progression, presence of tinnitus, pressure in the ears or
vertigo, historical exposure to ototoxic drugs and noise, and
other relevant clinical manifestations. Ten milliliter peripheral
venous blood sample was obtained from 45 participants. The
project was approved by the Ethics Committee of Chinese
PLA General Hospital.
A total of 35 family members (including two spouses) were
available for detailed clinical examination. After otoscopic
test, threshold audiograms were obtained with pure-tone audiometry at frequencies from 250 to 8000 Hz. All the family members demonstrated normal immittance testing and
bone conduction values that equal to the air conduction measurements, suggesting sensorineural hearing impairment. The
degree of HL was defined according to pure-tone averages
(PTA) based on the three frequencies of 500, 1000, and 2000
Hz. The hearing level of the better ear was labeled as mild
(2540 dB), moderate (4171 dB), severe (7195 dB), or
profound (>95 dB). A difference between left and right ear
air condition threshold >20 dB at least two frequencies out
of 500, 1000, 2000 Hz was indicated as asymmetric.
A family member was considered to be affected if (i) his/her
PTA of the better ear was worse than 25 dB; or (ii) he/she had
self-reported HL and had offspring(s) satisfying (i). A family
member was considered to be unaffected if (i) his/her PTA of
the better ear was better than 25 dB and older than 35 years
(the upper bound of age at onset for most patients) to account
for the late onset age; or (ii) he/she had no subjective hearing problems and unaffected parents. The phenotypes for the
remaining individuals were considered as uncertain (Fig. 1).
It should be noted that the phenotypic data were collected
on-site rather than in the sound-treated room. We found
that the conduction thresholds at 500, 1000, and 2000 Hz of
several apparently unaffected family members exceeded the
95 percentile of age- and gender-related threshold defined by
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the ISO 7029–2000 standard. Therefore, a relaxed threshold
on the PTA as above was used to define the affection status
to account for the measurement errors.
After the MYO6 mutation was confirmed as the causal variant, we used cross-sectional linear regression analysis to evaluate the progression of HL for the DFNA22 patients (Topsakal
et al., 2010). The mean binaural air conduction thresholds
at each measured frequency were regressed against ages. For
patients with asymmetric HL, only the better ear levels were
included in the analysis. The slope coefficient can be interpreted as annual threshold deterioration (ATD, in dB per
year). A significant slope implies the significant progression at
a particular frequency band. The extrapolated thresholds predicted by the linear model at the decades of 17 were used
to construct the age-related typical audiograms (ARTA). To
compare ARTA, the threshold feature array method proposed
by Huygen et al. (2003) was used.
Exome Capture and Massive Parallel Sequencing
Approximately 3 μg of genomic DNA for each selected
patient (III:13 and III:23) was extracted and purified from
peripheral blood leucocytes using QIAamp DNA blood kit
(Qiagen, Limburg, the Netherlands). DNA was sheared into
150250 base pair (bp) fragments (Covaris, Woburn, MA,
USA) and purified using MinElute PCR purification kit
(Qiagen). The quality of fragmentation and purification was
assessed by the Agilent 2100 Bioanalyzer (Agilent, Santa
Clara, CA, USA). The fragments were then end repaired,
adenylated, and ligated to adaptors (NEBNext, NEB, Ipswich,
MA, USA). The resulting DNA library was purified and PCR
amplified before hybridized to the biotinylated RNA baits of
SureSelect Human All Exon Kit V2 (Agilent). Captured genomic DNA was enriched using streptavidin-coated magnetic
Dynabeads (Invitrogen, Carlsbad, CA, USA). The wholeexome library of each patient was sequenced on two lanes
of the Illumina Genome Analyzer IIx using 71 bp paired-end
reads. Raw image data were analyzed using Illumina CASAVA
pipeline v1.7 to extract sequencing reads.
For prioritized variants, primers were designed to amplify
the encompassing genomic region. PCR products were sequenced in both forward and reverse directions on an ABI
3100 instrument using BigDye chemistry (Applied Biosystems, Foster City, CA, USA). Traces were aligned to the reference sequences and nucleotide changes were identified using
MutationSurveyor (SoftGenetics, State College, PA, USA).
