Genetic and functional analysis of monogenic inner ear dysfunction.

摘要

Ozzy is an ethylnitrosourea (ENU)-induced mouse mutant displaying headbobbing behaviour, indicative of inner ear dysfunction. CT scanning experiments revealed gross abnormalities in the three semicircular channels within the vestibulum. These defects were confirmed by vestibulo-ocular (VOR) and optokinetic (OKR) reflex tests, which determine the size of the nystagmus, a compensating movement of the eye as a reaction to movement of the body or the environment. Scanning electron microscopy revealed significant inner hair cell loss and outer hair cell duplications in Ozzy cochleae. However, the observed inner ear malformations of Ozzy mice only led to a moderate hearing loss in the middle frequencies.;The defects observed in Ozzy are inherited in an autosomal dominant pattern. Linkage analysis led to the identification of a region on mouse chromosome 2. A mutation in Jagged 1 was subsequently identified. Mutations in JAGGED1 in human cause Alagille syndrome (AGS), a syndrome characterized by liver disease in combination with at least two of the following characteristics: heart disease, eye, face or skeletal abnormalities. Sometimes these symptoms are accompanied by abnormalities in the kidney or ear. Growth delay or mental retardation may also be present. Ozzy was investigated for symptoms of AGS. An intrahepatic bile duct paucity was observed, in combination with the tetralogy of Fallot. The tetralogy of Fallot is a syndrome of the heart characterized by four malformations: (1) pulmonary stenosis, (2) an overriding aorta, (3) a ventricular septal defect and (4) right ventricular hypertrophy. The tetralogy of Fallot sometimes occurs as a symptom of AGS, and is the most frequent observed heart malformation in AGS patients. Furthermore, Ozzy mice suffered from growth retardation when compared with wild type mice. Therefore we concluded that Ozzy is not only a valuable tool for hearing research, but also a model to study several of the AGS characteristics.;In a second part of my thesis, I have performed functional studies on DFNA5. Mutations in DFNA5 cause an autosomal dominant, progressive, non-syndromic, sensorineural type of hearing loss. Not much is known concerning this gene's function. The genetics of DFNA5-related hearing impairment are particularly interesting. Over the years, four different families have been identified with DFNA5 mutations. These are different at the genomic DNA level, but all lead to skipping of exon 8 at the mRNA level. Moreover, an Iranian hearing-impaired family carried an inactivating mutation in exon 5 of DFNA5, which did not segregate with the observed hearing loss. Therefore exon 8 skipping seems to be the disease-causing mechanism in DFNA5-related hearing loss. We hypothesized that DFNA5-related hearing loss is caused by a very specific gain-of-function mutation, generating a truncated protein with a new, deleterious function.;To test this hypothesis, we have investigated the subcellular localization of wild-type and mutant DFNA5. Transfection experiments revealed that wild-type DFNA5 was distributed evenly over the cytoplasm, while mutant DFNA5 occurred in granular occlusions. Although mutant DFNA5 could still be observed in the cytoplasm, the mutant protein seemed to concentrate around the plasma membrane. Analysis by flow cytometry revealed that mutant DFNA5 caused massive cell death in mammalian cells in vitro, a phenomenon not observed in cells transfected with wild-type DFNA5. These experiments provided supportive evidence for our hypothesis about the new, deleterious function for the mutant DFNA5 protein.;The goal of the last part of my thesis was to identify the regulatory elements necessary for Dfna5 expression. This is not only an interesting objective from a hearing impairment scientist's point of view, but may be of interest to a larger research community as recently methylation of the DFNA5 5' flanking region has been implicated in the development of gastric, colorectal and breast cancer. (Abstract shortened by UMI.)