REVIEW

Value of Genetic Testing in the Otological Approach for
Sensorineural Hearing Loss
Tatsuo Matsunaga

Department of Otolaryngology, Laboratory of Auditory Disorders,National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan(Received for publication on January 13, 2009)(Revised for publication on May 10, 2009)(Accepted for publication on June 25, 2009)

Abstract
Sensorineural hearing loss (SNHL) is one of the most common disabilities in human, and genetics is an important aspect for SNHL, especially in children. In recent 10 years, our knowledge in genetic causes of SNHL has made a significant advance, and now it is used for diagnosis and other clinical practices. Hereditary hearing loss can be classified into syndromic andnonsyndromic hearing loss. As the nonsyndromic deafness genes, more than 100 loci for deafness genes have been determined, and more than 40 genes were identified. Furthermore, morethan 300 forms of syndromic hearing loss have been characterized, and each syndrome may
have several causative genes. In childhood hearing loss, early educational intervention is required in addition to medical intervention for normal development of speech and language. In
addition, even severe to profound hearing loss may be restored very effectively by hearing aids
or cochlear implants. Because of these features of SNHL, genetic testing has exceptionallyhigh value in the medical practice for hereditary hearing loss. Several strategies are used forgenetic testing of SNHL for accurate and efficient identification of the genetic causes, and theresults were used for explanation of the cause, prediction of auditory features, prevention ofdeafness, management of associated symptoms, determination of therapy, and genetic counseling. Identification of damaged cells in the inner ear and the underlying mechanism by genetic
testing undoubtedly facilitates development and introduction of novel and specific therapies todistinct types of SNHL. (Keio J Med 58 (4) : 216－222, December 2009)

Keywords: hereditary hearing loss, deafness gene, inner ear, cochlea
Introduction
Sensorineural hearing loss (SNHL) is one of the mostcommon disabilities in human, and genetics is an important aspect in research and clinical practice for SNHL.
One child in 1000 is born with bilateral SNHL, and50-70% of them have monogenic causes.1, 2 In addition,
10% of the people over 65 years have SNHL that interfere speech communication. 3 Although most of them
have polygenic causes associated with aging and various
environmental causes, some of them have monogenic
causes. In recent 10 years, our knowledge in monogenic

causes of SNHL has made a significant advance. The
knowledge of genetics in SNHL was originally established in the laboratory, but it is now used for genetic
testing and following clinical procedures for patientswith SNHL.
Classification of Hereditary Hearing Loss
Hereditary hearing loss can be classified into syndromic and nonsyndromic hearing loss. 4 Syndromic typewhich is associated with distinctive clinical features accounts for 30% of hereditary congenital hearing loss, andPresented at the 1589th meeting of The Keio Medical Society in Tokyo, October 14, 2008.
Reprint requests to: Tatsuo Matsunaga, MD, PhD, Department of Otolaryngology, Laboratory of Auditory Disorders, National Institute of Sensory Organs,
National Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro, Tokyo152-8902, Japan, E-mail: matsunagatatsuo@kankakuki.go.jp216Keio J Med 2009; 58 (4): 216－ 222

217

Deafness Genes

Table 1 Identified deafness genes
Autosomal dominant loci and genes
DFNA1
DIAPH1
DFNA2
Cx31/KCNQ4
DFNA3
Cx26/Cx30
DNFA4
MYH14
DFNA5
DFNA5
DFNA6/14
WFS1
DFNA8/12
TECTA
DFNA9
COCH
DFNA10
EYA4Autosomal recessive loci and genes
DFNB1
Cx26/Cx30
DFNB2
MYO7A
DFNB3
MYO15
DFNB4
SLC26A4DFNB6TMIEDFNB7/11
TMC1
DFNB8/10
TMPRSS3DFNB9OTOFDFNB12
CDH23
DFNB16STRC
DFNB18
USH1C
X-linked loci and genes
DFN3
POU3F4

