Figure 3 Common Intronic Variant Identified Causes Aberrant Splicing and POLE-Deficient Cells Show Deficiency of Polymerase Epsilon and Slowed S-phase Progression
(A) The c.1686+32C>G mutation causes aberrant splicing of intron 15 in subject cells. RT-PCR of POLE transcripts from primary fibroblasts. Primers indicated by arrows in schematic. P1, P3, POLE-deficient subjects; C1, C2, control subjects.
(B) Minigene assay demonstrating that aberrant splicing is a direct consequence of the c.1686+32C>G mutation. +ve control, point mutation in splice donor site, c.1686+1G>A. 5′ & 3′ indicate artificial vector-associated exons.
(C) POLE1 levels are markedly reduced in subject fibroblasts. Immunoblot of total cell extracts. POLE1 antibody raised against AA1-176. Vinculin, loading control. ∗ non-specific band.
(D and E) Fibroblast cells from affected individuals exhibit delayed S phase progression. Schematic, experimental set-up.
(D) Representative FACS plots.
(E) Quantification of n = 3 affected and n = 3 control cell lines from representative experiment (of n = 3 expts with n ≥ 2 biological replicates per group). Mid-S-phase mean (±SEM) BrdU-labeled cells, normalized to t = 0 time point are plotted for each group. p value, two-way ANOVA.