TTD Cells Research Articles

Evidence of altered Redox Homeostasis in Trichothiodystrophy

Trichothiodystrophy (TTD) is a rare hereditary disease whose prominent feature is brittle hair. Additional clinical signs are physical and neurodevelopmental abnormalities and in about half of the cases hypersensitivity to UV radiation. Although the mutations involved in this condition have been characterized, the correlation between the molecular defects and the plethora of clinical symptoms is not well understood. Recently the presence of a redox unbalance in TTD has been suggested although not clear evidences have been reported on this aspect. Therefore, in the present study, we evaluated the redox status of fibroblasts isolated from a TTD patient. In addition, to understand the ability of TTD cells to respond to oxidative insults, the cells were challenged with H2O2 and mitochondrial O2- and mitochondrial membrane potential were measured in different oxidative conditions. In addition, NRF2/BACH protein levels were also analysed in response to H2O2. The results suggested an aberrant mitochondrial response to oxidative stimuli, an increased baseline oxidative stress status in TTD and an altered NRF2/BACH level. This study emphasises that altered redox homeostasis might play a role in TTD pathogenesis and mitochondria functionality could represent an alternative therapeutic target for this condition to improve patients clinical features.

TFIIH mutations can impact on translational fidelity of the ribosome.

TFIIH is a complex essential for transcription of protein-coding genes by RNA polymerase II, DNA repair of UV-lesions and transcription of rRNA by RNA polymerase I. Mutations in TFIIH cause the cancer prone DNA-repair disorder xeroderma pigmentosum (XP) and the developmental and premature aging disorders trichothiodystrophy (TTD) and Cockayne syndrome. A total of 50% of the TTD cases are caused by TFIIH mutations. Using TFIIH mutant patient cells from TTD and XP subjects we can show that the stress-sensitivity of the proteome is reduced in TTD, but not in XP. Using three different methods to investigate the accuracy of protein synthesis by the ribosome, we demonstrate that translational fidelity of the ribosomes of TTD, but not XP cells, is decreased. The process of ribosomal synthesis and maturation is affected in TTD cells and can lead to instable ribosomes. Isolated ribosomes from TTD patients show an elevated error rate when challenged with oxidized mRNA, explaining the oxidative hypersensitivity of TTD cells. Treatment of TTD cells with N-acetyl cysteine normalized the increased translational error-rate and restored translational fidelity. Here we describe a pathomechanism that might be relevant for our understanding of impaired development and aging-associated neurodegeneration.

Reduced levels of prostaglandin I2 synthase: a distinctive feature of the cancer-free trichothiodystrophy

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I2 synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I2 synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.

Enhanced Human Immunodeficiency Virus-1 Replication in CD4+ T Cells Derived From Individuals With Latent Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV) coinfection increases mortality, accelerates progression to acquired immune deficiency syndrome, and exacerbates tuberculosis disease. However, the impact of pre-existing Mtb infection on subsequent HIV infection has not been fully explored. We hypothesized that Mtb infection creates an immunological environment that influences the course of HIV infection, and we investigated whether pre-existing Mtb infection impacts the susceptibility of CD4+ T cells to HIV-1 infection. Plasma and blood CD4+ T cells isolated from HIV-negative individuals across the Mtb infection spectrum and non-Mtb-infected control individuals were analyzed for inflammation markers and T-cell phenotypes. CD4+ T cells were infected with HIV-1 in vitro and were monitored for viral replication. We observed differences in proinflammatory cytokines and the relative proportion of memory T-cell subsets depending on Mtb infection status. CD4+ T cells derived from individuals with latent Mtb infection supported more efficient HIV-1 transcription, release, and replication. Enhanced HIV-1 replication correlated with higher percentages of CD4+ TEM and TTD cells. Pre-existing Mtb infection creates an immunological environment that reflects Mtb infection status and influences the susceptibility of CD4+ T cells to HIV-1 replication. These findings provide cellular and molecular insights into how pre-existing Mtb infection influences HIV-1 pathogenesis.

XPD/ERCC2 mutations interfere in cellular responses to oxidative stress.

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPD-mutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent γ-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.

Rheumatoid arthritis patients show a decline in peripheral blood CD4+CD161+ T lymphocytes

Background and objectivesAmple evidence suggests a role for T lymphocytes in the pathogenesis of rheumatoid arthritis (RA). CD161, a prototypic NK receptor was shown to confer tissue migratory properties to...

Comparative study of nucleotide excision repair defects between XPD-mutated fibroblasts derived from trichothiodystrophy and xeroderma pigmentosum patients

To get a clue to understand how mutations in the XPD gene result in different skin cancer susceptibilities in patients with xeroderma pigmentosum (XP) or trichothiodystrophy (TTD), a thorough understanding of their nucleotide excision repair (NER) defects is essential. Here, we extensively characterize the possible causes of NER defects in XP-D and in TTD fibroblasts. The 3 XP-D cell strains examined were similarly deficient in repairing UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) from genomic DNA. The severity of NER defects correlated with their UV sensitivities. Possible alterations of TFIIH (which consists of 10 subunits including XPD) were then examined. All XP-D cell strains were normal in their concentrations of TFIIH, and displayed normal abilities to recruit TFIIH to sites of UV-induced DNA damage. However, replication protein A (RPA; single-stranded DNA binding protein) accumulation at DNA damage sites, which probably reflects the in vivo XPD helicase activity of TFIIH, is similarly impaired in all XP-D cell strains. Meanwhile, all 3 TTD cell strains had ∼50% decreases in cellular TFIIH content. Importantly, 2 of the 3 TTD cell strains, which carry the major XPD mutations found in TTD patients, showed defective recruitment of TFIIH to DNA damage sites. Moreover, RPA accumulation at damage sites was impaired in all TTD cell strains to different degrees, which correlated with the severity of their NER defects. These results demonstrate that XP-D and TTD cells are both deficient in the repair of CPDs and 6-4PPs, but TTD cells have more multiple causes for their NER defects than do XP-D cells. Since TFIIH is a repair/transcription factor, TTD-specific alterations of TFIIH possibly result in transcriptional defects, which might be implication for the lack of increased incidence of skin cancers in TTD patients.

Structural basis for group A trichothiodystrophy

Patients with the rare neurodevelopmental repair syndrome known as group A trichothiodystrophy (TTD-A) carry mutations in the gene encoding the p8 subunit of the transcription and DNA repair factor TFIIH. Here we describe the crystal structure of a minimal complex between Tfb5, the yeast ortholog of p8, and the C-terminal domain of Tfb2, the yeast p52 subunit of TFIIH. The structure revealed that these two polypeptides adopt the same fold, forming a compact pseudosymmetric heterodimer via a beta-strand addition and coiled coils interactions between terminal alpha-helices. Furthermore, Tfb5 protects a hydrophobic surface in Tfb2 from solvent, providing a rationale for the influence of p8 in the stabilization of p52 and explaining why mutations that weaken p8-p52 interactions lead to a reduced intracellular TFIIH concentration and a defect in nucleotide-excision repair, a common feature of TTD cells.

Defective Transcription/Repair Factor IIH Recruitment to Specific UV Lesions in Trichothiodystrophy Syndrome

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFIIH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 6-4PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some TTD/XPD mutations affect the recruitment of TFIIH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFIIH complexes carrying an NH(2)-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFIIH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFIIH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

Persistence of repair proteins at unrepaired DNA damage distinguishes diseases withERCC2(XPD) mutations: cancer-prone xeroderma pigmentosum vs. non-cancer-prone trichothiodystrophy

Patients with xeroderma pigmentosum (XP) have a 1,000-fold increase in ultraviolet (UV)-induced skin cancers while trichothiodystrophy (TTD) patients, despite mutations in the same genes, ERCC2 (XPD) or ERCC3 (XPB), are cancer-free. Unlike XP cells, TTD cells have a nearly normal rate of removal of UV-induced 6-4 photoproducts (6-4PP) in their DNA and low levels of the basal transcription factor, TFIIH. We examined seven XP, TTD, and XP/TTD complex patients and identified mutations in the XPD gene. We discovered large differences in nucleotide excision repair (NER) protein recruitment to sites of localized UV damage in TTD cells compared to XP or normal cells. XPC protein was rapidly localized in all cells. XPC was redistributed in TTD, and normal cells by 3 hr postirradiation, but remained localized in XP cells at 24-hr postirradiation. In XP cells recruitment of other NER proteins (XPB, XPD, XPG, XPA, and XPF) was also delayed and persisted at 24 hr (p<0.001). In TTD cells with defects in the XPD, XPB, or GTF2H5 (TTDA) genes, in contrast, recruitment of these NER proteins was reduced compared to normals at early time points (p<0.001) and remained low at 24 hr postirradiation. These data indicate that in XP persistence of NER proteins at sites of unrepaired DNA damage is associated with greatly increased skin cancer risk possibly by blockage of translesion DNA synthesis. In contrast, in TTD, low levels of unstable TFIIH proteins do not accumulate at sites of unrepaired photoproducts and may permit normal translesion DNA synthesis without increased skin cancer.

Trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane pyrimidine dimers and (6-4)photoproducts.

A photosensitive form of trichothiodystrophy (TTD) results from mutations in the same XPD gene as the DNA-repair-deficient genetic disorder xeroderma pigmentosum group D (XP-D). Nevertheless, unlike XP, no increase in skin cancers appears in patients with TTD. Although the ability to repair ultraviolet (UV)-induced DNA damage has been examined to explain their cancer-free phenotype, the information accumulated to date is contradictory. In this study, we determined the repair kinetics of cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts (6-4PP) in three TTD cell strains using an enzyme-linked immunosorbent assay. We found that all three TTD cell strains are deficient in the repair of CPD and of 6-4PP. UV sensitivity correlated well with the severity of repair defects. Moreover, accumulation of repair proteins (XPB and proliferating cell nuclear antigen) at localized DNA damage sites, detected using micropore UV irradiation combined with fluorescent antibody labeling, reflected their DNA repair activity. Importantly, mutations of the XPD gene affected both the recruitment of the TFIIH complex to DNA damage sites and the TFIIH expression. Our results suggest that there is no major difference in the repair defect between TTD and XP-D and that the cancer-free phenotype in TTD is unrelated to a DNA repair defect.

DNA repair and cell survival of normal and xeroderma pigmentosum keratinocytes after UV irradiation

A normal level of UV-induced DNA-repair synthesis (UDS) was observed in fibroblasts from a patient affected by trichothiodystrophy (TTD) without photosensitivity. This finding indicates that the hypersensitivity to UV light and the reduced UDS due to the presence of xeroderma pigmentosum complementation group D mutation (XP-D), described in photosensitive TTD patients, are not constantly associated with TTD. Complementation analysis in heterokaryons, obtained by fusion of repair-proficient with repair-deficient TTD cells, demonstrates that cell from the patient showing normal photosensitivity are able to restore UDS in UV-hypertensitive TTD cells.

The Comet Assay as a Repair Test for Prenatal Diagnosis of Xeroderma Pigmentosum and Trichothiodystrophy

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are autosomal recessive diseases associated with extreme cutaneous photosensitivity, a defect in nucleotide excision repair (NER), and genetic complexity. Severe prognosis and lack of treatment led families at risk to request genetic counseling. Unscheduled DNA synthesis (UDS) is the classic method for diagnosis and requires 4 to 5 wk before conclusion. The use of the alkaline comet assay (single cell gel electrophoresis assay) is proposed as a simple repair test for earlier prenatal diagnosis. Amniotic or chorionic villus cells in two pregnancies at risk for XP and one for TTD were examined in comparison with skin fibroblasts of family members or with repair-proficient or -deficient control cells. The comet assay and the UDS test were performed in parallel. In repair-proficient cells, DNA strand breaks due to the incision of UV-induced DNA damage result in increased migration of high molecular weight DNA in the comet assay. Fetal cells demonstrate repair capacity similar to that of fibroblasts. In incision repair-deficient XP and TTD cells, after post-UV incubation, migration does not occur and comet moments are reduced. Two fetuses belonging to two XP families responded normally and were diagnosed as unaffected. Fetal cells in a TTD family had reduced comet moments and a low UDS. This fetus was diagnosed and confirmed later as affected. Heterozygotes had normal responses with both assays. The comet assay offers discrimination similar to that of the UDS assay in identifying NER-deficient phenotypes. Practical advantages in view of prenatal diagnosis include the reduced number of cells required, a 24-h delay in obtaining results, and no need for radioactivity.

Characteristics of UV-induced Mutation Spectra in Human XP-D/ERCC2 Gene-mutated Xeroderma Pigmentosum and Trichothiodystrophy Cells

To study the relationships between mutagenesis and carcinogenesis, we compared the mutations and their frequency induced by ultraviolet irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in nucleotide excision repair. Whereas XP-D patients develop early skin tumors, TTD patients do not exhibit abnormal levels of cancers.After verification of UV hypersensitivity and DNA repair defect of the immortalized cell lines XP-D and TTD compared with a wild-type cell line, UV-induced mutagenesis was studied with a new shuttle vector pR2, carrying the targetlacZ′ gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in XP-D cells (87%) and most mutations were targeted on dipyrimidine sites, chiefly on the cytosine at 5′-TC-3′ sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5′-TCPur3′ sites, while mutations in normal and TTD cells were mostly at 5′-TCC-3′ sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines.Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.

Stable SV40-transformation and characterisation of some DNA repair properties of fibroblasts from a trichothiodystrophy patient

To characterize nucleotide excision repair properties of cells from trichothiodystrophy (TTD) patients genetically-related to the xeroderma pigmentosum (XP) group D, TTD skin fibroblasts from two unrelated patients (TTD1VI and TTD2VI) belonging to the TTD/XPD group were transformed with a plasmid containing SV40 large T antigen-coding sequences and some DNA repair properties, such as unscheduled DNA synthesis (UDS), UV-survival, in vitro repair synthesis of cell extracts and reactivation of UV-irradiated reported plasmid were studied. Results showed that: a) both untransformed and transformed TTD cells present a reduced UV-survival, compared to wild-type cells, but at significantly less reduced levels that XP-D cells; b) reduced repair activities were detected in both TTD and XP-D transformed cells by using in vitro cell free extract repair and reactivation of UV-irradiated plasmid procedures, and those relative reduced extents correlated with respective UV-survival; c) surprisingly, near wild-type UDS levels were detected in TTD2VILas transformed cells at different passages after the crisis, suggesting a phenotypic reversion of this transformed cell line; d) fluoro-cytometric analysis of TTD2VILas cells revealed a strong increase of a cell population containing a DNA amount more than twice as high than that of untransformed cells; finally, e) when UDS data were normalized to the DNA content in TTD2VILas cells, it appeared that the repair efficiency was only slightly higher than in untransformed cells. This implies that in transformed cells DNA repair properties should be evaluated, taking into account additional parameters. We obtained an immortalized TTD cell line which maintains DNA repair properties similar to those of parental untransformed cells and may be used to characterize the TTD defect at genetic, molecular and biochemical levels.

Correction by the ERCC2 gene of UV sensitivity and repair deficiency phenotype in a subset of trichothiodystrophy cells.

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.

UV-induced mutations in a shuttle vector replicated in repair deficient trichothiodystrophy cells differ with those in genetically-related cancer prone xeroderma pigmentosum.

