Janin Lehmann

Xeroderma pigmentosum: Diagnostic procedures, interdisciplinary patient care, and novel therapeutic approaches

Xeroderma pigmentosum (XP) is an autosomal recessive disease, caused by a gene defect in the nucleotide‐excision‐repair (NER) pathway or in translesional DNA synthesis. At the age of eight, patients already develop their first skin cancers due to this DNA repair defect. In contrast, in the Caucasian population the first tumor formation in UV exposed skin regions occurs at a mean age of 60. The clinical picture among patients suffering from XP is highly diverse and includes signs of accelerated skin aging, and UV‐induced skin cancers, as well as ophthalmologic and neurological symptoms. Patients should therefore receive interdisciplinary care. This includes dermatologists, ophthalmologists, ENT specialists, neurologists, and human geneticists. Patients with XP are clinically diagnosed, but this may be supported by molecular‐genetic and functional analyses. These analyses allow pinpointing the exact disease‐causing gene defect (complementation group assignment, detection of the type and location of the mutation within the gene). The resulting information is already relevant to predict the course of disease and symptoms and probably will be utilized for individualized therapeutic approaches in the future. Recently, enhanced repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons by certain antibiotics could be demonstrated.

Clinical utility gene card for: Xeroderma pigmentosum

1.5 Mutational spectrum XPA: 122 known disease-causing mutations from 101 patients. Mutations: 77 (63%) G4C, 3 (2%) T4A, and 2 (1.5%) G4T transversions, 30 (25%) transitions (20 of these are c.682C4T stop codon mutations; GenBank accession number: NC_000009.11), 8 (7%) deletions, and 2 (1.5%) insertions. Consequences: frameshifts, protein truncations, and functional relevant amino-acid substitutions.1–3 XPB/ERCC3: eight known disease-causing mutations from five patients. Mutations: 1 (12.5%) C4A transversion, 1 (12.5%) A4C transversion, 1 (12.5%) G4A transition, 3 (37,5%) T4C transitions, 1 (12.5%) deletion (c.807-808delT_T), and 1 (12.5%) insertion (c.1421-1422insA). Consequences: two functional relevant amino-acid substitutions (c.296T4C (p.(Phe99Ser)) and c.3554C (p.(Thr199Pro))), and six frameshift/protein truncations (GenBank accession number: NC_000002.11).4 XPC: 51 known disease-causing mutations from 114 patients. Mutations: transitions, transversions, deletions, insertions, complex deletion/insertion mutations, and splice-site mutations. Consequences: frameshifts, protein truncations, and functional relevant amino-acid substitutions. Here, a phenotype correlation emerges indicating that XPC mutations only result in a classical XP phenotype.5–8 XPD/ERCC2: 48 known disease-causing mutations from 32 XP patients (36 trichothiodystrophy patients are excluded). Mutations: 43 (90%) base exchanges and 5 (10%) deletions. Consequences: frameshifts/protein truncations and functional relevant amino-acid substitutions. A genotype–phenotype correlation could already be established indicating that XPD mutations can result in six different clinical entities: classical XP, XP with neurological symptoms, trichothiodystrophy (TTD), XP/TTD complex, XP/Cockayne syndrome complex, or the cerebro-oculo-facio-skeletal syndrome (COFS).9–11 XPE/DDB2: nine known disease-causing mutations from 12 patients. Mutations: four (44%) transitions, two (23%) transversions, and three deletions (33%). Consequences: functional relevant amino-acid substitutions, frameshifts/protein truncations.12 XPF/ERCC4: 15 known disease-causing mutations from nine patients. Mutations: nine (60%) transitions and one (7%) transversion, four (26%) deletions, and one (7%) insertion. Consequences: functional relevant amino-acid substitutions, frameshifts/protein truncations.1–3 XPG/ERCC5: 25 known disease-causing mutations from 19 patients. Mutations: 12 (48%) transitions, 5 (20%) transversions, and 8 (32%) deletions. Consequences: Functional relevant amino-acid substitutions, frameshifts/protein truncations.13,14 POLH: 32 known disease-causing mutations from 22 patients (three patients are completely uncharacterized). Mutations: 5 (16%) transitions, 4 (13%) transversions, 18 (55%) deletions, and 5 (16%) insertions. Consequences: functional relevant amino-acid substitutions, frameshifts/protein truncations.15

An unusual mutation in the XPG gene leads to an internal in-frame deletion and a XP/CS complex phenotype.

