http://repub.eur.nl/res/aut/2469/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/30412/ 2008-05-03T00:00:00Z Laboratory diagnosis for DNA repair diseases has been performed in western Europe from the early seventies for xeroderma pigmentosum (XP) and from the mid-eighties for Cockayne syndrome (CS) and trichothiodystrophy (TTD). The combined data from the DNA repair diagnostic centres in France, (West) Germany, Italy, the Netherlands and the United Kingdom have been investigated for three groups of diseases: XP (including XP-variant), CS (including XP/CS complex) and TTD. Incidences in western Europe were for the first time established at 2.3 per million livebirths for XP, 2.7 per million for CS and 1.2 per million for TTD. As immigrant populations were disproportionately represented in the patients' groups, incidences were also established for the autochthonic western European population at: 0.9 per million for XP, 1.8 per million for CS and 1.1 per million for TTD. Perhaps contrary to general conceptions, compared to XP the incidence of CS appears to be somewhat higher and the incidence of TTD to be quite similar in the native West-European population. http://repub.eur.nl/res/pub/36714/ 2007-01-01T00:00:00Z Trichothiodystrophy (TTD) is a rare autosomal recessive disorder whose defining feature is brittle hair. Associated clinical symptoms include physical and mental retardation of different severity, ichthyosis, premature aging, and, in half of the patients, photosensitivity. Recently, C7orf11 (TTDN1) was identified as the first disease gene for the nonphotosensitive form of TTD, being mutated in two unrelated cases and in an Amish kindred. We have evaluated the involvement of TTDN1 in 44 unrelated nonphotosensitive TTD cases of different geographic origin and with different disease severity. Mutations were found in six patients, five of whom are homozygous and one of whom is a compound heterozygote. All five identified mutations are deletions that have not been described before. Three are deletions of a few bases, resulting in frameshifts and premature termination codons. The other two include the whole TTDN1 gene, suggesting that TTDN1 is not essential for cell proliferation and viability. The severity of the clinical features does not correlate with the type of mutation, indicating that other factors besides TTDN1 mutations influence the severity of the disorder. Since only a small proportion of the analyzed cases were mutated in TTDN1, the nonphotosensitive form of TTD is genetically heterogeneous. Mutations in TTDN1 do not affect the response to ultraviolet (UV) light or the steady state level of the repair/transcription factor IIH (TFIIH), which is central to the onset of the photosensitive form of TTD. http://repub.eur.nl/res/pub/3220/ 2004-07-01T00:00:00Z DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair-deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease. http://repub.eur.nl/res/pub/9478/ 2000-01-01T00:00:00Z When results of more than ten different studies on hormone-induced calcium signals in Sertoli cells are taken together, a wide variety of responses emerges. The reported changes range from increased concentrations, via no response at all, to decreased calcium concentrations. Minor variations in cell isolation techniques, culture conditions, or techniques for measuring the intracellular calcium could explain some of these differences. However, erratic variations in response are also observed within research groups under very similar experimental conditions. Such 'negative' findings are mainly reported orally and do not further penetrate the scientific community. As hormone-dependent calcium responses evidently may depend very much on the context of the cells, calcium transients would appear to be unreliable bioassay principles with which to detect the primary actions of FSH and effectors such as androgens on Sertoli cells. A more important biological question is whether these sometimes opposed calcium transients are connected with a particular cellular response. To date there is no evidence for such a tight coupling in Sertoli cells, implying that, at least under in vitro conditions, calcium signals might even be redundant altogether. Such calcium variability is probably not unique to Sertoli cells, and the aim of this commentary is to promote an open debate that may help to transform the current state of 'calcium confusion' into a better understanding of the intracellular calcium language. http://repub.eur.nl/res/pub/3109/ 1996-01-01T00:00:00Z Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP. http://repub.eur.nl/res/pub/3091/ 1995-05-01T00:00:00Z Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive. http://repub.eur.nl/res/pub/3079/ 1995-01-01T00:00:00Z Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%-40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly-->arg change at amino acid 675 in the allele inherited from the patient's mother and a -1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy. http://repub.eur.nl/res/pub/3085/ 1995-01-01T00:00:00Z To understand the heterogeneity in genetic predisposition to skin cancer in different nucleotide excision repair-deficient human syndromes, we studied repair of cyclobutane pyrimidine dimers (CPDs) and of