http://repub.eur.nl/res/aut/7276/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/29654/ 2008-09-24T00:00:00Z A novel mutation F826L located within the ligand binding domain (LBD) of the human androgen receptor (AR) was investigated. This mutation was found in a boy with severe penoscrotal hypospadias (classified as 46,XY DSD). The AR mutant F826L appeared to be indistinguishable from the wild-type AR, with respect to ligand binding affinity, transcriptional activation of MMTV-luciferase and ARE2-TATA-luciferase reporter genes, protein level in genital skin fibroblasts (GSFs), and sub-cellular distribution in transfected cells. However, an at least two-fold higher NH2-/COOH-terminal domain interaction was found in luciferase and GST pull-down assays. A two-fold increase was also observed for TIF2 (transcription intermediary factor 2) co-activation of the AR F826L COOH-terminal domain. This increase could not be explained by a higher stability of the mutant protein, which was within wild-type range. Repression of transactivation by the nuclear receptor co-repressor (N-CoR) was not affected by the AR F826L mutation. The observed properties of AR F826L would be in agreement with an increased activity rather than with a partial defective AR transcriptional activation. It is concluded that the penoscrotal hypospadias in the present case is caused by an as yet unknown mechanism, which still may involve the mutant AR. 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/35692/ 2007-12-01T00:00:00Z Objective: Evaluation of results in a consecutive series of 76 prenatal diagnoses for xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) made since 1977. Methods: UV-induced DNA repair synthesis was assessed by the autoradiographic measurement of the incorporation of3H-thymidine. Results: XP was diagnosed in 19 of the 76 investigated pregnancies at risk; cultured chorionic villus (CV) cells were used in 33 pregnancies with ten affected fetuses and cultured amniocytes in 43 pregnancies with nine affected fetuses. In four cases, CVS results were corroborated by subsequent investigation of amniocytes because maternal cell contamination in the CV cell culture was either present or could not be excluded. Uncertain results in two other cases with intermediate DNA repair capacity and severe maternal cell contamination required further investigation. Median time needed for cell culture and analysis was 25 days. To reduce intra-assay variations, a modification of the DNA repair synthesis assay has recently been developed. In this assay, patients and controls are investigated simultaneously in mixed cultures of cells labelled with polystyrene beads. Conclusion: Reliable prenatal diagnosis for XP and TTD can be made by the demonstration of clearly reduced UV-induced DNA repair synthesis due to defective global genome nucleotide excision repair. Copyright http://repub.eur.nl/res/pub/35561/ 2007-03-01T00:00:00Z Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1. 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/13522/ 2005-01-01T00:00:00Z Androgen insensitivity syndrome (AIS) is caused by defects in the androgen receptor (AR) that render the AR partially or completely inactive. As a result, embryonic sex differentiation is impaired. Here, we describe a novel mutation in the AR found in a patient with partial AIS. The mutation results in a substitution of a glutamine (Q) by a lysine (K) residue at position 902, Q902K. The AR Q902K mutation was investigated in vitro with respect to its functional properties. The equilibrium dissociation constants (K(d)s) of AR Q902K in the presence of either the synthetic androgen R1881 or the natural ligand DHT were slightly elevated. The R1881 dissociation rate (t(1/2)) was increased 3-fold for AR Q902K compared with wild type. Transcriptional activity was decreased to 85% of wild type, and the dose-response curve revealed that the sensitivity to hormone was decreased due to the mutation. Furthermore, the 114-kDa androgen-induced phosphorylated AR protein band was not detectable in genital skin fibroblasts. However, it could be detected in transfected CHO cells expressing the mutant receptor in the presence of 10 and 100 nm R1881. Functional interaction assays and a GST pull-down assay showed that the interaction between the NH2 and COOH terminus of AR Q902K was reduced to 50% of wild type. Furthermore, the transactivation by the coactivator TIF2 (transcriptional intermediary factor 2) was decreased 2- to 3-fold. The half-maximal response in both assays was shifted to a higher hormone concentration compared with wild type. These results indicate that residue Q902 is involved in TIF2 and NH2/COOH interaction and that the Q to K mutation results in a mild impairment of AR function, which can explain the partial AIS phenotype of the patient. http://repub.eur.nl/res/pub/3182/ 2001-03-20T00:00:00Z The xeroderma pigmentosum group D (XPD) helicase subunit of TFIIH functions in DNA repair and transcription initiation. Different mutations in XPD give rise to three ultraviolet-sensitive syndromes: the skin cancer-prone disorder xeroderma pigmentosum (XP), in which repair of ultraviolet damage is affected; and the severe neurodevelopmental conditions Cockayne syndrome (CS) and trichothiodystrophy (TTD). In the latter two, the basal transcription function of TFIIH is also presumed to be affected. Here we report four unusual TTD