Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss Jost Scho ¨nberger 1–4,8 , Libin Wang 1,2,8 , Jordan T Shin 5 , Sang Do Kim 1 , Frederic F S Depreux 1,2 , Hao Zhu 6 , Leonard Zon 6 , Anne Pizard 1 , Jae B Kim 1,2 , Calum A MacRae 5 , Andy J Mungall 7 , J G Seidman 1,2 & Christine E Seidman 1,2 We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles 1,2 cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193– amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six–mediated transcriptional regulation in normal heart function. We previously localized a gene mutated in autosomal dominant dilated cardiomyopathy (DCM) and SNHL in kindred MCE between loci D6S975 (centromeric boundary) and D6S292 (telomeric bound- ary) on chromosome 6q23–24 (ref. 3). Fine mapping narrowed the critical interval to between D6S975 and a CA repeat on BAC RP1-32B1 (AL023693; data not shown). This 2.0-Mb region contained eight known genes and several expressed-sequence tag clusters. We evalu- ated the cardiac and cochlear expression of these transcripts using cDNA libraries from both tissues. Only three of these genes, EYA4, SGK and TCF21 (also called epicardin), were expressed in both heart and cochlea. We determined the structures of these genes and all coding sequences in samples from two affected family members. Analyses of EYA4 identified a 4,846-bp deletion that encompassed the last nucleotide of exon 9, intron 9, exon 10 and part of intron 10. This deletion was present in all affected family members (Fig. 1a) and absent from 300 control chromosomes. No other mutations were identified in the remaining 19 exons of EYA4 or in TCF21 or SGK. We detected a short EYA4 transcript by RT-PCR of lymphocyte mRNA derived from affected individuals. Sequence analyses showed that this transcript lacked nucleotides 1,022–1,245, corresponding to exons 9 and 10, and was aberrantly spliced. The mutant transcript encoded a frameshift after amino acid residue 193, resulting in 29 new residues and a termination signal (Fig. 1b). We designated the mutant Eya4 protein E193. Three human EYA4 mutations have been pre- viously reported to cause dominant SNHL 1,2 ; none has an associated cardiac phenotype. EYA4 mutations that cause SNHL also truncate the peptide and encode only the N-terminal 342, 373 and 586 amino acid residues, respectively. E193 is 149 residues shorter than the shortest SNHL-associated Eya4 peptide (Fig. 1c). EYA4 (ref. 4) is one of four vertebrate orthologs (Eya1–Eya4) of the Drosophila melanogaster gene eyes absent (eya) 5 . Eya proteins are transcriptional coactivators that lack a DNA-binding domain but interact with the transcription factors Six and Dach 4–6 . Eya proteins contain an N-terminal variable region with transactivating function and a highly conserved C-terminal Eya domain that mediates inter- actions with members of the sine oculis family of proteins (Six1–Six6) 7–9 . Six binding by Eya proteins is required for nuclear translocation 7 . Phosphatase activity in the Eya domain releases Dach- Six–mediated transcriptional repression of genes 10–12 . Eya1-regulated genes include those that participate in cranial-facial, musculoskeletal and kidney development 10–12 , but a role for Eya proteins in the heart has not been recognized. We assessed cardiac expression of EYA4 by northern-blot analyses. EYA4 mRNA levels in the left ventricle were higher than in brain, placenta, lung, liver, kidney or pancreas, but lower than in skeletal muscle (Fig. 1d). Despite prominent EYA4 expression in skeletal muscle, clinical abnormalities in individuals with DCM and SNHL Published online 27 February 2005; doi:10.1038/ng1527 1 Harvard Medical School, Department of Genetics and 2 Howard Hughes Medical Institute, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. 3 University Hospital Wu ¨ rzburg, Department of Medicine and 4 Institute of Clinical Biochemistry and Pathobiochemistry, Josef-Schneider Strae 2, 97080 Wu ¨ rzburg, Germany. 5 Developmental Biology Laboratory and Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA. 6 Children’s Hospital Boston, Division of Pulmonary Medicine, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. 7 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. 8 These authors contributed equally to this work. Correspondence should be addressed to C.E.S. (cseidman@genetics.med.harvard.edu). 418 VOLUME 37 [ NUMBER 4 [ APRIL 2005 NATURE GENETICS LETTERS © 2005 Nature Publishing Group http://www.nature.com/naturegenetics