575055

research-article2015

AORXXX10.1177/0003489415575055Annals of Otology, Rhinology & LaryngologyMiyagawa et al

Article

Massively Parallel DNA Sequencing Successfully Identified Seven Families With Deafness-Associated MYO6 Mutations: The Mutational Spectrum and Clinical Characteristics

Annals of Otology, Rhinology & Laryngology 2015, Vol. 124(5S) 148S­–157S © The Author(s) 2015 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003489415575055 aor.sagepub.com

Maiko Miyagawa, MD, PhD1,2, Shin-ya Nishio, PhD1,2, Kozo Kumakawa, MD, PhD3, and Shin-ichi Usami, MD, PhD1,2

Abstract Objectives: To elucidate the involvement of MYO6 mutations, known to be responsible for DFNA22/DFNB37, in Japanese hearing loss patients through the use of genetic analysis. Methods: Genomic variations responsible for hearing loss were identified by massively parallel DNA sequencing (MPS) of 63 target candidate genes in 1120 Japanese hearing loss patients, and the detailed clinical features for the patients with MYO6 mutations were collected and analyzed. Results: Four mutations were successfully found in 7 families exhibiting autosomal dominant inheritance. All of the patients showed progressive hearing loss, but hearing type and onset age varied. Further, none of the affected patients showed any associated symptoms, such as hypertrophic cardiomyopathy or retinitis pigmentosa. Conclusions: MPS is powerful tool for the identification of rare causative deafness gene mutations, such as MYO6. The clinical characteristics noted in the present study not only confirmed the findings of previous reports but provided important new clinical information. Keywords MYO6, DFNA22, DFNB37, hearing loss, genetics, massively parallel DNA, next generation sequencing

Introduction Deafness is known to show a high degree of genetic heterogeneity. Autosomal recessive sensorineural hearing loss (ARSNHL) is the most common form of hereditary hearing loss, accounting for 80% of all inherited hearing loss. Autosomal dominant sensorineural hearing loss (ADSNHL) accounts for the remaining 20% of hereditary hearing loss.1 More than 80 genes responsible for hearing loss have already been reported. Among them, myosin genes are known to be involved in 10 types of syndromic and nonsyndromic hearing loss (MYO7A, DFNA11/DFNB2/USH1B; MYH9, DFNA17; MYH14, DFNA4; MYO6, DFNA22/ DFNB37; MYO3A, DFNB30; MYO15A, DFNB3). MYO6, located on chromosome 6q13, is known to be responsible for DFNA22 and DFNB372,3 and encodes mysoin VI, which is an actin-based motor responsible for the transport of intracellular cargos including proteins, vesicles, and organelles.4 The high concentration of myosin VI at the stereocilia rootlets of the inner and outer hair cells led to the suggestion that it has a crucial role in the maintenance of the cuticular-plate anchoring of stereocilia rootlets.5-7

Avraham et al8 were the first to report evidence for the involvement of myosin VI in the hearing process. In spite of its crucial function in the inner ear, genetic screening using Sanger sequencing for MYO6 has lagged, probably due to its huge size. To date, only 14 mutations in DFNA22 and 3 mutations in DFNB37 have been reported (Table 1). Consequently, the detailed clinical characteristics are not yet fully understood. Recent progress in MPS based on next-generation sequencing technology has resolved these difficulties and has successfully identified causative mutations in various rare genes.9 1

Department of Otorhinolaryngology, Shinshu University School of Medicine, Matsumoto, Japan 2 Department of Hearing Implant Sciences, Shinshu University School of Medicine, Matsumoto, Japan 3 Department of Otorhinolaryngology, Toranomon Hospital, Tokyo, Japan Corresponding Author: Shin-ichi Usami, MD, PhD, Department of Otorhinolaryngology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. Email: [email protected]

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Motor domain Motor domain Motor domain Motor domain

Motor domain

IQ motif

 9  9 10 13

19

  24

Globular domain NM_004999

Globular domain NM_004999 Globular domain NM_004999

Globular domain NM_004999 NM_004999 Motor domain NM_004999

Globular domain NM_004999

32

34 34

35  2  8

34

NM_004999 NM_004999 NM_004999

26 27 28

NM_004999

NM_004999 NM_004999

NM_004999

Between IQ and coil domain Coiled coil Coiled coil Coiled coil

25

NM_004999

Motor domain

 8

NM_004999 NM_004999 NA NM_004999

NM_004999

NM_No.

