Folate-sensitive Fragile Sites Research Articles

Implications of FRA16A Structure for the Mechanism of Chromosomal Fragile Site Genesis

Fragile sites are chemically induced nonstaining gaps in chromosomes. Different fragile sites vary in frequency in the population and in the chemistry of their induction. DNA sequences encompassing and including the rare, autosomal, folate-sensitive fragile site, FRA16A, were isolated by positional cloning. The molecular basis of FRA16A was found to be expansion of a normally polymorphic p(CCG)n repeat. This repeat was adjacent to a CpG island that was methylated in fragile site-expressing individuals. The FRA16A locus in individuals who do not express the fragile site is not a site of DNA methylation (imprinting), which suggests that the methylation associated with fragile sites may be a consequence and not a cause of their genesis.

中華民國小兒科醫學會第137屆學術演講會教育演講：先天性畸形－易脆X染色體徵候群

The fragile X syndrome is the most common inherited form of mental retardation. second only to Down syndrome, which is usually sporadic and not inherited. The condition segregates a X-linked dominant disorder with incomplete penetrance. Fully penetrant males exhibit moderate mental retardation along with a phenotype consisting of macro-orchidism (enlarged testes), substle facial dysmorphia, and mild connective tissue abnormalities. Female patients typically are less severely affected, showing little or no somatic signs and only borderline to mild mental retardation. The prevalence of affected male has been estimated approximately 1/1250, while that of nonpenetrant males has been estimated as 1/5000, leading to an overall prevalence of about 1/1000 among males. Among female carriers, approximately onethird havc some evidence of mental impairment or mental retardation The population prevalence of affected females is approximately 1/2000. Thus the overall prevalence of female carriers is estimiated to be about 1/700. On the basis of these estimates, it apperas that about 1/850 people carry the fra (X) chromosome. The vast majority of patients with the fragile X syndrome show a folate-sensitive fragile site at Xq27.3 (FRAXA) at thc cytogenetic level, and both amplification of the p (CGG) n repeat and hypermethylation of the CpG island in the S fragile X genc (FMRI) at the molecular level. The absence of FMRI message and its encoded protein FMRP is believed responsible for the phenotype of the fragile X syndrome. In addition to the common mutation change of repeat expansion, three variant patients with the clinical presentation of fragile X syndrome have been reported: two males with large deletions encompassing the FMRI locus and a severely affected male with a FMRI, lie^367→Asn missense mutation. The dynamic mutations caused by expanding trinucleotide repeats turned to be common causes of human disease. In the past two years they have been fingered as the culprits in three hereditary disorders besides fragile X syndrome: myotonic dystrophy. spinaobulbar muscular atrophy and Huntinton's disease. All in all, The trinucleotide repeat expansions have shaken the field of genetics almost as much as did Barbara McClintock's jumping genes, or transposons.

Physical linkage of the fragile site FRA11B and a Jacobsen syndrome chromosome deletion breakpoint in 11q23.3.

Autosomal fragile sites, unlike their X-linked counterparts, are not known to be associated with disease. However, one case report has highlighted a possible relationship between the inheritance of a rare folate-sensitive fragile site in band 11q23.3 (FRA11B) and the chromosome 11q23-->qter deletion in Jacobsen (11q-) syndrome. The mother and brother of the reported Jacobsen syndrome child are FRA11B carriers, suggesting that in vivo breakage at the fragile site during early development could have given rise to the chromosome deletion. We have tested this hypothesis by high resolution physical mapping of FRA11B and of the deletion chromosome breakpoint in the Jacobsen syndrome patient. A detailed restriction map of 600 kb of human chromosome band 11q23.3 has been assembled which covers the PBGD, CBL2 and THY1 genes. FISH experiments with YACs and cosmids from this region have localised FRA11B to an interval of approximately 100 kb containing the 5' end of the CBL2 gene, which includes a CCG trinucleotide repeat. This class of repeat is expanded in the four cloned examples of fragile site and therefore the CBL2 repeat is a candidate for the location of FRA11B. Further, it is shown that the chromosomal deletion breakpoint of the Jacobsen syndrome child maps within the same interval as the fragile site. The breakpoint has apparently been repaired and stabilised by the de novo addition of a telomere. These data are consistent with a role for an inherited fragile site in the aetiology of a chromosome deletion syndrome.

Segregation analysis of rare autosomal folate sensitive fragile sites.

We have studied 12 families with rare autosomal folate sensitive fragile sites (RAFSFS). Of these, 9 were informative for segregation analysis of fragile sites in order to assess differences in parental transmission. We identified 20 families with RAFSFS from the literature from 1985 to 1989; thirteen of these were informative for segregation analysis. Segregation analysis confirmed that paternal fragile site transmission rates deviated significantly from the expected 50% for a Mendelian co-dominant trait. Sex ratio comparisons showed a significant excess of transmitting females and a significant excess of males among fragile site non-carriers from the literature families. Comparison of the fragile site carriers with non-carriers in the combined data showed a non-significant excess of non-carriers. We confirmed a deficiency of offspring expressing fragile sites when transmission was through fathers, suggesting gametic selection or the phenomenon of parental genomic imprinting.

Bromodeoxyuridine induces chromosomal fragile sited in the canine genome

Peripheral blood lymphocytes from 3 clinically normal domestic dogs were cultured for bromodeoxyuridine (BrdU) induction of fragile site expression. BrdU induced fragile site expression in cells from all 3 dogs. The mean percent of cells with fragile sites and the mean number of fragile sites per cell were significantly increased in all BrdU incubated cultures compared to control cultures. The frequency of BrdU fragile site expression did not vary significantly among the dogs. Lymphocytes from all 3 dogs expressed BrdU induced autosomal fragile sites. Two BrdU induced fragile sites were identified on the long arm of chromosome 1, one of which was close to or coincident with a previously identified folate sensitive fragile site on this canine chromosome. Lymphocytes from the 2 female dogs also expressed BrdU induced fragile sites on the X chromosome, but BrdU failed to induce fragile sites on the X chromosome from the one male dog in the study. The 2 BrdU-induced fragile sites identified on the long arm of the X chromosome were close to, or coincident with 2 previously described folate-sensitive common fragile sites on the canine X chromosome. This is the first report of induction of BrdU-inducible fragile sites in the genome of the domestic dog.

Folate-sensitive and aphidicolin-inducible fragile sites are expressed in the genome of the domestic cat

Peripheral blood lymphocytes from three clinically normal domestic cats were cultured for folate-sensitive and aphidicolin-inducible fragile site expression. Induction of folate-sensitive fragile sites was accomplished by culturing cells with trimethoprim plus caffeine. Chromosomes from all cats expressed both folate-sensitive and aphidicolin-inducible breaks and gaps. There were no significant differences between the two methods of fragile site induction in the percentage of cells expressing chromosome breaks and gaps or the mean number of breaks and gaps per cell. All three cats expressed specific chromosome breaks resembling fragile sites at A1q21–22, A1p22, and B1q32. All three sites were induced by aphidicolin. The sites at A1q21–22 and B1q32 were also induced in folate-deficient medium. This is the first report of the induction of chromosomal fragile sites in a feline species.

Expressivity of a common fragile site, fra(3)(p14.2), in patients with cancer and other diseases

A population survey of a common folate-sensitive fragile site, fra(3)(p14.2), has been carried out on PHA-stimulated peripheral lymphocytes of patients with cancer and other diseases, under both culture conditions of folate deprivation and aphidicolin treatment. Overall findings regarding variability of expressivity due to age and sex were very similar to those obtained in a healthy population. The expression of fra(3)(p14.2) by folate deficient condition appeared hardly influenced by such exogenous factors as tobacco smoking habit, past histories of radiotherapy and chemotherapy, while it was associated with the unfavorable prognosis of cancers. Furthermore, proportion of those with higher expression was slightly but significantly larger in both lung and breast cancer patients. These findings suggest that some factors relevant to the expression of fra(3)(p14.2) may be associated with development and progression of certain kinds of cancer.

Experience with direct molecular diagnosis of fragile X.

The utility of the pfxa3 probe for direct molecular diagnosis of the fragile X (FRAXA) has been established. This probe detects amplification of an unstable DNA element consisting of variable length CCG repeats. The size of the amplified fragment is correlated with phenotype and was determined using PstI digested DNA in family members. In 35 families with the fragile X, there was correspondence in 183 cases between the presence of an amplified unstable element and the presence of the fragile X chromosome independently determined by cytogenetics, position in the pedigree, or linked DNA markers flanking the fragile X. There was also correspondence in 124 cases between the presence of the normal 1.0 kb PstI fragment and absence of the fragile X chromosome independently determined by linked flanking markers. Six additional families considered to be isolated cases of 'fragile X' had been diagnosed before recognition of FRAXD. The pfxa3 probe confirmed the cytogenetic diagnosis in three families, the other three being rediagnosed as non-fragile X. A further two families had consistent expression of a different folate sensitive fragile site, FRAXE, close to FRAXA but not associated with fragile X syndrome and not detectable with the pfxa3 probe. Subsequent referrals were received from additional family members or from members of new families for whom carrier status had not been predetermined by linked markers. Direct pfxa3 diagnosis for the 135 females within these 222 additional cases was confirmed by dosage analysis with the control probe pS8.(ABSTRACT TRUNCATED AT 250 WORDS)