DFNA11
DFNA13
DFNA15
DFNA17
DFNA20/26
DFNA22
DFNA28
DFNA36
DFNA48

MYO7A
COL11A2POU4F3
MYH9
ACTG1
MYO6
TFCP2L3
TMC1
MYO1A

DFNB21DFNB22DFNB23
DFNB28DFNB29DFNB30DFNB31DFNB36
DFNB37
DFNB67TECTA
OTOAPCDH15TRIOBP
CLDN14
MYO3A
WHRN
ESPN
MYO6
TMHS

Mitochondrial genes
12S rRNAtRNASer(UCN)

nonsyndromic type which is not associated with otherclinical features accounts for the other 70%. Nonsyndromic hearing loss can be classified into 4 groups by
the inheritance pattern, and relatively common clinicalfeatures have been noted for each inheritance patternwith a few exceptional genes, genotypes, and patients.
Patients with autosomal dominant inheritance typicallyshow progressive SNHL which begins in age 10-40, andthe degree of hearing loss is various while patients with
autosomal recessive inheritance most frequently show
congenital and severe hearing loss. Patients with mitochondrial inheritance tend to develop progressive SNHL
which begins in age 5-50, and the degree of hearing loss
is various. Autosomal recessive inheritance accounts for
80% of congenital nonsyndromic hereditary hearing loss,
and autosomal dominant inheritance accounts for mostof the other 20%. X-linked and mitochondrial inheritance accounts for only 1-2%. After aging, the prevalence of autosomal dominant inheritance and mitochondrial inheritance increases while that of autosomal recessive inheritance decreases. The precise prevalence ofeach inheritance pattern is not known for adults becauseof the difficulty in sampling and excluding the effect ofage-related hearing loss.

The first nonsyndromic deafness gene was discoveredin 1993.5 Since then, more than 100 loci for deafness
genes have been determined, and more than 40 genes
were identified (Table 1). Most of these genes play theirroles within the cochlea. Thus, hereditary hearing loss
almost exclusively features cochlear dysfunction.1, 2
Although many genes are known for nonsynromic
hearing loss, only a few genes including GJB2, GJB6,SLC26A4 accounts for over one third of patients withcongenital hearing loss. Mutations in GJB2 account for
50% of patients with autosomal recessive hearing loss,
i.e. 20% of all congenital hearing loss.6, 7 GJB2 encodes
connexin 26, a gap junction protein expressed in the cochlea. Gap junctions are intercellular channels allowing
recycling of potassium ions from hair cells to the stria
vascularis in the cochlea and maintains a high endocochlear potential which is of critical importance for normal hearing. Mutations in GJB2 show considerable phenotypic variation, but genotype-phenotype studies
showed that it is possible to predict the hearing loss associated with GJB2 mutations based on the specific genotype.8 Combination of mutations in GJB2 and closely
linked GJB6, in digenic transmission, accounts for about
8 % of deaf patients with GJB2.9 GJB6 is a gene with sequence similarity to GJB2, is also expressed in the cochlea, and its product, connexin 30, can form gap junction with connexin 26, explaining digenic transmissionof GJB2 and GJB6.
With regard to syndromic hearing loss, more than 300
forms have been characterized. In many forms, several
genes that can cause the same phenotype or a closely related phenotype have been identified. In syndromic hearing loss, hearing loss is most frequently caused by dysfunction of the cochlea but the middle ear and the outerear are also frequently involved. The most common formof syndromic hereditary SNHL is Pendred syndrome
which is characterized by SNHL, bilateral dilatation ofvestibular aqueduct with or without cochlear hypoplasia,
and goiter. Majority of patients with Pendred syndrome
have mutations in SLC26A4, and these mutations also
cause nonsyndromic SNHL.1 0, 11 Pendred syndrome accounts for 3 % of all congenital hearing loss and mutations in SLC26A4 including those causing nonsyndromic
SNHL account for 7 % of all deaf children at age of 4
years.2 SLC26A4 encodes a chloride-iodide cotransporter and is critical for maintaining endolymphatic ion homeostasis, which is essential to normal inner ear function.
Mutations in mitochondrial DNA are rarely detected incongenital hearing loss, but its prevalence in patientswith SNHL increases with aging. A1555G or A3243G
mitochondrial DNA mutations are found in approximately 6 % of adult patients with SNHL without known
causes, and both mutations cause cochlear dysfunc-

