Splice variants of the endonucleases XPF and XPG contain residual DNA repair capabilities and could be a valuable tool for personalized medicine.

The two endonucleases XPF and XPG are essentially involved in nucleotide excision repair (NER) and interstrand crosslink (ICL) repair. Defects in these two proteins result in severe diseases like xeroderma pigmentosum (XP). We applied our newly CRISPR/Cas9 generated human XPF knockout cell line with complete loss of XPF and primary fibroblasts from an XP-G patient (XP20BE) to analyze until now uncharacterized spontaneous mRNA splice variants of these two endonucleases. Functional analyses of these variants were performed using luciferase-based reporter gene assays. Two XPF and XPG splice variants with residual repair capabilities in NER, as well as ICL repair could be identified. Almost all variants are severely C-terminally truncated and lack important protein-protein interaction domains. Interestingly, XPF-202, differing to XPF-003 in the first 12 amino acids only, had no repair capability at all, suggesting an important role of this region during DNA repair, potentially concerning protein-protein interaction. We also identified splice variants of XPF and XPG exerting inhibitory effects on NER. Moreover, we showed that the XPF and XPG splice variants presented with different inter-individual expression patterns in healthy donors, as well as in various tissues. With regard to their residual repair capability and dominant-negative effects, functionally relevant spontaneous XPF and XPG splice variants present promising prognostic marker candidates for individual cancer risk, disease outcome, or therapeutic success. This merits further investigations, large association studies, and translational research within clinical trials in the future.

Functional and molecular genetic analyses of nine newly identified XPD-deficient patients reveal a novel mutation resulting in TTD as well as in XP/CS complex phenotypes.

The xeroderma pigmentosum (XP) group D protein is involved in nucleotide excision repair (NER) as well as in basal transcription. Determined by the type of XPD mutation, six different clinical entities have been distinguished: XP, XP with neurological symptoms, trichothiodystrophy (TTD), XP/TTD complex, XP/Cockayne syndrome (CS) complex or the cerebro-oculo-facio-skeletal syndrome (COFS). We identified nine new XPD-deficient patients. Their fibroblasts showed reduced post-UV cell survival, reduced NER capacity, normal XPD mRNA expression and partly reduced XPD protein expression. Six patients exhibited a XP phenotype in accordance with established XP-causing mutations (c.2079G>A, p.R683Q; c.2078G>T, p.R683W; c.1833G>T, p.R601L; c.1878G>C, p.R616P; c.1878G>A, p.R616Q). One TTD patient was homozygous for the known TTD-causing mutation p.R722W (c.2195C>T). Two patients were compound heterozygous for a TTD-causing mutation (c.366G>A, p.R112H) and a novel p.D681H (c.2072G>C) amino acid exchange, but exhibited different TTD and XP/CS complex phenotypes, respectively. Interestingly, the XP/CS patient's cells exhibited a reduced but well detectable XPD protein expression compared with hardly detectable XPD expression of the TTD patient's cells. Same mutations with different clinical outcomes in NER-defective patients demonstrate the complexity of phenotype-genotype correlations, for example relating to additional genetic variations (parental consanguinity), different allelic expression due to SNPs or differences in the methylation status.

Characterization of three XPG-defective patients identifies three missense mutations that impair repair and transcription.

Only 16 XPG-defective patients with 20 different mutations have been described. The current hypothesis is that missense mutations impair repair (xeroderma pigmentosum (XP) symptoms), whereas truncating mutations impair both repair and transcription (XP and Cockayne syndrome (CS) symptoms). We identified three cell lines of XPG-defective patients (XP40GO, XP72MA, and XP165MA). Patients' fibroblasts showed a reduced post-UVC cell survival. The reduced repair capability, assessed by host cell reactivation, could be complemented by XPG cDNA. XPG mRNA expression of XP165MA, XP72MA, and XP40GO was 83%, 97%, and 82.5%, respectively, compared with normal fibroblasts. XP165MA was homozygous for a p.G805R mutation; XP72MA and XP40GO were both compound heterozygous (p.W814S and p.E727X, and p.L778P and p.Q150X, respectively). Allele-specific complementation analysis of these five mutations revealed that p.L778P and p.W814S retained considerable residual repair activity. In line with the severe XP/CS phenotypes of XP72MA and XP165MA, even the missense mutations failed to interact with the transcription factor IIH subunits XPD and to some extent cdk7 in coimmunoprecipitation assays. Immunofluorescence techniques revealed that the mutations destabilized early recruitment of XP proteins to localized photodamage and delayed their redistribution in vivo. Thus, we identified three XPG missense mutations in the I-region of XPG that impaired repair and transcription and resulted in severe XP/CS.

Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 µm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner.