Elena Botta

A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A

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.

Analysis of Mutations in the XPD Gene in Italian Patients with Trichothiodystrophy: Site of Mutation Correlates with Repair Deficiency, but Gene Dosage Appears to Determine Clinical Severity

Xeroderma pigmentosum (XP) complementation group D is a heterogeneous group, containing patients with XP alone, rare cases with both XP and Cockayne syndrome, and patients with trichothiodystrophy (TTD). TTD is a rare autosomal recessive multisystem disorder associated, in many patients, with a defect in nucleotide-excision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repair-deficient TTD patients, the defect has been assigned to the XPD gene. The XPD gene product is a subunit of transcription factor TFIIH, which is involved in both DNA repair and transcription. We have determined the mutations and the pattern of inheritance of the XPD alleles in the 11 cases identified in Italy so far, in which the hair abnormalities diagnostic for TTD are associated with different disease severity but similar cellular photosensitivity. We have identified eight causative mutations, of which four have not been described before, either in TTD or XP cases, supporting the hypothesis that the mutations responsible for TTD are different from those found in other pathological phenotypes. Arg112his was the most common alteration in the Italian patients, of whom five were homozygotes and two were heterozygotes, for this mutation. The presence of a specifically mutated XPD allele, irrespective of its homozygous, hemizygous, or heterozygous condition, was always associated with the same degree of cellular UV hypersensitivity. Surprisingly, however, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect. The most severe clinical features were found in patients who appear to be functionally hemizygous for the mutated allele.

Genetic analysis of twenty-two patients with Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder with dwarfism, mental retardation, sun sensitivity and a variety of other features. Cultured CS cells are hypersensitive to ultraviolet (UV) light, and following UV irradiation, CS cells are unable to restore RNA synthesis rates to normal levels. This has been attributed to a specific deficiency in CS cells in the ability to repair damage in actively transcribed regions of DNA at the rapid rate seen in normal cells. We have used the failure of recovery of RNA synthesis, following UV irradiation of CS cells, in a complementation test. Cells of different CS donors are fused. Restoration of normal RNA synthesis rates in UV irradiated heterodikaryons indicates that the donors are in different complementation groups, whereas a failure to effect this recovery implies that they are in the same group. In an analysis of cell strains from 22 CS donors from several countries and different racial groups, we have assigned five cell strains to the CS-A group and the remaining 17 to CS-B. No obvious racial, clinical or cellular distinctions could be made between individuals in the two groups. Our analysis will assist the identification of mutations in the recently clonedCSA andCSB genes and the study of structure-function relationships.

Trichothiodystrophy: From basic mechanisms to clinical implications

Trichothiodystrophy (TTD) is an autosomal recessive disorder with symptoms affecting several tissues and organs. The most relevant features are hair abnormalities, physical and mental retardation, ichthyosis, signs of premature aging and cutaneous photosensitivity. The clinical spectrum of TTD varies widely from patients with only brittle, fragile hair to patients with the most severe neuroectodermal symptoms. To date, four genes have been identified as responsible for TTD: XPD, XPB, p8/TTDA, and TTDN1. Whereas the function of TTDN1 is still unknown, the former three genes encode subunits of TFIIH, the multiprotein complex involved in basal and activated transcription and in nucleotide excision repair (NER). Ongoing investigations on TTD are elucidating not only the pathogenesis of the disease, which appears to be mainly related to transcriptional impairment, but also the modalities of NER and transcription in human cells and how TFIIH operates in these two fundamental cellular processes.

Apoptosis and efficient repair of DNA damage protect human keratinocytes against UVB.

