Human hereditary deafness at the DFNB29 autosomal locus on chromosome 21q22.1 is caused by recessive mutations of CLDN14, encoding claudin 14. This tight junction protein is tetra-membrane spanning that localizes to the apical tight junctions of organ of Corti hair cells and in many other tissues. Typically, the DFNB29 phenotype is characterized by pre-lingual, bi-lateral, sensorineural hearing loss. The goal of this study was to define the identity and frequency of CLDN14 mutations and associated inner ear phenotypes in a cohort of 800 Pakistani families segregating deafness. Hearing loss in 15 multi-generational families was found to co-segregate with CLDN14-linked STR markers. The sequence of the six exons and regions flanking the introns of CLDN14 in these 15 families revealed five likely pathogenic alleles. Two are novel missense substitutions (p.Ser87Ile and p.Ala94Val) while p.Arg81His, p.Val85Asp and p.Met133ArgfsX23 have been reported previously. Haplotype analyses indicate that p.Val85Asp and p.Met133ArgfsX23 are founder mutations. The p.Val85Asp accounts for approximately 67% of the mutant alleles of CLDN14 in our cohort. Combined with previously reported data, CLDN14 mutations were identified in 18 of 800 Pakistani families (2.25%; 95% CI, 1.4-3.5%). Hearing loss in the affected individuals homozygous for CLDN14 mutations varied from moderate to profound. This phenotypic variability may be due to environmental factors (e.g. drug and noise exposure) and/or genetic modifiers.
CLDN14; claudin 14; DFNB29; mild hearing loss; profound deafness; Pakistan
Backscatter scanning electron microscopy and conventional whole cell patch-clamp experiments reveal a two-step mechanism for the regeneration of tip links, the crucial element of mechanotransduction machinery in the hair cells of the inner ear.
Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism's life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca2+-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.
The inner ear detects sound when stereocilia, the mechanosensory projections on the apical surface of the hair cells, are deflected and tug on tiny extracellular tip links. These links interconnect stereocilia and convey forces to the mechanosensitive transduction channels. Current models postulate a static composition of the tip link with protocadherin 15 (PCDH15) at the link's bottom end and cadherin 23 (CDH23) at the upper end. Tip links are subjected to substantial sound-induced forces. Although hair cells can renew (regenerate) disrupted tip links and restore hearing, the molecular details of this process are unknown. Our study provides mechanistic insight into tip link regeneration. We used backscatter scanning electron microscopy to monitor the distribution of immuno-gold labeled molecular components of the tip links during their re-formation and a conventional whole-cell patch-clamp technique to follow the concomitant recovery of mechano-electrical transduction. According to our data, the mechanotransduction machinery is initially re-established by the formation of functional (mechanotransduction-mediating) links of a previously unknown composition, PCDH15–PCDH15. Transition to the PCDH15–CDH23 composition underlies final maturation of mechanotransduction. This two-step mechanism of tip link regeneration was unexpected. As tip links are continuously stressed by loud sounds and regenerated throughout an organism's life, we provide a plausible molecular mechanism for the life-long maintenance of mechanosensory function in nonregenerating cochlear hair cells.
Hereditary deafness is genetically heterogeneous such that mutations of many different genes can cause hearing loss. This review focuses on the evidence and implications that several of these deafness genes encode actin-interacting proteins or actin itself. There is a growing appreciation of the contribution of the actin interactome in stereocilia development, maintenance, mechanotransduction and malfunction of the auditory system.
Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin–Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.
gentamicin; ototoxicity; transient receptor potential A1 channel; organ of Corti; reactive oxygen species; 4-hydroxynonenal
We compared cadherin 23 (Cdh23) mRNA and protein variants in the inner ear and retina of wild-type and mutant mice and primates to better understand the pleiotropic effects of Cdh23 mutations, and specifically to understand the absence of retinal degeneration in Cdh23 mutant mice.
Semiquantitative real-time PCR was used to compare the level of expression of Cdh23 alternative transcripts in the inner ear and retina of wild-type and homozygous Cdh23v-6J (waltzer) mice. Antibodies generated against CDH23 isoforms were used in immunohistochemistry, immunohistology, electron microscopy, and western blot analyses of mouse and primate inner ear and retina to study the distribution of these isoforms in various cellular compartments.
