Учебники / Genetic Hearing Loss Willems 2004

the stapes, and to fibrocytes within the cochlear duct (38). Notably, the subcellular localization of the protein shifts to the cytoplasm in areas of mesenchymal remodeling that will further develop to acellular structures, demonstrating a potential mechanism for crucial silencing of the gene during normal otic development.

1.Mutations in POU3F4 Lead to DFN3 X-Linked Mixed Deafness

Identification of the POU3F4 Mutations. The DFN3 locus (OMIM #304400) defines the third deafness locus to be identified on chromosome X. Although X-linked inheritance is relatively rare for nonsyndromic hearing loss (approximately 1% of inherited deafness), at least 50 DFN3 families have been identified. The DFN3 phenotype is variable, and at the very least it is characterized by profound sensorineural hearing loss. DFN3 is also often associated with conductive hearing loss and with stapes fixation. The first documented clinical description of a DFN3 family was described in 1967, and the gene for this locus was cloned almost 30 years later, in 1995 (40). This locus was initially described as a syndrome, since it is often associated with stapes fixation that may lead to a perilymphatic gusher upon surgery (gusherdeafness syndrome). Furthermore, as DFN3 maps to chromosome Xq21 in the region containing mental retardation and choroideremia as well, some patients with POU3F4 mutations have additional symptoms due to deletions of larger portions of the chromosome. In 1988, a number of reports were published describing the mapping of the DFN3 locus to chromosome Xq13– q21.1 (e.g., see Ref. 41). DFN3 was then localized to a 500-kb region on Xq21.1. containing the POU3F4 gene (40). Small mutations in five unrelated individuals were identified in the coding region of this transcription factor, leading to either protein truncations or nonconservative amino acid substitutions. This established POU3F4 as the causative gene for DFN3, and subsequently additional point mutations were identified (e.g., Ref. 42). But this left the larger deletions, duplications, and inversions of this region unexplained (43,44). These deletions account for a little over half of DFN3 mutations and many of them do not encompass the POU3F4 coding region (Fig. 4). A detailed molecular analysis of the region proximal to the POU3F4 gene revealed small deletions 900 kb proximal to the gene that are associated with DFN3 deafness (44). It was suggested that the DNF3 phenotype, without coding region mutations, could be caused by the loss of the POU3F4 enhancer, repressor, or promoter.

Clinical Phenotype of DFN3. All males with POU3F4 deafness have profound sensorineural hearing loss, which is often accompanied by conductive hearing loss. Obligate female carriers may or may not have hearing

at the time that the malformations seen are consistent with there being a space between the IAM and perilymph that may lead to the perilymphatic hydrops and gusher upon stapes surgery. Since then, however, the mouse knockout has revealed another reason for the hydrops, namely that it may be due to the loss of fibrocytes in the spiral limbus, as observed in the Pou3f4 knockout (see Sec. III.A.2). The observation of this Mondini-like dysplasia of the inner ear suggests that a defect occurs between embryonic days 25 and 47 of human inner ear development (43).

2.Mouse Models for Pou3f4 Mutations

Three mouse mutants are available for the study of the DFN3 ear phenotype. These mutants di er in both their phenotypes and the molecular mechanisms used for the silencing of the Pou3f4 gene. The first reported targeted null allele of the Pou3f4 gene was generated by replacing the coding sequence of the intronless Pou3f4 gene with a LacZ gene and a PGK-Neo resistance cassette, through homologous recombination of embryonic stem cells (ES) (46). The expression of the LacZ protein can be visualized using a biochemical assay based on 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside (X-gal). The insertion of a LacZ gene under the same regulatory elements of the replaced gene creates a powerful tool for studying the expression of the replaced gene. The resulting mutant lacked Pou3f4 gene expression in all mouse tissues in which it is known to be expressed. Shortly thereafter the same group reported the phenotype of an X-ray-induced mouse mutant, sex-linked fidget (slf).