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Matsunaga T: Genetic Testing in Otology

tion.1 2, 1 3 A3243G mitochondrial DNA mutation cause
not only nonsyndromic SNHL but also syndromic SNHL
such as MELAS (mitochondrial encephalopathy, lactic
acidosis, and stroke-like episodes) and MIDD (maternally inherited diabetes and deafness). A1555G mitochondrial DNA mutation causes rapidly progressive SNHL
which leads to severe degree in patients with the onset of
SNHL before age 10 and slowly progressive or nonprogressive SNHL which leads to mild to moderate degreein patients with the onset after age 10.1 4 A3243G mitochondrial DNA mutation causes progressive SNHL
which leads to moderate to severe degree in patients whodeveloped hearing loss during adulthood.
Unique Clinical Aspects of Hereditary Hearing Loss
Hereditary hearing loss is unique compared to other
hereditary diseases in the following three points. First, alarge number of genes are involved in hereditary hearing
loss, which makes it very difficult to identify causes andpathological mechanism in clinical practice. Second,
without speech and language rehabilitation, hearing loss
not only impedes audition but also hampers normal development of speech and language. Without speech andlanguage, it is almost impossible to maintain good social
relationship in the society of people with normal hearing.
Thus, educational intervention is required in addition to
medical intervention for children with SNHL. Third,
congenital deaf children can learn and manage to communicate with others if early diagnosis of hearing loss
followed by adequate rehabilitation can be made. Even
severe hearing loss can be restored very effectively by
hearing aids or cochlear implants coupled with early rehabilitative training in patients with hereditary hearing
loss.15 In most hereditary diseases, this level of functional restoration has not been possible yet. This feature leadto the worldwide implementation of universal newborn
hearing screening which aims to screen neonates forhearing loss immediately after birth or before hospitaldischarge so that intervention can be initiated to prevent
delayed language acquisition. Because of these uniqueclinical aspects of hereditary hearing loss, genetic testingof SNHL has high value in the otological approach tothis disorder. Identification of genetic causes provides a
key to understand the mechanism of hearing loss, leads
to better management of hearing loss, and facilitatesfunctional recovery by effective rehabilitation.
Strategy for Genetic Testing of Hearing Loss
Genetic testing of SNHL is conducted in several institutes worldwide including our institute, and the strategy
is various among different institutes. In our institute, it
consists of the following 3 steps; 1) identification of candidate patients who are suspected of having hereditary
hearing loss, 2) identification of candidate genes to betested, and 3) identification of causative mutations in thesuspected genes.
Our criteria for candidate patients are patients presenting with bilateral hearing loss without known causes except for heredity. Unilateral hearing loss is included only
when hearing loss is associated with specific types ofanomaly in the inner ear, middle ear, or outer ear.
Candidate genes for syndromic hearing loss are determined by clinical diagnosis of syndromic hearing loss
based on associated clinical symptoms. Usually, only
one or a few candidate genes are responsible for each
syndrome. Syndrome may be classified into subclasses
based on the different expression of phenotypes, and diagnosis of subclasses may further narrow down candidate genes. On the other hand, it is very difficult to determine candidate genes for nonsyndromic hearing loss,
and often impossible because of a large number of causative genes for a relatively undistinguishable phenotype,i.e. SNHL. Part of deafness genes for nonsyndromic
hearing loss demonstrates unique auditory features orother clinical features in CT imaging of inner ear, electrophysiological testing, or inheritance pattern. For thosegenes, we are making an algorithm indicating the genes
which should be tested and the order of the genetic tests
based on clinical features and the results of genetic tests.
After all the clinical examinations and tests for hearing
loss, we determine the candidate genes and the order ofgenetic analysis according to the established algorithm(Fig. 1). This strategy is named systematic genetic testing for deafness, and tentative algorithm is currently
used in our institutes to evaluate the sensitivity, specificity, and efficiency for clinical use.
Identification of causative mutations is mostly donewith direct sequencing of the candidate genes using
DNA extracted from blood samples. All exons and its
flanking short sequences in introns are sequenced andanalyzed for mutations. For large genes in which pathological mutations are mostly distributed within the restricted regions, sequencing may be done for the restricted region. In contrast, for several large genes with ubiquitous distribution of pathological mutations over entire
region, screening by degenerate HPLC are first conducted, and sequencing analysis can be done only for the regions which showed abnormal screening results. For afew genes in which mutations are limited to only one or
two frequent changes, restriction fragment length polymorphism PCR analysis is performed to detect the specific mutations. With the astonishing progress in the
speed of sequencing machines, sequencing of whole human genome will be practically available in several
years, first in laboratories, then in clinics. This may fundamentally change the way of genetic testing for SNHL.
Feedback to Patients
Discovery of many deafness genes had a significant

