Fragile X expression is decreased by 5-azacytidine and S-adenosylhomocysteine

A lymphoblastoid cell line derived from a patient with fragile X chromosome exhibited fragility only when 5-fluoro-2'-deoxyuridine (FUdR) was added to the culture medium. Addition of methionine with FUdR greatly increased the frequency of fragile X. Addition of 5-azacytidine (an inhibitor of methylation at the level of DNA) or S-adenosylhomocysteine (an inhibitor of the synthesis of the methyl group donor S-adenosylmethionine) reversed the effect of methionine as well as that of FUdR. It is proposed that DNA methylation is in some way involved in the mechanism of fragile X expression.     

The combined effects of FUdR addition and methionine depletion on the X-chromosome fragile site.

The fragile site on the X chromosome [fra(X)] associated with mental retardation is not normally seen in lymphocytes cultured in media containing folic acid or lacking methionine. The requirement for methionine has been taken to mean that amino acid metabolism or one-carbon transfer via S-adenosyl-methionine is most important in fra(X) expression. The inhibitory effect of folic acid can be overcome, as we have shown, by the addition of 5'-fluorodeoxyuridine (FUdR) to the culture medium, suggesting that depletion of dTMP available for DNA synthesis is most important in expression. We tested the combined effects of FUdR addition and methionine depletion on the fra(X). We found expression of the fra(X), indicating that methionine per se is not necessary for expression. Our work supports the suggestion that expression of the fra(X) is due to specific limitation of the dTMP pool available for DNA synthesis.     

Fragile sites and spontaneous abortions.

In this report we present the cytogenetic findings of the expression of fragile sites in 10 couples with two or more spontaneous abortions. These findings were compared with the results in a control group of 15 subjects with two normal offsprings. Individuals of couples experiencing early fetal losses carry fragile sites with significant higher frequency, moreover this frequency is markedly influenced by the number of spontaneous abortions. Eight fragile sites were significantly more expressed in individuals with miscarriages than in the controls. These eight fragile sites correspond with cancer breakpoints or sites of oncogenes. Hypothesis on the role of oncogene mutations in spontaneous abortions is proposed, based on the results of the nonrandom distribution of fragile sites on human chromosomes.     

The effect of methionine and 5-azacytidine on fragile X expression.

The cellular mechanism for the expression of the fragile site at Xq28 is unknown. We tested the effect of 5-azacytidine and methionine on fragile X expression in lymphocytes and lymphoblastoid cells in an attempt to determine if DNA methylation was involved. We were unable to demonstrate a consistent dosage effect of methionine on fragile X expression. While 5-azacytidine was found to inhibit the fragile X in both males and females, it did so only at relatively high concentrations. We conclude that the role, if any, of DNA methylation in fragile X expression is likely to be secondary, the primary effect being due to thymidylate depletion.     

Fragile Site Instability in Saccharomyces cerevisiae Causes Loss of Heterozygosity by Mitotic Crossovers and Break-Induced Replication

Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.     

Spontaneous expression of the chromosome fragile site fra(10)(q25).

We report the spontaneous expression of the chromosome fragile site at 10q25 in a child with progressive cerebellar ataxia and in her phenotypically normal brother and father. Expression of this fragile site was increased in all three individuals by addition of bromodeoxyuridine (BrdU) to the medium. This fragile site has previously been described only following growth of cells in BrdU.     

Structural chromosomal rearrangements in HpaII-treated human lymphocytes.

Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI-induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII-induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located.     

Chromosome breakage and recombination at fragile sites.

Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In the present report, fragile sites are shown to predispose to deletions and interchromosomal recombination. By use of somatic cell hybrids containing either human chromosome 3 or the fragile X chromosome, deletions and translocations were induced by FUdR or aphidicolin with breakpoints at the fragile sites Xq27 or 3p14.2 (FRA3B) or at points so close to the fragile sites as to be cytogenetically indistinguishable. Southern blot analysis of DNA from a panel of chromosome 3 deletion and translocation hybrids was then utilized to detect loss or retention of markers flanking FRA3B and to corroborate the cytogenetic evidence that the breakpoints were at this fragile site. One cell line with a reciprocal translocation between human chromosome 3 (with breakpoint at 3p14.2) and a hamster chromosome showed cytogenetically that the fragile site was expressed on both derivative chromosomes, supporting the hypothesis that the fragile site represents a repeated sequence. The approach described provides a means of generating specific rearrangements in somatic cell hybrids with a breakpoint at a fragile site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA

An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG/CGG_n trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human fragile-X probes on cattle chromosomes was however, medium-prominent sub-centromeric signal on two homologues. BrdU administration in 12 h before harvesting identified these homologues to be chromosome number 5. In addition retrospective slides of cattle and sheep chromosomes used for fragile site studies showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X.     

