Broken chromosomes heading into mitosis: More than one way to patch a flat tire.

A cell dealing with a broken chromosome in mitosis is like a driver dealing with a flat tire on the highway: damage repair must occur under non-ideal circumstances. Mitotic chromosome breaks encounter problems related to structures called micronuclei. These aberrant nuclei are linked to cell death, mutagenesis, and cancer. In the last few years, a flurry of studies illuminated two mechanisms that prevent mitotic problems related to micronuclei. One mechanism prevents micronuclei from forming during mitosis and involves DNA Polymerase Theta, a DNA repair regulator that patches up broken mitotic chromosomes. A second mechanism is activated after micronuclei form and then rupture, and involves CIP2A and TOPBP1 proteins, which patch micronuclear fragments to promote their subsequent mitotic segregation. Here, we review recent progress in this field of mitotic DNA damage and discuss why multiple mechanisms exist. Future studies in this exciting area will reveal new DNA break responses and inform therapeutic strategies.

A familial deletion of 10p12.1 associated with thrombocytopenia.

Thrombocytopenia can be inherited or acquired from a variety of causes. While hereditary causes of thrombocytopenia are rare, several genes have been associated with the condition. In this report, we describe an 18-year-old man and his mother, both of whom have congenital thrombocytopenia. Exome sequencing in the man revealed a 1006 kb maternally inherited deletion in the 10p12.1 region (arr[GRCh37] 10p12.1(27378928_28384564)x1) of uncertain clinical significance. This deletion in the THC2 locus includes genes ANKRD26, known to be involved in normal megakaryocyte differentiation, and MASTL, which some studies suggest is linked to autosomal dominant thrombocytopenia. In the family presented here, the deletion segregated with the congenital thrombocytopenia phenotype, suggesting that haploinsufficiency of one or both genes may be the cause. To our knowledge, this is the first report of a deletion of the THC2 locus associated with thrombocytopenia. Future functional studies of deletions of the THC2 locus may elucidate the mechanism for this phenotype observed clinically.

[Diagnosis status and genetic characteristics analysis of Fanconi anemia in China].

Objective: To analyze the clinical and molecular diagnostic status of Fanconi anemia (FA) in China. Methods: The General situation, clinical manifestations and chromosome breakage test and genetic test results of 107 pediatric FA cases registered in the Chinese Blood and Marrow Transplantation Registry Group (CBMTRG) and the Chinese Children Blood and Marrow Transplantation Registry Group (CCBMTRG) from August 2009 to January 2022 were analyzed retrospectively. Children with FANCA gene variants were divided into mild and severe groups based on the type of variant, and Wilcoxon-test was used to compare the phenotypic differences between groups. Results: Of the 176 registered FA patients, 69 (39.2%) cases were excluded due to lack of definitive genetic diagnosis results, and the remaining 107 children from 15 hospitals were included in the study, including 70 males and 37 females. The age at transplantation treatment were 6 (4, 9) years. The enrolled children were involved in 10 pathogenic genes, including 89 cases of FANCA gene, 7 cases of FANCG gene, 3 cases of FANCB gene, 2 cases of FANCE gene and 1 case each of FANCC, FANCD1, FANCD2, FANCF, FANCJ, and FANCN gene. Compound heterozygous or homozygous of loss-of-function variants account for 69.2% (72/104). Loss-of-function variants account for 79.2% (141/178) in FANCA gene variants, and 20.8% (37/178) were large exon deletions. Fifty-five children (51.4%) had chromosome breakage test records, with a positive rate of 81.8% (45/55). There were 172 congenital malformations in 80 children.Café-au-Lait spots (16.3%, 28/172), thumb deformities (16.3%,28/172), polydactyly (13.9%, 24/172), and short stature (12.2%, 21/172) were the most common congenital malformations in Chinese children with FA. No significant difference was found in the number of congenital malformations between children with severe (50 cases) and mild FANCA variants (26 cases) (Z=-1.33, P=0.185). Conclusions: FANCA gene is the main pathogenic gene in children with FA, where the detection of its exon deletion should be strengthened clinically. There were no phenotypic differences among children with different types of FANCA variants. Chromosome break test is helpful to determine the pathogenicity of variants, but its accuracy needs to be improved.