Cdh23 mRNA alternative splice variants were temporally and spatially regulated in the inner ear and retina. In the mature mouse retina, CDH23 isoforms were broadly expressed in various cellular compartments of the photoreceptor layer. The wild-type CDH23_V3 protein isoform, which has PDZ binding motifs but neither extracellular domains nor a transmembrane domain, localized exclusively to the outer plexiform layer of the retina containing photoreceptor cell synapses and to the synaptic region of auditory and vestibular hair cells. The longest CDH23 protein isoform, CDH23_V1, appeared by western blotting to be the only one affected by the Cdh23v-6J mutation; it was expressed in the wild-type mouse inner ear, but not in the mouse retina. However, CDH23_V1 was detected in western blot analyses of monkey and human retinas.
The time- and tissue-dependent expression patterns that we have shown for Cdh23 alternative transcripts suggest developmental roles and tissue-specific functions for the various transcripts. Many of these isoforms continue to be expressed in waltzer mice. The longest CDH23 isoform (CDH23_V1), however, is not expressed in mutant mice and is necessary for normal inner ear function. The longest isoform is expressed in the retinas of primates, but not detected in the mouse retina. This species difference suggests that the mouse may not be a suitable model for studying the retinitis pigmentosa phenotype of human Usher syndrome type 1D.
There are 68 sex-linked syndromes that include hearing loss as one feature and five sex-linked nonsyndromic deafness loci listed in the OMIM database. The possibility of additional such sex-linked loci was explored by ascertaining three unrelated Pakistani families (PKDF536, PKDF1132, PKDF740) segregating X-linked recessive deafness. Sequence analysis of POU3F4 (DFN3) in affected members of families PKDF536 and PKDF1132 revealed two novel nonsense mutations, p.Q136X and p.W114X, respectively. Family PKDF740 is segregating congenital blindness, mild to profound progressive hearing loss that is characteristic of Norrie disease (MIM#310600). Sequence analysis of NDP among affected members of this family revealed a novel single nucleotide deletion c.49delG causing a frameshift and premature truncation (p.V17fsX1) of the encoded protein. These mutations were not found in 150 normal DNA samples. Identification of pathogenic alleles causing X-linked recessive deafness will improve molecular diagnosis, genetic counseling, and molecular epidemiology of hearing loss among Pakistanis.
DFN3; Norrie disease; POU3F4; NDP; hearing loss; gushers
Mutations in human SLC26A4 are a common cause of hearing loss associated with enlarged vestibular aqueducts (EVA). SLC26A4 encodes pendrin, an anion-base exchanger expressed in inner ear epithelial cells that secretes HCO3– into endolymph. Studies of Slc26a4-null mice indicate that pendrin is essential for inner ear development, but have not revealed whether pendrin is specifically necessary for homeostasis. Slc26a4-null mice are profoundly deaf, with severe inner ear malformations and degenerative changes that do not model the less severe human phenotype. Here, we describe studies in which we generated a binary transgenic mouse line in which Slc26a4 expression could be induced with doxycycline. The transgenes were crossed onto the Slc26a4-null background so that all functional pendrin was derived from the transgenes. Varying the temporal expression of Slc26a4 revealed that E16.5 to P2 was the critical interval in which pendrin was required for acquisition of normal hearing. Lack of pendrin during this period led to endolymphatic acidification, loss of the endocochlear potential, and failure to acquire normal hearing. Doxycycline initiation at E18.5 or discontinuation at E17.5 resulted in partial hearing loss approximating the human EVA auditory phenotype. These data collectively provide mechanistic insight into hearing loss caused by SLC26A4 mutations and establish a model for further studies of EVA-associated hearing loss.
Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters’ cells in the Fgfr3P244R/+ mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s10162-010-0211-x) contains supplementary material, which is available to authorized users.
