TFIIH stabilization recovers the DNA repair and transcription dysfunctions in thermo-sensitive 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. The photosensitive form of TTD (PS-TTD) is most commonly caused by mutations in the ERCC2/XPD gene encoding a subunit of the transcription/DNA repair complex TFIIH. Here we report novel ERCC2/XPD mutations affecting proper protein folding, which generate thermo-labile forms of XPD associated with thermo-sensitive phenotypes characterized by reversible aggravation of TTD clinical signs during episodes of fever. In patient cells, the newly identified XPD variants result in thermo-instability of the whole TFIIH complex and consequent temperature-dependent defects in DNA repair and transcription. Improving the protein folding process by exposing patient cells to low temperature or to the chemical chaperone glycerol allowed rescue of TFIIH thermo-instability and a concomitant recovery of the complex activities. Besides providing a rationale for the peculiar thermo-sensitive clinical features of these new cases, the present findings demonstrate how variations in the cellular concentration of mutated TFIIH impact the cellular functions of the complex and underlie how both quantitative and qualitative TFIIH alterations contribute to TTD clinical features.

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.

Protein instability associated with AARS1 and MARS1 mutations causes trichothiodystrophy.

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.

Expansion of the clinical and molecular spectrum of an XPD-related disorder linked to biallelic mutations in ERCC2 gene.

Bi-allelic inactivation of XPD protein, a nucleotide excision repair (NER) signaling pathway component encoded by ERCC2 gene, has been associated with several defective DNA repair phenotypes, including xeroderma pigmentosum, photosensitive trichothiodystrophy, and cerebro-oculo-facio-skeletal syndrome. We report a pediatric patient harboring two compound heterozygous variants in ERCC2 gene, c.361-1G>A and c.2125A>C (p.Thr709Pro), affected by severe postnatal growth deficiency, microcephaly, facial dysmorphisms and pilocytic astrocytoma of the brainstem. Some of these features point to a DNA repair syndrome, and altogether delineate a phenotype differentiating from disorders known to be associated with ERCC2 mutations. The DNA repair efficiency following UV irradiation in the proband's skin fibroblasts was defective indicating that the new set of ERCC2 alleles impacts on NER efficiency. Sequencing analysis on tumor DNA did not reveal any somatic deleterious point variant in cancer-related genes, while SNP-array analysis disclosed a 2 Mb microduplication involving the 7q34 region, spanning from KIAA1549 to BRAF, and resulting in the KIAA1549:BRAF fusion protein, a marker of pilocytic astrocytoma. In conclusion, this report expands the clinical and mutational spectrum of ERCC2-related disorders.

#StayHomeStayFit: UNIMI's approach to online healthy lifestyle promotion during the COVID-19 pandemic.

The COVID-19 lockdown imposed radical changes in the lifestyles of the population through isolation measures, with considerable health, social, psychological and economic consequences. Lockdown measures may have exacerbated negative population behaviors regarding exercise and nutrition, with risk of weight gain and obesity, collectively predisposing to increased cardiometabolic risk and mortality. At particular risk of deleterious consequences were patients such as those affected by chronic non communicable diseases (CNCD). The benefits of regular exercise are evident at several levels of CNCD prevention, however, from a public health standpoint, it is important to consider they are also related to improved stress management, work/academic performance, and reduced illegal behavior, isolation and depression. Therefore, during enforced isolation, a primary goal for all individuals is to maintain energy balance. During lockdown, several lifestyle interventions were posted online, with the internet playing a major role in exercise and fitness promotion. Among these one must recognize the ambitious "#StayHomeStayFit" project by the University of Milan, providing useful general information and trustworthy advice regarding nutrition, physical activity, and psychological support, for the general population in a time of need. Data showed a total of 21224 views on various social media and webpages during the reference period, with a mean time of 4' 17'' spent per page/video. Given the health risks associated with population physical inactivity and unhealthy lifestyle, policymakers should evaluate the benefits of projects such as #StayHomeStayFit and consider how to maximize population perception and reach. After all, additional COVID-19 lockdowns might be implemented in the future.

Heterogeneity and overlaps in nucleotide excision repair disorders.

