Publications by authors named "Nicolaas G J Jaspers"

42 Publications

Trichothiodystrophy causative TFIIEβ mutation affects transcription in highly differentiated tissue.

Hum Mol Genet 2017 12;26(23):4689-4698

Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus MC, Rotterdam, The Netherlands.

The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEβ) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEβ strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.
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http://dx.doi.org/10.1093/hmg/ddx351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886110PMC
December 2017

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

Proc Natl Acad Sci U S A 2016 Mar 16;113(9):E1236-45. Epub 2016 Feb 16.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom;

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.
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http://dx.doi.org/10.1073/pnas.1519444113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780618PMC
March 2016

The Cerebro-oculo-facio-skeletal Syndrome Point Mutation F231L in the ERCC1 DNA Repair Protein Causes Dissociation of the ERCC1-XPF Complex.

J Biol Chem 2015 Aug 17;290(33):20541-55. Epub 2015 Jun 17.

From the Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands,

The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation.
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http://dx.doi.org/10.1074/jbc.M114.635169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536458PMC
August 2015

Attenuated XPC expression is not associated with impaired DNA repair in bladder cancer.

PLoS One 2015 30;10(4):e0126029. Epub 2015 Apr 30.

Department of Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands.

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126029PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4416023PMC
April 2016

Pollitt syndrome patients carry mutation in TTDN1.

Meta Gene 2014 Dec 30;2:616-8. Epub 2014 Aug 30.

Department of Bioinformatics, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.

Complete human genome sequencing was used to identify the causative mutation in a family with Pollitt syndrome (MIM #275550), comprising two non-consanguineous parents and their two affected children. The patient's symptoms were reminiscent of the non-photosensitive form of recessively inherited trichothiodystrophy (TTD). A mutation in the TTDN1/C7orf11 gene, a gene that is known to be involved in non-photosensitive TTD, had been excluded by others by Sanger sequencing. Unexpectedly, we did find a homozygous single-base pair deletion in the coding region of this gene, a mutation that is known to cause non-photosensitive TTD. The deleterious variant causing a frame shift at amino acid 93 (C326delA) followed the right mode of inheritance in the family and was independently validated using conventional DNA sequencing. We expect this novel DNA sequencing technology to help redefine phenotypic and genomic variation in patients with (mono) genetic disorders in an unprecedented manner.
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http://dx.doi.org/10.1016/j.mgene.2014.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287846PMC
December 2014

Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

PLoS Genet 2014 Oct 9;10(10):e1004686. Epub 2014 Oct 9.

Department of Genetics, Erasmus University Medical Center, Rotterdam, The Netherlands; Department of Vascular Surgery, Erasmus University Medical Center, Rotterdam, The Netherlands.

As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.
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http://dx.doi.org/10.1371/journal.pgen.1004686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191938PMC
October 2014

ERCC6 dysfunction presenting as progressive neurological decline with brain hypomyelination.

Am J Med Genet A 2014 Nov 22;164A(11):2892-900. Epub 2014 Sep 22.

NIH Undiagnosed Diseases Program, Common Fund, Office of the Director, NIH and National Human Genome Research Institute, NIH, Bethesda, Maryland.

Mutations in ERCC6 are associated with growth failure, intellectual disability, neurological dysfunction and deterioration, premature aging, and photosensitivity. We describe siblings with biallelic ERCC6 mutations (NM_000124.2:c. [543+4delA];[2008C>T]) and brain hypomyelination, microcephaly, cognitive decline, and skill regression but without photosensitivity or progeria. DNA repair assays on cultured skin fibroblasts confirmed a defect of transcription-coupled nucleotide excision repair and increased ultraviolet light sensitivity. This report expands the disease spectrum associated with ERCC6 mutations.
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http://dx.doi.org/10.1002/ajmg.a.36709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4205164PMC
November 2014

Mutations in ERCC4, encoding the DNA-repair endonuclease XPF, cause Fanconi anemia.

Am J Hum Genet 2013 May 25;92(5):800-6. Epub 2013 Apr 25.

Genome Instability and DNA Repair Group, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.

Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone marrow failure and predisposition to cancer. FA-associated gene products are involved in the repair of DNA interstrand crosslinks (ICLs). Fifteen FA-associated genes have been identified, but the genetic basis in some individuals still remains unresolved. Here, we used whole-exome and Sanger sequencing on DNA of unclassified FA individuals and discovered biallelic germline mutations in ERCC4 (XPF), a structure-specific nuclease-encoding gene previously connected to xeroderma pigmentosum and segmental XFE progeroid syndrome. Genetic reversion and wild-type ERCC4 cDNA complemented the phenotype of the FA cell lines, providing genetic evidence that mutations in ERCC4 cause this FA subtype. Further biochemical and functional analysis demonstrated that the identified FA-causing ERCC4 mutations strongly disrupt the function of XPF in DNA ICL repair without severely compromising nucleotide excision repair. Our data show that depending on the type of ERCC4 mutation and the resulting balance between both DNA repair activities, individuals present with one of the three clinically distinct disorders, highlighting the multifunctional nature of the XPF endonuclease in genome stability and human disease.
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http://dx.doi.org/10.1016/j.ajhg.2013.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644630PMC
May 2013

A Turkish trichothiodystrophy patient with homozygous XPD mutation and genotype-phenotype relationship.

J Dermatol 2012 Dec 5;39(12):1016-21. Epub 2012 Oct 5.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA.

Trichothiodystrophy (TTD) is a rare, recessive condition involving multiple organs and systems. Four genes associated with nuclear excision repair have been described in the molecular etiology of TTD. There is a significant heterogeneity of clinical and laboratory findings of TTD, even in individuals carrying the same mutation. Worldwide, approximately 120 cases have been reported, mostly from Western populations and the mutations are compound heterozygous. We herein present clinical and laboratory findings of a female patient with a homozygous mutation, R722W, in the XPD gene. To date, two patients who carry the same mutation have been reported. Our genotype-phenotype correlation study showed patients who carry R722W mutation have a more severe TTD phenotype than other types of mutations.
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http://dx.doi.org/10.1111/j.1346-8138.2012.01662.xDOI Listing
December 2012

SMARCAL1 deficiency predisposes to non-Hodgkin lymphoma and hypersensitivity to genotoxic agents in vivo.

Am J Med Genet A 2012 Sep 7;158A(9):2204-13. Epub 2012 Aug 7.

Child and Family Research Institute, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder with prominent skeletal, renal, immunological, and ectodermal abnormalities. It is caused by mutations of SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), which encodes a DNA stress response protein. To determine the relationship of this function to the SIOD phenotype, we profiled the cancer prevalence in SIOD and assessed if defects of nucleotide excision repair (NER) and nonhomologous end joining (NHEJ), respectively, explained the ectodermal and immunological features of SIOD. Finally, we determined if Smarcal1(del/del) mice had hypersensitivity to irinotecan (CPT-11), etoposide, and hydroxyurea (HU) and whether exposure to these agents induced features of SIOD. Among 71 SIOD patients, three had non-Hodgkin lymphoma (NHL) and one had osteosarcoma. We did not find evidence of defective NER or NHEJ; however, Smarcal1-deficient mice were hypersensitive to several genotoxic agents. Also, CPT-11, etoposide, and HU caused decreased growth and loss of growth plate chondrocytes. These data, which identify an increased prevalence of NHL in SIOD and confirm hypersensitivity to DNA damaging agents in vivo, provide guidance for the management of SIOD patients.
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http://dx.doi.org/10.1002/ajmg.a.35532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3429644PMC
September 2012

The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition.

Structure 2012 Apr 3;20(4):667-75. Epub 2012 Apr 3.

Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first HhH motif contacts the ssDNA phosphate backbone. One guanine base is flipped out of register and positioned in a pocket contacting residues from both HhH motifs of XPF. Comparison to other HhH-containing proteins indicates a one-residue deletion in the second HhH motif of XPF that has altered the hairpin conformation, thereby permitting ssDNA interactions. Previous nuclear magnetic resonance studies showed that ERCC1 in the XPF-ERCC1 heterodimer can bind dsDNA. Combining the two observations gives a model that underscores the asymmetry of the human XPF/ERCC1 heterodimer in binding at an ss/ds DNA junction.
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http://dx.doi.org/10.1016/j.str.2012.02.009DOI Listing
April 2012

A girl with an atypical form of ataxia telangiectasia and an additional de novo 3.14 Mb microduplication in region 19q12.

Eur J Med Genet 2012 Jan 27;55(1):49-55. Epub 2011 Aug 27.

Institut für Humangenetik, Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Mainz, Germany.

A 9-year-old girl born to healthy parents showed manifestations suggestive of ataxia telangiectasia (AT), such as short stature, sudden short bouts of horizontal and rotary nystagmus, a weak and dysarthric voice, rolling gait, unstable posture, and atactic movements. She did not show several cardinal features typical of AT such as frequent, severe infections of the respiratory tract. In contrast, she showed symptoms not generally related to AT, including microcephaly, profound motor and mental retardation, small hands and feet, severely and progressively reduced muscle tone with slackly protruding abdomen and undue drooling, excess fat on her upper arms, and severe oligoarthritis. A cranial MRI showed no cerebellar hypoplasia and other abnormalities. In peripheral blood samples she carried a de novo duplication of 3.14 Mb in chromosomal region 19q12 containing six annotated genes, UQCRFS1, VSTM2B, POP4, PLEKHF1, CCNE1, and ZNF536, and a de novo mosaic inversion 14q11q32 (96% of metaphases). In a saliva-derived DNA sample only the duplication in 19q12 was detected, suggesting that the rearrangements in blood lymphocytes were acquired. These findings reinforced the suspicion that she had AT. AT was confirmed by strongly elevated serum AFP levels, cellular radiosensitivity and two inherited mutations in the ATM gene (c.510_511delGT; paternal origin and c.2922-50_2940del69; maternal origin). This case suggest that a defective ATM-dependent DNA damage response may entail additional stochastic genomic rearrangements. Screening for genomic rearrangements appears indicated in patients suspected of defective DNA damage responses.
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http://dx.doi.org/10.1016/j.ejmg.2011.08.001DOI Listing
January 2012

SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype.

Nat Genet 2011 Feb 16;43(2):138-41. Epub 2011 Jan 16.

Department of Clinical Genetics, Vrije Universiteit (VU) Medical Center, Amsterdam, The Netherlands.

DNA interstrand crosslink repair requires several classes of proteins, including structure-specific endonucleases and Fanconi anemia proteins. SLX4, which coordinates three separate endonucleases, was recently recognized as an important regulator of DNA repair. Here we report the first human individuals found to have biallelic mutations in SLX4. These individuals, who were previously diagnosed as having Fanconi anemia, add SLX4 as an essential component to the FA-BRCA genome maintenance pathway.
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http://dx.doi.org/10.1038/ng.751DOI Listing
February 2011

Mislocalization of XPF-ERCC1 nuclease contributes to reduced DNA repair in XP-F patients.

PLoS Genet 2010 Mar 5;6(3):e1000871. Epub 2010 Mar 5.

Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV-induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPF(R153P)) were compared to an XP-causing mutation (XPF(R799W)) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPF(R153P)-YFP expressed in Xpf mutant cells. In addition, microinjection of XPF(R153P)-ERCC1 into the nucleus of XPF-deficient human cells restored nucleotide excision repair of UV-induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1.
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http://dx.doi.org/10.1371/journal.pgen.1000871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832669PMC
March 2010

A novel radiosensitive SCID patient with a pronounced G(2)/M sensitivity.

DNA Repair (Amst) 2010 Apr 15;9(4):365-73. Epub 2010 Jan 15.

Department of Toxicogenetics, Leiden University Medical Center, The Netherlands.

V(D)J rearrangement in lymphoid cells involves repair of double-strand breaks (DSBs) through non-homologous end joining (NHEJ). Defects in this process lead to increased radiosensitivity and severe combined immunodeficiency (RS-SCID). Here, a SCID patient, M3, is described with a T(-)B(+)NK(+) phenotype but without causative mutations in CD3delta, epsilon, zeta or IL7Ralpha, genes specifically involved in T cell development. Clonogenic survival of M3 fibroblasts showed an increased sensitivity to the DSB-inducing agents ionizing radiation and bleomycin, as well as the crosslinking compound, mitomycin C. We did not observe inactivating mutations in known NHEJ genes and results of various DSB-repair assays in G(1) M3 cells were indistinguishable from those obtained with normal cells. However, we found increased chromosomal radiosensitivity at the G(2) phase of the cell cycle. Checkpoint analysis indicated functional G(1)/S and intra-S checkpoints after irradiation but impaired activation of the "early" G(2)/M checkpoint. Together these results indicate a novel class of RS-SCID patients characterized by the specific absence of T lymphocytes and associated with defects in G(2)-specific DSB repair. The pronounced G(2)/M radiosensitivity of the RS-SCID patient described here, suggests a defect in a putative novel and uncharacterized factor involved in cellular DNA damage responses and T cell development.
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http://dx.doi.org/10.1016/j.dnarep.2009.12.004DOI Listing
April 2010

MicroRNA-mediated gene silencing modulates the UV-induced DNA-damage response.

EMBO J 2009 Jul 18;28(14):2090-9. Epub 2009 Jun 18.

Department of Cell Biology and Genetics, Erasmus MC, CA Rotterdam 3000, The Netherlands.

DNA damage provokes DNA repair, cell-cycle regulation and apoptosis. This DNA-damage response encompasses gene-expression regulation at the transcriptional and post-translational levels. We show that cellular responses to UV-induced DNA damage are also regulated at the post-transcriptional level by microRNAs. Survival and checkpoint response after UV damage was severely reduced on microRNA-mediated gene-silencing inhibition by knocking down essential components of the microRNA-processing pathway (Dicer and Ago2). UV damage triggered a cell-cycle-dependent relocalization of Ago2 into stress granules and various microRNA-expression changes. Ago2 relocalization required CDK activity, but was independent of ATM/ATR checkpoint signalling, whereas UV-responsive microRNA expression was only partially ATM/ATR independent. Both microRNA-expression changes and stress-granule formation were most pronounced within the first hours after genotoxic stress, suggesting that microRNA-mediated gene regulation operates earlier than most transcriptional responses. The functionality of the microRNA response is illustrated by the UV-inducible miR-16 that downregulates checkpoint-gene CDC25a and regulates cell proliferation. We conclude that microRNA-mediated gene regulation adds a new dimension to the DNA-damage response.
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http://dx.doi.org/10.1038/emboj.2009.156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718280PMC
July 2009

Contiguous gene deletion of ELOVL7, ERCC8 and NDUFAF2 in a patient with a fatal multisystem disorder.

Hum Mol Genet 2009 Sep 12;18(18):3365-74. Epub 2009 Jun 12.

Nijmegen Center for Mitochondrial Disorders at the Department of Pediatrics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Contiguous gene syndromes affecting the mitochondrial oxidative phosphorylation system have been rarely reported. Here, we describe a patient with apparent mitochondrial encephalomyopathy accompanied by several unusual features, including dysmorphism and hepatopathy, caused by a homozygous triple gene deletion on chromosome 5. The deletion encompassed the NDUFAF2, ERCC8 and ELOVL7 genes, encoding complex I assembly factor 2 (also known as human B17.2L), a protein of the transcription-coupled nucleotide excision repair (TC-NER) machinery, and a putative elongase of very long-chain fatty acid synthesis, respectively. Detailed evaluation of cultured skin fibroblasts revealed disturbed complex I assembly, depolarization of the mitochondrial membrane, elevated cellular NAD(P)H level, increased superoxide production and defective TC-NER. ELOVL7 mRNA was not detectable in these cells and no alterations in fatty acid synthesis were found. By means of baculoviral complementation we were able to restore the aberrations, thereby establishing causative links between genotype and cell-physiological phenotype. This first chromosomal microdeletion illustrates that beside primary defects in mitochondrial genes also additional genes possibly contribute to the disease phenotype, providing an additional explanation for the broad clinical symptoms associated with these disorders.
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http://dx.doi.org/10.1093/hmg/ddp276DOI Listing
September 2009

Neurological symptoms and natural course of xeroderma pigmentosum.

Brain 2008 Aug 21;131(Pt 8):1979-89. Epub 2008 Jun 21.

Department of Neurology, Turku University Central Hospital, PB 52, 20521 Turku, Finland.

We have prospectively followed 16 Finnish xeroderma pigmentosum (XP) patients for up to 23 years. Seven patients were assigned by complementation analysis to the group XP-A, two patients to the XP-C group and one patient to the XP-G group. Six of the seven XP-A patients had the identical mutation (Arg228Ter) and the seventh patient had a different mutation (G283A). Further patients were assigned to complementation groups on the basis of their consanguinity to an XP patient with a known complementation group. The first sign of the disease in all the cases was severe sunburn with minimal sun exposure in early infancy. However, at the time the diagnosis was made in only two cases. The XP-A patients developed neurological and cognitive dysfunction in childhood. The neurological disease advanced in an orderly fashion through its successive stages, finally affecting the whole nervous system and leading to death before the age of 40 years. Dermatological and ocular damage of the XP-A patients tended to be limited. The two XP-C patients were neurologically and cognitively intact despite mild brain atrophy as seen by neuroimaging. The XP-G patients had sensorineural hearing loss, laryngeal dystonia and peripheral neuropathy. The XP-C patients had severe skin and ocular malignancies that first presented at pre-school age. They also showed immunosuppression in cell-mediated immunity. Neurological disease appears to be associated with the complementation group and the failure of fibroblasts to recover RNA synthesis following UV irradiation, but not necessarily to the severity of the dermatological symptoms, the hypersensitivity of fibroblasts to UVB killing or the susceptibility of keratinocytes to UVB-induced apoptosis.
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http://dx.doi.org/10.1093/brain/awn126DOI Listing
August 2008

Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy.

DNA Repair (Amst) 2008 May 10;7(5):744-50. Epub 2008 Mar 10.

Department of Clinical Genetics, Erasmus Medical Centre, Rotterdam, The Netherlands.

Laboratory diagnosis for DNA repair diseases has been performed in western Europe from the early seventies for xeroderma pigmentosum (XP) and from the mid-eighties for Cockayne syndrome (CS) and trichothiodystrophy (TTD). The combined data from the DNA repair diagnostic centres in France, (West) Germany, Italy, the Netherlands and the United Kingdom have been investigated for three groups of diseases: XP (including XP-variant), CS (including XP/CS complex) and TTD. Incidences in western Europe were for the first time established at 2.3 per million livebirths for XP, 2.7 per million for CS and 1.2 per million for TTD. As immigrant populations were disproportionately represented in the patients' groups, incidences were also established for the autochthonic western European population at: 0.9 per million for XP, 1.8 per million for CS and 1.1 per million for TTD. Perhaps contrary to general conceptions, compared to XP the incidence of CS appears to be somewhat higher and the incidence of TTD to be quite similar in the native West-European population.
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http://dx.doi.org/10.1016/j.dnarep.2008.01.014DOI Listing
May 2008

The HhH domain of the human DNA repair protein XPF forms stable homodimers.

Proteins 2008 Mar;70(4):1551-63

Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands.

The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.
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http://dx.doi.org/10.1002/prot.21635DOI Listing
March 2008

Prenatal diagnosis of xeroderma pigmentosum and trichothiodystrophy in 76 pregnancies at risk.

Prenat Diagn 2007 Dec;27(12):1133-7

Department of Clinical Genetics, Erasmus University Medical Centre, Rotterdam, The Netherlands.

Objective: Evaluation of results in a consecutive series of 76 prenatal diagnoses for xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) made since 1977.

Methods: UV-induced DNA repair synthesis was assessed by the autoradiographic measurement of the incorporation of (3)H-thymidine.

Results: XP was diagnosed in 19 of the 76 investigated pregnancies at risk; cultured chorionic villus (CV) cells were used in 33 pregnancies with ten affected fetuses and cultured amniocytes in 43 pregnancies with nine affected fetuses. In four cases, CVS results were corroborated by subsequent investigation of amniocytes because maternal cell contamination in the CV cell culture was either present or could not be excluded. Uncertain results in two other cases with intermediate DNA repair capacity and severe maternal cell contamination required further investigation. Median time needed for cell culture and analysis was 25 days. To reduce intra-assay variations, a modification of the DNA repair synthesis assay has recently been developed. In this assay, patients and controls are investigated simultaneously in mixed cultures of cells labelled with polystyrene beads.

Conclusion: Reliable prenatal diagnosis for XP and TTD can be made by the demonstration of clearly reduced UV-induced DNA repair synthesis due to defective global genome nucleotide excision repair.
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http://dx.doi.org/10.1002/pd.1849DOI Listing
December 2007

Regulation of UV-induced DNA damage response by ubiquitylation.

DNA Repair (Amst) 2007 Sep 23;6(9):1231-42. Epub 2007 Mar 23.

MGC-CBG Department of Cell Biology and Genetics, Erasmus MC, P.O. Box 2040, 3000CA Rotterdam, The Netherlands.

