Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23)
Introduction
Cadherins comprise a large family of transmembrane proteins, which are crucial for basic cellular processes such as cell–cell adhesion, cell sorting, and cell migration (Tepass et al., 2000). Cadherins are defined by the presence of a variable number of repeat sequences, so-called ectodomains or cadherin motifs located in their extracellular domain. The ectodomain mediates cell–cell adhesion through the formation of Ca2+-dependent homophilic parallel and anti-parallel dimers (Yap et al., 1997). X-Ray diffraction analyses of the two N-terminal ectodomains of Cdh1 (E-cadherin) and Cdh2 (N-cadherin) revealed a barrel-like conformation, which is formed by seven β-strands and two α-helices (Shapiro et al., 1995). Four highly conserved short amino acid sequences are localized in the interface between two ectodomains where they form a complex with three calcium ions. Ca2+-binding is essential for linearization, rigidification and dimerization of the cadherin molecule (Nagar et al., 1996, Pertz et al., 1999).
Loss of function studies in Xenopus, Drosophila and mice implicated cadherins in gastrulation, pattern formation and carcinogenesis (Clark et al., 1995, Kim et al., 1998, Johnson, 1999). Recently, cadherins were also shown to play critical roles during the development of the mammalian inner ear. Mutations in cadherin 23 (Cdh23) and protocadherin 15 (Pcdh15) cause deafness in waltzer (Cdh23v) and ames waltzer (Pcdh15av) mice, respectively (Alagramam et al., 2001, Di Palma et al., 2001). In four alleles of waltzer (Cdh23v), loss of functional protein disrupts the highly organized stereocilia bundle of hair cells in the cochlea and the vestibule during late embryonic/early postnatal development (Di Palma et al., 2001, Wada et al., 2001). The coding sequence of Cdh23 encompasses 10,065 nucleotides (nt) and encodes a 3354 amino acid (aa) single transmembrane spanning protein, named otocadherin. Otocadherin contains 27 tandemly repeated ectodomains which account for 96% of the extracellular protein mass and 87% overall. The specific molecular function of otocadherin is presently unknown. The stereocilia disorganization and the restricted expression pattern suggest that otocadherin may be involved in structuring the hair bundle.
Mutations in the human homolog, CDH23, account for Usher syndrome type 1D, a recessive syndromic disorder characterized by congenital sensorineural hearing loss, retinitis pigmentosa and vestibular dysfunction (Bolz et al., 2001). In a partially consanguineous family, a loss of function mutation causes congenital hearing impairment and a severe retinopathy. However, a missense mutation (R1746Q) causes deafness but has only a mild ocular effect suggesting that auditory hair cells are more vulnerable than retinal cells to subtle changes in the ectodomain structure.
Age-related hearing loss (Ahl) is a characteristic of some inbred strains of mice such as BUB/BnJ, A/J and the widely used C57BL/6J strain (Zheng et al., 1999). In the latter strain Ahl starts with a moderate hearing impairment in 1-year-old mice, but progresses to a complete hearing loss comprising all frequencies as the mice age (Li and Borg, 1991). Histological analyses correlated Ahl with a gradual loss of hair cells, spiral ganglion cells and degeneration of fibrocytes in the spiral ligament (Shnerson et al., 1981, Hequembourg and Liberman, 2001). Linkage studies associated the Ahl with a locus, named Ahl, on chromosome 10 (Johnson et al., 2000). Ahl localizes to a 2 cM genetic region, between D10Mit60 and D10Mit130, which contains the Cdh23 locus (Johnson et al., 2000, Bryda et al., 2001). Mice from more than 20 other inbred strains develop a profound to complete hearing loss over time. Although there is significant variation with respect to the onset (4 weeks of age to 1 year), it is likely that these strains also carry an allele of Ahl (Zheng et al., 1999). Mice from wild-derived inbred strains (CAST/Ei), but also strains from Castle's mice (for example CBA/CaJ), retain good hearing throughout their life. Its map location and demonstrated role in the cochlea suggest Cdh23 as a candidate for Ahl. To gain further insight into the function of Cdh23, we established its complete genomic structure and studied various aspects of the gene in normal and mutant mice.
Section snippets
Elucidation of the intron/exon structure of Cdh23
A draft sequence of three RPCI-23 BAC clones (RP23-161B11, RP23-362C8 and RP23-438B23), containing the Cdh23v locus (Bryda et al., 2001), was generated by the Department of Chemistry and Biochemistry, The University of Oklahoma (GenBank Accession numbers: AC079818, AC079819 and AC079082) supported by the trans-NIH Mouse Genome Initiative. AC079818 is 239,658 bp long and consists of ten unordered pieces, AC079819 covers 180,148 bp in eight unordered contigs and AC079082 spans 207,749 bp in six
Genomic organization of the Cdh23 coding sequence
Otocadherin is encoded by 69 exons spread out over a genomic distance of at least 350 kb (Fig. 1). The exon size ranges from 3 bp (exon 33) to 927 bp (exon 69) and is on average 152 bp (Table 1). Exon 1 encodes the ATG initiation codon and the 66 nt signal sequence. The region encoding the 27 cadherin repeats spans from exon 2 through exon 60. Each ectodomain is encoded by at least two exons. The predicted helical transmembrane domain is encoded by exons 62 and 63. The intracellular
Discussion
This study was undertaken to investigate different aspects of the genomic structure of otocadherin in normal and mutant alleles. The locus comprises at least 71 exons, which cover a genomic distance of not less than 350 kb. Mouse and human loci are very similar in size and organization. The large size of the locus as well as the repetitive domain structure provide a rationale for the many mutant alleles in mouse and human. On the 5′-end, we identified two non-coding exons through database
Acknowledgements
We thank Ken Johnson for the v7J allele. We thank John Northup, Susan Sullivan and Heinz Arnheiter for their valuable comments on the manuscript and members of the lab for stimulating discussions. This work was supported by the National Institute on Deafness and Other Communication Disorders (NIDCD) intramural research project Z01 DC00036-02.
References (28)
- et al.
High-resolution genetic and physical mapping of modifier-of-deafwaddler (mdfw) and waltzer (Cdh23v)
Genomics
(2001) - et al.
Cell-cell interactions in early embryogenesis: a molecular approach to the role of calcium
Cell
(1981) - et al.
A major gene affecting age-related hearing loss is common to at least ten inbred strains of mice
Genomics
(2000) - et al.
mdfw: a deafness susceptibility locus that interacts with deaf waddler (dfw)
Genomics
(1997) - et al.
Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members
J. Mol. Biol.
(2000) - et al.
Age-related changes in the C57BL/6J mouse cochlea. II. Ultrastructural findings
Brain Res.
(1981) - et al.
A point mutation in a cadherin gene, Cdh23, causes deafness in a novel mutant, waltzer mouse Niigata
Biochem. Biophys. Res. Commun.
(2001) - et al.
Mutations in Cdh23 cause nonsyndromic hearing loss in waltzer mice
Genomics
(2001) - et al.
Teratocarcinoma cell adhesion: identification of a cell-surface protein involved in calcium-dependent cell aggregation
Cell
(1982) - et al.
Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses
Hear. Res.
(1999)