Chromosome Fragility Research Articles

FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

Fragile sites, chromosomal lesions, tandem repeats, and disease.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( 40 known), 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

Telomere length and chromosomal fragility increase in car painters exposed to organic solvents

ObjectiveTo comparing the chromosome fragility ratio and relative telomere length, of a group of car painters occupationally exposure to organic solvents with a group of non-exposed individuals in the city of Bogotá (Colombia). MethodsThis was a retrospective study of two cohorts (33 men each), matched by age (±2 years) at 1:1 ratio. The differences in the fragility ratio and relative telomere length distributions were determined for both groups. Case group was composed for adult males working in artisanal shops located in Bogotá (Colombia), who were occupationally exposed to organic solvents through automotive painting for a minimum of two years. Blood samples were analyzed. The chromosomal fragile sites were detected using cytogenetics techniques and DNA was extracted to determine the length of telomeres using qPCR (Cawthon’s method). Chromosomal fragility ratio and relative telomeric length were compared between car painters exposed to organic solvents and no-exposed individuals. ResultsStatistically significant differences in Chromosomal fragility ratio were found between exposed (0.645 ± 0.440, med = 0.520) and non-exposed (0.414 ± 0.217, med = 0.400) (p = 0.037, Wilcoxon one-tailed test). Also, in relative telomere length, in exposed (2.728 ± 5.581, med = 1.668) and non-exposed (1.835 ± 4.727, med = 0.732) individuals (p = 0.002, Wilcoxon one-tailed test). Low degree correlation for the two parameters, using Spearman's Rho, was found (0.21, p = 0.419). ConclusionsExposure to organic solvents increase both the telomere length and the rate of chromosomal sites fragile in the exposed group compared to the unexposed group. This study is a pioneer in the evaluation of the genotoxicity of exposure to organic solvents obtained the chromosomal fragility ratio and measuring the relative length of telomeres. We suggest applying this methodology to workers in other types of industries, for example carpentry shops, paint factories and stores where organic solvents are mixed and there is occupational exposure due to the inadequate use of protection measures or space conditioning.

Genetic landscape of a large cohort of Primary Ovarian Insufficiency: New genes and pathways and implications for personalized medicine.

SummaryBackgroundPrimary Ovarian Insufficiency (POI), a public health problem, affects 1-3.7% of women under 40 yielding infertility and a shorter lifespan. Most causes are unknown. Recently, genetic causes were identified, mostly in single families. We studied an unprecedented large cohort of POI to unravel its molecular pathophysiology.Methods375 patients with 70 families were studied using targeted (88 genes) or whole exome sequencing with pathogenic/likely-pathogenic variant selection. Mitomycin-induced chromosome breakages were studied in patients’ lymphocytes if necessary.FindingsA high-yield of 29.3% supports a clinical genetic diagnosis of POI. In addition, we found strong evidence of pathogenicity for nine genes not previously related to a Mendelian phenotype or POI: ELAVL2, NLRP11, CENPE, SPATA33, CCDC150, CCDC185, including DNA repair genes: C17orf53(HROB), HELQ, SWI5 yielding high chromosomal fragility. We confirmed the causal role of BRCA2, FANCM, BNC1, ERCC6, MSH4, BMPR1A, BMPR1B, BMPR2, ESR2, CAV1, SPIDR, RCBTB1 and ATG7 previously reported in isolated patients/families. In 8.5% of cases, POI is the only symptom of a multi-organ genetic disease. New pathways were identified: NF-kB, post-translational regulation, and mitophagy (mitochondrial autophagy), providing future therapeutic targets. Three new genes have been shown to affect the age of natural menopause supporting a genetic link.InterpretationWe have developed high-performance genetic diagnostic of POI, dissecting the molecular pathogenesis of POI and enabling personalized medicine to i) prevent/cure comorbidities for tumour/cancer susceptibility genes that could affect life-expectancy (37.4% of cases), or for genetically-revealed syndromic POI (8.5% of cases), ii) predict residual ovarian reserve (60.5% of cases). Genetic diagnosis could help to identify patients who may benefit from the promising in vitro activation-IVA technique in the near future, greatly improving its success in treating infertility.FundingUniversité Paris Saclay, Agence Nationale de Biomédecine.

Abstract 2021: Wilms tumor and pure erythroid leukemia arising from the same clone in an adolescent

Abstract Wilms tumor and pure erythroid leukemia (PEL) are distinct malignancies arising from different tissues. Here we describe a case of an adolescent with a constitutional pathogenic BRCA2 variant who was diagnosed with a Wilms tumor and developed PEL while undergoing treatment. We provide evidence that both malignancies are distinct and arose from the same clone. A 16-year-old boy presented with a diffusely anaplastic Wilms tumor metastatic to liver. He was treated with radical nephrectomy, partial hepatectomy, radiation, and multiagent chemotherapy. Six months into treatment he developed new lung and liver lesions along with cytopenias. A bone marrow aspirate confirmed a diagnosis of PEL. His disease progressed after one cycle of myeloid leukemia-directed chemotherapy and he died of disease shortly after. Samples from his initial renal tumor, blood at Wilms tumor diagnosis, and diagnostic bone marrow for PEL were characterized by whole genome sequencing and RNA sequencing. Two sequence variants were shared in both tumors but were not found in the constitutional tissue (blood at first presentation): a promoter variant in TERT (c.-124C>T) and a missense variant in PDE4DIP (p.Gln1045His). Although neither variant has been reported in Wilms tumor or PEL, the TERT variant has been well described in other tumor types and is associated with decreased survival in some carcinomas. Both tumors also had pathogenic TP53 variants albeit a different one in each tumor (p.Arg175His in the Wilms tumor, p.Asp228* in PEL). Loss of heterozygosity at TP53 was present in both malignancies via copy number loss of 17p with a shared breakpoint. Furthermore, while both malignancies had complex and distinct karyotypes, 7p loss and 22q loss with identical breakpoints were present in both. None of these variants or copy number variants were present in constitutional tissue. RNA sequencing data confirmed the histologic finding that these were distinct malignancies as the expression patterns for each malignancy were consistent with previously published expression data for Wilms tumors and myeloid leukemias respectively. Although the patient had a constitutional pathogenic BRCA2 variant, loss of heterozygosity at that locus was not found in either tumor. Chromosome fragility testing for Fanconi anemia was uninterpretable due to the concomitant use of chemotherapeutic agents at the time of testing, however the patient had no phenotypic features to support this diagnosis. In this case there are multiple lines of evidence suggesting that a Wilms tumor and PEL arose from the same clone including shared TERT variants and copy number variant breakpoints. Kidney and hematopoietic tissue both arise from the embryonic mesoderm suggesting that the earliest alterations occurred in this tissue. The contribution of each shared pathogenic variant (TERT, BRCA2, 17p loss) to tumorigenesis and the role of chemotherapy in PEL development are still under investigation. Citation Format: Anita Villani, Winnie Lo, Yisu Li, Mohamed Abdelhaleem, Mary Shago, Federico Comitani, My Linh Thibodeau, Sarah Alexander, Uri Tabori, David Malkin, Adam Shlien, Jack Brzezinski. Wilms tumor and pure erythroid leukemia arising from the same clone in an adolescent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2021.

Somatic mosaicism in patients with Fanconi anaemia: Proposal of alternative tissue for inconclusive diagnoses.

Fanconi anaemia (FA) is a rare genetic disorder marked by progressive bone marrow failure, chromosomal fragility, and increased cancer susceptibility. Laboratory diagnosis includes chromosomal instability test and mutation investigation. A total of 15%-25% of all patients may have somatic mosaicism, characterized by two distinct haematopoietic cell populations, one resistant and one sensitive to agents that induce chromosomal breakage, which complicates the diagnosis by a high incidence of reverted cells leading to inconclusive or false-negative results. The study aimed to evaluate the use of bone marrow stromal mesenchymal cells (BM-MSCs) as an alternative, non-haematopoietic tissue for diagnosis. Bone marrow mesenchymal stromal cells from 12 patients with positive diepoxybutane (DEB) tests were cultivated and analysed by cytogenetics and mutation investigation. The DEB test was performed at 0.1 and 0.01 μg/ml concentrations, with an index ranging from 0.24 to 1.00. At higher concentration, the metaphases number was lower, probably due to toxicity. Regarding the molecular investigation, all the mutations previously found in peripheral blood were identified on BM-MSC. This study demonstrated the possibility of using BM-MSCs as an alternative tissue for cytogenetic and molecular investigation. Future tests using an intermediate DEB concentration may lead to an optimal protocol that could be non-toxic to cells but provides conclusive results.

New Era of Mapping and Understanding Common Fragile Sites: An Updated Review on Origin of Chromosome Fragility

Common fragile sites (CFSs) are specific genomic loci prone to forming gaps or breakages upon replication perturbation, which correlate well with chromosomal rearrangement and copy number variation. CFSs have been actively studied due to their important pathophysiological relevance in different diseases such as cancer and neurological disorders. The genetic locations and sequences of CFSs are crucial to understanding the origin of such unstable sites, which require reliable mapping and characterizing approaches. In this review, we will inspect the evolving techniques for CFSs mapping, especially genome-wide mapping and sequencing of CFSs based on current knowledge of CFSs. We will also revisit the well-established hypotheses on the origin of CFSs fragility, incorporating novel findings from the comprehensive analysis of finely mapped CFSs regarding their locations, sequences, and replication/transcription, etc. This review will present the most up-to-date picture of CFSs and, potentially, a new framework for future research of CFSs.

Characterization of Chromosomal Instability in Glioblastoma.

Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with high chromosomal instability (CIN). Replication stress is a major cause of CIN that manifests as chromosome rearrangements, fragility, and breaks, including those cytologically expressed within specific chromosome regions named common fragile sites (CFSs). In this work, we characterized the expression of human CFSs in the glioblastoma U-251 MG cell line upon treatment with the inhibitor of DNA polymerase alpha aphidicolin (APH). We observed 52 gaps/breaks located within previously characterized CFSs. We found 17 to be CFSs in GBM cells upon treatment with APH, showing a frequency equal to at least 1% of the total gaps/breaks. We report that two CFSs localized to regions FRA2E (2p13/p12) and FRA2F (2q22) were only found in U-251 MG cells, but not lymphocytes or fibroblasts, after APH treatment. Notably, these glioblastoma-specific CFSs had a relatively high expression compared to the other CFSs with breakage frequency between ∼7 and 9%. Presence of long genes, incomplete replication, and delayed DNA synthesis during mitosis (MiDAS) after APH treatment suggest that an impaired replication process may contribute to this loci-specific fragility in U-251 MG cells. Altogether, our work offers a characterization of common fragile site expression in glioblastoma U-251 MG cells that may be further exploited for cytogenetic and clinical studies to advance our understanding of this incurable cancer.

Integrated genome and transcriptome analyses reveal the mechanism of genome instability in ataxia with oculomotor apraxia 2

Mutations in the SETX gene, which encodes Senataxin, are associated with the progressive neurodegenerative diseases ataxia with oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). To identify the causal defect in AOA2, patient-derived cells and SETX knockouts (human and mouse) were analyzed using integrated genomic and transcriptomic approaches. A genome-wide increase in chromosome instability (gains and losses) within genes and at chromosome fragile sites was observed, resulting in changes to gene-expression profiles. Transcription stress near promoters correlated with high GCskew and the accumulation of R-loops at promoter-proximal regions, which localized with chromosomal regions where gains and losses were observed. In the absence of Senataxin, the Cockayne syndrome protein CSB was required for the recruitment of the transcription-coupled repair endonucleases (XPG and XPF) and RAD52 recombination protein to target and resolve transcription bubbles containing R-loops, leading to genomic instability. These results show that transcription stress is an important contributor to SETX mutation-associated chromosome fragility and AOA2.

Transcription-Replication Collisions and Chromosome Fragility.

Accurate replication of the entire genome is critical for cell division and propagation. Certain regions in the genome, such as fragile sites (common fragile sites, rare fragile sites, early replicating fragile sites), rDNA and telomeres, are intrinsically difficult to replicate, especially in the presence of replication stress caused by, for example, oncogene activation during tumor development. Therefore, these regions are particularly prone to deletions and chromosome rearrangements during tumorigenesis, rendering chromosome fragility. Although, the mechanism underlying their “difficult-to-replicate” nature and genomic instability is still not fully understood, accumulating evidence suggests transcription might be a major source of endogenous replication stress (RS) leading to chromosome fragility. Here, we provide an updated overview of how transcription affects chromosome fragility. Furthermore, we will use the well characterized common fragile sites (CFSs) as a model to discuss pathways involved in offsetting transcription-induced RS at these loci with a focus on the recently discovered atypical DNA synthesis repair pathway Mitotic DNA Synthesis (MiDAS).

Fanconi-like anemia related to a FANCM mutation

Fanconi anemia is primarily inherited as an autosomal recessive genetic disorder with common delays in diagnosis and challenging treatments. Fanconi anemia patients have a high risk of developing solid tumors, particularly in the head and neck or anogenital regions.The diagnosis of Fanconi anemia is primarily based on the chromosomal breakage but FA gene sequencing is recommended in all patients with a positive chromosome fragility test.Here, we present a 32-year-old man with advanced tonsil squamous cell carcinoma and fatal toxicity after the first cycle of chemotherapy. No anemia was present. A recent variant mutation if the FANCM gene was detected (c1511_1515delGAGTA (pArg504AsnfsTer29)).Homozygous or double heterozygous pathogenic variants have been reported in FANCM and linked to azoospermia and primary ovarian failure without anemia. Alterations in this gene have also been associated with a genetic predisposition for solid tumors (breast and ovarian cancer) and hematological malignancies (B-cell acute lymphoblastic leukemia).Due to the hypersensitivity of these patients to DNA-damaging agents such as chemotherapy and radiotherapy, surgery is the best treatment option for malignant solid tumors. Dose reductions or alternative regimens of chemotherapy and/or radiotherapy are recommended in FA patients who develop a malignant tumor.

Mechanisms of Genome Instability in the Fragile X-Related Disorders.

The Fragile X-related disorders (FXDs), which include the intellectual disability fragile X syndrome (FXS), are disorders caused by expansion of a CGG-repeat tract in the 5′ UTR of the X-linked FMR1 gene. These disorders are named for FRAXA, the folate-sensitive fragile site that localizes with the CGG-repeat in individuals with FXS. Two pathological FMR1 allele size classes are distinguished. Premutation (PM) alleles have 54–200 repeats and confer the risk of fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X-associated primary ovarian insufficiency (FXPOI). PM alleles are prone to both somatic and germline expansion, with female PM carriers being at risk of having a child with >200+ repeats. Inheritance of such full mutation (FM) alleles causes FXS. Contractions of PM and FM alleles can also occur. As a result, many carriers are mosaic for different sized alleles, with the clinical presentation depending on the proportions of these alleles in affected tissues. Furthermore, it has become apparent that the chromosomal fragility of FXS individuals reflects an underlying problem that can lead to chromosomal numerical and structural abnormalities. Thus, large numbers of CGG-repeats in the FMR1 gene predisposes individuals to multiple forms of genome instability. This review will discuss our current understanding of these processes.

Graft rejection markers in children undergoing hematopoietic cell transplant for bone marrow failure

AbstractGraft rejection (GR) is a poorly understood complication of hematopoietic cell transplant (HCT). GR risk factors are well published, but there are no reliable biomarkers or therapies known. Fever is the most common symptom of GR, but no study has evaluated fever kinetics as a diagnostic marker of GR. The objectives of this study were to identify mechanisms, biomarkers, and potential therapies for GR after HCT. Chemokine ligand 9 (CXCL9), B-cell activating factor (BAFF), and complement markers (sC5b-9, C3a, and C5a) were measured in 7 patients with GR and compared with 15 HCT controls. All patients had a diagnosis of aplastic anemia, Fanconi anemia, or genetically undefined chromosomal fragility syndrome. All patients with GR were febrile during GR; therefore, control patients who underwent HCT were matched for diagnosis and early fevers after HCT. Patients withh GR had significantly higher CXCL9, BAFF, and sC5b-9 at the time of fever and GR compared with control patients who underwent HCT at the time of fever. The maximum fever was significantly higher and occurred significantly later in the transplant course in patients with GR compared with febrile HCT controls. These data support the use of CXCL9, BAFF, sC5b-9, and fever kinetics as GR markers. Two patients with GR underwent a second HCT that was complicated by high fevers. Both patients received interferon and complement blockers during their second HCT, and both preserved their graft. These laboratory and clinical findings support larger studies to evaluate the safety and efficacy of interferon, complement, and BAFF inhibitors for the prevention and treatment of GR after HCT.

Clinical consequences of BRCA2 hypomorphism

The tumor suppressor FANCD1/BRCA2 is crucial for DNA homologous recombination repair (HRR). BRCA2 biallelic pathogenic variants result in a severe form of Fanconi anemia (FA) syndrome, whereas monoallelic pathogenic variants cause mainly hereditary breast and ovarian cancer predisposition. For decades, the co-occurrence in trans with a clearly pathogenic variant led to assume that the other allele was benign. However, here we show a patient with biallelic BRCA2 (c.1813dup and c.7796 A > G) diagnosed at age 33 with FA after a hypertoxic reaction to chemotherapy during breast cancer treatment. After DNA damage, patient cells displayed intermediate chromosome fragility, reduced survival, cell cycle defects, and significantly decreased RAD51 foci formation. With a newly developed cell-based flow cytometric assay, we measured single BRCA2 allele contributions to HRR, and found that expression of the missense allele in a BRCA2 KO cellular background partially recovered HRR activity. Our data suggest that a hypomorphic BRCA2 allele retaining 37–54% of normal HRR function can prevent FA clinical phenotype, but not the early onset of breast cancer and severe hypersensitivity to chemotherapy.

Common Threads: Aphidicolin-Inducible and Folate-Sensitive Fragile Sites in the Human Genome.

The human genome has many chromosomal regions that are fragile, demonstrating chromatin breaks, gaps, or constrictions on exposure to replication stress. Common fragile sites (CFSs) are found widely distributed in the population, with the largest subset of these sites being induced by aphidicolin (APH). Other fragile sites are only found in a subset of the population. One group of these so-called rare fragile sites (RFSs) is induced by folate stress. APH-inducible CFSs are generally located in large transcriptionally active genes that are A + T rich and often enriched for tracts of AT-dinucleotide repeats. In contrast, all the folate-sensitive sites mapped to date consist of transcriptionally silenced CGG microsatellites. Thus, all the folate-sensitive fragile sites may have a very similar molecular basis that differs in key ways from that of the APH CFSs. The folate-sensitive FSs include FRAXA that is associated with Fragile X syndrome (FXS), the most common heritable form of intellectual disability. Both CFSs and RFSs can cause chromosomal abnormalities. Recent work suggests that both APH-inducible fragile sites and FRAXA undergo Mitotic DNA synthesis (MiDAS) when exposed to APH or folate stress, respectively. Interestingly, blocking MiDAS in both cases prevents chromosome fragility but increases the risk of chromosome mis-segregation. MiDAS of both APH-inducible and FRAXA involves conservative DNA replication and POLD3, an accessory subunit of the replicative polymerase Pol δ that is essential for break-induced replication (BIR). Thus, MiDAS is thought to proceed via some form of BIR-like process. This review will discuss the recent work that highlights the similarities and differences between these two groups of fragile sites and the growing evidence for the presence of many more novel fragile sites in the human genome.

Biology before the SOS Response-DNA Damage Mechanisms at Chromosome Fragile Sites.

The Escherichia coli SOS response to DNA damage, discovered and conceptualized by Evelyn Witkin and Miroslav Radman, is the prototypic DNA-damage stress response that upregulates proteins of DNA protection and repair, a radical idea when formulated in the late 1960s and early 1970s. SOS-like responses are now described across the tree of life, and similar mechanisms of DNA-damage tolerance and repair underlie the genome instability that drives human cancer and aging. The DNA damage that precedes damage responses constitutes upstream threats to genome integrity and arises mostly from endogenous biology. Radman’s vision and work on SOS, mismatch repair, and their regulation of genome and species evolution, were extrapolated directly from bacteria to humans, at a conceptual level, by Radman, then many others. We follow his lead in exploring bacterial molecular genomic mechanisms to illuminate universal biology, including in human disease, and focus here on some events upstream of SOS: the origins of DNA damage, specifically at chromosome fragile sites, and the engineered proteins that allow us to identify mechanisms. Two fragility mechanisms dominate: one at replication barriers and another associated with the decatenation of sister chromosomes following replication. DNA structures in E. coli, additionally, suggest new interpretations of pathways in cancer evolution, and that Holliday junctions may be universal molecular markers of chromosome fragility.

P–577 Impaired double strand DNA repair in isolated Primary Ovarian Insufficiency with homozygous nonsense mutation of SPIDR

Abstract Study question To identify the etiology of isolated Primary Ovarian Insufficiency (POI) in a patient from an Indian consanguineous family. Summary answer A homozygous nonsense mutation of SPIDR in the patient yielded chromosomal instability: first evidence of a role of this gene in DNA repair. What is known already POI, affecting 1% of women under 40, is a public health problem. To date ∼ 70% of cases remain idiopathic. The leap due to exome sequencing, led to the identification of ∼ 80 genes, often in single or few cases. SPIDR was recently identified as a scaffolding protein connecting RAD51, a central player in homologous recombination, to BLM, a helicase implicated in the integrity of the genome. But its precise role is still unknown. A SPIDR mutation was previously associated with POI. However, contradictory conclusions were reported on the mechanism of SPIDR action and on its pathogenic role in POI. Study design, size, duration Prospective genetic study of a cohort of 150 pateints with POI worldwide using a custom-made targeted next generation sequencing (NGS) panel comprising 60 known POI-causing genes. A single patient was found mutated in SPIDR. Cytogenetic studies were performed to analyse the consequences of the mutation on DNA repair and sister chromatide exchanges (SCE). Participants/materials, setting, methods The patient with SPIDR mutation had POI with primary amenorrhea, delayed puberty and streaks ovaries. She was born to consanguineous Indian parents. No other mutation was detected in our cohort of 150 patients with POI. Targeted NGS was performed in the proposita. Familial segregation was performed by Sanger sequencing. Mitomycin C (MMC)-induced chromosomal breakages were studied and a sister chromatid exchange (SCE) assay was performed in patient’s peripheral lymphocytes. Main results and the role of chance We identified a novel homozygous nonsense mutation in the exon 7 of SPIDR (KIAA0146) c.814C>T, R272*, predicted to yield either a truncated protein, or a non-sense-mediated mRNA decay. The patient’s cells display increased chromosomal fragility with high MMC-induced chromosomal breaks when compared to a control. Remarkably, there was no increased SCE. In the previous report of a SPIDR mutation in POI, no cytogenetic studies were performed, and contradictory results were obtained on a homologous recombination test between the two sisters, either enhanced or reduced. In conclusion, we show here that inactivation of SPIDR results in a defect of double strand DNA damage repair, similar to alteration of the RAD51 pathway. There was no increased SCE, the hallmark of the BLM pathway. This observation has major consequences for this patient’s care : indeed mutations of DNA-repair genes may also yield to tumors/cancers. A long follow-up of the patient is needed in a multidisciplinary team to detect possible comorbidities. Indeed, even in the absence of somatic symptomatology, the patient has enhanced chromosomal instability highlighted by cytogenetic studies, that may yield tumor-predisposition. Limitations, reasons for caution No other mutation of SPIDR in the replication cohort of 150 POI patients. SPIDR mutation are thus very rare world-wide. Wider implications of the findings: This is the first evidence of chromosomal instability associated with SPIDR defect, providing strong evidence for a role of SPIDR in double strand DNA damage repair in humans and for its causal role in POI. Our study improves the knowledge on SPIDR function and confirms its involvement in POI worldwide. Trial registration number Not applicable

Molecular Biology of the WWOX Gene That Spans Chromosomal Fragile Site FRA16D.

It is now more than 20 years since the FRA16D common chromosomal fragile site was characterised and the WWOX gene spanning this site was identified. In this time, much information has been discovered about its contribution to disease; however, the normal biological role of WWOX is not yet clear. Experiments leading to the identification of the WWOX gene are recounted, revealing enigmatic relationships between the fragile site, its gene and the encoded protein. We also highlight research mainly using the genetically tractable model organism Drosophila melanogaster that has shed light on the integral role of WWOX in metabolism. In addition to this role, there are some particularly outstanding questions that remain regarding WWOX, its gene and its chromosomal location. This review, therefore, also aims to highlight two unanswered questions. Firstly, what is the biological relationship between the WWOX gene and the FRA16D common chromosomal fragile site that is located within one of its very large introns? Secondly, what is the actual substrate and product of the WWOX enzyme activity? It is likely that understanding the normal role of WWOX and its relationship to chromosomal fragility are necessary in order to understand how the perturbation of these normal roles results in disease.

Two mechanisms of chromosome fragility at replication-termination sites in bacteria.

Chromosomal fragile sites are implicated in promoting genome instability, which drives cancers and neurological diseases. Yet, the causes and mechanisms of chromosome fragility remain speculative. Here, we identify three spontaneous fragile sites in the Escherichia coli genome and define their DNA damage and repair intermediates at high resolution. We find that all three sites, all in the region of replication termination, display recurrent four-way DNA or Holliday junctions (HJs) and recurrent DNA breaks. Homology-directed double-strand break repair generates the recurrent HJs at all of these sites; however, distinct mechanisms of DNA breakage are implicated: replication fork collapse at natural replication barriers and, unexpectedly, frequent shearing of unsegregated sister chromosomes at cell division. We propose that mechanisms such as both of these may occur ubiquitously, including in humans, and may constitute some of the earliest events that underlie somatic cell mosaicism, cancers, and other diseases of genome instability.

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.