Bioinformatics Analysis
Next generation sequencing data were analyzed using an
in-house bioinformatics pipeline. Short reads were mapped
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J. Cheng et al.
to the human reference genome (NCBI build 37) using
Burrow-Wheeler Aligner (v0.5.9) (Li & Durbin, 2009). Picard (v1.55, http://picard.sourceforge.net/) was used to mark
up duplicated reads and to evaluate sequencing completeness.
Realignment around indels and base quality recalibration were
performed using Genome Analysis Toolkit (GATK, v.1.4–9)
(DePristo et al., 2011) to produce analysis-ready alignments.
Depth of coverage was analyzed by the DepthOfCoverage
module of GATK. Sample identities were verified by matching aligned reads to the previously known single nucleotide
polymorphism (SNP) genotypes within the exome targets using verifyBamID (Jun et al., 2012). Single-nucleotide variants
(SNVs) and small insertion deletions (indels) were called by
GATK’s UnifiedGenotyper separately for each sample. Highquality variants were obtained by filtering out raw SNV calls
with the following empirically derived cutoffs: “QUAL<30
QD < 5.0 MQ < 40.0 FS > 60.0 HaplotypeScore
> 13 MQRankSum < -12.5 ReadPosRankSum < -8.0”
and filtering out raw indel calls with “QUAL<30 QC<2.0
ReadPosRankSum < -20.0 FS > 200.0.”
We used ANNOVAR (April 2012 version) (Wang et al.,
2010) to annotate the functional effects (missense, nonsense,
in-frame or frameshift indels, splicing, etc.) for each variant
based on the RefSeq gene annotation (March 2012). Allele frequencies in public databases were queried from dbSNP (build 135) and 1000 Genomes Project (Mar. 2012,
Phase I). The evolutionary conservation at each variant position was measured by Genome Evolutionary Rare Profiling
(GERP) (Cooper et al., 2005; Davydov et al., 2010) and
PhyloP (Siepel et al., 2005) downloaded from the UCSC
genome database. For missense SNVs, the pathogenic effect
predictions from four bioinformatics tools (PolyPhen2, SIFT,
MutationTaster, and LRT) were downloaded from dbNSFP
(Liu et al., 2011).
To identify the disease-causing mutation, we excluded the
variants whose allele frequency was >0.005 in any one of
the populations found in the public databases and variants
appearing more than once in 170 other in-house exomes
(Apr. 2012) using the same data processing protocol. Then
we focused on the functionally interpretable variants: SNVs
that were evolutionarily conserved (GERP>2.0) and causing
missense or nonsense changes, or SNVs that were located
within 2bp of intron-exon boundaries; and indels that caused
in-frame or frameshift alterations.
To test the possible consequence of the amino acid (AA)
substitution within the Limulus factor C, Coch-5b2 and the
Lgl1 (LCCL) domain of Cochlin, the 3D structure of the
human LCCL domain tertiary structure was modeled using SWISS-MO`DEL workspace (http://swissmodel.expasy.
org/). The effect of the AA substitution was evaluated by
DeepView (http://spdbv.vital-it.ch/).
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Annals of Human Genetics (2014) 78,410–423
Linkage and Haplotype Analysis
Thirty-two of the family members (indicated by “+” in
Fig. 1) were genotyped by the Affymetrix whole-genome
SNP 5.0 array which contains 443,818 SNP markers. The
genotype calls were made by the BRLMM-P algorithm using Affymetrix Genotyping Console (v4.0) under a stringent
confidence threshold (0.01). The raw genotypes were initially filtered to exclude SNPs with more than 10% missing
genotypes. Marker allele frequencies were calculated from
197 unrelated Chinese individuals in HapMap 3 (Altshuler
et al., 2010). To get a subset of SNPs with high heterozygosity, we kept only SNPs that had minor allele frequencies
>0.2 in genotyped samples and >0.15 in the Han Chinese
population. The remaining 124,825 SNPs were then thinned
to be evenly distributed at approximately 0.5 cM apart. The
resulting 6603 high-quality SNPs were used for subsequent
analyses. Genetic positions were linear interpolated from the
fine-scale recombination map (Myers et al., 2005).
