Molecula Medicine 8(10): 607–611, 2002
© 2002 No h Sho e-LIJ Resea ch Ins i u e
A 4bp-Inse ion in he eya-Homologous Region (eyaHR) o EYA4 Causes
Hea ing Impai men in a Hunga ian Family Linked o DFNA10
Ma kus Pfis e ,
1
Tímea Tó h,
2
Holge Thiele,
3,5
Bi gi Haack,
1,4
Nikolaus Blin,
4
Hans-Pe e Zenne ,
1
Is án Sziklai,
3
Pe e Nü nbe g,
3,5
Susan Kupka
1,4
1
Depa men o O ola yngology, Uni e si y o Tübingen, Tübingen, Ge many
2
Medical and Heal h Science Cen e , Depa men o O ola yngology, Uni e si y o Deb ecen, Hunga y
3
Gene Mapping Cen e , Max Delb ück Cen e o Molecula Medicine, Be lin-Buch, Ge many
4
Depa men o An h opology and Human Gene ics, Uni e si y o Tübingen, Tübingen, Ge many
5
Ins i u e o Medical Gene ics, Cha i é, Humbold Uni e si y, Be lin, Ge many
Accep ed Augus 23, 2002
Abs ac
Backg ound: He edi a y hea ing impai men (HHI) is a
he e ogeneous class o diso de s ha shows a ious pa -
e ns o inhe i ance and in ol es a mul i ude o di e en
genes. Mu a ions in he EYA4 gene a e esponsible o
pos lingual, p og essi e, au osomal dominan hea ing
loss a he DFNA10 locus. EYA4 is o hologous o he
D osophila gene eya (“eyes absen ”), a key egula o o eye
o ma ion. EYA4 plays an impo an ole in se e al de el-
opmen al p ocesses.
Ma e ial and Me hods: He e we epo a Hunga ian amily
displaying senso ineu al, p og essi e hea ing impai men .
The amily comp ising ou gene a ions wi h 11 a ec ed and
8 una ec ed membe s was subjec ed o genome-wide link-
age analysis and candida e gene sequencing.
Co espondence and ep in eques s should be add essed o:
Susan Kupka, Uni e si y o Tübingen, Depa men o
O ola yngology, Molecula Gene ics, El iede-Aulho n-S . 5,
72076 Tübingen, Ge many. Phone: (49) 7071-29-88168;
ax: (49) 7071-29-3311; e-mail: [email p o ec ed].
Resul s: By linkage analysis, he ch omosomal egion
6q22.3 was shown o seg ega e wi h he disease. Mu a ion
analysis o he EYA4 gene, which maps o 6q22.3, e ealed
an inse ion o 4 bp (1558insTTTG) in all a ec ed amily
membe s. This inse ion c ea es a ameshi and esul s in
a s op codon a posi ion 379. Hence, nea ly he comple e
“eya homologous egion” (eyaHR), which is essen ial o
he p o ein unc ion, would be dele ed in he mu an EYA4
p o ein i he ansc ip ion we e ound o be s able.
Conclusions: This amily is he hi d one linked o DFNA10
and e ealing a mu a ion in he EYA4 gene. In all h ee am-
ilies, he mu a ions a e localized in di e en egions o he
eyaHR, sugges ing ha his p o ein con ains se e al unc-
ional sub egions wi h di e en issue-specific impo ance.
In oduc ion
Nonsynd omic se e e o p o ound neu osenso y
hea ing impai men (NSHL) is one o he mos
common human senso y diso de , a ec ing 1 in
1000 child en wi h a leas 60% o cases being in-
he i ed (1,2). The mode o inhe i ance o nonsyn-
d omic hea ing diso de s can be dis inguished in
au osomal dominan (10–15%, DFNA), au osomal
ecessi e (70%, DFNB), X-linked (1–3%, DFN), and
mi ochond ial o ms. NSHL accoun s o up o 70%
o all inhe i ed senso ineu al hea ing de ec s. To
da e, 29 genes a e known o play a ole in NSHL (3).
Recen ly, Wayne e al. (4) showed he in ol e-
men o EYA4, a ansc ip ional ac i a o and mem-
be o he e eb a e EYA amily, in he de elopmen
o DFNA10 in one Belgian and one Ame ican amily.
EYA4 is a o holog o he D osophila gene eya (“eyes
absen ”), which is in ol ed in he o ma ion o
compound eyes (5). Flies wi h loss-o - unc ion
mu a ions o his gene de elop no eyes. The human
EYA4 encodes a ansc ip ional ac i a o ha in e -
ac s wi h membe s o he SIX and DACH p o ein
amilies in a conse ed ne wo k egula ing ea ly
emb yonic de elopmen . The p o ein con ains a
la ge, highly conse ed C- e minal domain, he eya
homology domain, and an alpha helical domain
o ming a leucine zippe (5).
