DFNA5: hearing impairment exon instead of hearing impairment gene?

Abstract

Background: Three mutations in the DFNA5 gene have been described in three families with autosomal dominant non-syndromic hearing impairment. Although these mutations are different at the genomic DNA level, they all lead to skipping of exon 8 at the mRNA level. We hypothesise that hearing impairment associated with DFNA5 is caused by a highly unusual mechanism, in which skipping of one specific exon leads to disease that is not caused by other mutations in this gene. We hypothesise that this represents a very specific ''gain of function'' mutation, with the truncated protein exerting a deleterious new function. Methods: We performed transfection experiments in mammalian cell lines (HEK293T and COS-1) with green fluorescent protein (GFP) tagged wildtype and mutant DFNA5 and analysed cell death with flow cytometry and fluorescence microscopy. Results: Post-transfection death of HEK293T cells approximately doubled when cells were transfected with mutant DFNA5-GFP compared with wildtype DFNA5-GFP. Cell death was attributed to necrotic events and not to apoptotic events. Conclusion:The transfection experiments in mammalian cell lines support our hypothesis that the hearing impairment associated with DFNA5 is caused by a ''gain of function'' mutation and that mutant DFNA5 has a deleterious new function.

The potential role of DFNA5, a hearing impairment gene, in p53-mediated cellular response to DNA damage

“…In the above-mentioned study, the ectopic expression of mutant DFNA5 led to cell-cycle arrest at the G1 or early S phase in yeast cells, whereas overexpression of wild-type DFNA5 did not reveal any change in the phenotype, implying that DFNA5 mutation results in a gain of function and that mutant DFNA5 may have a new deleterious function (Gregan et al 2003). Further, another study has recently demonstrated that overexpression of disease-associated mutant DFNA5 in mammalian cells induces cell death; however, wildtype DFNA5 does not (Van Laer et al 2004). Therefore, mutations of the DFNA5 gene that occur in patients with hearing impairment are unlikely to result in loss of function of wild-type DFNA5.…”

The DFNA5 gene, responsible for hearing loss and involved in cancer, encodes a novel apoptosis-inducing protein

“…[1][2][3][4][5] These findings have led to the hypothesis that DFNA5-associated HL is attributable to a highly specific gain-of-function mutation, in which skipping of one exon causes disease while mutations in other parts of this gene may not result in HL at all. Further experimental evidence for this hypothesis was provided by the finding that transfection of mutant DFNA5 causes cell death in both yeast 6 and mammalian 7 cells and by the discovery of a new DFNA5 mutation. 8 The latter mutation truncated the protein in the fifth exon, but did not segregate with HL and was present in family members with normal hearing.…”

“…The gating strategy and cell counting was as described previously. 7 All measurements were independently replicated at least three times, unless stated otherwise. Cell viability was determined as the ratio of cells showing no PI fluorescence to the total cell population.…”

“…7 Subcellular localization of selected constructs was performed using a Zeiss CLSM 510 confocal laser scanning microscope (Zeiss, Göttingen, Germany) equipped with an argon laser (excitation at 488 nm) and two helium/neon lasers (excitation at 543 and 630 nm, respectively). To test whether the mutant DFNA5 exon 9-exon 10 construct was localized in the endoplasmic reticulum (ER), double transfection with this construct and with pDsRed2-ER (Clontech, Mountain View, CA, USA) was performed in HEK293T cells.…”

“…Transfections were performed with a lipofectamine-plasmid emulsion as described previously. 7 Twenty-one hour post-transfection (16 h for apoptosis assays; only for HEK293T cells), cells were harvested using TrypLE Express (Invitrogen) and diluted in 2 ml DME. Following centrifugation, the resulting cell pellet was resuspended in 500 ml D-PBS (Dulbecco's Phosphate-buffered Saline; Invitrogen) and was subsequently used for flow cytometric evaluation.…”