Parental inheritance and psychological disability in fragile X females.

Studies of adult female carriers of the fragile X chromosome indicate that certain psychological problems occur with a greater frequency and severity than expected. This study examines the association of parental origin of the fragile X chromosome and of fragility detected in the karyotype with measures of social, educational, and psychological functioning in a group of adult fragile X females of normal intelligence. The results show that, as a group, women who inherit the fragile X chromosome from their mother and who demonstrate positive fragility in the karyotype (MI+ group = [maternal inheritance with positive fragility]) manifest significantly more impairment of social, educational, and psychological functioning when compared with fragile X females with paternal inheritance or negative fragility or with a matched control group comprising non-fragile X women. In particular, MI+ women show lower levels of both educational achievement and socioeconomic status and a greater degree of disturbance in communication, socialization, affect, and thought processes. These clinical findings are consistent with the recently advanced hypothesis which proposes that a two-stage process leading to chromosome imprinting in a preoogonial cell causes the fragile X syndrome.     

Micronucleus formation in lymphocytes of patients with fraXq27 syndrome

Human chromosome carry different fragile sites, but the most interesting one is the fragile X chromosome, especially in relation to mental retardation. In this report we present micronucleus formation in 5 individuals with the fragile X syndrome and 11 control subjects whose lymphocytes were cultured under 1) hydroxyurea treatment condition suitable for the increasing expression of the fraXq27, 2) control conditions. It has been found that hydroxyurea (HU) added to the final concentration 1.3 X 10(-5)M increases the frequency of the fraXq and micronucleus formation in comparison with culture without HU. The same concentration of HU has induced neither "nonspecific" "spontaneous" chromosomal breakage nor micronucleus formation in the control subjects.     

The fragile site (16)(q22)

Summary In the lymphocytes of heterozygous carriers of the rare autosomal fragile site (16)(q22) an exceptionally high frequency of sister chromatid exchanges was demonstrated at the induced fragile site by means of simultaneous berenil and BrdU treatment of the cultures. The rate of sister chromatid exchanges at q22 is also increased in the fragile chromosome 16 by treating the cells with BrdU alone. The possible reasons for the preferential occurrence of induced and spontaneous sister chromatid exchanges at fra (16)(q22) are discussed.     

Translocations of chromosome 12

Summary Human chromosome 12 has been used as a model for studying the distributions of sites of induced and spontaneous breaks. The breakpoints were determined from (1) translocations involving chromosome 12, (2) spontaneous breaks in untreated cultures, (3) radiation-induced breaks, and (4) spontaneous breaks in Fanconi's anaemia.Statistical analysis showed discordance in the results both between the eleven individual bands and between the four assessments. Also, the distribution of breaks for all bands was significantly diferent from random in each assessment. Certain bands added considerable bias to the results, and when analysed individually, only four bands (p11.1, q13, q24, and p13) showed distributions over the four assessments that were significantly different from random. These four bands are Giemsa-negative bands, and two (p13 and q24) are adjacent to telomeres, while p11.4 is adjacent to the centromere. The fourth band, q13, is a known fragile site.It is concluded that bands adjacent to centromeres, which are not C-banded, are peculiarly sensitive to breakage. Telomeric bands are variable in their response to different conditions of breakage, and both the physical structure of the telomere and the specific gene sequences of individual telomeres are probably of importance in determining this response. The fragile site q13 responds as if breakage at this site is due to the base composition of the DNA.     

