Fig. 1

Portraits of FA-Q patient 3104 at different age, and characterization of her genetic and cellular defects. a: Typical FA puppet-like face, small head, light brown hair, absence of the right and severe hypoplasia of the left thumb (arrows) at about 15 months. b: High slanting eye lids and beginning freckling of the face at age 12 years. c: Low-set auricles, early hair graying and actinic keratosis-like skin changes with erythema, atrophy and patchy pigmentation in her fifth decade. d: A heterozygous substitution of adenine with guanine (asterisk) 2 bp upstream of exon 5 in the XPF/ ERCC4/FANCQ gene was maternally inherited. e: The mutation shown in (d) leads to aberrant splicing (upper panel). Gel extraction and sequence analysis of a major splice product reveals skipping of exon 5 (lower panel). f: The second heterozygous FANCQ mutation c.1765C > T (asterisk) is located in exon 8 and was paternally inherited. g: 3104 fibroblasts transduced with mock vector (green line) or with vector containing XPF/ ERCC4/FANCQ with the missense mutation c.1765C > T (red line) are MMC-sensitive, though not quite to the degree of fibroblasts from an FA-B patient serving as a control (black line). Complementation of 3104 fibroblasts with wildtype FANCQ (FANCQ^WT, blue line) restores MMC resistance. Error bars designate SDs of three experiments. LC₅₀ levels are indicated by dotted lines of corresponding colors; they equal 42.6 ± 5.7 nM for 3104 + mock, 52.8 ± 2.7 nM for 3104 + FANCQ^MUT and 20.7 ± 2.4 nM for FA-B fibroblasts. The survival rates of 3104 + FANCQ^MUT and 3104 + mock are not significantly different, however the proportions for all concentrations (except 0) of these curves and 3104 + FANCQ^WT are different with p < 0.001. h: Immunoblot using fibroblast extracts from the FA-Q cell line 3104 and of the previously reported FA-Q cell line 1333 [9] with different FANCQ missense mutations showing immunoreactive residual FANCQ protein of normal size but reduced abundance in comparison to a normal (CON) and a FANCD2-deficient (FA-D2) control. One thousand three hundred thirty-three reveals an additional FANCQ band resulting from a truncating mutation on the second allele. Loading control: tubulin. i: Cell fractionation demonstrates residual mutant FANCQ protein in 3104 cells detectable in the chromatin fraction. j and k: Normal ERCC1-XPF interactions in 3104 transformed fibroblasts. ERCC1 (j) or XPF (k) were immunoprecipitated with antibodies against ERCC1 or XPF, respectively. Unspecific bands are marked by asterisks while the visible antibody heavy chain is indicated by an arrow