Nucleotide excision repair (NER) is an essential DNA repair pathway devoted to the removal of bulky lesions such as photoproducts induced by the ultraviolet (UV) component of solar radiation. Deficiencies in NER typically result in a group of heterogeneous distinct disorders ranging from the mild UV sensitive syndrome to the cancer-prone xeroderma pigmentosum and the neurodevelopmental/progeroid conditions trichothiodystrophy, Cockayne syndrome and cerebro-oculo-facio-skeletal-syndrome. A complicated genetic scenario underlines these disorders with the same gene linked to different clinical entities as well as different genes associated with the same disease. Overlap syndromes with combined hallmark features of different NER disorders can occur and sporadic presentations showing extra features of the hematological disorder Fanconi Anemia or neurological manifestations mimicking Hungtinton disease-like syndromes have been described. Here, we discuss the multiple functions of the five major pleiotropic NER genes (ERCC3/XPB, ERCC2/XPD, ERCC5/XPG, ERCC1 and ERCC4/XPF) and their relevance in phenotypic complexity. We provide an update of mutational spectra and examine genotype-phenotype relationships. Finally, the molecular defects that could explain the puzzling overlap syndromes are discussed.

Bi-allelic TARS Mutations Are Associated with Brittle Hair Phenotype.

Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEß), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.

Functional and clinical relevance of novel mutations in a large cohort of patients with Cockayne syndrome.

BACKGROUND: Cockayne syndrome (CS) is a rare, autosomal recessive multisystem disorder characterised by prenatal or postnatal growth failure, progressive neurological dysfunction, ocular and skeletal abnormalities and premature ageing. About half of the patients with symptoms diagnostic for CS show cutaneous photosensitivity and an abnormal cellular response to UV light due to mutations in either the ERCC8/CSA or ERCC6/CSB gene. Studies performed thus far have failed to delineate clear genotype-phenotype relationships. We have carried out a four-centre clinical, molecular and cellular analysis of 124 patients with CS. METHODS AND RESULTS: We assigned 39 patients to the ERCC8/CSA and 85 to the ERCC6/CSB genes. Most of the genetic variants were truncations. The missense variants were distributed non-randomly with concentrations in relatively short regions of the respective proteins. Our analyses revealed several hotspots and founder mutations in ERCC6/CSB. Although no unequivocal genotype-phenotype relationships could be made, patients were more likely to have severe clinical features if the mutation was downstream of the PiggyBac insertion in intron 5 of ERCC6/CSB than if it was upstream. Also a higher proportion of severely affected patients was found with mutations in ERCC6/CSB than in ERCC8/CSA. CONCLUSION: By identifying >70 novel homozygous or compound heterozygous genetic variants in 124 patients with CS with different disease severity and ethnic backgrounds, we considerably broaden the CSA and CSB mutation spectrum responsible for CS. Besides providing information relevant for diagnosis of and genetic counselling for this devastating disorder, this study improves the definition of the puzzling genotype-phenotype relationships in patients with CS.

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 (TFIIEß). 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 TFIIEß) 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.

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

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.

Reference genes for gene expression analysis in proliferating and differentiating human keratinocytes.

Abnormalities in keratinocyte growth and differentiation have a pathogenic significance in many skin disorders and result in gene expression alterations detectable by quantitative real-time RT-PCR (qRT-PCR). Relative quantification based on endogenous control (EC) genes is the commonly adopted approach, and the use of multiple reference genes from independent pathways is considered a best practice guideline, unless fully validated EC genes are available. The literature on optimal reference genes during in vitro calcium-induced differentiation of normal human epidermal keratinocytes (NHEK) is inconsistent. In many studies, the expression of target genes is compared to that of housekeeping genes whose expression, however, significantly varies during keratinocyte differentiation. Here, we report the results of our investigations on the expression stability of 15 candidate EC genes, including those commonly used as reference in expression analysis by qRT-PCR, during NHEK calcium-induced differentiation. We demonstrate that YWHAZ and UBC are extremely stable genes, and therefore, they represent optimal EC genes for expression studies in proliferating and calcium-induced differentiating NHEK. Furthermore, we demonstrate that YWHAZ/14-3-3-zeta is a suitable reference for quantitative comparison of both transcript and protein levels.

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

BACKGROUND: Trichothiodystrophy (TTD) is a group of rare autosomal recessive disorders that variably affect a wide range of organs derived from the neuroectoderm. The key diagnostic feature is sparse, brittle, sulfur deficient hair that has a 'tiger-tail' banding pattern under polarising light microscopy. PATIENTS AND METHODS: We describe two male cousins affected by TTD associated with microcephaly, profound intellectual disability, sparse brittle hair, aged appearance, short stature, facial dysmorphism, seizures, an immunoglobulin deficiency, multiple endocrine abnormalities, cerebellar hypoplasia and partial absence of the corpus callosum, in the absence of cellular photosensitivity and ichthyosis. Obligate female carriers showed 100% skewed X-chromosome inactivation. Linkage analysis and Sanger sequencing of 737 X-chromosome exons and whole exome sequencing was used to find the responsible gene and mutation. RESULTS: Linkage analysis localised the disease allele to a 7.75 Mb interval from Xq23-q25. We identified a nonsense mutation in the highly conserved RNF113A gene (c.901 C>T, p.Q301*). The mutation segregated with the disease in the family and was not observed in over 100,000 control X chromosomes. The mutation markedly reduced RNF113A protein expression in extracts from lymphoblastoid cell lines derived from the affected individuals. CONCLUSIONS: The association of RNF113A mutation with non-photosensitive TTD identifies a new locus for these disorders on the X chromosome. The extended phenotype within this family includes panhypopituitarism, cutis marmorata and congenital short oesophagus.

