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.     

The most common fragile site in man is 3p14

Summary In man a common fragile site is known to occur at 3p14. We studied the expression of this fragility in a group of 70 normal healthy subjects. Chromosome breaks, chromatid breaks and gaps at 3p14 could be observed in every examined individual, and in a total of 7000 metaphases they were seen in a mean of 4% of cells. Fluorescence studies in ten persons with chromosome No. 3 polymorphism showed that in all cases both Nos. 3 were about equally liable to breakage. A considerable variation in the fra 3p14 expression was found between individuals as well as in repeated cultures from the same person. Neither sex nor age influences could be detected. Cultures with a high percentage of lesions at 3p14 tended to have also a high number of lesions at other sites. Methotrexate and fluorodeoxyuridine markedly enhanced the expression of fra 3p14 and other fragilities. It is concluded that the chromosomal region at 3p14 represents man's most common fragile site, the expression of which seems to be influenced by environmental and heritable factors.     

Common fragile sites in man and three closely related primate species

The expression of common fragile sites was studied in peripheral lymphocytes of man, gorilla, chimpanzee, and orangutan after induction with aphidicolin, methotrexate, or fluorodeoxyuridine. As far as the chromosomal localization is concerned, it appears that many of these sites have been highly conserved during primate evolution. However, differences were found in the relative expression of certain sites. In all four species, mapping of approximately 500 lesions disclosed the most breakage-prone common fragile sites, at which about 90% of all induced aberrations were localized. Comparison of chromosome regions involved in evolutionary changes to fragile sites in the four primate species revealed 30 sites that were located at or close to the same chromosomal band. However, no correlation was found between the relative expression of a certain common fragile site in vitro and a potential involvement of this chromosomal site in evolutionary changes.     

Molecular basis for expression of common and rare fragile sites

Fragile sites are specific loci that form gaps, constrictions, and breaks on chromosomes exposed to partial replication stress and are rearranged in tumors. Fragile sites are classified as rare or common, depending on their induction and frequency within the population. The molecular basis of rare fragile sites is associated with expanded repeats capable of adopting unusual non-B DNA structures that can perturb DNA replication. The molecular basis of common fragile sites was unknown. Fragile sites from R-bands are enriched in flexible sequences relative to nonfragile regions from the same chromosomal bands. Here we cloned FRA7E, a common fragile site mapped to a G-band, and revealed a significant difference between its flexibility and that of nonfragile regions mapped to G-bands, similar to the pattern found in R-bands. Thus, in the entire genome, flexible sequences might play a role in the mechanism of fragility. The flexible sequences are composed of interrupted runs of AT-dinucleotides, which have the potential to form secondary structures and hence can affect replication. These sequences show similarity to the AT-rich minisatellite repeats that underlie the fragility of the rare fragile sites FRA16B and FRA10B. We further demonstrate that the normal alleles of FRA16B and FRA10B span the same genomic regions as the common fragile sites FRA16C and FRA10E. Our results suggest that a shared molecular basis, conferred by sequences with a potential to form secondary structures that can perturb replication, may underlie the fragility of rare fragile sites harboring AT-rich minisatellite repeats and aphidicolin-induced common fragile sites.     

Folate-sensitive fragile sites on the X-chromosome heterochromatin of the Indian mole rat, Nesokia indica

Folate-sensitive fragile sites have been demonstrated on the X chromosome of the Indian mole rat, Nesokia indica (subfamily Murinae), utilizing peripheral blood lymphocyte cultures. All normal female individuals expressed fragile sites on the constitutive heterochromatic long arm of one of their two X chromosomes (heterozygous expression); in contrast, no fragile sites were found on the single X chromosome of normal males. Preferential transmission of the maternal fragile X to the daughters is therefore suggested. Four sites have been detected so far: fra Xq1, fra Xq2, fra Xq3, and fra Xc (centromeric). It is significant that their location corresponds to the regions where constitutive heterochromatic deletions occur that result in a variety of polymorphic X chromosomes in natural populations of Nesokia. Thus there is a correlation between fragile sites, deletion sites, and karyotypic changes. In individuals that did not reproduce in the laboratory, there were more fragile sites on both X chromosomes of the females (homozygous/double heterozygous expression) and also on the X of the males (hemizygous expression). This difference in fragile site expression from the normal situation could be attributed to one or more new mutations. However, the mechanism by which fragile sites influence reproductive performance is unclear.     

Expression of fragile sites in human sperm and lymphocyte chromosomes

Summary Sperm and lymphocyte chromosome studies in a normal, fertile male have shown a high degree of coincidence between chromosome lesions and fragile sites in both types of cells. In this donor we also found that some fragile sites expressed in sperm chromosomes coincided with those expressed in lymphocyte chromosomes. These results indicate that the chromosome lesions expressed in sperm do not occur at random and that they are not technical artifacts. The fragility expression in sperm chromosomes could reflect in vivo conditions. The presence in some sperm metaphases of acentric fragments suggests that chromosome fragility can result in the loss of chromosome fragments or give rise to de novo structural rearrangements. However, the incidence of sperm with chromosomal abnormalities observed in this man was within the normal range.     