Pairwise sample relationships were verified using RELPAIR (Epstein et al., 2000). The analysis revealed that IV:16
was a half sib but not a full sib of IV:14,17,18,19,20 as reported by the family members. After correcting the pedigree structure, the likely genotyping errors were identified by
PedCheck (O’Connell & Weeks, 1998) and Merlin (Abecasis
et al., 2002). The cleaned genotype data were obtained by
masking out genotyping errors and excluding markers with
more than 10% missing genotypes.
To accommodate the large family sizes, the parametric linkage analysis using Merlin was performed with an ad hoc approach: three major branches of the pedigree as shown in
Figure 3 were treated as three unrelated families. The overall linkage evidence was then combined from three families.
In the initial round of analysis, a more stringent phenotyping criterion was used to assign affected/unaffected labels
to family members: the unaffected members were defined
using the same rule as described above but with PTA threshold set to 20 dB for both ears, whereas the PTA threshold for affected members was set to 30 dB. No chromosomal regions with suggestive linkage evidence (logarithm of
odds, LOD>2) could be found. After identification of the
disease-causing mutation, we excluded the phenocopy patient and performed the linkage scan again. The linkage evidence at the DFNA22 locus was also evaluated by a MonteCarlo method implemented in Simwalk2 (Sobel & Lange,
1996).
After prioritizing the candidate variants shared by the sequenced patients, we performed haplotype analysis to infer the segregation pattern of candidate variants within the
pedigree. Currently, there is no exact haplotyping method
that can directly handle large pedigrees as in our study.
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A New Chinese DFNA22 Family
Figure 3 Haplotype sharing analysis.
For the two candidate variants discovered from exome sequencing, we reconstructed the haplotypes around the 10 cM flanking region of
MYO6 (A) and COCH (B). The presumed disease haplotype is colored in black. Haplotypes for individuals without genotypes were
inferred from relatives. For each gene, nine flanking markers are displayed for visualization. The nondisease haplotypes for each founder are
colored in dark and light grays. Marker positions are given in mega base pairs. The positions of genes relative to the markers are indicated
by arrows.
We developed a customized solution using Merlin that first
inferred haplotypes on subsets of overlapping individuals
within the pedigree, and then merged those subsets based
on the consensus haplotypes on overlapping individuals.
Haplotypes were graphically visualized using HaploPainter
(Thiele & Nurnberg, 2005).
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Results
Clinical Description
After detailed otology testing and examining the feedback of
questionnaires from all participants, we identified 18 affected
and 11 unaffected (not including spouses) individuals using
Annals of Human Genetics (2014) 78,410–423
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J. Cheng et al.
the criteria given in MATERIALS AND METHODS. None
of the spouses reported subjective hearing impairment. The
details of the clinical information of the 35 family members
who underwent audiological examination are summarized in
Supplementary File 1. By medical history, HL was typically
noticed by patients in their 20s or early 30s. The proband
(IV:35) showed asymmetric HL. In her first visit to our clinic
at the age 36, the HL was 45 dB in the left ear at high
frequencies (2000 Hz), and 55 dB in the right ear from
middle to high frequencies (500 Hz). On her revisit five
years later, her audiogram showed that HL had progressed to
38 and 78 dB mainly at the intermediate frequencies
(5002000 Hz) for the left and right ears, respectively
(Fig. 2A). The HL of most other affected family members
was symmetric, could be characterized by flat or gently downsloping audiograms (examples are given in Figs. 2B and 2C).
One outlier patient (IV:40) was noted: this 38-year-old male
showed highly asymmetric HL starting from 12 years old
(Supplementary File 1). Most patients reported increased HL
severities with age, starting from noticeable HL at the high
frequencies and later involving all frequencies.
Comprehensive medical histories and clinical examination
of these individuals showed no other clinical abnormalities,
including diabetes, cardiovascular diseases, visual problems,
and neurological disorders. Computer tomography scan analysis of the proband also ruled out the presence of inner ear
malformation.
After the identification of the MYO6 mutation, ARTA
were constructed based on the cross-sectional analysis of 17
MYO6 mutation carriers (Fig. 2D). Significant progression
was demonstrated at all frequencies (P < 0.05). The average annual deterioration rate (ATD) was 0.90 dB per year.