FRA2B is distinct from inverted telomere repeat arrays at 2q13

Human chromosome 2 was formed by a telomere-to-telomere fusion of two ancestral ape chromosomes. The fusion point is localized in chromosomal band 2q13, which also contains the rare, folate-sensitive fragile site FRA2B. It has been hypothesized that this fragile site may be related to the presence of interstitial telomeric and subtelomeric sequences, which have come to lie in an inverted repeat arrangement as a result of the fusion event. Fluorescence in situ hybridization of a genomic cosmid c8.1, which spans the fusion point, was carried out on metaphase spreads of an individual who expressed the fragile site at 2q13. We show that the fusion point maps distal to this fragile site. Therefore, we conclude that the inverted arrays of telomeric and subtelomeric sequences found at this fusion point are unlikely to correspond to the rare fragile site at 2q13.

Animal model: Chromosomal fragile site expression in dogs: I. Breed specific differences

Peripheral blood lymphocytes from clinically normal Doberman pinscher and boxer dogs were cultured for folate-sensitive and, in preliminary studies, aphidicolin-inducible fragile site expression. Both autosomal and X chromosomal fragile sites were observed in canine cells cultured under folate/thymidine depletion and in cells cultured in medium containing aphidicolin. Results from the three dogs evaluated for both folate-sensitive and aphidicolin-inducible fragile site expression showed that the frequency of fragile site expression was significantly (P less than 0.05) greater in cells cultured in medium containing aphidicolin than in cells cultured in folate/thymidine-depleted medium. Cells from the boxer dog expressed a high percentage (66.67%) of aphidicolin-inducible fragile sites in contrast to the Doberman pinscher dog in which only 21.10% of the lymphocytes expressed aphidicolin-inducible fragile sites. The frequencies of spontaneous and folate-sensitive fragile site expression did not vary significantly by breed of dog. Age of dog was significantly and positively correlated with frequency of folate-sensitive fragile site expression in dogs of the boxer breed, but not in dogs of the Doberman pinscher breed. The dog X chromosome expressed three folate-sensitive and aphidicolin-inducible fragile sites. The G-band location of these three fragile sites showed homology with three recognized constitutive common fragile sites on the human X chromosome: Xp22, Xq21, and Xq27.2. Two specific autosomal fragile sites were identified, one on the distal end of the long arm of chromosome 1 and one on the distal end of the long arm of chromosome 8. Other autosomal fragile sites were also apparent but could not be assigned reliably to specific chromosomes.

A new folate sensitive fragile site at 1p21.3.

A new folate sensitive fragile site at 1p21.3.

Autosomal folate sensitive fragile sites in normal and mentally retarded individuals in Greece

The frequencies of autosomal folate sensitive fragile sites were compared in populations of mentally retarded fra(X) negative (N = 220) and normal children (N = 76) in Greece. In addition, the frequency of autosomal fragile sites was studied in 20 known fra(X) children in order to test if the fra(X) syndrome is associated with general chromosome instability. The frequencies of both common and rare autosomal fragile sites did not differ significantly between the mentally retarded and the normal children, although the rate of expression was considerably higher in the retarded group. Autosomal fragile sites were not increased in the fra(X) patients. Fra(3)(p14) was by far the most frequent one in all groups. The frequency of fra(6)(q26) was found to be considerably higher among the mentally retarded children, this difference being almost statistically significant. Further cytogenetic studies of normal and retarded individuals are required in order to elucidate this point further.

Telomeric associations involving Yq12 in a lymphoblastoid cell line

A number of apparent telomeric associations/fusions (TAs) involving Yq12 were observed in a lymphoblastoid cell line (GM6892A) established from a fragile-X-negative, phenotypically normal male with the fragile X gene mutation. The TAs were seen when these cells were grown for an extended period of time in medium 199, which is deficient in thymidine and folic acid. Because the low thymidine and folic acid condition of medium 199 is known to induce chromosome and chromatid gaps and breaks at folate-sensitive fragile sites, other fragile site-induction regimes were examined to determine if the TAs seen in GM6892A were due to a fragile site in the Yq12 band. No TAs were seen with any of the other regimes that were tried.

