Fig. 4: Experimental validation of LoF variants identified in PRDM1, STRA8 and MCMDC2.
From: Landscape of pathogenic mutations in premature ovarian insufficiency
a, Map of LoF variant locations relative to essential functional domains in PRDM1. b, Western blots of transiently expressed WT, p.Gly11Valfs*14, p.Tyr622* and p.Leu776Valfs*19 mutants of GFP-tagged PRDM1 in HEK293 cells. Data are representative of two independent experiments. c, Representative fluorescence microscopy images of transiently expressed WT, p.Gly11Valfs*14 and p.Tyr622* mutants of GFP-tagged PRDM1 in HeLa cells. Data are representative of three independent experiments. d, Top: western blots of WT, p.Tyr622* and p.Leu776Valfs*19 mutants of GFP-tagged PRDM1 at 0 hours, 4 hours, 8 hours and 12 hours in HEK293 cells from CHX chase assays. Bottom: quantification of PRDM1 protein levels normalized to β-actin. e, Map of c.258 + 1 G > A location relative to essential functional domain in STRA8. f, Schematic representation of mini-gene assay strategy and splicing mode of STRA8-WT and c.258 + 1 G > A. g, Agarose gel electrophoresis and Sanger sequencing chromatograms of cDNA after transfection of STRA8-WT or c.258 + 1 G > A into HeLa and 293T cells. Data are representative of two independent experiments in two cell lines. h, Representative fluorescence images of transiently expressed WT and p.Leu21_Lys86del (c.258 + 1 G > A) mutant of FLAG-tagged STRA8 in HeLa cells. Data are representative of three independent experiments. i, Map of LoF variant locations relative to essential functional domains in MCMDC2. j, Schematic diagram illustrating the principles of HR assays (Methods). k, Left: representative flow cytometry profiles measuring the proportion of cells with DNA repair by HR (GFP+ cells) after transfection with WT, p.Ala69Leufs*18 and p.Gln229* mutants of MCMDC2 among HEK293 cells with a GFP-based I-SceI-cleavable reporter. d,k, Representative data from n = 3 biological replicates. Data are shown as means ± s.e.m. Two-sided t-test was used to determine significance. The asterisk refers to P < 0.05 compared with WT. SET, SET domain; ZNF_C2H2, zinc fingers, C2H2 type; HLH, helix-loop-helix DNA-binding domain; MCM, MCM P-loop containing nucleoside triphosphate hydrolase domain; AAA-lid, AAA-lid domain found in MCM proteins.
