Учебники / Genetic Hearing Loss Willems 2004

to identify a genomic region that segregates with the otosclerosis phenotype. A computer algorithm then is used to calculate the likelihood that the genome segregation pattern and the clinical status are linked and not chance events. If a ‘‘linked’’ interval is found, it will contain the disease-causing gene. To minimize the size of the linked interval and thereby reduce the number of genes that must be considered, additional family members can be studied and additional markers can be used. Mutation screening of the genes in the candidate interval is used to find the disease-causing gene.

To date, only three otosclerosis-causing genes have been localized using three large families showing an autosomal dominant inheritance pattern. In 1998, we localized the first otosclerosis-causing gene, OTSCI, to a 14.5-cM interval on chromosome 15q between FES and D15S657 (32). An additional family originating from Tunisia also has been reported to be linked to this locus (33). In 2001, we mapped a second locus, OTSC2, flanked by two markers on chromosome 7q, D7S495 and D7S2426, that define a 16-cM interval (34). Recently, OTSC3 was mapped to chromosome 6p21-22 (35). None of the responsible genes has been cloned. Furthermore, the analysis of additional families segregating otosclerosis has provided evidence for further genetic heterogeneity; in addition to the three known loci, at least one additional otosclerosis locus must exist (36).

B.Complex Forms of Otosclerosis

Genome scans for complex diseases are carried out using a large collection of small families. Instead of the standard parametric linkage analysis used for monogenic conditions—where parameters such as inheritance pattern and penetrance need to be defined—nonparametric methods are used. The most frequently used nonparametric linkage method, a ected sib-pair (ASP) analysis, has been applied to many complex diseases besides otosclerosis. Examples include schizophrenia, bipolar disorder, diabetes, autism, multiple sclerosis, and lupus. For many of these diseases, significant linkage has been detected. A genome search by ASP analysis identifies the chromosomal regions that are likely to be shared between a ected sibs. Chromosomal regions resulting from the genome searches are large and contain a large number of genes.

Another tool for the analysis of complex diseases are association studies in a large number of a ected individuals and controls (case-control study) or in patient-parent trios. An allele is associated with a disease in a population if patients have this allele more often than the controls. The disease-associated allele may be the direct cause of the disease (causative e ect) or it may be very close to the disease-causing mutation so that the two are coinherited. This latter situation holds if the mutation occurred only once in the population

(founder e ect). An advantage of association studies is that they are more powerful to detect weak e ects. A major di erence with linkage is that association can be detected only over very small genetic distances (less than 1 cM). A total genome scan by association would require 10,000 markers and is not possible with current technology. Association studies are therefore limited to candidate genes. These genes can be selected on the basis of functional information or can be located in candidate regions from genome scans.

Results of nonparametric linkage analysis for otosclerosis have not yet been published. A single candidate-gene-based association study has been performed by McKenna et al. (37). Because of the clinical and histopathological similarities between otosclerosis and type I osteogenesis imperfecta, they investigated a possible association between otosclerosis and three collagen genes. Because mutations of type I collagen genes underlie the milder forms of osteogenesis imperfecta (38), the COL1A1, COL1A2, and COL2A1 genes were studied. The investigators found a statistically significant association between otosclerosis and COL1A1, while di erences in allele frequencies could not be detected between the otosclerosis group and controls for COL1A2 and COL2A1. This result indicates that the COL1A1 gene may play a role in the etiology of otosclerosis; however, further elucidation of a role for COL1A1 in the development of otosclerosis has not been reported.

VI. CONCLUSION

Based on available data, we hypothesize that most cases of otosclerosis represent a complex disease with a genetic basis acting on a complex genetic background and requiring an environmental trigger to become activated. Less frequent are the monogenic forms of otosclerosis. Currently, three autosomal dominant otosclerosis loci are mapped, but none of the genes has yet been cloned. The genes responsible for otosclerosis are likely to have specific roles in bone homeostasis in the otic capsule. Little is known about this process at the molecular level and the identification of these genes is the first step in the elucidation of mechanisms of bone turnover of the otic capsule. Elucidating the genetics of otosclerosis will have a significant impact on our understanding of this disease and may enable us to prevent a leading cause of hearing loss in the white adult population.

ACKNOWLEDGMENTS

This study was supported in part by grants from the University of Antwerp and from the Vlaams Fonds voor Wetenschappelijk Onderzoek (FWO) to

GVC and by NIH grant R01DC05218 to GVC and RJHS. KVDB holds a predoctoral research position with the Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaande- ren), and GVC holds a research position with the FWO.