A population cytogenetic study of a common fragile site, fra(3)(p14), in a healthy population

A population survey of a common folate-sensitive fragile site, fra(3)(p14), was carried out on PHA-stimulated peripheral lymphocytes of 1,078 healthy subjects. Fra(3)(p14) was expressed more frequently in the younger than in the older, and in males than in females. It also showed some seasonal variation. The age difference of the expression frequency was also observed by aphidicolin treatment. A positive correlation of the expression frequency was found, though not so strongly, between both culture conditions of folate deprivation and aphidicolin treatment, These findings suggest that the inter-individual variation in the expressivity of common fragile sites is not only ascribable to chance, but to some physiological conditions of blood donors such as response rate of lymphocyte to PHA stimulation and blood concentration of folic acid.

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

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.

Investigation of the effect of hydrogen peroxide on the chromosomes of young and elderly individuals

Active oxygen species have been considered to be responsible for the aging process and for the induction and initiation of neoplastic processes. The effect of hydrogen peroxide, an active oxygen species, was investigated in the chromosomes of three young women (20–21 years of age) and of three elderly women (73–79 years of age) in a culture medium favorable to the appearance of folate-sensitive fragile sites. Hydrogen peroxide at a final concentration of 5 × 10 6 during the final hours of culture caused a significant increase in hypodiploidy and structural aberrations, chromatid gaps in particular, only in the cultures from the three elderly women, suggesting that the chromosomes of older women are more sensitive to this agent than those of younger women. The preferential chromosome loss in both treated and untreated cultures from the elderly women involved chromosome X. The preferential sites for structural aberrations were 9p12, a constitutive heterochromatin site and 6q21, where the gene of mitochondrial superoxide dismutase, an enzyme involved in antioxidant processes in the cell, is located. Hydrogen peroxide significantly intensified the effect naturally occurring in the cells of elderly persons, such as hypodiploidy and increased structural aberrations, thus acting at the chromosome level in a manner similar to that of the natural aging process of the organism.

Possible association of rare autosomal folate sensitive fragile sites and idiopathic mental retardation: a blind controlled population study

The expression of folate sensitive fragile sites (FS) was assessed in cord blood lymphocyte cultures obtained from 790 newborns (NB) and in peripheral blood lymphocyte cultures from 326 institutionalized mentally retarded residents (MR). The mean rate of expression of common FS and the occurrence of rare FS was significantly higher in the MR population. Age, sex and history of chronic medication use did not appear to influence common FS expression in the MR population, 3/790 (0.38%) NB and 5/326 (1.53%) MR exhibited rare autosomal folate sensitive FS, a 4-fold difference in incidence (P = 0.009, Poisson test). Four of the five MR who expressed rare FS were considered to have idiopathic MR (4/179 or 2.2%). The occurrence of rare FS in 1/147 (0.68%) MR with known etiology is not significantly different from the frequency of occurrence in the NB population (P = 0.428, Poisson test). In this population, rare FS appear to be overrepresented in the idiopathic etiology MR group.

A cytogenetic study of male breast cancer

In a direct preparation from a male breast carcinoma two populations of cells were present, one hypodiploid (range 25–34) and the other hypertriploid (range 56–84). Twenty-two marker chromosomes were recognized. One of these, dic(5;11)(p14;q23) was present in one or two coples in every cell and has not been reported in any other case of breast cancer. There was a consistent monosomy of chromosome 7 and, in the hypertriploid cells, a gain of one to three copies of chromosome 3. The breakpoint 11q23 in a rare, folate-sensitive fragile site but was not expressed in peripheral blood cell lymphocytes from the patient.

Folate-sensitive fragile sites in Chinese hamster cell lines

The expression of fragile sites in three different Chinese hamster cell lines was studied. Results showed that folate-sensitive fragile sites were expressed in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 7 and in band 1q22. A comparison of the breakpoints involved in formation of chromosome rearrangements in some established Chinese hamster cell lines was also made. Results showed that while the specific type of rearrangement was random, the breakpoints were not. Three of the chromosomal sites most frequently involved in breaks were regions in which fragile sites were expressed.

Rapid detection of chromosome 16 inversion in acute nonlymphocytic leukemia, subtype M4: regional localization of the breakpoint in 16p

The pericentric inversion of chromosome 16 characteristic for acute nonlymphocytic leukemia, subtype M4, was detected in five patients by means of nonradioactive in situ hybridization of complete cosmids. First, five cosmids situated along the short arm of chromosome 16 were used to map the breakpoint of the inversion distal to the rare folate-sensitive fragile site FRA16A. Then, the use of two cosmids on either side of the breakpoint, combined with a probe specific for the centromeric region of chromosome 16, readily detected the inversion, even in poor metaphase spreads.