Genomic signature of Fanconi anaemia DNA repair pathway deficiency in cancer.

Fanconi anaemia (FA), a model syndrome of genome instability, is caused by a deficiency in DNA interstrand crosslink repair resulting in chromosome breakage1-3. The FA repair pathway protects against endogenous and exogenous carcinogenic aldehydes4-7. Individuals with FA are hundreds to thousands fold more likely to develop head and neck (HNSCC), oesophageal and anogenital squamous cell carcinomas8 (SCCs). Molecular studies of SCCs from individuals with FA (FA SCCs) are limited, and it is unclear how FA SCCs relate to sporadic HNSCCs primarily driven by tobacco and alcohol exposure or infection with human papillomavirus9 (HPV). Here, by sequencing genomes and exomes of FA SCCs, we demonstrate that the primary genomic signature of FA repair deficiency is the presence of high numbers of structural variants. Structural variants are enriched for small deletions, unbalanced translocations and fold-back inversions, and are often connected, thereby forming complex rearrangements. They arise in the context of TP53 loss, but not in the context of HPV infection, and lead to somatic copy-number alterations of HNSCC driver genes. We further show that FA pathway deficiency may lead to epithelial-to-mesenchymal transition and enhanced keratinocyte-intrinsic inflammatory signalling, which would contribute to the aggressive nature of FA SCCs. We propose that the genomic instability in sporadic HPV-negative HNSCC may arise as a result of the FA repair pathway being overwhelmed by DNA interstrand crosslink damage caused by alcohol and tobacco-derived aldehydes, making FA SCC a powerful model to study tumorigenesis resulting from DNA-crosslinking damage.

Gene therapy restores the transcriptional program of hematopoietic stem cells in Fanconi anemia.

Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-ß and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder.

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.

Natural gene therapy by reverse mosaicism leads to improved hematology in Fanconi anemia patients.

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.

Mosaicism in Fanconi anemia: concise review and evaluation of published cases with focus on clinical course of blood count normalization.

Fanconi anemia (FA) is a DNA repair disorder resulting from mutations in genes encoding for FA DNA repair complex components and is characterized by variable congenital abnormalities, bone marrow failure (BMF), and high incidences of malignancies. FA mosaicism arises from reversion or other compensatory mutations in hematopoietic cells and may be associated with BMF reversal and decreased blood cell sensitivity to DNA-damaging agents (clastogens); this sensitivity is a phenotypic and diagnostic hallmark of FA. Uncertainty regarding the clinical significance of FA mosaicism persists; in some cases, patients have survived multiple decades without BMF or hematologic malignancy, and in others hematologic failure occurred despite the presence of clastogen-resistant cell populations. Assessment of mosaicism is further complicated because clinical evaluation is frequently based on clastogen resistance in lymphocytes, which may arise from reversion events both in lymphoid-specific lineages and in more pluripotent hematopoietic stem/progenitor cells (HSPCs). In this review, we describe diagnostic methods and outcomes in published mosaicism series, including the substantial intervals (1-6 years) over which blood counts normalized, and the relatively favorable clinical course in cases where clastogen resistance was demonstrated in bone marrow progenitors. We also analyzed published FA mosaic cases with emphasis on long-term clinical outcomes when blood count normalization was identified. Blood count normalization in FA mosaicism likely arises from reversion events in long-term primitive HSPCs and is associated with low incidences of BMF or hematologic malignancy. These observations have ramifications for current investigational therapeutic programs in FA intended to enable gene correction in long-term repopulating HSPCs.

From exome analysis in idiopathic azoospermia to the identification of a high-risk subgroup for occult Fanconi anemia.

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.

Engraftment and in vivo proliferation advantage of gene-corrected mobilized CD34+ cells from Fanconi anemia patients.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34+ cells from FA-A patients with a therapeutic FANCA-lentiviral vector corrects the phenotype of in vitro cultured hematopoietic progenitor cells. Transplantation of transduced FA CD34+ cells into immunodeficient mice resulted in reproducible engraftment of myeloid, lymphoid, and CD34+ cells. Importantly, a marked increase in the proportion of phenotypically corrected, patient-derived hematopoietic cells was observed after transplantation with respect to the infused CD34+ graft, indicating the proliferative advantage of corrected FA-A hematopoietic repopulating cells. Our data demonstrate for the first time that optimized protocols of hematopoietic stem cell collection from FA patients, followed by the short and clinically validated transduction of these cells with a therapeutic lentiviral vector, results in the generation of phenotypically corrected HSPCs capable of repopulating and developing proliferation advantage in immunodeficient mice. Our results suggest that clinical approaches for FA gene therapy similar to those used in this study will facilitate hematopoietic repopulation in FA patients with gene corrected HSPCs, opening new prospects for gene therapy of FA patients.

Biallelic truncating FANCM mutations cause early-onset cancer but not Fanconi anemia.

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.

Individuals with FANCM biallelic mutations do not develop Fanconi anemia, but show risk for breast cancer, chemotherapy toxicity and may display chromosome fragility.

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.

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

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.

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair.

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

Savior siblings and Fanconi anemia: analysis of success rates from the family's perspective.

PURPOSE: The current curative treatment of Fanconi anemia is hematopoietic stem cell transplantation; this treatment has a higher rate of successful outcome when donors are compatible siblings. Therefore some families opt to have a healthy and compatible baby after selecting an embryo using preimplantation genetic diagnosis with human leukocyte antigen (HLA) typing. This study aims to estimate the success rate of this procedure from the family's perspective. METHODS: Genetic and embryology data were collected from genetic reports provided by the families. RESULTS: A total of 524 oocytes (14.1 oocytes/cycle) and 299 embryos were generated (8.0 embryos/cycle) after 38 in vitro fertilization cycles. Sixteen embryos were transferred to the uterus because they were non-Fanconi anemia and HLA matched. One baby was born. A younger couple delivered a healthy and HLA-compatible baby after four cycles. Therefore, the success rate per cycle is less than 5% (two babies from 42 trials). CONCLUSION: While Fanconi anemia per se does not worsen the probability of success, a critical factor is advanced maternal age; a late diagnosis leads to few transferrable embryos and high rates of aneuploidy. Families should be informed in advance of the many trials that they will probably need to undergo even if a haploidentical younger relative is available as an oocyte donor.

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells.

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

On the role of FAN1 in Fanconi anemia.

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 G(2) 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.

Evaluation of rare variants in the new fanconi anemia gene ERCC4 (FANCQ) as familial breast/ovarian cancer susceptibility alleles.

Recently, it has been reported that biallelic mutations in the ERCC4 (FANCQ) gene cause Fanconi anemia (FA) subtype FA-Q. To investigate the possible role of ERCC4 in breast and ovarian cancer susceptibility, as occurs with other FA genes, we screened the 11 coding exons and exon-intron boundaries of ERCC4 in 1573 index cases from high-risk Spanish familial breast and ovarian cancer pedigrees that had been tested negative for BRCA1 and BRCA2 mutations and 854 controls. The frequency of ERCC4 mutation carriers does not differ between cases and controls, suggesting that ERCC4 is not a cancer susceptibility gene. Interestingly, the prevalence of ERCC4 mutation carriers (one in 288) is similar to that reported for FANCA, whereas there are approximately 100-fold more FA-A than FA-Q patients, indicating that most biallelic combinations of ERCC4 mutations are embryo lethal. Finally, we identified additional bone-fide FA ERCC4 mutations specifically disrupting interstrand cross-link repair.

Origin, functional role, and clinical impact of Fanconi anemia FANCA mutations.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational "hot-spot" but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.

Telomeres and endocrine dysfunction of the adrenal and GH/IGF-1 axes.

Telomeres, located at the end of linear chromosomes, are essential to maintain genomic stability. Telomere biology has recently emerged as an important player in the fields of ageing and disease. To maintain telomere length (TL) and reduce its degradation after mitosis, the telomerase enzyme complex is produced. Genetic, epigenetic, hormonal and environmental factors can regulate telomerase function. These include stress hormones such as cortisol and growth factors. The hypothalamic-pituitary-adrenal (HPA) axis has been evaluated in psychiatric diseases where hypercortisolism and oxidative stress are often present. Some researches have linked TL shortening to increases in stress-related cortisol, but others have not. The effects of cortisol on the telomere system are complex and may depend on the intensity and duration of exposure. On the other hand, low levels of IGF-1 are associated with inflammation and ageing-related diseases (ischaemic heart disease, congestive heart failure). Both IGF-1 and TL diminish with age and are positively and strongly correlated with each other. It is not clear whether this positive correlation reflects a single association or a cause-effect relationship. Further research will ideally investigate longitudinal changes in telomeres and both these hormonal axes. To our knowledge, TL dysfunction has not been described in either endogenous hypercortisolism (Cushing's syndrome) or acromegaly where excessive amounts of GH and consequently IGF-1 are produced. This review focuses on the possible relationships between telomere dysfunction and the hypothalamic-pituitary-adrenal (HPA) axis and GH-IGF-1 system.

A self-repair history: compensatory effect of a de novo variant on the FANCA c.2778+83C>G splicing mutation.

Introduction: Fanconi anemia (FA) is a genome instability condition that drives somatic mosaicism in up to 25% of all patients, a phenomenon now acknowledged as a good prognostic factor. Herein, we describe the case of P1, a FA proband carrying a splicing variant, molecularly compensated by a de novo insertion. Methods and Results: Targeted next-generation sequencing on P1's peripheral blood DNA detected the known FANCA c.2778 + 83C > G intronic mutation and suggested the presence of a large deletion on the other allele, which was then assessed by MLPA and RT-PCR. To determine the c.2778 + 83C > G splicing effect, we performed a RT-PCR on P1's lymphoblastoid cell line (LCL) and on the LCL of another patient (P2) carrying the same variant. Although we confirmed the expected alternative spliced form with a partial intronic retention in P2, we detected no aberrant products in P1's sample. Sequencing of P1's LCL DNA allowed identification of the de novo c.2778 + 86insT variant, predicted to compensate 2778 + 83C > G impact. Albeit not found in P1's bone marrow (BM) DNA, c.2778 + 86insT was detected in a second P1's LCL established afterward, suggesting its occurrence at a low level in vivo. Minigene assay recapitulated the c.2778 + 83C > G effect on splicing and the compensatory role of c.2778 + 86insT in re-establishing the physiological mechanism. Accordingly, P1's LCL under mitomycin C selection preserved the FA pathway activity in terms of FANCD2 monoubiquitination and cell survival. Discussion: Our findings prove the role of c.2778 + 86insT as a second-site variant capable of rescuing c.2778 + 83C > G pathogenicity in vitro, which might contribute to a slow hematopoietic deterioration and a mild hematologic evolution.