Abstract
Background: We have established a mouse model of spontaneous deafness by sib-inbreeding over 10 years. The mouse was designated as kuru2 and has been previously reported in this Journal. Materials and Methods: In order to identify the genetic abnormality, the mouse was back-crossed to Mus musculus castaneus (CAST), and myosine 15 or myoXV on chromosome 11 was assumed to be the responsive gene. The background abnormality was identified by gene sequencing. Results: Deletion of 2446 base pairs occurred in the mouse (from 28795 to 31241 in the complete sequence of the Mus musculus unconventional myosin-15 gene; NCBI accession: AF144093). Discussion: The myosin ATP-binding site is present in the deleted area. Considering the function that the affected area regulates and previous reports, hearing loss of the examined mouse is attributable to the abnormality of the myoXV gene and this mouse might be another type of shaker-2 deaf mouse.
Hereditary deafness is a relatively frequent disease in newborn children and it occurs at the rate of one in 2,000 births (1). Some 70% of the incidence is non-syndromic and, although in most cases the etiologies are unknown, many genes have been identified as responsive abnormalities. Such elements contain genes that were not initially considered to be the cause of deafness and further explorations of the involved mechanisms are required. The genes relevant to deafness have been identified from the analysis of models of spontaneous deaf mice and many genes from such mice have been reported. We established a spontaneous deaf mouse more than 10 years ago and the mouse has been reported in this journal (2).
The specific mouse demonstrated hereditary congenital sensorineural deafness. It did not react to sound as high as 90 dB and the I wave was absent from the auditory brain-stem response (ABR). Head tossing, ataxia, and circling movement were evident and epileptic seizure with a spike discharge demonstrated by an electroencephalogram (EEG) was also observed under specific circumstances, such as shallow anesthesia. In histological examinations, while gross abnormalities did not stand out macroscopically, progressive degeneration of hair cells in the inner ear was disclosed by scanning electron microscopy (3). The stereocila of the cochlea started to degenerate on post-natal day 10 and subsequently the hair bundles continued to degenerate. On day 18, degeneration of the stereocila was complete. Degeneration and dropout of spiral ganglion cells were observed at a late age. A single autosomal recessive abnormality of the gene was deduced from the pedigree of the mouse. From these standpoints, linkage analysis of the gene locus was performed and the results indicated correlation of the disease with myoXV on chromosome 11 (4). In the current study, sequencing of the suspected gene revealed an abnormality of this element.
Materials and Methods
Animals were maintained on commercial breeding food (CA-1, Clea, Tokyo, Japan) and tap water at Jikei Medical University. To perform a linkage analysis of the responsive gene, Mus musculus castaneus (CAST) (Jackson Laboratories, Bar Harbor, Maine) was chosen for the back-cross since it is relatively distant from the Jcl:ICR strain and there is abundant information about its microsatellite markers. The mouse was bred with the mutant deaf mouse and offspring (the first generation, F1) mice were obtained. F1 mice were then back-crossed to the original mutant deaf mice and hybrid F2 generation mice were obtained. Following examinations of hearing acuity by ABR (Synax1200: NEC Medical Systems, Tokyo, Japan), these F2 mice were classified into normal and deaf groups. Correspondence to phenotype was also confirmed by observation of abnormal behaviors such as circling movement, head tossing, and ataxia. After reconfirmation of hearing impairment, deaf F2 generation mice were used for further DNA analysis. Under anesthesia, blood samples were collected from the mice and genomic DNA was extracted by the magnetic beads kit (MagExtractol-Genome, Toyobo, Osaka, Japan). Primers for linkage analysis were determined using the Mouse Genome Information (5). By comparing the lengths of the CAST and ICR (NOD) microsatellites, markers that differed by more than 6 base pairs in length were chosen, synthesized (Invitrogen Japan, Tokyo, Japan), and then used. As the first screening, microsatellites on both long and short arms, if possible, of each chromosome were selected. The Mit number of the markers is shown in Tables I and II. The markers for chromosome 11 were chosen in away that the whole chromosome was covered. The Mit number of the markers is shown in Table III. Microsatellite analyses of each chromosome were performed by polymerase chain reaction (PCR) and the genomic DNA of each deaf mouse was used as the template. Microsatellites were amplified with Phusion High-Fidelity DNA polymerase (Finnzymes, Thermo Fisher Scientific, Vantaa, Finland) and the products were electrophoresed in 5-20% gradient polyacrylamide gel (multi-gel mini (Sekisui Medical Co, Tokyo Japan). The length of each microsatellite was then analyzed. After the analysis, the gene considered to be a responsive element was sequenced by synthesizing the primers based on the obtained nucleotide alignment (BigDye terminator v3.1 cycle sequencing kit, Applied Biosystems, Lifetechnologies Japan, Tokyo). All the animal procedures were performed under the guidance of the Ethics Committee of the Animal Core Facility.
Results of microsatellite analysis (chromosomes 1-10).
