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to map a mouse deafness gene, identify the underlying mutation and develop a mouse model for human deafness.
genetic linkage cross and genome scan were used to map a novel mutation named hypoplasia of the membranous labyrinth (hml), which causes hearing loss in mutant mice.
1. hml was mapped on mouse Chr 10 (~43 cM from the centromere) suggests that the homologous human gene is on 12q22-q24, which was defined on the basis of known mouse-human homologies (OMIM, 2004). 2. This study has generated 25 polymorphic microsatellite markers, placed 3 known human genes in the correct order in a high-resolution mouse map and narrowed the hml candidate gene region to a 500kb area.
Genetic impairment of hearing affects about one of every 2000 children. Genetic analysis of mouse deafness mutations has already aided in the identification of human deafness genes. Depending on genetic background mutations of one gene can cause recessive or dominant, syndromic or nonsyndromic hearing loss. Mice homozygous for the shaker-1 mutation (sh1) are characterized by circling behavior and deafness. sh1 was shown by positional cloning to be a mutation of the Myo7a gene, which encodes an unconventional myosin-type protein. Subsequently, the homologous MYO7A gene in humans was shown to be responsible for both dominant (DFNA11) and recessive (DFNB2) forms of hearing impairment, as well as for Usher syndrome type1B.
Here we describe a newly discovered mutation that causes hearing loss in mice. The map position on mouse Chr 10 (~43 cM from the centromere) suggests that the homologous human gene is on 12q22-q24, on the basis of known mouse-human homologies (OMIM, 2004). A de novo deletion in a six-year-old boy with congenital hearing loss as well as mental and motor retardation provides a possible human homologue syndrome of hml at a critical interval to 13 cM in the 12q22-q24.1 region where DFNA25 resides. Proportional smaller body size represents a phenotype similar to hml. Thus, identification of the hml gene will provide insight into molecular mechanisms of inner ear development and formation.
Hypoplasia of the membranous labyrinth (hml) is a recessive spontaneous mutation that arose in a colony of inbred BALB/cByJ mice at The Jackson Laboratory (TJL), Bar Harbor, Maine. Homozygous hml mice can be identified at 10 days of age by their small size and unbalanced gait. The mouse strains CAST/Ei and BALB/cByJ-hml (abbreviation CBy-hml), their F1 hybrids, and F2 intercross progenies are maintained in our research colonies at TJL. All animal procedures were approved by the Animal Care and Use Committee (ACUC). Because neither sex of homozygotes CBy-hml/hml breed, the colony was maintained by progeny test and an outcross-intercross strategy was used for the mapping. Outcross: tested heterozygotes (CBy-hml/+) were outcrossed with CAST/Ei and half of the resulting F1 offspring were expected to carry the mutation - (CAST × CBy)-hml/+. F1 hybrids were mated with known CBy-hml/+ mice; if one or more offspring was born with a mutant phenotype, then the F1 was assumed to be an hml carrier. Intercross: F1 hybrids - (CAST × CBy)-hml/+ × (CAST × CBy)-hml/+ intercrosses were mated to generate F2 intercross progeny. One fourth of the F2s were expected to be homozygous for the wildtype allele (+/+), 1/2 heterozygous (hml/+) and 1/4 homozygous for the mutation (hml/hml). Phenotyping and the genome-wide scanning of these F2 progeny were carried out as described below.
Tail DNA was isolated according to Johnson. SSLP markers polymorphic between strains BALB/cByJ and CAST/Ei were selected at ~15 cM intervals across the mouse genome. A total of 123 SSLP markers were tested for the initial genome-wide screen. Additional markers were tested around loci that showed significant or suggestive linkage. For PCR amplification, 100 ng of DNA were used in a 10 µL volume containing 50 mmol KCl, 10 mmol Tris-HCl, pH 8.3, 2.5 mmol MgCl2, 0.2 mmol oligonucleotides, 200 µmol dNTP and 0.02 U AmpliTaq DNA polymerase. The reactions were subjected to the following temperature cycling program: initial denaturation for 2 min at 95°C; 20 s at 94°C, 30 s at 50°C, 40 s at 72°C for 49 cycles; followed by a 7 min extension at 72°C. PCR products were separated by electrophoresis on a 3% MetaPhor (FMC, Rockland, ME) agarose gel and visualized under UV light after staining with ethidium bromide.
BALB/cByJ (n = 8) and BALB/cByJ-hml/hml (n = 8) mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS) followed by Bouin’s fixative. The cochlea was removed and stored in Bouin’s fixative for 48 h. Serial sections (7 µm) of the cochlea were stained with hematoxylin and eosin (H&E) using standard procedures.
A computer-aided evoked potential system (Intelligent Hearing System, IHS; Miami, FL) was used to test mice for ABR thresholds as previously described. Mice were anesthetized with tribromoethanol (0.53 mg/g body wt i.p.). Subdermal needle electrodes were inserted at the vertex (active) and ventrolaterally to the right ear (reference) and to the left ear (ground). Specific acoustic stimuli were delivered binaurally through 1 cm plastic tubes channeled from high frequency transducers. Mice were tested with click stimuli and also with 8, 16 and 32 kHz tone pips at varying intensity, from low to high (10–90 dB SPL). An ABR threshold was determined for each stimulus frequency by identifying the lowest intensity which produced a recognizable ABR pattern (at least two consistent peaks).
