Blin, Nikolaus

Haack, Birgit

Kupka, Zsuzsanna

Nürnberg, Peter

Pfister, Markus

Sziklai, István

Thiele, Holger

Tóth, Tímea

Zenner, Hans-Peter

Molecular Medicine

University of Debrecen Electronic Archive

Molecular Medicine 8(10): 607–611, 2002
© 2002 North Shore-LIJ Research Institute
A 4bp-Insertion in the eya-Homologous Region (eyaHR) of EYA4 Causes
Hearing Impairment in a Hungarian Family Linked to DFNA10
Markus Pﬁster,1 Tímea Tóth,2 Holger Thiele,3,5 Birgit Haack,1,4 Nikolaus Blin,4 Hans-Peter Zenner,1
István Sziklai,3 Peter Nürnberg,3,5 Susan Kupka1,4
1Department of Otolaryngology, University of Tübingen, Tübingen, Germany
2Medical and Health Science Center, Department of Otolaryngology, University of Debrecen, Hungary
3Gene Mapping Center, Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany
4Department of Anthropology and Human Genetics, University of Tübingen, Tübingen, Germany
5Institute for Medical Genetics, Charité, Humboldt University, Berlin, Germany
Accepted August 23, 2002
Abstract
Background: Hereditary hearing impairment (HHI) is a
heterogeneous class of disorders that shows various pat-
terns of inheritance and involves a multitude of different
genes. Mutations in the EYA4 gene are responsible for
postlingual, progressive, autosomal dominant hearing
loss at the DFNA10 locus. EYA4 is orthologous to the
Drosophila gene eya (“eyes absent”), a key regulator of eye
formation. EYA4 plays an important role in several devel-
opmental processes.
Material and Methods: Here we report a Hungarian family
displaying sensorineural, progressive hearing impairment.
The family comprising four generations with 11 affected and
8 unaffected members was subjected to genome-wide link-
age analysis and candidate gene sequencing.
Correspondence and reprint requests should be addressed to: 
Susan Kupka, University of Tübingen, Department of
Otolaryngology, Molecular Genetics, Elfriede-Aulhorn-Str. 5,
72076 Tübingen, Germany. Phone: (49) 7071-29-88168; 
fax: (49) 7071-29-3311; e-mail: susankupka@yahoo.de.
Results: By linkage analysis, the chromosomal region
6q22.3 was shown to segregate with the disease. Mutation
analysis of the EYA4 gene, which maps to 6q22.3, revealed
an insertion of 4 bp (1558insTTTG) in all affected family
members. This insertion creates a frameshift and results in
a stop codon at position 379. Hence, nearly the complete
“eya homologous region” (eyaHR), which is essential for
the protein function, would be deleted in the mutant EYA4
protein if the transcription were found to be stable.
Conclusions: This family is the third one linked to DFNA10
and revealing a mutation in the EYA4 gene. In all three fam-
ilies, the mutations are localized in different regions of the
eyaHR, suggesting that this protein contains several func-
tional subregions with different tissue-speciﬁc importance.
Introduction
Nonsyndromic severe to profound neurosensory
hearing impairment (NSHL) is one of the most
common human sensory disorder, affecting 1 in
1000 children with at least 60% of cases being in-
herited (1,2). The mode of inheritance of nonsyn-
dromic hearing disorders can be distinguished in
autosomal dominant (10–15%, DFNA), autosomal
recessive (70%, DFNB), X-linked (1–3%, DFN), and
mitochondrial forms. NSHL accounts for up to 70%
of all inherited sensorineural hearing defects. To
date, 29 genes are known to play a role in NSHL (3).
Recently, Wayne et al. (4) showed the involve-
ment of EYA4, a transcriptional activator and mem-
ber of the vertebrate EYA family, in the development
of DFNA10 in one Belgian and one American family.
EYA4 is a ortholog to the Drosophila gene eya (“eyes
absent”), which is involved in the formation of 
compound eyes (5). Flies with loss-of-function 
mutations of this gene develop no eyes. The human
EYA4 encodes a transcriptional activator that inter-
acts with members of the SIX and DACH protein
families in a conserved network regulating early
embryonic development. The protein contains a
large, highly conserved C-terminal domain, the eya
homology domain, and an alpha helical domain
forming a leucine zipper (5).
Wayne et al. (4) found two EYA4 isoforms
expressed in human fetal cochlear cDNA. In the
same study, they identiﬁed mutations in the EYA4
gene that were responsible for postlingual, pro-
gressive, autosomal dominant hearing loss at the
DFNA10 locus.
Here we present a third family with sen-
sorineural hearing impairment also showing link-
age to DFNA10 and a mutated EYA4 gene.
Materials and Methods
Patients
The family is of Northeast Hungarian origin. The 
patients were recruited from the Department of 
Otolaryngology, University of Debrecen. To deter-
mine the etiology of hearing impairment, a detailed
otolaryngologic examination was performed with
