Improved technique for the expression of fragile-X in cultured amniotic fluid cells

Summary An improved technique for inducing fra(X) expression in cultured cells was obtained by using diazepam for mitotic arrest and 5-fluorodeoxyuridine (FUdR) for the induction of fra(X) expression. The method was developed using cultured fibroblast and urinary cells from fra(X) patients. Prenatal studies were performed on cultured amniotic fluid cells in five pregnancies at risk for fra(X). In two cases the cultured cells showed a 46,XY, fra(X) karyotype. One of the pregnancies was terminated and the diagnosis was confirmed by chromosome studies on several fetal tissues including chorionic villi and by histopathologic changes in the lymphatic vessels of the fetal testes. The fra(X) was also demonstrated in chorionic villi in a case in which amniotic fluid cells were not studied. Chorionic villi were isolated after a spontaneous abortion, the cultured cells had a 45,X karyotype and in addition 5% of the cells were fra(X) positive.     

Chromosomal characteristics of X-linked recessive mental retardation. I. The X chromosome

The X-chromosome was studied in blood lymphocytes of 68 males with aspecific mental retardation (MR), their 57 relatives and 15 intellectually normal males. The incidence of a fragile X-chromosome (fra(X)) was found to be 4.7% in an unselected group of 42 patients, 50% among 10 probands in which pedigree data were suggestive of X-linked MR diagnosis, and 75% in the group of 15 patients selected for phenotype characteristic of the fragile X syndrome. The fra(X) was present in 1-43% of metaphases in different individuals, no such marker being observed in cells of 15 normal individuals. No significant difference was found when the incidence of the fra(X) was compared in cells cultured in the medium 199 with low folic acid content and the Eagle's medium supplemented with 5-fluorodeoxyuridine (10.62 +/- 2.94 SEM and 13.53 +/- 2.85 SEM, respectively). The possibility of false-positive diagnosis of the fragile X syndrome was quantitatively appreciated. A half of the patients showing a fra(C) in conventionally stained chromosomes were found to have fragile 6 autosome as the only marker in these cells, and in patients with the evident fragile (X) syndrome the fra(6) constituted about one-third of the fra(X) frequency. Both culture media employed were similar in the fra(6) induction.     

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.     

Fragile (X) X-linked mental retardation. II. Frequency and replication pattern of fragile (X)(q28) in heterozygotes

The frequency of cytologic expression and the replication pattern of the fragile (X) [fra(X)] were investigated in 28 fra(X) heterozygotes, of which 25 agreed to psychological assessment. One-third of the heterozygotes in this study are mentally retarded. The intellectually impaired carriers had a higher frequency of fra(X) and a higher proportion of early-replicating fra(X) than the normally intelligent carriers. The early-replicating fra(X) accounted for 39% of the variability in IQ and the late-replicating fra(X) for 12%. Age had a minimal inverse effect on fra(X) expression and replication pattern. Thus, it appears that mental retardation in females heterozygous for the fra(X) may largely be a function of the proportion of cells with an early-replicating, active X chromosome possessing the fragile site.     

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.     

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.     

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.     

Spontaneous and induced chromosome instability in patients with fragile X syndrome

Spontaneous and degranol- and dimatiph-induced chromosomal instability in the lymphocyte culture of patients with fra-X syndrome was investigated. The cultures contained TC 199 and 5% FC serum. It was found that the frequency of spontaneous chromosomal aberrations (CA) was 7.3% in cells from patients with fra(X), 3.9% in patients with MR of unknown origin, and 1.3% in normal individuals. Spontaneous break-points in the patients with fra(X) were localized in 1p, 2q, 3p, 6q, 7q, 16 q more often than in normal individuals. No significant difference was found in SCEs. The cells of patients with fra(X) were not sensitive to the induction of CA by degranol. It was found that chromosomal telomeric changes (CTC) were mutagen-independent, remaining at the spontaneous level: in the patients with fra(X) CTC were 10.5% (9.5% fra-Xq27, and 1% autosomal telomeric changes); in normal individuals CTC were 0.1%.     

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.     

Fragile (X) expression induced by FUdR is transient and inversely related to levels of thymidylate synthase activity.