Loss of function of FIGNL1, a DNA damage response gene, causes human ovarian dysgenesis.

Ovarian dysgenesis (OD), an XX disorder of sex development, presents with primary amenorrhea, hypergonadotrophic hypogonadism, and infertility. In an Ashkenazi Jewish patient with OD, whole exome sequencing identified compound heterozygous frameshifts in FIGNL1, a DNA damage response (DDR) gene: c.189del and c.1519_1523del. Chromosomal breakage was significantly increased in patient cells, both spontaneously, and following mitomycin C exposure. Transfection of DYK-tagged FIGNL1 constructs in HEK293 cells showed no detectable protein in FIGNL1c.189del and truncation with reduced expression in FIGNL1c.1519_1523del (64% of wild-type [WT], P = .003). FIGNL1 forms nuclear foci increased by phleomycin treatment (20.6 ± 1.6 vs 14.8 ± 2.4, P = .02). However, mutant constructs showed reduced DYK-FIGNL1 foci formation in non-treated cells (0.8 ± 0.9 and 5.6 ± 1.5 vs 14.8 ± 2.4 in DYK-FIGNL1WT, P < .001) and no increase with phleomycin treatment. In conclusion, FIGNL1 loss of function is a newly characterized OD gene, highlighting the DDR pathway's role in ovarian development and maintenance and suggesting chromosomal breakage as an assessment tool in XX-DSD patients.

The abscission checkpoint senses chromatin bridges through Top2α recruitment to DNA knots.

In response to chromatin bridges, the abscission checkpoint delays completion of cytokinesis to prevent chromosome breakage or tetraploidization. Here, we show that spontaneous or replication stress-induced chromatin bridges exhibit "knots" of catenated and overtwisted DNA next to the midbody. Topoisomerase IIα (Top2α) forms abortive Top2-DNA cleavage complexes (Top2ccs) on DNA knots; furthermore, impaired Top2α-DNA cleavage activity correlates with chromatin bridge breakage in cytokinesis. Proteasomal degradation of Top2ccs is required for Rad17 localization to Top2-generated double-strand DNA ends on DNA knots; in turn, Rad17 promotes local recruitment of the MRN complex and downstream ATM-Chk2-INCENP signaling to delay abscission and prevent chromatin breakage. In contrast, dicentric chromosomes that do not exhibit knotted DNA fail to activate the abscission checkpoint in human cells. These findings are the first to describe a mechanism by which the abscission checkpoint detects chromatin bridges, through generation of abortive Top2ccs on DNA knots, to preserve genome integrity.

Inactivating TDP2 missense mutation in siblings with congenital abnormalities reminiscent of fanconi anemia.

Mutations in TDP2, encoding tyrosyl-DNA phosphodiesterase 2, have been associated with a syndromal form of autosomal recessive spinocerebellar ataxia, type 23 (SCAR23). This is a very rare and progressive neurodegenerative disorder described in only nine patients to date, and caused by splice site or nonsense mutations that result in greatly reduced or absent TDP2 protein. TDP2 is required for the rapid repair of DNA double-strand breaks induced by abortive DNA topoisomerase II (TOP2) activity, important for genetic stability in post-mitotic cells such as neurons. Here, we describe a sibship that is homozygous for the first TDP2 missense mutation (p.Glu152Lys) and which presents with clinical features overlapping both SCAR23 and Fanconi anemia (FA). We show that in contrast to previously reported SCAR23 patients, fibroblasts derived from the current patient retain significant levels of TDP2 protein. However, this protein is catalytically inactive, resulting in reduced rates of repair of TOP2-induced DNA double-strand breaks and cellular hypersensitivity to the TOP2 poison, etoposide. The TDP2-mutated patient-derived fibroblasts do not display increased chromosome breakage following treatment with DNA crosslinking agents, but both TDP2-mutated and FA cells exhibit increased chromosome breakage in response to etoposide. This suggests that the FA pathway is required in response to TOP2-induced DNA lesions, providing a possible explanation for the clinical overlap between FA and the current TDP2-mutated patients. When reviewing the relatively small number of patients with SCAR23 that have been reported, it is clear that the phenotype of such patients can extend beyond neurological features, indicating that the TDP2 protein influences not only neural homeostasis but also other tissues as well.