Wayne e al. (4) ound wo EYA4 iso o ms
exp essed in human e al cochlea cDNA. In he
same s udy, hey iden ified mu a ions in he EYA4
gene ha we e esponsible o pos lingual, p o-
g essi e, au osomal dominan hea ing loss a he
DFNA10 locus.
He e we p esen a hi d amily wi h sen-
so ineu al hea ing impai men also showing link-
age o DFNA10 and a mu a ed EYA4 gene.
Ma e ials and Me hods
Pa ien s
The amily is o No heas Hunga ian o igin. The
pa ien s we e ec ui ed om he Depa men o
O ola yngology, Uni e si y o Deb ecen. To de e -
mine he e iology o hea ing impai men , a de ailed
o ola yngologic examina ion was pe o med wi h
he a ec ed indi iduals and hei una ec ed ela i es.
The amily comp ised ou gene a ions and included
11 a ec ed and 8 una ec ed amily membe s. Ac-
co ding o he app o al o he E hics Comi ee o he
Uni e si y o Deb ecen, w i en in o med consen
was ob ained om all pa icipan s and om pa en s
o pa ien s younge han 18 yea s.
Audiologic Me hods
All amily membe s unde wen o oscopic and
audiome ic examina ions by using age-app op ia e
me hods. Th eshold audiog ams we e ob ained a e
o oscopic examina ion wi h pu e- one audiome y
in a sound- ea ed oom acco ding o cu en clinical
s anda ds. We used ai - and bone-conduc ion a 125,
250, 500, 1000, 2000, 4000, and 8000 Hz o all
a ec ed pa icipan s. The audiome ic configu a ion
was classified based on he defini ions o he
Eu opean Wo k G oup on Gene ics o Hea ing
Impai men .
Geno yping
Genomic DNA was ex ac ed om pe iphe al blood
lymphocy es by s anda d echniques. Indi iduals
we e geno yped in a genomewide linkage analysis
using 384 mic osa elli e ma ke s wi h an a e age
spacing o 11 cM. PCR eac ions we e pe o med
using manu ac u e s’ p o ocols. Semiau oma ed
geno yping was pe o med by a MegaBACE-1000
analysis sys em. Da a we e analyzed by Gene ic
P ofile So wa e 1.5. Two-poin LOD sco e calcula-
ion was pe o med wi h he LINKAGE 5.2
p og am package (6). Mos likely haplo ypes we e
cons uc ed wi h Simwalk2 2.82 (7).
Sequencing
Sequencing was pe o med using p ime s desc ibed
elsewhe e (4). PCR p oduc s we e gel ex ac ed (Gel
Ex ac ion Ki , Qiagen) and sequenced wi h co e-
sponding p ime s on an ABI 377 au oma ed fluo es-
cen sequence machine. De ec ed mu a ions we e
confi med a leas wo imes and on bo h DNA
s ands. Sequences we e compa ed wi h NCBI-
Accession numbe 13642856 using he DNAsis so -
wa e (MWG).
Resul s
Clinical Da a
The hea ing diso de was senso ineu al, p og es-
si e, and bila e al in all a ec ed amily membe s. A
onse , hea ing impai men was de ec ed a he mid-
and low- equencies, deg ading o a p o ound hea -
ing impai men in ol ing all equencies.
Linkage Analysis
Mic osa elli e analysis e ealed significan linkage
o ma ke D6S1009 wi h he disease pheno ype
608 Molecula Medicine, Volume 8, Numbe 10, Oc obe 2002
(max. wo-poin LOD sco e Z
max
⫽4.73 a ⌰
max
⫽
0.00). By haplo ype analysis, a c i ical in e al o
36.8 cM was de e mined be ween ma ke s D6S262
and D6S305, which co esponds o he ch omosomal
egion 6q23.2-q26 and con ains he candida e gene
EYA4 (Fig. 1).
Sequencing
Sequencing o all 21 EYA4 exons e ealed an inse -
ion o ou bases (TTTG) in exon 13 (1558insTTTG)
(Fig. 2). This inse ion was de ec ed in all a ec ed am-
ily membe s (da a no shown). Mu a ion 1558 insTTTG
causes a ameshi beginning in codon 373, ollowed
by amino acid subs i u ions and a p ema u e e mina-
ion codon (PTC) a posi ion 379. This PTC is likely o
cause deg ada ion o he mu an ansc ip by non-
sense-media ed decay (5), o he wise i would esul
in a nea ly comple e dele ion o he eya homologous
egion o EYA4.