Interestingly, while there is no Pou3f4 expression in the inner ear of these mice, Pou3f4 expression in the neural tube remains normal (47). This silencing of inner ear expression is caused by a chromosomal inversion whose breakpoint lies at least a 6–10-kb stretch upstream of the Pou3f4 coding sequence, most probably separating the Pou3f4 gene from its inner ear– specific transcriptional regulation elements. The above two models showed indistinguishable inner ear phenotypes consisting of mild hearing loss and vertical head bobbing. The mutant mice su ered a few cochlear and temporal bone abnormalities (Fig. 6), including a constricted superior semicircular canal, widening of the internal auditory meatus, thinning of various structures in the temporal bone, a misshaped stapes footplate, and a shortening of the cochlea, demonstrated as a reduction in the number of cochlear coils in most of the mutants. In addition, the mice had a generally hypoplastic cochlea, with widening of the scala tympani, and dysplasia of fibrocytes in the spiral limbus, which may lead to the hydrops observed in these animals. Notably, no structural abnormalities were observed in the organ of Corti. Bearing in mind that the Pou3f4 protein is expressed in mesenchymal tissue (38), the authors concluded that the widened structures result from disruption

di erentiation of all six mechanosensory neurons, and loss of function mutations in unc-86 result in a loss of the animal’s ability to respond to mechanical stimulation. Since that time, class IV POU-domain proteins have been found in Xenopus (51), metazoans (33), and zebrafish (52). The mammalian POU-domain class IV consists of three members, namely Pou4f1 (also named Brn3a or Brn3.0), Pou4f2 (also named Brn3b or Brn3.2), and Pou4f3 (also named Brn3c or Brn3.1). This family of transcription factors was isolated using a degenerate reverse transcriptase (RT)-PCR approach (27) and then shown to be encoded by three independent genes (53,54). The class IV POU-domain proteins share high sequence similarity in their DNAbinding domains and recognition properties (53). However, in their N- terminal transactivation domains, they diverge substantially.

These three transcription factors are all expressed in cells from a neuronal origin, including the dorsal root ganglia, the retina, the inner ear, and the spinal cord. The cell type in which each gene is first expressed is associated with the most severe phenotype for that gene (54). Thus, Pou4f1, as the first gene within the group to be expressed in the spiral, vestibular, and geniculate ganglia, has a severe and perinatally lethal phenotype in knockout mice, including ine ective swallowing and mechanosensory defects, di erential loss of neurons, defects in their migration, and reduction of neuronal soma size in these three ganglia (55–57). Pou4f2 is first among the three to be expressed in the retinal ganglion cells and the knockout phenotype consists of a loss of approximately 60–70% of the retinal ganglion cells and defective retinal axon pathfinding (54,58,59). Mutations in the Pou4f3 gene, which is the first reported gene among this group to be expressed in the inner ear hair cells (58), result in profound hearing loss and circling (Fig. 7).

The high similarity of these transcription factors and the critical role of their spatiotemporal control are further delineated by the fact that at least in the retina, all three class IV POU-domain proteins have a similar ability to promote retinal ganglion cell development upon misexpression (60). Nevertheless, although all three transcription factors of this group are expressed in the mammalian inner ear, to date, only mutations in the POU4F3 gene have been associated with human hereditary hearing loss or human disease overall.

1.Mutations in POU4F3 Lead to DFNA15 Autosomal Dominant Deafness

Identification of the POU4F3 Mutation. The DFNA15 locus (OMIM #602459) defines the fifteenth dominant deafness locus to be identified, based on a large Israeli Jewish family with autosomal dominant nonsyndromic sensorineural hearing loss. A mutation in the POU4F3 gene is associated with hearing loss in this family, named Family H (Fig. 8). A ected members of

determine if deafness in Family H mapped to a chromosomal region already known to harbor a gene for hearing loss. Linkage was found to 5q31–q33, close to DFNA1, the first locus mapped for dominant, nonsyndromic deafness (62), but further genotyping confirmed linkage of deafness in Family H to markers distal (toward the telomere) to DFNA1.

Therefore, the new deafness locus was defined as DFNA15. Examination of this chromosomal region in humans did not reveal any interesting candidates. However, an ideal candidate was identified on mouse chromosome 18 in the region of homology to 5q31. There were two compelling reasons why the Pou4f3 gene was a good candidate for deafness in Family H. First, targeted deletion of the entire Pou4f3 gene leads to vestibular dysfunction and profound deafness in the knockout mice (58,63). Second, expression of murine Pou4f3 is restricted to the cochlear and vestibular hair cells of the inner ear. Primers spanning the human gene were designed and DNA from both una ected and a ected Family H members was amplified. In hearingimpaired members of Family H, an 8-bp deletion was identified in the 3V portion of human POU4F3. This deletion leads to a frameshift, causing a stop codon to be formed prematurely (Fig. 9). Presumably a truncated protein is formed, which might still have the capacity to bind to DNA, though at a lower a nity owing to the loss of the POU-specific domain and a large portion of the POU homeodomain.

