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 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.     

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.     

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.     

Expression of aphidicolin, FUdR and caffeine‐induced fragile sites in lymphocytes of healthy Turkish individuals

The expression of common fragile sites (c‐fra) and frequency of chromosomal aberrations were studied in peripheral lymphocytes of 50 healthy Turkish individuals (26 males and 24 females from 1 to 87 years of age) after induction with aphidicolin (APC), 5′‐fluorodeoxyuridine (FUdR), and caffeine. A correlation was seen between age and the frequency of chromosomal aberrations in APC and caffeine treated cultures, but there were no significant differences in the frequencies of chromosomal aberrations between males and females in any of the treatments. The mean frequency of aberrations induced by FUdR was significantly higher than that induced by APC and caffeine. A chromosome aberration is defined as a fragile site when present in 1% of the cells analyzed from each culture and in at least 50% of the individuals studied. Using these criteria, 12 c‐fra were observed in the three treatments: 1p21, 1q21, 2p11‐q11, 3p14, 4q31, 6q26, 7q22, 7q32, 8q24, 11q23, 16q23, and Xp22. Sites 3p14, 16q23, and Xp22 were the most frequently observed c‐fra, with only the frequency of Xp22 being significantly increased in females in APC treated cultures. The results of these studies are important as a base against which the effects of other clastogenic and environmental agents, as well as genetic background, can be compared. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

Pseudodeficiency of arylsulfatase A: a common genetic polymorphism with possible disease implications

Summary The distribution and frequency of aphidicolin-induced common fragile sites were studied in chromosomes of cultured skin fibroblasts and PHA-stimulated lymphocytes from five normal individuals; 0.2 M aphidicolin was added for the last 26 h of culture. Skin fibroblasts from five fra(X)-positive patients were also studied in the same manner. Fragile sites most frequently found in fibroblasts from normal individuals were 3q26.2, 7q11.23, 16q23, 1p31, 10q11.2, 12q23 and 7q31, whereas those in lymphocytes from the same individuals were 3p14, 16q23, Xp22, 7q32 and 14q24. The distribution of fragile sites in fibroblasts from fra(X)-positive patients was essentially identical with that in normal individuals. The average number of gaps and breaks in 100 metaphases was 36.8 in fibroblasts from normal individuals, 113.8 in those from fra(X)-positive patients, and 279 in lymphocytes from normal individuals. Their rates of chromosome-type breaks and gaps were 7.9%, 29.7% and 54.5%, respectively. Thus, the distribution and frequency of aphidicolin-induced fragile sites were different between skin fibroblasts and lymphocytes, possibly reflecting differences in their DNA replication sequence or gene activity.     

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).     

体外培养的绒毛细胞中普通脆点的研究

体外培养绒毛细胞,用缺叶酸培养基或在培养基中加入大量鸟苷或胸苷,造成脱氧核苷酸库的不平衡,导致染色体断裂和普通脆点的表达。大量鸟苷对普通脆点的诱导作用较强,而大量胸苷的作用则稍缓和。大量鸟苷与缺叶酸培养基合并使用时,诱导作用减弱,其可能原因是:缺乏叶酸使鸟苷生成受阻,脱氧核苷酸库的不平衡状态减轻。     

Fragile X expression and X inactivation

Summary The major concept of fragile X pathogenesis postulates that the fragile site at band Xq27.3 [fra(X)] represents the primary defect. The expression of fra(X) is predicted to be an intrinsic property of the mutated chromosome and, hence, should not be suppressed by X inactivation in females or induced by X-linked trans-acting factors. We made fibroblast clones of a fra(X)-positive female. Monoclonality was demonstrated using the DNA methylation assay at DXS255. The mutated X chromosomes and their states of genetic activity in the different clones were also defined by molecular methods. Five clones were selected to induce expression of fra(X) by 10^-7 M FUdR; two carried an active mutated X chromosome, in the other three the mutated X chromosome was inactivated. Fra(X) was found expressed in both types of clones. The percentages of positive cells were as high as 7–10%, regardless of the genetic activity of the mutated X chromosomes. DNA replicating patterns, obtained by BUdR labelling, demonstrated that expression occurred only on the mutated X chromosomes previously identified by molecular methods. The concept that the fragile site represents the primary mutation is now strongly supported by experimental evidence. The expression of fra (X) in females is independent of X inactivation and other trans-acting factors.     