Motor domain

Domain

 8

Exon

c.3496C>T

c.3667G>A c.36-37insT c.647A>T

c.3496C>T c.3610C>T

c.3361A>T

c.2752insA c.2944C>T c.2971C>T

c.2545C>T

c.2417-1758T>G c.2473C>T

c.1975C>T

c.737A>G c.862_865delACAA c.884_893delGCAAAGTCC c.1325C>T

c.614G>A

c.613C>T

Nucleotide Change

p.R1166X

p.E216V

p.D1223N

p.R1166X p.R1204W

p.K1121X

p.Q918fsX941 p.Q982X p.R991X

p.R849X

p.R825X

p.R659X

p.H246R p.D288DfsX17 p.R295LfsX13 p.C442Y

p.R205Q

p.R205X

Amino Acid Change

Table 1.  MYO6 Pathogenic Mutations in Autosomal Dominant Sensorineural Hearing Loss.

Mild Moderate to profound NA Profound Severeprofound Profound

Mild to moderate Mild to severe NA NA Moderate to profound Mild to profound Mild to severe Moderate to profound Moderate to profound Moderate NA Mild to moderate Mild

NA

Audiogram Configration

Korean

Family Origin

Firstdecade/50 y. Korean/ Japanese 5-54 y American NA American Prelingual German Childhood Italian

NA

Onset Age

NA

NA NA NA

High frequency involved Flat All frequencies

Flat NA Down-sloping

All frequencies

Congenital

NA Congenital Congenital

29_35y.o. 5y

26 y

Fifth decade NA Second decade

21 y

Pakistani

American Pakistani Pakistani

Japanese Dutch

Japanese

Korean Chinese Korean

Danish

Flat/high frequency First/second/fifth Japanese involved decade U-shaped or flat Third decade Down-sloping NA Korean

Flat/high frequencies High frequency involved Down-sloping NA Flat Down-sloping

Significant Progression

 3

28  3  3

This study 25

This study

22 32 22

24

31 22

9, this study

23 28 29 30

22, this study

27

Reference

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Table 2.  Clinical Features of the 7 Families. Family No. Patient ID

Nucleotide Amino Acid Change Change Age Onset Age

1 2 3 3 3 4 5 6 7

c.614G>A c.1975C>T c.1975C>T c.1975C>T c.1975C>T c.3361A>T c.3496C>T c.3496C>T c.3496C>T

4461 3667 4536 4535 4538 JHLB1235 JHLB0530 JHLB0193 JHLB0315

p.R205Q p.R659X p.R659X p.R659X p.R659X p.K1121X p.R1166X p.R1166X p.R1166X

65 76 15 43 13 41 41 32 37

50 50 9 28 Precritical 26 29 31 35

Audiogram Configuration

Hearing Level (dB)

Progression

Intervention

High frequencies High frequencies Low frequencies Flat Normal High frequencies Flat High frequencies High frequencies

61.3 102.5 50 70 17.5 43.8 42.5 33.8 25

Progressive Progressive Progressive Progressive Unknown Progressive Progressive Progressive Progressive

Hearing aid Cochlear implant Hearing aid Hearing aid None NA Hearing aid None None

In this study, we used MPS technology to identify and characterize a number of Japanese families with MYO6 mutations. Herein, we present the detailed clinical characteristics of the patients with MYO6 mutations and discuss appropriate forms of intervention.

Subjects and Methods Subjects A total of 1120 Japanese hearing loss patients (ADSNHL, 266; ARSNHL, 600; unknown, 254) from 53 otolaryngology departments nationwide participated in this study. Written informed consent was obtained from all subjects (or from their next of kin, caretaker, or guardian in the case of minors/children) prior to enrollment in the project. This study was approved by the Shinshu University Ethical Committee as well as the respective ethical committees of the other participating institutions.

Amplicon Library Preparation Amplicon libraries were prepared using an Ion AmpliSeq Custom Panel (Applied Biosystems, Life Technologies, Carlsbad, California, USA), in accordance with the manufacturer’s instructions, for 63 genes reported to cause nonsyndromic hearing loss. The detailed protocol has been described elsewhere.10 After preparation, the amplicon libraries were diluted to 20 pM, and equal amounts of 6 libraries for 6 patients were pooled for 1 sequence reaction.

