Impaired B-cell function in ERCC2 deficiency.

Background: Trichothiodystrophy-1 (TTD1) is an autosomal-recessive disease and caused by mutations in ERCC2, a gene coding for a subunit of the TFIIH transcription and nucleotide-excision repair (NER) factor. In almost half of these patients infectious susceptibility has been reported but the underlying molecular mechanism leading to immunodeficiency is largely unknown. Objective: The aim of this study was to perform extended molecular and immunological phenotyping in patients suffering from TTD1. Methods: Cellular immune phenotype was investigated using multicolor flow cytometry. DNA repair efficiency was evaluated in UV-irradiation assays. Furthermore, early BCR activation events and proliferation of TTD1 lymphocytes following DNA damage induction was tested. In addition, we performed differential gene expression analysis in peripheral lymphocytes of TTD1 patients. Results: We investigated three unrelated TTD1 patients who presented with recurrent infections early in life of whom two harbored novel ERCC2 mutations and the third patient is a carrier of previously described pathogenic ERCC2 mutations. Hypogammaglobulinemia and decreased antibody responses following vaccination were found. TTD1 B-cells showed accumulation of γ-H2AX levels, decreased proliferation activity and reduced cell viability following UV-irradiation. mRNA sequencing analysis revealed significantly downregulated genes needed for B-cell development and activation. Analysis of B-cell subpopulations showed low numbers of naïve and transitional B-cells in TTD1 patients, indicating abnormal B-cell differentiation in vivo. Conclusion: In summary, our analyses confirmed the pathogenicity of novel ERCC2 mutations and show that ERCC2 deficiency is associated with antibody deficiency most likely due to altered B-cell differentiation resulting from impaired BCR-mediated B-cell activation and activation-induced gene transcription.

Cerebro-oculo-facio-skeletal syndrome caused by the homozygous pathogenic variant Gly47Arg in ERCC2.

DNA damage repair is a pivotal mechanism in life. The nucleotide excision repair pathway protects the cells against DNA damage and involves XPD, an ATP dependent helicase that is part of the multisubunit protein complex TFIIH. XPD is encoded by the excision repair cross-complementation group 2 gene (ERCC2). Only three patients with cerebro-oculo-facio-skeletal syndrome (COFS), caused by mutations in ERCC2, have been published so far. This report describes a boy with the homozygous amino acid change p.Gly47Arg in XPD. He presented with profound microcephaly, psychomotor retardation, failure to thrive, cutaneous photosensitivity, a bilateral hearing deficit and optic atrophy, thrombocytopenia, and recurrent episodes of pneumonia. We report the first homozygous occurrence of the pathogenic variant Gly47Arg in the ERCC2 gene. Occurring homozygous, this variant was associated with COFS syndrome, leading to early death of the patient at the age of 21 months.

Frequent retrotransposition of endogenous genes in ERCC2-deficient cells derived from a patient with xeroderma pigmentosum.

BACKGROUND: Retrotransposition of protein-coding genes is thought to occur due to the existence of numerous processed pseudogenes in both animals and plants. Unlike retrotransposons including Alu and LINE-1, direct evidence of such retrotransposition events has not been reported to date. Even if such an event occurs in a somatic cell, it is almost impossible to detect it using bulk of cells as a sample. Single-cell analyses or other techniques are needed. METHODS: In order to examine genetic stability of stem cells, we have established induced pluripotent stem cell (iPSC) lines from several patients with DNA repair-deficiency disorders, such as ataxia telangiectasia and xeroderma pigmentosum, along with healthy controls. Performing whole-exome sequencing analyses of these parental and iPSC lines, we compiled somatic mutations accumulated by the deficiency of DNA repair mechanisms. Whereas most somatic mutations cannot be detected in bulk, cell reprogramming enabled us to observe all the somatic mutations which had occurred in the cell line. Patterns of somatic mutations should be distinctive depending on which DNA repair gene is impaired. RESULTS: The comparison revealed that deficiency of ATM and XPA preferentially gives rise to indels and single-nucleotide substitutions, respectively. On the other hand, deficiency of ERCC2 caused not only single-nucleotide mutations but also many retrotranspositions of endogenous genes, which were readily identified by examining removal of introns in whole-exome sequencing. Although the number was limited, those events were also detected in healthy control samples. CONCLUSIONS: The present study exploits clonality of iPSCs to unveil somatic mutation sets that are usually hidden in bulk cell analysis. Whole-exome sequencing analysis facilitated the detection of retrotransposition mutations. The results suggest that retrotranspositions of human endogenous genes are more frequent than expected in somatic cells and that ERCC2 plays a defensive role against transposition of endogenous and exogenous DNA fragments.

XPD localizes in mitochondria and protects the mitochondrial genome from oxidative DNA damage.

Xeroderma pigmentosum group D (XPD/ERCC2) encodes an ATP-dependent helicase that plays essential roles in both transcription and nucleotide excision repair of nuclear DNA, however, whether or not XPD exerts similar functions in mitochondria remains elusive. In this study, we provide the first evidence that XPD is localized in the inner membrane of mitochondria, and cells under oxidative stress showed an enhanced recruitment of XPD into mitochondrial compartment. Furthermore, mitochondrial reactive oxygen species production and levels of oxidative stress-induced mitochondrial DNA (mtDNA) common deletion were significantly elevated, whereas capacity for oxidative damage repair of mtDNA was markedly reduced in both XPD-suppressed human osteosarcoma (U2OS) cells and XPD-deficient human fibroblasts. Immunoprecipitation-mass spectrometry analysis was used to identify interacting factor(s) with XPD and TUFM, a mitochondrial Tu translation elongation factor was detected to be physically interacted with XPD. Similar to the findings in XPD-deficient cells, mitochondrial common deletion and oxidative damage repair capacity in U2OS cells were found to be significantly altered after TUFM knock-down. Our findings clearly demonstrate that XPD plays crucial role(s) in protecting mitochondrial genome stability by facilitating an efficient repair of oxidative DNA damage in mitochondria.

[The metabolic and molecular bases of Cockayne syndrome]. / Las bases metabólicas y moleculares del síndrome de Cockayne.

Cockayne is a segmental progeroid syndrome that has autosomal recessive inheritance pattern. It is mainly characterized by Intrauterine growth retardation, severe postnatal growth deficiency, cachectic dwarfism, microcephaly, wizened face, sensorineural hearing loss, cataracts, dental caries, cardiac arrhythmias, hypertension, atherosclerosis, proteinuria, micropenis, renal failure, skeletal abnormalities, skin photosensitivity, decreased subcutaneous adipose tissue, cerebral atrophy, dementia, basal ganglia calcifications, ataxia and apraxia. It has a complex phenotype given by genetic heterogeneity. There are five gene responsible for this syndrome: CSA, CSB, XPB, XPD and XPG, in which various mutations have been found. The biochemical effect of these mutations includes dysfunctional protein of the repair system for oxidative damage to DNA, the complex coupled to transcription and the nucleotide excision repair system. Considering the role played for these proteins and its effects on clinical phenotype when they are deficient, we suggest that these genes might be candidates for analyzing susceptibility to common chronic degenerative diseases related to oxidative stress and aging.

UV-mediated NF-kappaB activation is abolished in deficient XPC/D primary fibroblasts.

Genetic pathologies involving deficits in DNA repair, like xeroderma pigmentosum (XP), show recurrent cell death, tissue degeneration and oncogenesis due to high sensitivity to ultraviolet radiation (UV). Various inducers including UV activate NF-kappaB, a pathway largely involved in cell proliferation and apoptosis. However, the mechanism(s) involving NF-kappaB activation by UV are poorly understood. To improve this knowledge, we examined NF-kappaB in two XP cell groups (XPC and XPD/TTD). XPC/D primary fibroblasts possess functional NF-kappaB dimers, and pro-inflammatory cytokines consistently activate NF-kappaB pathway. Contrarily, UV-mediated NF-kappaB activation is practically absent, whereas kappaB-specific DNA binding and transcriptional activity are dramatically undermined. These results indicate that lack of UV responsiveness at the NF-kappaB level is a common feature of XPC/D cells, suggesting that XP proteins might act upstream on NF-kappaB activity induced by UV. These observations help us to better understand the UV sensitivity and compromised survival of XP deficient cells.

Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells.

Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase delta/epsilon inhibitor, aphidicolin, but not 2',3'-dideoxythymidine triphosphate (ddTTP), a polymerase beta inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.

Accelerated aging pathology in ad libitum fed Xpd(TTD) mice is accompanied by features suggestive of caloric restriction.

Trichothiodystrophy (TTD) patients with a mutation in the XPD gene of nucleotide excision repair (NER) have a short life span and show various features of premature aging, thereby linking DNA damage to the aging process. Xpd(TTD) mutant mice share many features with TTD patients, including a shorter life span, accompanied by a segmental progeroid phenotype. Here we report new pathology features supportive to the premature aging phenotype of Xpd(TTD) mice. Strikingly, accelerated aging pathology is accompanied by signs suggestive of caloric restriction (CR), a condition usually linked to retardation of age-related pathology and life extension. Accelerated aging symptoms in Xpd(TTD) mice are most likely due to accumulation of endogenously generated DNA damage and compromised transcription leading to cell death, whereas CR symptoms may reflect the need of Xpd(TTD) mice to reduce metabolism (ROS production) in an attempt to extend their life span. Our current findings in Xpd(TTD) mice further strengthen the link between DNA damage, repair and aging.