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Fanconi anemia (FA) is characterized by chromosome fragility, bone marrow failure (BMF) and predisposition to cancer. As reverse genetic mosaicism has been described as "natural gene therapy" in patients with FA, we sought to evaluate the clinical course of a cohort of FA mosaic patients followed at referral centers in Spain over a 30-year period. This cohort includes patients with a majority of T cells without chromosomal aberrations in the DEB-chromosomal breakage test. Relative to non-mosaic FA patients, we observed a higher proportion of adult patients in the cohort of mosaics, with a later age of hematologic onset and a milder evolution of (BMF). Consequently, the requirement for hematopoietic stem cell transplant (HSCT) was also lower. Additional studies allowed us to identify a sub-cohort of mosaic FA patients in whom the reversion was present in bone marrow (BM) progenitor cells leading to multilineage mosaicism. These multilineage mosaic patients are older, have a lower percentage of aberrant cells, have more stable hematology and none of them developed leukemia or myelodysplastic syndrome when compared to non-mosaics. In conclusion, our data indicate that reverse mosaicism is a good prognostic factor in FA and is associated with more favorable long-term clinical outcomes.

PurposePatients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients’ characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies.Methods68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies.ResultsWe identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as ‘affecting functions’ are SNPs. Deep analysis of sequencing data revealed patients’ true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations)ConclusionWES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.

© 2021 the Author(s)., Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders.

Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34+ cells/μL required to initiate apheresis. A median of 21.8 CD34+ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34+ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34+ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34+ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34+ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols., Graphical abstract, Mobilization and collection of HSPCs from FA patients with sufficient HSPC reserve is a safe and efficient procedure, incorporating filgrastim and plerixafor as mobilization agents. Adequate HSPC mobilization correlates with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher BM CD34+ cell numbers.

Altres ajuts: MSSSI/FIS PI12/00070 PurposeMonoallelic germ-line mutations in the BRCA1/FANCS, BRCA2/FANCD1 and PALB2/FANCN genes confer high risk of breast cancer. Biallelic mutations in these genes cause Fanconi anemia (FA), characterized by malformations, bone marrow failure, chromosome fragility, and cancer predisposition (BRCA2/FANCD1 and PALB2/FANCN), or an FA-like disease presenting a phenotype similar to FA but without bone marrow failure (BRCA1/FANCS). FANCM monoallelic mutations have been reported as moderate risk factors for breast cancer, but there are no reports of any clinical phenotype observed in carriers of biallelic mutations.MethodsBreast cancer probands were subjected to mutation analysis by sequencing gene panels or testing DNA damage response genes.ResultsFive cases homozygous for FANCM loss-of-function mutations were identified. They show a heterogeneous phenotype including cancer predisposition, toxicity to chemotherapy, early menopause, and possibly chromosome fragility. Phenotype severity might correlate with mutation position in the gene.ConclusionOur data indicate that biallelic FANCM mutations do not cause classical FA, providing proof that FANCM is not a canonical FA gene. Moreover, our observations support previous findings suggesting that FANCM is a breast cancer-predisposing gene. Mutation testing of FANCM might be considered for individuals with the above-described clinical features.

Summary Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34+ cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.

XPF endonuclease is one of the most important DNA repair proteins. Encoded by XPF/ERCC4, XPF provides the enzymatic activity of XPF-ERCC1 heterodimer, an endonuclease that incises at the 5’ side of various DNA lesions. XPF is essential for nucleotide excision repair (NER) and interstrand crosslink repair (ICLR). XPF/ERCC4 mutations are associated with several human diseases: Xeroderma Pigmentosum (XP), Segmental Progeria (XFE), Fanconi Anemia (FA), Cockayne Syndrome (CS), and XP/CS combined disease (XPCSCD). Most affected individuals are compound heterozygotes for XPF/ERCC4 mutations complicating the identification of genotype/phenotype correlations. We report a detailed overview of NER and ICLR functional studies in human XPF-KO (knock-out) isogenic cells expressing six disease-specific pathogenic XPF amino acid substitution mutations. Ultraviolet (UV) sensitivity and unscheduled DNA synthesis (UDS) assays provide the most reliable information to discern mutations associated with ICLR impairment from mutations related to NER deficiency, whereas recovery of RNA synthesis (RRS) assays results hint to a possible role of XPF in resolving R-loops. Our functional studies demonstrate that a defined cellular phenotype cannot be easily correlated to each XPF mutation. Substituted positions along XPF sequences are not predictive of cellular phenotype nor reflect a particular disease. Therefore, in addition to mutation type, allelic interactions, protein stability and intracellular distribution of mutant proteins may also contribute to alter DNA repair pathways balance leading to clinically distinct disorders.

