Research in the Section on Neurogenetics focuses on the identification and characterization of genes that are critical for structure and function of sensory hair cells in the cochlea. Spontaneous mutations that cause hearing loss are ideal tools to identify genes important for the functioning of the inner ear and to elucidate their role in these sensory systems. Towards this end research efforts concentrated on three lines of experimental investigation: (1) the ahl5 quantitative trait locus in the Black Swiss strain, (2) a high-frequency hearing loss phenotype in the NIH Swiss line and (3) hearing loss in the Tail short (Ts) mutant mouse. (1) Heterogeneous strains represent a genetic spectrum different from the common inbred strains and show a greater degree of allelic heterogeneity. They are models to isolate genetic interactions and to identify new deafness alleles. We found that mice of the Black Swiss strain develop early-onset slowly progressing hearing loss. The segregation pattern in intercrosses and backcrosses was consistent with a polygenic inheritance. Genome-wide linkage analyses on backcross and intercross populations localized two quantitative trait loce (QTLs) underlying hearing loss in Black Swiss mice. A major QTL localized to chromosome 10 (named ahl5) and a second small-effect QTL localized to chromosome 18 (ahl6). To fine-map ahl5, (Black Swiss x CAST/Ei) F1 hybrids were backcrossed to Black Swiss for ten consecutive generations. Recombination events in two congenic lines delimit the ahl5 critical interval to a 2-Mb region. Through direct sequencing of genes in the interval we identified mutations in the Gipc3 gene causing the hearing loss and audiogenic seizure phenotype in the Black Swiss strain. We localized Gipc3 to the inner ear sensory hair cells and spiral ganglion. We determined that the missense mutation in the PDZ domain of Gipc3 has an attenuating effect on mechanotransduction and the aquisition pf mature inner hair cell potassium currents. We also established a causative correlation between magnitude and temporal progression of wave I amplitude of afferent neurons with progression of audiogenic seizures. Lastly, we identified pathologic mutations in human GIPC3 causing recessive deafness DFNB15 and DFNB95. In summary, our analysis of progressive hearing loss in Blackswiss revealed a novel deafness gene and suggests a pivotal role for Gipc3 in acoustic signal aquisition and propagation in cochlear hair cells. (2) The NIH Swiss outbred strain was originally derived from a population of Swiss albino mice in Lausanne, Switzerland. Previous ABR measurements in a population of thirty NIH Swiss mice showed a wide range of thresholds from normal to completely absent responses. This wide range of thresholds offered the opportunity to select for specific phenotypes such as hearing loss affecting only the higher frequencies. The cochlea is tonotopically organized from the base to the apex, where higher frequencies are recognized at the base and lower frequencies at the apex. The exact molecular mechanisms that underlies this frequency-recognition gradient is not known although some data suggest that location-specific alternative splice-forms of the Ca2+-activated K+ channel Kcnma1 might play a role. As most types of hearing impairment ultimately affect all frequencies, a mouse model that exhibits a frequency-specific hearing loss could provide insights into how frequency-selectivity in the cochlea is attained. Starting from a phenotypically mixed population, we selected mice that showed a high-frequency hearing loss (HFHL) and hearing loss across all frequencies (AFHL)at eight weeks of age. By quantitative trait loci analyses of these two lines, we localized QTLs on chromosomes 7, 8, and 10 that showed a significant effect on hearing function. The loci on chromosome 7 and 8 (HFHL1 and HFHL2) are previously unrecognized loci and adversely affect hearing at the higher frequencies, only. This frequency-specific hearing loss suggests that HFHL1 and HFHL2 may affect tonotopic development. The study lays the groundwork for the molecular characterization of HFHL1 and HFHL2, which may prove relevant in identifying common forms of hearing impairment in the human population, such as age- or noise-related hearing loss. (3) Tail Short is a semi-dominant mutation, which was discovered by Walter Morgan in 1950. The mutation arose spontaneously on the BALB/c background and maps to distal chromosome 11. Homozygous mutants die before or at the time of gestation between 3.5 and 5.5 days post-coitum. The most obvious phenotype in heterozygotes is the short, kinked, and curled tail. This is the result of skeletal abnormalities that occur along the vertebral column, which include fusion of two or three successive vertebrae, dyssymphyses, and additional vertebrae and ribs. These skeletal malformations can be traced back to an absent or dysmorphic notochord, a thinner neural tube and a marked anemia, which is first observed in 8-day old embryos. Embryonic lethality in heterozygotes is due to neural tube defects including exencephaly and spina bifida. The known effect of the neural tube on inner ear development and the previously recognized link between neural tube defects and planar cell polarity defects of cochlea hair cells prompted us to study the hearing phenotype in the Ts mutant. By genetic means, we show that the Ts phenotypes arise from an 18kb deletion/insertion of the Rpl38 gene, encoding a ribosomal protein of the large subunit. We show that Ts mutants exhibit significantly elevated auditory-brain stem response (ABR) thresholds and reduced distortion-product otoacoustic emissions (DPOAEs), in the presence of normal endocochlear potentials and typical inner ear histology suggestive of a conductive hearing impairment. We locate the cause of the hearing impairment to the middle ear, demonstrating over-ossification at the round window ridge, ectopic deposition of cholesterol crystals in the middle ear cavity, enlarged Eustachian tube, and chronic otitis media with effusion all beginning at around three weeks after birth. Using specific antisera, we demonstrate that Rpl38 is an 8 kDa protein that is predominantly expressed in mature erythrocytes. Finally, using an Rpl38 cDNA transgene we rescue the Ts phenotypes. Together, these data present a previously uncharacterized combination of interrelated middle ear pathologies and suggest Rpl38-deficiency as a model to dissect the causative relationships between neo-ossification, cholesterol crystal deposition, and Eustachian tube in the etiology of otitis media.