Two mutant mice were observed in the progeny from a mating pair in the BALB/cByJ (CBy) inbred strain colony at The Jackson Laboratory. The mutation arose spontaneously, and neither parent was affected. The mutants were originally noticed by their small body size and distinctively fail-to-thrive behavior. Once they were placed on the back, it took up to a minute to upright themselves. Some mutants died perinatally through two months of age. Once they passed two months of age they could live up to 18 months of age. Even though neither sex bred, sperm shape and activities were checked and no abnormalities were found. Unlike their normal littermates, the mutant mice were unable to swim and did not respond to tapping or clicking sounds, indicating that they also had hearing loss. Initially the mutant mouse was named Hypoplasia of the membranous labyrinth (hml). In the mapping cross (CAST × CBy)-hml/hml F2 mutant showed vigorous life and variable phenotypes.
All CBy-hml/hml mice were severely hearing impaired: 21/24 were deaf (no ABR response at maximal stimuli) and 3/24 exhibited severe hearing loss (70–90 dB SPL) as early as 19 days of the tested ages. In contrast, all 78 of the littermate controls and 6 tested carriers (hml/+) had normal ABR thresholds (<45 dB SPL for any tested stimulus from click, 8, 16 to 32 kHz). (CAST × CBy)-hml/hml showed incomplete penetrance: 15 out of 20 hml/hml were deaf. One hml/hml had nearly normal hearing (20–40 dB SPL).
Inner ear histology (Fig.1) shows extensive hypoplasia of the membranous labyrinth, including a rudimentary tectorial membrane. The sizes of basilar membrane, Reisner’s membrane, stria vascularis, scala media and scala tympani were significantly reduced and even completely lost. The scala vestibuli was greatly enlarged. Sixteen inner ears from 8 mutant mice from 17 to 23 days of age were histologically examined; results showed some variation however a constant relationship of pathological severity to age has not been determined. Four hml/+ ears at 4 months of age showed normal structure at light microscopy level, which suggested no heterozygous effect.
The hml mutation was initially localized to the central region of Chr 10 as shown in Fig 2. hml maps near Ap3d, the gene mutated in mocha mice that causes a progressive hearing loss. Ap3d is eliminated as a candidate for hml because its localization is proximal to D10Mit42, whereas the hml is located distal to D10Mit42 Jittery (ji), another neurological mouse mutation located on central Chr 10, is also eliminated as a candidate for hml because it is located proximal to D10Mit42. Ames waltzer (av), a mouse mutation causing deafness, also maps to the middle region of Chr10 but has been localized proximal to D10Mit91. By directly testing for allelism, all other hearing-related genes or mutations are excluded in the region where hml maps, this mutation definces a new deafness gene. A total of 342 F2 mice(= 684 meioses) were genotyped with 22 additional microsatellite markers, and hml was localized between D10Mit65 and D10Mit132, with 2 recombinant events, which define an interval of 0.3cM.
A total of 2300 F2 progeny (4600 meioses) was accumulated (see Fig 3.). The minimal genetic interval containing hml was narrowed to a region smaller than 0.1 cm, between markers D10Mit208 and D10Mit261 with 43 self-designed markers. Twenty-two animals with crossovers between D10Mit208 and D10Mit261 were generated and the candidate region was further narrowed down to 0.07 cM, which is a 500kb region between self-designed markers 18L357 and 261L453. We searched and compared our mapping data with both private and public databases. The human homologous region of the critical genetic interval containing hml was defined to be 12q22-24. Tmp3 and Igf1 were excluded, candidate gene test is still underway. Sequence information of self-designed markers is available upon request.
This study has generated 25 polymorphic microsatellite markers, placed 3 known human genes in the correct order in a high-resolution mouse map and narrowed the hml candidate gene region to a 500kb area. The identification of hml gene is critical to the progression of genetic hearing impairment research: A novel locus for autosomal dominant nonsyndromic deafness (DFNA25) mapped to human chromosome 12q24-qter is in the homologous region to our hml area. Identification of the hml gene could potentially facilitate the identification of the underlining gene for DFNA25 . A de novo deletion in a six-year-old boy with congenital hearing loss as well as mental and motor retardation provides a possible human homologue syndrome of hml at a critical interval to 13 cM in the 12q22-q24.1 region where DFNA25 resides. DFNA25 that maps in the 12q22-q24.1 region are included in the deletion that is homologous to hml in the mouse chr 10. Magnetic Resonance Tomography showing a consecutive dilatation of the left lateral ventricle, proportional smaller body size, represents a phenotype similar to hml. A mouse model for human DFNA25 will aid in the understanding of the molecular bases and the development of a potential treatment for the human disease.
This work was supported by National Institutes of Health (NIH) grants DC04376 and DC05846 to QYZ. We thank Dr. Doris K. Wu from NIDCD for her advice for research design.
Biography: Qing Yin Zheng MD, male, born at Zoucheng City, Shandong in 1963, Research scientist .Tel: +1 2072886609; Fax: +1 2072886149;gro.xaj@zyq