The breast cancer susceptibility gene BRCA2 is required for the maintenance of telomere homeostasis

Inactivating mutations in the breast cancer susceptibility gene BRCA2 cause gross chromosomal rearrangements. Chromosome structural instability in the absence of BRCA2 is thought to result from defective homology-directed DNA repair. Here, we show that BRCA2 links the fidelity of telomere maintenance with genetic integrity. Absence of BRCA2 resulted in signs of dysfunctional telomeres, such as telomere shortening, erosions, and end fusions in proliferating mouse fibroblasts. BRCA2 localized to the telomeres in S phase in an ATR-dependent manner, and its absence resulted in the accumulation of common fragile sites, particularly at the G-rich lagging strand, and increased the telomere sister chromatid exchange in unchallenged cells. The incidence of common fragile sites and telomere sister chromatid exchange increased markedly after treatment with replication inhibitors. Congruently, telomere-induced foci were frequently observed in the absence of Brca2, denoting activation of the DNA damage response and abnormal chromosome end joining. These telomere end fusions constituted a significant portion of chromosome aberrations in Brca2-deficient cells. Our results suggest that BRCA2 is required for telomere homeostasis and may be particularly important for the replication of G-rich telomeric lagging strands.     

Chromosome characteristics of X-linked mental retardation. II. Autosomes

The frequencies of chromosome and chromatid breaks and gaps were studied in blood lymphocytes of three groups of individuals: 21 males with X-linked mental retardation characterized by fragile X chromosome; 52 males with non-differentiated X-linked mental retardation having no fra(X) chromosome in their cells; 15 intellectually normal males. The lymphocytes were cultured both in medium 199 and in Eagle's medium supplemented with fluoro-deoxyuridine. The significantly higher frequencies of various autosomal lesions were observed in the individuals with the fragile X chromosome syndrome and in those with mental retardations without fra(X) chromosome, in comparison with normal males. The significant difference in some autosome lesions was also found between both groups of the patients. The distribution of chromosome lesions in autosomes of different groups was significantly higher in chromosomes A and lower in groups B, E, F and G, than expected in accordance with their relative length in the haploid set. In all the groups of individuals studied, the predominant localization of chromosome and chromatid breaks and gaps was observed in fragile sites 1p31, 3p14, 6q26 and 16q23.     

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.     

Regional localization for HLA by recombination with a fragile site at 6p23.

A family with a fragile site on chromosome 6 at band p23 was examined for recombination between the fragile site and HLA. Recombination was observed in four of the 20 offspring in whom it could occur. The estimate of the genetic length of chromosome between the fragile site and HLA is 20 centimorgans (cM) with a lower 95% probability limit of 8.5 cM, placing HLA proximal to the midpoint of 6p22. The most likely regional localization is at 6p21.3, which agrees closely with methods that do not involve recombination with the fragile site. This fragile site does not measurably disrupt recombination frequency, and the allele predisposing to expression of the fragile site is situated at the fragile site.     

A BrdU-enhanceable fragile site or viral modification site at 11q23.1 in lymphoblastoid cultures

Chromosomes prepared from EBV-transformed lymphoblastoid cell lines show an achromatic gap or fragile site-like lesion at 11q23.1. The low spontaneous expression of this lesion is greatly enhanced by BrdU and n-butyric acid. The lesion was expressed homozygously in all 16 cell lines examined. It is suggested that the lesion is a viral chromosome modification site.     

猕猴、白眉长臂猿、人类染色体普通型脆性部位和G-带的比较研究

以BrdU、FdU、MTX诱导猕猴、白眉长臂猿和人类染色体普通型脆性部位的表达,并对染色体脆性部位和染色体进化的关系以及三种灵长类染色体的同源性进行了比较分析。结果表明,近缘动物染色体同源区内的脆性部位在进化上是保守的,可作为染色体具有共同起源的标志,结合G-带的比较,可以用以阐明近缘动物染色体的同源性和染色体进化。     

The determination of chromosome fragile sites in the peripheral blood lymphocytes of patients with colorectal cancer taking into account the cancer pathology predisposition in the familial anamnesis

Results of comparative study of spontaneous and 5-bromdeoxyuridine-induced fragility of peripheral blood lymphocytes chromosomes in 9 patients with colorectal adenocarcinoma were presented. It was shown the increase of average spontaneous level of chromosomal fragility in patients with tumor aggregation in family as well as without it to 4.5 +/- 1.0 and 5.3 +/- 1.1 per 100 tested cells, accordingly. The increase of average level of damaged chromosomes in spectrum of rare sites to 12.5 +/- 2.6 in the patients with tumor aggregation in pedigree comparing to the patients without oncopathology in family 8.0 +/- 1.7 was observed. The most number of rare fragile sites was observed in 1q21 site of the chromosome 1. Possible connection between fragile sites of chromosomes in normal cells and malignant processes in the patients with colorectal cancer is discussed.