Учебники / Genetic Hearing Loss Willems 2004

V.POSITIONAL CLONING OF DFNB12

We used a positional cloning strategy to identify the DFNB12 gene. Consanguineous families segregating SNHL were used to refine the linked interval of DFNB12 on chromosome 10q21–q22 (15). One DFNB12 family, PKSR46a, had recombinations in two individuals that reduced the DFNB12 interval to 0.5 cM. The gene for DFNB12 was positionally cloned based on an evaluation of genomic DNA sequence of 18 genes from the critical chromosomal interval, and missense mutations of CDH23 were identified in five DFNB12 families (15). Since the DFNB12 and USH1D loci overlapped and might be allelic, we also sequenced the CDH23 gene in six USH1D families. We identified nonsense and splice-site mutations cosegregating with the Usher syndrome phenotype, demonstrating allelism. Simultaneously and independently, mutations in the mouse ortholog (Cdh23) were identified in waltzer mice (26), which led to the identification of CDH23 mutations in USH1D patients (16). CDH23 is one of three known examples of a gene in which mutations can cause nonsyndromic deafness or Usher syndrome. Mutations of MYO7A are associated with DFNA11, DFNB2, and USH1B (22,27–29), and mutations in USH1C are associated with both USH1C and DFNB18 (23,24,30).

VI. CDH23 MUTANT ALLELES

CDH23 homozygous missense mutations were identified in children of consanguineous marriages segregating hearing loss and exhibiting linkage to the DFNB12 locus (15). CDH23 nonsense, splice-site, frameshift, and missense mutations were identified in USH1D families (15,16). We proposed a genotype-phenotype correlation where some amino acid replacements in cadherin 23 were presumed to cause partial loss of function and nonsyndromic deafness while more disabling mutations and functional null alleles of CDH23 cause RP and vestibular dysfunction in addition to deafness (15). This genotype-phenotype correlation was further examined in a larger cohort of nonsyndromic deafness and Usher syndrome type I patients. More than 100 probands with nonsyndromic deafness or USH1 were screened for CDH23 mutations (31) and the type of mutant alleles of CDH23 characterized was consistent with the genotype-phenotype correlation.

The 19 CDH23 mutations identified in DFNB12 families are homozygous missense mutations or compound heterozygous missense mutations in the putative extracellular domain of cadherin 23. Many of the nonsyndromic deafness families have missense mutations that occur in the conserved calcium-binding motifs of the extracellular EC domains (15,31,31a). This suggests that Ca2+ chelation by the EC domains of cadherin 23 is required in

the organ of Corti but may not be necessary in the retina. However, we do know that some ‘‘nonsyndromic deafness families’’ have presymptomatic signs of RP revealed by ERG and fundus examinations (31), suggesting that even missense mutations of CDH23 may have a subclinical e ect on the retina.

Nonsense mutations, insertions, deletions, splicing variants, and missense mutations have all been identified in USH1 probands, many of whom have a typical USH1 phenotype. Not surprisingly, some families with CDH23 mutations exhibit an atypical USH1 phenotype. A ected members of family PKSR7a were found to be homozygous for the IVS66+1G > A mutation at the splice donor site of an exon-intron boundary encoding the putative cytoplasmic domain of cadherin 23. This Pakistani family was originally diagnosed with nonsyndromic deafness, but reevaluation of the two oldest a ected individuals (27 and 28 years old) by an ophthalmologist revealed early-stage RP (15).

The identification of late-onset RP demonstrates the importance of monitoring DFNB12-a ected individuals for presymptomatic development of RP. Likewise, three branches of a Cuban family were reported to have variable degrees and ages of onset of retinal degeneration depending on their CDH23 genotypes; homozygous R1746Q, homozygous Q1496H (a putative splice-site mutation), or compound heterozygous R1746Q/Q1496H (16). Moreover, additional a ected individuals have been diagnosed with atypical Usher syndrome by virtue of an absent, mild, and/or late-onset phenotype (31).

To date, 27 missense, five nonsense, two insertions, seven deletions, and 10 splice-site mutations of CDH23 have been identified and are associated with a range of hearing and retinal phenotypes (summarized in Ref. (31)). All reported CDH23 alleles identified in nonsyndromic deafness patients have been missense mutations (15,31), with the exception of one profoundly deaf individual. She is compound heterozygous for a DFNB12 missense mutation and a truncating mutation in the region of CDH23 encoding the extracellular domain, and has normal vestibular and visual function at age 26 (JM Schultz and AJ Gri th, unpublished data, 2003). This suggests that a single hypomorphic missense allele is su cient for a normal retinal phenotype. However, until we understand the function of cadherin 23 in the retina and auditory system, and the role of modifier genes, we cannot accurately predict the phenotype of an individual who is compound heterozygous for a hypomorphic and a more severe mutant allele of CDH23.

