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Daisuke Iejima, Kazushige Tsunoda, Takeshi Iwata; Functional Analysis of RP1L1 R45W Mutant Responsible for Occult Macular Dystrophy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):173.
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© 2017 Association for Research in Vision and Ophthalmology.
Occult macular dystrophy (OMD) is an autosomal dominant form of macular dystrophy characterized by progressive decrease of central visual acuity due to macular dysfunction without abnormal fundus appearance and abnormal full field electroretinogram. The disease causing mutations R45W and W960R were identified in Retinitis Pigmentosa 1-Like 1 (RP1L1) (Akahori et al., Am J Hum Genet 2010). However, both RP1L1 function and OMD pathogenesis by RP1L1 gene mutation has not been fully understood. To investigate the biological role of RP1L1, we constructed both wildtype RP1L1 and mutant R45W expression vectors and analyzed the localization within the cell.
Human RP1L1 cDNA was amplified in two separate fragments at the 5' end (3.6 kbp) and 3' end (4.0 kbp) using human retinal cDNA library (Clontech). Two fragments were cloned into pCMV-HA-N vector (Life Technologies) and fused to full cDNA of 7.6 Kbp. The full length RP1L1 and was further mutated for R45W using KOD-plus-Mutagenesis inverse PCR kit (TOYOBO) from 364 bp (forward) to 363 bp (reverse) at 5’ end of RP1L1 cloned vector. Both wildtype and mutant expression vectors were transfection into HEK293T cells using ViaFect Transfection Reagent (Promega) for expression analysis and protein localization.
Both wildtype and mutant RP1L1 were successfully expressed showing 260 kDa band by western blot analysis. The localization of wildtype RP1L1 was observed around the nucleus while R45W mutant was detected diffused in the cell cytoplasm.
The full 7.6 Kbp cDNA for human RP1L1 was cloned for the first time and mutant R45W constructed for protein expression in HEK293 cell line. Mislocalization of mutant R45W in the cell may have association with the disease onset.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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