The ATD was slowest at 250 Hz with 0.74 dB per year
and fastest at 8000 Hz with 1.25 dB per year. The expected threshold per decade in ARTA showed a mild HL
starting at age 2030 years and evolving to moderate after
40 years.
Genetic Findings
Two patients from the third generation (III:13 and III:23) were
selected for whole-exome sequencing. Both patients showed
similar audiogram profiles suggesting the same genetic etiology. We found that the 26 known ADNSHL genes were well
represented in the exome library; 94.6% of their functional
territories were encompassed by the designed target intervals. About 810 Giga base pairs (Gbp) of raw sequences
were generated for each sample, such that approximately 90%
(80%) of the exome targets were covered by at least 10 (20)
nonduplicated reads with high mapping quality (Table 1). The
coverage on the ADNSHL genes was similar to the over-
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Annals of Human Genetics (2014) 78,410–423
Table 1 The coverage statistics for whole-exome sequencing of the
two affected family members.
Parameter
III:13
III:23
Total sequencing yield (Gbp)
∗
Usable base on targets (Gbp)
Mean depth of coverage† on target
regions
Coverage of exome targets (10X)
Coverage of exome targets (20X)
Mean depth of coverage on ADNSHL
gene regions‡
Coverage of ADNSHL gene regions
(10X)
Coverage of ADNSHL gene regions
(20X)
7.79
3.71
79.5
10.14
4.80
102.6
89.4%
81.9%
78.3
91.4%
85.9%
99.4
87.8%
89.8%
80.2%
82.5%
∗
Refers to the number of bases of aligned duplication-removed reads
that mapped on exome targets.
†
The depth of coverage is defined as the number of high- quality mapped reads (mapping quality >>=17) overlapping the target
bases.
‡
Gene regions are defined as all coding exons plus 10bp intron-exon
boundaries.
all targets. Per-target depth of coverage was highly correlated
between two samples (Pearson correlation coefficient = 0.98).
We identified more than 33,000 and 1800 high-quality
SNVs and indels, respectively, in each patient. Variants were
prioritized after a series of filtering (Table 2). Then we focused
on 200 very rare genic variants that caused alterations to encoded protein sequences in each patient. Three heterozygous
variants in known ADNSHL genes were found, all of which
were absent in both public and in-house databases and subsequently validated by Sanger sequencing. Two of them were
shared by the sequenced patients (Table 3). One missense
variant c.328C>A in exon 5 of COCH (DFNA9) resulted
in a substitution of Glutamine with Lysine at AA position
110. The other was a deletion c. 2814_2815delAA in exon
26 of MYO6 (DFNA22) which caused a frameshift alteration
p. R939Tfs∗ 2 that resulted in a truncated protein. Different
filtering strategies did not provide further candidate variants
for investigation.
Given the initial failure to identify any region with suggestive linkage evidence (LOD>2), we first used haplotype
analysis on whole-genome SNP genotypes to infer the cosegregation of these two candidate variants with phenotypes.
The haplotypes tagging the candidate variants was assumed to
be shared identity-by-descent by two sequenced individuals.
Among 23 genotyped individuals with confirmed phenotypes
(16 affected and seven unaffected), we found that the MYO6
mutation was carried by all except one affected and by no
unaffected individuals (Fig. 3A). The COCH mutation was
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A New Chinese DFNA22 Family
Table 2 Number of variants after each step of filtering.
III:13
III:23
Shared
Filtering step
SNVs
Indels
SNVs
Indels
SNVs
Indels
All high-quality variants
∗
After filtering public variation databases
†
After filtering in-house exome database
After functional effects filtering‡
Variants disrupting ADNSHL genes
33,169
914
601
204
1
1869
526
36
12
1
33,443
922
632
185
2
2065
644
47
16
1
24,196
299
86
36
1
1488
483
10
5
1
∗
Excluding variants having alternative allele frequencies 0.005 in any one of the populations in dbSNP and 1000 Genomes.
Excluding variants appearing at least once in 170 unrelated in-house exomes.