the affected individuals and their unaffected relatives.
The family comprised four generations and included
11 affected and 8 unaffected family members. Ac-
cording to the approval of the Ethics Comittee of the
University of Debrecen, written informed consent
was obtained from all participants and from parents
of patients younger than 18 years.
Audiologic Methods
All family members underwent otoscopic and 
audiometric examinations by using age-appropriate
methods. Threshold audiograms were obtained after
otoscopic examination with pure-tone audiometry
in a sound-treated room according to current clinical
standards. We used air- and bone-conduction at 125,
250, 500, 1000, 2000, 4000, and 8000 Hz for all 
affected participants. The audiometric conﬁguration
was classiﬁed based on the deﬁnitions of the 
European Work Group on Genetics of Hearing 
Impairment.
Genotyping
Genomic DNA was extracted from peripheral blood
lymphocytes by standard techniques. Individuals
were genotyped in a genomewide linkage analysis
using 384 microsatellite markers with an average
spacing of 11 cM. PCR reactions were performed 
using manufacturers’ protocols. Semiautomated
genotyping was performed by a MegaBACE-1000
analysis system. Data were analyzed by Genetic 
Proﬁler Software 1.5. Two-point LOD score calcula-
tion was performed with the LINKAGE v5.2 
program package (6). Most likely haplotypes were
constructed with Simwalk2 v2.82 (7).
Sequencing
Sequencing was performed using primers described
elsewhere (4). PCR products were gel extracted (Gel
Extraction Kit, Qiagen) and sequenced with corre-
sponding primers on an ABI 377 automated ﬂuores-
cent sequencer machine. Detected mutations were
conﬁrmed at least two times and on both DNA
strands. Sequences were compared with NCBI-
Accession number 13642856 using the DNAsis soft-
ware (MWG).
Results
Clinical Data
The hearing disorder was sensorineural, progres-
sive, and bilateral in all affected family members. At
onset, hearing impairment was detected at the mid-
and low-frequencies, degrading to a profound hear-
ing impairment involving all frequencies.
Linkage Analysis
Microsatellite analysis revealed signiﬁcant linkage
of marker D6S1009 with the disease phenotype
608 Molecular Medicine, Volume 8, Number 10, October 2002
(max. two-point LOD score Zmax  4.73 at max 
0.00). By haplotype analysis, a critical interval of
36.8 cM was determined between markers D6S262
and D6S305, which corresponds to the chromosomal
region 6q23.2-q26 and contains the candidate gene
EYA4 (Fig. 1).
Sequencing
Sequencing of all 21 EYA4 exons revealed an inser-
tion of four bases (TTTG) in exon 13 (1558insTTTG)
(Fig. 2). This insertion was detected in all affected fam-
ily members (data not shown). Mutation 1558 insTTTG
causes a frameshift beginning in codon 373, followed
by amino acid substitutions and a premature termina-
tion codon (PTC) at position 379. This PTC is likely to
cause degradation of the mutant transcript by non-
sense-mediated decay (5), otherwise it would result
in a nearly complete deletion of the eya homologous
region of EYA4.
Discussion
Autosomal dominant inherited hearing impairment
is a genetically heterogenous disorder. So far, 41
chromosomal loci have been linked to this disease
and 17 genes have been reported (3). Recently,
Wayne et al. (4) identiﬁed mutations in the EYA4
gene that were responsible for postlingual, progres-
sive, autosomal dominant loss at the DFNA10 locus.
EYA4 is orthologous to the Drosophila gene eya (“eyes
absent”), and is localized on 6q23 (6). The EYA4
gene consists of 21 exons, of which some are alter-
natively spliced creating several isoforms. The en-
coded protein contains a highly conserved 271 
amino acid C-terminus called the eya-homologous
region (eyaHR, eya domain) and a more divergent
proline-serine-threonine (PST)-rich transactivation
domain at the N-terminus (6).
We report a DFNA10 family displaying sen-
sorineural, progressive hearing impairment and
linkage to 6q23. The results of the detailed audio-
metric analysis of a Belgian DFNA10 family coin-
cide with our clinical ﬁndings (9). To our knowl-
edge, this is the third DFNA10 family revealing a
mutation in the EYA4 gene. The detected insertion
of 4 bp (1558insTTTG) creates a frameshift and re-
sults in a PTC at position 379. The effect is either a
complete degradation of the mutant messenger or a
nearly complete deletion of the eyaHR in the EYA4
protein. The eyaHR is essential for members of
the EYA protein family regarding their interaction
with PAX, SIX, and DACH proteins in a genetic net-
work which is conserved across species (10). First
described in Drosophila as a key regulator of eye for-
mation, this network and its function in several de-
velopmental processes had also been demonstrated
