12q13 fragility in a family with recurrent spontaneous abortions: expression of the fragile site under different culture conditions

A fragile site at the 12q13 band was found in metaphases from lymphocyte cultures of three members of a family. A comparison of the frequency and expression of the fragile site was carried out on cells cultured in RPM-I 1640 with and without BrdU and in 199 media. The fragile site was not typically folate-sensitive, being expressed in standard medium as well as in cultures after exposure to BrdU.     

Heritable fragile sites on human chromosomes. V. A new class of fragile site requiring BrdU for expression.

A new fragile site at 10q25 is described, representing a new class of fragile site that requires bromodeoxyuridine (BrdU) in the culture medium for expression. This new fragile site is present in approximately one in 30 of the Australian population; it has only been observed in heterozygotes.     

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.     

Induction of the fragile X on BrdU-substituted chromosomes with direct visualization of sister chromatid exchanges on banded chromosomes

Summary After incorporation of BrdU for one or more replication cycles, the fragile site at Xq27 [fra(X)] was induced by a late pulse with excess thymidine (dT), resulting in the simultaneous visualization of G bands and differentially stained sister chromatids. The degree of BrdU substitution (uni- vs bifilarly substituted DNA) did not affect the expression of the fra(X). Without addition of dT, expression was the same in M1, M2, and M3 cells. With the addition of dT, expression was reduced in M1 cells and increased in M2 and M3 cells. One way to explain this fact would be an increased repair of the fragile site in M1 cells by illegitimate G:BrdU pairing under dCTP-deficient conditions. A preferential depletion of M3 cells, and to a lesser extent also M2 cells, could suggest a synergistic toxic effect of BrdU substitution and dCTP depletion. With this technique, sister chromatid exchanges (SCEs) could be directly localized at band level, facilitating a more detailed study of SCEs at the Xq27 fragile site.     

Heritable fragile sites on human chromosomes. VII. Children homozygous for the BrdU-requiring fra(10)q25 are phenotypically normal

A brother and sister have been detected who are homozygous for the bromodeoxyuridine (BrdU)-requiring fragile site at 10q25. The children are phenotypically normal, indicating that homozygosity for this fragile site is harmless, at least during childhood.     

Manifestation of the fragile site Xq27 in fibroblasts

A protocol is reported which allows the efficient induction of bromodeoxyuridine (BrdU)-induced R-type replication patterns in fibroblast cultures prepared to demonstrate the fragile site fra(X)(q27). The technique includes partial synchronization of the culture by fluorodeoxyuridine (FdU) blocking at the G1/S transition. This block does not impair the induction of the fragile site in medium 199 containing methotrexate. The marked increase of the mitotic index in the synchronized culture may be an advantage in the study of folic acid sensitive fragile sites in fibroblasts. Adding BrdU after block removal leads to an efficient labeling of replicating chromosomes without severely impairing the manifestation of fra(X)(q27).     

Heritable fragile sites on human chromosomes. IX. Population cytogenetics and segregation analysis of the BrdU-requiring fragile site at 10q25

The frequencies of the bromodeoxyuridine (BrdU)-requiring fragile site at 10q25 in 1,026 unselected neonates, 901 patients referred for chromosome studies, and 87 institutionalized retardates were not significantly different from each other. The gene frequency was .013, and the population was in Hardy-Weinberg equilibrium. Segregation analysis confirmed that the fragile site followed codominant inheritance. This fragile site and its nonfragile allelomorph can be considered to constitute the first true chromosomal polymorphism to be described in man.     

New classes of common fragile sites induced by 5-azacytidine and bromodeoxyuridine

Summary Two new classes of common fragile site seen in chromosomes from blood lymphocyte cultures are reproted. The first cláss is induced in bands 1q42 and 19q13 by 5-azacytidine (5-AZA). Maximum induction of these fragile sites occurs when the 5-AZA is added 5–8 h prior to harvest. The second class is induced in bands 6q13, 9p21, and 10q21 by bromodeoxyuridine (BrdU). In this instance maximum induction occurred if the BrdU was added 4–6h prior to harvest. The known fragile sites, both rare and common, are summarised.     

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).     

Heritable fragile sites on human chromosomes. XI. Factors affecting expression of fragile sites at 10q25, 16q22, and 17p12.

The fragile sites at 10q25, 16q22, and 17p12 can all be induced in lymphocyte culture by BrdU or BrdC added 6-12 hrs prior to harvest. Without induction, fra(10)(q25) is rarely expressed spontaneously, whereas fra(16)(q22) is frequently expressed spontaneously. Fra(17)(p12) is frequently expressed spontaneously but is probably expressed only after induction in some individuals. Distamycin A, netropsin, and Hoechst 33258 induced high levels of expression of fra(16)(q22) and fra(17)(p12) but did not enhance expression of fra(10)(q25). The mechanisms of induction of fra(16)(q22) by BrdU and distamycin A appear to be different, since the time of induction by BrdU reaches a maximum about 12 hrs prior to harvest whereas induction by distamycin A requires much longer exposure. The fragile sites at 10q25 and 16q22 were both induced in fibroblast culture by BrdU. Fra(17)(p12) is accepted as a fragile site because preliminary studies show that it behaves similarly in lymphocyte culture to fra(16)(q22); however, there is only limited evidence for fragility at 17p12.     

