Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress

Nucleotide excision repair (NER) is the most flexible of all known DNA‐repair mechanisms, and XPG is a 3′‐endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP‐G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP‐G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV‐irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV‐ but not oxidative‐stress‐induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.     

Retinitis Pigmentosa Mutations of SNRNP200 Enhance Cryptic Splice‐Site Recognition

Mutations in SNRP200 gene cause autosomal‐dominant retinal disorder retinitis pigmentosa (RP). The protein product of SNRNP200 is BRR2, a DExD/H box RNA helicase crucial for pre‐mRNA splicing. In this study, we prepared p.S1087L and p.R1090L mutations of human BRR2 using bacterial artificial chromosome recombineering and stably expressed them in human cell culture. Mutations in BRR2 did not compromise snRNP assembly and both mutants were incorporated into the spliceosome just as the wild‐type (wt) protein. Surprisingly, cells expressing RP mutants exhibited increased splicing efficiency of the LDHA gene. Next, we found that depletion of endogenous BRR2 enhanced usage of a β‐globin cryptic splice site while splicing at the correct splice site was inhibited. Proper splicing of optimal and cryptic splice sites was restored in cells expressing BRR2‐wt but not in cells expressing RP mutants. Taken together, our data suggest that BRR2 is an important factor in 5′‐splice‐site recognition and that the RP‐linked mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) affect this BRR2 function.     

Correlation of Phenotype/Genotype in a Cohort of 23 Xeroderma Pigmentosum‐Variant Patients Reveals 12 New Disease‐Causing POLH Mutations

Xeroderma pigmentosum variant (XP‐V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XP‐V patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV‐hypersensitivity in the presence of caffeine, a signature of the XP‐V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal‐cell carcinoma was 13 per patient, six for squamous‐cell carcinoma, and five for melanoma. XP‐V is due to defects in the translesion‐synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life‐cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP‐Vs from sun exposure.     

Characterisation of novel mutations in Cockayne syndrome type A and xeroderma pigmentosum group C subjects

We report that a subject with Cockayne syndrome type A (CS3BE) was a compound heterozygote for mutations in CKN1, the gene encoding the CSA protein (MIM 216400). CS3BE displayed a novel missense mutation (A160V) and a previously described nonsense mutation (E13X). Although residing between the second and third WD-40 repeats characteristic of the CSA protein, A160 is completely conserved in all species that possess a CKN1 homologue. We also describe a mutation in a previously uncharacterised xeroderma pigmentosum group C subject (XP8CA) in the XPC gene (MIM 278720). XP8CA was homozygous for a 2 bp TG deletion in codon 547 resulting in premature termination at codon 572. Immunoblotting of XP8CA extracts confirmed the absence of full-length XPC protein that was present in unaffected cell lines.     

DNA damage and nucleotide excision repair capacity in healthy individuals

Interindividual differences in DNA repair capacity (DRC) represent an important source of variability in genome integrity and thus influence health risk. In the last decade, DRC measurement has attracted attention as a potential biomarker in cancer prediction. Aim of the present exploratory study was to characterize the variability in DNA damage and DRC on 100 healthy individuals and to identify biological, lifestyle, or genetic factors modulating these parameters. The ultimate goal was to obtain reference data from cancer‐free population, which may constitute background for further investigations on cancer patients. The endogenous DNA damage was measured as a level of DNA single‐strand breaks and DRC, specific for nucleotide excision repair (NER), was evaluated using modified comet assay, following the challenge of peripheral blood mononuclear cells with benzo[a]pyrene diolepoxide. Additionally, genetic polymorphisms in NER genes (XPA, XPC, XPD, and XPG) were assessed. We have observed a substantial interindividual variability for both examined parameters. DNA damage was significantly affected by gender and alcohol consumption (P = 0.003 and P = 0.012, respectively), whereas DRC was associated with family history of cancer (P = 0.012). The stratification according to common variants in NER genes showed that DNA damage was significantly modulated by the presence of the variant T allele of XPC Ala499Val polymorphism (P = 0.01), while DRC was modulated by the presence of the A allele of XPA G23A polymorphism (P = 0.048). Our results indicate the range of endogenous DNA single‐strand breaks and capacity of NER in healthy volunteers as well as the role of potentially relevant confounders. Environ. Mol. Mutagen. 2011. © 2011 Wiley‐Liss, Inc.     

