RESUMO
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.
Assuntos
Síndrome de Cockayne/genética , Regulação da Expressão Gênica , Mutação , Síndromes de Tricotiodistrofia/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/genética , Adolescente , Adulto , Criança , Pré-Escolar , Reparo do DNA , Feminino , Fibroblastos/metabolismo , Estudos de Associação Genética , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Adulto JovemRESUMO
Patients belonging to xeroderma pigmentosum (XP) complementation group C comprise one-third of all XP patients. Only four major reports compiled larger groups of XP-C patients from southern Europe (12 pts), North America (16 pts) and Africa (14 and 56 pts) as well as their genetic background (46 XPC mutations). We identified 16 XP-C patients from Germany. Interestingly, only five patients exhibited severe sun sensitivity. The mean age of XP diagnosis was 9.4 years, and the median age of the first skin cancer was 7 years. Neurological symptoms were absent in all but two patients. Primary fibroblasts from all 16 patients showed reduced post-UV cell survival (mean: 50% vs 93% in normal cells) and reduced reactivation of an UV-treated luciferase reporter gene (mean: 6.4% vs 30.7% in normal cells). XPC mRNA expression was also greatly reduced compared with normal cells (mean: 14.3%; range 8.3-25.7%) except in XP47MA (274.1%). All patients carried homozygous XPC mutations. Four mutations have been described previously: c.1747_1748delTG (found in 4/16), c.567 C>T (4/16), c.1839 C>T (1/16) and a complex insertion/deletion mutation in exon 9 (1/16). The novel frameshift mutations c.446_447delAG (2/16), c.1525insA (1/16) and c.2271delC (1/16) lead to truncated XPC proteins as does the novel nonsense mutation c.843C>T (1/16). XP47MA carries an interesting mutation (c.2538_2540delATC; p.Ile812del) resulting in an in-frame single amino acid deletion. This mutation results in a classical XP phenotype, a non-functional XPC protein, but elevated XPC mRNA expression. Our study indicates that extrinsic factors may contribute to XP-C symptom severity due to nonsense-mediated message decay.
Assuntos
Análise Mutacional de DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Fenótipo , Neoplasias Cutâneas/genética , Xeroderma Pigmentoso/genética , Adolescente , Adulto , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Criança , Pré-Escolar , Códon sem Sentido , Proteínas de Ligação a DNA/metabolismo , Meio Ambiente , Feminino , Fibroblastos/efeitos da radiação , Mutação da Fase de Leitura , Alemanha , Humanos , Mutação INDEL , Masculino , Doenças do Sistema Nervoso/complicações , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/etiologia , Queimadura Solar/etiologia , Queimadura Solar/prevenção & controle , Luz Solar/efeitos adversos , Xeroderma Pigmentoso/complicações , Adulto JovemRESUMO
Unlike other immunosuppressive drugs including everolimus, cyclosporin A causes a dramatic increase of UV-induced skin cancer, a feature that is reminiscent of xeroderma pigmentosum (XP), where defective nucleotide excision repair (NER) of UV-induced DNA damage results in cutaneous carcinogenesis. The molecular basis of the clinically important differential activities of cyclosporin A and everolimus is still unclear. We measured post-UV cell survival of cyclosporin A- and everolimus-treated human fibroblasts and lymphoblasts using a cell proliferation assay (MTT). The cellular NER capacity was assessed by host cell reactivation. Using an ELISA and specific antibodies, cyclobutane pyrimidine and pyrimidine-6,4-pyrimidone photoproduct removal from the cellular genome was measured. The effect of calcineurin on NER was investigated using a calcineurin A expression vector and specific RNAi. Cyclosporin A led to a dose dependent decrease in post-UV cell survival, inhibited NER and blocked photoproduct removal. In contrast, none of these effects where seen in everolimus-treated cells. Overexpression of calcineurin A resulted in increased NER and complemented the Cyclosporin A-induced reduction of NER. Downregulation of calcineurin using RNAi inhibited NER comparable to cyclosporin A-treatment. We conclude that cyclosporin A, but not everolimus, leads to an increased skin cancer risk via a calcineurin signalling-dependent impairment of NER.
Assuntos
Calcineurina/metabolismo , Ciclosporina/farmacologia , Reparo do DNA/efeitos dos fármacos , Terapia de Imunossupressão/efeitos adversos , Neoplasias/etiologia , Sirolimo/análogos & derivados , Calcineurina/genética , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ciclosporina/efeitos adversos , Everolimo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Humanos , Imunossupressores/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/efeitos da radiação , Fosforilação/efeitos dos fármacos , Dímeros de Pirimidina/metabolismo , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Sirolimo/farmacologia , Transfecção , Raios UltravioletaRESUMO
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.
