Sequencing Micronuclei Reveals the Landscape of Chromosomal Instability

Genome instability (GIN) is a main contributing factor to congenital and somatic diseases, but its sporadic occurrence in individual cell cycles makes it difficult to study mechanistically. One profound manifestation of GIN is the formation of micronuclei (MN), the engulfment of chromosomes or chromosome fragments in their own nuclear structures separate from the main nucleus. Here, we developed MN-seq, an approach for sequencing the DNA contained within micronuclei. We applied MN-seq to mice with mutations in Mcm4 and Rad9a, which disrupt DNA replication, repair, and damage responses. Data analysis and simulations show that centromere presence, fragment length, and a heterogenous landscape of chromosomal fragility all contribute to the patterns of DNA present within MN. In particular, we show that long genes, but also gene-poor regions, are associated with chromosome breaks that lead to the enrichment of particular genomic sequences in MN, in a genetic background-specific manner. Finally, we introduce single-cell micronucleus sequencing (scMN-seq), an approach to sequence the DNA present in MN of individual cells. Together, sequencing micronuclei provides a systematic approach for studying GIN and reveals novel molecular associations with chromosome breakage and segregation.

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

Graft 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 GR patients and compared to 15 HCT controls. All patients had a diagnosis of aplastic anemia, Fanconi anemia or genetically undefined chromosomal fragility syndrome. All GR patients were febrile during GR, therefore control HCT patients were matched for diagnosis and early fevers after HCT. GR patients had significantly higher CXCL9, BAFF and sC5b-9 at the time of fever and GR compared to control HCT patients at the time of fever. The maximum fever was significantly higher and occurred significantly later in the transplant course in GR patients compared to febrile HCT controls. These data support the use of CXCL9, BAFF, sC5b-9 and fever kinetics as GR markers. Two GR patients underwent a 2nd HCT that was complicated by high fevers. Both patients received interferon and complement blockers during their 2nd 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.

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.

A NEW METHODOLOGY FOR DIAGNOSIS OF FANCONI ANEMIA BASED ON BIOLOGICAL DOSIMETRY

Fanconi Anemia (FA) is a syndrome associated with chromosomal fragility. Current laboratory tests to diagnose this disease are based on the scoring of chromosomal aberrations induced in peripheral blood lymphocytes by clastogenic chemical agents, mainly: diepoxybutane (DEB) or mitomycin C (MMC). This study evaluated an alternative test for the diagnosis of FA, in which ionizing radiation replaces DEB/MMC. Two groups were studied: normal and DEB-sensitive individuals. From each individual, samples of peripheral blood were irradiated using an electron linear accelerator. Following lymphocyte cultures, and slide preparation, metaphases were scored based on the same methodology for biological dosimetry, according to recommendations of the International Atomic Energy Agency. Our results emphasized a pattern of distribution of dicentrics, fragments, as well as abnormal chromosomal arrangements. The methodology of analysis here proposed permitted to distinguish normal from DEB-sensitive subjects.

Alternative Donor and Disease-Specific Conditioning Regimen Hematopoietic Stem Cell Transplantation for Inherited Bone Marrow Failure Syndromes