‡
Keeping SNVs that are evolutionarily conserved and cause missense, nonsense changes, or potentially disrupt splice sites; keeping indels that
result in in-frame or frameshift alterations.
†
Table 3 Prioritized variants that are predicted to disrupt ADNSHL genes.
Patients
Gene
III:13,
COCH
III:23
III:13,
MYO6
III:23
III:23
WFS1
only
Variants
effects
change
change
Nonsynonymous
SNV
∗
score Predictions
14:31348105
Missense
NM_004086:exon5:c.328C>A p.Q110K
53
C>A
SNV
6:76599925
Frame-shift NM_004999:exon26:
p.R939Tfs∗ 2
GAA>G
deletion
c.2814_2815delAA
4:6296873 A>C Missense
NM_006005:exon7:c.818A>C p.E273A
107
SNV
-–++
++++
PhyloP† GERP†
4.81
2.83
3.37
1.56
3.76
1.51
∗
Results from four non-synonymous SNV effect prediction algorithms, from left to right: PolyPhen2, SIFT, LRT, MutationTaster. +:
deleterious or damaging, -: benign
†
Measures of evolutionary constraint. PhyloP is the –log10 of P-value for testing the null hypothesis of neutral evolution, based on 46-way
whole-genome alignment of vertebrates. GERP score can be interpreted as the substitutions expected under neutrality minus the number of
substitutions observed at the position, which was derived from 35-way whole-genome alignment of mammals and had a theoretical maximum
of 6.18. For deletions, the score is the average of deleted bases.
carried by 10 affected but also two unaffected (III:5 and IV:33)
individuals (Fig. 3B). Direct genotyping of all samples in the
pedigree showed a consistent segregation pattern with haplotype analysis on the overlapping samples (Fig. 1). The only
patient who did not carry the MYO6 mutation turned out to
be the outlier patient IV:40 who had a distinct HL phenotype
suggesting a different etiological cause. We did not investigate the genetic causes for the HL in patient IV:40 in this
study but considered him as a phenocopy possibly resulting
from nongenetic factors. A recent study also suggested a very
low genetic diagnostic rate for persons with asymmetric HL
(Shearer et al., 2013).
After excluding the phenocopy patient IV:40, we found
that the linkage evidence at the DFNA22 locus increased from
1.60 to 4.51, which was also confirmed by a second method
based on Monte-Carlo simulation (Sobel & Lange, 1996).
The negative finding from the initial linkage scan was likely
caused by the sensitivity of multipoint linkage analysis due
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to model misspecification (Risch & Giuffra, 1992). Therefore, we consider the MYO6 mutation as disease-causing in
this pedigree. The result also revealed a female carrier with
uncertain phenotype (V:12) who still had subjective normal
hearing for her age.
Discussion
In the current study, exome sequencing was applied to resolve the genetic cause in a pedigree in which initial linkage analysis was hindered by possible measurement errors
in phenotyping and by the existence of a phenocopy. The
exome sequencing approach allows an unbiased search for
coding variants directly but also introduces new challenges
in variant interpretation. The discovery of two novel variants in known ADNSHL genes shared by two sequenced
patients in this family was worth noting. The missense mutation in COCH (DFNA9) may also be pathogenic. The
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J. Cheng et al.
mutation changed a highly conserved nucleotide across vertebrate species (PhyloP>4, GERP>2). The resulting AA
substitution Q110K was located within the LCCL domain, where most DFNA9-causing mutations were found
(Supplementary File 2: Fig. A). Molecular modeling suggested
that the affected residue was located on a beta sheet and was
solvent-accessible. The substitution can abolish the hydrogen
bond of glutamine to a nearby aspartic acid at AA position
105 (Supplementary File 2: Figs. B and C). Two substitutions
in the neighboring residue have been reported in DFNA9
families before [I109N (Kamarinos et al., 2001) and I109T
(Pauw et al., 2007)]. Highly variable ages of onset (from 20
to 70) were observed for DFNA9 (Hildebrand et al., 2009);
so the two unaffected carriers of the COCH mutations in our
family (39-year-old female IV:33 and 62-year-old female
III:5) might yet reach their onset ages. Since the mutation was also carried by patient IV:40 who did not carry
the MYO6 mutation, we could tentatively suggest that two
disease-causing variants could collectively account for the
phenotypes of all patients in the family.