Results
Firstly, the hearing acuities of CAST and the model mice were examined by ABR and non-hearing impairment of CAST and deafness of kuru2 mice were confirmed. Next, hybrid F1 mice were obtained. All the F1 mice revealed a normal ABR pattern and there were no hearing-impaired mice born. Furthermore, there were no animals with abnormal behaviors (data not shown). The pedigree suggested the heredity pattern of autosomal recessiveness of the disease and the results confirmed is prediction. Next, hearing impairment and abnormal behaviors of back-crossed F2 mice were examined. The results demonstrated a ratio of almost 1:1 between normal and impaired mice. There was also no gender difference. The results indicated that there is only one gene corresponding to the abnormality, or, if multiple genes are involved, they are linked on the same chromosome. The 127 deaf F2 animals demonstrated by ABR were further analyzed by microsatellite linkage examination. The ratio of homozygous animals whose microsatellites both derived from ICR (ICR/ICR), and the heterozygote (ICR/CAST) in whom one microsatellite derived from ICR and the other from CAST, are listed in Table I (chromosome 1-10) and Table II (chromosome 11-19, X).
When a responsive gene for hearing loss exists, it can be expected that the gene is linked to a nearby microsatellite due to the recessive inheritance of the disease. Such a gene would exist near the microsatellites with almost 100% homozygosity. As shown in Table II, the responsive gene was thought to exist on chromosome 11 (l1 Mit51 homo/sample=110/127, 11 mit263 homo/sample=98/127).
Results of microsatellite analysis (chromosomes 11-19 and X).
Results of microsatellite analysis (chromosome 11).
Genetic deletion of myoXV in kuru2 mouse. The genomic and transcript structures of myoXV are demonstrated in the upper part of the figure. In kuru2, nucleotides from 28795 to 31241 are deleted. This area corresponds to the five exons indicated and the last exon encodes the ATP-binding site of the protein.
To identify the detailed region of the responsive gene on chromosome 11, 33 primers were used and their linkage was analyzed. The results of 22 microsatellites are shown in Table III. Consequently, the location of the gene was predicted to be between 20 and 34 cM on the chromosome. The results revealed that the responsive lesion was located near the myoXV gene. Accordingly, to examine whether or not the gene has an abnormality, genomic sequences of myoXV in kuru2 were determined. As a result, a relatively large deletion was found from 28795 to 31241 in the genome (Figure 1). The deleted region corresponded to exon 3-7 for myoXV mRNA variant 1 (NCBI accession: NM_010862 XM_994684) and 3 (NCBI accession: NM_001103171), and exon 2-6 for variant 2 (ncbi accession: NM_182698 XM_910636). This region defined a part of the head or motor domain of the myoXV protein and the last exon of the deletion encoded the ATP-binding site of the motor domain.
Discussion
Due to abnormalities of mouse myoXV, shaker-2 and shaker-2J mice have been known to have non-syndromic deafness (6). These mice were models for human hereditary deafness caused by mutations of Deafness, autosomal recessive 3 (DFNB3) (7). We believe that a large deletion of the myoXV gene is responsible for the phenotype of the ICR kuru2 mouse. The reasons include the findings that the cause of anomalies derives from a single abnormality on chromosome 11, the phenotype is closely associated with 20 to 34 cM of the same chromosome, the area does not contain other important genes related to known hearing-impairments, the identified deleted sequence corresponds to the motor domain of the myoXV protein, the spoiled exons are relevant not only to variant 1 but also to variants 2 and 3 of the mRNA, and the last exon of the deletion contains the vital ATP-binding site.
A critical mutation of the actin binding site is the determinant for shaker-2 (8), and hindward deletion of 14.7 kb of the myoXV gene is the determinant for the shaker 2J lesion (6). The deletion removes the last six exons from the 3’-terminus of myoXV and encodes the F, ezrin, radixin and moesin (FERM) domain which interacts with integral membrane proteins. In addition, the abnormality presented in kuru2 is similar to that reported in the shaker-2 mouse. Both mice demonstrate similar phenotypes, such as circling, head-tossing and profound deafness. These mice do not have onset of symptoms in heterozygotes and the trait appears only in homozygotes. In kuru2, no mutation or deletion of the actin binding site, or of the FERM domain was found. Moreover, since the flanking region contained CTGTGG sequences, it is possible that the deletion occurred between these sequences. A histological abnormality in the organ of Corti has been previously reported (3). The morphology and pattern of degeneration of the hair cells was also similar to those reported for shaker-2 (9-12). However, some phenotypes, such as epileptic tendency, differed between shaker-2, in which the etiology was evidently known to be a critical mutations of myoXV, and kuru2, suggesting that there might be different gene(s) or background between the two mice. Although abnormal circling behavior and deafness in the kuru2 mouse suggested a lesion in the vestibule-cochlear system, hair cells in the vestibules were not severely impaired. More studies are required to explain the differences. At any rate, a deletion of the motor domain of the myoXV gene was demonstrated in the kuru2 mouse. This animal might be another type of shaker-2 deaf mouse and could be useful for further studies aimed at understanding the function of unconventional myoXV.
Acknowledgements
This work was partly supported by a grant from the Takeda Science Foundation. The tables used in the article are modifications of the previous results published in Japanese (Oto-Rhino-Laryngology, Tokyo). We include them with the permission of the publisher.
- Received April 12, 2012.
- Revision received June 25, 2012.
- Accepted June 26, 2012.
- Copyright © 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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