How common are common fragile sites: variation of aphidicolin-induced chromosomal fragile sites in a population of the deer mouse (Peromyscus maniculatus)

Aphidicolin (APC)-induced chromosomal gaps and breaks were analyzed for ten deer mice (Peromyscus maniculatus) from a natural population. The FSM statistical methodology was used to identify fragile sites as chromosomal loci exhibiting significantly non-random numbers of gaps/breaks in each individual and enabled an assessment of variation in fragile sites among the individuals. The individual deer mice exhibited as few as 7 to as many as 19 of the populational total of 34 sites. Two sites were fragile in all individuals and 13 sites were fragile in single individuals only. Defined by populational frequencies of greater than 50%, high-frequency fragile sites constituted 26% of the populational total. Approximately 35% of the total fragile sites were fragile in 20–40% of the population (low-frequency fragile sites) and about 38% were fragile in single individuals only. Analysis of the data pooled over all individuals identified significantly non-random breakage at 80 sites, 47 of which were not identified as fragile in any single individual. It appears, therefore, that fragile site identifications from pooled data have fostered an inflated estimate of the numbers and frequencies of common fragile sites. Comparison of the fragile site and spontaneous breakage (control) data suggest that APC-induced fragile sites represent regions of chromosomes that experience elevated levels of somatic mutation. Additionally, the occurrence of APC-induced fragile sites at or near the interstitial breakpoints of two pericentric-inversion polymorphisms in this population supports the hypothesis that fragile sites experience an increased rate of meiotic chromosomal mutation and are predisposed to undergo phylogenetic rearrangement. Received: 22 January 1997 / Accepted: 24 February 1997     

Identifying chromosomal fragile sites from individuals: a multinomial statistical model

The inability to identify fragile sites from data for single individuals remains the major obstacle to determining whether these chromosomal loci are predisposed to cancer-causing and evolutionary rearrangements. We describe a novel statistical model that is amenable to data from single individuals and that establishes site-specific chromosomal breakage as nonrandom with respect to the distribution of total breakage. Our method tests incrementally smaller subsets of the data for homogeneity under a multinomial model that assigns equal probabilites to a maximal set of nonfragile sites and unrestricted probabilities to the remaining fragile sites with significantly higher numbers of breaks. We show how standardized Pearson's chi-square (X ²) and likelihood-ratio (G ²) statistics can be appropriately used to measure goodness-of-fit for sparse contingency (individual-based) data in this model. A sample application of this approach indicates extensive variation in fragile sites among individuals and marked differences in fragile-site inferences from pooled as opposed to per-individual data.     

The complex basis underlying common fragile site instability in cancer

Common fragile sites (CFSs) were characterized almost 30 years ago as sites undergoing genomic instability in cancer. Recently, in vitro studies have found that oncogene-induced replication stress leads to CFS instability. In vivo, CFSs were found to be preferentially unstable during early stages of cancer development and to leave a unique signature of instability. It is now increasingly clear that, along the spectrum of replication features characterizing CFSs, failure of origin activation is a common feature. This and other features of CFSs, together with the replication stress characterizing early stages of cancer development, lead to incomplete replication that results in genomic instability preferentially at CFSs. Here, we review the shared and unique characteristics of CFSs, their underlying causes and their implications, particularly with respect to the development of cancer.     

Family study of common fragile sites

Summary The frequency of folate-sensitive common fragile sites (1p31, 1q44, 3p14, 3q26.2, 6q26, 16q23, Xp22.3) was determined in 19 healthy individuals from four families. The individuals consisted of 12 males and 7 females from 1 to 59 years of age. The frequency showed intrafamilial variation, but we were unable to demonstrate that the frequency was inherited in a Mendelian codominant fashion. In eight subjects whose chromosome 3 homologues could be distinguished by Q-band polymorphism, breakages at 3p14 occurred with equal frequencies on the homologues. Our study suggests that common fragile sites are a part of normal chromosome structure, and the frequency of their expression largely depends on environmental factors.     

Arabinofuranosyl nucleosides induce common fragile sites

Summary The capacities for fragile site induction of three inhibitors of semiconservative DNA synthesis and DNA repair synthesis, aphidicolin, arabinofuranosyl cytosine, and arabinofuranosyl adenosine were compared. Aphidicolin is known to induce type 4 fragile sites, the largest recognized group of common fragile sites. Although the modes of action of these inhibitors vary, both arabinofuranosyl analogs induce type 4 aphidicolin-sensitive fragile sites. An analysis of variance demonstrates that the three inhibitors are not equally capable of inducing significant breakage (P<0.01) at all type 4 fragile sites. Induction of type 4 fragile sites appears to be a general consequence of inhibition of DNA polymerization.     

Replication timing of two human common fragile sites: FRA1H and FRA2G

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.     

