A mutation in OTOF, encoding otoferlin, a FER-1-like protein, causes DFNB9, a nonsyndromic form of deafness

Approximately 1 in 1,000 children is affected by deafness at birth or before 2 years of age (that is, in the prelingual period). Although many syndromes including hearing loss have been described, hearing loss is the sole symptom in most cases; these forms are referred to as isolated or nonsyndromic deafness. In developed countries, approximately two-thirds of prelingual nonsyndromic deafness cases are of genetic origin, 85% of which are inherited in an autosomal recessive mode (DFNB forms). These forms most frequently cause severe or profound hearing loss, which impedes speech acquisition (for review, see 1). DFNB forms have been predicted to be monogenic diseases that are genetically highly heterogenous; 20 different DFNB loci have been reported to date. Five of the corresponding genes have been identified: GJB2 (or CX26), encoding the gap junction protein connexin 26 (DFNB1; ref. 2); MYO7A and MYO15, encoding two unconventional myosins, myosin VIIA and myosin XV (DFNB2 and DFNB3, respectively; refs 3,4 and 5); PDS, encoding pendrin, a putative sulfate⁶ or iodide⁷ transporter (DFNB4); and TECTA, encoding a component of the tectorial membrane, -tectorin⁸ (DFNB21). With the exception of DFNB1, which accounts for approximately one-half of all non-syndromic, prelingual, inherited deafness cases in the populations studied^{9, 10, 11} (France, Italy, Spain, United Kingdom and New Zealand), only one or a few affected families have been reported for each deafness locus. As a result, in most instances the defined chromosomal candidate gene interval is too large to undertake the search for the corresponding gene exclusively by a positional cloning strategy. To circumvent this difficulty, a candidate gene approach based on the isolation of genes specifically or preferentially expressed in the inner ear has been developed, as the proteins encoded by these genes are likely to have a role in auditory function¹. Here, using a combination of the candidate gene strategy that we previously described^{12, 13} and a positional cloning strategy, we report the identification of the gene underlying DFNB9 (OMIM 601071).

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We previously reported the segregation of the DFNB9 locus in a consanguineous family (family F) living in northern Lebanon and affected with profound sensorineural prelingual hearing loss¹⁴. The causative gene had been assigned to a 2-cM interval, delimited by D2S2303 and D2S174, on chromosome 2 at p23.1. A recent genetic linkage study of 30 Lebanese families presenting with deafness and large enough to provide significant lod scores, led us to identify three additional unrelated consanguineous DFNB9-affected families (AB, K1 and K2). Families F, AB and K1 were living in geographically distinct regions in northern Lebanon, and family K2 in southern Lebanon. The 10 affected members of family F, as well as the 11 affected members of the 3 other families, suffered from a prelingual severe to profound form of sensorineural deafness. The results of the segregation analysis performed in family AB ( Fig. 1), combined with previous mapping data obtained in family F, permitted the refinement of the DFNB9 interval to between D2S158 and D2S174 (in a region of 1 cM; Figs 1 and 2).

Figure 1. Genetic linkage analysis of the DFNB9-affected family AB. Individuals with prelingual deafness are indicated by filled symbols and unaffected individuals by open symbols. Segregation analysis with the microsatellite polymorphic markers located on chromosome 2p23.1 limited the DFNB9 candidate interval to between D2S158 and D2S165. The haplotype associated with the mutated DFNB9 allele in this family (AB) is indicated by a vertical bar. Combining these data with the previous mapping data obtained in family F (ref. 14) permitted the narrowing of the candidate gene interval to between markers D2S158 and D2S174 (a distance of 1 cM). Full Figure and legend (40K)

