Identification of three novel hearing loss mouse strains with mutations in the Tmc1 gene.

We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.

An ENU-induced mutation of Cdh23 causes congenital hearing loss, but no vestibular dysfunction, in mice.

Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea-induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca(2+)-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23(V2360E/V2360E) mutation alters the structural conformation of the protein and affects Ca(2+)-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12.

Fat aussie--a new Alström syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis.

Mutations in the human ALMS1 gene are responsible for Alström syndrome, a disorder in which key metabolic and endocrinological features include childhood-onset obesity, metabolic syndrome, and diabetes, as well as infertility. ALMS1 localizes to the basal bodies of cilia and plays a role in intracellular trafficking, but the biological functions of ALMS1 and how these relate to the pathogenesis of obesity, diabetes, and infertility remain unclear. Here we describe a new mouse model of Alström syndrome, fat aussie, caused by a spontaneous mutation in the Alms1 gene. Fat aussie (Alms1 foz/foz) mice are of normal weight when young but, by 120 d of age, they become obese and hyperinsulinemic. Diabetes develops in Alms1 foz/foz mice accompanied by pancreatic islet hyperplasia and islet cysts. Female mice are fertile before the onset of obesity and metabolic syndrome; however, male fat aussie mice are sterile due to a progressive germ cell loss followed by an almost complete block of development at the round-to-elongating spermatid stage of spermatogenesis. In conclusion, Alms1 foz/foz mouse is a new animal model in which to study the pathogenesis of the metabolic and fertility defects of Alström syndrome, including the role of ALMS1 in appetite regulation, pathogenesis of the metabolic syndrome, pancreatic islet physiology, and spermatogenesis.

Detection of mutations in genes associated with hearing loss using a microarray-based approach.

Knowing the etiology of hearing loss in a person has implications for counseling and management of the condition. More than 50% of cases of early onset, nonsyndromic sensorineural hearing loss are attributable to genetic factors. However, deafness is a genetically heterogeneous condition and it is therefore currently not economically and practically feasible to screen for mutations in all known deafness genes. We have developed a microarray-based hybridization biochip assay for the detection of known mutations. The current version of the hearing loss biochip detects nine common mutations in the connexin 26 gene, four mutations in the pendrin gene, one mutation in the usherin gene, and one mutation in mitochondrial DNA. The biochip was validated using DNA from 250 people with apparent nonsyndromic, moderate to profound sensorineural hearing loss. The hearing loss biochip detected with 100% accuracy the mutations it was designed for. No false-positives or false-negative results were seen. The biochip can easily be expanded to test for additional mutations in genes associated with hearing impairment or other genetic conditions.

Clinical and molecular features of encephalomyopathy due to the A3302G mutation in the mitochondrial tRNA(Leu(UUR)) gene.

BACKGROUND: The mitochondrial DNA mutation A3302G in the tRNA(Leu(UUR)) gene causes respiratory chain complex I deficiency. The main clinical feature appears to be a progressive mitochondrial myopathy with proximal muscle weakness. OBJECTIVE: To report on clinical and molecular features in 4 novel patients with the A3302G mutation. DESIGN: Case reports. PATIENTS: Four patients (3 of whom are from the same family) with a myopathy caused by the A3302G mitochondrial DNA mutation. MAIN OUTCOME MEASURE: Identification of the A3302G mutation by DNA sequencing. RESULTS: All 4 patients had an adult-onset progressive mitochondrial myopathy with proximal muscle weakness, resulting in exercise intolerance. In 2 unrelated patients, upper limb reflexes were absent with preservation of at least some lower limb reflexes. Other features including hearing loss, recurrent headaches, ptosis, progressive external ophthalmoplegia, and depression were present. CONCLUSION: While the dominant clinical features of the A3302G mutation were exercise intolerance and proximal muscle weakness, other features of mitochondrial encephalomyopathies, previously not described for this mutation, were present.

Characterization of a novel ENU-generated myosin VI mutant mouse strain with congenital deafness and vestibular dysfunction.

Myosin VI (Myo6) is known to play an important role in the mammalian auditory and vestibular systems. We have identified a novel N-ethyl-N-nitrosourea mutagenised mouse strain, charlie, carrying an intronic Myo6 splice site mutation. This mutation (IVS5+5G > A) results in skipping of exon 5, and is predicted to cause a frameshift and premature termination of the protein. We detected essentially no Myo6 transcript in tissue from charlie homozygous mutant mice (Myo6(chl/chl)). Myo6(chl/chl) mice exhibit vestibular dysfunction and profound hearing impairment when first tested at four weeks of age. Analysis of vestibular and cochlear hair cells by scanning electron microscopy and immunohistochemistry revealed highly disorganised hair bundles with irregular orientation and kinocilium position at postnatal stage P2-P3. Within a few weeks, the majority of hair cell stereocilia are missing, or fused and elongated, and degeneration of the sensory epithelium occurs. This novel mouse strain will be an important resource in elucidating the role myosin VI plays in the mammalian auditory system, as well as its non-auditory functions.