Keio J Med 2009; 58 (4): 216－ 222

219

Fig. 1 Our original algorithm for systematic genetic testing for deafness in patients who are suspected of nonsyndromic SNHL
(sensorineural hearing loss). Based on the clinical or genetic test results shown in the left column, candidate mutations or genes listed
in the right column are determined. Corresponding mutations or genes for each clinical or genetic category are indicated by horizontal
arrows. Genetic tests start from Step 1. If causative mutations are not determined or an indicated category does not fit for a patient,
genetic tests proceed to the next step until causative mutations are determined or no appropriate category is found. Genes examined forspecific exons, regions or domains are described with short explanatory tags, and those examined for all exons are described without
explanation. Numbers in parenthesis indicate periods of age at onset of SNHL.

impact on the otological approach to patients with
SNHL. First, explanation of the cause of SNHL to deaf
patients or parents of deaf children has become possible
in many cases. Without definite explanation, patientstend to visit other hospitals seeking for explanation andrepeat redundant tests or treatments and feel anxiety
about what is related to deafness of themselves or theirchildren and whether other disability is also present but
not detected. These lead to delay of rehabilitation whichshould be initiated immediately after diagnosis of hearing loss for effective acquisition of language andspeech.16 Thus, early and definite explanation by genetic
tests facilitates rehabilitation.
Second, identification of causative mutations helps
doctors to predict auditory features such as audiogram ofthe patients and prognosis of their hearing, especially inchildren who cannot cooperate with subjective hearing
tests. This provides valuable information in making adequate planning of clinical follow-up, estimation of hearing levels for fitting hearing aids, and selection of occupation by patients.17
Third, prevention of deafness can be done by avoiding
use of specific drugs or specific activities in geneticallysusceptible patients. As an example, patients with
A1555G mitochondrial DNA mutation should avoid
aminoglycosides which induce or aggravate SNHL byeven one injection in subjects with this mutation.1 8 Another example is that detection of SLC26A4 suggests
dilatation of vestibular aqueduct even in neonates who
are usually not tested for inner ear anomaly by CT or
MRI. Patients with this mutation should have temporalbone CT and patients who are found to have dilatation ofvestibular aqueduct should avoid activities in which
physical shock on their head is likely to occur. This isbecause such a shock tends to cause aggravation of
SNHL in these patients.
Fourth, identification of causative mutations in patientswith syndromic hearing loss enables prevention or early
detection of associated symptoms. These examples include diabetes mellitus in patients with A3243G mitochondrial DNA mutation and goiter in patients with
SLC26A4 mutations. Early detection and managementof these associated symptoms help to prevent disorders
related to the associated symptoms such as diabetic retinopathy for diabetes mellitus, and facilitate early recovery from symptoms such as hypothyroidism. Because

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occurrence of associated symptoms may delay more than
10 years after the onset of SNHL, many patients andeven doctors who see those patients cannot notice the association of the symptoms with SNHL, and unnecessaryor even harmful tests tend to be done for the diagnosis.
Thus, it would be worthwhile to understand the associated symptoms and prepare the risk of manageable disorders at the time of diagnosis of SNHL. In addition, genetic tests may be valuable in substituting more stressful
tests. For an example, renal biopsy and/or skin biopsy
are currently necessary for diagnosis of Alport syndrome
which is a hereditary nephritis associated with SNHL,
and this procedure usually requires hospitalization andhas a certain physical risk. Mutations in COL4A3,
COL4A4, COL4A5, and MYH9 are known causes ofAlport syndrome, but genetic tests of these genes are
currently rarely available as a clinical test mainly because of an extremely high cost. Several laboratories in
the world including my laboratory offer these tests as a
research basis. With remarkable advances in genetics, increase of sensitivity and specificity and decrease of costs
for genetic analysis are in progress. In the near future,
diagnosis of Alport syndrome may be first done by clinical genetic tests, and renal and skin biopsy may be
avoided in many patients.19
Fifth, identification of causative mutations clarifies thecell types and nature of damages which are responsible
for SNHL, which is especially important for indicationof cochlear implant surgery. Because spiral ganglion
neurons which are necessary for successful cochlear implants are well preserved in most types of hereditary
hearing loss, identification of mutations in the deafness
genes usually indicates good indication for cochlear implant surgery. This is most helpful in babies who cannot
corporate detailed audiological tests for evaluation of
SNHL.
Identification of causative mutations is also importantfor clinical management of patients with auditory neuropathy. Auditory neuropathy is a distinct type of SNHL
which features normal outer hair cell function and abnormal activities of auditory neurons, and a relatively frequent cause of congenital SNHL (~15 %). Developmentof speech and language cannot be expected by hearing
aids in congenital auditory neuropathy because of poor
speech recognition inherent in this disorder. Either innerhair cells or spiral ganglion neurons are affected, but current clinical tests cannot distinguish these two types. Because normal spiral ganglion neurons are necessary forsuccess of cochlear implants, pathology underlying
SNHL needs to be determined in order to evaluate theindication of cochlear implant surgery. Recent studies
have shown that mutations in OTOF cause auditory neuropathy by inner hair cell dysfunction and that spiralganglion neurons are normal in patients with these mutations.2 0 In agreement with the pathological mechanismof mutations in OTOF, results of cochlear implants have

