David C. Kohrman

Mutation Detection in the med and medJ Alleles of the Sodium Channel Scn8a UNUSUAL SPLICING DUE TO A MINOR CLASS AT-AC INTRON

Analysis of a transgene-induced mutation at the mouse med locus led to the identification of the novel voltage-gated sodium channel gene Scn8a (Burgess, D. L., Kohrman, D. C., Galt, J., Plummer, N. W., Jones, J. M., Spear, B., and Meisler, M. H. (1995) Nat. Genet. 10, 461-465). We now report the identification of splicing defects in two spontaneous mutations of Scn8a. The original med mutation was caused by insertion of a truncated LINE element into exon 2 of Scn8a. The med transcript is spliced from exon 1 to a cryptic acceptor site in intron 2. A 4-base pair deletion within the 5′ donor site of exon 3 in the medJ allele results in splicing from exon 1 to exon 4. Both mutant transcripts have altered reading frames with premature stop codons close to the N terminus of the protein. Loss of Scn8a expression results in progressive paralysis and early death. Intron 2 of Scn8a is flanked by minor class AT-AC splice sites. The observed splicing patterns of the med and medJ mutant transcripts provide the first evidence for preferential in vivo splicing between donor and acceptor sites of the same class. The apparent functional incompatibility may be a consequence of the different composition of spliceosomes bound to major and minor splice sites.

Mutations in a Novel Gene, TMIE, Are Associated with Hearing Loss Linked to the DFNB6 Locus

We have identified five different homozygous recessive mutations in a novel gene, TMIE (transmembrane inner ear expressed gene), in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6. The mutations include an insertion, a deletion, and three missense mutations, and they indicate that loss of function of TMIE causes hearing loss in humans. TMIE encodes a protein with 156 amino acids and exhibits no significant nucleotide or deduced amino acid sequence similarity to any other gene.

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.

Insertional mutation of the motor endplate disease (med) locus on mouse chromosome 15

Homozygous transgenic mice from line A4 have an early-onset progressive neuromuscular disorder characterized by paralysis of the rear limbs, muscle atrophy, and lethality by 4 weeks of age. The transgene insertion site was mapped to distal chromosome 15 close to the locus motor endplate disease (med). The sequence of mouse DNA flanking the insertion site junctions was determined. A small (< 20 kb) deletion was detected at the insertion site, with no evidence of additional rearrangement of the chromosomal DNA. Noncomplementation of the transgene-induced mutation and med was demonstrated in a cross with medJ/+mice. The new allele is designated medTgNA4Bs (medtg). The homologous human locus MED was assigned to chromosome 12. Synaptotagmin 1 and contactin 1 were eliminated as candidate genes for the med mutation. The transgene-induced allele provides molecular access to the med gene, whose function is required for synaptic transmission at the neuromuscular junction and long-term survival of cerebellar Purkinje cells.

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

A Frameshift Mutation in GRXCR2 Causes Recessively Inherited Hearing Loss

More than 360 million humans are affected with some degree of hearing loss, either early or later in life. A genetic cause for the disorder is present in a majority of the cases. We mapped a locus (DFNB101) for hearing loss in humans to chromosome 5q in a consanguineous Pakistani family. Exome sequencing revealed an insertion mutation in GRXCR2 as the cause of moderate‐to‐severe and likely progressive hearing loss in the affected individuals of the family. The frameshift mutation is predicted to affect a conserved, cysteine‐rich region of GRXCR2, and to result in an abnormal extension of the C‐terminus. Functional studies by cell transfections demonstrated that the mutant protein is unstable and mislocalized relative to wild‐type GRXCR2, consistent with a loss‐of‐function mutation. Targeted disruption of Grxcr2 is concurrently reported to cause hearing loss in mice. The structural abnormalities in this animal model suggest a role for GRXCR2 in the development of stereocilia bundles, specialized structures on the apical surface of sensory cells in the cochlea that are critical for sound detection. Our results indicate that GRXCR2 should be considered in differential genetic diagnosis for individuals with early onset, moderate‐to‐severe and progressive hearing loss.

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio).

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long‐term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing‐related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post‐fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (∼37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well. Developmental Dynamics 237:941–952, 2008.

Age-related changes in auditory nerve–inner hair cell connections, hair cell numbers, auditory brain stem response and gap detection in UM-HET4 mice

This study compared the timing of appearance of three components of age-related hearing loss that determine the pattern and severity of presbycusis: the functional and structural pathologies of sensory cells and neurons and changes in gap detection (GD), the latter as an indicator of auditory temporal processing. Using UM-HET4 mice, genetically heterogeneous mice derived from four inbred strains, we studied the integrity of inner and outer hair cells by position along the cochlear spiral, inner hair cell-auditory nerve connections, spiral ganglion neurons (SGN), and determined auditory thresholds, as well as pre-pulse and gap inhibition of the acoustic startle reflex (ASR). Comparisons were made between mice of 5-7, 22-24 and 27-29 months of age. There was individual variability among mice in the onset and extent of age-related auditory pathology. At 22-24 months of age a moderate to large loss of outer hair cells was restricted to the apical third of the cochlea and threshold shifts in the auditory brain stem response were minimal. There was also a large and significant loss of inner hair cell-auditory nerve connections and a significant reduction in GD. The expression of Ntf3 in the cochlea was significantly reduced. At 27-29 months of age there was no further change in the mean number of synaptic connections per inner hair cell or in GD, but a moderate to large loss of outer hair cells was found across all cochlear turns as well as significantly increased ABR threshold shifts at 4, 12, 24 and 48 kHz. A statistical analysis of correlations on an individual animal basis revealed that neither the hair cell loss nor the ABR threshold shifts correlated with loss of GD or with the loss of connections, consistent with independent pathological mechanisms.

Gene therapy for deafness.

Hearing loss is the most common sensory deficit in humans and can result from genetic, environmental or combined etiologies that prevent normal function of the cochlea, the peripheral sensory organ. Recent advances in understanding the genetic pathways that are critical for the development and maintenance of cochlear function, as well as the molecular mechanisms that underlie cell trauma and death, have provided exciting opportunities for modulating these pathways to correct genetic mutations, to enhance the endogenous protective pathways for hearing preservation and to regenerate lost sensory cells with the possibility of ameliorating hearing loss. A number of recent animal studies have used gene-based therapies in innovative ways toward realizing these goals. With further refinement, some of the protective and regenerative approaches reviewed here may become clinically applicable.

Alleles that modulate late life hearing in genetically heterogeneous mice

A genetically heterogeneous population of mice was tested for hearing at 8, 18, and 22 months by auditory brainstem response (ABR), and genotyped at 128 markers to identify loci that modulate late life hearing loss. Half of the test mice were exposed to noise for 2 hours at age 20 months. Polymorphisms affecting hearing at 18 months were noted on chromosomes 2, 3, 7, 10, and 15. Most of these loci had effects only on responses to 48 kHz stimuli, but a subset also influenced the auditory brainstem response at lower frequencies. Loci on chromosomes 4, 10, 12, and 14 had significant effects on hearing at 22 months in noise-exposed mice, and loci on chromosomes 10 and 11 had effects on mice not exposed to noise. Outer hair cell loss was modulated by polymorphisms on chromosomes 10, 11, 12, 17, and 19. Resistance to age-related hearing loss is thus modulated by a set of genetic effects, some age-specific, some frequency specific, some dependent on prior exposure to noise, and some of which compromise survival of cochlear hair cells.