by Edward Wilcox, Ph.D., NICHD, and Marci Lesperance, M.D., Children's National Medical Center, Washington, D.C.
Investigators in the Section on Linkage Studies and Molecular Cloning, Laboratory of Molecular Genetics, NIDCD-and our collaborators at the Department of Otolaryngology at Vanderbilt University in Nashville-have discovered a novel locus for nonsyndromic hereditary hearing impairment on chromosome 4p in the region of the Huntington's disease gene. This locus causes a dominant, progressive low-frequency hearing loss (LFHL) in a large U.S. family. The region of linkage spans a distance of approximately 1.7 megabases (Mb), and we are hoping to narrow this region as additional polymorphic markers are examined. There is a possibility that mutations in known sequences, mapped during the efforts to clone the Huntington's disease locus, are responsible for this phenotype.
Genetic factors account for most cases of hearing impairment in young children. Among individuals with genetic hearing loss, approximately 74% have an autosomal-recessive mode of inheritance, about 25% have an autosomal-dominant type, and the remaining 1% having X-linked or mitochondrial types of hereditary hearing impairment. One-third of individuals with hereditary hearing impairment have other associated symptoms recognizable as a syndrome. The other two-thirds have no known associated findings and are classified as having "nonsyndromic" hereditary hearing impairment (NSHHI) (1,2). In many types of inherited hearing loss, morphologic or neuroepithelial defects are observed in inner ear structures (3). At the present time, however, little is known about the molecular mechanisms involved in the development and homeostasis of the inner ear.
Linkage studies have identified seven dominant gene loci and seven recessive loci for autosomal NSHHI. One of these loci is also the cause of Usher syndrome type 1B and is linked to chromosome 11q13.5 (4,5). Mutations in this gene, a novel myosin VIIA gene (6), are apparently able to cause Usher's syndrome in some families and recessive NSHHI with vestibular malfunction in others.
Two NSHHI loci map to the X chromosome (7,8). Mutations in the Pou3F4 gene have been shown to cause X-linked fixation of the stapes with perilymphatic gusher (9), a rare congenital defect leading to mixed hearing loss. Despite this progress in our understanding of the genetic causes of hearing loss, many families are plagued by inherited hearing loss in which the gene responsible is still unknown.
Haplotype analysis of family with nonsyndromic hereditary hearing
Parentheses indicate inferred genotype. Question marks indicate unknown genotype. Markers genotyped are displayed vertically, from top to bottom: D4S43, D4S127, D4S412, D4S126, and D4S432. Boxes indicate inheritance of the chromosome linked to the disease. Dashed lines indicate that the affected parent is uninformative for that marker.
Mouse strains that exhibit the NSHHI phenotype may help in identifying novel genes important in human NSHHI. The deafness, or dn, mouse is among the most intriguing of a couple dozen such models. The responsible dn gene maps to mouse chromosome 19 (D19MIT14, 60, and 41), which is syntenic with human chromosome 9q21 and therefore represents an additional potential location for a human NSHHI-related gene (10). Tilted mice, named for their characteristic tilted heads and discovered at Jackson Laboratory in Bar Harbor, Maine, are another possible model. The defective gene in tilted mice maps to the syntenic mouse chromosome 5 and may be the mouse version of the human dominant NSHHI gene that our lab recently localized to a region on chromosome 4p that is bounded by the Huntington's disease locus. The tilted-mouse mutant has a recessive cochleo-vestibular loss recognizable by the mouse's tilted head and inability to swim (11). Although in mice it is not easy to detect loss of low-frequency hearing that may be associated with the tilted mutant gene, this promises to be a valuable model.
Mutations in genes that are homologous in mice and humans sometimes share a similar but not identical phenotype. This point is well illustrated by the relationship between the shaker-1 mouse-in which mutations in the myosin VIIA gene result in hearing loss and a vestibular abnormality-and human Usher syndrome type 1B-in which mutations in the same gene result in retinal degeneration, in addition to hearing loss and vestibular abnormality. Several other mouse strains could serve as potential models for NSHHI, even though these mice also have altered vestibular function in addition to hearing loss (12-15).
In our current study of an extended family in the United States with more than 100 members, the majority of patients have a bilateral and symmetric hearing loss involving frequencies of 250, 500, and 1000 Hz at the onset of symptoms. The progression of the hearing loss follows one of three patterns: 1) confined to the low frequencies, 2) involving all frequencies and producing a flat audiogram, or 3) involving low and high frequencies while sparing the middle frequencies (2000 Hz). As a result of this comparatively mild pattern of hearing loss, few of the family members use hearing aids, and none have cochlear implants. The age of onset is generally in the second decade of life; no family members showed hearing loss before age 5, but all affected members had developed hearing loss by age 15 (16).
The family was genotyped for D4S432, D4S412, D4S127, D4S43, and D4S126 markers on human chromosome 4p. These five markers span a genetic distance of approximately 5 centimorgans (17). The region has a much higher recombination rate than would be expected for its physical size (less than 3 Mb) (18).
Four recombinants were identified by haplotype analysis (see figure). Individual 111-21 has a recombination between D4S127 and D4S412. Unaffected individuals 111-17 and 111-19 have recombinations between D4S126 and D4S432. Since 111-11 is an affected individual with the same recombinant haplotype, we postulate that the breakpoints for 111-11 vs. 111-17 and 111- 19 lie on opposite sides of the gene for hearing loss. D4S126 is not a fully informative marker; the affected parents of the recombinants are homozygous at this locus, preventing detection of recombinants in their children. Thus the most likely location for the gene is between D4S412 and D4S432, a distance of 1.7 Mb (19). The maximum LOD score was 5.05 at q = 0.05 for D4S412. Given the marker order of D4S412 - D4S126 - D4S432, the multipoint mapping yielded a maximum LOD score of 6.5 when the disease gene was placed in the interval between D4S126 and D4S432.
This region on chromosome 4 has been well mapped. More than 20 genes were identified in the course of the lengthy search for the Huntington's disease gene, HD or IT15 (20), which was finally located at 4p16.3 in 1993 by the Huntington's Disease Collaborative Research Group. Expression of IT15 has been detected in all areas of the cerebral cortex, predominantly in neurons (21). Hearing loss has not been described as a clinical feature of Huntington's disease; however, to our knowledge, there has been no study that specifically screens for more subtle forms of hearing loss in patients with Huntington's disease. The multipoint data for our NSSHI locus suggest a location proximal to IT15 and D4S126, a region that includes the gene for the alpha-2C-adrenergic receptor (ADRA2C) (22). Alpha-2-adrenergic receptors are widely expressed in the brain, especially in regions with high dopamine content. Another candidate gene in this region is the alpha-2- macroglobulin receptor-associated protein (A2MRAP) also known as the low- density-lipoprotein receptor-related, protein-associated protein (LRPAP1) (23,24). Although the function of this protein is unknown, its corresponding receptor is important in proteinase inhibition and lipoprotein metabolism.
Now that we have documented these tantalizing potential connections to the Huntington's disease gene (25) and the tilted-mouse gene, our laboratory would like to screen any and all the candidate genes that have been mapped to the human 4p16.3 region. We are planning to breed the tilted mice to test their hearing. If these mice are good candidate models, we would expect to find close syntenic relationships in the mapping of mouse chromosome 5 and the human 4p16.3 region.
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