Discussion
Au osomal dominan inhe i ed hea ing impai men
is a gene ically he e ogenous diso de . So a , 41
ch omosomal loci ha e been linked o his disease
and 17 genes ha e been epo ed (3). Recen ly,
Wayne e al. (4) iden ified mu a ions in he EYA4
gene ha we e esponsible o pos lingual, p og es-
si e, au osomal dominan loss a he DFNA10 locus.
EYA4 is o hologous o he D osophila gene eya (“eyes
absen ”), and is localized on 6q23 (6). The EYA4
gene consis s o 21 exons, o which some a e al e -
na i ely spliced c ea ing se e al iso o ms. The en-
coded p o ein con ains a highly conse ed 271
amino acid C- e minus called he eya-homologous
egion (eyaHR, eya domain) and a mo e di e gen
p oline-se ine- h eonine (PST)- ich ansac i a ion
domain a he N- e minus (6).
We epo a DFNA10 amily displaying sen-
so ineu al, p og essi e hea ing impai men and
linkage o 6q23. The esul s o he de ailed audio-
me ic analysis o a Belgian DFNA10 amily coin-
cide wi h ou clinical findings (9). To ou knowl-
edge, his is he hi d DFNA10 amily e ealing a
mu a ion in he EYA4 gene. The de ec ed inse ion
o 4 bp (1558insTTTG) c ea es a ameshi and e-
sul s in a PTC a posi ion 379. The e ec is ei he a
comple e deg ada ion o he mu an messenge o a
nea ly comple e dele ion o he eyaHR in he EYA4
p o ein. The eyaHR is essen ial o membe s o
he EYA p o ein amily ega ding hei in e ac ion
wi h PAX, SIX, and DACH p o eins in a gene ic ne -
wo k which is conse ed ac oss species (10). Fi s
desc ibed in D osophila as a key egula o o eye o -
ma ion, his ne wo k and i s unc ion in se e al de-
elopmen al p ocesses had also been demons a ed
o play an impo an ole in e eb a es. D osophila
eya plays a c i ical ole in mo phogenesis o a numbe
o issues sepa a ely, du ing ea ly eye o ma ion
(11). By analyzing D osophila eya gene mu a ions
Ma kus Pfis e e al.: A 4bp-Inse ion in he eya-Homologous Region (eyaHR) 609
Fig. 1. Seg ega ion o he mu an allele in Family “U30”. (dash ⫽missing geno ype)
1
1
3
3
3
4
4
III/1 III/2
IV/1 IV/3 IV/6IV/2
V/1 V/2 V/3
IV:4
V/4 V/5
IV/5
V/6 V/7 V/8
VI/1
III/3
IV/8IV:7
V/9
II/1 II/2
III/5 III/6III/4
IV/9 IV/10
I/2
II/3
I/1
VI/2
D6S1040 (129.1)
D6S262 (129.8)
D6S292 (138.2)
D6S1009 (138.8)
D6S308 (145.5)
D6S305 (166.6)
D6S1277 (173.4)
1
7
6
5
1
7
3
1
8
7
5
4
8
-
2
10
6
9
3
7
-
D6S1040 (129.1)
D6S262 (129.8)
D6S292 (138.2)
D6S1009 (138.8)
D6S308 (145.5)
D6S305 (166.6)
D6S1277 (173.4)
2
10
6
9
3
8
3
1
7
6
5
1
4
4
1
1
3
3
3
4
4
2
10
6
9
3
8
3
1
8
7
5
4
8
-
1
1
3
3
3
4
-
EYA4
1
1
3
3
3
1
3
1
7
6
5
3
10
5
1
1
3
3
3
1
3
2
10
6
9
3
1
1
2
7
4
2
2
1
1
1
7
6
5
3
10
5
3
7
3
9
4
9
1
2
3
1
2
3
9
7
1
1
3
3
3
1
3
1
7
6
5
1
7
3
3
7
-
9
4
9
1
1
7
-
5
1
1
3
1
7
3
3
3
7
3
2
3
1
2
3
9
7
3
2
2
2
4
8
4
3
2
4
6
3
6
5
D6S1040 (129.1)
D6S262 (129.8)
D6S292 (138.2)
D6S1009 (138.8)
D6S308 (145.5)
D6S305 (166.6)
D6S1277 (173.4)
1
7
3
3
3
9
7
1
7
3
3
3
9
7
3
2
2
2
4
6
5
3
2
2
2
4
8
4
1
1
3
3
3
1
3
4
7
4
2
3
7
4
4
7
4
2
3
7
4
3
6
4
7
3
9
4
4
2
2
2
3
4
4
2
7
4
4
3
4
4
1
1
3
3
3
4
4
1
2
4
4
3
4
4
I
II
III
IV
V
VI
EYA4
EYA4
Bui e al. (12) showed ha he loss o he en i e eya
domain esul s in eya inac i i y, whe eas alleles wi h
unca ions wi hin he eya domain display pa ial
unc ion.
