Abstract
Purpose: :
Mutations in cilia genes cause syndromic disorders. Common clinical features in these ciliopathies include renal cystic disease, retinitis pigmentosa. We recently found that mutations in the novel cilia protein TTC21B cause Meckel and Jeune syndromes and nephronophthisis. We performed analyses of the locations of multiple mutant TTC21B proteins in cultured renal cells and the retina in vivo to investigate the mechanisms by which the mutations cause cilia dysfunction and disease.
Methods: :
Mutant and wt cDNAs were cloned into an entry vector, and moved to aV5-tag destination vector. The plasmids were transfected into mIMCD3 cells and neonatal rat retinas using lipofection and in vivo electroporation, respectively. The location of TTC21B in mIMCD3 and retinal cells were determined by expressing V5-epitope tagged mutants. The cellular location of the wt and mutant proteins was evaluated by immunoflourecence using anti-V5 Ab. The location of the proteins in photoreceptor sensory cilia was evaluated in 3D reconstructions of confocal image stacks obtained from immunostained vibratome sections of transfected retina. The levels of expression of the mutant proteins were evaluated using Western blotting.
Results: :
The wt TTC21B protein localized in the transition zone of mIMCD3 and rat photoreceptor. Seventeen missense mutations were identified in patients with cilia disorders. The locations of all 17 mutant proteins were evaluated in mIMCD3 cells. The location of 6 mutants was evaluated in rat retinas in vivo. Five of the mutants were mislocalized in both the mIMCD3 and photoreceptor cells in vivo. Three mutants were grossly mislocalized in the photoreceptor cell bodies. Two were partially mislocalized, but retained some transition zone localization. Seven mutations resulted in reduced levels of protein.
Conclusions: :
The results suggest that a subset of TTC21B mutations cause disease by resulting in mislocalization of the mutant in ciliated cells. An additional group of mutations may cause disease by reduced protein level. Evaluation of the ability of mutant proteins to localize correctly in specific cell structures can be a useful method for assessing the pathogenicity of potentially disease-causing sequence variants.
Keywords: proteins encoded by disease genes • photoreceptors • mutations