Emulsion PCR and Sequencing Emulsion PCR and sequencing were performed according to the manufacturer’s instructions. The detailed protocol has been described elsewhere.10 MPS was performed with an Ion Torrent Personal Genome Machine (PGM) system using an Ion PGM 200 Sequencing Kit and an Ion 318 Chip (Life Technologies).

Tinnitus Vertigo + + + − − + − − −

− + − − − − − − −

Base Call and Data Analysis The sequence data were mapped against the human genome sequence (build GRCh37/hg19) with a Torrent Mapping Alignment Program. After sequence mapping, the DNA variant regions were piled up with Torrent Variant Caller plug-in software. After variant detection, their effects were analyzed using ANNOVAR software.11,12 The missense, nonsense, insertion/deletion, and splicing variants were selected from among the identified variants. Variants were further selected as less than 1% of (1) the 1000 genome database,13 (2) the 6500 exome variants,14 (3) the Human Genetic Variation Database (data set for 1208 Japanese exome variants),15 and (4) the 269 in-house Japanese normal hearing controls. The pathogenicity of missense variants was predicted using the following functional prediction software; PhyloP,16 Sorting Intolerant from Tolerant (SIFT),17 Polymorphism Phenotyping (PolyPhen2),18 LRT,19 MutationTaster,20 and GERP++.21 Candidate mutations were confirmed by Sanger sequencing, and the responsible mutations were identified by segregation analysis using samples from among each patient’s family members.

Results Detected Mutations Three nonsense mutations, c.1975C>T (p.R659X), c.3361A>T (p.K1121X), and c.3496C>T (p.R1166X), and 1 missense mutation, p.R205Q (c.614G>A), were detected in the MYO6 gene in the 7 ADSNHL families (Table 2). All detected mutations were confirmed by Sanger sequencing and were predicted to be pathological by several software programs. Segregation analysis confirmed them to be plausible diseasecausing mutations. Among the mutations identified in this study, p.R659X (c.1975C>T) in family 3 had been reported in our previous study.9 In the present study, we found 1 additional family

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Family 1

I 1

2

II 1

2

3

5

4

6

4461

III 1

2

3

c.[614G>A];[=]

4

5

6

IV 1

B

2

3

C CAGTCGATTTG

Frequency (Hz)

-20

CAGTCRATTTG

125

250

500

1,000 2,000 4,000 8,000

-10

Hearing threshold (dB)

0

WT

c.614G>A

10 20 30 40 50 60 70 80 90 100 110 120

III-2; 65 y.o.

Figure 1.  Family 1. (A) The family tree of Patient 4461 (a 65-year-old female). (B) The results of Sanger sequencing. (C) Audiogram of the family members, showing high frequency–involved progressive hearing loss.

carrying the same mutation (family 2). Two mutations (c.614G>A [p.R205Q] and c.3496C>T [p.R1166X]) had already been reported; c.614G>A (p.R205Q) as a cause of DFNA1022 and c.3496C>T (p.R1166X) as a cause of DFNB37.3 c. 3361A>T (p.K1121X) was identified as a novel causative mutation.

Clinical Findings The clinical features for the 7 families are described in Figures 1, 2, 3, and 4 and Table 2. All pedigrees showed a typical autosomal dominant inheritance pattern, and all affected patients displayed progressive, symmetrical sensorineural hearing loss.

Family 1 (Figure 1: ID 4461). Patient 4461 (a 65-year-old female) had a heterozygous missense mutation, c.614G>A (p.R205Q). The pedigree showed a completely autosomal dominant hereditary pattern. Due to the rapid progression of her hearing loss from 50 years of age, Patient 4461 used hearing aids. She had experienced no associated episodes of vertigo but had a history of tinnitus. Family 2 (Figure 2: ID 3667). Patient 3667 (a 76-year-old female) possessed a c.1975C>T (p.R659X) mutation. Hearing in her left ear had been lost as the result of measles when she was 3 years of age, and she noticed hearing loss in her right ear at around 50 years. She began to experience

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Family 2 I 1

2

3667

II 1

2

3

4

c.[1975C>T];[=]

B

C

6

7

8

9

Frequency (Hz) -20

125

250

500

1,000 2,000 4,000 8,000

-10

Hearing threshold (dB)

AACTTCGAAGT AACT T Y G A A G T

5

0 10 20 30 40 50 60 70 80 90

100

WT

c.1975C>T

110 120

II-4; 76 y.o.