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.     

A common fragile site at Xq27: theoretical and practical implications.

The fragile site at Xq27 (FRAXA) is associated with a common form of X-linked mental retardation (Martin-Bell syndrome). It is induced in culture by conditions of thymidylate stress and is generally considered a rare fragile site found only in association with an X-linked form of mental retardation. Using a somatic cell hybrid system, we previously demonstrated that fragile-X expression can be induced by thymidylate stress in normal X chromosomes at low levels (4%-5%). In the present report, significantly higher levels of fragile-X expression (6%-28%) have been induced in lymphocytes or lymphoblasts of all seven control males using high doses of aphidicolin (1.5 microM). Similar high levels of expression (10%-12%) were observed in both of two normal male chimpanzees (Pan troglodytes). These data demonstrate that Xq27 contains a common fragile site (FRAXD) that is ancestral to the divergence of man and the chimpanzee. Presence of a common and a rare fragile site in the same metaphase chromosome band does not prove that they are identical and may, in fact, represent two unrelated fragile sites. However, the possibility exists that the common fragile site at Xq27 may be the substrate for unequal recombination events that produces the rare fragile site associated with Martin-Bell syndrome. In addition, presence of a common fragile site at Xq27 may explain the occasional observation of low-frequency fragile-X expression in normal control individuals. Caution is therefore warranted in the interpretation of low-level fragile-X expression in diagnostic and prenatal diagnostic settings.     

Evolutionary conservation of fragile sites induced by 5-azacytidine and 5-azadeoxycytidine in man,gorilla, and chimpanzee

Summary Lymphocyte cultures from man, gorilla, and chimpanzee were treated with 5-azacytidine and 5-azadeoxycytidine. These cytidine analogues induce common fragile sites in the chromosome bands 1q42 and 19q13 of man. A rare fragile site is induced by 5-azadeoxycytidine in the band 1q24. The optimum conditions required for inducing these new fragile sites were determined by a series of experiments. The common fragile site in human chromosome 1q42 also exists in the gorilla and chimpanzee in the homologous band 1q32. The fragile site in human chromosome 19q13 was demonstrated in the gorilla in the homologous chromosome band 20q13. These are the first examples found of evolutionary highly conserved fragile sites in homologous chromosome bands in related primate species. The interaction between 5-azacytidine, 5-azadeoxycytidine, and chromosomal DNA; the evolutionary conservation of genes located within or closely adjacent to the fragile sites in the chromosome 1 of Hominoidea; and the phylogenetic origin of the two new common fragile sites are discussed.     

Correlation of fragile sites and bleomycin induced chromosome break points

Bleomycin (Blm) induced break points in human chromosome preparations were compared with the known fragile sites. A total of 136 breaks were observed from 100 well spread G-banded plates (1.3 bps/cell). These correspond to a total of 57 break prone sites. Of these 57 sites, 24 correspond to the known fragile sites, 5 to sites of protooncogenes and neoplasia, 26 sites correspond to more than one known site of fragility, protooncogene, neoplasia or reciprocal translocation sites, and 2 unknown sites. The findings suggest that fragile sites, either commonly expressed or induced, might be a predisposing factor for chromosome aberrations in human. The expression of fragile sites induced by Blm and their correlation with the known cancer chromosome break points, oncogenes and reciprocal translocation, suggest that the fragile sites are prone to mutagenic action.     

Fragile×expression and×inactivation

Summary The inactive fragile×chromosomes of a 47,fra(X),fra(X),Y male with a typical fragile×phenotype were successfully separated from the active homologues by means of somatic cell hybridization. It was shown by FUdR-induction and caffein-posttreatment that the separated inactive×chromosomes expressed their fragile sites and that the presence of an active mutated \sxchromosome was not a prerequisite for fragile X expression. The fragility seems to be an intrinsic property of the individual fragile site. This result is in favour of the classical concept that the fragile site at Xq27.3 has a primary pathogenetic function in this syndrome, although the fragility itself could represent a secondary phenomenon related to an unknown alteration of the DNA in this chromosome region. It is also concluded that inactivation of the fragile\sxchromosome in females is not responsible for either false negative fragile\sxfindings or the observation of fragile\sxnegative colonies isolated from fragile\sxpositive fibroblasts in heterozygotes.     

Human chromosome fragility

Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis. They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci. Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites. From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.     

The effect of caffeine on fragile X expression

Summary Caffeine has been reported to enhance the expression of the fragile X [fra(X)] and common fragile sites in peripheral blood lymphocyte cultures (PBLC) treated with 5-fluorodeoxyuridine (FUdR). One of the effects of caffeine on replicating cells is inhibition of DNA repair suggesting that fragile sites may be regions of DNA with a high rate of misreplication under the conditions of thymidylate stress induced by FUdR. We have studied the effect of caffeine on the expression of the fra(X) and common folate-dependent fragile sites in PBLC from two fra(X) expressing individuals and in five lymphoblastoid cell lines (LCL) established from individuals in families in which the fra(X) is segregating. Caffeine did not enhance the expression of the fra(X) in the PBLC or in the three LCL from fra(X) expressing individuals nor did it elicit fra(X) expression in LCL from a non-expressing obligate-carrier female and a transmitting male. However, in all cultures there was a marked increase of common fragile site expression due to caffeine treatment. These data suggest that the mechanism of expression of the common fragile sites and the fra(X) may be quite different.     

