Nucleotide Excision Repair Genes XPA Research Articles

CRISPR-Cas9 Knockout Screens Identify DNA Damage Response Pathways and BTK as Essential for Cisplatin Response in Diffuse Large B-Cell Lymphoma

The recurrence of diffuse large B-cell lymphoma (DLBCL) has been observed in 40% of cases. The standard of care for refractory/relapsed DLBCL (RR-DLBCL) is platinum-based treatment prior to autologous stem cell transplantation; however, the prognosis for RR-DLBCL patients remains poor. Thus, to identify genes affecting the cisplatin response in DLBCL, cisplatin-based whole-genome CRISPR-Cas9 knockout screens were performed in this study. We discovered DNA damage response (DDR) pathways as enriched among identified sensitizing CRISPR-mediated gene knockouts. In line, the knockout of the nucleotide excision repair genes XPA and ERCC6 sensitized DLBCL cells to platinum drugs irrespective of proliferation rate, thus documenting DDR as essential for cisplatin sensitivity in DLBCL. Functional analysis revealed that the loss of XPA and ERCC6 increased DNA damage levels and altered cell cycle distribution. Interestingly, we also identified BTK, which is involved in B-cell receptor signaling, to affect cisplatin response. The knockout of BTK increased cisplatin sensitivity in DLBCL cells, and combinatory drug screens revealed a synergistic effect of the BTK inhibitor, ibrutinib, with platinum drugs at low concentrations. Applying local and external DLBCL cohorts, we addressed the clinical relevance of the genes identified in the CRISPR screens. BTK was among the most frequently mutated genes with a frequency of 3–5%, and XPA and ERCC6 were also mutated, albeit at lower frequencies. Furthermore, 27–54% of diagnostic DLBCL samples had mutations in pathways that can sensitize cells to cisplatin. In conclusion, this study shows that XPA and ERCC6, in addition to BTK, are essential for the response to platinum-based drugs in DLBCL.

Time-Dependent Induction of the Nucleotide Excision Repair Gene XPA and RAD51 in Homologous Recombination in Human Lymphocytes Exposed to Low Doses of Ionizing Radiation

Introduction: The aim of the present study was to understand the effect of low-doses of ionizing radiation (LDIR) on repair genes expression in blood samples that were taken from healthy donors. The next purpose was to examine the time-effect on the modified gene expression caused by low-doses of ionizing radiation. Material and method: The RNA of peripheral blood lymphocytes (PBLs) taken from four healthy donors was isolated at different time points after exposure included 4, 24, 48, 72, and 168 hours and then cDNA was synthesized. Modification of XPA and RAD51 expression levels due to LDIR (2, 5, 10 cGy) were evaluated by relative quantitative reverse transcription-polymerase chain reaction.Results: Significant up-regulation of both repair genes was observed at the 4 and 168 h following to 10 cGy. Also, this dose could increase expression levels of RAD51 at 48 and 72 h after radiation. For lower doses at 5 cGy, only XPA levels were significantly up-regulated after 168 h. A significant regression was found between the XPA levels and the dose, at 168 h after irradiation to PBLs that can represent a new potential biomarker for biological dosimetry purposes.Conclusion: The results of this study could support the hypothetical role of the different DNA repair pathways in response to LDIR. This led us to propose a molecular biodosimetry method for ionizing radiation in the range of LDIR.

Nucleotide Excision Repair Factor XPC Ameliorates Prognosis by Increasing the Susceptibility of Human Colorectal Cancer to Chemotherapy and Ionizing Radiation.

Nucleotide excision repair (NER) is a DNA damage repair mechanism in mammals, but the relationship between NER and human colorectal cancer (HRC) progression has not been clarified yet. In this study, the expression of the NER genes XPA, XPC, XPF, XPG, ERCC1, and XPD was measured in normal and cancerous human colorectal tissue. Among them, only the XPC gene expression was significantly increased in colorectal cancer tissue. To establish the role of XPC in colorectal cancer, small interference RNA (siRNA) targeting XPC was used to knockdown the expression of XPC in HRC cell lines. In addition, an expression vector plasmid containing the XPC cDNA was constructed and stably transfected into HRC cell lines to overexpress the XPC gene. Interestingly, MTT and apoptosis assay demonstrated that XPC gene overexpression significantly increased the susceptibility of HRC cell lines to cisplatin and X-ray radiation. In order to study the relationship between XPC expression and the progression of HRC, XPC expression was measured in 167 patients with colorectal cancer. The results showed that patients with high XPC expression had longer survival time. Cox regression analysis showed that high XPC expression might be a potential predictive factor for colorectal cancer. In conclusion, XPC plays a key role in the susceptibility of colorectal cancer to chemotherapy and ionizing radiation and is associated with a good patients' prognosis.