Figure 2.  Family 2. (A) The family tree of Patient 3667 (a 76-year-old female). (B) The results of Sanger sequencing. (C) Audiogram of Patient 3667.

vertigo at around age 60. She eventually underwent cochlear implantation in the right ear at the age of 71. Her mother also had progressive hearing loss. Family 3 (Figure 3: ID4536, 4535, and 4538).  Patient 4536 (a 15-year-old female) had a heterozygous c.1975C>T (p.R659X) mutation. Her hearing loss was first detected during a school physical examination at 9 years of age, and she was diagnosed with sensorineural hearing loss involving the low frequencies. The patient’s hearing thereafter deteriorated over a 6-year period (Figure 3B). Average hearing (500, 1000, 2000, and 4000 Hz) fell from 33.8 dB at age 9 to 50 dB at age 15. Her otoacoustic emission (OAE) response was normal at age 11 but had disappeared by age 15. She showed normal vestibular function as evaluated by caloric test and vestibular evoked myogenic potential (cVEMP) (Figure 3C). Because of the progressiveness of her hearing loss, she started using a hearing aid at 14 years of age. Her father (patient 4535, II-2) also had progressive hearing loss. His average hearing (500, 1000, 2000, and 4000 Hz) fell from 61.3 dB at age 39 to 70 dB at age 43. He did not experience vertigo, but his caloric test and cVEMP showed bilateral areflexia (Figure 3C). Her grandfather (I-2) possessed the same

mutation, and anamnestic evaluation also revealed progressive hearing loss. Those 3 patients became aware of the progressive nature of their hearing loss around the second or third decade, and subsequently started wearing hearing aids. Her grandfather developed profound hearing loss at the age of 65. Her younger brother (patient 4538, III-2) carried the same mutation, but his audiogram appeared to be normal. Family 4 (Figure 4: JHLB1235). A 41-year-old female (JHLB1235) was identified with a c.3361A>T (p.K1121X) mutation that was compatible with autosomal dominant hearing loss. Her hearing loss had started when she was 26 years old and thereafter slowly progressed to involve the high frequencies. Her mother also had high frequency– involved hearing loss. Family 5 (Figure 5: JHLB0530). Patient JHLB0530 (a 41year-old female) experienced the onset of hearing loss at age 29 and started to wear a hearing aid at age 39 due to the progressive nature of her hearing loss. Her mother and her younger sister also suffered hearing loss. c.3496C>T (p.R1166X) mutations were identified in all affected members of the family.

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Family 3 I 2

1

c.[1975C>T];[=]

3

4535

4

4537

II 2

1

3

c.[1975C>T];[=]

4

c.[=];[=]

4536

4538

III 2

1

c.[1975C>T];[=]

B

Frequency (Hz)

Frequency (Hz) 125

250

500

1,000 2,000 4,000 8,000

-20

125

250

Frequency (Hz)

1,000 2,000 4,000 8,000

-20 -10

0

0

0

10

10

10

20 30 40 50 60 70 80

Hearing threshold (dB)

-10

20 30 40 50 60 70 80

60 70 80 90

100

110

110

110

120

120

120

250

500

II -3

Frequency (Hz)

1,000 2,000 4,000 8,000

-20

125

250

500

1,000 2,000 4,000 8,000

-20

-10

-10

0

0

0

10

10

10

20 30 40 50 60 70 80

Hearing threshold (dB)

-10

Hearing threshold (dB)

Hearing threshold (dB)

125

20 30 40 50 60 70 80

250

500

1,000 2,000 4,000 8,000

30 40 50 60 70 80 90

90

100

100

100

110

110

110

120

120

120

III-1; 15 y.o.

125

Frequency (Hz)

20

90

III-1; 9 y.o.

1,000 2,000 4,000 8,000

50

100

-20

500

40

100

II-2; 43 y.o.

250

30

90

II-2; 39 y.o.

125

20

90

Frequency (Hz)

C

500

-10

Hearing threshold (dB)

Hearing threshold (dB)

-20

c.[1975C>T];[=]

III-2; 11 y.o.

4536 n23

Left

Right

n23

p13 p13

Stim

Right

100µV

Left 2 sec

10m sec

4535 Right

Left

Right Stim

Left 50µV 10m sec

2 sec

Figure 3.  Family 3. (A) The family tree of Patient 4536 (a 15-year-old female), her grandfather, father, and younger brother. (B) Audiograms of the family members. (C) Vestibular examination of the patient and her father (Patient 4536 and 4535).