DEAR EDITOR, The nucleotide excision repair (NER) pathway repairs ultraviolet (UV)-induced photoproducts. According to the respective mutated genes (XPA–XPG) and a variant form with a defect in translesion synthesis (Pol H), seven xeroderma pigmentosum (XP) complementation groups (XP-A to XP-G) have been identified. Patients belonging to XP complementation groups B, D, F and G can exhibit XP symptoms, including photosensitivity, freckling and a high increase in the risk of skin cancer, combined with Cockayne syndrome (CS) symptoms, which include photosensitivity, neurological abnormalities and failure to thrive, but no preponderance for skin cancer (XP/CS complex phenotype). This indicates a role of the respective proteins in DNA repair, as well as in basal transcription. The human XPG/ERCC5 gene (OMIM: 278780) encodes an 1186-amino acid protein and is located on chromosome 13q32 3-q33 1. During NER, XPG performs the 30 incision of the damage-containing strand and stabilizes the basal transcription factor IIH (TFIIH) by two interaction

Identification and Functional Testing of ERCC2 Mutations in a Multi-national Cohort of Patients with Familial Breast- and Ovarian Cancer.

The increasing application of gene panels for familial cancer susceptibility disorders will probably lead to an increased proposal of susceptibility gene candidates. Using ERCC2 DNA repair gene as an example, we show that proof of a possible role in cancer susceptibility requires a detailed dissection and characterization of the underlying mutations for genes with diverse cellular functions (in this case mainly DNA repair and basic cellular transcription). In case of ERCC2, panel sequencing of 1345 index cases from 587 German, 405 Lithuanian and 353 Czech families with breast and ovarian cancer (BC/OC) predisposition revealed 25 mutations (3 frameshift, 2 splice-affecting, 20 missense), all absent or very rare in the ExAC database. While 16 mutations were unique, 9 mutations showed up repeatedly with population-specific appearance. Ten out of eleven mutations that were tested exemplarily in cell-based functional assays exert diminished excision repair efficiency and/or decreased transcriptional activation capability. In order to provide evidence for BC/OC predisposition, we performed familial segregation analyses and screened ethnically matching controls. However, unlike the recently published RECQL example, none of our recurrent ERCC2 mutations showed convincing co-segregation with BC/OC or significant overrepresentation in the BC/OC cohort. Interestingly, we detected that some deleterious founder mutations had an unexpectedly high frequency of > 1% in the corresponding populations, suggesting that either homozygous carriers are not clinically recognized or homozygosity for these mutations is embryonically lethal. In conclusion, we provide a useful resource on the mutational landscape of ERCC2 mutations in hereditary BC/OC patients and, as our key finding, we demonstrate the complexity of correct interpretation for the discovery of “bonafide” breast cancer susceptibility genes.

A novel mutation in the XPA gene results in two truncated protein variants and leads to a severe XP/neurological symptoms phenotype.

The nucleotide excision repair (NER) pathway repairs UV‐induced DNA lesions in an accurate fashion and prevents UV‐irradiated areas of the skin from tumour formation. The XPA protein plays a major role in DNA damage demarcation as well as stabilization of other NER factors and was found to be defective in xeroderma pigmentosum (XP) complementation group A patients.