Thymidylate synthase (TS) activity was monitored in fluorodeoxyuridine (FUdR)-treated lymphoblasts from individuals carrying the fragile (X) [fra(X)] chromosome. Fra(X) expression and levels of TS activity were measured over a 72-hr period at different cell densities. TS activity was 80%-90% inhibited immediately after exposure to FUdR and remained suppressed for the first 24 hrs. Fra(X) expression was not found until 6-8 hrs after FUdR treatment, and at 24 hrs, reached a maximum expression of approximately 50%. At 48 and 72 hrs, however, increasing levels of TS activity paralleled a dramatic drop in fra(X) expression. High fra(X) expression at 48 and 72 hrs could be maintained by rechallenging cultures with increasing doses of FUdR. At low cell densities, fra(X) expression was maintained at high levels for a much longer period of time. In two lymphoblastoid cell lines from obligate carriers, which either expressed at very low levels or did not express the fra(X) in routine cultures, TS activity was also 90% inhibited but with no corresponding fra(X) expression 12 or 24 hrs after FUdR treatment. We conclude that: FUdR inhibits TS activity immediately and induces fra(X) expression 6-8 hrs later, FUdR-induced fra(X) expression and TS activity are inversely related, the FUdR effect on fra(X) expression and TS activity is time and cell-density dependent, and inhibition of TS activity is a necessary but not sufficient condition for fra(X) expression.     

Low frequencies of apparently fragile X chromosomes in normal control cultures: a possible explanation

Low frequencies of apparently fragile X [fra(X)] chromosomes have been reported in normal control, short-term, whole blood cultures, and they have been noted in both amniocyte and fetal blood cultures. However, there is currently no universal agreement on the lowest frequency for fra(X)(q27) that is diagnostic for the fragile X syndrome. Here, we present our observations on low levels of apparently fra(X) chromosomes in normal samples. We observed frequencies of 0.5% in short-term whole blood cultures and 0.9% in amniotic fluid cell cultures. In 1982, Steinbach et al. described nonspecific telomeric structural changes (TSC) and suggested that such low frequencies of apparently fra(X) chromosomes in normal material may be occurring by the same mechanism that is responsible for TSC formation. To determine if TSC formation can explain the significant baseline frequencies of fra(X) in normal controls, 10,457 cells were screened from 178 individuals referred for fra(X) analysis. Our findings indicated that TSC are not randomly distributed across chromosomes but tend to occur at specific sites. Based on our observations, we offer the hypothesis that the low frequency of apparent fra(X) in normal individuals may be due to nonrandom TSC distribution.     

Neurobehavioral effects of the fragile X premutation in adult women: a controlled study.

Although previous studies have suggested that the fragile X premutation (fra [X] pM) does not cause deleterious effects, methodological constraints have prevented more definitive conclusions from being reached. In this report, we describe the neuropsychiatric and cognitive-neuropsychological status of 34 adult women with the fra (X) pM, as compared with a well-matched control group of 41 mothers of fra (X)-negative children with developmental disability. The results indicate that there are no meaningful differences between adult women with the fra (X) pM and control subjects with respect to cognitive abilities or profile, neuropsychological function, psychiatric diagnoses or symptoms, and self-rated personality profile. No measure for either group showed evidence of functioning outside the normal range except for a high lifetime prevalence of major depression in both groups. Additional exploratory analyses within the fra (X) group showed no significant effect of either the size of the fra (X) insert or X chromosome inactivation pattern in leukocytes, on any measure of neurobehavioral function. These findings provide additional information to professionals providing genetic counseling to, and assessment of, fra (X) families.     

Cytogenetic investigations in mentally retarded and normal males from 14 families with the fragile site at Xq28

Summary Lymphocyte cultures from 27 mentally retarded males aged 1 year to 77 years, and from 11 normal brothers from a total of 14 families with the fragile X segregating have been examined cytogenetically employing three different culture methods including methods for induction of fra(X) by FUdR (fluorodeoxyuridine) or MTX (methotrexate). All mentally retarded males showed unequivocal fra(X) expression. No statistically significant correlation between fra(X) expression and age could be demonstrated. No enhancement with FUdR was observed. Fibroblast cultures from 10 retarded males expressed fra(X) in a dose-response relationship to increasing concentrations of FUdR. None of the normal males showed fra(X). In vivo folic acid treatment of seven mentally retarded males resulted in marked reduction in fra(X) expression in lymphocyte cultures grown in medium 199. However, reinduction was achieved by FUdR or MTX, except in one case who temporarily received very high doses of folic acid.     

