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

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. Re e ences 1. F ase GR. (1971) The gene ics o congeni al dea ness. O ola yngol. Clin. No h Am. 4: 227–247. 2. Mo on NE. (1991) Gene ic epidemiology o hea ing impai - men . Ann N Y Acad Sci 630: 16–31. 3. Van Camp G, Smi h RJ. He edi a y hea ing loss homepage. A ailable om: URL: h p://dnalab-www.uia.ac.be/dnalab/ hhh/. Re ie ed Augus , 2002. 4. Wayne S, Robe son NG, DeClau F, e al. (2001) Mu a ions in he ansc ip ional ac i a o EYA4 cause la e-onse dea ness a he DFNA10 locus. Hum. Mol. Gene . 10: 195–200. 5. Hen ze MW, Kulozik AE. (1999) A pe ec message: RNA su - eillance and nonsense-media ed decay. Cell. 96: 307–310. 6. Bo sani G, DeG andi A, Ballabio A, e al. (1999) EYA4, a no el e eb a e gene ela ed o D osophila eyes absen . Hum. Mol. Gene . 8: 11–23. Fig. 2. Pa o he sequence o EYA4 exon 13. (A) Nona - ec ed amily membe (V:1) displaying wo no mal alleles. (B) A ec ed amily membe (V:2) showing he he e ozygo e 4-bp inse ion. Ma kus Pfis e e al.: A 4bp-Inse ion in he eya-Homologous Region (eyaHR) 611 13. Heanue TA, Da is RJ, Rowi ch, e al. (2002) Dach1, a e eb a e homologue o D osophila dachshund, is exp essed in he de- eloping eye and ea o bo h chick and mouse and is egula ed independen ly o Pax and Eya genes. Mech. De . 111: 75–87. 14. Xu PX, Adams J, Pe e s H, B own MC, Heaney S, Maas R. (1999) Eya1-deficien mice lack ea s and kidneys and show ab- no mal apop osis o o gan p imo dia. Na . Gene . 23: 113–117. 15. Cla k SW, Fee BE, Cle eland JL. (2002) Misexp ession o he eyes absen amily igge s he apop o ic p og am. J. Biol. Chem. 277: 3560–3567. 16. Mo ishi a T, Makishima T, Kaneko C, e al. (2001) Dea ness due o degene a ion o cochlea neu ons in caspase-3-deficien mice. Biochem. Biophys. Res. Commun. 284: 142–149. 17. Takahashi K, Kamiya K, U ase K, e al. (2001) Caspase- 3-deficiency induces hype plasia o suppo ing cells and de- gene a ion o senso y cells esul ing in he hea ing loss. B ain Res. 894: 359–367. 7. La h op G, Lalouel J. (1984) Easy calcula ions o lod sco es and gene ic isks on small compu e s. Am. J. Hum. Gene . 36: 460–465. 8. Sobel E, Lange K. (1996) Descen g aphs in pedig ee analy- sis: applica ions o haplo yping, loca ionsco es, and ma ke - sha ing s a is ics. Am. J. Hum. Gene . 58: 1323–1337. 9. Ve s eken M, DeClau F, Scha eman I, e al. (2000) Audio- me ic analysis o a Belgian amily linked o he DFNA10 locus. Am. J. O ola yngol. 21: 675–681. 10. Heanue TA, Reshe R, Da is RJ, e al. (1999) Syne gis ic eg- ula ion o e eb a e muscle de elopmen by Dach2, Eya2, and Six1, homologs o genes equi ed o D osophila eye o - ma ion. Genes De . 13: 3231–3243. 11. Bonini NM, Leise son WM, Benze S. (1998) Mul iple oles o he eyes absen gene in D osophila. De . Biol. 196: 42–57. 12. Bui QT, Zimme man JE, Liu H, Bonini NM. (2000) Molecula analysis o D osophila eyes absen mu an s e eals ea u es o he conse ed Eya domain. Gene ics 155: 709–720.