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.     

Induction of distamycin A-inducible rare fragile sites and increased sister chromatid exchanges at the fragile site

Summary Expression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(pl2), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did nto induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra (16)(pl2) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.     

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.     

Increased genetic instability of the common fragile site at 3p14 after integration of exogenous DNA.

We determined previously that the selectable marker pSV2neo is preferentially inserted into chromosomal fragile sites in human x hamster hybrid cells in the presence of an agent (aphidicolin) that induces fragile-site expression. In contrast, cells transfected without fragile-site induction showed an essentially random integration pattern. To determine whether the integration of marker DNA at fragile sites affects the frequency of fragile-site expression, the parental hybrid and three transfectants (two with pSV2neo integrated at the fragile site at 3p14.2 [FRA3B] and specific hamster fragile sites [chromosome 1, bands q26-31, or mar2, bands q11-13] and one with pSV2neo integrated at sites that are not fragile sites) were treated with aphidicolin. After 24 h the two cell lines with plasmid integration at FRA3B showed structural rearrangements at that site; these rearrangements accounted for 43%-67% of the total deletions and translocations observed. Structural rearrangements were not observed in the parental cell line. After 5 d aphidicolin treatment, the observed excess in frequency of structural rearrangements at FRA3B in the cell lines with pSV2neo integration at 3p14 over that in the two lines without FRA3B integration was less dramatic, but nonetheless significant. Fluorescent in situ hybridization (FISH) analysis of these cells, using a biotin-labeled pSV2neo probe, showed results consistent with the gross rearrangements detected cytogenetically in the lines with FRA3B integration; however, the pSV2neo sequences were frequently deleted concomitantly with translocations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sister chromatid exchanges are preferentially induced at expressed and nonexpressed common fragile sites

Summary To investigate the relationship between common fragile sites and sister chromatid exchange (SCE), lymphocyte cultures were treated with aphidicolin and bromodeoxyuridine (BrdU) and analyzed using a sequential GSCE staining protocol. A total of 1163 SCEs were mapped to their corresponding G-band sites, which were assigned to one of the following four categories: fragile sites expressed; fragile sites nonexpressed; nonfragile sites with breaks; or nonfragile sites with no breaks. The designated common fragile sites were found to be preferred locations for SCE formation, not only when these sites were expressed as visible gaps or breaks, but even when they were nonexpressed in the cell. SCEs were also more likely to occur at nonfragile sites with breaks than at nonfragile with no break sites. Further, SCEs were found to be distributed nonrandomly across fragile sites and nonfragile sites, and among the fragile sites, the high frequency SCE sites were highly correlated with the high frequency breakage sites. These data support the hypothesis of common steps in the mechanism of aphidicolin-induced SCE formation and common fragile site expression.     

Population cytogenetics of rare fragile sites in Japan

Summary A population cytogenetic study of three groups of rare fragile sites defined in Human Gene Mapping 8 (HGM8, Berger et al. 1985) has been conducted using peripheral blood lymphocytes of healthy Japanese subjects. We have examined 1,022 blood donors for folate-sensitive and bromodeoxyuridine (BrdU)-requiring, and 845 for distamycin A-inducible fragile sites. Out of 17 rare autosomal fragile sites defined in HGM8, the following six were identified in Japan; folate-sensitive fra(2)(q11), fra(11)(q13) and fra(11)(q23), distamycin A-inducible fra(16)(q22) and fra(17)(p12), and BrdU-requiring fra(10)(q25). The incidences of distamycin A-inducible fra(16)(q22) (1.42%) and fra(17)(p12) (3.08%) were considerably higher than those of the other sites in Japan. Furthermore, a folate-sensitive fra(17)(p12) and a distamycin A-inducible fra(8)(q24.1) have been newly found in the present study. Their incidences were 0.10% (1/1,022) and 0.71% (6/845), respectively. Since the expression of this fra(17)(p12) was induced by fluorodeoxyuridine, supressed by thymidine, but not induced by distamycin A, it can be classified as a folate-sensitive site. The expression of the new distamycin A-inducible fra(8)(q24.1) was also enhanced by treatment with Hoechst 33258, berenil and 4,6-diamidino-2-phenylindole (DAPI). This fragile site fulfils all four classical criteria suggested by Sutherland (1979) and also new criteria for a rare fragile site defined in HGM8 (Berger et al. 1985).     

Heritable fragile sites on human chromosomes I. Factors affecting expression in lymphocyte culture.