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new HCs can be produced in adult mammalian vestibular organs following ototoxic damage (14–17). In the embryonic mammalian cochlea, laserablated HCs in cultured organs of Corti can be replaced by new HCs (18) and at the neonatal stage, there is some evidence that HCs can repair themselves after sublethal mechanical injury (19) and that regenerative capacities are present (20,21). However, in the adult mammalian cochlea, HC loss is currently considered irreversible.

Although mammals have a very limited capacity for replacing lost HCs, continuous HC turnover and ability to regenerate HCs are established features of the mechanosensory organs of many lower vertebrates. The processes of HC development and regeneration may share common molecular pathways, and many of the genes involved in both inner ear and HC development are likely to be responsible for inherited deafness (22). Genes implicated in ear morphogenesis and HC di erentiation include various transcription factors, secreted factors, receptor/tyrosine kinases, cyclindependent kinase inhibitors, and membrane-bound signaling proteins. We review here (a) the current knowledge of the mechanisms of HC production or regeneration and of the identity of HC progenitors and (b) the various genes implied in HC development and regeneration.

II.MECHANISMS OF HAIR CELL PRODUCTION OR REGENERATION AND CHARACTERIZATION OF HC PROGENITORS

A.Nonmammalian Vertebrates

Studies on birds, amphibians, and fishes led to the suggestion that newly produced HCs result from SC proliferation (for review, see Ref. 23–25). In fishes and amphibians, this perpetual production of HCs leads to a continual increase in the number of HCs, which at least in fishes results in an increase of the size of the sensory epithelium (1,2). Birds appear to be the only warm-blooded animals that demonstrate a robust HC regenerative response beyond the embryonic stages. Jorgensen and Mathiesen (6) first demonstrated that ongoing production of HCs occurs in the vestibular epithelium of adult birds. This regenerative process does not result in an increase in the total number of BrdU-labeled cells over time because there is also a spontaneous and continuous apoptosis of vestibular HCs (7). This ongoing vestibular HC death is postulated to drive the production of new HCs (26). In the basilar papilla (i.e., the auditory portion of the inner ear of birds), new HCs are produced only in response to experimental injury (27). The appearance of new HCs and SCs is preceded by mitosis in the sensory epithelium; the mitotic pool of cells gives rise to new HCs and SCs

(10,11,28–30). It has recently been shown in birds by lineage studies that HCs and SCs are generated from a common pool of precursors and that two morphological types of SCs may exist: a di erentiated one and an immature one that could be a precursor cell for both HCs and di erentiated SCs (31). There is also evidence that HCs can arise by transdi erentiation of SCs i.e., without an intermediate mitosis, in the lateral line of axolotl and in the basilar papilla of birds (32–34).

B.Mammalian Inner Ear

In the vestibular portion of the mammalian inner ear, di erent mechanisms for HC recovery following injury, such as ototoxicity induced by aminoglycosides, have been postulated: (a) proliferation of HC progenitors through mitotic division, (b) transdi erentiation of SCs into HCs, and (c) repair of partly damaged HCs. It has been shown that the vestibular epithelium of the mature mammalian inner ear has the ability to produce new HCs by renewed mitotic activity in response to aminoglycosides injury in vitro and in vivo (14–17). However, there is an apparent discrepancy between the number of proliferating cells and the number of immature HCs, characterized by immature stereocilia bundles, which may indicate that other mechanisms are involved in mammalian adult vestibular HC regeneration. HCs were also shown to arise from SCs or from another unidentified cell type of the sensory epithelium by direct transdi erentiation, i.e., without going through a mitotic cycle (35,36). A third possibility is that immature bundles represent a repair process whereby nonlethally damaged HCs replace stereocilia bundles that have been lost due to some traumatic event (37). In addition, experiments with cultured organs of Corti also suggest the possibility of self-repair of the stereociliary bundles after partial damage to HCs (38,39).

In the adult mammalian cochlea, loss of HCs does not induce regeneration or repair but rather results in an epithelial scar whereby SCs contact each other at sites where HCs are missing (40). However, in the developing organ of Corti several data suggest that undi erentiated cells can function as progenitors for new HCs. Laser ablation experiments have demonstrated that regenerated HCs can arise in vitro in the embryonic mouse organ of Corti possibly through direct transdi erentiation of preexisting cells that change from their normal developmental fate (18). HC production has also been suggested to occur in immortalized cell lines derived from organs of Corti of the H2kbtsA58 transgenic mouse (Immortomouse), which carries a conditionally expressed, temperature-sensitive immortalizing gene that perpetuates cell division (41). Isolated organ of Corti cells growing at 33 jC proliferated rapidly and when moved to 39 jC,

the cells reduced their rate of proliferation while expressing some specific HC markers such as brn-3c, OCP2, and myosin VIIa (42,43). However, despite the acquisition of HC markers, no demonstration was provided that these cells had functional or ultrastructural features of sensory cells. Production of supernumerary HCs has also been shown in cultured P0 rat organ of Corti explants (44,45). However, in these in vitro experiments, new HCs were never demonstrated to arise after a proliferative phase (46,47).