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

Chromosomal breakage tests in the differential diagnosis of Fanconi anemia and aplastic anemia.

BACKGROUND: FA patients are hypersensitive to preconditioning of bone marrow transplantation. OBJECTIVE: Assessment of the power of mitomycin C (MMC) test to assign FA patients. METHODS: We analysed 195 patients with hematological disorders using spontaneous and two types of chromosomal breakage tests (MMC and bleomycin). In case of presumed Ataxia telangiectasia (AT), patients' blood was irradiated in vitro to determine the radiosensitivity of the patients. RESULTS: Seven patients were diagnosed as having FA. The number of spontaneous chromosomal aberrations was significantly higher in FA patients than in aplastic anemia (AA) patients including chromatid breaks, exchanges, total aberrations, aberrant cells. MMC-induced ≥10 break/cell was 83.9 ± 11.4% in FA patients and 1.94 ± 0.41% in AA patients (p < .0001). The difference in bleomycin-induced breaks/cell was also significant: 2.01 ± 0.25 (FA) versus 1.30 ± 0.10 (AA) (p = .019). Seven patients showed increased radiation sensitivity. Both dicentric + ring, and total aberrations were significantly higher at 3 and 6 Gy compared to controls. CONCLUSIONS: MMC and Bleomycin tests together proved to be more informative than MMC test alone for the diagnostic classification of AA patients, while in vitro irradiation tests could help detect radiosensitive-as such, individuals with AT.

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks.

Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG)70 or (CTG)70 repeat tract. When the ssDNA template was a CTG sequence, there were increased repeat contractions and a fragile site was created leading to large-scale deletions. When the CTG sequence was on the resected strand, resection was inhibited, resulting in repeat expansions. Increased nucleolytic processing by deletion of Rad9, the ortholog of 53BP1, rescued repeat instability and chromosome breakage. Loss of Rad51 increased contractions implicating a protective role for Rad51 on ssDNA. Together, our work implicates structure-forming repeats as an impediment to resection and gap-filling which can lead to mutations and large-scale deletions.

Chromosomal fragile site breakage by EBV-encoded EBNA1 at clustered repeats.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with several cancers of lymphocytic and epithelial origin1-3. EBV encodes EBNA1, which binds to a cluster of 20 copies of an 18-base-pair palindromic sequence in the EBV genome4-6. EBNA1 also associates with host chromosomes at non-sequence-specific sites7, thereby enabling viral persistence. Here we show that the sequence-specific DNA-binding domain of EBNA1 binds to a cluster of tandemly repeated copies of an EBV-like, 18-base-pair imperfect palindromic sequence encompassing a region of about 21 kilobases at human chromosome 11q23. In situ visualization of the repetitive EBNA1-binding site reveals aberrant structures on mitotic chromosomes characteristic of inherently fragile DNA. We demonstrate that increasing levels of EBNA1 binding trigger dose-dependent breakage at 11q23, producing a fusogenic centromere-containing fragment and an acentric distal fragment, with both mis-segregated into micronuclei in the next cell cycles. In cells latently infected with EBV, elevating EBNA1 abundance by as little as twofold was sufficient to trigger breakage at 11q23. Examination of whole-genome sequencing of EBV-associated nasopharyngeal carcinomas revealed that structural variants are highly enriched on chromosome 11. Presence of EBV is also shown to be associated with an enrichment of chromosome 11 rearrangements across 2,439 tumours from 38 cancer types. Our results identify a previously unappreciated link between EBV and genomic instability, wherein EBNA1-induced breakage at 11q23 triggers acquisition of structural variations in chromosome 11.

Dicentric chromosome breakage in Drosophila melanogaster is influenced by pericentric heterochromatin and occurs in nonconserved hotspots.

Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

Effects of various environments on epigenetic settings and chromosomal damage.