CD44; FGFR3; cochlea; outer pillar cells; deafness
Genetic analysis of an inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at θ=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23–q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.
hereditary deafness; DFNB79; DFNB33; Pakistan; chromosome 9q34.3
Genetic analysis of inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at θ=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23-q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.
hereditary deafness; DFNB79; DFNB33; Pakistan; chromosome 9q34.3
Inner ear sensory hair cells convert mechanical stimuli into electrical signals. This conversion happens in the exquisitely mechanosensitive hair bundle that protrudes from the cell's apical surface. In mammals, cochlear hair bundles are composed of 50-100 actin-filled stereocilia, which are organized in three rows in a staircase fashion. Stereocilia actin filaments are uniformly oriented with their barbed ends toward stereocilia tips. During development, the actin core of each stereocilium undergoes elongation due to addition of actin monomers to the barbed ends of the filaments. Here we show that in the mouse cochlea the barbed end capping protein twinfilin 2 is present at the tips of middle and short rows of stereocilia from postnatal day (P) 5 onward, which correlates with a time period when these rows stop growing. The tall stereocilia rows, which do not display twinfilin 2 at their tips, continue to elongate between P5 and P15. When we expressed twinfilin 2 in LLC/PK1-CL4 (CL4) cells, we observed a reduction of espin-induced microvilli length, pointing to a potent function of twinfilin 2 in suppressing the elongation of actin filaments. Overexpression of twinfilin 2 in cochlear inner hair cells resulted in a significant reduction of stereocilia length. Our results suggest that twinfilin 2 plays a role in the regulation of stereocilia elongation by restricting excessive elongation of the shorter row stereocilia thereby maintaining the mature staircase architecture of cochlear hair bundles.
Auditory; Cochlea; Cytoskeleton; Actin; Mechanosensory; Cochlea
Mutations of PCDH15, encoding protocadherin 15, can cause either combined hearing and vision impairment (type 1 Usher syndrome; USH1F) or nonsyndromic deafness (DFNB23). Human PCDH15 is reported to be comprised of 35 exons and encodes a variety of isoforms with 3 to 11 ectodomains (EC), a transmembrane domain and a carboxy-terminal cytoplasmic domain (CD). Building on these observations we describe an updated gene structure that has four additional exons of PCDH15 and isoforms that can be subdivided into four classes. Human PCDH15 encodes three alternative, evolutionarily conserved unique cytoplasmic domains (CD1, CD2 or CD3). Families ascertained on the basis of prelingual hearing loss were screened for linkage of this phenotype to markers for PCDH15 on chromosome 10q21.1. In seven of twelve families segregating USH1 we identified homozygous mutant alleles (1 missense, 1 splice site, 3 nonsense and 2 deletion mutations) of which six are novel. One family was segregating nonsyndromic deafness DFNB23 due to a homozygous missense mutation. To date in our cohort of 557 Pakistani families, we have found 11 different PCDH15 mutations that account for deafness in 13 families. Molecular modeling provided mechanistic insight into the phenotypic variation in severity of the PCDH15 missense mutations. We did not find pathogenic mutations in five of the twelve USH1 families linked to markers for USH1F, which suggest either the presence of mutations of yet additional undiscovered exons of PCDH15, mutations in the introns or regulatory elements of PCDH15, or an additional locus for type I USH at chromosome 10q21.1.
DFNB23; Usher syndrome; protocadherin 15; PCDH15; deafness; retinitis pigmentosa
Usher syndrome (USH) is a hereditary disorder associated with sensorineural hearing impairment, progressive loss of vision attributable to retinitis pigmentosa and variable vestibular function. Three clinical types have been described with type I (USH1) being the most severe. To date six USH1 loci have been reported. We ascertained two large Pakistani consanguineous families segregating profound hearing loss, vestibular dysfunction, and retinitis pigmentosa, the defining features of USH1. In these families we excluded linkage of USH to the 11 known USH loci, and subsequently performed a genome-wide linkage screen. We found a novel USH1 locus designated USH1H that mapped to chromosome 15q22-23 in a 4.92 cM interval. This locus overlaps the non-syndromic deafness locus DFNB48 raising the possibility that the two disorders may be caused by allelic mutations.
deafness; DFNB48; retinitis pigmentosa; Usher syndrome; USH1H; vestibular dysfunction; 15q22-23
Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome.