Xeroderma pigmentosum: low prevalence of germline XPA mutations in a Brazilian XP population.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder characterized by DNA repair defects that cause photophobia, sunlight-induced cancers, and neurodegeneration. Prevalence of germline mutations in the nucleotide excision repair gene XPA vary significantly in different populations. No Brazilian patients have been reported to carry a germline mutation in this gene. In this study, the germline mutational status of XPA was determined in Brazilian patients exhibiting major clinical features of XP syndrome. The study was conducted on 27 unrelated patients from select Brazilian families. A biallelic inactivating transition mutation c.619C>T (p.Arg207Ter) was identified in only one patient with a history of neurological impairment and mild skin abnormalities. These findings suggest that XP syndrome is rarely associated with inherited disease-causing XPA mutations in the Brazilian population. Additionally, this report demonstrates the effectiveness of genotype-phenotype correlation as a valuable tool to guide direct genetic screening.

Association of the genetic variants in the nucleotide excision repair genes XPA and XPC with cisplatin-induced hearing loss in patients with osteosarcoma.

10077 Background: Cisplatin is a widely used and effective chemotherapeutic agent in the treatment of osteosarcoma. However, cisplatin-induced ototoxicity is a serious problem, affecting more than 60% of patients and compromising language and cognitive development. Unfortunately, individuals at risk to develop ototoxicity cannot be identified upfront. Genetic variants in genes involved in the metabolism of cisplatin may predispose to cisplatin-induced hearing loss and help to identify patients at risk. Methods: In a candidate gene pathway approach, we selected 224 SNPs in 30 candidate genes related to Platinum-DNA repair pathways and genotyped for a discovery group of 105 patients with osteosarcoma for these variants. Cisplatin-induced ototoxicity (n = 47), defined as the development of grade 2–4 hearing impairment using Common Terminology Criteria for Adverse Events (CTCAE version 3), showing a hearing loss of >25 dB at frequencies of 4–8 kHz, was associated with genetic variation. A replication study was performed in a independent cohort of 51 patients with osteosarcoma. Genotyping was performed using the Illumina GoldenGate assay. Association analysis and meta-analysis were performed using the whole genome association analysis toolset PLINK. Results: In the discovery cohort a total of 13 SNPs were significantly (p value < 0.05) associated with ototoxicity. Upon meta-analysis, addition of the replication set resulted in lower p-values for 2 SNPs. The two SNPs showing a strong association with hearing loss in patients with osteosarcoma were rs2805835 in the gene XPA (p-value 0.01, OR=2.7 (95%CI: 1.20-6.15) and rs2227999 in XPC (p-value 0.02; OR=3.2 (95% CI:1.19-8.80). Conclusions: The Nucleotide Excision Repair (NER) genes XPA and XPC form an important molecular mechanism by which cisplatin DNA adducts can be repaired and it has recently been shown that these genes have a high expression in the cochlea. Our data suggest that genetic variants in these genes, may contribute to cisplatin ototoxicity. This study should be viewed as the first step in the development of genetic markers to predict cisplatin-induced ototoxicity in individual patients.