Eeyore: a novel mouse model of hereditary deafness.

Animal models that recapitulate human disease are proving to be an invaluable tool in the identification of novel disease-associated genes. These models can improve our understanding of the complex genetic mechanisms involved in disease and provide a basis to guide therapeutic strategies to combat these conditions. We have identified a novel mouse model of non-syndromic sensorineural hearing loss with linkage to a region on chromosome 18. Eeyore mutant mice have early onset progressive hearing impairment and show abnormal structure of the sensory epithelium from as early as 4 weeks of age. Ultrastructural and histological analyses show irregular hair cell structure and degeneration of the sensory hair bundles in the cochlea. The identification of new genes involved in hearing is central to understanding the complex genetic pathways involved in the hearing process and the loci at which these pathways are interrupted in people with a genetic hearing loss. We therefore discuss possible candidate genes within the linkage region identified in eeyore that may underlie the deafness phenotype in these mice. Eeyore provides a new model of hereditary sensorineural deafness and will be an important tool in the search for novel deafness genes.

Inner ear morphology is perturbed in two novel mouse models of recessive deafness.

Human MYO7A mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the Myo7a gene with distinct phenotypes. The mutation in Myo7a(I487N/I487N) ewaso is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in Myo7a(F947I/F947I) dumbo. These mice show a less severe hearing loss than in Myo7a(I487N/I487N) ewaso; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The Myo7a(F947I/F947I) dumbo strain is therefore the first reported Myo7a mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.

A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart.

BACKGROUND: Hearing impairment is the most common sensory impairment in humans, affecting 1:1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade. METHODOLOGY/PRINCIPAL FINDINGS: The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2(N538K) mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2(N538K/N538K)) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent. CONCLUSIONS/SIGNIFICANCE: Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.

Molecular characterization and expression of maternally expressed gene 3 (Meg3/Gtl2) RNA in the mouse inner ear.

The pathways responsible for sound perception in the cochlea involve the coordinated and regulated expression of hundreds of genes. By using microarray analysis, we identified several transcripts enriched in the inner ear, including the maternally expressed gene 3 (Meg3/Gtl2), an imprinted noncoding RNA. Real-time PCR analysis demonstrated that Meg3/Gtl2 was highly expressed in the cochlea, brain, and eye. Molecular studies revealed the presence of several Meg3/Gtl2 RNA splice variants in the mouse cochlea, brain, and eye. In situ hybridizations showed intense Meg3/Gtl2 RNA staining in the nuclei of type I spiral ganglion cells and in cerebellum near the dorsal vestibular region of the cochlea. In embryonic mouse head sections, Meg3/Gtl2 RNA expression was observed in the otocyst, brain, eye, cartilage, connective tissue, and muscle. Meg3/Gtl2 RNA expression increased in the developing otocyst and localized to the spiral ganglion, stria vascularis, Reissner's membrane, and greater epithelial ridge (GER) in the cochlear duct. RT-PCR analysis performed on cell lines derived from the organ of Corti, representing neural, supporting, and hair cells, showed significantly elevated levels of Meg3/Gtl2 expression in differentiated neural cells. We propose that Meg3/Gtl2 RNA functions as a noncoding regulatory RNA in the inner ear and that it plays a role in pattern specification and differentiation of cells during otocyst development, as well as in the maintenance of a number of terminally differentiated cochlear cell types.

Expression and mutation analysis of the Wilms' tumor 1 gene in human neural tumors.

The Wilms' tumor 1 gene, WT1, encodes a zinc-finger protein that is implicated in the development of Wilms' tumor. Mutant or aberrantly expressed WT1 isoforms have also been described in desmoplastic small round cell tumor, acute leukemias, mesothelioma, breast tumors and melanoma. During early development, WT1 is expressed in the brain and spinal cord, however its role in the malignancies that affect these tissues has not been previously investigated. In our study we have examined neural tumors including brain tumors and neuroblastomas for WT1 expression and for mutations affecting the zinc-fingers. Although WT1 expression was detected in gliomas, medulloblastomas and neuroblastomas, neither zinc-finger region mutations nor splicing anomalies affecting the KTS site were detected. We therefore conclude that WT1 does not play a significant role in the etiology of human neural tumors.