VII. CDH23 EXPRESSION ANALYSIS

Northern blot analysis of human RNA, from dissected ocular tissues and brain, probed with the portion of CDH23 encoding the unique cytoplasmic

domain, demonstrates a f9.5–10 kb mRNA expressed in the retina, but not in the ciliary body, retinal pigmented epithelium-choroid, lens, iris, or occipital cortex (15). The 488-bp probe of our Northern blot was derived from a portion of exons 66–69 and could be amplified from a human cochlear cDNA library, demonstrating its expression in the cochlea. No Northern hybridization signal was observed for human RNA from heart, brain, placenta, lung, liver, skeletal muscle, or kidney using the same probe derived from the cytoplasmic domain. However, a 1.35-kb mRNA was detected in human pancreas but has not been further characterized (15). CDH23 message was detected in RNA from human retina, brain, kidney, skeletal muscle, and blood by RT-PCR analysis using primers that hybridize to the region of the gene encoding the twenty-second and twenty-third EC domains (16).

Using RT-PCR, Cdh23 expression in the mouse was found in the brain, heart, kidney, eye, and ear (26). In the auditory system, in situ hybridization experiments demonstrate Cdh23 expression in the mouse cochlear neuroepithelium with specific staining of neurosensory cells (26,32). Using embryonic day 18 tissue, Wilson and co-workers reported a more extensive in situ expression analysis with a Cdh23 probe showing hybridization in the hair cells of the vestibular sensory epithelium and cells comprising Reissner’s membrane, as well as the cochlear inner and outer hair cells (32). Cdh23 expression was also observed in the tongue and olfactory bulbs (32). For both of these studies, it would be interesting to know whether their in situ probes would fail to detect Cdh23 message in a Cdh23-null mouse.

The localization of the CDH23/Cdh23 mRNA expression is not consistent among these studies and appears to be dependent upon the assay used to detect the message. The probes for RT-PCR and in situ hybridization were derived from regions of the extracellular domain that also encode EC repeats, which may not be specific for CDH23/Cdh23, while the probe for our Northern blot analysis was directed against the novel cytoplasmic domain. RT-PCR is a more sensitive method of detecting low levels of message in comparison to Northern blot hybridization analyses. However, the physiological relevance of these potentially low mRNA levels in tissues other than the inner ear and retina has not been determined.

VIII. MOUSE MODEL FOR STUDYING

CADHERIN 23 FUNCTION

Nonsense, splice-site, and frameshift mutations of Cdh23 have been identified in 10 strains of waltzer mice, which have sensorineural deafness and

vestibular defects (26,32–34). Each mutant allele is predicted to be a functional null. Cdh23 mRNA localizes to the cochlear hair cells by in situ hybridization (26,32). Scanning electron microscopy of the cochlear inner and outer hair cells of mice with Cdh23 mutations reveals disorganized stereocilia (26,33,35). Unlike humans with CDH23-null alleles, no retinal phenotype was observed for waltzer mice with Cdh23-null mutations. Spe- cies-specific variations may account for the di erences in the retinal phenotype of humans and mice. The mouse retina may express another cadherin that is able to compensate for the loss of cadherin 23, or the retinal phenotype may be late-onset or subtle. Alternatively, since the 10 Cdh23 alleles are not on the same genetic background, some waltzer strains may harbor a modifier gene that suppresses retinal degeneration but not hearing loss.

IX. FUNCTION OF CADHERIN 23

Although there is no published supporting data, perhaps cadherin 23 is involved in the organization of hair cell stereocilia through homophilic interactions on adjacent stereocilia. The stereocilia of hair cells are arranged in rows of decreasing height and are interconnected by tip links, shaft connectors, side links, and ankle links that have been identified in ultrastructural studies (36,37). Perhaps cadherin 23 is a molecular correlate of one or more of these structures.

In other tissues, some cadherins are bound to h-catenin or plakoglobin, which in turn binds to other proteins such as a-catenin, vinculin, and Z0–1 (2). Cadherin 23 likely does not bind any of these proteins since the cytoplasmic domain does not contain these protein-binding motifs. CDH23 has an alternatively spliced exon in the region encoding the cytoplasmic domain. Mutations in this alternate exon 68 have not yet been identified in DFNB12 or USH1D probands. RT-PCR experiments conducted with RNA from adult mice show that the cadherin 23 isoform containing exon 68 is expressed in the inner ear but not in the retina (38). Yeast two-hybrid and biochemical assays demonstrate an interaction of the cytoplasmic domain of cadherin 23 with the PDZ domains of harmonin, the USH1C gene product (38,39).

In summary, missense mutations of CDH23 with presumed subtle functional disablements of cadherin 23 are associated with nonsyndromic deafness, while mutant alleles of CDH23 with a more severe e ect cause deafness accompanied by vestibular and progressive retinal dysfunction. Studies of the phenotypic variation caused by di erent mutations of CDH23 coupled with the assignment of functions to cadherin 23 within the inner ear and retina may eventually permit the rational design of therapies.

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