ARMS2 gene is not annotated in the mouse genome, and
ARMS2 transcripts are not detectable in mouse eye or other tissues. We therefore chose MEFs to examine the effects of variants on exogenous
ARMS2 transcripts while avoiding potential interference of endogenous
ARMS2 in human cells. MEFs were transfected with three minigene plasmid vectors including pcDNA3.1-
ARMS2-WT, pcDNA3.1-
ARMS2-indel, and pcDNA3.1-
ARMS2-R38X. The differences among these vectors were only at the variant R38X or the indel. Because a haplotype with minor alleles at both R38X and the indel was not identified in a large dataset (1169 AMD cases and 707 controls) in our previous studies, a minigene carrying combinative R38X and the indel was not included in this experiment.
19 Recently, the
ARMS2 gene transcription start site was found 220 bp upstream from the annotation. A novel alternative splice isoform of
ARMS2 (isoform B) was identified through characterization of
ARMS2 transcription in human eyes by our group.
20 We designed primers for amplifying
ARMS2 transcripts of both isoforms. Polymerase chain reaction results displayed only one specific band in MEFs after transfection for 48 hours (
Fig. 2A). The sequence of this band perfectly matched the annotated
ARMS2 transcripts (isoform A) (data not shown), suggesting that there was no alternative splicing of exogenous
ARMS2 in MEFs. As shown in
Figure 2A, we first applied RT-PCR to evaluate
ARMS2 transcripts in MEFs. Semiquantification of the PCR bands indicated that there was no significant difference between minigenes of WT and the indel by normalization with either housekeeping gene
GAPDH or renilla luciferase transcribed from cotransfected psiCHECK2 vectors (
Fig. 2B). In contrast, the level of
ARMS2 transcripts was lower in the R38X group than in the WT group (
P < 0.01). Quantitative RT-PCR further verified that R38X, not the indel, correlated with a much lower level of transcripts in MEFs (
P < 0.01;
Fig. 2C).