However, there were also several lines of evidence against
the pathogenicity of the COCH mutation. First, the only
patient that cannot be explained by the MYO6 mutation
showed a very early age of onset and an asymmetric audiogram, which were also incompatible with the DFNA9
phenotypes. Second, Q110K is located in the LCCL domain of the cochlin protein; familial patients with pathogenic
mutations in this domain have invariably shown different
extents of vestibular symptoms (Hildebrand et al., 2009).
However, none of the affected patients in our family reported an inability to walk in the dark or felt unsteady on
uneven ground, or reported sporadic periods of dizziness.
Only one patient, III:19, had a history of Ménière’s disease but had fully recovered 20 years ago. The vestibular
function of the proband was evaluated by oculomotor test,
computerized dynamic posturography, and vestibular evoked
myogenic potential. No abnormality was detected. Third,
although the affected residue Q110 was highly conserved
across mammals, the mutant allele K matched to the wildtype allele of Limulus Factor C; in contrast, none of the previously reported pathogenic mutations showed this property
(Supplementary File 2: Fig. A). For all DFNA22 patients in
this pedigree, the carrier status of the COCH mutation was
also not a significant predictor of HL level after accounting for the age effect (P > 0.1 across the frequency spectrum by linear regression). Taken together, we would consider the Q110K variant had no clinical significance at this
stage.
We also found a private missense variant p.E273A of the
WFS1 gene (encoding Wolframin) in III:23 (Table 3). The
variant was carried by three affected members (III:19. III:23,
IV:35) and by one with self-reported normal hearing but
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Annals of Human Genetics (2014) 78,410–423
without detailed audiogram (III:25) (Supplementary File 3).
So the co-segregation information alone was not enough
to evaluate the pathogenicity of this variant. We noted
that heterozygous missense mutations in WFS1 are known
to cause DFNA6/14/38 characterized by low-frequency
nonsyndromic HL (LFNSHL) at the onset of the first or
second decade. However, none of the family members who
carried the WFS1 variant was diagnosed or had self-reported
LFNSHL. The known LFNSHL-causing mutations were also
preferentially clustered in the C-terminal domains (WFS1
Gene Mutation and Polymorphism Database: http://www.
khri.med.umich.edu/research/lesperance lab/low freq.php),
whereas the identified variant p.E273A was located in the
N-terminal region outside any functional domain. Therefore,
pathogenicity of the WFS1 mutation could also be rejected.
The MYO6 gene encodes unconventional myosin VI
which is required for structural integrity and proper functioning of inner ear hair cells (Sweeney & Houdusse, 2007).
The myosin VI protein can be divided into three regions:
an N-terminal motor domain, followed by a neck domain
consisting of a linker domain and a single IQ motif, and a Cterminal tail region with a coiled-coil (Sweeney & Houdusse,
2007). The frameshift deletion discovered in this study can
result in a truncated protein at the middle of the coiled-coil
domain (Fig. 4A).