Clinical Phenotype of DFNA15. A clinical study of the auditory phenotype of a ected members of Family H was performed that indicated that POU4F3 mutation-associated deafness cannot be identified through clinical evaluation, but only through molecular analysis (42,64). Hearing was measured by pure-tone audiometry (Fig. 10) on all participating relatives of Family H for whom the genome scan was performed. The thresholds were compared to the ageand gender-related median of normal-hearing standards. All individuals with a POU4F3 mutation and who were above the age of 40 exhibited sensorineural bilateral hearing loss, ranging from mild to severe hearing loss. The shape of the audiograms ranged from flat to a sloping curve. There was no one typical audiogram for a ected members of this family.

No sequential audiograms were available, although the a ected individuals complained of progressive hearing loss. The rate of progression, designated as annual threshold deterioration (ATD), was calculated using cross-sectional linear regression analyses to be approximately 1.1 dB/year at 0.25–1 kHz and about 2.1 dB/year at 2–4 kHz. Correction of the hearing threshold for (median) presbyacusis was performed as well, and the results show that the hearing loss caused by the POU4F3 mutation is progressive beyond (normal) presbyacusis. Normal ABR suggests a functional central

auditory pathway in hearing-impaired individuals with POU4F3 mutations. Furthermore, otoacoustic emission testing (OAE) suggests that the OHCs are malfunctioning, which is consistent with POU4F3 expression in the OHC. The clinical parameters tested demonstrate that the hearing loss due to the Family H POU4F3 mutation is progressive, bilateral, and sensorineural. However, intrafamilial variability suggests that other genetic or environmental factors may modify the age of onset and rate of progression.

2.Mouse Models for Pou4f3 Mutations

While very little is known to date about the bona fide targets of Pou4f3, the biology of the mutants of this gene can teach us a great deal about the potential cellular roles and biochemical interactions of this protein. Within the inner ear, the mammalian Pou4f3 is a hair-cell-specific protein. Pou4f3 protein expression is first noticed in scattered cells of the presumptive sensory epithelium as early as mouse E12.5 (63). By E14.5 and E15.5, when all sensory epithelia are formed (organ of Corti, saccule, utricle, and crista ampullaris), it is expressed in the hair cells of these epithelia, limited only to postmitotic hair cells. This expression pattern persists through adulthood (65).

Four mouse models are available for the study of the Pou4f3 gene. Three groups have generated Pou4f3 knockout mice (54,58,63), on top of an existing spontaneous mouse mutant, the dreidel (ddl) mouse. The ddl mouse mutant arose in 1995 as a phenotypic deviant at the Jackson Laboratory (Bar Harbor, Maine, USA) and was mapped to the same genetic location as the Pou4f3 gene (http://www.informatics.jax.org/). A ‘‘TG’’ dinucleotide deletion was found, resulting in a premature stop codon upstream of the Pou4f3 DNA-binding domains.

The Pou4f3 mutants su er from congenital deafness and profound vestibular dysfunction, demonstrated by no auditory brainstem response at any level up to 114 dB SPL, hyperactivity, circling, and an inability to walk on a horizontal drum or tumbling when placed in water. The Pou4f3 knockout phenotype is recessive, and heterozygous animals are indistinguishable from the wild types. Their viability is reported to be normal; however, most mutants are 10–20% smaller than wild type and some laboratories report that the animals su er from low fertility (63). The most striking feature in the inner ears of the Pou4f3 mutant mice is missing hair bundles in all of the sensory patches (Fig. 11). In the adult mice it is caused from a lack of hair cells in these patches. Instead, the sensory epithelium of the cochlea consist of a single layer of supporting cells, most probably of Hensen cells, rudimentary or variable presentation of Deiter’s and Pillar cells. The vestibular system is missing the type I hair cells and both systems su er from a secondary loss of innervation of the sensory epithelium and myelinated axons. The bony