Assuntos
Ciclosporina/efeitos adversos , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/antagonistas & inibidores , Endonucleases/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Proteína de Xeroderma Pigmentoso Grupo A/antagonistas & inibidores , Calcineurina/genética , Inibidores de Calcineurina , Linhagem Celular , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endonucleases/deficiência , Endonucleases/genética , Endonucleases/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Imunossupressores/efeitos adversos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/etiologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplantes/efeitos adversos , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismoRESUMO
We examined the clinical, molecular and genetic features of a 16-year-old boy (XP2GO) with xeroderma pigmentosum (XP) and progressive neurological symptoms. The parents are not consanguineous. Increased sun sensitivity led to the diagnosis of XP at 2 years of age and a strict UV protection scheme was implemented. Besides recurrent conjunctivitis and bilateral pterygium, only mild freckling was present on his lips. He shows absent deep tendon reflexes, progressive sensorineural deafness and progressive mental retardation. MRI shows diffuse frontal cerebral atrophy and dilated ventricles. Symptoms of trichothiodystrophy (brittle hair with a tiger-tail banding pattern on polarized microscopy) or Cockayne syndrome (cachectic dwarfism, cataracts, pigmentary retinopathy and spasticity) were absent. XP2GO fibroblasts showed reduced post-UV cell survival (D(37) = 3.8 J/m(2)), reduced nucleotide excision repair, reduced expression of XPD mRNA and an undetectable level of XPD protein. Mutational analysis of the XPD gene in XP2GO revealed two different mutations: a common p.Arg683Trp amino acid change (c.2047C>T) known to be associated with XP and a novel frameshift mutation c.2009delG (p.Gly670Alafs*39). The latter mutation potentially behaves as a null allele. While not preventing neurological degeneration, early diagnosis and rigorous sun protection can result in minimal skin disease without cancer in XP patients.
Assuntos
Deleção de Genes , Pele/efeitos da radiação , Luz Solar/efeitos adversos , Proteína Grupo D do Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologia , Adolescente , Análise Mutacional de DNA , Reparo do DNA , Fibroblastos/metabolismo , Humanos , Masculino , Mutação , Fenótipo , Neoplasias Cutâneas/prevenção & controle , Raios UltravioletaRESUMO
Nucleotide excision repair (NER) is the most versatile and best studied DNA repair system in humans. NER can repair a variety of bulky DNA damages including UV-light induced DNA photoproducts. NER consists of a multistep process in which the DNA lesion is recognized and demarcated by DNA unwinding. Then, an approximately 28 bp DNA damage containing oligonucleotide is excised followed by gap filling using the undamaged DNA strand as a template. The consequences of defective NER are demonstrated by three rare autosomal-recessive NER-defective syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). XP patients show severe sun sensitivity, freckling in sun exposed skin, and develop skin cancers already during childhood. CS patients exhibit sun sensitivity, severe neurologic abnormalities, and cachectic dwarfism. Clinical symptoms of TTD patients include sun sensitivity, freckling in sun exposed skin areas, and brittle sulfur-deficient hair. In contrast to XP patients, CS and TTD patients are not skin cancer prone. Studying these syndromes can increase the knowledge of skin cancer development including cutaneous melanoma as well as basal and squamous cell carcinoma in general that may lead to new preventional and therapeutic anticancer strategies in the normal population.
Assuntos
Síndrome de Cockayne/genética , Reparo do DNA , Doenças do Cabelo/genética , Neoplasias/genética , Xeroderma Pigmentoso/genética , Síndrome de Cockayne/complicações , Síndrome de Cockayne/patologia , Doenças do Cabelo/complicações , Doenças do Cabelo/patologia , Humanos , Neoplasias/etiologia , Síndrome , Xeroderma Pigmentoso/complicações , Xeroderma Pigmentoso/patologiaRESUMO
Xeroderma pigmentosum (XP) patients exhibit a 1000-fold increased risk for developing skin cancers including malignant melanoma. We investigated the role of three variant alleles of the DNA repair gene XPC and one variant allele of the XPG gene in a hospital-based case-control study of 294 Caucasian patients from Germany with malignant melanoma and 375 healthy control individuals from the same area matched by sex. The polymorphisms G1580A (XPC exon 8; Arg492His), T1601C (XPC exon 8; Val499Ala), G2166A (XPC exon 10; Arg687Arg), and C3507G (XPG exon 15; Asp1104His) were not in linkage disequilibrium. The allele frequencies (cases: controls) were for 1580A 6.29%: 5.63%, for 1601C 79.08%: 78.28%, for 2166A 26.19%: 28.13%, and for 3507G 79.86%: 78.61%. We found no association of the homozygous 1580A, 1601C, 2166A, and 3507G genotypes with increased risks of melanoma: OR 1.254 (95% CI: 0.486-3.217), OR 1.108 (95% CI: 0.629-1.960), OR 0.817 (95% CI: 0.490-1.358), and OR 1.168 (95% CI: 0.670-2.044), respectively. Exploratory analyses of subgroups of melanoma patients compared to all controls indicated no association of these genotypes with increased risks for development of multiple primary melanomas (n = 28), a negative family history for melanoma (n = 277), melanomas in individuals with a low number of nevi (n = 273), melanomas in individuals older than 55 years (n = 142), and melanomas thicker than 1 mm (n = 126).