The MYO6 gene is known to be responsible for both
ADNSHL (DFNA22) and ARNSHL (DFNB37). To date,
eleven disease-causing mutations of MYO6 have been reported in different DFNA22 families worldwide; seven
of those had detailed clinical descriptions (Table 4). It
is interesting to note that eight of them were splicing/nonsense/frameshift mutations that led to truncated
protein products. The truncation points seemed randomly
distributed across the protein structure (Fig. 4A). Those mutations could trigger the nonsense mediated decay (NMD)
mechanism to partially eliminate the mutant mRNAs (Hilgert
et al., 2008; Volk et al., 2013). The remaining truncated
myosin VI could not form dimers with the wild-type protein to carry out normal functions (Sweeney & Houdusse,
2007). Therefore, current data suggest the decreased level of
myosin VI or haplo-insufficiency as the major pathogenic
mechanism for DFNA22, which was also discussed by other
authors (Topsakal et al., 2010; Volk et al., 2013). In supporting this notion, Becker et al. (2012) recently described two
patients with 6q14 deletion syndrome who had only one copy
of the functioning MYO6 gene. Neither patient had HL at
birth, but following up one female patient revealed HL in
her 20s (Becker et al., 2012). Seven other newborn patients
carrying copy number deletions overlapping the MYO6 gene
were also reported in the literature (Becker et al., 2012). Although only two of them were diagnosed with HL at birth,
no phenotypes at their adult ages were available. Furthermore,
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2014 John Wiley & Sons Ltd/University College London
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AD
AD
AD
AD
AD
AD
American
American‡
Italian
Japan‡
Belgian
Danish
AD
AD
Chinese‡
Dutch
AD
AD
Korea‡
Chinese
AD
German
Inheritance
p.H246R
p.R205∗
p.A185Efs∗ 17†
Aminoacid
change
c.3610C>T
c.2944C>T
c.2814_2815delAA
c.2545C>T
c.2416+2428T>G
c.1975C>T
c.1325G>A
p.R1204W
p.Q982∗
p.R939Tfs∗‡
p.R849∗
p.K807Vfs∗ 19
p.R659∗
p.C442Y
c.866_869delAACA p.K289Rfs∗ 17
c.737A>G
c.613C>T
c.554–1G>A
Nucleotide
change
Age of
onset
-
2nd and 3rd
decade
839 years old
Third decade
-
810 years old
-
Progressive, mild mean onset age
to severe or
38 years,
profound
range 26–46
Moderate to
profound
Moderate to
severe
Progressive, mild
to severe
Progressive,
moderate at
mid-30s
Progressive,
mostly
moderate
-
-.
Progressive, mild School age
to profound
Mild to
moderate
Progressive, mild First decade
to moderate
Severity
of hearing
impairment
Flat,
down-sloping
later in life
Typically
down-sloping
or flat
Gently
down-sloping
or flat
-
U-shaped or flat
Flat
Down-sloping
Flat, Gently
down-sloping
or ascending
-
Down-sloping
or flat
Flat
Shape of
audiogram
No major
abnormalities
-
No subjective
symptom
No subjective
dysfunction
No
-
No
-
-
-
No
Vestibular
symptoms
The resulting protein change is predicted assuming exon 7 skipping. Volk et al. (2013) indeed reported four possible consequences of the splicing change, most of which
resulted in frameshift changes starting at p.A185.
‡
These four families were discovered from recent large-scale targeted resequencing studies.
†
Yang et al.
(2013)
Oonk et al.
(2013)
This study
Shearer et al.
(2010)
Melchionda et
al. (2001)
Miyagawa et al.
(2013)
Hilgert et al.
(2008)
Sanggaard et al.
(2008)
Volk et al.
(2013)
Choi et al.
(2013)
Mohiddin et al.
(2004)
Reference
Family of
origin
Table 4 A summary of reported mutations causing DFNA22.
A New Chinese DFNA22 Family
Annals of Human Genetics (2014) 78,410–423
419
J. Cheng et al.
A
B
Figure 4 Previously reported DFNA22-causing mutations.
(A) The schematic representation of the myosin VI protein consisting of motor, IQ, and coiled-coil
domains. The relative positions of the protein alterations predicted by the reported disease causing
mutations are shown mapped on to the protein structure. Details for each mutation are given in
Table 4. Three missense mutations are shown in bold. The mutation discovered in this study is
enclosed in a bounding box. The corresponding DFNA22 family summarized by Topsakal et al.
(2010) was labeled in parentheses following the mutation. (B) The threshold feature arrays of ARTA
of DFNA22 families following Topsakal et al. (2010). The cell counts are plotted above the table. The
curves for the five previously reported pedigrees are shown in gray. One of the families (BEL2) used
in this comparison was reported by Hilgert et al. (2008) (the second family in that study) for which
they found complete linkage at the DFNA22 locus but no mutation in the coding region of the
MYO6 gene.
