Hemophagocytic syndrome in a patient with Fanconi anemia and VACTERL association / Síndrome hemofagocítica em paciente com anemia de Fanconi e associação VACTERL

INTRODUCTION: Hemophagocytic syndrome results from hyperactivity of histiocytes and lymphocytes, triggered by infections, mainly viral by cytomegalovirus, Epstein-Barr and herpes. Fanconi anemia (FA) is a rare genetic disease with heterogeneous symptoms common to other diseases such as VACTERL, a disease of unknown etiology in which there are several congenital malformations. The concomitance of Fanconi and VACTERL anemia occurs in 5 to 30% of FA patients. REPORT: A 14-month-old male infant was admitted to investigate fever, hepatosplenomegaly, and granulopenia. The patient was diagnosed with hemophagocytic syndrome due to hyperferritinemia, bone marrow hemophagocytosis, transaminase elevation, decreased fibrinogen, and cytomegalovirus (CMV) infection confirmed by serology and PCR. The test with mitomycin C (MMC) showed chromosomal fragility. The patient was diagnosed with a VACTERL/FA association for having a clinic and a test compatible with both FA and VACTERL. CONCLUSION: The VACTERL/FA association is seldom described, but is present in pediatric medical practice. This study presented the main clinical-laboratory aspects and reviewed the main aspects of the concurrence of this pathology.

INTRODUÇÃO: A síndrome hemofagocítica decorre da hiperatividade de histiócitos e linfócitos e é desencadeada por infeções, principalmente virais por citomegalovírus, Epstein-barr e herpes. A anemia de Fanconi (AF) é uma doença genética rara com sintomas heterogêneos em comum a outras doenças como a associação VACTERL, uma doença de etiologia desconhecida na qual existe diversas mal formações congênitas. A concomitância da anemia de Fanconi e VACTERL é descrita em 5 a 30% dos pacientes AF. RELATO: Lactente de 14 meses, sexo masculino, admitido para investigar um quadro de febre, hepatoesplenomegalia e granulopenia. Os exames laboratoriais mostraram a hiperferritemia, elevação da transaminases, medula óssea com hemofagocitose e, sorologia e PCR positivos para citomegalovírus (CMV). O paciente foi diagnosticado com síndrome hemofagocítica por citomegalovírus. Como havia também hipoplasia do polegar esquerdo, presença de hemivértebra, agenesia renal e teste positivo de fragilidades cromossômicas com mitomicina C (MMC), o paciente foi diagnosticado com associação VACTERL/AF. CONCLUSÃO: O citomegalovírus quando infecta pacientes com problemas de imunidade como AF, apresenta risco de desencadear a síndrome hemofagocítica. A associação VACTERL/AF é pouco descrita, mas presente na prática médica da pediatria. Esse estudo descreveu os principais aspectos clínicos-laboratoriais e revisou os aspectos fundamenais descritos sobre a concomitância dessas patologias.

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites.

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

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.

[Genetic analysis of an individual with a fragile site at 16q22].

OBJECTIVE: To analyze a patient with infertility and a fragile site found at 16q22 by using cytogenetic methods. METHODS: Peripheral blood sample was taken from the patient and subjected to chromosomal karyotyping and single nucleotide polymorphism microarray (SNP-array) analysis. RESULTS: The patient was found to be a mosaicism for a fragile site at 16q22, which has a variable morphology and cannot be induced by folic acid treatment. No abnormality was found by SNP-array analysis. CONCLUSION: A rare fragile site, which can be induced without folic acid treatment, has been identified at 16q22. The strategy of assisted reproduction for such individuals is yet to be explored.

FANCD2 modulates the mitochondrial stress response to prevent common fragile site instability.

Common fragile sites (CFSs) are genomic regions frequently involved in cancer-associated rearrangements. Most CFSs lie within large genes, and their instability involves transcription- and replication-dependent mechanisms. Here, we uncover a role for the mitochondrial stress response pathway in the regulation of CFS stability in human cells. We show that FANCD2, a master regulator of CFS stability, dampens the activation of the mitochondrial stress response and prevents mitochondrial dysfunction. Genetic or pharmacological activation of mitochondrial stress signaling induces CFS gene expression and concomitant relocalization to CFSs of FANCD2. FANCD2 attenuates CFS gene transcription and promotes CFS gene stability. Mechanistically, we demonstrate that the mitochondrial stress-dependent induction of CFS genes is mediated by ubiquitin-like protein 5 (UBL5), and that a UBL5-FANCD2 dependent axis regulates the mitochondrial UPR in human cells. We propose that FANCD2 coordinates nuclear and mitochondrial activities to prevent genome instability.