Ear; Inner; MPSS; Transcription; Genetic; ESTs, expressed sequence tags; GEO, gene expression omnibus; MoCR, mouse organ of Corti MPSS library; MoSV, mouse stria vascularis MPSS library; MoVB, mouse vestibular sensory epithelia MPSS library; MPSS, massively parallel signature sequencing; MRT, mouse reference transriptome; SAGE, serial analysis of gene expression; tpm, transcripts per million
To determine whether cadherin 23 and protocadherin 15 can substitute for one another in the maintenance of the retina and other tissues in the mouse. Does homozygosity for both v and av mutant alleles (i.e., a double homozygous mouse) cause retinal degeneration or an obvious retinal histopathology?
We generated mice homozygous for both Cdh23v-6J and Pcdh15av-Jfb alleles. The retinal phenotypes of double heterozygous and double homozygous mutant mice were determined by light microscopy and electroretinography (ERG). Histology on 32 different tissues, scanning electron microscopy of organ of Corti hair cells as well as serum biochemical and hematological examinations were evaluated.
ERG waves of double heterozygous and double homozygous mice showed similar shape, growth of the amplitude with intensity, and implicit time for both rod and cone pathway mediated responses. Mice homozygous for both Cdh23v-6J and Pcdh15av-Jfb mutations showed no sign of retinitis pigmentosa or photoreceptor degeneration but, as expected, were deaf and had disorganized hair cell sensory bundles.
The simultaneous presence of homozygous mutant alleles of cadherin 23 and protocadherin 15 results only in deafness, not retinal degeneration or any other additional obvious phenotype of the major organ systems. We conclude that in the mouse cadherin 23 or protocadherin 15 appear not to compensate for one another to maintain the retina.
Sensorineural hearing loss is genetically heterogeneous. Here we report that mutations in CIB2, encoding a Ca2+- and integrin-binding protein, are associated with nonsyndromic deafness (DFNB48) and Usher syndrome type 1J (USH1J). There is one mutation of CIB2 that is a prevalent cause of DFNB48 deafness in Pakistan; other CIB2 mutations contribute to deafness elsewhere in the world. In rodents, CIB2 is localized in the mechanosensory stereocilia of inner ear hair cells and in retinal photoreceptor and pigmented epithelium cells. Consistent with molecular modeling predictions of Ca2+ binding, CIB2 significantly decreased the ATP-induced Ca2+ responses in heterologous cells, while DFNB48 mutations altered CIB2 effects on Ca2+ responses. Furthermore, in zebrafish and Drosophila, CIB2 is essential for the function and proper development of hair cells and retinal photoreceptor cells. We show that CIB2 is a new member of the vertebrate Usher interactome.
Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-14C]galactose to 14CO2, whereas strains deficient in other enzymes of the Leloir pathway cannot do so.
Many proteins necessary for sound transduction have been discovered through positional cloning of genes that cause deafness1–3. In this study, we report that mutations of LRTOMT are associated with profound non-syndromic hearing loss at the DFNB63 locus on human chromosome 11q13.3-q13.4. LRTOMT has two alternative reading frames and encodes two different proteins, LRTOMT1 and LRTOMT2, that are detected by Western blot analyses. LRTOMT2 is a putative methyltransferase. During evolution, novel transcripts can arise through partial or complete coalescence of genes4. We provide evidence that in the primate lineage LRTOMT evolved from the fusion of two neighboring ancestral genes, which exist as separate genes (Lrrc51and Tomt) in rodents.
Cilia are highly specialized microtubule-based organelles that have pivotal roles in numerous biological processes, including transducing sensory signals. Defects in cilia biogenesis and transport cause pleiotropic human ciliopathies. Mutations in over 30 different genes can lead to cilia defects, and complex interactions exist among ciliopathy-associated proteins. Mutations of the centrosomal protein 290 kDa (CEP290) lead to distinct clinical manifestations, including Leber congenital amaurosis (LCA), a hereditary cause of blindness due to photoreceptor degeneration. Mice homozygous for a mutant Cep290 allele (Cep290rd16 mice) exhibit LCA-like early-onset retinal degeneration that is caused by an in-frame deletion in the CEP290 protein. Here, we show that the domain deleted in the protein encoded by the Cep290rd16 allele directly interacts with another ciliopathy protein, MKKS. MKKS mutations identified in patients with the ciliopathy Bardet-Biedl syndrome disrupted this interaction. In zebrafish embryos, combined subminimal knockdown of mkks and cep290 produced sensory defects in the eye and inner ear. Intriguingly, combinations of Cep290rd16 and Mkksko alleles in mice led to improved ciliogenesis and sensory functions compared with those of either mutant alone. We propose that altered association of CEP290 and MKKS affects the integrity of multiprotein complexes at the cilia transition zone and basal body. Amelioration of the sensory phenotypes caused by specific mutations in one protein by removal of an interacting domain/protein suggests a possible novel approach for treating human ciliopathies.
