Patient UV^SS1VI is shown at age 8 (A and B) and age 13 (C and D) presenting extreme sun sensitivity with erythema and freckling. No cutaneous tumors have been observed to date.

Genetic analysis of the repair defect in patient UV^SS1VI by complementation and response to UV of normal and CS-A cells expressing the EGFP-CSAtrp361cys fusion protein. (A) Complementation analysis in heterodikaryons obtained by fusion of UV^SS1VI fibroblasts with cells representative of different excision repair-deficient groups (CS-A, CS6PV; CS-B, CS1PV; UV^SS, TA24). The partners in each fusion were labeled with latex beads of different sizes, and the RRS 24 h after UV was analyzed 73 h after fusion. The columns indicate the mean number of autoradiographic grains per nucleus in homodikaryons (patient, white column; reference strain, black column) and in heterodikaryons (dotted column). The bars indicate SE. The horizontal lines indicate the grain number per nucleus in the corresponding unirradiated samples analyzed in parallel. (B) RRS after UV irradiation in UV^SS1VI primary fibroblasts after transfection with the plasmid pEGFP-CSA. Nuclear accumulation of the green fluorescent signal of the ectopic CSA protein (cell indicated by the white arrow, Right) is paralleled by a substantial increase in the number of autoradiographic grains, i.e., in the RRS levels, compared with nontransfected cells (Left). (C) RRS after UV irradiation in C3PV, UV^SS1VI, and UV^SS1VI expressing the EGFP-CSA fusion protein. The frequency distributions of nuclei with different grain numbers and the mean values of RRS ± SE are shown. (D) RRS after UV irradiation in normal (C3PV) and CS-A (CS3BR) primary fibroblasts after transfection with the plasmid pEGFP-CSAtrp361cys. The columns indicate the mean number of autoradiographic grains per nucleus in cells before (unirradiated: white columns; irradiated: shaded columns) and after transfection with the plasmid pEGFP-CSAtrp361cys (unirradiated: black columns; irradiated: dotted columns). The bars indicate SE.

CSA protein and inactivating amino acid changes caused by the mutations found in 20 patients with CS, 8 of which are affected by the classical form of CS (code in black), and 3 by the severe form of CS (code in black bold). The clinical form of CS is not reported in the remaining 9 cases (code in gray). The diagram shows the CSA protein with the 7 WD repeats (black boxes). The amino acid changes are shown boxed, with the change in black on white. The numbers 1 and 2 after the patient code denote the different alleles. The change on the second CSA allele of the patient CS3BE refers to our unpublished observations; the mutation reported in Ridley et al. (23) is present on the genomic DNA and at the transcript level results in the deletion of exon 5 (c. 400_481del). The 8 Brazilian patients studied by Bertola et al. (16) are marked by *. Mutation nomenclature follows the format indicated at www.hgvs.org/mutnomen and refers to the cDNA sequence NM_000082.3 and protein sequence NP_000073.1. For cDNA numbering, +1 corresponds to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1.

Response to UV irradiation. Fibroblast strains were from 2 normal donors (C3PV and C405VI; □) or patients UV^SS1VI (●), XP11PV (XP-A) and XP202VI (XP-C) (dotted lines), and CS177VI and CS1PV (CS-B) (dashed lines). (A) UV-induced DNA repair synthesis (UDS) expressed as mean number of autoradiographic grains/nucleus. Bars indicate the SE. (B) RRS 24 h after UV irradiation. The mean numbers of autoradiographic grains per nucleus in irradiated samples are expressed as percentages of those in unirradiated cells. (C) Sensitivity to the lethal effects of UV light in proliferating fibroblasts labeled with ³H-thymidine 48 h after irradiation. Incorporation values in irradiated samples are expressed as percentages of those in unirradiated cells. The values in B and C were calculated from at least 2 independent experiments with SE <10% in all cases. (D) Cell cycle analysis by fluorescence-activated cell sorter (FACS) of fibroblasts from patient UV^SS1VI, normal donor C405VI, and CS-B patient CS177VI. Dot plots of exponentially growing fibroblasts 24 h after exposure to 0 or 10 J/m² of UV. Cells were pulse-labeled with 30 μM BrdU for 3 h, as described in Materials and Methods. (E) Quantitative assessment of the percentage of cells in S as compared with G₀/G₁ at the indicated UV doses.

Sensitivity to the lethal effects of oxidative stress. (A) Response of primary fibroblasts from patient UV^SS1VI (●), normal donor C3PV (□), UV^SS patient Kps3 (dotted line, ▵), and 2 CS patients (dashed lines; CS7PV: CS-A, ○; CS1AN: CS-B, △) to KBrO₃ treatment. Survival values in treated samples are expressed as percentages of those in untreated cells. The reported values are the mean of at least 2 independent experiments. The bars indicate SE. (B) Response of SV40-transformed fibroblasts from patient UV^SS1VI (●), normal donor C405VI (□), CS-A patient CS3BE (○), and UV^SS patient Kps3 (△) to menadione treatment. Clonogenic survival is expressed as percentage of that for untreated cells. (C and D) Response of SV40-transformed CS3BE cells stably expressing WT CSA (●), the mutated CSAtrp361cys protein (○), or GFP alone (■) to UV irradiation (C) or menadione treatment (D). Survival values in treated samples are expressed as percentages of those in untreated cells. The reported values are the mean of at least 3 independent experiments.