Xeroderma pigmentosum group C protein interacts with histones: regulation by acetylated states of histone H3

In the mammalian global genome nucleotide excision repair pathway, two damage recognition factors, XPC and UV‐DDB, play pivotal roles in the initiation of the repair reaction. However, the molecular mechanisms underlying regulation of the lesion recognition process in the context of chromatin structures remain to be understood. Here, we show evidence that damage recognition factors tend to associate with chromatin regions devoid of certain types of acetylated histones. Treatment of cells with histone deacetylase inhibitors retarded recruitment of XPC to sites of UV‐induced DNA damage and the subsequent repair process. Biochemical studies showed novel multifaceted interactions of XPC with histone H3, which were profoundly impaired by deletion of the N‐terminal tail of histone H3. In addition, histone H1 also interacted with XPC. Importantly, acetylation of histone H3 markedly attenuated the interaction with XPC in vitro, and local UV irradiation of cells decreased the level of H3K27ac in the damaged areas. Our results suggest that histone deacetylation plays a significant role in the process of DNA damage recognition for nucleotide excision repair and that the localization and functions of XPC can be regulated by acetylated states of histones.     

Functional SNP in 3′‐UTR MicroRNA‐Binding Site of ZNF350 Confers Risk for Age‐Related Cataract

Many studies have suggested that individual susceptibility to age‐related cataract (ARC) may be associated with DNA sequence polymorphisms affecting gene regulation. As DNA repair is implicated in ARC pathogenesis and single‐nucleotide polymorphisms (SNPs) in the 3′‐terminal untranslated region (3′‐UTR) targeted by microRNAs (miRNAs) can alter the gene function, we hypothesize that the miRNA‐binding SNPs (miRSNPs) in DNA double‐strand break repair (DSBR) and nucleotide excision repair (NER) pathways might associate with ARC risk. We genotyped nine miRSNPs of eight genes in DSBR and NER pathways in Chinese population and found that ZNF350‐ rs2278414:G>A was significantly associated with ARC risk. Even though the Comet assay of cellular DNA damage indicated that all the subtypes of ARC patients had more DNA breaks in peripheral lymphocytes than the controls independent of rs2278414 genotypes, individuals carrying the variant A allele (AA and AG) had lower ZNF350 mRNA levels compared with individuals with GG genotype. Moreover, the in vitro experiment indicated that miR‐21‐3p and miR‐150‐5p specifically downregulated luciferase reporter expression in the cell lines transfected with rs2278414 A allele compared with rs2278414 G. These results suggested that the association of SNP rs2278414 with ARC might involve an altered miRNA regulation of ZNF350.     

Alternative splicing and retinal degeneration

Alternative splicing is highly regulated in tissue‐specific and development‐specific patterns, and it has been estimated that 15% of disease‐causing point mutations affect pre‐mRNA splicing. In this review, we consider the cis‐acting splice site and trans‐acting splicing factor mutations that affect pre‐mRNA splicing and contribute to retinal degeneration. Numerous splice site mutations have been identified in retinitis pigmentosa (RP) and various cone‐rod dystrophies. Mutations in alternatively spliced retina‐specific exons of the widely expressed RPGR and COL2A1 genes lead primarily to X‐linked RP and ocular variants of Stickler syndrome, respectively. Furthermore, mutations in general pre‐mRNA splicing factors, such as PRPF31, PRPF8, and PRPF3, predominantly cause autosomal dominant RP. These findings suggest an important role for pre‐mRNA splicing in retinal homeostasis and the pathogenesis of retinal degenerative diseases. The development of novel therapeutic strategies to modulate aberrant splicing, including small molecule‐based therapies, has the potential to lead to new treatments for retinal degenerative diseases.     