Several cell types are HC progenitor candidates. Some observations suggest that Deiters’ cells of the organ of Corti might have kept a potential to di erentiate into HCs, as treatment of young rats with ototoxic concentrations of amikacin leads to the replacement of lost HCs by Deiters’ cells with early di erentiating stereocilia bundles (48–51). An overproduction of HCs was also observed in vitro at the level of the greater epithelial ridge of cultured organ of Corti when an overexpression of Math1 transcription factor was induced (52). Very recently, we have shown that cultured immature nestin positive cells present in the newborn rat organ of Corti can proliferate and subsequently di erentiate into HCs and SCs (53). Interestingly, in these newborn rat organs of Corti, nestin-positive cells are located in the region of the greater epithelial ridge.

III.PRINCIPAL GENES IMPLICATED IN DEVELOPMENT AND REGENERATION OF HC

During both spontaneous and damage-induced HC regeneration, the cellular organization of the sensory epithelium remains precisely maintained. Very little is known about the molecules that control the formation of the correct number, types, and pattern of cells during HC regeneration and which mechanisms are lacking in nonor poorly regenerating systems. The identification of the genes involved in HC development could provide some leads to understand regeneration. Actually, several genes are known to be implicated in inner ear morphogenesis and HC development and di erentiation, including genes coding for membrane-bound signaling proteins, various transcription factors, cyclin-dependent inhibitors, and secreted factors, and these genes may also be involved in HC regeneration. Here, we summarize these findings in the prospect of HC regeneration (Table 1).

A.Notch Signaling Pathway

It has been proposed that the formation of the precise array of HCs and SCs is regulated by lateral inhibition that is mediated in several biological systems by the lin-12/Notch family of transmembranous receptors and their

ligands, Delta and Serrate or Delta and Jagged, respectively, in birds and mammals. In that paradigm, nascent HCs express a Notch ligand or ligands and thereby inhibit their immediate neighbors in the developing sensory patch, which express Notch, from di erentiating in the same way (54,55). Delta-Notch signaling was first implicated in lateral inhibition in sensory cell di erentiation based on studies in zebrafish. Misregulation of delta genes and a Serrate homolog have been observed in the zebrafish mind bomb mutant where the ear sensory patches consist solely of HCs (56). There is also a deltaA zebrafish mutant, which has increased numbers of HCs (57). Mouse mutations have made it possible to determine the roles of the various players in this pathway. Mice homozygous for a targeted null mutation of Jagged2 (Jag2) develop supernumerary HCs (58), two mice mutant for Jagged1 (Jag1) gene, i.e., slalom and headturner (Htu) mice, show reduced number of outer HCs (59,60), whereas a mutation in lunatic fringe (Lfng), which encodes an extracellular modulator of the Notch signaling pathway, partly suppresses the e ect of the Jag2 mutation (61). Moreover, the expression patterns of Notch and ligands are consistent with their presumed role in lateral inhibition resulting in an ordered array of HCs and SCs (Table 2). In addition, Delta-Notch signaling has been involved during avian HC regeneration. Early after gentamicin-induced HC injury, Delta1 and Notch are highly upregulated in areas of cell proliferation in the basilar papilla, which correspond to HCs progenitors (62).

B.Transcription Factors and HC Differentiation

A number of transcription factors are known to be expressed during inner ear development. In addition, several genetic disorders of inner ear development have recently been identified in human and mouse as being caused by mutations of genes controlling transcription factors. Furthermore, the targeted mutation of several transcription factor genes influences the development of specific inner ear cell types, and suggests a critical role for these factors in controlling inner ear cell lineages.

1.POU-Domain Transcription Factors

The POU proteins are a large family of transcriptional regulators that contains a bipartite DNA recognition domain consisting of a variant homeodomain and a POU-specific domain. Among the POU factors, those of the POU-IV or Brn-3 subclass appear to be especially involved in inner ear development. In mammals, this subclass includes three genes, Brn-3a,

Brn-3b, and Brn-3c (also referred to as Brn-3.0, Brn-3.2, and Brn-3.1, respectively), all of which are homologs of the C. elegans developmental