From laboratory tests to functional characterisation of Cockayne syndrome.

The significant progress made over the last few years on the pathogenesis of Cockayne syndrome (CS) greatly improved our knowledge on several aspects crucial for development and ageing, demonstrating that this disorder, even if rare, represents a valuable tool to clarify key aspects of human health. Primary cells from patients have been instrumental to elucidate the multiple roles of CS proteins and to approach the dissection of the complex interplay between repair and transcription that is central to the CS clinical phenotype. Here we discuss the results of the cellular assays applied for confirmation of the clinical diagnosis as well as the results of genetic and molecular studies in DNA repair defective patients. Furthermore, we provide a general overview of recent in vivo and in vitro studies indicating that both CSA and CSB proteins are involved in distinct aspects of the cellular responses to UV and oxidative stress, transcription and regulation of gene expression, chromatin remodelling, redox balance and cellular bioenergetics. In light of the literature data, we will finally discuss how inactivation of specific functional roles of CS proteins may differentially affect the phenotype, thus explaining the wide range in type and severity of symptoms reported in CS patients.

XPD mutations in trichothiodystrophy hamper collagen VI expression and reveal a role of TFIIH in transcription derepression.

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

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

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 TTD24PV's 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.

Mutations in the C7orf11 (TTDN1) gene in six nonphotosensitive trichothiodystrophy patients: no obvious genotype-phenotype relationships.

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.

Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome.

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 gamma-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.

Two new XPD patients compound heterozygous for the same mutation demonstrate diverse clinical features.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are both rare autosomal recessive disorders with defects in DNA repair. They are usually distinct both clinically and genetically but in rare cases, patients exhibit the clinical characteristics of both diseases concurrently. We report two new phenotypically distinct cases of XP with additional features of CS (xeroderma pigmentosum and Cockayne syndrome crossover syndrome (XP/CS)) carrying an identical mutation (G47R) in the XPD gene within the N terminus of the protein. Both patients had clinical features of XP and CS but only one fulfilled most criteria for diagnosing CS. Unusually, patient 1 developed early skin cancer, in contrast to patient 2, who never developed any malignancies. Cells from both these patients have repair defects typical of xeroderma pigmentosum complementation group D (XPD) cells, but also had the phenotype of uncontrolled DNA breakage found specifically in XPD/CS cells and similarly reduced levels of TFIIH. Despite these similarities between our two patients, their clinical features are quite different and the clinical severity correlates with other cellular responses to ultraviolet irradiation.

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.

True XP group E patients have a defective UV-damaged DNA binding protein complex and mutations in DDB2 which reveal the functional domains of its p48 product.

Xeroderma pigmentosum (XP) is a skin cancer-prone autosomal recessive disease characterized by inability to repair UV-induced DNA damage. The major form of XP is defective in nucleotide excision repair (NER) and comprises seven complementation groups (A-G). The genes defective in all groups have been identified unambiguously with the exception of group E. The cells of some XP-E patients are deficient in a protein complex (consisting of two subunits: p127/DDBI and p48/DDB2) which binds to UV-damaged DNA (UV-DDB) and is specifically involved in the removal of photoproducts from the non-transcribed regions of the genome. However, other XP-E patients have been reported not to lack UV-damaged DNA binding activity (DDB(+)). Here we describe several genetically unrelated XP-E patients, not previously analyzed in depth, each carrying two mutated alleles for DDB2, causing either a single amino acid change or a protein truncation or internal deletion. These defects result in a severe decrease of detectable p48 protein, abolish interaction with the p127 subunit, and produce a deficiency in UV-DDB binding activity (DDB(-)). The role of p48 in the repair defect of these patients was demonstrated in vivo and in vitro. Investigation of four DDB(+) cell strains from patients previously assigned to XP-E, allowed us to reclassify all of them into other groups and to show that they do not share the molecular and biochemical features typical for XP-E. Besides confirming that the true XP-E phenotype is DDB(-), resulting from defects in a single gene, DDB2, our results identify the functional domains of the corresponding p48 protein.