Trichothiodystrophy (TTD) is a rare genetic disease associated in approximately 50% of patients with DNA repair deficiency analogous to that found in xeroderma pigmentosum group D (XP-D) patients. Although XP-D patients exhibit a very high level of skin cancer on sun-exposed parts, TTD is not associated with cancer. We analysed UV-induced mutations in TTD cells and compared them to data in XP-D in order to determine if the molecular mechanisms of mutagenesis can explain the discrepancies between these two syndromes. We first immortalized a fibroblast TTD line with an ori(-)-SV40 plasmid. To investigate the kinds of mutations induced in TTD cells, we used an UV-irradiated (at 254 nm) shuttle vector carrying the supF tRNA gene as a target. We compared our data with those published by others with the same pZ189 vector in normal and XP-D fibroblast lines (Bredberg et al., Proc. Natl. Acad. Sci. USA, 83, 8273-8277; Seetharam et al., J. Clin. Invest., 80, 1613-1617). The frequency of mutants increased linearly with UV dose and the slope was > 4 times steeper in TTD cells than that observed in normal cells. The mutation frequency was almost identical between XP-D and TTD cells. Sequence analysis of the supF tRNA gene showed that 96% of mutations obtained in TTD cells are base substitutions. Single base substitutions were found in 62% of mutants in TTD cells while they corresponded to 86% in XP-D cells. The frequency of multiple mutations in TTD cells (26%) was similar to that in normal cells (27%) and much higher than that in XP-D cells (9%). Despite the fact that the same gene is mutated in TTD and XP patients, the molecular characteristics of mutagenesis are not identical. The fact that the frequency of mutations in TTD and XP cells are similar shows that a high level of UV-induced mutations is therefore not always directly related to cancer-proneness. Other factors such as catalase activity and immuno-surveillance may intervene in cancer incidence.

Genetic heterogeneity of the excision repair defect associated with trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare autosomal recessive disease characterized by brittle hair with reduced sulfur content, mental and physical retardation, a peculiar face and ichthyosis. Photosensitivity has been reported in approximately 20% of the cases in the literature. DNA repair investigations demonstrated that clinical photosensitivity is usually associated with an enhancement of the cellular UV-sensitivity and that the repair defect is in the same gene as in patients from group D of xeroderma pigmentosum (XP). In this paper we describe the characterization of 13 further TTD patients; a defect in the nucleotide-excision repair was observed in fibroblast strains from 10 patients, confirming that TTD is frequently associated with DNA repair defects. Genetic analysis based on complementation studies demonstrated the presence of the XP-D defect in seven repair-defective TTD cases, indicating definitively that the concurrence of TTD with XP-D is not a sporadic or casual event. However, three further cell strains (TTD4VI and TTD6VI from two French siblings and TTD1BR from an English patient) showed restoration of normal UV-induced DNA repair synthesis after fusion with XP or TTD cells belonging to XP group D. These observations, which give the first indication that TTD is associated with repair defects behaving differently in the functional test of complementation, suggest some kind of causal connection between defective excision-repair factors and clinical features diagnostic for TTD. A peculiar aspect of TTD in which repair deficiencies are not related to an increased susceptibility to cancer is confirmed also in all the repair-defective TTD patients investigated in this paper.

DNA repair investigations in nine Italian patients affected by trichothiodystrophy

Trichothiodystrophy (TTD) is a rare autosomal recessive disorder characterized by brittle hair, mental and growth retardation, peculiar face, ichthyosis, and in 20% of the reported cases photosensitivity. Cellular photosensitivity due to the same genetic defect present in xeroderma pigmentosum group D (XP-D) has been described in several patients.Nine patients with clinical symptoms diagnostic for TTD have been identified in Italy to date. We report the results of DNA repair investigations performed in cultured fibroblasts from these patients and 8 TTD parents.Survival, DNA repair synthesis and RNA synthesis following UV irradiation were all normal in the 8 TTD heterozygous cell strains. Among the 9 TTD-affected individuals, normal cellular UV sensitivity was observed in the 2 patients without signs of clinical photosensitivity. In contrast, the other 7 TTD cell strains showed a notable reduction in UV-induced DNA repair synthesis (UDS) levels, ranging between 40% and 5–15% of normal values. Complementation analysis indicated that in the repair-deficient TTD cell strains the genetic defect is the same as that present in XP-D cells.The biochemical heterogeneity of the XP-D defect in TTD patients characterized by different degrees of defective UDS results in different patterns of response to the killing effect of UV light in non-proliferating cells.