Abstract
Purpose :
Three sequence variants (V1 - V3) in the enhancer of PRDM13 were reported to underly North Carolina Macular Dystrophy (NCMD)[1]. In the past [2] we reported further sequence variants (M1 - M5) we identified in patients with macular degeneration.
Here we report on an improved assay to evaluate these sequence variants by heterologous expression of a reporter system in HEK293 cells.
Methods :
The core promotor of PRDM13 (720 bp) was cloned into a firefly luciferase reporter pGL4.10 [luc2] vector (Promega). This core promotor construct (PROM) was used as reference of basic promotor activity. A reference sequence (1139 bp) was reported to affect PRDM13 transcription [1]. This enhancer reference sequence was inserted into PROM to create an enhancer reporter vector (WT). WT was used as reference to deduce the influence of the enhancer on transcriptional activity onto PRDM13. WT was modified by site directed mutagenesis PCR for sequence variants previously reported (M1-M5, V1, V3 with M3=V2) [1,2]. In addition corresponding enhancer sequence from two patients with combined variants was directly inserted into PROM (2K3 (M2 + M4), 3K4 (M3 + M5) [2]. All constructs were transiently expressed in HEK293 cells in at least triplicate. Whole RNA was isolated from each set and amplified with luciferase (reporter gene) and GAPDH (refrence) specific primers by quantitative PCR (qPCR) after reverse transcription using oligo-dT primers. qPCR was evaluated using the 2-ΔΔCt method.
Results :
The assay showed higher expression of the basic reporter gene construct PROM compared to construct WT. The constructs covering previvously reported sequence variants [1] also presented an increase in expression of the reporter gene (PROM > M3/V2 > V1 > V3). As well as the constructs covering the sequence variants identified in our patient cohort (PROM > M1 > M2 > M3 = M4 > M5). Construct 2K3 including sequence variant M2 and M4 resulted in decreased expression of the reporter gene while Construct 3K4 including sequence variants M3 and M5 resulted in increased expression of the reporter gene equal to V1 but less than V2.
Conclusions :
We confirmed our previous results that the enhancer region of the PRDM13 gene which is affected by sequence variants in NCMD is a gene silencer. The majority of sequence variants in the enhancer region override the effect of the reference enhancer thus leading to increased expression of PRDM13.
1. PubMED: 26507665
2. ARVO 2019, Abstract 395
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.