The clinical impact of deficiency in DNA non-homologous end-joining

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models, since radiation potently induces DSBs. The process of V(D)J recombination functions during the development of the immune response, and involves the introduction and rejoining of programmed DSBs to generate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJ deficiency confers (severe) combined immunodeficiency – (S)CID – due to a failure to carry out V(D)J recombination efficiently. NHEJ also functions in class switch recombination, another step enhancing T and B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patients revealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syndromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have been identified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCID patients frequently display additional characteristics including microcephaly, dysmorphic facial features and growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our current understanding of the underlying biology.     

Reprint of “The clinical impact of deficiency in DNA non-homologous end-joining”

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models, since radiation potently induces DSBs. The process of V(D)J recombination functions during the development of the immune response, and involves the introduction and rejoining of programmed DSBs to generate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJ deficiency confers (severe) combined immunodeficiency – (S)CID – due to a failure to carry out V(D)J recombination efficiently. NHEJ also functions in class switch recombination, another step enhancing T and B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patients revealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syndromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have been identified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCID patients frequently display additional characteristics including microcephaly, dysmorphic facial features and growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our current understanding of the underlying biology.     

Cellular and clinical impact of haploinsufficiency for genes involved in ATR signaling

Ataxia telangiectasia and Rad3-related (ATR) protein, a kinase that regulates a DNA damage-response pathway, is mutated in ATR-Seckel syndrome (ATR-SS), a disorder characterized by severe microcephaly and growth delay. Impaired ATR signaling is also observed in cell lines from additional disorders characterized by microcephaly and growth delay, including non-ATR-SS, Nijmegen breakage syndrome, and MCPH1 (microcephaly, primary autosomal recessive, 1)-dependent primary microcephaly. Here, we examined ATR-pathway function in cell lines from three haploinsufficient contiguous gene-deletion disorders--a subset of blepharophimosis-ptosis-epicanthus inversus syndrome, Miller-Dieker lissencephaly syndrome, and Williams-Beuren syndrome--in which the deleted region encompasses ATR, RPA1, and RFC2, respectively. These three genes function in ATR signaling. Cell lines from these disorders displayed an impaired ATR-dependent DNA damage response. Thus, we describe ATR signaling as a pathway unusually sensitive to haploinsufficiency and identify three further human disorders displaying a defective ATR-dependent DNA damage response. The striking correlation of ATR-pathway dysfunction with the presence of microcephaly and growth delay strongly suggests a causal relationship.     

Nbs1 is required for ATR-dependent phosphorylation events

Nijmegen breakage syndrome (NBS) is characterised by microcephaly, developmental delay, characteristic facial features, immunodeficiency and radiosensitivity. Nbs1, the protein defective in NBS, functions in ataxia telangiectasia mutated protein (ATM)-dependent signalling likely facilitating ATM phosphorylation events. While NBS shares overlapping characteristics with ataxia telangiectasia, it also has features overlapping with ATR-Seckel (ATR: ataxia-telangiectasia and Rad3-related protein) syndrome, a subclass of Seckel syndrome mutated in ATR. We show that Nbs1 also facilitates ATR-dependent phosphorylation. NBS cell lines show a similar defect in ATR phosphorylation of Chk1, c-jun and p-53 in response to UV irradiation- and hydroxyurea (HU)-induced replication stalling. They are also impaired in ubiquitination of FANCD2 after HU treatment, which is ATR dependent. Following HU-induced replication arrest, NBS and ATR-Seckel cells show similarly impaired G2/M checkpoint arrest and an impaired ability to restart DNA synthesis at stalled replication forks. Moreover, NBS cells fail to retain ATR in the nucleus following HU treatment and extraction. Our findings suggest that Nbs1 functions in both ATR- and ATM-dependent signalling. We propose that the NBS clinical features represent the result of these combined defects.     

A novel radiosensitive SCID patient with a pronounced G2/M sensitivity

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 CD3δ, ?, ζ or IL7Rα, 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 G₁ M3 cells were indistinguishable from those obtained with normal cells. However, we found increased chromosomal radiosensitivity at the G₂ phase of the cell cycle. Checkpoint analysis indicated functional G₁/S and intra-S checkpoints after irradiation but impaired activation of the “early” G₂/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 G₂-specific DSB repair. The pronounced G₂/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.     

DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.

DNA ligase IV functions in DNA nonhomologous end-joining and V(D)J recombination. Four patients with features including immunodeficiency and developmental and growth delay were found to have mutations in the gene encoding DNA ligase IV (LIG4). Their clinical phenotype closely resembles the DNA damage response disorder, Nijmegen breakage syndrome (NBS). Some of the mutations identified in the patients directly disrupt the ligase domain while others impair the interaction between DNA ligase IV and Xrcc-4. Cell lines from the patients show pronounced radiosensitivity. Unlike NBS cell lines, they show normal cell cycle checkpoint responses but impaired DNA double-strand break rejoining. An unexpected V(D)J recombination phenotype is observed involving a small decrease in rejoining frequency coupled with elevated imprecision at signal junctions.     

Microcephalin: A Causal Link Between Impaired Damage Response Signalling and Microcephaly

Seckel Syndrome (SS) and Primary Microcephaly (MCPH) are disorders exhibiting marked microcephaly with a head circumference less than 3 standard deviations below the mean. ATR-Seckel Syndrome is conferred by mutations in ataxia and telangiectasia and Rad3 related (ATR), a kinase that activates a DNA damage signalling response. Cell lines from additional SS patients, who are normal for ATR, show defective ATR signalling, suggesting that they carry mutations in other components of the ATR pathway. Primary Microcephaly is distinct from SS since patients displaying solely microcephaly without accompanying growth delay. MCPH1, the first Primary Microcephaly causative gene identified, encodes three BRCT domains, similar to other damage response proteins. Recent studies employing MCPH1 siRNA or exploiting cell lines from MCPH1 patients have shown that MCPH1 functions in the ATR-dependent DNA damage response pathway. Additionally, MCPH1 has a function in the regulation of mitotic entry that is ATR-independent and confers a characteristic phenotype of premature chromosome condensation. Recent studies will be reviewed and their relationship to the aetiology of microcephaly discussed.     

Absence of PTPN11 mutations in 28 cases of cardiofaciocutaneous (CFC) syndrome

CFC (cardiofaciocutaneous) syndrome (MIM 115150) has been considered by several authors to be a more severe expression of Noonan syndrome. Affected patients present with congenital heart defects, cutaneous abnormalities, Noonan-like facial features and severe psychomotor developmental delay. We have recently demonstrated that Noonan syndrome can be caused by missense mutations in PTPN11(MIM 176876), a gene that encodes the non-receptor protein tyrosine phosphatase SHP-2. In this report, we have evaluated the possible involvement of mutations in PTPN11 in CFC syndrome. A cohort of 28 CFC subjects rigorously assessed as having CFC based on OMIM diagnostic criteria was examined for mutations in the PTPN11 coding sequence by using DHPLC analysis. The results showed no abnormalities in the coding region of the PTPN11 gene in any CFC patient, nor any evidence of major deletions within the gene suggesting that mutations in other gene(s) are responsible for this syndrome.     

Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency

The V(D)J recombination process insures the somatic diversification of immunoglobulin and antigen T cell receptor encoding genes. This reaction is initiated by a DNA double-strand break (dsb), which is resolved by the ubiquitously expressed DNA repair machinery. Human T-B-severe combined immunodeficiency associated with increased cellular radiosensitivity (RS-SCID) is characterized by a defect in the V(D)J recombination leading to an early arrest of both B and T cell maturation. We previously mapped the disease-related locus to the short arm of chromosome 10. We herein describe the cloning of the gene encoding a novel protein involved in V(D)J recombination/DNA repair, Artemis, whose mutations cause human RS-SCID. Protein sequence analysis strongly suggests that Artemis belongs to the metallo-beta-lactamase superfamily.     

