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.     

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.     

Chromosomal fragile sites in schizophrenic patients

Schizophrenia is a common and complex mental disorder. Cytogenetic and molecular studies have shown that genetic factors play an important role in the etiology of schizophrenia. As a preliminary step in the search for chromosomal location of a susceptible gene predisposing to schizophrenia, cytogenetic screening of patients might be useful. Therefore, this report is aimed at studying the relationship between chromosomal fragile sites (FS) (gaps, breaks, triradial figures, and several rearrangements) and the etiology of schizophrenia. Because of this, we compared the frequencies of folate-sensitive FS from schizophrenic patients and normal individuals in short-term whole-blood cultures. The rate of FS expression in the patients was considerably higher than in the controls. We determined 15 common FS (cFS) (1q21, 1q32, 2q21, 2q31, 3p14, 4q31, 5q31, 6q21, 6q26, 7q22, 7q32, 10q22, 13q32, Xp22, and Xq22), six rare FS (rFS) (6p21, 8q22, 11q23, 12q24, 16q22, and Xq26), and two previously unknown FS (3p25 and 5q22). Among these expressed FS, there was a significantly higher frequency of 12 FS at 2q31, 3p25, 3p14, 5q31, 6q21, 7q22, 7q32, 10q22, 11q23, 12q24, Xq22, and Xq26 in patient group than in controls by x ²-test (P between 0.0001 to 0.036). Sites 3p14, 5q31, and 7q22 were also the most frequently observed cFS. Males exhibited twice as many FS as females, but no age effects were observed. The potential relationship between increased FS frequency and the occurrence of schizophrenia in these patients is discussed. The text was submitted by the authors in English.     

Sister chromatid exchange in immature haemopoietic cells,T-and B-lymphocytes

Summary Human lymphocytes from 18 healthy adults were cultured in minimal essential medium without folic acid. The addition of 4mM cytidine or 1 mM guanosine to cultures 24h prior to harvest produced a statistically significant increase in hot point 3p14 breaks. An excess of 3mM adenosine or thymidine had no such effect on the hot point. The mechanism of the effects of nucleosides on hot points was discussed.     

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.     

Family study of common fragile sites

Summary The frequency of folate-sensitive common fragile sites (1p31, 1q44, 3p14, 3q26.2, 6q26, 16q23, Xp22.3) was determined in 19 healthy individuals from four families. The individuals consisted of 12 males and 7 females from 1 to 59 years of age. The frequency showed intrafamilial variation, but we were unable to demonstrate that the frequency was inherited in a Mendelian codominant fashion. In eight subjects whose chromosome 3 homologues could be distinguished by Q-band polymorphism, breakages at 3p14 occurred with equal frequencies on the homologues. Our study suggests that common fragile sites are a part of normal chromosome structure, and the frequency of their expression largely depends on environmental factors.     

Chromosomal fragile sites in schizophrenic patients

Schizophrenia is a common and complex mental disorder. Cytogenetic and molecular studies have shown that genetic factors play an important role in the etiology of schizophrenia. As a preliminary step in the search for chromosomal location of a susceptible gene predisposing to schizophrenia, cytogenetic screening patients might be useful. Therefore, this report is aimed at studying the relationship between chromosomal fragile sites (FS: gaps, breaks, triradial figures, and several rearrangements) and the etiology of schizophrenia. Because of this, we were compared the frequencies of folate-sensitive FS from schizophrenic patients and normal individuals in short-term whole blood cultures. The rate of FS expression in the patients was considerably higher than in the controls. We determined 15 common FS (cFS) (1q21, 1q32, 2q21, 2q31, 3p14, 4q31, 5q31, 6q21, 6q26, 7q22, 7q32, 10q22, 13q32, Xp22 and Xq22), 6 rare FS (rFS) (6p21, 8q22, 11q23, 12q24, 16q22, and Xq26) and 2 previously unknown FS (3p25 and 5q22). Among these expressed FS, there was a significantly higher frequency of 12 FS at 2q31, 3p25, 3p14, 5q31, 6q21, 7q22, 7q32, 10q22, 11q23, 12q24, Xq22 and Xq26 in patient group than in controls by chi2 test (P = between 0.0001 to 0.036). Sites 3p14, 5q31 and 7q22 were also the most frequently observed cFS. Males exhibited twice as many FS as females, but no age effects were observed. The potential relationship between increased FS frequency and the occurrence of schizophrenia in these patients is discussed.     

Synergistic effect of aphidicolin and ethanol on the induction of common fragile sites

Summary The effect of ethanol on the frequency of aphidicolin-induced common fragile sites was studied using lymphocyte cultures from two normal women. Aphidicolin was added to the cultures at a final concentration of 0.2 M and ethanol at 0.02%, 0.1%, 0.2%, 0.5%, and 1%, both during the last 26 h of culture. The frequency of common fragile sites increased from 296% in subject 1 and 201% in subject 2 with aphidicolin plus 0.02% ethanol, to 765% and 823%, respectively, with aphidicolin plus 1% ethanol. Ethanol alone added to cultures did not induce common fragile sites. The gaps and breaks induced by aphidicolin plus ethanol were highly nonrandom. Altogether, 35 common fragile sites were identified. The addition of 1% ethanol to aphidicolin increased both random and nonrandom gaps and breaks as compared with that of 0.02% ethanol. Dimethyl sulfoxide added to culture at final concentrations of 0.02% to 1% did not change the frequency of aphidicolin-induced fragile sites. The frequency of fluorodeoxyuridine-induced fragile sites was not affected by the addition of 0.02% to 1% ethanol. It was thus concluded that ethanol enhances the aphidicolin-induced fragile sites, possibly inhibiting the repair mechanism of gaps and breaks induced by aphidicolin.     