Figure 2. Physical map of the DFNB9 region. The candidate region is delimited by loci D2S158 and D2S174. Polymorphic markers corresponding to D2S2223 and D2S2350 are homozygous in all deaf individuals from the DFNB9-affected families AB, F, K1 and K2. YACs, BACs and PACs are represented by hatched, grey and dotted lines, respectively. PCR amplification using primers derived from the 3´ ends of cDNAs permitted the mapping of 4 genes, namely PPIL1 (encoding peptidylprolyl isomerase (cyclophilin)-like 1), HADHA and HADHB (encoding hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase (trifunctional protein) and subunits), and CENPA (encoding centromere protein A) to this contig. ESTs RH57018, RH20012, RH1296, RH25229, RH12053 and RH26192 were also assigned to this contig. The ten corresponding genes were ordered, except HADHA /RH20012, HADHB/RH1296 and CENPA/RH26192. The position of HADHA and of the gene encoding RH20012 with regard to D2S158 was not determined. RH12053 corresponds to the 3´ end of the OTOF cDNA. PCR amplification performed on the BACs of the contig oriented the 5´ end of the gene centromeric to its 3´ end. The deduced amino acid sequence corresponding to RH57018 was similar to those of ras-related GTP binding proteins. Full Figure and legend (21K)

A contig of yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and P1 phage artificial chromosomes (PACs) covering the defined chromosomal interval was constructed. From the size of the various inserts, and particularly that of YAC 876b12 (720 kb), which covers the entire interval, the candidate interval was estimated to extend less than 700 kb (Fig. 2). Two genes, HADHB (encoding trifunctional protein subunit) and CENPA (encoding centromere protein A), and expressed sequence tags (ESTs) RH1296, RH25229, RH12053 and RH26192, belonging to different transcription units, were assigned to this interval. The two genes were not considered as candidate genes for deafness due to the putative functions of their encoded proteins. The ESTs were submitted to round(s) of extension by 5´-RACE-PCR on total fetus mRNA. The predicted amino acid sequences they encode were compared with those derived from clones previously isolated from two subtracted mouse cochlear cDNA libraries^{12, 13}. The deduced amino acid sequence of one of these extended clones, RH12053 (isolated from infant brain), showed 89.7% identity and 97.1% similarity with the predicted sequence (of 205 aa) encoded by mouse cDNA clone 75A2MH. These two homologous sequences detected a single 9.5-kb and 9.0-kb band on EcoR1-digested human and mouse DNA, respectively. This indicates that RH12053 and 75A2MH are derived from orthologous genes. We thus considered the corresponding human gene a candidate for DFNB9.

Figure 3. Sequence and structure of human otoferlin. a, Deduced amino acid sequence of human otoferlin. The predicted C-terminal transmembrane domain is indicated in bold. The three predicted C2 domains are underlined. The five aspartyl residues that presumably bind Ca2+ in the three C2 domains20 are indicated in bold; two are located in the loop between predicted strands 2 and 3, and three others in the loop between predicted strands 6 and 7. The C2A, C2B and C2C domains share only 20% amino acid identity and 50% amino acid similarity on average. For the C2A (aa 196-329) and C2B (aa 709-833) domains, the similarity with Syt-1 C2A does not extend to the 1 and 8 strands; however, 1 and 8 strands are predicted by the secondary-structure analysis software. The C2C domain (aa 949-1,104) shows similarity with Syt-1 C2A along the eight strands, except for the longer loop between 5 and 6 (37 aa instead of 14 aa). The position of the premature stop codon (Y730) in DFNB9 patients is indicated (arrowhead). b, Schematic representation of otoferlin, dysferlin and FER-1. The transmembrane domain is indicated by a vertical bar. Dashed boxes indicate the sequences related to C2 domains, partial or full. The C2 domains with putative Ca2+-binding motifs are indicated (asterisk). The three proteins are mainly similar in their C-terminal regions, where otoferlin (aa 196 to C-terminus) showed 39.3% and 30.1% identity and 65.6% and 57.5% similarity with dysferlin (aa 1,135 to C-terminus) and FER-1 (aa 954 to C-terminus), respectively. The N-terminal 195-aa fragment of otoferlin is weakly similar to dysferlin (aa 697-889) and to FER-1 in two different regions (aa 13-217 and 550-745). In dysferlin, the 2 N-terminal sequences related to C2 domains are located at aa 1-108 and 363-504, respectively. In FER-1, the three C-terminal sequences related to C2 domains show only 16.4-21.9% identity and 48.4-49.2% similarity with Syt-1 C2A. Full Figure and legend (107K)