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry.     

MCM3AP and POMP Mutations Cause a DNA‐Repair and DNA‐Damage‐Signaling Defect in an Immunodeficient Child

Immunodeficiency patients with DNA repair defects exhibit radiosensitivity and proneness to leukemia/lymphoma formation. Though progress has been made in identifying the underlying mutations, in most patients the genetic basis is unknown. Two de novo mutated candidate genes, MCM3AP encoding germinal center‐associated nuclear protein (GANP) and POMP encoding proteasome maturation protein (POMP), were identified by whole‐exome sequencing (WES) and confirmed by Sanger sequencing in a child with complex phenotype displaying immunodeficiency, genomic instability, skin changes, and myelodysplasia. GANP was previously described to promote B‐cell maturation by nuclear targeting of activation‐induced cytidine deaminase (AID) and to control AID‐dependent hyperrecombination. POMP is required for 20S proteasome assembly and, thus, for efficient NF‐κB signaling. Patient‐derived cells were characterized by impaired homologous recombination, moderate radio‐ and cross‐linker sensitivity associated with accumulation of damage, impaired DNA damage‐induced NF‐κB signaling, and reduced nuclear AID levels. Complementation by wild‐type (WT)‐GANP normalized DNA repair and WT‐POMP rescued defective NF‐κB signaling. In conclusion, we identified for the first time mutations in MCM3AP and POMP in an immunodeficiency patient. These mutations lead to cooperative effects on DNA recombination and damage signaling. Digenic/polygenic mutations may constitute a novel genetic basis in immunodeficiency patients with DNA repair defects.     

Influence of nucleotide excision repair on N-hydroxy-2-acetylaminofluorene-induced mutagenesis studied in λlacZ-transgenic mice

To study the influence of nucleotide excision repair (NER) on mutagenesis in vivo, ERCC1+/−, XP−/−, and wild-type (ERCC1+/+ and XP+/+, respectively) λlacZ-transgenic mice were treated i.p. with N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and lacZ mutant frequencies were determined in liver. No significant effect of the treatment on the mutant frequency in wild-type or ERCC1-heterozygous mice was observed. The liver mutant frequency appeared to be significantly increased in treated XP−/− mice only. To distinguish N-OH-AAF-induced from spontaneous mutations, lacZ mutants derived from treated XP−/− mice were subjected to DNA-sequence analysis and the spectrum obtained was compared to that established for lacZ mutants in liver of PBS-treated λlacZ-transgenic mice of the parentstrain 40.6. The N-OH-AAF-induced mutation spectrum appeared to be significantly different from the spontaneous mutation spectrum: the former consisted of mainly (19/22) single bp substitutionstargeted at G, of which the majority (12/19) were G:C → T:A transversions, suggesting that N-(deoxyguanosin-8-yl)-2-aminofluorene [dG-C8-A], the major DNA adduct in N-OH-AAF-treated mice, is the premutagenic lesion. After analysis of 21 spontaneous mutants, only ten single bp substitutions targeted at G were found, of which five were G:C → T:A transversions. This study with XP−/− λlacZ-transgenic mice shows that one of the components of NER, that is, the XPA protein, suppresses mutagenesis in vivo. Environ. Mol. Mutagen. 31:41–47, 1998. © 1998 Wiley-Liss, Inc.     

Xeroderma pigmentosum: from symptoms and genetics to gene-based skin therapy

Xeroderma pigmentosum (XP) is a rare, recessively inherited genodermatosis prone to ultraviolet (UV)-induced skin neoplasms from keratinocyte origin, i.e. basal and squamous cell carcinoma. Cells from classic XP patients fail to properly eliminate UV-induced DNA lesions by the nucleotide excision repair (NER) mechanism. A variant form of XP, called XP-V suffers from faulty translesion synthesis. We review here recent data on XP gene products whose alterations affect NER and result in one of the 7 complementation groups of XP. Encouraging results of retrovirus-based genetic correction of XP keratinocytes are summarized and support realistic prospects of gene therapy for the XP-C complementation group.