Purpose: in about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these "occult" FA cases is often a solid cancer and cancer treatment-related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing. - Methods: (1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test. - Results: ES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell-only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test. - Conclusion: we report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for "occult" FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.

Fanconi anemia (FA) is a DNA repair syndrome generated by mutations in any of the 22 FA genes discovered to date1,2. Mutations in FANCA account for more than 60% of FA cases worldwide3,4. Clinically, FA is associated with congenital abnormalities and cancer predisposition. However, bone marrow failure is the primary pathological feature of FA that becomes evident in 70–80% of patients with FA during the first decade of life5,6. In this clinical study (ClinicalTrials.gov, NCT03157804 ; European Clinical Trials Database, 2011-006100-12), we demonstrate that lentiviral-mediated hematopoietic gene therapy reproducibly confers engraftment and proliferation advantages of gene-corrected hematopoietic stem cells (HSCs) in non-conditioned patients with FA subtype A. Insertion-site analyses revealed the multipotent nature of corrected HSCs and showed that the repopulation advantage of these cells was not due to genotoxic integrations of the therapeutic provirus. Phenotypic correction of blood and bone marrow cells was shown by the acquired resistance of hematopoietic progenitors and T lymphocytes to DNA cross-linking agents. Additionally, an arrest of bone marrow failure progression was observed in patients with the highest levels of gene marking. The progressive engraftment of corrected HSCs in non-conditioned patients with FA supports that gene therapy should constitute an innovative low-toxicity therapeutic option for this life-threatening disorder. In an early-phase lentiviral gene therapy trial, gene-corrected autologous hematopoietic stem cells show sustained engraftment and phenotypic correction in non-conditioned patients with Fanconi anemia.

Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only curative treatment for the bone marrow failure in Fanconi anemia (FA) patients. However, recent advances in lentiviral-mediated gene therapy have shown that corrected FA HSCs develop an in vivo proliferation advantage, facilitating the engraftment of corrected HSCs in non-conditioned FA patients. Based on these observations, we proposed that gene editing might constitute a promising alternative to correct patients' hematopoietic stem and progenitor cells (HSPCs) in this disorder. Since non-homologous end joining (NHEJ) is the most frequent repair pathway in HSCs, particularly in FA-HSCs, we aimed at exploiting this DNA repair mechanism to remove/compensate specific mutations in different FANC genes by the use of CRISPR/Cas9 system, thus mimicking spontaneous genetic reversions observed in FA mosaic patients. Our results in lymphoblastic cell lines from five different complementation groups (FANCA, FANCB, FANCC, FANCD2 and FANCD1/BRCA2) demonstrated the efficiency of this approach to generate potentially corrective events in all the different complementation groups studied. Importantly, corrected cells showed a marked proliferative advantage after in vitro culture and the analysis by next generation sequencing confirmed the expansion of cells harboring therapeutic events. Functional studies showing the reversion of mitomycin C sensitivity, FANCD2 foci formation and chromosomal instability supported the phenotypic correction of different mutations by NHEJ-mediated gene editing. Moving towards the clinical application of NHEJ-mediated repair we focused on improving the gene editing efficiency in HSCs. To this aim, chemically modified small guide RNAs (MS-sgRNAs) enabled us to increase the editing efficacy 8-fold compared to efficacies obtained with in vitro transcribed sgRNAs, reaching up to 89% indels in healthy donor hematopoietic stem/progenitor cells. Moreover, the CRISPR/Cas9 system demonstrated high editing capacity in the primitive HSCs capable of engrafting immunodeficient NSG mice, confirming the efficacy of NHEJ-editing to correct the phenotype of long-term repopulating HSCs. Finally, studies conducted in mobilized peripheral blood and bone marrow CD34+ cells from FA patients demonstrated the feasibility to correct FA HSCs by NHEJ-mediated gene editing and confirmed the proliferative advantage of NHEJ-mediated corrected cells both in vitro and in vivo. Our results suggest that NHEJ-mediated gene editing should constitute a versatile and simple therapeutic approach to efficiently correct specific mutations in FA and other monogenic disorders of the hematopoietic system. Disclosures Sevilla: Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Rio:Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects’ cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-strand breaks, and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here we describe for the first time the possibility to correct mutations in FA genes by generating compensatory indels that restore their coding frame. The efficacy of the strategy was first verified in FA lymphoblastic cell lines and then in primary FA CD34 cells, which acquired proliferative advantage and correction of their characteristic FAcell phenotype. Importantly, in contrast to homologous directed repair (HDR), the efficacy of NHEJ to edit primitive human HSCs was comparable to that observed in more mature progenitor cells, indicating that NHEJ-editing approaches should constitute a good approach for the editing of long-term repopulating HSCs and consequently for the treatment of FA and other monogenic hematopoietic diseases.

