Isosomppi, J., Västinsalo, H., Geller, S. F., Heon, E., Flannery, J. G., & Sankila, E. M. | Molecular Vision | 2009 Sep 8 | Vol. 15 | pgs. 1806-18| ncbi.nlm.nih.gov/pmc/articles/PMC2742642/
Abstract
Purpose
Mutations of clarin 1 (CLRN1) cause Usher syndrome type 3 (USH3). To determine the effects of USH3 mutations on CLRN1 function, we examined the cellular distribution and stability of both normal and mutant CLRN1 in vitro. We also searched for novel disease-causing mutations in a cohort of 59 unrelated Canadian and Finnish USH patients.
Methods
Mutation screening was performed by DNA sequencing. For the functional studies, wild-type (WT) and mutant CLRN1 genes were expressed as hemagglutinin (HA) tagged fusion proteins by transient transfection of BHK-21 cells. Subcellular localization of CLRN1-HA was examined by confocal microscopy. The N-glycosylation status of CLRN1 was studied by using the N-glycosidase F (PNGase F) enzyme and western blotting. Cycloheximide treatment was used to assess the stability of CLRN1 protein.
Results
We found three previously reported pathogenic mutations, p.A123D, p.N48K, and p.Y176X, and a novel sequence variant, p.L54P, from the studied USH patients. The WT HA-tagged CLRN1 was correctly trafficked to the plasma membrane, whereas mutant CLRN1-HA proteins were mislocalized and retained in the endoplasmic reticulum. PNGase F treatment of CLRN1-HA resulted in an electrophoretic mobility shift consistent with sugar residue cleavage in WT and in all CLRN1 mutants except in p.N48K mutated CLRN1, in which the mutation abolishes the glycosylation site. Inhibition of protein expression with cycloheximide indicated that WT CLRN1-HA remained stable. In contrast, the CLRN1 mutants showed reduced stability.
Conclusions
WT CLRN1 is a glycoprotein localized to the plasma membrane in transfected BHK-21 cells. Mutant CLRN1 proteins are mislocalized. We suggest that part of the pathogenesis of USH3 may be associated with defective intracellular trafficking as well as decreased stability of mutant CLRN1 proteins.
Introduction
Usher syndrome (USH) describes a group of autosomal recessive diseases with bilateral sensorineural hearing loss and visual impairment phenotypically similar to retinitis pigmentosa (RP) [1-4]. Prevalence of USH in different populations is estimated to range from 3.5 to 6.2 per 100,000, thus making it the most frequent cause of combined deaf-blindness worldwide [5]. The condition has been classified into three clinical subtypes (USH1, USH2, and USH3), based on the severity and progression of the hearing impairment, presence or absence of vestibular dysfunction, and the age of onset of RP [1]. This classification remains in clinical use, although recent progress on the molecular genetics and clinical research of USH has revealed broad genetic and clinical heterogeneity [3,6]. Atypical forms of USH have been identified within all three clinical types, and there is considerable overlap of symptoms among the subtypes. A distinguishing feature of USH3 is the wide spectrum of nonlinear progressive hearing impairment, which ranges from a near normal to a severe audiometric phenotype [7]. USH3 patients may also have either normal or decreased vestibular responses [8]. The rate of visual loss in USH3 is similar to other USH subtypes [9], with the most recent analyses suggesting that retinal degeneration in USH3 progresses more rapidly than in USH2A [10,11]. The variable phenotype may cause USH3 to be under-diagnosed and it may be more prevalent than previously indicated [6].
To date, nine USH gene products have been identified: the molecular motor myosin VIIa (USH1B) [12]; the cell adhesion proteins cadherin 23 (USH1D) [13] and protocadherin 15 (USH1F) [14,15]; the scaffold proteins harmonin (USH1C) [16], SANS (USH1G) [17], and whirlin (USH2D) [18]; the G-protein-coupled 7-transmembrane receptor VLGR1b (USH2C) [19]; two isoforms of the extracellular matrix connected protein usherin (USH2A) [20,21]; and the four-pass transmembrane domain protein clarin 1 (USH3) [22,23]. There is growing evidence suggesting that these proteins form a network, which is critical for the development and maintenance of the sensorineural cells in the inner ear and the retina [3,4,24-28].
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