Modulation of nucleotide excision repair in human lymphocytes by genetic and dietary factors

Gene-environment interactions determine inter-individual variations in nucleotide excision repair (NER) capacity. Oxidative stress was previously found to inhibit NER, thus supplementation with dietary antioxidants could prevent this inhibition, especially in genetically susceptible subjects. To study the effects of genetic polymorphisms in NER-related genes and dietary intake of antioxidants on an individual's NER capacity, lymphocytes of 168 subjects were isolated before and after a 4-week blueberry and apple juice intervention. Twelve genetic polymorphisms in NER genes XPA, XPC, ERCC1, ERCC2, ERCC5, ERCC6 and RAD23B were assessed by multiplex PCR with single base extension. Based on specific genotype combinations, a subset of individuals (n 36) was selected for phenotypical assessment of NER capacity, which was significantly affected by the total sum of low-activity alleles (P = 0.027). The single polymorphism XPA G23A was the strongest predictor of NER capacity (P = 0.002); carriers of low-activity alleles AA had about three times lower NER capacity than XPA GG carriers. NER capacity assessed before and after intervention correlated significantly (R(2) 0.69; P < 0.001), indicating that inter-individual differences in NER capacity are maintained over 4 weeks. Although the intervention increased plasma trolox equivalent antioxidant capacity from 791 (SE 6.61) to 805 (SE 7.90) microm (P = 0.032), on average it did not affect NER capacity. Nonetheless, carriers of twelve or more low-activity alleles seemed to benefit from the intervention (P = 0.013). Among these, carriers of the variant allele for RAD23B Ala249Val showed improved NER capacity upon intervention (P = 0.020). In conclusion, improved NER capacity upon dietary intervention was detected in individuals carrying multiple low-activity alleles. The XPA G23A polymorphism might be a predictor for NER capacity.

Single Nucleotide Polymorphisms in Nucleotide Excision Repair Genes XPA, XPD, XPG and ERCC1 in Advanced Colorectal Cancer Patients Treated with First-Line Oxaliplatin/Fluoropyrimidine

Background/Aims: Oxaliplatin damages the DNA, leading to apoptosis. XPA, XPD, ERCC1 and XPG genes are involved in DNA repair, and single nucleotide polymorphisms (SNPs) in these genes can influence the efficacy of oxaliplatin. We examined SNPs in these genes and correlated the results with time to progression (TTP), overall survival and response to oxaliplatin in 42 advanced colorectal cancer patients (CRC) treated with first-line oxaliplatin/fluoropyrimidine. Methods: DNA was obtained from peripheral blood cells, and the allelic discrimination assay was used to analyze the XPA 5′UTR T/C, XPD Lys751Gln, ERCC1 Lys259Thr and XPG, C/T. Results: Patients with XPG C/C genotype had a longer survival (p = 0.001) and TTP (p = 0.009) than patients with XPG C/T or T/T genotypes, and patients with both XPG C/C and XPA T/C or C/C genotypes had a longer survival (p = 0.0001) and TTP (p = 0.0001) than patients with other genotypes. XPG (CC) combined with XPA (TC/CC) genotypes showed an independent role for TTP (relative risk, RR = 6.38; p = 0.0001) and survival (RR = 34; p = 0.0005). Conclusion: Polymorphism in XPG combined with XPA may be an important prognosticator of clinical outcome following oxaliplatin/ fluoropyrimidine chemotherapy. Further studies in larger patient cohorts are warranted to confirm their role in CRC.

Nucleotide excision repair and the degradation of RNA pol II by the Caenorhabditis elegans XPA and Rsp5 orthologues, RAD-3 and WWP-1

The Caenorhabditis elegans rad-3 gene was identified in a genetic screen for radiation sensitive (rad) mutants. Here, we report that the UV sensitivity of rad-3 mutants is caused by a nonsense mutation in the C. elegans orthologue of the human nucleotide excision repair gene XPA. We have used the xpa-1/rad-3 mutant to examine how a defect in nucleotide excision repair (NER) perturbs development. We find that C. elegans carrying a mutation in xpa-1/rad-3 are hypersensitive and hypermutable in response to UV irradiation, but do not display hypersensitivity to oxidative stress or show obvious developmental abnormalities in the absence of UV exposure. Consistent with these observations, non-irradiated xpa-1 mutants have a similar lifespan as wild type. We further show that UV irradiated xpa-1 mutants undergo a stage-dependent decline in growth and survival, which is associated with a loss in transcriptional competence. Surprisingly, transcriptionally quiescent dauer stage larvae are able to survive a dose of UV irradiation, which is otherwise lethal to early stage larvae. We show that the loss of transcriptional competence in UV irradiated xpa-1 mutants is associated with the degradation of the large RNA polymerase II (RNA pol II) subunit, AMA-1, and have identified WWP-1 as the putative E3 ubiquitin ligase mediating this process. The absence of wwp-1 by itself does not cause sensitivity to UV irradiation, but it acts synergistically with a mutation in xpa-1 to enhance UV hypersensitivity.