Variation in the expression of aphidicolin-induced fragile sites in human lymphocyte cultures

Summary A correlation between specific fragile sites and cancer breakpoints has been suggested raising the question of fragile site expression as a predisposing factor in the occurrence of cancer in some persons. Before addressing the question of increased fragility among patients at high risk for cancer, we analyzed the variability of aphidicolin-induced fragile sites among nine normal persons and also among repeated samples from three of these individuals. Considerable variation in both the frequency and location of these fragile sites was observed and the data strongly suggest the significant variation of 6 of the 16 selected sites to be primarily due to sampling differences. These findings indicate that the use of fragile sites as a screening tool for patients at high risk of cancer should be carefully monitored relative to the variation inherent in both culture and individual expression.     

Common fragile sites: mechanisms of instability revisited

Common fragile sites (CFSs) are large chromosomal regions prone to breakage upon replication stress that are considered a driving force of oncogenesis. CFSs were long believed to contain sequences blocking fork progression, thus impeding replication completion and leading to DNA breaks upon chromosome condensation. However, recent studies show that delayed completion of DNA replication instead depends on a regional paucity in initiation events. Because the distribution and the timing of these events are cell type dependent, different chromosomal regions can be committed to fragility in different cell types. These new data reveal the epigenetic nature of CFSs and open the way to a reevaluation of the role played by these sites in the formation of chromosome rearrangements found in tumors from different tissues.     

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.Subject terms: Cancer, DNA damage and repair     

WWOX,large common fragile site genes,and cancer

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.     

DAPI-inducible common fragile sites

DAPI, a compound specific for the AT bases of DNA, causes gaps and breaks in three human chromosome sites, at the 1q41-1q42 interface, 2q31, and 7p22. It also induces undercondensation of a chromosome site at the 13q21-13q22 interface. The first three sites have the characteristics of "common fragile sites" and are present as gaps and breaks on the chromosomes of seven individuals.     

Population cytogenetics of folate-sensitive fragile sites

Summary The location and frequency of folate-sensitive common fragile sites (CFS) were studied in three populations: (1) 111 mentally retarded children of school age, (2) 240 mentally subnormal children attending special schools, and (3) 85 healthy children attending normal schools. Common fragile sites were found at 54 chromosomal bands including also the band Xq27, where gaps and breaks were detected in 4% of the children. The most frequent CFS were FRA3B (at 3p14.2), FRA6E (at 6q26), and FRA16D (at 16q23) seen in 73%, 65%, and 58% of the individuals totally studied. The frequencies of CFS-positive individuals did not differ among the populations. The variation found in the distribution of CFS among the populations was primarily assumed to be due to sampling differences and study method. The rate of expression of the most frequent CFS varied significantly among the individuals, seeming to suggest that polymorphism exists at those CFS.     

Common fragile sites

Aphidicolin-induced common fragile sites are site-specific gaps or breaks seen on metaphase chromosomes after partial inhibition of DNA synthesis. These fragile sites were first recognized during the early studies of the fragile X syndrome and are induced by the same conditions of folate or thymidylate stress used to induce the fragile X site. Common fragile sites are normally stable in cultured human cells. However, following induction with replication inhibitors, they display a number of characteristics of unstable and highly recombinogenic DNA. From the many studies that have cloned and characterized fragile sites, it is now known that these sites extend over large regions, are associated with genes, exhibit late or delayed replication, and contain regions of high flexibility but are otherwise unremarkable in sequence. Studies showing that fragile sites and their associated genes are frequently deleted or rearranged in cancer cells have clearly demonstrated their importance in genome instability in tumorigenesis. Yet until recently, very little was known about the molecular mechanisms involved in their stability. Recent findings showing that the key checkpoint genes ATR and BRCA1 are critical for genome stability at fragile sites have shed new light on these mechanisms and on the biological significance of common fragile sites.     

Rodent common fragile sites: Are they conserved? Evidence from mouse and rat

Nineteen fragile sites induced by aphidicolin in lymphocyte cultures from the laboratory mouse are documented. These sites are compared with previously described fragile sites induced in mouse fibroblast systems, and then with those reported on chromosomes which have been evolutionarily conserved between the mouse and the laboratory rat. Of a total of 38 fragile sites thus far identified in mouse fibroblasts and lymphocytes, only 4 sites are common to the two cell types; 34 sites show no correspondence of loci. The reason for this discrepancy is unclear, but it is possible that these data may indicate some degree of tissue specificity of fragile site expression in the mouse. Eight autosomes in the mouse and rat retain straightforward and nearly complete banding homology. To test the hypothesis that fragile sites are conserved between the two species, we compared these eight autosomes with regard to number and distribution of fragile site loci. A total of 30 fragile sites is distributed over the conserved chromosomes. Only 4 (possibly 5) are common to both species; 18 are found in the rat but not the mouse, and 4 are found in the mouse but not the rat. Of the 4 shared sites, notable differences in frequency of expression exist. Our comparisons show that: (1) a small numer of fragile sites is conserved; (2) a large number of fragile sites is not conserved, and (3) some sites which are conserved are quite different in the frequency at which they are expressed in the two species, indicating that the sites themselves may have undergone evolutionary change. The chromosomes compared between mouse and rat are widely conserved among murid rodents and thereby offer further opportunities to investigate fragile site phenomena in diverse species.     