pyrimidine(6-4)pyrimidone (6-4PP) photoproducts in cells from trichothiodystrophy (TTD) patients. TTD is not associated with increased incidence of skin cancer, although 50% of the patients are photosensitive and carry a defect in the nucleotide excision repair pathway, similar to Xeroderma pigmentosum patients. However, in striking contrast to TTD, Xeroderma pigmentosum is highly prone to cancer. To address this apparent paradox, two types of studies were conducted: (a) reactivation of UV-irradiated plasmids harboring actively transcribed reporter genes, with or without photolyase treatment before transfection of SV40-transformed fibroblasts; and (b) the kinetics of removal of UV-induced CPDs and 6-4PPs in genomic DNA by immunoblot analysis using lesion-specific mAbs in SV40-transformed and untransformed fibroblasts representative of all genetic TTD complementation groups. Results showed that all cell lines from photosensitive TTD patients efficiently express Cat or luciferase genes in transfected plasmids carrying non-CPD lesions, including 6-4PP, and display wild-type or near-wild-type (50-70% in 3 cell lines) 6-4PP repair in the overall genome after immunoblot analysis. However, CPD lesions (the repair of which is defective in the overall genome) also block the expression of the reporter gene in transfected plasmids. Two cell lines from nonphotosensitive TTD patients showed wild-type levels of repair for both photoproducts in overall genome. A model on the lesion-specific repair in the context of the molecular defect in TTD is proposed. The implication of the defective CPD repair and efficient 6-4PP repair subpathways in cancer prevention in TTD patients is discussed. http://repub.eur.nl/res/pub/3059/ 1994-01-01T00:00:00Z Trichothiodystrophy (TTD) is a rare genetic disease with heterogeneous clinical features associated with specific deficiencies in nucleotide excision repair. Patients have brittle hair due to a reduced content of cysteine-rich matrix proteins. About 50% of the cases reported in the literature are photosensitive. In these patients an altered cellular response to UV, due to a specific deficiency in nucleotide excision repair, has been observed. The majority of repair-defective TTD patients have been assigned by complementation analysis to group D of xeroderma pigmentosum (XP). Recently, the human excision repair gene ERCC2 has been shown to correct the UV sensitivity of XP-D fibroblasts. In this work we describe the effect of ERCC2 on the DNA repair deficient phenotype of XP-D and on two repair-defective TTD cell strains (TTD1VI and TTD2VI) assigned by complementation analysis to group D of XP. ERCC2 cDNA, cloned into a mammalian expression vector, was introduced into TTD and XP fibroblasts via DNA-mediated transfection or microneedle injection. UV sensitivity and cellular DNA repair properties, including unscheduled DNA synthesis and reactivation of a UV-irradiated plasmid containing the chloramphenicol acetyltransferase reporter gene (pRSVCat), were corrected to wild-type levels in both TTD and XP-D cells. These data show that a functional ERCC2 gene is sufficient to reestablish a wild-type DNA repair phenotype in TTD1VI and TTD2VI cells, confirming the genetic relationship between TTD and XP-D. Furthermore, our findings suggest that mutations at the ERCC2 locus are responsible for causing a similar phenotype in TTD and XP-D cells in response to UV irradiation, but produce quite different clinical symptoms. http://repub.eur.nl/res/pub/3049/ 1993-01-01T00:00:00Z The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. We report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associated with the clinical features of TTD argues strongly for an involvement of repair proteins in hair-shaft development. http://repub.eur.nl/res/pub/3034/ 1992-01-01T00:00:00Z A workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic Centre South-West, The Netherlands (MGC), c/o Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden (The Netherlands). Local organizers were: D. Bootsma (chairman), W. Ferro, J.H.J. Hoeijmakers, A.R. Lehmann, P.H.M. Lohman, L. Mullenders, and A.A. van Zeeland (secretarial assistance: Mrs. C. Escher-van Heerden and Mrs. R. Bontre). Over 190 scientists participated, and the format of the meeting followed that of the 1987 workshop on the 'Molecular Aspects of DNA Repair' (Friedberg et al., 1987). Plenary review talks in the mornings were followed, in the afternoon, by poster viewing in three or four parallel sessions. Groups of 15-20 posters were discussed in detail, and later on, in plenary sessions, chairpersons of the poster discussions reviewed the afternoons' posters. The principal themes of the meeting were the isolation and characterisation of repair genes and proteins, repair in specific sequences, consequences of defective DNA repair, and new methods for detecting DNA damage and repair. Remarkable progress has been made recently in all of these areas, and many exciting new results were presented. It is impossible to summarize all contributions to this (intensive) one-week meeting. Therefore, and for the sake of coherence, presentations that did not fit easily into any of the general themes of the meetings have not been included. http://repub.eur.nl/res/pub/3026/ 1991-09-10T00:00:00Z