Assuntos
Proteínas de Ligação a DNA/genética , Endonucleases/genética , Éxons/genética , Melanoma/etiologia , Proteínas Nucleares/genética , Polimorfismo Genético/genética , Neoplasias Cutâneas/etiologia , Fatores de Transcrição/genética , Xeroderma Pigmentoso/genética , Substituição de Aminoácidos/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Xeroderma Pigmentoso/complicaçõesRESUMO
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.
Assuntos
Síndrome de Cockayne/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Mutação de Sentido Incorreto/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Xeroderma Pigmentoso/genética , Sequência de Aminoácidos , Linhagem Celular , Síndrome de Cockayne/patologia , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Endonucleases/análise , Endonucleases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Genótipo , Heterozigoto , Homozigoto , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Fenótipo , Fatores de Transcrição/análise , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta , Xeroderma Pigmentoso/patologiaRESUMO
OBJECTIVES: The efficacy of temozolomide (TMZ) or dacarbazine (DTIC) in melanoma treatment depends on low O-6-methylguanine-DNA-methyltransferase (MGMT) repair and on high mismatch repair. The aim of this study was to identify individual host markers for hematologic side effects and the treatment efficacy of TMZ or DTIC in melanoma treatment. METHODS: Fifty-one Caucasian patients with metastasized melanoma were recruited. In each patient, the mRNA expression of MGMT and two essential mismatch repair genes, MLH1 and MSH2, was measured in peripheral blood. The coding gene regions, including splice sites, were sequenced to identify genetic variants, and the promoter methylation status of the genes was determined. RESULTS: Both constitutively low and high mRNA expression of MGMT, MLH1, and MSH2 were significantly associated with reduced hematologic side effects (P = 0.008-0.020), but did not correlate with treatment efficacy. We identified five variants in the MGMT gene, 13 variants in MLH1, and seven variants in MSH2, including five novel genetic variants in MLH1. Variations of the hosts' gene expression of MGMT, MLH1, and MSH2 did not result from promoter methylation. Of note, one variant in MSH2 (rs2303428) was associated with increased hematologic side effects and showed a tendency for better treatment response. CONCLUSION: Our results indicate that either low or high host expression of MGMT, MLH1, and MSH2 may serve as a marker for reduced hematologic side effects of TMZ or DTIC, but not for treatment efficacy in melanoma. The genetic variant rs2303428 (MSH2) might serve as a predictive marker for hematologic side effects and treatment response.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Reparo do DNA/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/genética , População Branca/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Melanoma/enzimologia , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/genética , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Temozolomida , Células Tumorais CultivadasRESUMO
Individuals with the rare DNA repair deficiency syndrome xeroderma pigmentosum (XP) are sensitive to the sun and exhibit a 1000-fold increased risk for developing skin cancers, including cutaneous melanoma. Inherited polymorphisms of XP genes may contribute to subtle variations in DNA repair capacity and genetic susceptibility to melanoma. We investigated the role of three polymorphic alleles of the DNA repair gene XPC in a hospital-based case-control study of 294 Caucasian patients from Germany who had cutaneous melanoma and 375 healthy cancer-free sex-matched Caucasian control subjects from the same area. We confirmed that the XPC intron 9 PAT+, intron 11 -6A, and the exon 15 2920C polymorphisms are in a linkage disequilibrium. Only 1.6% of the 669 donors genotyped were discordant for these three polymorphisms. The allele frequencies (cases: controls) were for intron 9 PAT+ 41.7%:36.9%, for intron 11 -6A 41.8%:37.0% and for exon 15 2920C 41.3%:37.3%. Using multivariate logistic regression analyses to control for age, skin type and number of nevi, the three polymorphisms were significantly associated with increased risks of melanoma: OR 1.87 (95% CI: 1.10-3.19; P = 0.022), OR 1.83 (95% CI: 1.07-3.11; P = 0.026), and OR 1.82 (95% CI: 1.07-3.08; P = 0.026), respectively. Exploratory multivariate analyses of distinct subgroups revealed that these polymorphisms were associated with increased risks for the development of multiple primary melanomas (n = 28). The results of our case-control study support the hypothesis that the intron 9 PAT+, intron 11 -6A and exon 15 2920C haplotype may contribute to the risk of developing cutaneous melanoma by increasing the rate of an alternatively spliced XPC mRNA isoform that skips exon 12 and leads to reduced DNA repair. Our results should be validated in independent samples in order to guard against false positive findings.