Figure 1

Figure 1 Origin, location, segregation, and implications of the FAAP100 variant.

(A) Pedigree of a fetus with FA-suggestive phenotype (III-2, affected individual; solid and slashed circle, marked with a red arrow, ID no. 1176) and family. Squares denote males; circles denote females. Genotyped individuals of the core family are indicated by an asterisk, unassessed genotypes by a question mark, and proven or obligate heterozygotes by half-filled symbols. (B) Five FA (prefix FANC) and FA-associated (prefix FAAP) genes in major autozygous regions (black blocks) on 3 different chromosomes (Chr) in the affected III-2 (1176) are shown. (C) Sanger electropherograms identified a homozygous A>C variant sequence, heterozygous sequence, or WT sequence at FAAP100 position c.1624 (highlighted by a red frame), predicting p.(T542P), in relatives of the core family as indicated. (D) Cell-cycle analysis of cultured amniotic fibroblasts by flow cytometry. Exemplary individual measurements. Histograms of cells from a normal control (non-FA; top), a fetus with FA (positive control; middle), and fetus III-2 (1176; bottom). Black histograms are from untreated cultures and superimposed gray or gray/red histograms are from cultures exposed to MMC (10 ng/mL, 48 hours), shown individually in Supplemental Figure 1C. The percentage of cells in G₂ phase is indicated. Red coloring and arrows denote an increased G₂ compartment size. Variability and significance of G₂ phase arrest in repeated measurements are shown in Supplemental Figure 4A. (E) Titration experiments show reduced levels of FAAP100 protein in FAAP100^T542P-mutant 1176 cells from affected III-2 cells (approximately 3 steps of 1:3 dilution each lower than in non-FA cells). Reduced levels of FANCL are also suggested in 1176 cells (approximately 2 steps of 1:3 dilution lower than in non-FA cells). Wedges represent dilutions at the ratios indicated above the blots. FA-A, cells from an individual with FA, subtype A. Vinculin was used as a loading control.