Matsunaga T: Genetic Testing in Otology

been successful.21 According to the recent studies, OTOF
mutations may account for majority of congenital auditory neuropathy.22 Thus, the genetic test for OTOF mutations in patients with auditory neuropathy is of high clinical importance.
Sixth, identification of causative mutations significantly helps to provide adequate genetic counseling whichprimarily concerns planning of pregnancy and delivery
with the information of a recurrence risk. Prenatal genetic diagnosis of nonsyndromic SNHL is not conducted inmost countries because of ethical issues. For syndromic
SNHL which is associated with severe symptoms otherthan SNHL, prenatal diagnosis may be considered.
Future Expectation of the Use of
Genetic Testing in Therapeutics
Although hearing aids or cochlear implants can significantly restore hearing in patients with SNHL, quality of
restored hearing is quite different from original or normal hearing. These instruments are made to help remaining functions of the damaged inner ear, but future therapeutics aims at complete recovery of the inner ear. Because current clinical diagnostic modalities cannot identify which parts or cells in the inner ear is damaged,
therapeutic approach targeting at specific parts or cells in
the inner ear has not been used. Identification of damaged cells in the inner ear and the underlying mechanism
by genetic testing undoubtedly facilitates development
and introduction of novel and specific therapies to distinct types of SNHL.
As one of such therapies, we have established novel
therapeutic approaches targeting at cochlear fibrocytes
which are essential for normal hearing and involved invarious type of SNHL including certain types of hereditary SNHL, age-related SNHL, noise-induced SNHL,
and Meniere’s disease. A rat model of SNHL due specificto cochlear fibrocytes was made by treatment with a mitochondrial toxin, 3-nitropropionic acid (3-NP), at around window of inner ear.23 Histological and molecularanalysis in this model revealed caspase-mediated apoptosis in the cochlear fibrocytes.2 4 As the therapy during
acute phase of SNHL due to damages on cochlear fibrocytes, we used a general administration of caspase inhibitor, Z-VAD-FMK, to inhibit apoptosis.25 This chemical,
when administered before 3-NP treatment, almost completely inhibited 3-NP induced apoptosis of cochlear fibrocytes without obvious side effects and significantly
improved the hearing level. Administration of Z-VADFMK after 3-NP treatment also showed significant inhibition of apoptosis and improvement of hearing. As thetherapy during chronic phase of SNHL due to damages
on cochlear fibrocytes, we used transplantation of bonemarrow-derived mesenchymal stem cells into the inner
ear in this animal model.2 6 Histological examination of
the transplanted rats demonstrated that transplanted stem

Keio J Med 2009; 58 (4): 216－ 222

cells survived, migrated to the damaged area, and apparently substituted the damaged cochlear fibrocytes. Those
stem cells made a connection with the surrounding fibrocytes and expressed connexins which are essential forreestablishment of potassium recycling pathway mediated by cochlear fibrocytes within the cochlea. Evaluationof hearing by auditory brainstem responses in the transplanted rats revealed significant improvement of hearing
compared to control rats. These animal experiments indicate that therapeutic strategy for genetic SNHL may be
personalized, based on the cause of SNHL, using chemicals targeting at specific molecules or stem cells targeting at specific tissues for regenerative therapy. In addition, novel therapies developed for genetic SNHL may
be applicable to other types of SNHL with similar pathological features.

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9.

10.

11.

Acknowledgements
I thank Prof. Kaoru Ogawa, Dr. Kimitaka Kaga, andDr. Hidenobu Taiji for help and advices in conducting
genetic testing and counseling at Keio University Hospital, National Tokyo Medical Center, and National Centerfor Child Health and Development, respectively. This
work was supported by a Network Research Grants for
Disorders of Sensory Organs from the National Hospital
Organization and Health Science Research Grants from
the Ministry of Health, Labor, and Welfare of Japan.

12.

13.
14.

15.

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