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Common fragile sites (CFSs) are difficult-to-replicate regions of eukaryotic genomes that are sensitive to replication stress and that require resolution by the MUS81–EME1 endonuclease to re-initiate POLD3-dependent DNA synthesis in early mitosis; this study defines the specific pathway of events causing the CFS fragility phenotype. Inbuilt chromosome fragility Common fragile sites (CFSs) are regions of eukaryotic genomes that are sensitive to DNA replication stressors. They are stably maintained and replicated in the human genome most of the time, but have been associated with some cancers and genetic disease. CFS 'expression' was shown recently to be a programmed event promoted by the MUS81 endonuclease, rather than disruption of the chromosome structure caused by mechanical forces in mitosis. Ian Hickson and colleagues have now defined the specific chain of events causing CFSs. They describe a pathway of unscheduled DNA synthesis regulated by the MUS81 endonuclease, the POLD3 subunit of DNA polymerase delta and the prophase pathway in early mitosis. Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis 1 . Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs) 2 , 3 , 4 , 5 . CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS ‘expression’), particularly when cells have been exposed to replicative stress 6 . The MUS81–EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress 7 , 8 , 9 . Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN + ) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.

The instability of chromosome fragile sites is implicated as a causative factor in several human diseases, including cancer [for common fragile sites (CFSs)] and neurological disorders [for rare fragile sites (RFSs)]. Previous studies have indicated that problems arising during DNA replication are the underlying source of this instability. Although the role of replication stress in promoting instability at CFSs is well documented, much less is known about how the fragility of RFSs arises. Many RFSs, as exemplified by expansion of a CGG trinucleotide repeat sequence in the fragile X syndrome-associated FRAXA locus, exhibit fragility in response to folate deficiency or other forms of “folate stress.” We hypothesized that such folate stress, through disturbing the replication program within the pathologically expanded repeats within FRAXA, would lead to mitotic abnormalities that exacerbate locus instability. Here, we show that folate stress leads to a dramatic increase in missegregation of FRAXA coupled with the formation of single-stranded DNA bridges in anaphase and micronuclei that contain the FRAXA locus. Moreover, chromosome X aneuploidy is seen when these cells are exposed to folate deficiency for an extended period. We propose that problematic FRAXA replication during interphase leads to a failure to disjoin the sister chromatids during anaphase. This generates further instability not only at FRAXA itself but also of chromosome X. These data have wider implications for the effects of folate deficiency on chromosome instability in human cells.

PICH is a SNF2 family DNA translocase that binds to ultra-fine DNA bridges (UFBs) in mitosis. Numerous roles for PICH have been proposed from protein depletion experiments, but a consensus has failed to emerge. Here, we report that deletion of PICH in avian cells causes chromosome structural abnormalities, and hypersensitivity to an inhibitor of Topoisomerase II (Topo II), ICRF-193. ICRF-193-treated PICH −/− cells undergo sister chromatid non-disjunction in anaphase, and frequently abort cytokinesis. PICH co-localizes with Topo IIα on UFBs and at the ribosomal DNA locus, and the timely resolution of both structures depends on the ATPase activity of PICH. Purified PICH protein strongly stimulates the catalytic activity of Topo II in vitro . Consistent with this, a human PICH −/− cell line exhibits chromosome instability and chromosome condensation and decatenation defects similar to those of ICRF-193-treated cells. We propose that PICH and Topo II cooperate to prevent chromosome missegregation events in mitosis. During mitosis the translocase PICH binds to ultrafine bridges formed from DNA catenanes that are unresolved by topoisomerase II. In this study, the authors show that PICH stimulates toposiomerase II activity and that they cooperate to resolve these structures.