In about 10% of patients affected by Fanconi anemia (FA) the diagnosis is delayed until adulthood, and the presenting symptom in these "occult" FA cases is often a solid cancer and cancer treatment-related toxicity. Highly predictive clinical parameter(s) for diagnosing such an adult-onset cases are missing.(1) Exome sequencing (ES), (2) Sanger sequencing of FANCA, (3) diepoxybutane (DEB)-induced chromosome breakage test.ES identified a pathogenic homozygous FANCA variant in a patient affected by Sertoli cell-only syndrome (SCOS) and in his azoospermic brother. Although they had no overt anemia, chromosomal breakage test revealed a reverse somatic mosaicism in the former and a typical FA picture in the latter. In 27 selected SCOS cases, 1 additional patient showing compound heterozygous pathogenic FANCA variants was identified with positive chromosomal breakage test.We report an extraordinarily high frequency of FA in a specific subgroup of azoospermic patients (7.1%). The screening for FANCA pathogenic variants in such patients has the potential to identify undiagnosed FA before the appearance of other severe clinical manifestations of the disease. The definition of this high-risk group for "occult" FA, based on specific testis phenotype with mild/borderline hematological alterations, is of unforeseen clinical relevance.

PurposeMutations in genes involved in Fanconi anemia (FA)/BRCA DNA repair pathway cause cancer susceptibility diseases including familial breast cancer and Fanconi anemia (FA). A single FA patient with biallelic FANCM mutations was reported in 2005 but concurrent FANCA pathogenic mutations precluded assignment of FANCM as an FA gene. Here we report three individuals with biallelic FANCM truncating mutations who developed early-onset cancer and toxicity to chemotherapy but did not present congenital malformations or any hematological phenotype suggestive of FA.MethodsChromosomal breakages, interstrand crosslink sensitivity, and FANCD2 monoubiquitination were assessed in primary fibroblasts. Mutation analysis was achieved through Sanger sequencing. Genetic complementation of patient-derived cells was performed by lentiviral mediated transduction of wild-type FANCM complementary DNA followed by functional studies.ResultsPatient-derived cells exhibited chromosomal fragility, hypersensitivity to interstrand crosslinks, and impaired FANCD2 monoubiquitination. We identified two homozygous mutations (c.2586_2589del4; p.Lys863Ilefs*12 and c.1506_1507insTA; p.Ile503*) in FANCM as the cause of the cellular phenotype. Patient-derived cells were genetically complemented upon wild-type FANCM complementary DNA expression.ConclusionLoss-of-function mutations in FANCM cause a cancer predisposition syndrome clinically distinct from bona fide FA. Care should be taken with chemotherapy and radiation treatments in these patients due to expected acute toxicity.

Fanconi anemia (FA) and ataxia telangiectasia (AT) share common traits such chromosomal instability and proneness to hematological cancers. Both AT and FA cell lines, and patients, are characterized by abnormally high levels of oxidative stress markers. The key FA protein FANCD2 is phosphorylated on Ser 222 by ATM after ionizing radiation (IR), thus allowing normal activation of the S-phase checkpoint, and ATM cells are known to be hypersensitive to oxidative damage. In this work we show that FANCD2 deficient cells have a defective S-phase checkpoint after Hydrogen Peroxide (H(2)O(2)) induced oxidative damage. ATM dependent phosphorylation of FANCD2 at the S222 residue is necessary for normal S-phase checkpoint activation after oxidative stress, while FANCD2 monoubiquitination at K561 is dispensable. We also show that FANCD2 is not required for base excision repair of 8-oxoG and other DNA lesions (abasic sites, uracils), while treatments that exclusively induce 8-oxoG, but not DNA double strand breaks, fail to activate FANCD2 monoubiquitination, thus indicating that the known accumulation of 8-oxoG in FA cells reflects an overproduction of ROS rather than defective processing of oxidized bases. We conclude that the handling of DNA damage after H(2)O(2)-induced oxidative stress requires the coordinated action of FANCD2 and ATM.