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Barrier-to-autointegration-factor (Banf1) modulates DNA double-strand break repair pathway choice via regulation of DNA-dependent kinase (DNA-PK) activity

DNA repair pathways are essential to maintain the integrity of the genome and prevent cell death and tumourigenesis. Here, we show that the Barrier-to-Autointegration Factor (Banf1) protein has a role in the repair of DNA double-strand breaks. Banf1 is characterized as a nuclear envelope protein and mutations in Banf1 are associated with the severe premature aging syndrome, Néstor–Guillermo Progeria Syndrome. We have previously shown that Banf1 directly regulates the activity of PARP1 in the repair of oxidative DNA lesions. Here, we show that Banf1 also has a role in modulating DNA double-strand break repair through regulation of the DNA-dependent Protein Kinase catalytic subunit, DNA-PKcs. Specifically, we demonstrate that Banf1 relocalizes from the nuclear envelope to sites of DNA double-strand breaks. We also show that Banf1 can bind to and directly inhibit the activity of DNA-PKcs. Supporting this, cellular depletion of Banf1 leads to an increase in non-homologous end-joining and a decrease in homologous recombination, which our data suggest is likely due to unrestrained DNA-PKcs activity. Overall, this study identifies how Banf1 regulates double-strand break repair pathway choice by modulating DNA-PKcs activity to control genome stability within the cell.     

DNA double-strand break repair: a relentless hunt uncovers new prey

A major pathway for repair of DNA double-strand breaks is nonhomologous end-joining (NHEJ). In this issue of Cell, and report the discovery of a new NHEJ factor called Cernunnos-XLF. Both groups report that this protein is mutated in a rare inherited human syndrome characterized by severe immunodeficiency, developmental delay, and hypersensitivity to agents that cause DNA double-strand breaks.     

Evidence for a distinct region causing a cat-like cry in patients with 5p deletions.

The cri-du-chat syndrome is a contiguous gene syndrome that results from a deletion of the short arm of chromosome 5 (5p). Patients present with a cat-like cry at birth, which is usually considered diagnostic of this syndrome. Additional features of the syndrome include failure to thrive, microcephaly, hypertelorism, epicanthal folds, hypotonia, and severe mental retardation. We report on four families in which patients with 5p deletions have only the characteristic cat-like cry, with normal to mildly delayed development. The precise locations of the deletions in each family were determined by FISH using lambda phage and cosmid clones. All of the deletion breakpoints map distal to a chromosomal region that is implicated with the facial features and severe mental and developmental delay in the cri-du-chat syndrome. DNA clones mapping in the chromosomal region associated with the cat-like cry feature will be useful diagnostic tools. They will allow for the distinction between 5p deletions that will result in the severe delay observed in most cri-du-chat syndrome patients and those deletions that result in the isolated cat-like cry feature, which is associated with a better prognosis.     

Greig syndrome associated with an interstitial deletion of 7p: confirmation of the localization of Greig syndrome to 7p13

Summary An 11-month-old infant with Greig cephalopolysyndactyly syndrome and mild developmental delay is described. High-resolution chromosomal analysis showed a de novo interstitial deletion of chromosome 7p with breakpoints located at p13 and p14. Cytogenetic analysis of polymorphisms of the heterochromatin in the pericentromeric region suggested the deleted chromosome was of paternal origin. This case confirms the localization of Greig syndrome to 7p13 and emphasizes the importance of performing cytogenetic studies on patients with Mendelian disorders who have unusual findings or cognitive abnormalities in a disorder usually associated with normal intellect. Review of clinical features in published reports of patients with a deletion involving 7p13 showed a number to have features overlapping with Greig syndrome. Because of this, we suggest that cytogenetic aberrations, particularly chromosomal microdeletions, may represent a significant etiology for Greig syndrome.     

Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis^−/−, DNA ligase 4^−/− (LIG4^−/−), and LIG4^−/−/Artemis^−/− double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.     

Allelic heterogeneity in the COH1 gene explains clinical variability in Cohen syndrome

Cohen syndrome is a rare autosomal recessive disorder with a variable clinical picture mainly characterized by developmental delay, mental retardation, microcephaly, typical facial dysmorphism, progressive pigmentary retinopathy, severe myopia, and intermittent neutropenia. A Cohen syndrome locus was mapped to chromosome 8q22 in Finnish patients, and, recently, mutations in the gene COH1 were reported in patients with Cohen syndrome from Finland and other parts of northern and western Europe. Here, we describe clinical and molecular findings in 20 patients with Cohen syndrome from 12 families, originating from Brazil, Germany, Lebanon, Oman, Poland, and Turkey. All patients were homozygous or compound heterozygous for mutations in COH1. We identified a total of 17 novel mutations, mostly resulting in premature termination codons. The clinical presentation was highly variable. Developmental delay of varying degree, early-onset myopia, joint laxity, and facial dysmorphism were the only features present in all patients; however, retinopathy at school age, microcephaly, and neutropenia are not requisite symptoms of Cohen syndrome. The identification of novel mutations in COH1 in an ethnically diverse group of patients demonstrates extensive allelic heterogeneity and explains the intriguing clinical variability in Cohen syndrome.     

Increased RPA1 gene dosage affects genomic stability potentially contributing to 17p13.3 duplication syndrome

A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression.     

A review and appraisal of the DNA damage theory of ageing

Given the central role of DNA in life, and how ageing can be seen as the gradual and irreversible breakdown of living systems, the idea that damage to the DNA is the crucial cause of ageing remains a powerful one. DNA damage and mutations of different types clearly accumulate with age in mammalian tissues. Human progeroid syndromes resulting in what appears to be accelerated ageing have been linked to defects in DNA repair or processing, suggesting that elevated levels of DNA damage can accelerate physiological decline and the development of age-related diseases not limited to cancer. Higher DNA damage may trigger cellular signalling pathways, such as apoptosis, that result in a faster depletion of stem cells, which in turn contributes to accelerated ageing. Genetic manipulations of DNA repair pathways in mice further strengthen this view and also indicate that disruption of specific pathways, such as nucleotide excision repair and non-homologous end joining, is more strongly associated with premature ageing phenotypes. Delaying ageing in mice by decreasing levels of DNA damage, however, has not been achieved yet, perhaps due to the complexity inherent to DNA repair and DNA damage response pathways. Another open question is whether DNA repair optimization is involved in the evolution of species longevity, and we suggest that the way cells from different organisms respond to DNA damage may be crucial in species differences in ageing. Taken together, the data suggest a major role of DNA damage in the modulation of longevity, possibly through effects on cell dysfunction and loss, although understanding how to modify DNA damage repair and response systems to delay ageing remains a crucial challenge.     

Epigenetics and Neural Developmental Disorders

Neural developmental disorders, such as autism, Rett Syndrome, Fragile X syndrome, and Angelman syndrome manifest during early postnatal neural development. Although the genes responsible for some of these disorders have been identified, how the mutations of these genes affect neural development is currently unclear. Emerging evidence suggest that these disorders share common underlying defects in neuronal morphology, synaptic connectivity and brain plasticity. In particular, alterations in dendritic branching and spine morphology play a central role in the pathophysiology of most mental retardation disorders, suggesting that common pathways regulating neuronal function may be affected. Epigenetic modulations, mediated by DNA methylation, RNA-associated silencing, and histone modification, can serve as an intermediate process that imprints dynamic environmental experiences on the “fixed” genome, resulting in stable alterations in phenotypes. Disturbance in epigenetic regulations can lead to inappropriate expression or silencing of genes, causing an array of multi-system disorders and neoplasias. Rett syndrome, the most common form of mental retardation in young girls, is due to germline mutation of MECP2, encoding a methylated DNA binding protein that translates DNA methylation into gene repression. Angelman syndrome is due to faulty genomic imprinting or maternal mutations in UBE3A. Fragile X Syndrome, in most cases, results from the hypermethylation of FMR1 promoter, hence the loss of expression of functional FMRP protein. Autism, with its complex etiology, may have strong epigenetic link. Together, these observations strongly suggest that epigenetic mechanisms may play a critical role in brain development and etiology of related disorders. This report summarizes the scientific discussions and major conclusions from a recent conference that aimed to gain insight into the common molecular pathways affected among these disorders and discover potential therapeutic targets that have been missed by looking at one disorder at a time.