Fig. 2: Loss-of-function variants in ERG are responsible for primary lymphoedema.
From: Genetic association analysis of 77,539 genomes reveals rare disease etiologies
a, Pedigrees for the four probands with loss-of-function variants in the canonical transcript of ERG, ENST00000288319.12. Hom. ref., homozygous reference. b, Truncated bar chart showing the distribution of the number of reads supporting the p.S182Afs*22 alternate allele in the 100KGP. The embedded windows show the read pileups at this position in the two affected members of the family with the variant encoding p.S182Afs*22 (het., heterozygous genotype call). The reads supporting the reference allele are in blue and those supporting the variant allele are in red. c, Schematic showing the effects of each variant at the cDNA and amino acid level and on the protein product with respect to the canonical transcript. PNT, pointed ___domain; ETS, Erythroblast Transformation Specific DNA binding ___domain; AA, amino acid. d, Reverse transcription PCR amplification of ERG mRNA in HDLECs relative to HUVECs. Data are normalized to GAPDH. Statistical significance was assessed using a two-sided Student’s t test. NS, not significant (P = 0.39). e, Immunoblot (representative of two replicates) of HUVEC and HDLEC protein lysates identified several bands corresponding to ERG isoforms expressed at similar intensities in both cell types. f, Immunofluorescence microscopy (representative of three replicates) of HDLECs shows ERG (green) nuclear colocalization with the lymphatic endothelial cell nuclear marker PROX1 (violet) and the nuclear marker DAPI (blue). HDLEC junctions are shown using an antibody to VE-cadherin (yellow). Scale bar, 50 µm. g, En face immunofluorescence confocal microscopy (representative of five replicates) of mouse ear skin. Vessels are stained with antibodies to the lymphatic marker PROX1 (violet) and ERG (green). Scale bar, 100 µm. h, Exemplar immunofluorescence microscopy image of HEK293 cells overexpressing wild-type ERG and the p.T224Rfs*15 variant ERG. Cells were stained for ERG (green) and nuclear marker DAPI (blue). Scale bars, 20 μm. The brightness is optimized for print. i, Dot plot of the estimated proportion of ERG not overlapping the nuclear marker DAPI in each of a set of immunofluorescence microscopy images of HEK293 cells overexpressing different ERG cDNAs (20 replicates for the wild type (WT), 17 replicates per tested mutant). The estimated proportions were significantly higher in each of the variants compared with WT: P = 1.52 × 10−11, 4.10 × 10−13 and 3.03 × 10−5 for each of p.S182Afs*22, p.T224Rfs*15 and p.A447Cfs*19, respectively (two-sided Student’s t tests).