to play an important role in vertebrates. Drosophila
eya plays a critical role in morphogenesis of a number
of tissues separately, during early eye formation
(11). By analyzing Drosophila eya gene mutations
Markus Pﬁster et al.: A 4bp-Insertion in the eya-Homologous Region (eyaHR) 609
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Bui et al. (12) showed that the loss of the entire eya
domain results in eya inactivity, whereas alleles with
truncations within the eya domain display partial
function.
Recently, Heanue et al. (13) studied expression
of the Dach/Pax/Eya network in mice and chicken.
They showed that DachI and Eya1 expression overlap
in the developing ear and Pax2 and Eya1 are
required for normal ear development and they 
suggested that Drosophila Pax/Eya/Dach network
may be evolutionarily conserved such that Pax
genes, Eya1, and Dach1 may function together in ver-
tebrates to regulate neural development.
By studying eya1-deﬁcient mice, Xu et al. (14)
showed that eya1 controls critical, early inductive
signaling events involved in ear and kidney forma-
tion. Eya1 heterozygotes () showed renal ab-
normalities and a conductive hearing loss similar to
human branchiootorenal dysplasia (BOR; OMIM
113650) syndrome, which is caused by mutations 
in EYA1. Eya1 homozygotes () lacked ears and 
kidneys due to defective inductive tissue interac-
tions and apoptotic regression of the organ primordia.
Inner ear development in Eya1 null mice arrested 
at the otic vesicle stage, and all components of the
inner ear and speciﬁc cranial sensory ganglia failed
to form. The authors concluded that the evolu-
tionary conserved pax-eya-six regulatory hierarchy
is used in mammalian inner ear and kidney
development (14).
So far, all three EYA4 mutations detected in
DFNA10 families result in PTCs, presumably entail-
ing nonsense-mediated decay of the mRNA (5) or,
alternatively, deletions of parts of the eyaHR. Based
on the observations mentioned, this region is of ex-
ceptional importance for the function of the protein.
Therefore, it would not be surprising that, even if
the mutant proteins were present in the cells, inter-
rupting mutations within the eyaHR would lead to
610 Molecular Medicine, Volume 8, Number 10, October 2002
haploinsufﬁciency. Interestingly, the phenotype is
obviously not depending on the position of the PTC
which is commensurate with an instable mutant mes-
senger. In our Hungarian family, and in the American
family, almost the complete eyaHR is deleted,
whereas in the Belgian family only a small part at the
C-terminus is absent (4). These observations support
the presumption that the eyaHR contains several
functional subregions with different tissue-speciﬁc
importance. This would also serve as an explanation
for the limited phenotype of DFNA10, showing no
congenital abnormalities, despite the wide range of
expression in early embryogenesis.
In addition to their developmental functions,
members of the eya gene family were shown to act as
a pro-apoptotic signal by Clark et al. (15). When
overexpressed eya can directly activate the apoptotic
program, and this function is conserved between ﬂy,
mouse, and human eya proteins. Eya-induced cell
death has many features typical for apoptosis, in-
cluding plasma and mitochondrial membrane
changes and caspase activities. Furthermore, eya
appears to induce apoptosis by triggering both
caspase-dependent and caspase-independent path-
ways (15). Caspase-dependent apoptosis occurs in the
inner ear, suggesting an important role in the func-
tioning of the auditory system. Caspase-3 knockout
mice show progressive severe hearing impairment,
hyperplasia of supporting cells, and degeneration of
sensory hair cells (16,17). Progressive hearing im-
pairment is a characteristic feature in DFNA10-linked
families. Therefore, the apoptotic function of EYA4
may be relevant for the hearing process. Thus muta-
tions within the EYA4 gene may not only provoke
hearing impairment due to developmental failures but
also because of apoptotic deﬁciencies.
Acknowledgments
This study was supported by grants of Else-Kröner-
Fresenius-Stiftung, the Ministry of Education, Depart-
ment for Research in Hungary (TeT 40/2000), and
the Hungarian Basic Research Foundation (OTKA-
T037255) and Dr. Karl Kuhn-Stiftung. We thank all
patients for their cooperation in the study.
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A 4bp-insertion in the eya-homologous region (eyaHR) of EYA4 causes hearing impairment in a Hungarian family linked to DFNA10

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A 4bp-insertion in the eya-homologous region (eyaHR) of EYA4 causes hearing impairment in a Hungarian family linked to DFNA10

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