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© 2000 Oxford University Press Human Molecular Genetics, 2000, Vol. 9, No. 8 1171–1175 Identical mutations in the CSB gene associated with either Cockayne syndrome or the DeSanctis– Cacchione variant of xeroderma pigmentosum Stefano Colella, Tiziana Nardo, Elena Botta, Alan R. Lehmann1 and Miria Stefanini+ Istituto di Genetica Biochimica ed Evoluzionistica CNR, Via Abbiategrasso, 207-27100 Pavia, Italy and 1MRC Cell Mutation Unit, Sussex University, Falmer, Brighton BN1 9RR, UK Received 26 November 1999; Revised and Accepted 13 March 2000 Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two hereditary disorders in which photosensitivity is associated with distinct clinical and cellular phenotypes and results from genetically different defects. We have identified the primary molecular alteration in two patients in whom clinical manifestations strongly reminiscent of a severe form of XP were unexpectedly associated with the CS cellular phenotype and with a defect in the CSB gene. Sequencing of the CSB-coding region in both cDNA and genomic DNA showed that these patients had identical alterations to those in a patient with the clinical features of the classical form of CS. These data, together with fluorescence in situ hybridization analysis, demonstrated that the two siblings with XP as well as the CS patient were homozygous for the same CSB mutated allele, containing a silent C2830T change and a nonsense mutation C2282T converting Arg735 to a stop codon. The finding that the same inactivating mutation underlies different pathological phenotypes indicates that there is no simple correlation between the molecular defect and the clinical features. Therefore, alterations in the CSB gene give rise to the same repair defect at the cellular level but other genetic and/or environmental factors determine the pathological phenotype. INTRODUCTION Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are two autosomal, recessive disorders in which hypersensitivity to UV light is associated with distinct clinical and cellular phenotypes and results from genetically different defects (see ref. 1 for a recent review). XP is characterized by hypersensitivity to sun-exposure, pigmentary alterations and premalignant lesions in sun-exposed areas of the skin, and an extremely high incidence of skin cancer. Approximately 20% of XP patients show neurological abnormalities of varying severity due to primary neuronal degeneration. The biochemical defect in most XP patients is in nucleotide-excision repair (NER) and typically results in a reduced capability to perform +To UV-induced DNA repair synthesis (UDS). A defect in postreplication repair is present in a minority of cases, the so-called XP variant (XP-V) group characterized by a defect in the ability to synthesize intact daughter DNA strands after UV irradiation. Genetic analysis by somatic cell hybridization has led to the identification in the NER-defective form of XP of seven complementation groups, designated XP-A to XP-G. CS is a multisystem disorder characterized by postnatal growth failure, progressive neurological dysfunction due to demyelination, premature ageing and otherwise clinically heterogeneous features which commonly include cutaneous photosensitivity but not cancer. Cells from CS patients are often hypersensitive to UV light and are unable to recover normal RNA synthesis rates following UV irradiation, despite having normal levels of UDS. Two complementation groups, CS-A and CS-B, have been identified; both groups are specifically defective in the sub-pathway of NER which rapidly removes damage from the transcribed strand of active genes (2). Rare patients have been described in which the cutaneous alterations of XP are combined with some of the major clinical symptoms of CS, namely pigmentary retinal degeneration, primary demyelination, calcification of the basal ganglia and cachetic dwarfism. All these patients, assigned to a clinical entity designated XP/CS, show the cellular phenotype typical of XP, i.e. reduced UDS level, and they have been assigned to the XP-B, XP-D and XP-G groups (1). Clinical features reminiscent of the DeSanctis–Cacchione syndrome (DSC), the form of XP with severe neurological involvement, have been recently described in three siblings from an Hispanic family. Detailed DNA repair investigations in fibroblast strains from the two youngest patients, coded GM10903 and GM10905, surprisingly showed that the cellular response to UV light in both siblings was similar to that typically described in CS without any evidence of defects indicative either of