SMC1 inhibition results in FRA3B expression but has no effect on its delayed replication

Cellular processes involved in fragile site expression have been investigated by studying the effect on the replication pattern of the commonest fragile site FRA3B of RNA interference (RNAi)-mediated sister maintenance chromosome 1 (SMC1) inhibition in normal human fibroblasts. Replication timing of FRA3B in G2 was studied by bromodeoxyuridine (BrdU) labeling for the final 2h of cell culture whereas in the S phase was investigated by a fluorescence in situ hybridization (FISH)-based approach through the analysis of clones spanning the FRA3B region. Results showed that FRA3B is normally late replicated even though it is not expressed in untreated cells. On the other hand, SMC1 inhibition leads to FRA3B expression even if the percent of late replicated cells is comparable to control cells. These results obtained by analysing the commonest fragile site suggest that SMC1 plays a role in protecting late replicating regions from stresses occurring in the final steps of genome replication and that delayed replication is necessary but not sufficient for inducing fragile site expression.     

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.     

Heterozygous female carriers of the marker-X-chromosome: IQ estimation and replication status of fra(X)(q)

Summary The IQ levels of 18 female carriers with the marker X chromosome were evaluated, and cytogenetic studies after BrdU incorporation were performed. A highly significant correlation between mental capacity and replication pattern of the X chromosomes could be demonstrated. Heterozygous females with normal intelligence showed a clear tendency to carry the fragile site at the late replicating X chromosome, while other female carriers with lower intelligence or mental impairment expressed their fragile site mainly with the early replicating X chromosome. This observation could be interpreted as an expression of Lyonisation.     

Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome

The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of S?o Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo.     

Segregation analysis of rare autosomal fragile sites

Summary Segregation analyses were performed on pedigrees with rare autosomal fragile sites. The results of the analysis of pedigrees with folate sensitive fragile sites, including 2q1, 6p23, 7p11, 8q22, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, and 20p11, suggested that expression of the gene depended on the carrier parent: it was only 50% penetrant when transmitted by a carrier father, but fully penetrant when transmitted by a carrier mother. Pedigrees with the bromodeoxyuridine (BrdU) fragile site, fra(10)(q25), showed the same trend but the results were not statistically significant. In addition, 38 of the 44 probands with folate sensitive or BrdU-sensitive fragile sites received the gene from their carrier mother and only six received it from their father. In contrast, the analysis of pedigrees with the distamycin A-inducible site, fra(16)(q22), gave the results expected for a simple codominant trait with complete penetrance. Probands with this fragile site received the gene equally from mothers or fathers. The genetic implications of these results are discussed.     

A new rare distamycin a-inducible fragile site,fra(11)(p15.1), found in two acute nonlymphocytic leukemia (ANLL) patients with t(7;11)(p15-p13;p15)

Summary Fragile sites were analyzed in normal peripheral lymphocytes from two acute nonlymphocytic leukemia patients with t(7;11)(p15-p13;p15) leukemic cells. To induce expression of fragile sites, cultures were exposed to folate deprivation (M-F10), BrdU, distamycin A, or Hoechst 33258. Fragility at 11p15.1 was induced by distamycin A and Hoechst 33258 but was not seen in M-F10, BrdU, and control cultures. Fra(11)(p15.1) was found neither in healthy Japanese subjects (0 in 845) nor in patients with leukemia or other hematologic disorders without the t(7;11) (0 in 126). From these results, fra(11)(p15.1) can now be calssified as a rare distamycin A-inducible fragile site. Furthermore, this fra(11)(p15.1) coincided with one of the breakpoints of the t(7;11)(p15-p13;p15).     

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.     

Fragile (X) expression: Relationship to the cell cycle

Summary The fragile (X) chromosome demonstrable in individuals with one type of X-linked mental retardation is seldom, if ever, seen in more than 50% of cells of affected individuals. We have devised a model to explain this apparent 50% maximum, one essential feature of which is that the fragile (X) will not be seen in cells in their first division in thymidine-depleted media. The validity of our model was tested on lymphoblastoid cell lines from affected males by treating the cells with fluorodeoxyuridine (FUdR) to induce the marker and/or bromodeoxyuridine (BrdU) to determine the cell cycle. We have evidence that the fragile (X) is present in cells in the first and subsequent these observations our model is not valid and the 50% expression of the fragile site at Xq(28) and other unusual properties of this region of the X chromosome remain unexplained.This work was supported by Grant HD 07879 from the National Institutes of Health     

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.     

大熊猫繁育障碍与染色体脆性位点的相关性研究

通过研究建立起适合大熊猫染色体脆性位点表达的BrdU诱导体系。以大熊猫外周血淋巴细胞为材料,通过较长时间培养（96h）,采用低浓度BrdU(10μg/ml）,短时间（4h）诱导,并结合复制带技术将大熊猫染色体脆性位点的高发生区段准确地定位在No.2和No.12号染色体着丝粒区域。经生物统计学分析发现,No.2和No.12号染色体脆性位点表达频率在个体中有明显差异,而且前者与大熊猫个体的子代存活率呈负相关（r=-0.772).研究结果提示,No.2染色体着丝粒处高效表达的脆性位点地大熊猫个体繁育及其后代的存活是不利的.