The role of nucleotides in human fragile site expression

Fragile sites are points on chromosomes which tend to break non-randomly when exposed to specific chemical agents or conditions of tissue culture. There are 3 groups of rare fragile sites, and carriers of these range in incidnece from about 1 in 20 to 1 in several thousand individuals. Rare fragile sites are essentially chromosome variants with no known phenotypic consequence, except for the fragile X which is associated with the commonest inherited form of mental retardation in man. There are also 3 groups of common fragile sites, carried by all or most individuals. These are part of normal chromosomal architecture. Expression of most of the groups of gragile sites is mediated by perturbations of the nucleotide pool and these, as they relate to each group of fragile sites, are discussed. The rare folate-sensitive fragile sites are expressed when thymidylate or deoxycytidine are in limited supply during DNA synthesis. Other rare fragile sites are induced by bromodeoxyuridine (BrdU). Sets of common fragile sites are induced by BrdU, 5-azacytidine and aphidicolin. Various hypotheses on the molecular nature of fragil sites are considered.     

Liability to chromosome damage in lymphocytes of "cancer family" subjects: a study of spontaneous and induced chromosomal fragility

Spontaneous chromosomal fragility was detected in seven tumor patients and one healthy member from two families with a high recurrence of cancer. Major chromosome lesions, such as terminal deletions and rearranged chromosomes, were found at levels significantly higher than those reported for control individuals. The prevalence of these aberrations in comparison to minor ones (chromosome gaps and chromatid breaks) in this group of patients seems to indicate that the fragility observed is the end-point of a process of chromosomal instability, which may have already been brought to expression. Study of other parameters of genetic instability in the most unstable karyotypes showed that the chromosome damage observed was neither paralleled by abnormal SCE frequency nor sustained by defective DNA repair mechanisms or expression of inherited or constitutional fragile sites. As all the subjects investigated here had previously been shown to display intraindividual variations in the C-banded region of chromosome 1, it is possible that spontaneous fragility and acquired C-heterochromatin polymorphism may be markers that, combined with chromosomal instability, create genetic predisposition to cancer.     

Expression of fragile sites in childhood acute lymphoblastic leukemia patients and normal controls

Summary A high concordance has been reported between fragile sites and breakpoints involved in chromosomal rearrangements in cancer. A prospective study on the role of fragile sites in the etiology of childhood acute lymphocytic leukemia (ALL), with appropriate comparisons to results obtained from normal controls, analyzed fluorodeoxyuridine-, aphidicolin-, and caffeine-induced fragile sites in the peripheral blood of seven ALL patients (three with cytogenetically normal karyotype and four with pseudodiploid karyotype) and eight normal controls. While extensive variations in the number and distribution of fragile sites was observed within each group, there was no significant difference in the mean total fragile sites and mean fragile sites per cell between the two groups (P>0.05) in all three treatments. Similarly, within the ALL patients, the two karyotypic groups did not exhibit any significant difference in fragility (P>0.05).     

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.     

Thymidylate synthetase inhibitors and fragile site expression in lymphocytes.

The ability of three thymidylate synthetase inhibitors, fluorodeoxyuridine, fluorodeoxycytidine, and trifluorothymidine, to induce the expression of eight different folate-sensitive fragile sites has been investigated in 22 patients and compared with the efficacy of simple folate deprivation for inducing fragile site expression. Fluorodeoxyuridine and fluorodeoxycytidine were equal in their ability to elicit fragile site expression but fluorodeoxycytidine proved less cytotoxic under comparable culture conditions. Both fluorodeoxyuridine and fluorodeoxycytidine were found to be more efficient than trifluorothymidine at comparable concentrations but less efficient than simple folate deprivation in eliciting fragile site expression in lymphocytes. Since the three inhibitors induced expression of eight different folate-sensitive fragile sites, it is likely that all folate-sensitive fragile sites have a common underlying mechanism of expression. The practical application of thymidylate synthetase inhibitors in the routine detection of heritable fragile sites is discussed.     

129 The molecular mechanism of breakage at fragile site FRA16D

Replication stress induces physical breakage at discrete loci in chromosomes, which can be visualized on a metaphase chromosome spread. These common fragile sites (CFS) are conserved across species and are hotspots for sister chromatid recombination, viral integration, rearrangements, translocations, and deletions (Glover et al 2005). Despite multiple theories, the molecular mechanisms of CFS expression and genomic instability are still not well understood. The fragile site FRA16D is of special interest because it is the second most highly expressed fragile site and is located within the WWOX tumor suppressor gene. Previous data identified a polymorphic AT repeat within a FRA16D subregion called F1 that causes chromosome fragility and replication fork stalling in a yeast model (Zhang and Freudenreich 2007). Recently, we have found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeat in the context of F1 forms a secondary structure, making the region more vulnerable to breakage.