the NER-defective or the variant form of XP (3). Following genetic analysis they have been assigned to the complementation group B of CS (4). Therefore, they represent the first XP cases assigned to this group, which comprises about 30 subjects with CS features (5–10). The cloning of the CSB gene has enabled mutations to be identified in patients classified in the CS-B group (11). In the 22 CS cases previously analysed by us, many of the inactivating whom correspondence should be addressed. Tel: +39 0382 546330; Fax +39 0382 422286; Email: [email protected] 1172 Human Molecular Genetics, 2000, Vol. 9, No. 8 Figure 1. Mutations found in the CSB cDNA and resulting protein alterations in the GM10903 and GM10905 patients. The diagram shows the CSB cDNA [white area: open reading frame (ORF); black areas: untranslated regions] and the CSB protein with the predicted functional domains (11). Causative mutations are indicated in bold. mutations resulted in severely truncated polypeptides because of either stop codons, frameshifts or splice abnormalities (9,10). In this paper we report the results of the molecular analysis of the CSB gene in the patients GM10903 and GM10905. RESULTS AND DISCUSSION Sequence analysis of the CSB gene was performed on fibroblast strains obtained from the siblings GM10903 and GM10905 (3). Genetic analysis by somatic cell hybridization assigned both siblings to the CS-B complementation group (4). To identify the molecular alterations, the sequence of the coding region of the CSB gene was analysed by RT–PCR followed by direct sequencing (nucleotides 80–4558 in ref. 11). Both patients showed in the whole of the amplified cDNA population a silent C2830T change (Gly917) and a nonsense mutation C2282T, which converts Arg735 to a stop codon (opal), resulting in the synthesis of a truncated protein of 734 amino acids (Fig. 1). We showed previously that the Turkish patient CS1TAN was homozygous and the Caucasian patient 25627 heterozygous for these same mutations at the cDNA level (9). Both these cases showed the clinical features of the classical form of CS. The patient CS1TAN was born from a consanguineous marriage whereas consanguinity between GM10903 and GM10905 parents could only be inferred from the recurrence of the same family name in both maternal and paternal lines and from the fact that both families had lived in the same small Mexican village for many years (3). Since cells from the parents were not available, we took different approaches to demonstrate that the inactivating mutation was present on both the CSB alleles of the patients. As detailed in Materials and Methods, the identity of the cell strains was checked by both karyotypic and fingerprint analysis. Sequencing of the relevant genomic DNA region showed only the mutant T allele at position 2282 in all three patients (GM10903, GM10905 and CS1TAN) suggesting that they were all homozygous for the C2282T mutation (Fig. 2). To confirm that this was indeed the case and to exclude the possibility of a deletion including the CSB gene in one of the two alleles, we carried out fluorescence in situ hybridization (FISH) analysis with a probe specific for the CSB gene. In all three cases we obtained two positive signals on nuclei and specific hybridization on two chromo- Figure 2. Mutations found in the CSB genomic DNA of the patients GM10903 and CS1TAN. Autoradiograph of the sequencing gel of the PCR products obtained after amplification of the DNA region containing the C2282T mutation in a control, GM10903 and CS1TAN. somes (Fig. 3). These observations rule out the possibility of a deletion in this region and confirm that GM10903, GM10905 and CS1TAN patients carry two CSB alleles, both containing the same inactivating mutation. Therefore, the intriguing genotype–phenotype relationship in the siblings GM10903 and GM10905 cannot be related simply either to the specific CSB mutation or to the dosage of the mutated allele. The mutation in the CSB alleles in these patients results in an altered cellular response to UV, typical of CS but it underlies a clinical phenotype suggestive of DSC. As detailed by Moriwaki et al. (12), DSC cases can be distinguished from those with CS by the absence of retinal degeneration, decreased to absent deep tendon reflexes, primary neuronal degeneration, and absence of calcification of the basal ganglia and other brain structures. Patients with CS, in contrast, do not show XPtype skin freckling or skin cancer but do have increased or normal deep tendon reflexes, signs of primary demyelination and often calcification of the brain. As well as showing the absence of the major clinical