Gilles de la Tourette syndrome (GTS) is a complex neurodevelopmental disorder characterized by motor and vocal tics. Most of the GTS individuals have comorbid diagnoses, of which obsessive-compulsive disorder (OCD) and attention deficit-hyperactivity disorder (ADHD) are the most common. Several neurotransmitter systems have been implicated in disease pathogenesis, and amongst these, the dopaminergic and the serotonergic pathways are the most widely studied. In this study, we aimed to investigate whether the serotonin transporter (SERT) gene (SLC6A4) was differentially expressed among GTS individuals compared to healthy controls, and whether DNA variants (the SERT-linked polymorphic region 5-HTTLPR, together with the associated rs25531 and rs25532 variants, and the rare Ile425Val variant) or promoter methylation of SLC6A4 were associated with gene expression levels or with the presence of OCD as comorbidity. We observed that SLC6A4 expression is upregulated in GTS individuals compared to controls. Although no specific genotype, allele or haplotype was overrepresented in GTS individuals compared to controls, we observed that the LAC/LAC genotype of the 5-HTTLPR/rs25531/rs25532 three-locus haplotype was associated with higher SLC6A4 mRNA expression levels in GTS individuals, but not in the control group.

FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.

FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.

Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis (1). Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs) (2-5). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress (6). The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress (7-9). Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability ([CIN.sup.+]) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

Chromothripsis is a mutational mechanism leading to complex and relatively clustered chromosomal rearrangements, resulting in diverse phenotypic outcomes depending on the involved genomic landscapes. It may occur both in the germ and the somatic cells, resulting in congenital and developmental disorders and cancer, respectively. Asymptomatic individuals may be carriers of chromotriptic rearrangements and experience recurrent reproductive failures when two or more chromosomes are involved. Several mechanisms are postulated to underlie chromothripsis. The most attractive hypothesis involves chromosome pulverization in micronuclei, followed by the incorrect reassembly of fragments through DNA repair to explain the clustered nature of the observed complex rearrangements. Moreover, exogenous or endogenous DNA damage induction and dicentric bridge formation may be involved. Chromosome instability is commonly observed in the cells of patients with DNA repair disorders, such as ataxia telangiectasia, Nijmegen breakage syndrome, and Bloom syndrome. In addition, germline variations of TP53 have been associated with chromothripsis in sonic hedgehog medulloblastoma and acute myeloid leukemia. In the present review, we focus on the underlying mechanisms of chromothripsis and the involvement of defective DNA repair genes, resulting in chromosome instability and chromothripsis-like rearrangements.

There may be sex-based disparities in intensive care unit (ICU) management and outcomes. We compared baseline variables, interventions, and outcomes of immunocompromised critically ill men and women. We performed a post hoc analysis of the Efraim study, a prospective multinational cohort study of immunocompromised adults with acute hypoxemic respiratory failure admitted to one of 68 ICU in 16 countries between November 2015 and July 2016. We compared in unadjusted and adjusted analyses baseline variables, ICU interventions, and outcomes between men and women. We included 1536 immunocompromised adults (922 men, 614 women) in this study. Women and men had similar age, BMI, and diagnoses leading to immunosuppression; hematopoietic cell transplant was more common in men. On the first ICU day, SOFA score was higher in men vs. women (7 [IQR 4–10] vs 6 [4–10]), p = 0.0005). The use of ICU supportive interventions, including mechanical ventilation, vasopressors, renal replacement, bronchoalveolar lavage, and ARDS adjuncts, were similar between men and women; as were mortality in ICU, in hospital, and at 90 days. After adjustment, female sex (sub-hazard ratio 1.19, 95 % CI 1.05–1.36, p = 0.007), SOFA score on ICU day 1 (sHR 1.16, 95 % CI 1.12–1.19, p < 0.001) and chronic kidney disease (sHR 0.74, 95 % CI 0.59–0.93, p = 0.009) were associated with mechanical ventilation. Age, performance status and SOFA score on ICU day 1 were associated with hospital mortality. In this post hoc analysis of immunocompromised adult ICU patients with hypoxemic respiratory failure, women and men received similar ICU interventions, and had similar outcomes. •We performed a post hoc analysis of Efraim, a prospective multinational cohort study of immunocompromised adults with AHRF•We compared baseline variables, interventions, and outcomes of critically ill men and women•Men and women received similar ICU supportive interventions and had similar outcomes•After adjustment, female sex, SOFA score on ICU day 1, and chronic kidney disease were associated with MV•Age, performance status and SOFA score on ICU day 1 were associated with hospital mortality