Hilgert et al. (2008) also reported a Belgian DFNA22 family
for which they found no mutation in the coding region, but
quantitative assessment of the mRNA levels showed overexpression of the MYO6 gene in the patients. Combining
420
Annals of Human Genetics (2014) 78,410–423
the data from this Belgian family, it suggests that the MYO6
gene has a dosage sensitive function in the inner ear for maintaining normal hearing. Further ascertainment of DFNA22
families and follow-up otology examinations for newborn
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2014 John Wiley & Sons Ltd/University College London
A New Chinese DFNA22 Family
patients with copy number alterations at the MYO6 locus
will be useful to test this hypothesis.
Regarding the genotype-phenotype correlations, it was
previously suggested that HLs caused by the dosage changes
of myosin VI often exhibit a less severe phenotype than
HLs resulting from missense mutations in the motor domain, which presumably act in a dominant negative manner
(Topsakal et al., 2010). The HLs in patients of our family
were found to be sensorineural, mostly symmetrical, with
onset at 2035yrs, progressing to moderate to severe at older
ages. This was fully consistent with observations in three
other families caused by mutations predicted to result in
truncated proteins lacking the myosin tail (Table 4). We also
compared the ARTA of our family with those of five other
DFNA22 families summarized by Topsakal et al. (2010) using the threshold feature array method (Huygen et al., 2003)
(Fig. 4B). χ 2 tests in pairwise comparisons showed no significant differences (P > 0.1) for all except the Italian family
(Melchionda et al., 2001) in which HL was caused by a
missense mutation at the motor domain. Comparison to a
recently reported German family (Volk et al., 2013) is difficult, because only six patients with the most prominent
phenotypes were shown. However, the data seemed to suggest that a truncation at the motor domain could lead to early
onset ages and severe HL levels. More data would be required
to delineate the genotype–phenotype correlation due to protein truncation mutations.
Conclusions
In summary, our exome sequencing study has led to the discovery of a new DFNA22 family, and supported the emerging
genotype–phenotype correlations for DFNA22. The discovery of two novel mutations in ADNSHL genes also illustrated
that although exome sequencing is a powerful tool for molecular diagnosis of genetically heterogeneous disease, correct interpretation of novel variants can still be challenging and can
require considerable skills.
Author Contributions
HY, DH, and MQZ designed the study. YL, BH, and YZ
collected the phenotype data and blood samples. LL performed the exome capture and high-throughput sequencing
experiments. KC contributed reagents. X Zhou performed
the bioinformatics and statistical analysis. JC and YL carried out the Sanger sequencing validation and co-segregation
analyses. JC and X Zhou interpreted the results under the
supervision of YH, MQZ, and X Zhang. X Zhou drafted
the manuscript with input from JC and YL. X Zhang and
MQZ revised the manuscript. All authors read and approved
the final manuscript.
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2014 John Wiley & Sons Ltd/University College London
Acknowledgements
We sincerely thank all the family members for their participation in this study. These investigations were supported
by NSFC Key Project (81030017), National Science Fund
for Distinguished Young Scholars (81125008) to HY, NSFC
grant (81271091) to JC, the National Basic Research Program (2013CB945402 to DH and HY, 2012CB316503,
2012CB316504 to X Zhang and MQZ), and NSFC grant
(91010016) to X Zhang and MQZ.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Supplementary File 1 - A summary of clinical data for 35
family members who underwent otology examination.
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2014 John Wiley & Sons Ltd/University College London
Supplementary File 2 – Reported DFNA9-causing
mutations and molecular modeling of COCH p.
Q110K.
(A) Multiple sequence alignment of the LCCL domain of
the cochlin. The locations of the α-helix and β-strands are
shown at the bottom following (Liepinsh et al., 2001). Positions of previously reported disease causing mutations are
shown above the sequence and indicated by the red arrows.
Those mutations were previously reviewed by Hildebrand et
al. (2009) in their Table 1. The position of Q110K identified in this study is indicated by a blue arrow. (B) The
ribbon representations of the LCCL structures superimposed
by the Q110 residue and its interaction partner D105 on
the wild-type domain. (B) The substituted residue K110
has no hydrogen bond to D105 or to other neighboring
residues.
Supplementary File 3 – Segregation of the WFS1 variant in
the pedigree.
Pedigree symbols are the same as Figure 1.
Received: 4 June 2014
Accepted: 28 July 2014
Annals of Human Genetics (2014) 78,410–423
423