Air pollution is a dominant environmental exposure factor with significant health consequences. Unexpectedly, research in a heavily polluted region of the Czech Republic, with traditional heavy industry, revealed repeatedly the lowest frequency of micronuclei in the season with the highest concentrations of air pollutants including carcinogenic benzo[a]pyrene (B[a]P). Molecular findings have been collected for more than 10 years from various locations of the Czech Republic, with differing quality of ambient air. Preliminary conclusions have suggested adaptation of the population from the polluted locality (Ostrava, Moravian-Silesian Region (MSR)) to chronic air pollution exposure. In this study we utilize the previous findings and, for the first time, investigate micronuclei (MN) frequency by type: (i) centromere positive (CEN+) MN, representing chromosomal losses, and (ii) centromere negative (CEN-) MN representing chromosomal breaks. As previous results indicated differences between populations in the expression of XRCC5, a gene involved in the non-homologous end-joining (NHEJ) repair pathway, possible variations in epigenetic settings in this gene were also investigated. This new research was conducted in two seasons in the groups from two localities with different air quality levels (Ostrava (OS) and Prague (PG)). The obtained new results show significantly lower frequencies of chromosomal breaks in the OS subjects, related to the highest air pollution levels (p < 0.001). In contrast, chromosomal losses were comparable between both groups. In addition, significantly lower DNA methylation was found in 14.3% of the analyzed CpG loci of XRCC5 in the population from OS. In conclusion, the epigenetic adaptation (hypomethylation) in XRCC5 involved in the NHEJ repair pathway in the population from the polluted region, was suggested as a reason for the reduced level of chromosomal breaks. Further research is needed to explore the additional mechanisms, including genetic adaptation.

Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1.

Repeat-mediated deletions (RMDs) are a type of chromosomal rearrangement between two homologous sequences that causes loss of the sequence between the repeats, along with one of the repeats. Sequence divergence between repeats suppresses RMDs; the mechanisms of such suppression and of resolution of the sequence divergence remains poorly understood. We identified RMD regulators using a set of reporter assays in mouse cells that test two key parameters: repeat sequence divergence and the distances between one repeat and the initiating chromosomal break. We found that the mismatch repair factor MLH1 suppresses RMDs with sequence divergence in the same pathway as MSH2 and MSH6, and which is dependent on residues in MLH1 and its binding partner PMS2 that are important for nuclease activity. Additionally, we found that the resolution of sequence divergence in the RMD product has a specific polarity, where divergent bases that are proximal to the chromosomal break end are preferentially removed. Moreover, we found that the domain of MLH1 that forms part of the MLH1-PMS2 endonuclease is important for polarity of resolution of sequence divergence. We also identified distinctions between MLH1 versus TOP3α in regulation of RMDs. We suggest that MLH1 suppresses RMDs with sequence divergence, while also promoting directional resolution of sequence divergence in the RMD product.

Chromosomal breaks at the origin of small tandem DNA duplications.

Small tandem DNA duplications in the range of 15 to 300 base-pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9-based experimental data from different model systems. Recent work points to fill-in synthesis at double-stranded DNA breaks with complementary sequences, regulated by end-joining mechanisms, to account for small tandem duplications. We will review the prevalence of small tandem duplications in the population, and we will speculate on the potential sources of DNA damage that could give rise to this mutational signature. With the development of novel algorithms to analyse sequencing data, small tandem duplications are now more frequently detected in the human genome and identified as oncogenic gain-of-function mutations. Understanding their origin could lead to optimized treatment regimens to prevent therapy-induced activation of oncogenes and might expose novel vulnerabilities in cancer.