XPF knockout via CRISPR/Cas9 reveals that ERCC1 is retained in the cytoplasm without its heterodimer partner XPF

The XPF/ERCC1 heterodimeric complex is essentially involved in nucleotide excision repair (NER), interstrand crosslink (ICL), and double-strand break repair. Defects in XPF lead to severe diseases like xeroderma pigmentosum (XP). Up until now, XP-F patient cells have been utilized for functional analyses. Due to the multiple roles of the XPF/ERCC1 complex, these patient cells retain at least one full-length allele and residual repair capabilities. Despite the essential function of the XPF/ERCC1 complex for the human organism, we successfully generated a viable immortalised human XPF knockout cell line with complete loss of XPF using the CRISPR/Cas9 technique in fetal lung fibroblasts (MRC5Vi cells). These cells showed a markedly increased sensitivity to UVC, cisplatin, and psoralen activated by UVA as well as reduced repair capabilities for NER and ICL repair as assessed by reporter gene assays. Using the newly generated knockout cells, we could show that human XPF is markedly involved in homologous recombination repair (HRR) but dispensable for non-homologous end-joining (NHEJ). Notably, ERCC1 was not detectable in the nucleus of the XPF knockout cells indicating the necessity of a functional XPF/ERCC1 heterodimer to allow ERCC1 to enter the nucleus. Overexpression of wild-type XPF could reverse this effect as well as the repair deficiencies.

Research on genodermatoses using novel genome‐editing tools

Genodermatoses comprise a clinically heterogeneous group of mostly devastating disorders affecting the skin. To date, treatment options have in general been limited to symptom relief. However, the recent technical evolution in genome editing has ushered in a new era in the development of causal therapies for rare monogenetic diseases such as genodermatoses. The present review revisits the advantages and drawbacks of engineered nuclease tools currently available: zinc finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), meganucleases, and – the most innovative – clustered regularly interspaced short palindromic repeats (CRISPR)‐associated (Cas) nuclease 9 (CRISPR/Cas9) system. A mechanistic overview of the different modes of action of these programmable nucleases as well as their significance for causal therapy of genodermatoses is presented. Remaining limitations and challenges such as efficient delivery and off‐target activity are critically discussed, highlighting both the past and future of gene therapy in dermatology.

Photosensitive form of trichothiodystrophy associated with a novel mutation in the XPD gene

Department of Dermatology, Venereology and Allergology, University Medical Center, Georg August University, G€ ottingen, Germany. Clinic for Dermatology and Venereology, University Medical Center Rostock, Rostock, Germany. DWI – Leibniz Institute for Interactive Materials, Aachen, Germany. Interdisciplinary Pediatric Center for Children with Developmental Disabilities and Severe Chronic Disorders, University Medical Center, Georg August University, G€ ottingen, Germany. Lower Saxony Institute of Occupational Dermatology, University Medical Center G€ ottingen and University of Osnabr€ uck, Osnabr€ uck, Germany.

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.

Forschung zu Genodermatosen durch neue Genom-Editing -Methoden: Genodermatosen und Genom-Editing-Instrumente

Genodermatosen umfassen eine klinisch heterogene Krankheitsgruppe mit meist schwerwiegenden Auswirkungen auf die Haut. Bis heute beschränken sich die Behandlungsmöglichkeiten auf die Linderung der Symptome. Jüngste technische Fortschritte im Rahmen des Genome‐Editing leiten eine neue Ära in der Entwicklung kausaler Therapien seltener monogenetischer Erkrankungen wie Genodermatosen ein. Diese Übersichtsarbeit behandelt die Vor‐ und Nachteile der derzeit verfügbaren Nuklease‐basierten Methoden: Zinkfinger‐Nukleasen (ZFNs), transcription activator‐like effector nucleases (TALENs), Meganukleasen und das innovative clustered regularly interspaced short palindromic repeats (CRISPR)‐associated (Cas) nuclease 9 (CRISPR/Cas9) system. Ein mechanistischer Überblick über die verschiedenen Wirkmechanismen dieser programmierbaren Nukleasen, sowie deren Bedeutung für die Kausaltherapie von Genodermatosen wird vorgestellt. Fortbestehende Grenzen und Herausforderungen, wie das effiziente Eindringen und Off‐Target‐Aktivität, werden kritisch diskutiert, um die Vergangenheit und Zukunftsaussichten der Gentherapie in der Dermatologie deutlich zu machen.