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Discussion Frequency

Family 4

MPS is the most powerful method available for screening genetic causes in small families. Using this technology, we successfully identified 4 MYO6 mutations in 7 families. In this study, MYO6 mutations were found in 0.6% (7/1120) of bilateral hearing loss probands and 2.6% (7/266) of autosomal dominant hearing loss patients. The frequency of the patients with MYO6 mutations suggested that these mutations are not common, like GJB2 or SLC26A4, but are also not extremely rare, as multiplex families were found in our series. Our results provide evidence of the benefits of MPS in identifying rare causative gene mutations such as MYO6.

I 2

1

JHLB-1235

II 1

c.[3361A>T];[=]

B

ACAAGAAGAGA ACAAGWAGAGA

WT

C

c.3361A>T

Frequency (Hz) -20

125

250

500

1,000 2,000 4,000 8,000

-10

Hearing threshold (dB)

0 10 20 30 40 50 60 70 80 90 100 110 120

II-1; 41 y.o.

Figure 4.  Family 4. (A) The family tree of Patient JHLB1235 (a 41-year-old male). (B) The results of Sanger sequencing. (C) Audiograms of Patient JHLB1235.

Family 6 (Figure 5: JHLB0193). Patient JHLB0193 (a 32year-old male) possessed a c.3496C>T (p.R1166X) heterozygous mutation and first noticed his hearing impairment when he was 31 years old. His audiogram showed high frequency–involved mild hearing loss. His mother also started wearing hearing aids when she was 40 years old. Family 7 (Figure 5: JHBL0315). Patient JHBL0315 (a 37year-old male) showed mild progressive hearing loss from age 35. He was identified with a c.3496C>T (p.R1166X) mutation. His pedigree is compatible with an autosomal dominant inheritance pattern. Hearing loss in the affected members of his family generally began in the fourth or fifth decade and progressed slowly thereafter.

Mutational Spectrum Three out of the 4 mutations identified in this study have already been reported as causative mutations3,9,22; c.1975C>T (p.R659X) from a Japanese family, c.614G>A (p.R205Q) from a Korean family, and c.3496C>T (p.R1166X) from a Pakistani family. Further study to clarify whether they represent mutational hot spots or possess a common ancestor is required. The 4 mutations found in this study were not found in publicly available databases (the 1000 genome database or the 6500 exome variants). The missense mutation, c.614G>A (p.R205Q), was predicted to be pathologic by several software programs (PhyloP, 2.396; SIFT, 1; Polyphen2_HDIV, 1; LRT, 0; Mutation Taster, 1; GERP++, 5.13). Seventeen mutations have already been reported in the MYO6 gene. As shown in Table 1, the majority of mutations are nonsense mutations, with 3 out of the 4 mutations found in this study also found to be nonsense mutations. c.3496C>T (p.R1166X) has been reported to be present in autosomal recessive families. Ahmed et al3 reported 5 patients from a consanguineous family with profound sensorineural hearing loss who were identified with homozygous c.3496C>T (p.R1166X). However, 4 family members with heterozygous c.3496C>T (p.R1166X) were described as normal. In the present study, the same mutation was found in 3 ADSNHL families (Families 5, 6, and 7). Although the size of the families was insufficient for conclusive analysis, segregation analysis suggested that it is a plausible diseasecausing mutation. The phenotype of the affected family members is similar to that other patients with DFNA22, supporting the pathogenicity of this mutation. The discrepancy between these 2 studies remains to be clarified.

Clinical Characteristics In previous studies, hearing loss caused by MYO6 was found to be progressive in nature; however, no detailed

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Family 5

I

1

2

3

6

4

7

5

8

c.[3496C>T];[=]

c.[=];[=]

JHLB-0530

II 1

2

3

c.[3496C>T];[=]

Family 6

4

c.[3496C>T];[=]

Family 7

I

I 1

2

1

2

2

3

II

II 1

3

2

1

4

JHLB-0193

JHLB-0315

III

III 1

c.[3496C>T];[=]

2

3

1

2

c.[3496C>T];[=]

B TGAACCGACAG

TGAACYGACAG

WT

c.3496C>T

C

Family 5 125

250

Frequency (Hz) 500

Family 6

Frequency (Hz)

1,000 2,000 4,000 8,000

-20

125

250

500

Family 7

Frequency (Hz)

1,000 2,000 4,000 8,000

-20

-10

-10

0

0

0

10

10

10

20 30 40 50 60 70 80

Hearing threshold (dB)

-10

Hearing threshold (dB)

Hearing threshold (dB)

-20

20 30 40 50 60 70 80

50 60 70 80

90

90 100

110

110

110

III-1; 32 y.o.