© The Author 2015. Published by Oxford University Press. All rights reserved. Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p. Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p. Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Fanconi anemia (FA) is a rare, clinically heterogeneous autosomal recessive or X-linked genetic disease characterized by chromosome fragility, congenital malformations and cancer susceptibility. FA patients are usually radiosensitive when exposed to radiotherapy but the role of the FA in response to ionizing radiation (IR) is controversial. Here we have investigated IR-induced activation of the FA pathway by systematically analyzing monoubiquitination of the central protein FANCD2 and subsequent recruitment to stalled replication forks in primary fibroblasts. We developed an immunolabelling method to simultaneously visualize IR-induced FANCD2 and γH2AX foci in S-phase. We observed FANCD2 foci formation in a subset of IR-induced γH2AX foci in S-phase cells. This was observed at doses of IR ranging from 0.1 to 5.0Gy in a dose dependent non-threshold fashion. Our results indicate that minimum doses of IR can produce replication fork stalling and FA pathway activation during S-phase in primary cells.

Fanconi anemia (FA) is characterized by bone marrow failure, malformations, and chromosome fragility. We review the recent discovery of FA genes and efforts to develop genetic therapies for FA in the last five years. Because current data exclude FANCM as an FA gene, 15 genes remain bona fide FA genes and three (FANCO, FANCR and FANCS) cause an FA like syndrome. Monoallelic mutations in 6 FA associated genes (FANCD1, FANCJ, FANCM, FANCN, FANCO and FANCS) predispose to breast and ovarian cancer. The products of all these genes are involved in the repair of stalled DNA replication forks by unhooking DNA interstrand cross-links and promoting homologous recombination. The genetic characterization of patients with FA is essential for developing therapies, including hematopoietic stem cell transplantation from a savior sibling donor after embryo selection, gene therapy, or genome editing using genetic recombination or engineered nucleases. Newly acquired knowledge about FA promises to provide therapeutic strategies in the near future.

The S3 ribosomal protein of Drosophila melanogaster possesses various DNA repair activities, including the capacity to incise at apurinic/apyrimidinic (AP) sites and 8-oxo-7,8-dihydroguanine (8-oxoG) residues. We have recently hypothesized that this multifunctional protein may improve the efficiency of DNA base excision repair (BER) in mammalian cells. We have investigated the effect of pure GST-tagged Drosophila S3 on BER of different endogenous lesions performed by human and mouse cell extracts. Drosophila S3 significantly accelerated the BER of 8-oxoG (initiated by the bifunctional glycosylase OGG1). The stimulating effect was linked to the capacity of S3 to remove the 8-oxoG lesion and cleave the resulting AP site, rather than acceleration of downstream steps of the BER pathway (e.g., removal of 3′ blocking fragments). No stimulating effect was observed on the BER of uracil, natural AP sites, and β-lyase-cleaved AP sites. Heterologous expression of Drosophila S3 may be used to enhance 8-oxoG repair in human cells. Environ. Mol. Mutagen. 42:50–58, 2003. © 2003 Wiley-Liss, Inc.

Fanconi anaemia (FA) is a rare autosomal recessive disease characterized by increased spontaneous and DNA crosslinker-induced chromosome instability, progressive pancytopenia and cancer susceptibility. An increasing number of genes are involved in FA, including the breast cancer susceptibility gene BRCA2. Five of the FA proteins (FANCA, FANCC, FANCE, FANCF and FANCG) assemble in a complex that is required for FANCD2 activation in response to DNA crosslinks. Active FANCD2 then interacts with BRCA1 and forms discrete nuclear foci. FANCD2 is independently phosphorylated by ATM (the protein whose gene is mutated in ataxia telangiectasia) in response to ionizing radiation. In addition, the FA proteins are interconnected with other nuclear and cytoplasmic factors all related to cellular responses to carcinogenic stress and to caretaker and gatekeeper functions. In this review, the most recently published data on the molecular biology of the FA pathway and its molecular crosstalk with ATM, BRCA1 and BRCA2, proteins involved in xenobiotic and reactive oxygen species metabolism, apoptosis, cell cycle control and telomere stability, are summarized. The currently available data indicate that FA is a central node in a complex nuclear and cytoplasmic network of tumour suppressor and genome stability pathways fully committed to prevent cancer.

Deregulation of control of the apoptotic process in Fanconi anaemia (FA) appears to be one of the main features of this disease at the cellular level. We show here that FA cells are resistant to treatments with rhodamine-1,2,3 and doxycycline, which both interfere with mitochondrial functionality by different mechanisms. In contrast, normal lymphoblastoid cells are severely affected by these treatments, which result in acute ATP depletion and a significant enhancement of the fraction of cells undergoing apoptotic cell death. FA cells are very sensitive to the action of 2-deoxy-D-glucose (2dG) and iodoacetic acid (IAA), two inhibitors of glycolytic metabolism. The ability of FA cells to sustain metabolic insults interfering with energy production and balance may be linked with the pathological manifestations of the disease, including susceptibility to acute myeloid leukemia. These findings suggest that FA genes may be involved in a pathway that mediates a protective response to stress. We suggest that a peculiar metabolic regulation in FA cells could explain both defective apoptosis and susceptibility to oxidative stress.

This study investigates the timing of p53 mutations detected in the malignant cells of a Burkitt's lymphoma cell line (BRG-P) with respect to other maturation or transforming events. The BRG-P cell line, derived from an AIDS patient, was of special value since it displayed subclones that had undergone an isotype switch from IgM to IgA, (BRG-M and BRG-A cells). BRG-M and BRG-A cells were characterized by the same monoclonal c-myc and VDJ rearrangements and by the expression of Ig receptors with specificity for a 45 kDa protein of human breast cells. Analysis of p53 mutations in the different BRG subclones showed that I) BRG-M cells displayed 2 different p53 mutations in trans; since the original BL cells also showed the same mutations, this finding indicated that both occurred in vivo; 2) one of the p53 alleles of BRG-A cells was lost, while the other showed a mutation different from those seen in BRG-M cells; and 3) all 3 mutations observed in BRG-M or BRG-A cells resulted in the functional inactivation of the transcriptional activation function of p53. Together, our data demonstrate that p53 mutations were relatively late events during lymphomagenesis. Moreover, in view of the role of p53 in cell apoptosis, it is conceivable that BRG cells were subjected to a strong selective pressure that favored p53 inactivation. Such inactivation was possibly required to counterbalance other potentially apoptotic events, including the presence of a deregulated c-myc oncogene and signals delivered by the host environment in situ.

BFanconi 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.

Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1 and 6 additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3′- and 5′-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell survival and G2 phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand crosslink repair compared with true FA genes and exclude FAN1 as a novel FA gene.

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination.

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AX(S136A/S139A)) indicate that phosphorylated H2AX (gammaH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to gammaH2AX is BRCA1-dependent and cells deficient or depleted of H2AX show an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX-deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage-induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.

Studies on cancer-prone and rare human genetic disorders often lead to significant advances in our understanding of the complex network of genome stability and DNA repair pathways that have evolved in the human genome to prevent the harmful effects of exposure to DNA damaging agents. One such disorder is Fanconi Anemia, an autosomal recessive disease characterized by an increased spontaneous and DNA cross-linkers induced chromosome instability, progressive pancytopenia and cancer susceptibility. At least eleven genes are involved in Fanconi anemia, including the breast cancer susceptibility gene BRCA2. Six of the Fanconi anemia proteins (FANCA, C, E, F, G and L) assemble in a complex that is required for FANCD2 activation by monoubiquitination in response to DNA damage or during S-phase progression. Active FANCD2 then colocalizes with the product of the breast cancer susceptibility gene BRCA1 in discrete nuclear foci. FANCD2 is also independently phosphorylated by ATM in response to ionising radiation and interacts with the MRE1 l/Rad50/NBSl complex, which is directly involved in homologous recombination DNA repair pathway and in cell cycle checkpoint response to DNA damage. Available data indicate that FANCD2 is involved in cell cycle regulation and DNA repair. Our current knowledge on the functional significance of FA pathway and more specifically FANCD2 and its interacting proteins in pathways of genomic surveillance and maintenance will be discussed in this chapter.

Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure and hypersensitivity to DNA crosslinks. Nine FA genes have been isolated so far. The biochemical function(s) of the FA proteins remain(s) poorly determined. However, a large consensus exists on the evidence that, to cope with DNA cross-links, a cell needs a functional FA pathway. In this review, we resume current understanding of how the FA pathway works in response to DNA damage and how it is integrated in a complex network of proteins involved in the maintenance of the genetic stability.

The breast cancer predisposing genes BRCA1 and BRCA2 appear to be involved in DNA repair. In particular, the sensitivity of BRCA2-deficient mouse embryonic fibroblasts to ionizing radiation and the demonstrated interaction of the BRCA2 protein with Rad51, a major factor in recombinational repair, indicate that BRCA2 is important for double strand break repair. The human BRCA2-deficient human cell line Capan-1, whilst being sensitive to ionizing radiation, is also sensitive to the alkylating agent methymethanesulfonate. The major lesions induced by this agent are methylated bases which are removed primarily by the base excision repair (BER) pathway. We have investigated the efficiency of BER in Capan-1 cells by an in vitro assay in which plasmid substrates containing a single lesion are repaired by mammalian cell extracts. In comparison to the control cell lines BxPC-3, T24 and MCF7, Capan-1 cells exhibited a reduced rate of DNA ligation during both the single-nucleotide insertion and PCNA-dependent pathways of BER. The reduced rate of DNA ligation exhibited by Capan-1 cell extracts was complemented by addition of bacteriophage T4 DNA ligase or human DNA ligase III. BRCA2-mutant Capan-1 cells may possess reduced DNA ligase activity during BER.

The presence of the 4977 bp deletion ('common deletion') in the mitochondrial DNA (mtDNA) is associated with defects in the metabolic machinery acquired during ageing as a hallmark of a degenerative phenotype. We analysed 27 samples (18 from surgical patients and nine from autopsy cases) of DNA extracted from smooth muscle cells of abdominal aorta fragments affected by atherosclerotic lesions. The deletion was detected by PCR amplification gel electrophoresis and characterized by sequencing of the PCR product. The mtDNA 'common deletion' was detected in all analysed samples. However, its levels were not particularly high, which may be ascribed to the fact that smooth muscle cells in atherosclerotic lesions have a lower energy requirement and an appreciable proliferation rate, as compared for instance with cardiac myocytes. When the subjects were divided into two numerically equivalent age classes (60-72 years plus a 45-year-old subject versus 73-95 years), the deletion had significantly higher levels in the older subjects. Conversely, its presence did not correlate with source (surgical or autoptic), sex, cigarettes consumption, other clinical and anamnestic parameters or with the levels of adducts and 8-hydroxy-2'-deoxyguanosine measured in the nuclear DNA of the same samples. A previously unreported deletion of 5111 bp was additionally found in the mtDNA from a 45-year-old woman. The origin of this lesion seems to be compatible with the slipped mispairing model proposed for the 'common deletion'.

This study investigates the timing of p53 mutations detected in the malignant cells of a Burkitt's lymphoma cell line (BRG-P) with respect to other maturation or transforming events. The BRG-P cell line, derived from an AIDS patient, was of special value since it displayed subclones that had undergone an isotype switch from IgM to IgA1 (BRG-M and BRG-A cells). BRG-M and BRG-A cells were characterized by the same monoclonal c-myc and VDJ rearrangements and by the expression of Ig receptors with specificity for a 45 kDa protein of human breast cells. Analysis of p53 mutations in the different BRG subclones showed that 1) BRG-M cells displayed 2 different p53 mutations in trans; since the original BL cells also showed the same mutations, this finding indicated that both occurred in vivo; 2) one of the p53 alleles of BRG-A cells was lost, while the other showed a mutation different from those seen in BRG-M cells; and 3) all 3 mutations observed in BRG-M or BRG-A cells resulted in the functional inactivation of the transcriptional activation function of p53. Together, our data demonstrate that p53 mutations were relatively late events during lymphomagenesis. Moreover, in view of the role of p53 in cell apoptosis, it is conceivable that BRG cells were subjected to a strong selective pressure that favored p53 inactivation. Such inactivation was possibly required to counterbalance other potentially apoptotic events, including the presence of a deregulated c-myc oncogene and signals delivered by the host environment in situ.

In the framework of a project investigating the possible involvement of cancer biomarkers in human atherogenesis, we evaluated the occurrence of K-ras mutations in the DNA extracted from smooth muscle cells of abdominal aorta atherosclerotic lesions. The molecular analysis of the DNA from 32 surgical specimens, using PCR-based denaturing gradient gel electrophoresis (DGGE), did not reveal any variant in K-ras codons 12 and 13, which are the most frequently involved codons among the ras genes mutated in various types of human tumors. Analysis of the DNA extracted from four cell lines carrying known K-ras mutational alleles showed typically positive DGGE patterns. Thus, on the whole, the conclusions of this study and of previous studies using the same biological material are consistent with the occurrence of DNA adducts in human atherosclerotic lesions but in the absence of p53 involvement or of K-ras mutations in codons 12 and 13. The search for candidate genes which may possibly be involved in the atherogenetic process warrants further studies.

The p53 tumour suppressor gene has an important role in the the maintenance of genome stability and its mutational inactivation may be at the origin of aneuploidy in cancer cells. The aim of this study was to determine whether p53 mutations were associated to DNA aneuploidy, as assessed by flow cytometry, in colorectal adenocarcinomas. Analysis of p53 mutations spectrum of the sorted nuclei was done by Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing. Overall, we studied 20 adenocarcinomas, the corresponding control mucosa, and 7 lymph node metastases.Five tumours (25%) were DNA diploid, while 15 tumours (75%) were composed of DNA aneuploid and diploid subpopulations. DNA diploid control mucosa and adenocarcinomas showed no p53 mutations, while 60% of the tumours with DNA aneuploidy had p53 mutations. Therefore, p53 mutations occurred significantly more often in DNA aneuploid than in DNA diploid tumours (p< 0.04, Fisher’s exact test). Incidences of DNA aneuploidy and p53 mutations in lymph node metastases were 60 and 86%, respectively. In all tumours showing a p53 mutation, the wild-type allele was not or only bearly visible in DNA aneuploid cells suggesting that, in such cells, aneuploidy is accompanied by complete p53 functional inactivation.The present observations suggest that p53 mutations may have a role in the origin of aneuploidy at late stages of colorectal carcinogenesis.

The oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG), the product of deamination of cytosine uracil (U), and the sites of base loss [abasic (AP) sites] are among the most frequent mutagenic lesions formed in the human genome under physiological conditions. In human cells, the enzymatic activities initiating DNA base excision repair (BER) of 8-oxoG, U and AP sites are the 8-oxoG DNA glycosylase (hOGG1), the U-DNA glycosylase (UNG) and the major hydrolytic AP endonuclease (APE/HAP1), respectively. In recent work, we observed that BER of the three lesions occurs in human cell extracts with different efficacy. In particular, 8-oxoG is repaired on average 4-fold less efficiently than U, which, in turn, is repaired 7-fold slower than the natural AP site. To discriminate whether the different rates of repair may be linked to different expression of the initiating enzymes, we have determined the amount of hOGG1, UNG and APE/HAP1 in normal human cell extracts by immunodetection techniques. Our results show that a single human fibroblast contains 123 000 +/- 22 000 hOGG1 molecules, 178 000 +/- 20 000 UNG molecules and 297 000 +/- 50 000 APE/HAP1 molecules. These limited differences in enzyme expression levels cannot readily explain the different rates at which the three lesions are repaired in vitro. Addition to reaction mixtures of titrated amounts of purified hOGG1, UNG and APE/HAP1 variably stimulated the in vitro repair replication of 8-oxoG, U and the AP site respectively and the increase was not always proportional to the amount of added enzyme. We conclude that the rates of BER depend only in part on cellular levels of initiating enzymes.

8-oxo-7,8-dihydroguanine (8-oxoG) is a potent mutagenic lesion that forms at elevated levels in cellular DNA and is repaired with low efficiency in human cells. Unlike its human counterpart, the Drosophila S3 ribosomal/repair protein is endowed with a vigorous 8 oxoG repair activity that is associated to β,δ-elimination AP lyase activity. We have recently observed that pure GST-tagged Drosophila S3 protein can significantly accelerate the in vitro repair of 8 oxoG performed by human and mouse cell extracts [Cappelli et al., unpublished data]. In this work, we have transfected Chinese hamster cells with mammalian expression vectors containing the Drosophila S3 cDNA. The cells synthesized both S3 mRNA and protein but no improved repair of 8 oxoguanine was observed. Factors important for the proper expression of Drosophila genes in mammalian cells are discussed. Teratogenesis Carcinog. Mutagen. Suppl. 1:113–121, 2003. © 2003 Wiley-Liss, Inc.