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Markus N Preising, Marc Platschek, Birgit Lorenz; Testing for North Carolina Macular Dystrophy (NCMD) Variations. Invest. Ophthalmol. Vis. Sci. 2019;60(9):395.
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© ARVO (1962-2015); The Authors (2016-present)
North Carolina Macular Dystrophy (NCMD) is a hereditary macular degeneration with autosomal dominant inheritance. Recently 3 point mutations in the enhancer region controlling the promotor of the PRDM13 gene were reported. Here we report a reporter gene assay to evaluate a set of variations identified in patients with macular scars indicating NCMD.
Fifty index cases with macular scars were screened by Sanger sequencing of PCR products for sequence variations in the enhancer region of the PRDM13 promotor using primers previously reported .For evaluation of sequence changes a firefly luciferase reporter assay was established in a pGL4.10[luc2] vector. (Promega) The basic promotor and the enhancer region of PRDM13 were isolated by long range PCR to construct the vector with the basic promotor to drive luciferase expression and the enhancer region to control the basic promotor. Enhancer sequences with variations were amplified from patient DNA.The empty vector (pGL4.10), a vector with the basic promotor to drive luciferase expression (pGL4.10-Prom), and vector constructs including the basic promotor and the enhancer (pGL4.10-PE) were used to transfect HEK293T cells in triplicate for each construct in a 24-well plate. For expression of luciferase over night the cells in each well were split 6 times after 8 h into a 96-well plate and incubated over night. Subsequently cell culture medium was changed for Luciferase Firefly Luc Assay reagent (NanoLight, Pinetop AZ, USA) and luciferase activity was recorded on a 96-well plate reader (TECAN Inifinite M1000 Pro, Crailsheim, Germany)
Sequence variations were identified in 23 index patients including 2 index patients carrying previously reported variant V2 . Segregation could be confirmed in available relatives of 7 index cases.The basic promotor created a strong increase in luciferase activity. The enhancer construct (pGL4.10-PE) testing the wildtype sequence reduced luciferase activity. Variant V2 further reduced luciferase activity compared to wildtype. Detailed data on the other variants identified will be presented including novel variations probably underlying NCMD.
We developed an assay to test PRDM13 enhancer variants by luciferase activity. NCMD-associated enhancer variant V2 was shown to reduce PRDM13 promotor activity thus indicating reduced activity of PRDM13 as a cause of NCMD.1. Small KW et al. Ophthalmology 2016; 123(1):9-18
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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