Background: The outcomes of alternative donor haematopoietic stem cell Transplantation(HSCT)for Inherited bone marrow failure syndromes(IBMFS) have not been well described,especially the conditioning regimens are major challenges ,each disease type has different characteristics, whether engraftment can be achieved with less toxicity. Method : With this background,we retrospectively analyzed alternative donor HSCT for 42 patients with IBMFS in our single center from November 2012 to August 2018. Results: 27 cases were Fanconia anemia(FA),7 cases were dyskeratosis congenital(DC), 8 cases were severe congenital neutropenias(SCN). The median age at diagnosis and transplantation were 4(1 to 25 ) years and 10(1.9 to 26)years respectively. Male to female was 28 :14. All patients were confirmed to have BMF and disease-specific pathogenesis-related gene mutations. 16 cases had disease specific congenital anomalies, 10 patients had family history. Chromosomal fragility test was positive in 8 cases of FA group. Indication of HSCT for FA and DC patients which were 30 patients had BMF or transfusion dependency at transplantation;4 cases had clonal disease (2 cases myelodysplasia, 2 cases acute myeloid leukaemia). Indication of HSCT for SCN patients were uncontrollable severe infection .FA received low dose Busulfan (Bu;total dose of 6.4 mg/kg, IV), Fludarabine (Flu; total dose of 120 mg/m2, IV) , Cyclophosphamide (Cy; total dose of 2.0 g/m2, IV) based-reduced intensity conditioning(RIC) ; DC patients received low dose TBI (total 300cGy, Special position, supine) , Flu(total dose of 120 mg/m2, IV) , Cy( total dose of 3.0 g/m2, IV) based-RIC, while SCN patients had Bu(total dose of 12.8 mg/kg, IV),Cy( total dose of 3.6 g/m2, IV) or Flu(total dose of 160 mg/m2, IV) based -myeloablative conditioning(MA); and all patients combinated either of 2 different rabbit ATG ,ATG-T , rabbit anti-human thymocyte immunoglobulin, total dose 5.0 -10 mg/kg in 26 cases or ATG-F ,rabbit anti-human lymphocyte immunoglobulin, total dose 20 mg/kg in 14 cases. Campath-1, Anti-CD52 mAb was accepted with total dose 1mg/kg in 2 cases. Donor types were matched unrelated donor(MUD) in 22 patients ,Haploidentical donor (HID) in 17 patients,unrelated cord blood (UCB) in three cases. Unmanipulated stem cells were used for all patients. The Haplo-HSCT cohort received granulocyte colony-stimulating factor (G-CSF)-primed BM combined with peripheral blood stem cells (PBSCs) , The MUD HSCT cohort only received G-CSF PBSCs. The UCB HSCT cohort received one unit CB . No primary graft failure was observed. The median myeloid engraftment time was 14 (range, 10 to 21) days.Survivor median follow-up time was 38 months (range, 9-63 months), the overall survival in all patients was 76.1% ,in FA,DC,SCN were 72.4% ,100%,53.0% respectively. Cumulative incidence of 100 days acute graft-versus-host disease(GVHD) was 48.1%,Cumulative incidence 1 year and 3 years of chronic GVHD were 35.0% and 69.3% respectively. The positive chromosomal fragility test was the only independent adverse prognostic factor in multivariate analysis for FA patients rather than age ,donor type and graft source. Main causes of death were GVHD (50%) and infection (20%).No secondary malignancies occurred after HSCT till the last follow up time. Conclusion: In our study, alternative donor and disease-specific conditioning regimen HSCT for IBMFS showed promising prognosis especially for DC patients. Chromosomal fragility test positive was the only independent adverse prognostic factor in HSCT for FA patients. Disclosures No relevant conflicts of interest to declare.

Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

Common fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences

Tandem DNA repeats derived from the ancestral (TTAGGG)n run were first detected at chromosome ends of the majority of living organisms, hence the name telomeric DNA repeats. Subsequently, it has become clear that telomeric motifs are also present within chromosomes, and they were suitably called interstitial telomeric sequences (ITSs). It is well known that telomeric DNA repeats play a key role in chromosome stability, preventing end-to-end fusions and precluding the recurrent DNA loss during replication. Recent data suggest that ITSs are also important genomic elements as they confer its karyotype plasticity. In fact, ITSs appeared to be among the most unstable microsatellite sequences as they are highly length polymorphic and can trigger chromosomal fragility and gross chromosomal rearrangements. Importantly, mechanisms responsible for their instability appear to be similar to the mechanisms that maintain the length of genuine telomeres. This review compares the mechanisms of maintenance and dynamic properties of telomeric repeats and ITSs and discusses the implications of these dynamics on genome stability.

Rare case of hereditary aplastic anemia [Fanconis anemia]

Fanconi anaemia is rare autosomal recessive disease characterized by pancytopenia, varied phenotypic abnormalities, hyperpigmentation and developmental delay1. It is diagnosed by Mitomycin C sensitive chromosomal fragility test of blood lymphocytes2. We are reporting a rare case of Fanconi anaemia in a 12-year-old child. She has grossly hyperpigmentation over face, tongue, neck, intertriginous area upper and lower limb. Elder sister also had hyperpigmentation all over the body. Peripheral smear revealed macrocytic hypochromic anaemia with few ovalocytes, leukopenia and thrombocytopenia with elevated red blood cell mean corpuscular volume. Fanconi anaemia was diagnosed by doing Mitomycin c stress cytogenetic test for Fanconis anaemia. Hematopoietic stem cell transplantation is the only curative therapy for the hematologic abnormalities in fanconi anaemia3.