The expression of heritable fragile sites on human chromosomes has been shown to be dependent upon composition of the tissue medium for sites at 2q11, 10q23, 11q13, 16p124, 20p11 and Xq27 or 28 but not for the site at 16q22. Expression of the fragile sites is inhibited by folic acid, thymidine, folinic acid, and probably bromodeoxyuridine, and induced by methrotrexate. In addition, there is a correlation between frequency of expression of the sites and pH of the culture medium for the sites on 2q, 10q and Xq. Possible reasons for these findings are discussed, and a definition and classification of fragile sites is proposed.     

Nonrandom distribution of methotrexate-induced aberrations on human chromosomes. Detection of further folic acid sensitive fragile sites

Summary Eleven folic acid sensitive fragile sites (3p14, 7p13, 7q31.1, 7q32, 9q32, 11p13, 14q23, 15q22, 16q23, Xp22.2, Xq22) were detected in one individual, eight of them previously unknown. These sites seem to bear each its specific sensitivity to folic acid deficiency. Six of the sites were observed simultaneously on both homologous chromosomes in at least one cell. Each of these 11 sites was also found in at least one among 12 individuals further examined. Some of these individuals showed six of these 11 sites. The fragile site 3p14 was detected in all individuals examined. The homologous sites 3p14 of one individual differed from each other in their frequency of lesions induced by methotrexate as well as fluorodeoxyuridine. This observation suggests that folic acid sensitivity is a property inherent in the chromatin of an individual chromosome at the site involved in fragility. This property seems to be responsible for the nonrandom fragility at that site and also for the individual sensitivity of each chromosomal site.     

Three infants having null acute lymphoblastic leukemia with chromosome rearrangements at 11q23: no involvement of a heritable fragile site in this band

Three families are presented in which an infant with null acute lymphoblastic leukemia had a karyotype rearrangement involving a break at 11q23. Peripheral blood was obtained, where possible, from both parents and from the child during periods of remission. The blood was stimulated with phytohemagglutinin and cultured under conditions that enhance expression of heritable folate-sensitive fragile sites. In all individuals studied very low levels of fra(11)(q23.3) were observed. These levels were far below those recorded for expression of the heritable folate-sensitive site fra(11)(q23.3) but are comparable with expression of the common fragile site fra(11)(q23.3) under these conditions.     

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 mouse-human hybrid cell panel for mapping human chromosome 16

A mouse-human hybrid cell panel for human chromosome 16 was constructed from human cell lines with breakpoints on chromosome 16 at p13.11, q13, q22 and q24. Fusions with the human fibroblast line GM3884, t(X;16)(q26;q24) allowed the isolation of clones with either the derivative X or the derivative 16 as the only human chromosome. This was a consequence of both the genes APRT and HPRT being involved in the translocation. The breakpoints of the line GM3884 were confirmed by aphidicolin induction of the common fragile site at 16q23. The results of the fusions with this line suggest a localisation of the APRT gene at 16q24 and confirm the localisation of HPRT to Xq26 to Xq27.3. These hybrid cell lines enable the localisation of genes and DNA fragments to six clearly defined regions. Further localisation within three of these regions is possible by use of the three fragile sites on chromosome 16. In situ hybridisation with the probe pBLUR confirmed that of three lines tested all contained a single human chromosome.     

Neither age nor sex influence the expression of folate sensitive common fragile sites on human chromosomes

Summary The expression of folate sensitive common fragile sites was investigated in 82 normal healthy males and females of various ages. In 100 studied metaphases of each of these controls, between 0 and 56 lesions were detected (mean 18.3 ± 10.3 SD). No significant difference was found between the mean number of expressed lesions in females and males. No age-effects were observed. Two new common fragile sites were discovered at 6p21 and 17q21. Their fragile site status, however, needs to be confirmed.     

Heritable fragile sites on human chromosomes II. Distribution, phenotypic effects, and cytogenetics.

Individuals and families have been documented in which there are a number of fragile sites on chromosomes. These include sites at 2q11, 10q23, 11q13, 16p124, 16q22, 20p11, and Xq27 or 28. Fragile sites reported in the literature are compiled. The cytogenetics of the sites is discussed. The phenotypic effects of the sites are considered, and it is speculated that homozygosity of the autosomal sites might be deleterious as is hemizygosity of the site on Xq. These sites are used in the previous report which documents the effect of tissue medium components on their expression.     