Like many other cellular processes, regulation of the DNA damage response (DDR) is regulated at different levels, ranging from transcriptional control to an array of distinct post-translational modifications. Involvement of ubiquitylation and the ubiquitin proteasome system in adjusting DDR are such protein modifications that were receiving increasing attention in the field. In this review we summarize and discuss a few recent key publications addressing the issue of DDR factor ubiquitylation, focusing on UV-induced DDR. We discuss the implications of these modifications to allow swift adaptation and regulation of genome surveillance factors.
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http://dx.doi.org/10.1016/j.dnarep.2007.01.012DOI Listing
September 2007

First reported patient with human ERCC1 deficiency has cerebro-oculo-facio-skeletal syndrome with a mild defect in nucleotide excision repair and severe developmental failure.

Am J Hum Genet 2007 Mar 29;80(3):457-66. Epub 2007 Jan 29.

Department of Genetics, Erasmus Medical Center, Rotterdam, The Netherlands.

Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.
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http://dx.doi.org/10.1086/512486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1821117PMC
March 2007

A new progeroid syndrome reveals that genotoxic stress suppresses the somatotroph axis.

Nature 2006 Dec;444(7122):1038-43

Center for Biomedical Genetics Medical Genetic Center Department of Cell Biology and Genetics, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands.

XPF-ERCC1 endonuclease is required for repair of helix-distorting DNA lesions and cytotoxic DNA interstrand crosslinks. Mild mutations in XPF cause the cancer-prone syndrome xeroderma pigmentosum. A patient presented with a severe XPF mutation leading to profound crosslink sensitivity and dramatic progeroid symptoms. It is not known how unrepaired DNA damage accelerates ageing or its relevance to natural ageing. Here we show a highly significant correlation between the liver transcriptome of old mice and a mouse model of this progeroid syndrome. Expression data from XPF-ERCC1-deficient mice indicate increased cell death and anti-oxidant defences, a shift towards anabolism and reduced growth hormone/insulin-like growth factor 1 (IGF1) signalling, a known regulator of lifespan. Similar changes are seen in wild-type mice in response to chronic genotoxic stress, caloric restriction, or with ageing. We conclude that unrepaired cytotoxic DNA damage induces a highly conserved metabolic response mediated by the IGF1/insulin pathway, which re-allocates resources from growth to somatic preservation and life extension. This highlights a causal contribution of DNA damage to ageing and demonstrates that ageing and end-of-life fitness are determined both by stochastic damage, which is the cause of functional decline, and genetics, which determines the rates of damage accumulation and decline.
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http://dx.doi.org/10.1038/nature05456DOI Listing
December 2006

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

Hum Mutat 2007 Jan;28(1):92-6

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (CNR), Pavia, Italy.

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.
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http://dx.doi.org/10.1002/humu.20419DOI Listing
January 2007

Prenatal diagnosis of the Cockayne syndrome: survey of 15 years experience.

Prenat Diagn 2006 Oct;26(10):980-4

Department of Clinical Genetics, Erasmus University Medical Centre, Rotterdam, The Netherlands.

Objective: Evaluation of results in a consecutive series of 29 prenatal diagnoses for the Cockayne syndrome.

Methods: Recovery of DNA-synthesis in UV-irradiated cultured fetal cells was measured by scintillation counting of incorporated (3)H-thymidine. Semiquantitative autoradiographic assessment of the recovery of RNA-synthesis (RecRS) was used as an adjunctive method.

Results: In 26 of the 29 pregnancies at risk, a definite diagnosis was directly made, based on normal (n = 23) or clearly reduced (n = 3) recovery of DNA-synthesis in UV-irradiated cultured chorionic villus (CV) cells (n = 23) or amniocytes (n = 3). Adjunctive studies were performed in several pregnancies to corroborate the initial results. On three occasions initial results were unreliable, which required investigation of the recovery of RNA-synthesis (n = 2) or even additional amniocentesis (n = 1) to achieve a firm diagnosis. Thus, four affected fetuses were diagnosed in 29 pregnancies at risk (13.8%).

Conclusion: Reliable prenatal diagnosis of the Cockayne syndrome can be made by the demonstration of a strongly reduced recovery of DNA-synthesis in UV-irradiated cultured chorionic villus cells or amniocytes. Assessment of the recovery of RNA-synthesis was needed as an adjunctive method in rare cases of poor cell growth and DNA-synthesis.
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http://dx.doi.org/10.1002/pd.1541DOI Listing
October 2006

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2.

Mutat Res 2006 Oct 21;601(1-2):191-201. Epub 2006 Aug 21.