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.     

Distribution of spontaneous chromosome breaks in human chromosomes

Summary Localization of chromosome breaks in human chromosomes was analyzed in 264 peripheral lymphocyte cultures. Three hundred and sixty-nine chromosome breaks could be exactly localized to a chromosome band or region of the Paris Conference nomenclature. The distribution of breaks in the chromosome regions was found to be nonrandom. Chromosome 3 alone had 23% of the breaks and region 3p2 had 13% of the total breaks. Some other chromosome regions, such as 5p1, 9q1, 14q2, and 16q2 also displayed clustering of breaks. Sex chromosomes had less breaks than expected. Spontaneous chromosome breaks were almost exclusively located in the lightly stained G bands.Supported by grants from the Foundation for Pediatric Research and Research Foundation of Orion Corporation Ltd.     

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.     

DAPI-inducible common fragile sites

DAPI, a compound specific for the AT bases of DNA, causes gaps and breaks in three human chromosome sites, at the 1q41-1q42 interface, 2q31, and 7p22. It also induces undercondensation of a chromosome site at the 13q21-13q22 interface. The first three sites have the characteristics of "common fragile sites" and are present as gaps and breaks on the chromosomes of seven individuals.     

DNA polymerase α inhibition by aphidicolin induces gaps and breaks at common fragile sites in human chromosomes

Summary Aphidicolin, a specific inhibitor of DNA polymerase , is known to induce chromosomal aberrations. At concentrations that did not greatly affect mitotic index, aphidicolin induced a striking number of chromosome gaps and breaks distributed in a highly nonrandom manner in cultured human lymphocytes. Specific chromosome bands, especially 2q31, 3p14, 6q26, 7q32, 16q23, and Xp22 were preferentially damaged in lymphocytes from each of 12 subjects studied. Total and site-specific damage was dose dependent and greatly increased when folic acid was removed from the medium. The sites most sensitive to aphidicolin damage include the hot spots seen under conditions of thymidylate stress and in studies of spontaneous chromosomal damage. The fragile X site, which can also be induced by thymidylate stress, was not induced by aphidicolin in lymphocytes, suggesting a separate mechanism for its induction. Aphidicolin represents a novel tool for detection of hot spots on human chromosomes through the mechanism of DNA polymerase inhibition. The hot spots induced by aphidicolin represent a new class of fragile sites which we term common fragile sites.     

45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp

Background

In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported.

Methods and Results

During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH) using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions) at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region.

Conclusions

The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.     

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.     

Chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis)

The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.     

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.     

Population cytogenetics of aphidicolin-induced fragile sites

Summary Chromosome fragile sites are inducible by aphidicolin in cultured human lymphocytes. To assess the frequency and distribution of these common fragile sites in the general population, a cytogenetic survey was performed on 126 subjects, 59 males and 67 females, whose age ranged from 1 day to 72 years. Common fragile sites, induced by aphidicolin, were widespread and showed a remarkably different sensitivity among individuals; age influenced the overall frequency of fragile sites. Moreover, both age and sex seemed to modulate the expression of specific fragile sites. In our population, the most common fragile sites were: 3p14, 16q23, Xp22, 6q26, 1p31, 4q31, 1p22, 7q22, 2q33, 3q27, 2q31, 7q32, 14q24, 10q22, 5q31, 2q37, 6p21.     

Frequency and distribution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced structural chromosome aberrations in fibroblasts from sarcoma and non-Hodgkin's lymphoma patients

The frequency and distribution of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced structural aberrations, i.e., chromatid and chromosome gaps, breaks, and exchanges, were studied in fibroblasts from 16 patients with sarcoma, 15 with non-Hodgkin's lymphoma (NHL), and 14 controls. The mean frequencies of aberrant cells, and gap, break, and gap + break events per 100 cells were 22.9, 5.1, 28.6, and 33.7 in the sarcoma group; 19.1, 5.0, 22.5, and 27.5 in the NHL group; and 23.5, 6.1, 33.5, and 39.6 in the control group. None of the differences between the groups were statistically significant. The distribution of MNNG-induced aberrations was non-random (P less than 0.001) in all 3 groups. Eight, 11, and 17 chromosome bands in the sarcoma, NHL, and control groups, respectively, were particularly break-prone. Only 2 hot spots in the sarcoma group (1p32, 11q23), and 3 in the NHL group (1p36, 3q25, 6p21), coincided with the 25 and 60 bands known to be involved in primary rearrangements in sarcomas and NHL. We conclude that neither the frequency nor the distribution of MNNG-induced chromosomal aberrations indicates any latent chromosomal instability in sarcoma and NHL patients.     

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.