Human chromosome hot points

Summary Peripheral lymphocytes from 16 healthy adults, 9 pregnant women, and 3 fragile X syndrome patients were cultured in Eagle's minimum essential medium without folic acid (MEM-FA). The addition of 2mM, 4mM, or 8mM uridine 24h or 72h prior to harvest resulted in increases of chromosome gaps or breaks, especially at hot points 3p14, 16q23-24, and at fragile site Xq27. Pregnant women showed higher frequencies of 3p14 breaks and total chromosome breaks than men and non-pregnant women. The other chromosome regions, such as 6q26, 7q23, 7q35, 6p25, Xp22, 14q23 and 11p13, also frequently showed gaps or breaks. The results indicated that the unbalance of nucleotide pools was one of the causes of chromosome breakages. The higher frequencies of chromosome gaps and breaks under the condition of thymidylate stress may be due to the misincorporation of uracil instead of thymine into DNA.     

The characterization of the common fragile site FRA16D and its involvement in multiple myeloma translocations

Fragile sites appear as breaks, gaps, or decondensations on metaphase chromosomes when cells are grown under specific culture conditions. The breaks are nonrandom, appearing in defined, conserved locations throughout the mammalian genome. Common fragile sites, as their name implies, are present in virtually all individuals. With three common fragile sites cloned, their mechanism of expression and the role, if any, they play in human disease are still unclear. We have assembled a BAC contig of >1 Mb across the second most active common fragile site, FRA16D (16q23.2). We fluorescently labeled these BACs and used them as probes on metaphases from aphidicolin-induced lymphocytes and demonstrated that FRA16D decondensation/breakage occurs over a region of at least 1 Mb. Thus, this is the largest common fragile site cloned to date. Microsatellite markers that map within FRA16D show a very high loss in prostate, breast, and ovarian tumors, indicating that loss within this fragile site may be important in the development or progression of these tumors. In addition, a common t(14q32;16q23) translocation is observed in up to 25% of all multiple myelomas (MM). We localized four of four such cloned t(14;16) MM breakpoints within the FRA16D region. This work further demonstrates that the common fragile sites may play an important role in cancer development.     

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.     

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.     

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).     

Aphidicolin-Induced FRA3B Breakpoints Cluster in Two Distinct Regions

The common fragile site at chromosomal band 3p14.2 (FRA3B) is the most sensitive single site in the human genome to induced chromosomal lesions. This fragile site may predispose chromosome 3p to breakage that is commonly observed in lung, renal, and many other cancers. We previously used aphidicolin induction of FRA3B expression in a chromosome 3-only somatic cell hybrid to generate a series of hybrids with breakpoints in the 3p14.2 region. These breakpoints were localized to two distinct clusters, separated by 200 kb, that lie on either side of a region of frequent breakage within FRA3B as observed by FISH analysis. Seven proximal aphidicolin-induced breakpoints were localized at or near the end of a THE element. The THE-1 element is flanked by LINE andAlurepetitive elements. The eight distal aphidicolin-induced breakpoints clustered in a region capable of forming multiple hairpin-like structures. Thus repetitive elements and hairpin-like structures may be responsible for chromosome fragility in this region.     

Fragile sites induced by FUdR,caffeine, and aphidicolin

Summary The frequencies of common fragile sites (c-fra) induced in peripheral blood lymphocytes by fluorodeoxyuridine (FUdR), aphidicolin, or caffeine, in eight healthy controls were studied. There was a significantly higher frequency of breaks (P<0.05) in the latter two treatments than the former. Also, significant variation in total number of breaks was observed among the eight individuals within the three treatments. The relative frequency of a fragile site in relation to the total number of fragile sites in an individual rather than its expression in total cells was considered important. Use of a frequency of 4% or more of total fragile sites was proposed to eliminate apparent random breaks that were observed. Using these criteria, a total of 31 c-fra were observed in the three treatments. The distribution of the fragile sites was different in FUdR-treated cells as opposed to caffeine- and aphidicolin-treated cells. Sites 3p14 and 16q23 and Xp22 were the three most frequently observed c-fra. The higher frequency of expression of some fragile sites in normal controls, as observed here, suggests that any relationship between fragile sites and neoplastic transformation has to be carefully evaluated. A classification based on frequency in the population, rather than mode of induction, is suggested.     

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.     

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.