criteria for the diagnosis of CS, extensive clinical evaluation of the siblings GM10903 and GM10905 strongly suggested they have DSC (3). The DSC patients so far described all show the cellular phenotype typical of XP and are mutated in the XPA or Human Molecular Genetics, 2000, Vol. 9, No. 8 1173 Figure 3. Fluorescence in situ hybridization with the cDNA of the CSB gene on mitotic cells and interphase nuclei from the patients GM10903 (upper panels) and CS1TAN (lower panels). The hybridization signals correspond to the yellow spots. Arrows point to the chromosomes showing the hybridization signal. XPD gene. Cellular and genetic DNA repair investigations in the siblings GM10903 and GM10905 argue against the presence of an additional mutation in one of the XP genes identified so far (3,4). Therefore, GM10903 and GM10905 represent the first cases in which the severe neurological alterations diagnostic for DSC are associated with mutations in the CSB gene. Interestingly, despite the presence of the neurological anomalies typical of XP, in the patients GM10903 and GM10905, the cutaneous symptoms of XP are limited to actinic skin changes. Therefore, in these patients, as in CS patients, the presence of mutated CSB alleles does not result in any increased incidence of cancer. It has been suggested recently that the CSB protein might have an additional function in transcription beyond its involvement in coupling repair to blocked transcription (13,14). We are tempted to speculate that mutations in the CSB gene might interfere with some of the steps leading to neoplastic transformation and progression, which may be dependent on the transcriptional role of CSB protein. Although there are a few reports in the literature of the same mutation being linked to two different clinical entities (e.g. refs 15,16), this is unprecedented for syndromes associated with DNA repair defects. Molecular analysis of NER-defective XP patients defective in the XPB, XPD and XPG genes has shown that specific mutations are associated with particular pathological phenotypes. This holds true for patients with trichothiodystrophy (TTD) or XP/CS altered in the XPB gene (17,18), for XP, TTD and XP/CS cases altered in the XPD gene (19–27), and for XP and XP/CS altered in the XPG gene (28,29). The patients reported in this paper provide the first evidence that the same inactivating mutation in the CSB gene underlies distinct pathological phenotypes diagnostic, respectively, for CS (CS1TAN) and for a severe form of XP (GM10903 and GM10905). These observations imply that there is not any obvious and direct correlation between the molecular defect in the CSB gene and the clinical features. Our previous molecular analysis of the CSB gene in several CS patients with clinically heterogeneous features has already provided data suggesting that other factors, besides the site of mutation, influence the type and severity of the CS pathological phenotype (9,10). This notion is further extended by the results reported in this paper showing that the same set of mutated CSB alleles is associated with distinct clinical entities. What might the alterations be in the genetic background of the GM10903 and GM10905 patients that hamper the expression of the mutated CSB alleles at the phenotypic but not at the cellular level? Despite the presence of the neurological abnormalities typical of XP, two pieces of experimental evidence argue against the presence of an altered XP gene. The patients show the clinical symptoms but not the cellular features of XP, as clearly demonstrated by the results reported in ref. (3). Furthermore, at least in the transgenic mouse system, the association of XP and CS defects (CSB–/–XPA–/– and CSB–/–XPC–/– double knock-out mice) has a strong synergistic effect at the phenotypic level and leads to dramatically pronounced CS features (30; G.T.H. van der Host, unpublished data). The GM10903 and GM10905 patients might be mutated in a gene, or a panel of genes, that is able to confer neurological abnormalities indistinguishable from those resulting from XP defects but does not have any effect on repair. In addition, as suggested above, the absence of a functional CSB protein might per se be a sufficient condition to prevent carcinogenesis. Current hypotheses propose that the neurological abnormalities typical of CS result from subtle transcription alterations and/or from an inability to repair oxidative damage (reviewed in ref. 31). It is possible that the genetic background in the GM10903 and GM10905 patients suppresses or compensates for the deleterious effects of the CSB defect on transcription or repair of oxidative damage, thereby preventing expression of the neurological abnormalities typical of CS. MATERIALS AND METHODS Cells and culture conditions The study was performed on cells from patients GM10903 and GM10905 purchased from the NIGMS Human Genetic Mutant Cell Repository (Camden, NJ). Fibroblasts were routinely grown in Ham’s F-10 medium (Gibco-BRL, Rockville, MD) supplemented with 12% fetal calf serum (FCS; Irvine, Santa Ana, CA) and subcultured by trypsinization. The identity of the cell strains was ascertained both at the cellular and molecular levels. The analysis of four hypervariable regions in the genomic DNA samples used for sequencing showed that GM10903 and GM10905 have the same set of FGA and TH01 alleles (23–26 and 7–8, respectively) and share one allele of VWA (GM10903, 18–19; GM10905, 15–18) and D21S11 (GM10903, 31.2–32.2; GM10905, 30–32.2). A different combination of alleles for all the four analysed regions was observed in CS1TAN, namely FGA, 20–24; TH01, 8–9; VWA, 17–18 and D21S11, 29–31.2. Molecular analysis of a locus allowing for sex-discrimination (amelogenin) and karyotype analysis on cell samples parallel to those used for FISH indicated that GM10903 is a female whereas CS1TAN and GM10905 are males, as expected. 1174 Human Molecular Genetics, 2000, Vol. 9, No. 8 Identification of the molecular alteration ACKNOWLEDGEMENTS RNA extraction, cDNA synthesis and PCR amplification of the cDNA of the CSB gene were carried out using protocols and primers described in detail in ref. (9). Briefly, 2 µg of RNA in a total volume of 10 µl were heated to 90°C for 2 min and incubated at 37°C for 1 h following addition of 30 µl of a mix containing 1× first strand cDNA buffer (Gibco-BRL), 10 mM DTT, 1mM dNTPs, 25 ng oligo(dT)15 (Promega, Madison, WI) and 200 U Moloney Murine Leukemia Virus Reverse Transcriptase (M-MuLV RT, Gibco-BRL). Samples were then heated to 95°C for 5 min, made up to 100 µl with water and stored at –20°C. cDNA was amplified by PCR into six overlapping fragments; PCR mixtures contained 5–25 µl of cDNA, 1× Gene Amp buffer II (Perkin Elmer, Norwalk, CT), 2 mM MgCl2, 0.2 mM dNTPs, 20 pmol each of the required primers and 2U AmpliTaq (Perkin Elmer) in a total volume of 50 µl. Amplification was carried out for 35 cycles of 94°C for 1 min, annealing at 65°C (fragment I), 63°C (fragment II) or 67°C (fragment III–VI) for 1.5 min and 72°C for 3 min. Genomic DNA was isolated from samples of 5 × 106 fibroblasts by phenol extraction and isopropanol precipitation, following an overnight incubation at 37°C with 6 ml lysis buffer (150 mM NaCl, 10 mM Tris–HCl, pH 8.0; 1 mM EDTA, 150 µg/ml proteinase K, 0.5% SDS) and RNase treatment for 1 h at 37°C. Genomic DNA amplification of the region containing the identified mutation was carried out on 0.5 µg samples using the primer pairs A (5′-TAATGTTCCCTTCTCTGCTCTTATTAAAGG-3′ in Intron 10) and B (5′-ATCTGGCAAAGAAAGGCTCATCTTGACATC-3′ in Exon 11). The fragment was amplified using AmpliTaq Gold (Perkin Elmer) and the following conditions: 1 cycle at 95°C for 4 min and 35 cycles at 95°C for 1 min, 60°C for 10 min. PCR products were purified by agarose gel electrophoresis and directly sequenced using the ThermoSequenase cycle sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden). We are grateful to Dr Wim Vermeulen (Erasmus University, Rotterdam) for providing us with the pSLME6 plasmid and to Dr Carlo Previderè (Department of Legal Medicine, University, Pavia) for fingerprint analysis. This work was supported in part by Telethon grant E.550 to M.S. and EC contracts BMH4-CT98-3045 and QLG1-1999-00181 to A.R.L. and M.S. Fluorescence in situ hybridization The pSLME6 plasmid containing the whole of the CSB cDNA (11) was used as probe. Plasmid DNA was labelled with biotin-16-dUTP by nick translation according to the supplier’s instructions (Gibco-BRL) and purified through a Sephadex G50 column (Amersham Pharmacia Biotech). Hybridization was carried out according to methods described by Lichter and Cremer (32). Metaphase spreads and nuclei were denatured for 3 min at 70°C in 70% formamide, 2× SSC and then the slides were dehydrated at 4°C. The labelled probe (200 ng/slide) was resuspended in a hybridization mixture (30 µl/slide) containing 50% deionized formamide, 2× SSC, 10% dextran sulfate, 100× excess of salmon sperm DNA, denatured at 70°C for 10 min and then applied to slides under a 24 × 50 mm coverslip. After incubation for 16 h at 37°C the slides were washed three times for 5 min in 50% formamide, 2× SSC and three times for 5 min in 2× SSC. All washes were at 42°C. 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