Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss

Identification and characterisation of new models for age-related hearing loss

Age-related hearing loss (ARHL), or Presbycusis, is the most prevalent sensory impairment observed in the elderly. It is a progressive, symmetrical, age-related sensorineural hearing loss, most pronounced at higher frequencies. ARHL is a multifactorial disease, with contribution from both environmental and genetic factors. To date, little progress has been made in determining the genetic loci involved. The aim of my doctorate studies is to elaborate upon the genetics underlying ARHL through the identification and characterization of ENU-induced mouse models of ARHL. This approach has identified trombone, a recessive model of ARHL arising from the Harwell Ageing Screen. Recurrent auditory phenotyping at 2, 6, 9 and 12 months of age shows that affected animals display elevated ABR thresholds from 9 months of age, when compared to littermates, and these are further increased at 12 months of age. Genome mapping studies identified a 12.5Mb critical region on chromosome 2 and next generation sequencing identified a T>C mutation in the novel deafness gene Slc4a10, causing a leucine to proline substitution in the encoded protein. Immunohistochemical staining of cochlear sections demonstrates that Slc4a10 is expressed in the type II and V fibrocytes of the spiral ligament of wildtype mice, whereas no labelling is observed in Slc4a10trmb/trmb mice. In addition, ultrastructural studies show progressive sensory hair cell loss (inner and outer) in the Slc4a10trmb/trmb mice from >6 months of age. Furthermore, histological assessment of the lateral wall identified strial thinning in the Slc4a10trmb/trmb mice. Given the expression pattern and morphological changes observed, endocochlear potentials were measured in these mice. This identified that Slc4a10trmb/trmb mice have a chronically low endocochlear potential compared to their wildtype and heterozygous littermates. My findings establish the presence of Slc4a10 in the inner ear and suggest an important role for this sodium-coupled bicarbonate transporter in normal auditory function. I hypothesize that trombone is a novel model of strial presbycusis and further functional characterization of this model promises to increase our understanding of the pathobiology associated with age-related hearing loss

Identification and characterisation of new models for age-related hearing loss

Age-related hearing loss (ARHL), or Presbycusis, is the most prevalent sensory impairment observed in the elderly. It is a progressive, symmetrical, age-related sensorineural hearing loss, most pronounced at higher frequencies. ARHL is a multifactorial disease, with contribution from both environmental and genetic factors. To date, little progress has been made in determining the genetic loci involved. The aim of my doctorate studies is to elaborate upon the genetics underlying ARHL through the identification and characterization of ENU-induced mouse models of ARHL. This approach has identified trombone, a recessive model of ARHL arising from the Harwell Ageing Screen. Recurrent auditory phenotyping at 2, 6, 9 and 12 months of age shows that affected animals display elevated ABR thresholds from 9 months of age, when compared to littermates, and these are further increased at 12 months of age. Genome mapping studies identified a 12.5Mb critical region on chromosome 2 and next generation sequencing identified a T&amp;GT;C mutation in the novel deafness gene Slc4a10, causing a leucine to proline substitution in the encoded protein. Immunohistochemical staining of cochlear sections demonstrates that Slc4a10 is expressed in the type II and V fibrocytes of the spiral ligament of wildtype mice, whereas no labelling is observed in Slc4a10trmb/trmb mice. In addition, ultrastructural studies show progressive sensory hair cell loss (inner and outer) in the Slc4a10trmb/trmb mice from &amp;GT;6 months of age. Furthermore, histological assessment of the lateral wall identified strial thinning in the Slc4a10trmb/trmb mice. Given the expression pattern and morphological changes observed, endocochlear potentials were measured in these mice. This identified that Slc4a10trmb/trmb mice have a chronically low endocochlear potential compared to their wildtype and heterozygous littermates. My findings establish the presence of Slc4a10 in the inner ear and suggest an important role for this sodium-coupled bicarbonate transporter in normal auditory function. I hypothesize that trombone is a novel model of strial presbycusis and further functional characterization of this model promises to increase our understanding of the pathobiology associated with age-related hearing loss. </p

Identification and characterisation of new models for age-related hearing loss

Age-related hearing loss (ARHL), or Presbycusis, is the most prevalent sensory impairment observed in the elderly. It is a progressive, symmetrical, age-related sensorineural hearing loss, most pronounced at higher frequencies. ARHL is a multifactorial disease, with contribution from both environmental and genetic factors. To date, little progress has been made in determining the genetic loci involved. The aim of my doctorate studies is to elaborate upon the genetics underlying ARHL through the identification and characterization of ENU-induced mouse models of ARHL. This approach has identified trombone, a recessive model of ARHL arising from the Harwell Ageing Screen. Recurrent auditory phenotyping at 2, 6, 9 and 12 months of age shows that affected animals display elevated ABR thresholds from 9 months of age, when compared to littermates, and these are further increased at 12 months of age. Genome mapping studies identified a 12.5Mb critical region on chromosome 2 and next generation sequencing identified a T&GT;C mutation in the novel deafness gene Slc4a10, causing a leucine to proline substitution in the encoded protein. Immunohistochemical staining of cochlear sections demonstrates that Slc4a10 is expressed in the type II and V fibrocytes of the spiral ligament of wildtype mice, whereas no labelling is observed in Slc4a10trmb/trmb mice. In addition, ultrastructural studies show progressive sensory hair cell loss (inner and outer) in the Slc4a10trmb/trmb mice from &GT;6 months of age. Furthermore, histological assessment of the lateral wall identified strial thinning in the Slc4a10trmb/trmb mice. Given the expression pattern and morphological changes observed, endocochlear potentials were measured in these mice. This identified that Slc4a10trmb/trmb mice have a chronically low endocochlear potential compared to their wildtype and heterozygous littermates. My findings establish the presence of Slc4a10 in the inner ear and suggest an important role for this sodium-coupled bicarbonate transporter in normal auditory function. I hypothesize that trombone is a novel model of strial presbycusis and further functional characterization of this model promises to increase our understanding of the pathobiology associated with age-related hearing loss. </p

Clarin‐2 is essential for hearing by maintaining stereocilia integrity and function

International audienceHearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2 clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our indepth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance