Cockayne syndrome complementation group B associated with xeroderma pigmentosum phenotype

Two siblings have been reported whose clinical manifestations (cutaneous photosensitivity and central nervous system dysfunction) are strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum (XP), a severe form of XP. Fibroblasts from the siblings showed UV sensitivity, a failure of recovery of RNA synthesis (RRS) after UV irradiation, and a normal level of unscheduled DNA synthesis (UDS), which were, unexpectedly, the biochemical characteristics usually associated with Cockayne syndrome (CS). However, no complementation group assignment in these cells has yet been performed. We here report that these patients can be assigned to CS complementation group B (CSB) by cell fusion complementation analysis. To our knowledge, these are the first patients with defects in the CSB gene to be associated with an XP phenotype. The results imply that the gene product from the CSB gene must interact with the gene products involved in excision repair and associated with XP.     

Xeroderma pigmentosum complementation group G associated with Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two rare inherited disorders with a clinical and cellular hypersensitivity to the UV component of the sunlight spectrum. Although the two traits are generally considered as clinically and genetically distinct entities, on the biochemical level a defect in the nucleotide excision-repair (NER) pathway is involved in both. Classical CS patients are primarily deficient in the preferential repair of DNA damage in actively transcribed genes, whereas in most XP patients the genetic defect affects both "preferential" and "overall" NER modalities. Here we report a genetic study of two unrelated, severely affected patients with the clinical characteristics of CS but with a biochemical defect typical of XP. By complementation analysis, using somatic cell fusion and nuclear microinjection of cloned repair genes, we assign these two patients to XP complementation group G, which previously was not associated with CS. This observation extends the earlier identification of two patients with a rare combined XP/CS phenotype within XP complementation groups B and D, respectively. It indicates that some mutations in at least three of the seven genes known to be involved in XP also can result in a picture of partial or even full-blown CS. We conclude that the syndromes XP and CS are biochemically closely related and may be part of a broader clinical disease spectrum. We suggest, as a possible molecular mechanism underlying this relation, that the XPGC repair gene has an additional vital function, as shown for some other NER genes.     

DNA repair in Cockayne syndrome.

Cockayne syndrome (CS) is a rare recessive genetic disease characterized in part by premature ageing and photosensitive skin. Because of the latter characteristic, this syndrome was considered to be an example of a UV-sensitive DNA repair-defective human disorder. We demonstrated normal levels of UV-induced unscheduled DNA synthesis (UDS) in four unrelated CS patients that show hypersensitivity to both UV and Mitomycin C (MMC). At low UV exposure, CS DNA shows a dose-dependent decrease in size. By contrast, heterozygotes appear to have a threshold below which there is little change in size of single strand DNA. Immediately following UV or MMC treatment, CS DNA is deficient in high molecular weight species, but undergoes a normal transition to larger DNA during a chase interval in the presence or absence of caffeine. This suggests a defect in replication or excision repair and no defect in post-replication repair (PRR). Pulse studies performed in the presence of hydroxyurea (HU) also reveal a deficient production of large DNA, suggesting the defect is in repair. As these cells have normal UDS and normal PRR, the basis for their UV sensitivity must be distinct from that observed in xeroderma pigmentosum (XP).     

Xeroderma pigmentosum and the role of UV-induced DNA damage in skin cancer

Xeroderma pigmentosum (XP) is a rare, autosomal recessive disease that is characterized by the extreme sensitivity of the skin to sunlight. Compared to normal individuals, XP patients have a more than 1000-fold increased risk of developing cancer on sun-exposed areas of the skin. Genetic and molecular analyses have revealed that the repair of ultraviolet (UV)-induced DNA damage is impaired in XP patients owing to mutations in genes that form part of a DNA-repair pathway known as nucleotide excision repair (NER). Two other diseases, Cockayne syndrome (CS) and the photosensitive form of trichothiodystrophy (TTD), are linked to a defect in the NER pathway. Strikingly, although CS and TTD patients are UV-sensitive, they do not develop skin cancer. The recently developed animal models that mimic the human phenotypes of XP, CS and TTD will contribute to a better understanding of the etiology of these diseases and the role of UV-induced DNA damage in the development of skin cancer.     

Cerebro-Oculo-Facio-Skeletal Syndrome with a Nucleotide Excision–Repair Defect and a Mutated XPD Gene, with Prenatal Diagnosis in a Triplet Pregnancy

Cerebro-oculo-facio-skeletal (COFS) syndrome is a recessively inherited rapidly progressive neurologic disorder leading to brain atrophy, with calcifications, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low to normal birth weight, growth failure, brain dysmyelination with calcium deposits, cutaneous photosensitivity, pigmentary retinopathy and/or cataracts, and sensorineural hearing loss. Cultured CS cells are hypersensitive to UV radiation, because of impaired nucleotide-excision repair (NER) of UV-induced damage in actively transcribed DNA, whereas global genome NER is unaffected. The abnormalities in CS are caused by mutated CSA or CSB genes. Another class of patients with CS symptoms have mutations in the XPB, XPD, or XPG genes, which result in UV hypersensitivity as well as defective global NER; such patients may concurrently have clinical features of another NER syndrome, xeroderma pigmentosum (XP). Clinically observed similarities between COFS syndrome and CS have been followed by discoveries of cases of COFS syndrome that are associated with mutations in the XPG and CSB genes. Here we report the first involvement of the XPD gene in a new case of UV-sensitive COFS syndrome, with heterozygous substitutions-a R616W null mutation (previously seen in patients in XP complementation group D) and a unique D681N mutation-demonstrating that a third gene can be involved in COFS syndrome. We propose that COFS syndrome be included within the already known spectrum of NER disorders: XP, CS, and trichothiodystrophy. We predict that future patients with COFS syndrome will be found to have mutations in the CSA or XPB genes, and we document successful use of DNA repair for prenatal diagnosis in triplet and singleton pregnancies at risk for COFS syndrome. This result strongly underlines the need for screening of patients with COFS syndrome, for either UV sensitivity or DNA-repair abnormalities.     

Comparative studies of host-cell reactivation, cellular capacity and enhanced reactivation of herpes simplex virus in normal, xeroderma pigmentosum and Cockayne syndrome fibroblasts

Host-cell reactivation (HCR) of UV-irradiated herpes simplex virus type 2 (HSV-2), capacity of UV-irradiated cells to support HSV-2 plaque formation and UV-enhanced reactivation (UVER) of UV-irradiated HSV-2 were examined in fibroblasts from 4 patients with Cockayne syndrome (CS), 5 with xeroderma pigmentosum and 5 normals. All UV-survival curves for HSV-2 plaque formation showed 2 components. HCR was similar to normal for the XP variant strain and the 2 CS strains tested, but substantially reduced in the 4 excision-deficient XP strains. The capacity of UV-irradiated fibroblasts to support HSV-2 plaque formation was determined by UV-irradiating fibroblast monolayers with various doses of UV and 48 h later, infecting the monolayers with unirradiated HSV-2. The D37 values for the delayed-capacity curves so obtained were in the range 8.6-12.4 J/m2 for the normal strains, 2.8-3.2 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.3 and 1.5 for the XP excision-deficient strains tested. These results indicate that delayed capacity for HSV-2 plaque formation is a more sensitive assay than HCR in the detection of cellular DNA-repair deficiency for XP and CS. For the examination of UVER, fibroblasts were irradiated with various UV doses and subsequently infected with either unirradiated or UV-irradiated HSV and scored for plaque formation 2 days later. UVER expression was maximum when the delay between UV-irradiation of the cells and HSV infection was 48 h. The magnitude of UVER expression was also found to be dependent on the UV dose to the cells and increased with increasing UV dose to the virus. Using a UV dose to the virus resulting in a plaque survival of about 10(-2) on unirradiated cells, the the maximum UVER factor had a mean value of 1.3 for the normal strains following a dose of 15 J/m2 to the cells. Somewhat higher UVER values were found for all the patient strains tested and resulted from lower UV doses to the cells than for normal strains. Maximum UVER factors for the CS strains ranged from 2.2 to 3.3 at a dose of 5 J/m2 to the cells, for the XP excision-deficient strains; 2.1 to 2.6 at doses of 0.5 to 2.5 J/m2 to the cells and for the XP variant strain tested; 2.5 at UV dose of 10 J/m2 to the cells.     

Clinical and biochemical studies in three patients with severe early infantile Cockayne syndrome

Summary We present clinical and biochemical data from three patients with severe Cockayne syndrome (CS) of very early onset. Unlike in classic CS, signs became evident in the first weeks of life and led to unusually early death. Fibroblasts from two of the patients showed a complete defect of the repair of UV-induced thymine dimer lesions. They were unable to remove thymine dimer lesions from their DNA, had a severe reduction of the RNA synthesis rates after UV irradiation, and showed no reactivation of an UV-inactivated indicator gene and no DNA recondensation after UV irradiation. DNA repair investigated in these two fibroblast cell strains resembled that of xeroderma pigmentosum cells of complementation group A. In contrast, fibroblasts from the third patient showed the same in vitro repair characteristics as classic CS cells.     

Nucleotide excision repair and cancer

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, a ~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-rezessive 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.     

Studies on p53 and Bax protein expression in Cockayne syndrome cells after UV irradiation and interferon-beta treatment

Human interferon (HuIFN) has a protective effect against ultraviolet (UV)-induced killing of Cockayne syndrome (CS) and xeroderma pigmentosum (XP) cells. Irradiation with ultraviolet (UV) resulted in nuclear accumulation of p53 in normal human fibroblast cells, and this accumulation was suppressed by treatment with HuIFN-beta. On the other hand, a large amount of p53 was found in both nuclear and cytoplasmic fractions of one SV40-transformed XP and two SV40-transformed CS cell strains irrespective of UV irradiation. Treatment with HuIFN-beta reduced the level of pro-apoptotic Bax protein without suppression of nuclear accumulation of p53 in the CS cells but not in the XP cells. These findings suggest that there are different mechanisms of UV-refractoriness caused by HuIFN-beta in UV-sensitive CS and XP cells.     

UV damage causes uncontrolled DNA breakage in cells from patients with combined features of XP-D and Cockayne syndrome

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. Defects in NER result in three different human disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS). Two cases with the combined features of XP and CS have been assigned to the XP-D complementation group. Despite their extreme UV sensitivity, these cells appeared to incise their DNA as efficiently as normal cells in response to UV damage. These incisions were, however, uncoupled from the rest of the repair process. Using cell-free extracts, we were unable to detect any incision activity in the neighbourhood of the damage. When irradiated plasmids were introduced into unirradiated XP-D/CS cells, the ectopically introduced damage triggered the induction of breaks in the undamaged genomic DNA. XP-D/CS cells thus have a unique response to sensing UV damage, which results in the introduction of breaks into the DNA at sites distant from the damage. We propose that it is these spurious breaks that are responsible for the extreme UV sensitivity of these cells.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

用DNA介导的基因转移法提高XP细胞对紫外辐射损伤的修复能力

本文用两种DNA介导的基因转移法(DNA—磷酸钙共沉淀法和电脉冲刺激法),将具有切除修复功能的人HeLaS3细胞的DNA,植入切除修复缺陷的着色性干皮症(XP)细胞中。实验结果表明:植入HeLaS3 DNA后,可以部份恢复XP细胞DNA切除修复的功能,提高其对紫外线辐射损伤的抗性。表现为转化细胞在UV_(254)照射后存活率的显著升高和非周期DNA合成能力的增强。     

Induced mutagenic effects in the nucleotide excision repair deficient Drosophila mutant mus201(D1), expressing a truncated XPG protein

Defects in nucleotide excision repair (NER) as defined by the UV sensitivity of xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) patients has lead to the identification of most of the genes involved: XPA through XPG, CSA and CSB. Whereas XP patients often show an increased risk for skin cancer after exposure to sunlight, this is not the case for patients with CS and TTD. Several CS patients have been shown to carry a defect in the XPG gene. The XPG, a structure specific endonuclease makes the incision 3' of damage and is also involved in the subsequent 5'incision during the NER process. In addition, XPG plays a role in the removal of oxidative DNA damage. The Drosophila XPG gene was isolated and based on the molecular defect of a spontaneous (insertion) and an EMS induced mutant, it was shown that a mutated XPG is responsible for the Drosophila mutagen-sensitive mutants mus201. One of these mutants, mus201(D1) has been used extensively in studies of the effects and mechanisms of many chemical mutagens as well as X-rays. The results of these studies are discussed in the light of the finding that mus201p is the Drosophila homologue of XPG.     

Routine autoradiographic analysis of DNA excision-repair. Report of prenatal and postnatal diagnosis in eleven families.

DNA excision-repair of UV induced damages was investigated by unscheduled DNA synthesis and quantitative autoradiography. The method has been routinely used on lymphocytes for postnatal diagnosis of xeroderma pigmentosum and PIBIDS syndrome. Ten XP-families including 13 clinical XP patients and 9 XP-risk children, and one family with one clinical PIBIDS case and one PIBIDS-risk child were screened. Each of the 14 affected patients were biologically ascertained with a significant excision-repair defect. Among the 9 XP-risk children without clinical manifestations, the DNA excision-repair was defected in 4 cases considered as biological XP, and normal in 5 cases considered as biologically normal subjects. Likewise the PIBIDS-risk child exhibited a normal excision-repair. According to the age of the XP or PIBIDS-risk children, and the delay of appearance of clinical manifestations, the method should not present neither false positive nor false negative results and allows the infraclinical diagnosis. The protocol was extended for prenatal diagnosis on amniocytes and fetal cord blood. Excision-repair analysis on normal cultivated chorionic villi cells has been performed allowing a further first trimester prenatal diagnosis.     

Ultraviolet light-induced chromosomal aberrations in cultured cells from Cockayne syndrome and complementation group C xeroderma pigmentosum patients: lack of correlation with cancer susceptibility.

Both Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are inherited diseases with defective repair of damage induced in DNA by UV. Patients with XP, but not those with CS, have an increased susceptibility to formation of sunlight-induced skin tumors. We determined the frequency of UV-induced chromosomal aberrations in cultured lymphoblastoid cell lines from five CS patients and three complementation-group-C XP patients to determine whether such aberrations were abnormally increased only in the XP cells. We found that CS cells had the same abnormally increased number of induced aberrations as the XP cells, indicating that the number of UV-induced aberrations in XP group C cells does not account for the susceptibility of these XP patients to sunlight-induced skin cancer.     

Repair of damaged DNA by extracts from a xeroderma pigmentosum complementation group A revertant and expression of a protein absent in its parental cell line.

Cells derived from individuals with mutations in the xeroderma pigmentosum complementation group A gene (XP-A gene) are hypersensitive to UV light and have a severe defect in nucleotide excision repair of damaged DNA. UV-resistant revertant cell lines can arise from XP-A cells in culture. Cells of one such revertant, XP129, were previously shown to remove (6-4) photoproducts from irradiated DNA, but to have poor repair of cyclobutane pyrimidine dimers. To analyze the biochemical nature of the reversion, whole cell extracts were prepared from the SV40-immortalized fibroblast cell lines XP12RO (an XP-A cell line), the revertant XP129 (derived from XP12RO), and 1BR.3N (from a normal individual). The ability of extracts to carry out repair synthesis in UV-irradiated DNA was examined, and immunoblots were performed using antiserum that recognizes XP-A protein. XP12RO extracts exhibited a very low level of repair and no detectable XP-A protein, but repair activity could be conferred by adding purified XP-A protein to the reaction mixture. XP129 extracts have essentially normal repair synthesis consistent with the observation that most repair of UV-irradiated DNA by extracts appears to occur at (6-4) photoproducts. An XP-A polypeptide of normal size was present in XP129, but in reduced amounts. The results indicate that in XP129 a mutational event has converted the inactive XP12RO XP-A gene into a form which expresses an active XP-A protein.