patients with fever-dependent reversible deterioration of TTD features such as brittle hair. Cells from these patients show an in vivo temperature-sensitive defect of transcription and DNA repair due to thermo-instability of TFIIH. Our findings reveal the clinical consequences of impaired basal transcription and mutations in very fundamental processes in humans, which previously were only known in lower organisms. http://repub.eur.nl/res/pub/9738/ 2001-01-01T00:00:00Z Androgen insensitivity syndrome encompasses a wide range of phenotypes, which are caused by numerous different mutations in the AR gene. Detailed information on the genotype/phenotype relationship in androgen insensitivity syndrome is important for sex assignment, treatment of androgen insensitivity syndrome patients, genetic counseling of their families, and insight into the functional domains of the AR. The commonly accepted concept of dependence on fetal androgens of the development of Wolffian ducts was studied in complete androgen insensitivity syndrome (CAIS) patients. In a nationwide survey in The Netherlands, all cases (n = 49) with the presumptive diagnosis androgen insensitivity syndrome known to pediatric endocrinologists and clinical geneticists were studied. After studying the clinical phenotype, mutation analysis and functional analysis of mutant receptors were performed using genital skin fibroblasts and in vitro expression studies. Here we report the findings in families with multiple affected cases. Fifty-nine percent of androgen insensitivity syndrome patients had other affected relatives. A total of 17 families were studied, seven families with CAIS (18 patients), nine families with partial androgen insensitivity (24 patients), and one family with female prepubertal phenotypes (two patients). No phenotypic variation was observed in families with CAIS. However, phenotypic variation was observed in one-third of families with partial androgen insensitivity resulting in different sex of rearing and differences in requirement of reconstructive surgery. Intrafamilial phenotypic variation was observed for mutations R846H, M771I, and deletion of amino acid N682. Four newly identified mutations were found. Follow-up in families with different AR gene mutations provided information on residual androgen action in vivo and the development of the prepubertal and adult phenotype. Patients with a functional complete defective AR had some pubic hair, Tanner stage P2, and vestigial Wolffian duct derivatives despite absence of AR expression. Vaginal length was functional in most but not all CAIS patients. The minimal incidence of androgen insensitivity syndrome in The Netherlands, based on patients with molecular proof of the diagnosis is 1:99,000. Phenotypic variation was absent in families with CAIS, but distinct phenotypic variation was observed relatively frequent in families with partial androgen insensitivity. Molecular observations suggest that phenotypic variation had different etiologies among these families. Sex assignment of patients with partial androgen insensitivity cannot be based on a specific identified AR gene mutation because distinct phenotypic variation in partial androgen insensitivity families is relatively frequent. In genetic counseling of partial androgen insensitivity families, this frequent occurrence of variable expression resulting in differences in sex of rearing and/or requirement of reconstructive surgery is important information. During puberty or normal dose androgen therapy, no or only minimal virilization may occur even in patients with significant (but still deficient) prenatal virilization. Wolffian duct remnants remain detectable but differentiation does not occur in the absence of a functional AR. In many CAIS patients, surgical elongation of the vagina is not indicated. http://repub.eur.nl/res/pub/9393/ 1999-01-01T00:00:00Z Palmitoyl-protein thioesterase (PPT) deficiency was recently shown to be the primary defect in infantile neuronal ceroid lipofuscinosis (INCL). The available enzyme assay is complicated and impractical for diagnostic use and is, in practice, unavailable. We have developed a new fluorimetric assay for PPT based on the sensitive fluorochrome 4-methylumbelliferone. This PPT assay is simple, sensitive, and robust and will facilitate the definition of the full clinical spectrum associated with a deficiency of PPT. PPT activity was readily detectable in fibroblasts, leucocytes, lymphoblasts, amniotic fluid cells, and chorionic villi, but was profoundly deficient in these tissues from INCL patients. Similarly, a deficiency of PPT was shown in patients with the variant juvenile NCL with GROD. These results show that rapid pre- and postnatal diagnosis can be performed with this new enzyme assay for PPT. 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/3058/ 1994-01-01T00:00:00Z The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with ther remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in the vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here we report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of > 40 years. Analysis of the frequency of hprt- mutant T-lymphocytes in blood samples suggests a relatively low in vivo mutation frequency in these patients. Factors in addition to NER deficiency may be required for the development of cutaneous tumors. http://repub.eur.nl/res/pub/26293/ 1973-09-26T00:00:00Z