1,000 2,000 4,000 8,000

40

100 120

500

30

90

II-4; 41 y.o.

250

20

100 120

125

120

III-2; 37 y.o.

Figure 5.  Families 5, 6, and 7. (A) The family trees of Patient JHLB0530 (Family 5), JHLB0193 (Family 6), and JHLB0315 (Family 7). (B) The results of Sanger sequencing. (C) Audiograms of the patients.

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clinical details were available. In this study, progressive hearing loss was proven by serial audiograms. The rate of progression was about 2.7 dB/y for Patient 4536 (15 years old) and 2.2 dB/y for Patient 4535 (43 years old). Patient 3667 (76 years old) with the same mutation received cochlear implantation (CI) and showed profound hearing loss, indicating that hearing loss in patients with MYO6 mutations may be progressive in nature. There is striking evidence that the OAE response deteriorates with age. This is the first objective evidence to show both a gradual decline in outer hair cell function and that this hair cell dysfunction may lead to progressive hearing loss. Previous studies presented variations in audiogram configuration (flat, U-shaped, down-sloping), onset age, and associated symptoms (Table 1). The 9 patients reported showed various audiogram configurations and did not share any particular tendencies. With regard to genotype-phenotype correlations, no particular correlations were found in this study. For example, the c.614G>A (p.R205Q) mutation reported in the Korean population showed first decade onset and a U-shaped or flat audiogram.22 Patient 4461 with the same mutation, however, showed late onset (around 50 years of age) and high frequency–involved hearing loss. MYO6 is also expressed in the vestibular endorgans and Myo6 mutant mice have vestibular dysfunction.8,23 However, none of the patients reported in the previous literature showed vestibular symptoms, except 1 family (DFNB37).3 In this study, only 1 aged patient (Patient 3667, 76 years old) had vestibular symptoms. We succeeded in performing a detailed vestibular examination for 2 patients. Caloric test and cVEMP failed to reveal any abnormal findings in Patient 4536 (15 years old) with mild hearing loss. Her father (4535, 43 years old), however, had severe hearing loss and showed bilateral vestibular dysfunction. Vestibular symptoms are often masked by vestibular compensation, and the present results suggested that vestibular dysfunction may become evident when associated with progressive hearing loss. Due to a lack of evidence, further investigation is still required to clarify the existence of vestibular involvement in patients with MYO6 mutations.

Intervention Patient 4536 received CI at age 71. Due to her associated diseases, Parkinson’s disease and dementia, the outcome of the CI was not satisfactory (26% in monosyllable test at 70 dBSPL). However, a review of the literature indicates that the outcomes for CI are generally good.24,25 As MYO6 is predominantly expressed within the cochlea, an intracochlear etiology appears to be compatible with a good outcome for CI.

Associated Symptoms MYO6 is also expressed in the retina and heart.23,26 Although rare, associated symptoms, such as hypertrophic cardiomyopathy in a DFNA22 family23 and retinitis pigmentosa in a DFNB37 family,3 have been reported. Most of the patients with MYO6 were found to have nonsyndromic hearing loss. In this study, none of the affected patients suffered retinitis pigmentosa or cardiac disorders. In conclusion, the present findings provide additional clinical information for patients with deafness-associated MYO6 mutations. Furthermore, next-generation sequencing-based genetic testing was shown to be a valuable tool for the identification of causative mutations in relatively rare gene mutations such as MYO6. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a Health and Labour Sciences Research Grant for Research on Rare and Intractable Diseases (H24Nanchiou(Nan)-Ippan-032) and Comprehensive Research on Disability Health and Welfare (H25-Kankaku-Ippan-002) from the Ministry of Health, Labour and Welfare of Japan (S.U.) and by a Grant-in-Aid for Scientific Research (A) (22249057) from the Ministry of Education, Science and Culture of Japan (S.U.).

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