Multiple common fragile sites are expressed in the genome of the laboratory rat

Splenic lymphocytes from Sprague Dawley and Fischer 344 rats were exposed to two chemicals known to induce common fragile site expression in man: fluorodeoxyuridine (in conjunction with the enhancing effects of caffeine) and aphidicolin. Of 39 sites that were significantly damaged in excess, 12 meet the criteria for fragility proposed in this investigation. Rat fragile sites appear to differ from those in man in that no common hierarchical frequency of expression is evident from the two methods of induction. In addition, a comparison of published cancer-specific chromosome breakpoints from a variety of rat tumors reveals little or no apparent concordance with the identified fragile sites. The rat is an animal model in which multiple common fragile sites can be induced and, as such, will be valuable for testing hypotheses concerning the biological basis of chromosomal fragility.     

Inhibition of topoisomerase I prevents chromosome breakage at common fragile sites

Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the DNA polymerase inhibitor, aphidicolin. We and others have identified several cell cycle checkpoint and DNA repair proteins that influence common fragile site stability. However, the initial events underlying fragile site breakage remain poorly understood. We demonstrate here that aphidicolin-induced gaps and breaks at fragile sites are prevented when cells are co-treated with low concentrations of the topoisomerase I inhibitor, camptothecin. This reduction in breakage is accompanied by a reduction in aphidicolin-induced RPA foci, CHK1 and RPA2 phosphorylation, and PCNA monoubiquitination, indicative of reduced levels of single stranded DNA. Furthermore, camptothecin reduces spontaneous fragile site breakage seen in cells lacking ATR, even in the absence of aphidicolin. These data from cultured human cells demonstrate that topoisomerase I activity is required for DNA common fragile site breaks and suggest that polymerase–helicase uncoupling is a key initial event in this process.     

Chromosomal instability at common fragile sites in Seckel syndrome

Seckel syndrome (SCKL) is a rare, genetically heterogeneous disorder, with dysmorphic facial appearance, growth retardation, microcephaly, mental retardation, variable chromosomal instability, and hematological disorders. To date, three loci have been linked to this syndrome, and recently, the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) was identified as the gene mutated at the SCKL1 locus. The ATR mutation affects splicing efficiency, resulting in low levels of ATR in affected individuals. Elsewhere, we reported increased instability at common chromosomal fragile sites in cells lacking the replication checkpoint gene ATR. Here, we tested whether cells from patients carrying the SCKL1 mutation would show increased chromosome breakage following replication stress. We found that, compared with controls, there is greater chromosomal instability, particularly at fragile sites, in SCKL1-affected patient cells after treatment with aphidicolin, an inhibitor of DNA polymerase alpha and other polymerases. The difference in chromosomal instability between control and patient cells increases at higher levels of aphidicolin treatment, suggesting that the low level of ATR present in these patients is not sufficient to respond appropriately to replication stress. This is the first human genetic syndrome associated with increased chromosome instability at fragile sites following replication stress, and these findings may be related to the phenotypic findings in patients with SCKL1.     

Population cytogenetics of aphidicolin-induced fragile sites

Summary Chromosome fragile sites are inducible by aphidicolin in cultured human lymphocytes. To assess the frequency and distribution of these common fragile sites in the general population, a cytogenetic survey was performed on 126 subjects, 59 males and 67 females, whose age ranged from 1 day to 72 years. Common fragile sites, induced by aphidicolin, were widespread and showed a remarkably different sensitivity among individuals; age influenced the overall frequency of fragile sites. Moreover, both age and sex seemed to modulate the expression of specific fragile sites. In our population, the most common fragile sites were: 3p14, 16q23, Xp22, 6q26, 1p31, 4q31, 1p22, 7q22, 2q33, 3q27, 2q31, 7q32, 14q24, 10q22, 5q31, 2q37, 6p21.     

Induction of sister chromatid exchanges at common fragile sites.

Experiments were performed to gain further insight into chromosome structure and behavior at common fragile sites by testing the hypothesis that gaps at these sites predispose to intrachromosomal recombination as measured by sister chromatid exchanges (SCEs). Human lymphocytes were concurrently treated with aphidicolin, for determination of fragile site expression, and with 5-bromodeoxy-uridine, for SCE analysis. Aphidicolin induced chromosome gaps nonrandomly, with the great majority of gaps occurring at common fragile sites. On average, 66% of gaps were accompanied by an SCE at the site of the lesion. Analysis of two specific common fragile sites at 3p14 and 16q23 showed the same pattern; that is, on average 70% of gaps at these sites were accompanied by an SCE. These results show that common fragile sites are hot spots not only for chromosomal lesions such as gaps but also for SCE formation.