A missense mutation of Gipc3 was previously reported to cause age-related hearing loss in mice. Point mutations of human GIPC3 were found in two small families, but association with hearing loss was not statistically significant. Here, we describe one frameshift and six missense mutations in GIPC3 cosegregating with DFNB72 hearing loss in six large families that support statistically significant evidence for genetic linkage. However, GIPC3 is not the only nonsyndromic hearing impairment gene in this region; no GIPC3 mutations were found in a family cosegregating hearing loss with markers of chromosome 19p. Haplotype analysis excluded GIPC3 from the obligate linkage interval in this family and defined a novel locus spanning 4.08 Mb and 104 genes. This closely linked but distinct nonsyndromic hearing loss locus was designated DFNB81.
Inner ear hair cells detect sound through deflection of mechanosensory stereocilia. Each stereocilium is supported by a paracrystalline array of parallel actin filaments that are packed more densely at the base, forming a rootlet extending into the cell body. The function of rootlets and the molecules responsible for their formation are unknown. We found that TRIOBP, a cytoskeleton-associated protein mutated in human hereditary deafness DFNB28, is localized to rootlets. In vitro, purified TRIOBP isoform 4 protein organizes actin filaments into uniquely dense bundles reminiscent of rootlets, but distinct from bundles formed by espin, an actin cross-linker in stereocilia. We generated mutant Triobp mice (TriobpΔex8/Δex8) that are profoundly deaf. Stereocilia of TriobpΔex8/Δex8 mice develop normally, but fail to form rootlets and are easier to deflect and damage. Thus, F-actin bundling by TRIOBP provides durability and rigidity for normal mechanosensitivity of stereocilia and may contribute to resilient cytoskeletal structures elsewhere.
Recessive mutations of fibroblast growth factor 3 (FGF3) can cause LAMM syndrome (OMIM 610706), characterized by fully penetrant complete labyrinthine aplasia, microtia and microdontia.
We performed a prospective molecular genetic and clinical study of families segregating hearing loss linked to FGF3 mutations. Ten affected individuals from three large Pakistani families segregating FGF3 mutations were imaged with CT, MRI, or both to detect inner ear abnormalities. We also modeled the three dimensional structure of FGF3 to better understand the structural consequences of the three missense mutations.
Two families segregated reported mutations (p.R104X and p.R95W) and one family segregated a novel mutation (p.R132GfsX26) of FGF3. All individuals homozygous for p.R104X or p.R132GfsX26 had fully penetrant features of LAMM syndrome. However, recessive p.R95W mutations were associated with nearly normal looking auricles and variable inner ear structural phenotypes, similar to that reported for a Somali family also segregating p.R95W. This suggests that the mild phenotype is not entirely due to genetic background. Molecular modeling result suggests a less drastic effect of p.R95W on FGF3 function compared with known missense mutations detected in fully penetrant LAMM syndrome. Since we detected significant intrafamilial variability of the inner ear structural phenotype in the family segregating p.R95W, we also sequenced FGF10 as a likely candidate for a modifier. However, we did not find any sequence variation, pointing out that a larger sample size will be needed to map and identify a modifier. We also observed a mild to moderate bilateral conductive hearing loss in three carriers of p.R95W, suggesting either a semi-dominant effect of this mutant allele of FGF3, otitis media, or a consequence of genetic background in these three family members.
We noted a less prominent dental and external ear phenotype in association with the homozygous p.R95W. Therefore, we conclude that the manifestations of recessive FGF3 mutations range from fully penetrant LAMM syndrome to deafness with residual inner ear structures and, by extension, with minimal syndromic features, an observation with implications for cochlear implantation candidacy.