OTOF extends over 21 kb and contains 28 coding exons, a 5´ UTR exon and a 1,018-bp 3´ UTR exon. Primers flanking each of the coding exons and adjacent splicing sites were selected (Table 1). All OTOF coding exons were amplified and sequenced in family F. We detected a transversion in exon 18 at position 2,416 (TA) that substitutes a stop codon for a tyrosine codon (Y730X; Fig. 4). This nonsense mutation is expected to lead to a truncated protein (of 729 aa), and was also detected in families AB, K1 and K2. The mutation was homozygous in all affected individuals (21 individuals) and heterozygous in their parents (11 individuals). It was not detected in 106 unaffected individuals living in Lebanon who were unrelated to these families. These results identify OTOF as the causative gene for DFNB9. The existence of the same mutation in four unrelated DFNB9 families with different geographical origins indicates a single mutation may have spread in the Middle East. Therefore, the only other DFNB9 family reported, which originates from eastern Turkey¹⁷, might carry the same mutation.

Figure 4. Sequence analysis of the mutation present in OTOF exon 18 in family F. Genomic sequence of a control individual (a), a heterozygous parent (b) and an affected individual (c). The position of the TA transversion, which results in a premature stop codon at aa 730, is indicated (arrowhead). Full Figure and legend (63K)

Table 1. Primers for PCR amplification of human OTOF exons Full Table

Analysis of the deduced amino acid sequence of otoferlin reveals a highly hydrophobic C terminus (of 33 aa), including a stretch of leucine residues predicted to form a transmembrane domain (aa 1,198-1,214; Fig. 3a). No leader peptide or any other protein-targeting signal was detected. The rest of the protein (aa 1-1,197) was predicted to have a cytoplasmic location. We found 4 putative N-glycosylation sites and 13 potential protein kinase C phosphorylation sites. Sequence comparisons showed that otoferlin is homologous not only to the nematode FER-1 protein¹⁶, but also to a newly identified human protein, dysferlin (encoded by DYSF). DYSF has recently been reported to underly Miyoshi myopathy (MM) and limb-girdle muscular dystrophy type 2B (LGMD2B; refs 18,19). Otoferlin is 38.1% and 28.0% amino acid identical and 64.0% and 52.9% amino acid similar to dysferlin and FER-1, respectively, but dysferlin (2,080 aa) and FER-1 (2,034 aa) are longer than human otoferlin. The three proteins are mainly similar in their C-terminal regions (aa 196 to the C-terminus of otoferlin), whereas the amino-terminal sequence (of 195 aa) of otoferlin presents only weak similarity with dysferlin and FER-1 (Fig. 3b). Three sequences with homology to C2 domains were recognized in otoferlin, namely C2A (aa 196-329), C2B (aa 709-833) and C2C (aa 949-1,104; Fig. 3). Approximately 100 C2 domains have now been described. According to the three-dimensional structures established for four of them, C2 domains are composed of two four-stranded sheets with high structural homology (although two distinct topologies have been reported^{20, 21}). The C2A domain of rat synaptotagmin-1 (Syt-1 C2A) is the most extensively characterized. The three otoferlin C2 domains showed 25.0-29.7% identity and 55.4-58.6% similarity with Syt-1 C2A (ref. 22). Each contains five aspartyl residues located at positions similar to those that bind Ca²⁺ in Syt-1 C2A (Fig. 3; Refs 20,21).

Figure 5. RT-PCR analysis of Otof expression in mouse tissues. a, Primers used for Otof were 75A2MH-P5 (3´-UTR exon) for reverse transcription and 75A2MH-P1 (exon 27) and 75A2MH-P3 (exon 25) for PCR. b, Gapd was used as a positive control. '+/-' indicates presence or absence of reverse transcriptase in the RT reaction. Full Figure and legend (54K)

Figure 6. In situ hybridization analysis of Otof expression in mouse inner ear. Cochlea (a,b) and vestibule (c-f) are shown. a, At P2, Otof is expressed in the inner hair cells (ihc). A weak signal is seen in the outer hair cells (ohc) and spiral ganglion (sg) neurons. b, Otof is detected in the inner hair cells at P12. No signal is observed in the outer hair cells and supporting cells. OTOF is expressed in the neuroepithelia of the utricle (c) and lateral and superior crista ampullaris (d) at P2. The orientation of the sections is indicated in (d). A, anterior; P, posterior; D, dorsal; V, ventral. The detail of the utricular neuroepithelium boxed in (c) shows Otof labelling in some hair cells (e) and myosin VIIA staining in hair cells ( f). Otof is highly expressed in type I hair cells (arrowheads), whereas only weak labelling is observed in type II hair cells (arrows). The quenching of the fluorescence signal in type I cells is due to co-localization of Otof and Myo7a labellings. rm, Reissner's membrane; sv, stria vascularis. Bar, 50 m (a), 20 m (b), 70 m (c ,d), 35 m (e,f). Full Figure and legend (195K)

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Otoferlin is the second human protein described that is related to the C. elegans FER-1 protein. The first one, dysferlin, was identified on the basis of its implication in two muscular dystrophies^{18, 19}. According to sequence analysis, these two human proteins are predicted to be C-terminal membrane-anchored cytosolic proteins containing C2 domains. We identified three full C2 domains in otoferlin. The re-examination of the dysferlin sequence in light of the deduced characteristics of otoferlin also revealed the presence of three full C2 domains at corresponding positions. Some C2 domains bind Ca²⁺, and this binding has been shown to primarily involve aspartyl side chains that act as bidentate ligands for these ions. In otoferlin and dysferlin, the three common C2 domains possess these five aspartyl residues, suggesting that their interactions with other molecules are Ca²⁺-dependent^{20, 21}. Dysferlin has two additional sequences related to C2 domains in its N-terminal end; the most N-terminal one is only partial (lack of the predicted 1 strand) and both are missing some aspartyl residues. FER-1 protein presents substantial differences with otoferlin and dysferlin. Three C2-related sequences, detected at the same positions as C2A, C2B and C2C in otoferlin and dysferlin, show only weak similarity with C2 domains, and lack most of the aforementioned Ca²⁺-binding aspartyl residues (Fig. 3b); moreover, the cysteine-rich region located near the C terminus of FER-1 is absent from the human proteins. Based on the existence of at least two additionalhuman ESTs with FER-1 similarity, the mammalian FER-1-like family should comprise several other members¹⁶; their study should help clarify the relationship between the mammalian protein family and FER-1.

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Human OTOF cDNA was obtained by 5´-RACE-PCR on total fetus poly(A)⁺ RNA according to the supplier's instructions (Marathon cDNA amplification kit, Clontech); random hexamers or cDNA-specific primers were used in reverse transcription. The reconstruction of the complete ORF of OTOF was checked by RT-PCR performed on total human fetus mRNA using the primers RH12053-NGSP-5´ (5´-TCCTCATGATGACCGATACTCAGG-3´) and RH12053-NGSP-3´ (5´-AGGGTTGAGGAACCAGACGAAGG-3´), located in the 5´ and 3´ UTR, respectively. The amplification product was cloned into pGEM-T vector (Promega) and sequenced using the Thermo Sequenase dye terminator cycle sequencing pre-mix kit version 2.0 (Amersham Life Science) on an ABI 377 DNA sequencer. YAC clone 876b12 was subcloned into a gt11 vector and direct sequencing of the exons and flanking introns was performed using primers derived from the OTOF cDNA, thus allowing the determination of the exon-intron structure of the gene (manuscript in preparation).

In situ hybridization was performed using digoxigenin-11-UTP-labelled RNA probes⁴⁹. The mouse Otof cDNA fragment from 75A2MH was cloned into pGEM-T vector (Promega). Sense and antisense probes were transcribed using SP6 and T7 RNA polymerases after appropriate linearization. After DNase I digestion, the probes were ethanol-precipitated twice with LiCl (0.4 M). Mouse inner ears were fixed for 1 h at 4 °C in 4% paraformaldehyde-PBS. After three washes in PBS, they were immersed in 20% sucrose overnight at 4 °C. Cryostat sections (10-14 m) were postfixed and rinsed in PBS. Following prehybridization at RT for at least 3 h, they were hybridized overnight at 56 °C in a humid chamber. The sections were then washed and incubated with sheep anti-digoxigenin antibody coupled to alkaline phosphatase. Staining by NBT/BCIP (Boehringer) was performed for 2 h at 37 °C and overnight at RT. Some sections were directly mounted in Aquatex (Merck) and observed using an optical microscope (Leica). Other sections were also stained by immunofluorescence with an antibody to myosin VIIA, as described⁴⁷. The care of experimental animals was in accordance with institutional European guidelines.

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