Учебники / Genetic Hearing Loss Willems 2004

and that expression of both eya and so is initiated by ey (eyeless) (10,11). In vertebrates, members of the Six gene family (the orthologs of so) bind to Eya proteins to induce nuclear translocation of the resultant protein complex (12). Amino-terminal transcriptional activation activity has been demonstrated for the Drosophila eya and murine Eya1-3 gene products, suggesting that EYA interactions and pathways are conserved across species (9–17).

C.EYA4 Structure and Predicted Effects of DFNA10 Causing Mutations

The structure of EYA4, as deduced from its cDNA sequence, conforms to the basic pattern established by EYA1-3. The EYA4 coding sequence is comprised of 20 exons. The mutations we identified in EYA4 are predicted to a ect the eyaHR. The 1468insAA mutation in the American family causes a frameshift and subsequent novel stop codon in exon 14. Since the eyaHR of EYA4 is encoded by the 3V-most 6 bp of exon 12 through the 5V-most 78 bp of exon 21, this mutation e ectively eliminates the entire eyaHR (Fig. 6B). The 2200C!T mutation in the Belgian family creates a premature stop codon, eliminating 52 amino acids from the C-terminal end of the eyaHR (Fig. 6B). Given the importance of the eyaHR to EYA protein function, it is not surprising that these mutations have a phenotypic correlate.

D.EYA4 Alternative Splicing

Alternative splicing of EYA4 mRNA has been reported for exons 5, 16, and 20 and results in several isoforms (6) (Fig. 7). Exon 5 may be spliced in or out; the first 68 bp of exon 16 may be spliced out by use of a cryptic splice acceptor site within the exon that results in a predicted truncated protein of 452 amino acids; and exon 20 may be substituted for exon 19, equal in length and with 69.3% nucleic acid identity and 66.7% amino acid identity.

1.EYA4 Splice Variants in the Cochlea and Brain (3)

To determine which splice variants are expressed in the cochlea, RT-PCR and sequencing was performed on human fetal cochlear RNA, using primers flanking exons 5, 16, and 19/20. Human fetal and adult brain cDNA libraries were screened as well. Neither the exon 5–containing isoform of EYA4 nor the shorter form of exon 16 was detected in the cochlea, but both exon 19– and exon 20–containing splice variants could be amplified in human fetal cochlear cDNA. Both of the latter two isoforms also were present in

Figure 7 Alternative splicing of EYA4 mRNA involving (A) exon 5, (B) exon 16, and (C) exon 19/20. Numbered boxes correspond to exons, thin solid lines to introns, and alternative splicing patterns are represented by heavy solid and dotted lines. (Adapted from Ref. 6.)

human brain cDNA libraries, although only the exon 19–containing variant was found in fetal brain. Adult brain had minimally detectable exon 19–containing EYA4 transcripts and abundant expression of the exon 20– containing variant. Adult rat cochlea had only the exon 20–containing variant.

V.Eya GENE EXPRESSION

Eya genes are expressed in a wide range of tissues early in embryogenesis, and although each Eya gene has a unique expression pattern, there is extensive overlap. For example, murine studies have shown that Eya1, Eya2, and Eya4 are all expressed in the presomitic mesoderm and head mesenchyme, but only Eya1 and Eya4 are expressed in the otic vesicle (12). Eya3 expression is restricted to craniofacial and branchial arch mesenchyme, in regions underlying or surrounding the Eya1-2-or-4 expressing cranial placodes (12,15).

A.Eya4 Inner Ear Expression

In situ hybridization, performed on embryonic and postnatal rat cochleae with a probe derived from mouse Eya4 cDNA, revealed strong Eya4 mRNA expression in the neuroepithelium of the developing rat inner ear (Fig. 8). On embryonic day 14 (e14.5) and e16.5, moderate expression was present primarily in the upper epithelium of the cochlear duct, a region that gives rise to Reissner’s membrane and the stria vascularis. Low-level expression was observed in the mesenchyme surrounding the duct. The highest levels of expression were seen on e18.5 and were found in areas of the cochlear duct destined to become the spiral limbus, organ of Corti, and spiral prominence,

Figure 8 In situ hybridization for Eya4 mRNA in the developing rat cochlea. Expression is greatest in the epithelium of the cochlear duct (CD). At e14.5 and e16.5, this expression is greatest in the upper half of the duct, cells destined to form the stria vascularis and Reissner’s membrane. Concurrently, weak expression is observed in the mesenchyme adjacent to the cochlear duct. Cochlear expression of Eya4 mRNA peaks at e18.5 and is found preferentially in the lower half of the duct epithelium, in the greater (arrows) and lesser (arrowheads) epithelial ridges, especially in the basal turn. At older ages, expression becomes restricted to cells derived from the spiral limbus, organ of Corti, and spiral prominence. For the first 2 weeks after birth, strong expression is also observed in cells of the developing bony cochlear capsule, as illustrated at p12. (From Ref. 3.)

especially its more rapidly maturing basal turn. These areas continued to express Eya4 as total expression decreased. Strong expression was also observed in the developing cochlear capsule during the period of ossification, from shortly after birth until p14. In the developing vestibular system, expression was observed primarily in the developing sensory epithelia.

In situ hybridization studies in developing rodent inner ears thus reveal a spatial variability in Eya4 expression not seen with Eya1 expression. Both Eya1 and Eya4 are expressed early in the otic vesicle (6,18). However, after di erentiation of the otic vesicle into auditory and vestibular components, Eya4 is concentrated in the upper cochlear duct within cells that develop into the stria vascularis and Reissner’s membrane, while Eya1 is expressed in the floor of the cochlear duct, an area that gives rise to the organ of Corti. Throughout development of the inner ear, Eya1 expression is maintained in derivatives of the neuropithelium of the cochlear duct floor; Eya4 expression shifts from the upper cochlear duct to the neuroepithelim of the cochlear duct floor only at stage e18.5.

VI. WHY IS DFNA10 HEARING LOSS POSTLINGUAL?

These data suggest an apparent disjunction between the early expression of Eya4 and the late-onset hearing loss characteristic of DFNA10; however, it is not unusual for genes to play di erent roles at di erent times in development. Eya4 is present in the adult rodent inner ear, where expression of the exon 20–containing splice variant has been documented. Based on the in situ data and the DFNA10 phenotype, it is tempting to speculate that Eya4 plays a developmental role in embryogenesis and a survival role in the mature system. Although the neuroepithelial cell types that express Eya4 have not been characterized, the apparent overlap in expression of Eya1 and Eya4 in embryogenesis suggests that some functions of Eya4 may be redundant to Eya1 during development. Creating and studying a mouse with a targeted mutation of Eya4 would resolve many of these issues.

VII. WHY IS DFNA10 HEARING LOSS NONSYNDROMIC?

The association of late-onset hearing loss with developmentally important transcriptional activators is not unprecedented, as mutations in POU4F3 are known to cause postlingual hearing loss at the DFNA15 locus (14). What is surprising, however, is the limited DFNA10 phenotype, especially when one considers the clinical impact of EYA1 mutations. Like the DFNA10-causing EYA4 mutations, the BOR syndrome-causing mutations in EYA1 nearly all

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Figure 1 The extended Dutch family with hearing impairment linked to the DFNA5 locus. The pedigree represents only those family members from whom DNA was obtained during our molecular studies in the period 1995–1998. DFNA5 genotyping defines the a ection status of each family member. A ected individuals are represented by solid symbols. Spouses are omitted. Branches 1, 4, and 5 are excluded, as these contain no additional patients.

2C). Generally, most individuals use hearing aids as soon as their losses exceed 40 dB in the speech frequency range. Occasionally, CT scans have been made; however, no abnormalities have been detected. Recently, speech recognition scores were analyzed in 34 a ected individuals. Surprisingly, in spite of the severity of the hearing impairment, speech recognition scores were relatively good. At age 70, the extrapolated maximum score was still more than 50%. In comparison with two other well-characterized types of progressive hearing impairment, the maximum phoneme scores for DFNA5 were between those for DFNA2 and DFNA9, DFNA9 showing the worst scores, while typical DFNA5 threshold levels were fairly similar to DFNA2 and DFNA9 threshold levels (7). Although none of the a ected family members had vestibular problems, four of them were subjected to extensive vestibular testing, but the response parameter values were normal with few exceptions (8).

Figure 2 The progression of the hearing impairment in the DFNA5 family. Air conduction is represented by open circles. (A) Initially, high-tone losses increase, but low-frequency thresholds remain normal. (B) When the high-tone thresholds further deteriorate beyond 80 dB, the lower frequencies become a ected as well. (C) Finally, the endstage is reached.