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.     

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.     

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.     

Segregation analysis of autosomal fragile sites in three families with the fragile X chromosome.

Fragile sites on chromosomes 9, at 9p21, 10, at 10q25 and 12, at 12q24, were found in the lymphocytes of some members of three families during the study for detection of a fragile X chromosome. The sites were found to be heritable and folato-sensitive. The genetic implications of these results are discussed.     

Lead acetate and arsenic trioxide induce instability of microsatellites at three different fragile sites (6q21, 9q32-9q33 and 15p14) within the genome of the rat

Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.     

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.     

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.     

Synergistic effect of DAPI and thymidylate stress conditions on the induction of common fragile sites

When supplied to human leukocytes grown in complete medium (RPMI 1640), DAPI, a nonintercalating compound specific for the AT bases of DNA, induces the appearance of three common fragile sites (CFRA) mapped at 1q42, 2q31, and 7p22. The same treatment with DAPI in a medium deficient in folic acid and thymidine (199 M) considerably increases the expression of these sites and induces the appearance of a further 16 CFRA sites at 1q24, 2p25, 4p16, 4q25, 5p15.3, 6p21.3, 6p25, 6q13, 9p24, 16p13.3, 16q23, 17q21, 18q23, 20q13.1, 21q21, and Xq28. The results point to the existence of a synergism between DAPI and thymidylate-stress culture conditions in inducing site-specific chromosome damage. The results also agree with the hypothesis that DAPI-induced CFRA sites are DNA late-replicating chromosomal areas rich in AT bases.     

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.     

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.     

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.     

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.     

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.     

Heritable fragile sites on human chromosomes

Summary The chromosomal gaps associated with fragile sites at 2q11, 10q23, 11q13, 16q22, 20p11 and Xq27 do not stain with silver nitrate as do NOR regions of the acrocentric chromosomes.     

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.     

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.     

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.     

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.     

A male with two different familial autosomal fragile sites and a cytogenetically abnormal offspring

During the course of routine prenatal cytogenetics, a male with 2 different autosomal fragile sites (FS) was detected. The FS were at 9p21 and 12q13 and his sister also had both fragile sites, inherited from an obligate carrier father. He was the father of a foetus with an abnormal chromosome complement 46,XY/47,XY,+frag. The origin of the fragment could not be determined.     

How common are common fragile sites in humans: interindividual variation in the distribution of aphidicolin-induced fragile sites

To obtain an estimate of the variation in common fragile sites (CFSs) among individuals, aphidicolin (APC)-induced chromosomal breakage data were analyzed for 20 karyotypically normal adult humans. As it is specifically designed to meet the analytical requirements for considering fragile sites as presence/absence characters in single individuals, the FSM methodology (B?hm et al., 1995) was used to statistically distinguish fragile from nonfragile sites. These analyses indicated that the APC-induced fragile sites are not ubiquitous but vary extensively among individuals; the per-individual number of fragile sites ranged from as few as seven to as many as 20. Of the 45 different sites identified as fragile, 19 (42%) occurred in more than half of the individuals, but only two sites (3p14 and 16q23) were fragile in all of the individuals; 12 (27% of the total) were fragile in single individuals only. Although these analyses provide statistical confirmation (and initial estimates of population variation) for 43 of the 88 APC-inducible fragile sites currently recognized as occurring among humans, they are consistent with the hypothesis that many of the currently recognized human CFSs have been erroneously identified. These results indicate the need for per-individual statistical identification of CFSs for larger samples of individuals and that studies of particular fragile sites should be conducted on individuals documented to be fragile at the loci under consideration.