Polymorphisms in Nucleotide Excision Repair Genes, Arsenic Exposure, and Non-Melanoma Skin Cancer in New Hampshire

BackgroundArsenic exposure may alter the efficiency of DNA repair. UV damage is specifically repaired by nucleotide excision repair (NER), and common genetic variants in NER may increase risk for non-melanoma skin cancer (NMSC).ObjectiveWe tested whether polymorphisms in the NER genes XPA (A23G) and XPD (Asp312Asn and Lys751Gln) modify the association between arsenic and NMSC.MethodsIncident cases of basal and squamous cell carcinoma (BCC and SCC, respectively) were identified through a network of dermatologists and pathology laboratories across New Hampshire. Population-based controls were frequency matched to cases on age and sex. Arsenic exposure was assessed in toenail clippings. The analysis included 880 cases of BCC, 666 cases of SCC, and 780 controls.ResultsThere was an increased BCC risk associated with high arsenic exposure among those homozygous variant for XPA [odds ratio (OR) = 1.8; 95% confidence interval (CI), 0.9–3.7]. For XPD, having variation at both loci (312Asn and 751Gln) occurred less frequently among BCC and SCC cases compared with controls (OR = 0.8; 95% CI, 0.6–1.0) for both case groups. In the stratum of subjects who have variant for both XPD polymorphisms, there was a 2-fold increased risk of SCC associated with elevated arsenic (OR = 2.2; 95% CI, 1.0–5.0). The test for interaction between XPD and arsenic in SCC was of borderline significance (p < 0.07, 3 degrees of freedom).ConclusionsOur findings indicate a reduced NMSC risk in relation to XPD Asp312Asn and Lys751Gln variants. Further, these data support the hypothesis that NER polymorphisms may modify the association between NMSC and arsenic.

Xeroderma pigmentosum group A gene action as a protection factor against 4-nitroquinoline 1-oxide-induced tongue carcinogenesis.

To test the hypothesis that nucleotide excision repair (NER) plays a protective role in chemical carcinogenesis in internal organs, xeroderma pigmentosum group A gene-deficient (XPA(-/-)) mice, heterozygous (XPA(+/-)) and wild-type (XPA(+/+)) mice were orally administered 0.001% 4-nitroquinoline 1-oxide (4NQO) in their drinking water and compared. After 50 weeks of 4NQO exposure, tongue squamous cell carcinomas (SCCs) occurred in XPA(-/-) mice only, no tumors being observed in XPA(+/-) and XPA(+/+) animals. Of the XPA(-/-) mice 86% had tumors and 100% demonstrated multiple foci of dysplastic epithelium in the tongue. Accumulation of p53 protein was immunohistochemically detected in 56% of the SCCs. Mutational analysis of the p53 gene (exons 4-10) in carcinoma DNA revealed missense mutations in exons 5 and 9 in four of 20 samples. Our results clearly demonstrate that the NER gene XPA acts as a defensive factor against 4NQO-induced tongue carcinogenesis in vivo.

Mice deficient in the nucleotide excision repair gene XPA have elevated sensitivity to benzo[a]pyrene induction of lung tumors

Mice deficient in the nucleotide excision repair gene XPA have elevated sensitivity to benzo[a]pyrene induction of lung tumors

Mice deficient in the nucleotide excision repair gene XPA have elevated sensitivity to benzo[ a ]pyrene induction of lung tumors

This study is focused on chemical induction of lung tumors in xeroderma pigmentosum group A gene (XPA)-deficient mice to clarify the role of nucleotide excision repair (NER) in internal organs. Six-week-old female XPA-/-, XPA(+/-) and XPA(+/+) mice were instilled intratracheally with benzo[a] pyrene (B[a]P). A total of 68 surviving XPA mice treated with B[a]P were examined at month 16. The pulmonary adenoma incidence in XPA(-/-) mice was significantly higher than that in XPA(+/+) mice (71 versus 35%). Similarly, tumor multiplicity was elevated and, in addition, only XPA(-/-) mice had lung carcinomas. These results provide the first evidence that a deficiency in the NER gene XPA leads to enhanced tumorigenesis in the lung after exposure to B[a]P.