Mutagenic Evaluation of Fluoxetine and Fluoxetine-Galactomannan Complex Through the Analysis of Chromosomal Aberrations in Human Peripheral Leukocytes and Salmonella typhimurium/Microssome Assay / Avaliação Mutagênica de Fluoxetina e Complexo Fluoxetina-Galactomanana Através da Análise de Aberrações Cromossômicas em Leucócitos Periféricos Humanos e em Salmonella typhimurium / Ensaio Microssômico

Objectives: The purpose of this study was to evaluate the mutagenic potential of fluoxetine and fluoxetine-galactomannan. Methods: Chromosomal aberration test and Salmonella typhimurium/microsome mutagenicity assay. Results: The results showed that fluoxetine (250 µg/mL) can cause chromosomal breaks of treated leukocytes and increase the frequency of reversion of the tester strains of S. typhimurium / microsome assay only at the highest concentration (5 mg/mL), while fluoxetine encapsulated in galactomannan did not cause these changes (leukocytes and S. typhimuriums strains). Conclusion: In summary, fluoxetine showed a mutagenic effect detectable only at high concentrations in both eukaryotic and prokaryotic models. Furthermore, the fluoxetine/galactomannan complex, in this first moment, prevented the mutagenicity attributed to fluoxetine, emphasizing that the present encapsulation process can be an alternative in preventing these effects in vitro.

Objetivos: avaliar o potencial mutagênico da fluoxetina e da fluoxetina-galactomanana. Métodos: Teste de aberração cromossômica e ensaio de mutagenicidade de Salmonella typhimurium /microssoma. Resultados: a fluoxetina (250 µg/mL) pode causar quebras cromossômicas de leucócitos tratados e aumentar a frequência de reversão das cepas testadoras de S. typhimurium /microssoma apenas na concentração mais alta (5 mg/mL), enquanto a fluoxetina encapsulada em galactomanano não causou essas alterações (leucócitos e cepas de S. typhimurium). Conclusão: a fluoxetina mostrou um efeito mutagênico detectável apenas em altas concentrações em modelos eucarióticos e procarióticos. Além disso, o complexo fluoxetina/galactomanan, neste primeiro momento, evitou a mutagenicidade atribuída à fluoxetina, ressaltando que o presente processo de encapsulamento pode ser uma alternativa na prevenção desses efeitos in vitro.

O-GlcNAc transferase is important for homology-directed repair.

O-Linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) to serine or threonine residues is a reversible and dynamic post-translational modification. O-GlcNAc transferase (OGT) is the only enzyme for O-GlcNAcylation, and is a potential cancer therapeutic target in combination with clastogenic (i.e., chromosomal breaking) therapeutics. Thus, we sought to examine the influence of O-GlcNAcylation on chromosomal break repair. Using a set of DNA double strand break (DSB) reporter assays, we found that the depletion of OGT, and its inhibition with a small molecule each caused a reduction in repair pathways that involve use of homology: RAD51-dependent homology-directed repair (HDR), and single strand annealing. In contrast, such OGT disruption did not obviously affect chromosomal break end joining, and furthermore caused an increase in homology-directed gene targeting. Such disruption in OGT also caused a reduction in clonogenic survival, as well as modifications to cell cycle profiles, particularly an increase in G1-phase cells. We also examined intermediate steps of HDR, finding no obvious effects on an assay for DSB end resection, nor for RAD51 recruitment into ionizing radiation induced foci (IRIF) in proliferating cells. However, we also found that the influence of OGT on HDR and homology-directed gene targeting were dependent on RAD52, and that OGT is important for RAD52 IRIF in proliferating cells. Thus, we suggest that OGT is important for regulation of HDR that is partially linked to RAD52 function.

Genome characterization and CRISPR-Cas9 editing of a human neocentromere.

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

To indel or not to indel: Factors influencing mutagenesis during chromosomal break end joining.

Chromosomal DNA double-strand breaks (DSBs) are the effective lesion of radiotherapy and other clastogenic cancer therapeutics, and are also the initiating event of many approaches to gene editing. Ligation of the DSBs by end joining (EJ) pathways can restore the broken chromosome, but the repair junctions can have insertion/deletion (indel) mutations. The indel patterns resulting from DSB EJ are likely defined by the initial structure of the DNA ends, how the ends are processed and synapsed prior to ligation, and the factors that mediate the ligation step. In this review, we describe key factors that influence these steps of DSB EJ in mammalian cells, which is significant both for understanding mutagenesis resulting from clastogenic cancer therapeutics, and for developing approaches to manipulating gene editing outcomes.