Genetic analysis of an individual with a fragile site at 16q22 / 中华医学遗传学杂志

OBJECTIVE@#To analyze a patient with infertility and a fragile site found at 16q22 by using cytogenetic methods.@*METHODS@#Peripheral blood sample was taken from the patient and subjected to chromosomal karyotyping and single nucleotide polymorphism microarray (SNP-array) analysis.@*RESULTS@#The patient was found to be a mosaicism for a fragile site at 16q22, which has a variable morphology and cannot be induced by folic acid treatment. No abnormality was found by SNP-array analysis.@*CONCLUSION@#A rare fragile site, which can be induced without folic acid treatment, has been identified at 16q22. The strategy of assisted reproduction for such individuals is yet to be explored.

The epicenter of chromosomal fragility of Fra14A2, the mouse ortholog of human FRA3B common fragile site, is largely attached to the nuclear matrix in lymphocytes but not in other cell types that do not express such a fragility.

Common fragile sites (CFSs) correspond to chromosomal regions susceptible to present breaks, discontinuities or constrictions in metaphase chromosomes from cells subjected to replication stress. They are considered as genomic regions intrinsically difficult to replicate and they are evolutionary conserved at least in mammals. However, the recent discovery that CFSs are cell-type specific indicates that DNA sequence by itself cannot account for CFS instability. Nevertheless, the large gene FHIT that includes FRA3B, the most highly expressed CFS in human lymphocytes, is commonly deleted in a variety of tumors suggesting a tumor suppressor role for its product. Here, we report that the epicenter of fragility of Fra14A2/Fhit, the mouse ortholog of human FRA3B/FHIT that like its human counterpart is the most highly expressed CFS in mouse lymphocytes, is largely attached to the nuclear matrix compartment in naive B lymphocytes but not in primary hepatocytes or cortical neurons that do not express such a CFS. Our results suggest a structural explanation for the difficult-to-replicate nature of such a region and so for its common fragility in lymphocytes, that is independent of the possible tumor suppressor role of the gene harboring such CFS.

Genetic Assays to Study Repeat Fragility in Saccharomyces cerevisiae.

Trinucleotide repeats are common in the human genome and can undergo changes in repeat number and cause length-dependent chromosome fragility. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), with variations that allow the role of transcription to be evaluated. The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC). The second fragility assay relies on the fact that a chromosomal break stimulates recombination-mediated repair. A PCR-based assay can be used to evaluate instability of the repeat in the same conditions used to measure repeat fragility. These assays have contributed to understanding the genetic mechanisms that cause chromosome breaks and tract-length changes at unstable trinucleotide repeats.

Chromosome fragility in the buccal epithelium in patients with Fanconi anemia.

Fanconi anemia (FA) is a rare genome instability syndrome characterized by progressive bone marrow failure and predisposition to cancer, especially head and neck squamous cell carcinoma. Surgical resection is the standard of care for solid tumors, as patients with FA do not tolerate genotoxic chemotherapies or radiation, leading to poor prognosis. It is therefore imperative to develop chemoprevention strategies such as the identification of novel biomarkers to detect the formation of the tumor before its emergence and to use them in clinical trials aimed to counteract genome instability of patients with FA in tissues at risk. Micronuclei (MN) are chromosome fragments that are left behind in anaphase and appear in daughter cells as small additional nuclei. In this work, we analyzed MN frequencies in exfoliated buccal cells from 40 patients with FA and 24 controls. We found that MN frequency was significantly increased in the FA cohort indicating that we can detect chromosome fragility in patients with FA in basal conditions and in a tissue that is divided in vivo. Consequently, the MN assay in exfoliated buccal cells of patients with FA could be used in cancer risk studies and clinical trials aimed to identify cancer chemopreventive drugs.

Biallelic germline BRCA1 mutations in a patient with early onset breast cancer, mild Fanconi anemia-like phenotype, and no chromosome fragility.

BACKGROUND: Biallelic BRCA1 mutations are regarded either embryonically lethal or to cause Fanconi anemia (FA), a genomic instability syndrome characterized by bone marrow failure, developmental abnormalities, and cancer predisposition. We report biallelic BRCA1 mutations c.181T > G (p.Cys61Gly) and c.5096G > A (p.Arg1699Gln) in a woman with breast cancer diagnosed at the age of 30 years. The common European founder mutation p.Cys61Gly confers high cancer risk, whereas the deleterious p.Arg1699Gln is hypomorphic and was suggested to confer intermediate cancer risk. METHODS AND RESULTS: Aside from significant toxicity from chemotherapy, the patient showed mild FA-like features (e.g., short stature, microcephaly, skin hyperpigmentation). Chromosome fragility, a hallmark of FA patient cells, was not present in patient-derived peripheral blood lymphocytes. We demonstrated that the p.Arg1699Gln mutation impairs DNA double-strand break repair, elevates RAD51 foci levels at baseline, and compromises BRCA1 protein function in protecting from replication stress. Although the p.Arg1699Gln mutation compromises BRCA1 function, the residual activity of the p.Arg1699Gln allele likely prevents from chromosome fragility and a more severe FA phenotype. CONCLUSION: Our data expand the clinical spectrum associated with biallelic BRCA1 mutations, ranging from embryonic lethality to a mild FA-like phenotype and no chromosome fragility.

Loss of a Fragile Chromosome Region leads to the Screwy Phenotype in Paramecium tetraurelia.

A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.

The processivity factor Pol32 mediates nuclear localization of DNA polymerase delta and prevents chromosomal fragile site formation in Drosophila development.

The Pol32 protein is one of the universal subunits of DNA polymerase δ (Pol δ), which is responsible for genome replication in eukaryotic cells. Although the role of Pol32 in DNA repair has been well-characterized, its exact function in genome replication remains obscure as studies in single cell systems have not established an essential role for Pol32 in the process. Here we characterize Pol32 in the context of Drosophila melanogaster development. In the rapidly dividing embryonic cells, loss of Pol32 halts genome replication as it specifically disrupts Pol δ localization to the nucleus. This function of Pol32 in facilitating the nuclear import of Pol δ would be similar to that of accessory subunits of DNA polymerases from mammalian Herpes viruses. In post-embryonic cells, loss of Pol32 reveals mitotic fragile sites in the Drosophila genome, a defect more consistent with Pol32's role as a polymerase processivity factor. Interestingly, these fragile sites do not favor repetitive sequences in heterochromatin, with the rDNA locus being a striking exception. Our study uncovers a possibly universal function for DNA polymerase ancillary factors and establishes a powerful system for the study of chromosomal fragile sites in a non-mammalian organism.

Fragile DNA contributes to repeated evolution.

Sequence features that affect DNA fragility might facilitate fast, repeated evolution by elevating mutation rates at genomic hotspots.

The role of fork stalling and DNA structures in causing chromosome fragility.

Alternative non-B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single-stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT-rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance.

Mrc1 and Tof1 prevent fragility and instability at long CAG repeats by their fork stabilizing function.

Fork stabilization at DNA impediments is key to maintaining replication fork integrity and preventing chromosome breaks. Mrc1 and Tof1 are two known stabilizers that travel with the replication fork. In addition to a structural role, Mrc1 has a DNA damage checkpoint function. Using a yeast model system, we analyzed the role of Mrc1 and Tof1 at expanded CAG repeats of medium and long lengths, which are known to stall replication forks and cause trinucleotide expansion diseases such as Huntington's disease and myotonic dystrophy. We demonstrate that the fork stabilizer but not the checkpoint activation function of Mrc1 is key for preventing DNA breakage and death of cells containing expanded CAG tracts. In contrast, both Mrc1 functions are important in preventing repeat length instability. Mrc1 has a general fork protector role that is evident at forks traversing both repetitive and non-repetitive DNA, though it becomes crucial at long CAG repeat lengths. In contrast, the role of Tof1 in preventing fork breakage is specific to long CAG tracts of 85 or more repeats. Our results indicate that long CAG repeats have a particular need for Tof1 and highlight the importance of fork stabilizers in maintaining fork integrity during replication of structure-forming repeats.

Phenotypic Spectrum of Epidermolysis Bullosa: The Paradigm of Syndromic versus Non-Syndromic Skin Fragility Disorders.

The heritable forms of epidermolysis bullosa (EB), a phenotypically heterogeneous group of skin fragility disorders, is currently associated with mutations in as many as 21 distinct genes. EB is primarily a disorder affecting the epithelial layers of skin and mucous membranes, without extracutaneous manifestations, and thus is nonsyndromic. However, recent demonstrations of skin blistering in multisystem disorders with single gene defects highlight the concept of syndromic EB. Here, we review the phenotypic and genotypic features of syndromic forms of EB to delineate the concept of syndromic versus nonsyndromic skin fragility disorders.

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.

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.

Genome Organization Drives Chromosome Fragility.

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.