Accelerated accumulation of somatic mutations in mice deficient in the nucleotide excision repair gene XPA.

Inheritable mutations in nucleotide excision repair (NER) genes cause cancer-prone human disorders, such as xeroderma pigmentosum, which are also characterized by symptoms of accelerated ageing. To study the impact of NER deficiency on mutation accumulation in vivo, mutant frequencies have been determined in liver and brain of 2-16 month old NER deficient XPA-/-, lacZ hybrid mice. While mutant frequencies in liver of 2-month old XPA-/-, lacZ mice were comparable to XPA+/-, lacZ and the lacZ parental strain animals, by 4 months of age mutant frequencies in the XPA-deficient mice were significantly increased by a factor of two and increased further until the age of 16 months. In brain, mutant frequencies were not found to increase with age. These results show that a deficiency in the NER gene XPA causes an accelerated accumulation of somatic mutations in liver but not in brain. This is in keeping with a higher incidence of spontaneous liver tumors reported earlier for XPA-/- mice after about 15 months of age.

XPA-deficiency in hairless mice causes a shift in skin tumor types and mutational target genes after exposure to low doses of U.V.B.

Xeroderma pigmentosum (XP) patients with a defect in the nucleotide excision repair gene XPA, develop tumors with a high frequency on sun-exposed areas of the skin. Here we describe that hairless XPA-deficient mice also develop skin tumors with a short latency time and a 100% prevalence after daily exposure to low doses of U.V.B. Surprisingly and in contrast to U.V.B.-exposed repair proficient hairless mice who mainly develop squamous cell carcinomas, the XPA-deficient mice developed papillomas with a high frequency (31%) at a U.V. dose of 32 J/m2 daily. At the highest daily dose of 80 J/m2 mainly squamous cell carcinomas (56%) and only 10% of papillomas were found in XPA-deficient hairless mice. p53 gene mutations were examined in exons 5, 7 and 8 and were detected in only 3 out of 37 of these skin tumors, whereas in tumors of control U.V.B.-irradiated wild type littermates this frequency was higher (45%) and more in line with our previous data. Strikingly, a high incidence of activating ras gene mutations were observed in U.V.B.-induced papillomas (in 11 out of 14 tumors analysed). In only two out of 14 squamous cell carcinomas we found similar ras gene mutations. The observed shift from squamous cell carcinomas in wild type hairless mice to papillomas in XPA-deficient hairless mice, and a corresponding shift in mutated cancer genes in these tumors, provide new clues on the pathogenesis of chemically- versus U.V.B.-induced skin carcinogenesis.

Enhanced XPA mRNA levels in cisplatin-resistant human ovarian cancer are not associated with XPA mutations or gene amplification.

Enhanced expression of the nucleotide excision repair gene XPA is associated with resistance to cisplatin treatment in human ovarian cancer. Understanding the cause of enhanced XPA expression will provide new molecular targets for therapy directed at overcoming chemoresistance. Enhanced gene expression in cancer cells is often caused by mutations or gene amplification. Molecular analyses of the XPA genes in human ovarian cancers indicate that gene mutation and amplification are not the cause of enhanced XPA mRNA levels in ovarian cancers overexpressing XPA. Altered nucleotide excision repair (NER) gene regulation in chemoresistant tumors is discussed.

Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA.

Xeroderma pigmentosum patients with a defect in the nucleotide-excision repair gene XPA are characterized by, for example, a > 1,000-fold higher risk of developing sunlight-induced skin cancer. Nucleotide-excision repair (NER) is involved in the removal of a wide spectrum of DNA lesions. The XPA protein functions in a pre-incision step, the recognition of DNA damage. To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells. The XPA-/-mice appear normal, at least until the age of 13 months. XPA-/-mice are highly susceptible to ultraviolet (UV)-B-induced skin and eye tumours and to 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumours. We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum.