Manifestation of the fragile site Xq27 in fibroblasts

Summary Fibroblasts from a heterozygous carrier for the Martin-Bell syndrome, who manifests the fragile site Xq27, were cloned to separate the population carrying the primary defect on the active X chromosome from the population with this defect on the inactive X. Clones with this defect on the active X manifest the fra(X)(q27) whereas clones from the other population are fra(X)-negative (Steinbach et al. 1983b). In this project, the replication status of the X chromosome manifesting the fra(X)(q27) was studied in seven clones with this defect on the active X.The results obtained on the clones were very similar to the results obtained from uncloned fibroblasts and lymphocytes. In the clones the fragile site was found manifested on the early replicating X in 73 cells and on the late replicating X in four cells.Since the defect is located on the active X chromosome of these cells the manifestation of the fragile site on the late replicating X suggests that the defect and the fragile site cannot be identical. It is concluded that there is no obligate synteny of this defect and the manifested fragile site.     

Heterozygous female carriers of the marker-X-chromosome: IQ estimation and replication status of fra(X)(q)

Summary The IQ levels of 18 female carriers with the marker X chromosome were evaluated, and cytogenetic studies after BrdU incorporation were performed. A highly significant correlation between mental capacity and replication pattern of the X chromosomes could be demonstrated. Heterozygous females with normal intelligence showed a clear tendency to carry the fragile site at the late replicating X chromosome, while other female carriers with lower intelligence or mental impairment expressed their fragile site mainly with the early replicating X chromosome. This observation could be interpreted as an expression of Lyonisation.     

Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome

The cytogenetic study of 182 river buffalo (Bubalus bubalis L., 2n = 50) of Murrah, Mediterranean and Jaffarabadi breeds, from the State of S?o Paulo, was carried out to characterize their chromosomes and to detect possible chromosomal abnormalities. The karyotypes were indistinguishable with conventional staining as well as with C and replication R banding techniques. In about 44% of the sample (8 males and 72 females), an X marker chromosome due to a fragile site was shown. The frequency of metaphases expressing the fragility site on the X was highly variable, from 2.86 to 41.03%. In females, the fragile site, rarely appeared on both X chromosomes. Most of the metaphases showed only 1 marker chromosome. In R-banded metaphases using 5-bromodeoxyuridine (BrdU) treatment, it corresponded in general to the late replicating X chromosome. No correlation between the X fragile site and altered phenotype was found. Structural and numerical chromosome rearrangements were ruled out in the present sample of buffalo.     

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.     

Replication status of the fragile X chromosome,fra(X)(q27), in three heterozygous females

Summary Investigation of lymphocyte cultures from three females heterozygous for fra(X)(q27) shows widely differing proportions of early and late replicating X chromosomes having the fragile site, and suggests that the replication status of the fragile X may be related to the mental capacity of the patient. The study has utilised a sequential staining technique to reduce ascertainment bias, and evidence is presented to suggest that the expression of the fragile site is independent of the differential incorporation of BUdR into the early and late replicating X chromosomes.     

Replication pattern in XXY cells with fra(X)

Summary Clinical and cytogenetic aspects, including replication studies, of a Klinefelter's patient with fra(X) are reported. In the majority of metaphases the fragile site was observed at the early replicating X chromosome.     

Frequency of the fragile X syndrome in institutionalized mentally retarded females in Japan

Summary The fragile X [fra(X)] syndrome was screened on 190 Japanese institutionalized females with moderate to severe mental retardation. Two inmates with severe mental retardation (IQ 20) had the fra(X) chromosome in 26% and 15% of the cells examined, indicating that the prevalence of the fra(X) syndrome was about 1% in all female inmates and was about 3.27% in severely mentally retarded females with known causes. However, no female with fra(X) syndrome was found in 35 moderately retarded females. Both had brothers with the fra(X) syndrome and the prevalence was 10% in females with a family history of mental retardation. In addition, the replication study of the fra(X) chromosome in the patients supported the proposal that an excess of the early replicated fra(X) chromosome is related to the mental capacity in heterozygous females. Therefore, the fra(X) syndrome should not be ignored even in severely mentally retarded females with a family history, though the heterozygotes are commonly normal to subnormal in their mental development. in addition, the replication study of the fra(X) chromosome may help to estimate mental development in the carrier children.     

Replication patterns of the fragile X in heterozygous carriers: analysis by a BrdUrd antibody method.

The replication status of the fragile X chromosomes was studied in short-term cultures of lymphocytes from six female heterozygous carriers. The fragile X was induced by adding 0.1 microM fluorodeoxyuridine during the last 24 h of culturing. The replication status of the X chromosomes was studied using a bromodeoxyuridine (BrdUrd) antibody method. BrdUrd was added (1) at a final concentration of 0.2 micrograms/ml during the early S phase of chromosome replication (16-10 h before harvest), (2) at 0.2 microgram/ml during the late S phase (the last 6 h of culturing), (3) at 20 micrograms/ml during the early S phase, and (4) at 20 micrograms/ml during the late S phase. BrdUrd that was incorporated into replicating chromosomes was detected by using a nuclease and BrdUrd monoclonal antibody. The frequency of the fragile X was reduced by BrdUrd treatment. The degree of reduction was more severe in the 20 micrograms/ml than in the 0.2 microgram/ml series and was more severe with late S than with early S treatment. Of the early- and late-replicating fragile X chromosomes, those which were actively replicating during a BrdUrd treatment were more reduced than the others. Thus, the average rate of early and late S treatment with 0.2 microgram BrdUrd/ml was assumed to be the closest reflection of the situation in vivo. There was no correlation between the average rate of the early replicating, active fragile X and the intelligence of the heterozygous carriers studied.     

Late replication patterns in adult and embryonic mice carrying Searle's X-autosome translocation

Bromodeoxyuridine-dye technique analysis of X chromosome DNA synthesis in female adult and fetal mice carrying the balanced form of the T(X; 16) 16H translocation demonstrated that the structurally normal X chromosome was late replicating (and hence presumably inactive) in 93% of the adult cells and 99% of the 9-day embryo cells, with the X¹⁶ chromosome late replicating in the remaining cells. We conclude from these results that in T16H/+ females either there is preferential inactivation of the normal X chromosome or that, if inactivation is random, cell selection takes place before 9 days of development. Two 9-day female embryos with an unbalanced karyotype were also studied; both had two late-replicating chromosomes in most of their cells, one being the chromosome 16^X, the other a normal X chromosome. These results, together with the presence of a late-replicating X¹⁶ chromosome in T16H/+ adult and fetal mice, support the concept that more than one inactivation center is present on the X chromosome of the mouse because the X¹⁶ and the 16^x chromosomes can be late replicating.     

Activity of the fragile X in heterozygous carriers

Chromosome analyses with conventional stain, Q- and G-banding, and R-banding with 5-bromodeoxyuridine (BrdU) incorporation were performed on the lymphocytes of two sisters who are heterozygous for the fragile X chromosome and clinically diagnosed as slow learners. Two heterozygous relatives with normal intelligence were used as controls. The frequencies of the active fragile X for the "slow" females were 100/129 (77.5%) and 85/120 (70.8%) compared with 40/78 (51.3%) and 10/32 (31.3%) for controls, the difference being highly significant. These observations are consistent with the Lyon hypothesis: activity of the abnormal X could account for the reduction in mental ability of some heterozygous females. Similar to retarded males with the fragile X chromosome, our slow learners had verbal scores that were lower than performance scores.     

Cell fusion-induced quick change in replication time of the inactive mouse X chromosome: an implication for the maintenance mechanism of late replication.

It is unknown how and why the genetically inactivated mammalian X chromosome replicates late in S phase. There are also occasional inactive X chromosomes characterized by an opposite behavior replicating early in S phase. Two clonal cell lines, MTLB3 and MTLH8, isolated from a cultured murine T-cell lymphoma have an allocyclic X chromosome of the latter type. This precociously replicating X chromosome was judged to be genetically inactive as the late replicating one. Immediately after fusion with another cell line, the precociously replicating X chromosome from these cells starts to replicate late in S phase. This finding seems to suggest that late replication characterizing the inactive X chromosome is actively maintained by a trans-acting factor in female somatic cells, and that its lack entails a switch from late replication to precocious replication. It remains unknown whether this presumptive factor also modifies the autosomal replication pattern.     

Parental inheritance and psychological disability in fragile X females.

Studies of adult female carriers of the fragile X chromosome indicate that certain psychological problems occur with a greater frequency and severity than expected. This study examines the association of parental origin of the fragile X chromosome and of fragility detected in the karyotype with measures of social, educational, and psychological functioning in a group of adult fragile X females of normal intelligence. The results show that, as a group, women who inherit the fragile X chromosome from their mother and who demonstrate positive fragility in the karyotype (MI+ group = [maternal inheritance with positive fragility]) manifest significantly more impairment of social, educational, and psychological functioning when compared with fragile X females with paternal inheritance or negative fragility or with a matched control group comprising non-fragile X women. In particular, MI+ women show lower levels of both educational achievement and socioeconomic status and a greater degree of disturbance in communication, socialization, affect, and thought processes. These clinical findings are consistent with the recently advanced hypothesis which proposes that a two-stage process leading to chromosome imprinting in a preoogonial cell causes the fragile X syndrome.     

Fragile (X) expression: Relationship to the cell cycle

Summary The fragile (X) chromosome demonstrable in individuals with one type of X-linked mental retardation is seldom, if ever, seen in more than 50% of cells of affected individuals. We have devised a model to explain this apparent 50% maximum, one essential feature of which is that the fragile (X) will not be seen in cells in their first division in thymidine-depleted media. The validity of our model was tested on lymphoblastoid cell lines from affected males by treating the cells with fluorodeoxyuridine (FUdR) to induce the marker and/or bromodeoxyuridine (BrdU) to determine the cell cycle. We have evidence that the fragile (X) is present in cells in the first and subsequent these observations our model is not valid and the 50% expression of the fragile site at Xq(28) and other unusual properties of this region of the X chromosome remain unexplained.This work was supported by Grant HD 07879 from the National Institutes of Health     

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).     

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.     

Variability in the expression of the fragile site of the (fra)X chromosome in 2 consecutive cell cycles

The number and morphology of X chromosomes were analysed in tetraploid cells induced with colcemid in cultured blood lymphocytes obtained from a patient with fra(X) syndrome of mental retardation. In contrast to diploid cells containing fra(X) chromosome in 22.7% of cells, the marker X was found in 51.6% of tetraploids, each cell containing only one fragile X out of the two expected ones. The data obtained indicate an extreme lability of the expression of fragile site (X) (q 27) in consecutive lymphocyte generation.     

Late replicating X chromosomes in human triploidy.

The status of X-chromosome replication was studied in twenty-seven 69,XXY and nine 69,XXX human triploids in which the parental origin of the additional haploid set was known from the study of chromosome heteromorphisms. Among the 69,XXY triploids, fourteen had no late replicating X, two had one late replicating X in all cells examined, and eleven had two populations of cells, one with late replicating X chromosome, and one without any. Among the 69,XXX triploids, four had a single late replicating X, and five had two populations of cells, one with one late replicating X, and one with two late replicating X chromosomes. There was no correlation between the parental origin of the triploidy and the type of X-chromosome inactivation. However the number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue when compared with those grown from extra-embryonic tissue. In cultures derived from extra-embryonic tissue there was a significant correlation between the gestational age of the sample and the proportion of late replicating X chromosomes. The older the specimen, the greater the number of late replicating X chromosomes.     

Enhanced sensitivity of lymphoblastoid cells from individuals carrying the mutation for the fragile X syndrome to the clastogenic effects of FUdR

Individuals known to carry the mutation for the fragile X syndrome can sometimes be identified cytogenetically by the presence of a fragile site on the X chromosome at q27.3. The frequency of cells bearing this fragile site is known to be enhanced by culturing the cells in folic acid deficient medium and/or by introducing folic acid metabolism inhibitors such as FUdR. In this study FUdR induction of chromosomal aberrations other than the fragile X was investigated. Lymphoblastoid cells from an obligate carrier, a mentally retarded male and a control were cultured in folic acid deficient medium in the presence of FUdR and harvested at various times after culture initiation. The frequency of chromosome and chromatid breaks was found to be higher in cells from the individuals carrying the mutation for the fragile X syndrome. The frequency of micronuclei, an indirect index of chromosome breakage, was also more elevated in cells from these individuals than in cells from the control. These findings are of potential importance to carrier detection of this common genetic disorder.     

A recombinant X chromosome in a short statured girl resulting from a maternal pericentric inversion

Summary a 73/4-year-old girl with short stature was found to have a recombinant (X), dup q chromosome resulting from an apparently unique pericentric inversion (X)(p11.2q26) present in her mother and maternal grandmother. The recombinant X chromosome was shown to be late replicating and the inversion X chromosome to be randomly inactivated. This appears to be only the eighth report (7 female, 1 male) of a recombinant resulting from an X pericentric inversion despite all diagnosed females having mild clinical abnormalities. Reasons for the rarity of such recombinant X chromosomes in man are examined.     

Sex chromosome-autosome translocations in the leaf-nosed bats, family Phyllostomidae. I. Mitotic analyses of the subfamilies Stenodermatinae and Phyllostominae

Mitotic analyses using RBA- and C-banding were performed on Stenodermatine bats with X-autosome (XY1Y2) and X- and Y- autosome (neo-XY) translocations. RBA-banded metaphases of females revealed differential replication of the inactive X chromosome. An early replicating band comprises the short arm of the X, and an intermediate replicating band is located interstitially on the long arm. The early replicating short arm has a homologous counterpart either in the form of a free autosome (the Y2) or as part of the Y. Both the "autosomal" short arm of the X and its homologue fused to the Y are C-band negative and behave autonomously from the remainder of the sex chromosomes. They are separated from X and Y chromatin by centromeric heterochromatin which presumably acts as a barrier. The intermediate replicating region of the long arm of the X is also present in the subfamily Phyllostominae. In both subfamilies this region lacks a homologous counterpart. However, it may also represent a translocated autosome which, unlike the short arm of the X, is not separated from the inactive X by centromeric heterochromatin. Its intermediate replication time may represent a retarded replication due to its juxtaposition to late replicating X chromatin. These data are discussed in light of the theory of the evolution of sex chromosome heteromorphism, specifically as it applies to mammals.