Recen ly, Heanue e al. (13) s udied exp ession
o he Dach/Pax/Eya ne wo k in mice and chicken.
They showed ha DachI and Eya1 exp ession o e lap
in he de eloping ea and Pax2 and Eya1 a e
equi ed o no mal ea de elopmen and hey
sugges ed ha D osophila Pax/Eya/Dach ne wo k
may be e olu iona ily conse ed such ha Pax
genes, Eya1, and Dach1 may unc ion oge he in e -
eb a es o egula e neu al de elopmen .
By s udying eya1-deficien mice, Xu e al. (14)
showed ha eya1 con ols c i ical, ea ly induc i e
signaling e en s in ol ed in ea and kidney o ma-
ion. Eya1 he e ozygo es (⫹Ⲑ⫺) showed enal ab-
no mali ies and a conduc i e hea ing loss simila o
human b anchioo o enal dysplasia (BOR; OMIM
113650) synd ome, which is caused by mu a ions
in EYA1. Eya1 homozygo es (⫺Ⲑ⫺) lacked ea s and
kidneys due o de ec i e induc i e issue in e ac-
ions and apop o ic eg ession o he o gan p imo dia.
Inne ea de elopmen in Eya1 null mice a es ed
a he o ic esicle s age, and all componen s o he
inne ea and specific c anial senso y ganglia ailed
o o m. The au ho s concluded ha he e olu-
iona y conse ed pax-eya-six egula o y hie a chy
is used in mammalian inne ea and kidney
de elopmen (14).
So a , all h ee EYA4 mu a ions de ec ed in
DFNA10 amilies esul in PTCs, p esumably en ail-
ing nonsense-media ed decay o he mRNA (5) o ,
al e na i ely, dele ions o pa s o he eyaHR. Based
on he obse a ions men ioned, his egion is o ex-
cep ional impo ance o he unc ion o he p o ein.
The e o e, i would no be su p ising ha , e en i
he mu an p o eins we e p esen in he cells, in e -
up ing mu a ions wi hin he eyaHR would lead o
610 Molecula Medicine, Volume 8, Numbe 10, Oc obe 2002
haploinsu ficiency. In e es ingly, he pheno ype is
ob iously no depending on he posi ion o he PTC
which is commensu a e wi h an ins able mu an mes-
senge . In ou Hunga ian amily, and in he Ame ican
amily, almos he comple e eyaHR is dele ed,
whe eas in he Belgian amily only a small pa a he
C- e minus is absen (4). These obse a ions suppo
he p esump ion ha he eyaHR con ains se e al
unc ional sub egions wi h di e en issue-specific
impo ance. This would also se e as an explana ion
o he limi ed pheno ype o DFNA10, showing no
congeni al abno mali ies, despi e he wide ange o
exp ession in ea ly emb yogenesis.
In addi ion o hei de elopmen al unc ions,
membe s o he eya gene amily we e shown o ac as
a p o-apop o ic signal by Cla k e al. (15). When
o e exp essed eya can di ec ly ac i a e he apop o ic
p og am, and his unc ion is conse ed be ween fly,
mouse, and human eya p o eins. Eya-induced cell
dea h has many ea u es ypical o apop osis, in-
cluding plasma and mi ochond ial memb ane
changes and caspase ac i i ies. Fu he mo e, eya
appea s o induce apop osis by igge ing bo h
caspase-dependen and caspase-independen pa h-
ways (15). Caspase-dependen apop osis occu s in he
inne ea , sugges ing an impo an ole in he unc-
ioning o he audi o y sys em. Caspase-3 knockou
mice show p og essi e se e e hea ing impai men ,
hype plasia o suppo ing cells, and degene a ion o
senso y hai cells (16,17). P og essi e hea ing im-
pai men is a cha ac e is ic ea u e in DFNA10-linked
amilies. The e o e, he apop o ic unc ion o EYA4
may be ele an o he hea ing p ocess. Thus mu a-
ions wi hin he EYA4 gene may no only p o oke
hea ing impai men due o de elopmen al ailu es bu
also because o apop o ic deficiencies.
Acknowledgmen s
This s udy was suppo ed by g an s o Else-K öne -
F esenius-S i ung, he Minis y o Educa ion, Depa -
men o Resea ch in Hunga y (TeT 40/2000), and
he Hunga ian Basic Resea ch Founda ion (OTKA-
T037255) and D . Ka l Kuhn-S i ung. We hank all
pa ien s o hei coope a ion in he s udy.
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