Studies on three rare fragile sites

Summary Three fragile sites 2q13, 12q13, and 17p12 were found in one family. In the index case, who was first investigated in 1969 for low birth weight and bilateral inguinal hernia, three tissues were examined, blood, marrow, and skin. Three of the family have been reinvestigated after 17 years. Cultures for sister chromatid exchange (SCE) and the effects of aphidicolin, fluorodeoxyuridine (FUdR), bromodeoxyuridine (BrdU), and methotrexate on the frequency of the fragilities were studied. The mother of the index case who is an obligate carrier for the fragile 2q13 does not express it in folate/thymidine deficient medium. Further studies on her using a lymphoblastoid cell line, showed that there was a reduced level of fragility of 12q13 and 17p12 in B-lymphocytes compared to T-lymphocytes. Excess thymidine and FUdR when added to the lymphoblastoid cell line did not induce the 2q13. These studies also confirm the induction of a range of common fragile sites by treatment with aphidicolin, showing in addition homozygosity for at least 3p14, 6q26, 16q23, and Xp22. There were no detectable increases in the SCE rate between individuals with fragile sites and the five controls tested. There was no history of cancer or phenotypic abnormalities in the family.     

fra(1) (p11), fra(1) (q22) and r(1) (p11q22) in a retarded girl.

A mentally retarded girl with a 46,XX/47, XX+r(1) (p11q22q22p11)/47, XX+r(1) (p11q22) fra(1) (p31) fra(1) (p11) fra(1) (q22) karyotype who inherited the fragile sites from the normal mother was studied. The conicidence of fra(1) (p11) and fra(1) (q22) with the ring chromosome breakpoints strongly suggests a cause-effect relationship. This finding agrees with other reported associations between fragile sites and structural chromosome abnormalities and constitutes the fourth reported of a de novo structurally abnormal chromosome as a consequence of presumed in vivo fragile sites instability. Although risk figures for chromosome anomalies and cancer associated with fragile sites are lacking, carriers of fra (1) (p11) may have a higher risk for abnormalities of chromosome 1 in somatic and gonadal cells than the general population.     

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.     

Increased frequencies of sister chromatid exchanges at common fragile sites (1)(q42) and (19)(q13)

Summary Human lymphocyte cultures were treated with 5-azadeoxycytidine for the induction of the common fragile sites at 1q42 and 19q13 and with 5-bromodeoxyuridine for differential sister chromatid staining. A remarkably high frequency of sister chromatid exchanges was observed directly at the gaps of both fragile sites. In addition, the rate of sister chromatid exchanges occurring at the region corresponding to 1q42 with-out a concurrent visible gap was also increased. This confirms previous data on increased intrachromosomal recombination in common and rare fragile sites of various categories.     

The fragile site (16) (q22)

Summary The rare fragile site at 16q22 was experimentally induced in lymphocyte cultures with various AT-specific, non-intercalating DNA-ligands. The optimum conditions for the induction of fra (16)(q22) were determined. The best expression of fra (16)(q22) was found with the aromatic diamidine berenil which is recommended for further studies on this fragile site. The results indicate that fra (16)(q22) is a region with AT-rich, late replicating DNA. The simultaneous treatment of lymphocytes with berenil and aphidicolin (inhibitor of DNA polymerase ) induces both the rare fra (16) (q22) and the common fra (16) (q23) within the same chromosome. A population study on 350 unselected individuals showed that fra (16)(q22) is the most common of all rare autosomal fragile sites in man. The frequency of individuals heterozygous for fra (16)(q22) is 5.1% no homozygosity for fra (16) (q22) was detected. Statistical analysis indicates that the population is in Hardy-Weinberg equilibrium with respect to the fragile and non-fragile chromosomes 16.     

Autosomal fragile sites

It is possible to distribute the 17 autosomic fragile sites presently known in three categories according to their sensitivity: BrdU-sensitive sites (10q25, 16q22, 17p12), distamycin A-sensitive sites (16q22, 17p12) and folate- and thymidilate-sensitive sites (2q11-q14, 3p14, 6p23, 7p11, 8q22, 9p21, 9q32, 10q23, 11q13, 11q23, 12q13, 16p12, 16q23, 17p12, 20p11). Four fundamental problems are discussed, first the relation between the presence of a fragile site and the phenotype, secondly the incidence of autosomic sites, third the origin of fragility (particularity of DNA structure, defect of the DNA/proteins binding and abnormal arrangement of chromatin, abnormality of the metaphasic scaffold) and fourth the localization of fragile sites.