Since the eighties it is well known that keratinocytes are more resistant to the lethal effects of UV light than fibroblasts, but the mechanism behind this phenomenon is still unknown. In the present study we investigated cell survival, apoptosis, cell cycle progression, UV photoproduct induction and repair, p53 gene response following UVB exposure in primary cultures of keratinocytes, and we compared the response with that of primary fibroblasts from the same skin biopsy. Keratinocytes are the primary target for UVB-induced human cutaneous malignancies, thus epidermal cells might have specific strategies to mantain genomic integrity. Nucleotide excision repair (NER) is a major defense mechanism against the deleterious effects of pyrimidine dimers (CPD) and 6-4 photoproducts (6-4 PP), the most biologically relevant damage induced by UV into DNA. The NER system has two distinct subpathways: global genome repair (GGR) that repairs lesions throughout the genome, and transcriptioncoupled repair (TCR) that operates on lesions in the transcribed strand of active genes (reviewed in Balajee and Bohr). To efficiently repair damage, cells transiently arrest their growth at different points of the cell cycle (reviewed in Bartek and Lukas). To limit the survival in the presence of irreparable DNA damage, cells die by apoptosis (reviewed in Kulms and Schwarz). This phenomenon is evident in skin with the appearance of sunburn cells. In keratinocytes, UV-induced apoptosis is p53-dependent. The colony-forming ability of primary keratinocytes and fibroblasts from two independent skin biopsies was measured after UVB exposure (Figure 1a). Keratinocytes were more resistant to the lethal effects of UVB than the fibroblasts (D37 of 1000 J/m and 500 J/m for keratinocytes and fibroblasts, respectively). Cell death can occur via different mechanisms including apoptosis. Apoptosis was measured by TUNEL assay at different times after cell exposure to 1000 J/m of UVB (Figure 1b). The number of apoptotic keratinocytes increased significantly at 24 and 72 h after UVB exposure whereas at the same dose fibroblasts were completely refractory to apoptosis. The activation of an apoptotic response by UVB in keratinocytes was confirmed by fluorimetric detection of caspase-3 (data not shown). Therefore, keratinocytes although more resistant to the lethal effects of UVB are more susceptible to UVB-induced apoptosis than fibroblasts. The differential sensitivity to UVB of keratinocytes and fibroblasts might be because of differences in the level or repair of DNA damage in the two cell types. Fibroblasts and keratinocytes were exposed to 1000 J/m of UVB and the amount of CPD and 6-4 PP was determined on the extracted DNA by ELISA using the specific antibodies. The yield of both DNA lesions was approximately 1.5-fold higher in fibroblasts than in keratinocytes but the ratio of CPD to 6-4 PP was similar in the two cell types (data not shown). In general, the loss of 6-4 PP was more rapid than that of CPD, as expected on the basis of their half-life (Figure 1c) (reviewed in Balajee and Bohr). The repair rate of 6-4 PP was similar in both cell types. In contrast, CPD were repaired at a significant faster rate in keratinocytes than in fibroblasts. After 24 h irradiation, only 20% of the initial CPD were left in keratinocyte DNA whereas over 50% of the initial lesions remained in fibroblast DNA. The higher efficiency in repair of CPD by keratinocytes cannot be ascribed to the lower level of initial DNA damage since the repair kinetics in fibroblasts following a dose of 500 J/m was similar to that reported after a dose of 1000 J/m UVB (data not shown). To address the question of whether cell cycle progression is differentially affected in the two cell types, cells were exposed to UVB and cell cycle position was determined 24 and 48 h after irradiation. A representative cell cycle distribution at 24 h post-irradiation is displayed in Figure 1d. In fibroblasts, at both UVB doses, a G1–S phase arrest was observed whereas in keratinocytes the cell cycle distribution was substantially unaltered. The level of the stress response protein p53 was determined after irradiation. Both fibroblasts and keratinocytes responded to UVB damage (1000 J/m) with stabilization of the p53 protein (Figure 1e). However, while in fibroblasts p53 displayed a significant increase at 12 h after irradiation and continued to accumulate up to 24 h, in keratinocytes p53 level reached a peak at 6 h and then drastically decreased to background at 12 h. Moreover, higher levels of UVB-induced p53 protein were observed in fibroblasts than in keratinocytes. In keratinocytes, a rapid but transitory p53 response to UVB was observed also after 2000 J/m (data not shown). From this study, apoptosis and an efficient DNA repair machinery for UV photoproducts emerged as the major defense mechanisms of keratinocytes against the deleterious effects of UVB. Keratinocytes undergo apoptosis at UVB doses that are ineffective in fibroblasts. It has been proposed that the stalling of the transcription machinery at CPD leads to activation of p53, thus initiating apoptosis. This model is strongly supported by the finding that in TCR-defective fibroblasts, derived from patients with Cockayne syndrome, p53 and apoptosis are induced at UVC doses that are Cell Death and Differentiation (2003) 10, 754–756 & 2003 Nature Publishing Group All rights reserved 1350-9047/03

A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity

25.00

Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect.

A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.

Transcription-Associated Breaks in Xeroderma Pigmentosum Group D Cells from Patients with Combined Features of Xeroderma Pigmentosum and Cockayne Syndrome

Significance Xeroderma pigmentosum (XP) is a genetic disorder caused by defective repair of DNA damage. Affected patients are mutated in one of eight genes and develop skin pigmentation changes, skin cancers, ocular surface abnormalities, and, in some cases, acute sunburn and neurodegeneration. The XP proteins are involved in different steps in the repair of DNA damage. Examination of 89 patients, the largest reported cohort under long-term follow-up, by the same multidisciplinary team of clinicians and scientists has revealed unexpected clinical heterogeneity dependent on the affected gene and the exact mutation. Our findings provide new insights into the mechanisms of carcinogenesis, ocular surface disease, and neurodegeneration, as well as providing improved clinical management and more definitive prognostic predictions. Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins.

Genotype-phenotype relationships in trichothiodystrophy patients with novel splicing mutations in the XPD gene

ABSTRACT Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either γ-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.

A novel X-linked trichothiodystrophy associated with a nonsense mutation in RNF113A

Trichothiodystrophy (TTD) is a rare, autosomal recessive neurodevelopmental disorder most commonly caused by mutations in ERCC2 (XPD), a gene that encodes a subunit of the transcription/repair factor IIH (TFIIH). Here, we describe two TTD cases in which detailed biochemical and molecular investigations offered a clue to explain their moderately affected phenotype. Patient TTD22PV showed new mutated XPD alleles: one contains a nonsense mutation (c.1984C>T) encoding a nonfunctional truncated product (p.Gln662X) whereas the second carries a genomic deletion (c.2191‐18_c.2213del) that affects the splicing of intron 22 and generates multiple out‐of‐frame transcripts from codon 731. XPD mRNA from the second allele corresponds to 20% of the total. The predicted proteins, which are longer than normal, affect the cellular repair activity but only partially interfere with TFIIH stability, suggesting that the observed changes in the C‐ter region of XPD cause minor structural changes that do not drastically compromise the transcriptional activity of TFIIH. Patient TTD24PV was compound heterozygous for a typical TTD allele (c.2164C>T, p.Arg722Trp) and for a new XPD allele with a mutation that partially affects intron 10 splicing, resulting in both mutated and normal XPD transcripts (that together represent 15% of the total XPD mRNA). Compared to the previously described TTD compound heterozygotes for the Arg722Trp change, Patient TTD24PVs cells show similar level of TFIIH but increased repair activity, suggesting that even low amounts of normal XPD subunits are able to partially rescue the functionality of TFIIH complexes. Hum Mutat 0, 1–8, 2008.