Department of Toxicogenetics, Leiden University Medical Center, Building 2, Postzone S-6-P, 2300 RC, Leiden, The Netherlands.

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.
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http://dx.doi.org/10.1016/j.mrfmmm.2006.07.003DOI Listing
October 2006

An Xpd mouse model for the combined xeroderma pigmentosum/Cockayne syndrome exhibiting both cancer predisposition and segmental progeria.

Cancer Cell 2006 Aug;10(2):121-32

Medical Genetics Center, Department of Cell Biology and Genetics, Center of Biomedical Genetics, Cancer Genomics Center, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands.

Inborn defects in nucleotide excision DNA repair (NER) can paradoxically result in elevated cancer incidence (xeroderma pigmentosum [XP]) or segmental progeria without cancer predisposition (Cockayne syndrome [CS] and trichothiodystrophy [TTD]). We report generation of a knockin mouse model for the combined disorder XPCS with a G602D-encoding mutation in the Xpd helicase gene. XPCS mice are the most skin cancer-prone NER model to date, and we postulate an unusual NER dysfunction that is likely responsible for this susceptibility. XPCS mice also displayed symptoms of segmental progeria, including cachexia and progressive loss of germinal epithelium. Like CS fibroblasts, XPCS and TTD fibroblasts from human and mouse showed evidence of defective repair of oxidative DNA lesions that may underlie these segmental progeroid symptoms.
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http://dx.doi.org/10.1016/j.ccr.2006.05.027DOI Listing
August 2006

Inhibition of DNA synthesis by ionizing radiation: a marker for an S-phase checkpoint.

Methods Mol Biol 2006 ;314:51-9

Department of Genetics, Erasmus Medical Center, Rotterdam, The Netherlands.

Inhibition of replicative DNA synthesis by ionizing radiation is partly caused by an active, signal-mediated response termed the "S-phase checkpoint." Defects in this checkpoint were first discovered in the human inherited disorder ataxia-telangiectasia (AT). gamma-Irradiated cells from AT patients consistently display a diminished inhibition of DNA synthesis, a feature called "radioresistant DNA synthesis" (RDS). RDS has been widely used as a diagnostic marker for AT, in postnatal as well as prenatal material. The regulation and control of the S-phase checkpoint is complex and multifaceted; it is not restricted to ionizing radiation, but can occur after many genotoxic stressors. Defects in both upstream control functions, such as ATM, NBS1, and MRE11, as well as downstream modulators can provoke an RDS phenotype. Here a simple, accurate and highly reproducible experimental protocol is presented for the generation of DNA synthesis inhibition curves from cells in culture.
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http://dx.doi.org/10.1385/1-59259-973-7:051DOI Listing
June 2006

A new type of radiosensitive T-B-NK+ severe combined immunodeficiency caused by a LIG4 mutation.

J Clin Invest 2006 Jan 15;116(1):137-45. Epub 2005 Dec 15.

Department of Immunology and Department of Cell Biology and Genetics, Erasmus Medical Center, Rotterdam, The Netherlands.

V(D)J recombination of Ig and TCR loci is a stepwise process during which site-specific DNA double-strand breaks (DSBs) are made by RAG1/RAG2, followed by DSB repair by nonhomologous end joining. Defects in V(D)J recombination result in SCID characterized by absence of mature B and T cells. A subset of T-B-NK+ SCID patients is sensitive to ionizing radiation, and the majority of these patients have mutations in Artemis. We present a patient with a new type of radiosensitive T-B-NK+ SCID with a defect in DNA ligase IV (LIG4). To date, LIG4 mutations have only been described in a radiosensitive leukemia patient and in 4 patients with a designated LIG4 syndrome, which is associated with chromosomal instability, pancytopenia, and developmental and growth delay. The patient described here shows that a LIG4 mutation can also cause T-B-NK+ SCID without developmental defects. The LIG4-deficient SCID patient had an incomplete but severe block in precursor B cell differentiation, resulting in extremely low levels of blood B cells. The residual D(H)-J(H) junctions showed extensive nucleotide deletions, apparently caused by prolonged exonuclease activity during the delayed D(H)-J(H) ligation process. In conclusion, different LIG4 mutations can result in either a developmental defect with minor immunological abnormalities or a SCID picture with normal development.
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http://dx.doi.org/10.1172/JCI26121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1312018PMC
January 2006