Neuroanatomy in fragile X females: the posterior fossa.

The relative homogeneity of the neuropsychiatric phenotype in individuals with fragile (fra) X syndrome suggests that there are consistent central nervous system (CNS) abnormalities underlying the observed cognitive and behavioral abnormalities. In this study, the neuroanatomy of the posterior fossa and other selected CNS regions in 12 young fra X females were compared with those of a group of 12 age-, sex-, and IQ-matched females without evidence of the fra X syndrome. Fra X females were shown to have decreased size of the posterior cerebellar vermis and increased size of the fourth ventricle, findings that are identical to those previously reported for fra X males. When compared with fra X male and nonfra X control groups, the distribution of the posterior-vermis and fourth-ventricle variables for the fra X female group was intermediate. These results support the hypothesis that the fra X genetic abnormality leads to hypoplasia of the posterior cerebellar vermis, a neuroanatomical variation of potential importance to both developmental and neuropsychiatric syndromes.     

Multilocus analysis of the fragile X syndrome

Summary A multilocus analysis of the fragile X (fra(X)) syndrome was conducted with 147 families. Two proximal loci, DXS51 and F9, and two distal loci, DXS52 and DXS15, were studied. Overall, the best multipoint distances were found to be DXS51-F9, 6.9%, F9-fra(X), 22.4%; fra(X)-DXS52, 12.7%; DXS52-DXS15, 2.2%. These distances can be used for multipoint mapping of new probes, carrier testing and counseling of fra(X) families. Consistent with several previous studies, the families as a whole showed genetic heterogeneity for linkage between F9 and fra(X).     

Further evidence for genetic heterogeneity in the fragile X syndrome

Summary The X-linked fragile X[fra(X)] syndrome, associated with a fragile site at Xq27.3, is the most common Mendeban inherited form of mental deficiency. Approximately 1 in 1060 males and 1 in 677 females carry the fra (X) chromosome. However, diagnosis of carrier status can be difficult since about 20% of males and 44% of females are nonpenetrant for mental impairment and/or expression of fra (X). We analyzed DNA from 327 individuals in 23 families segregating fra (X) for linkage to three flanking polymorphic probes: 52A, F9, and ST14. This allowed probable nonpenetrant, transmitting males and carrier females to be identified. A combined linkage analysis was conducted using these families and published probe information on F9 in 27 other families, 52A in six families, and ST14 in five families. The two-point recombination fraction for 52A-F9 was 0.13 (90% confidence interval, 0.10–0.16), for F9-fra(X) was 0.21 (0.17–0.24), and for fra(X)-ST14 was 0.12 (0.07–0.17). Tight linkage between F9 and fra(X) was observed in some families; in others loose linkage was seen suggesting genetic linkage heterogeneity. Risk analysis of carrier status using flanking DNA probes showed that probable nonpenetrant transmitting males were included in families showing both tight and loose linkage. Thus, in contrast to our previous conclusions, it appears that the presence or absence of nonpenetrant, transmitting males in a family is not an indicator of heterogeneity. To determine if heterogeneity was present, we employed the admixture test. Evidence for linkage heterogeneity between F9 and fra(X) was found, significant at P<0.0005. Nonsignificant heterogeneity was seen for 52A-F9 linkage. No heterogeneity was found for fra(X)-ST14. The frequency of fra(X) expression was significantly lower in families with tight F9-fra(X) linkage than in families with loose linkage. Cognition appeared to relate to linkage type: affected males in tight linkage families had higher IQs than those in loose linkage families. These findings of genetic heterogeneity can account in part for the high prevalence and apparent high new mutation rate of fra(X). They will affect genetic counseling using RFLPs. An understanding of the basis for genetic heterogeneity in fra(X) will help to clarify the nature of the unusual pattern of inheritance seen in this syndrome.     

The combined effects of FUdR addition and methionine depletion on the X-chromosome fragile site.

The fragile site on the X chromosome [fra(X)] associated with mental retardation is not normally seen in lymphocytes cultured in media containing folic acid or lacking methionine. The requirement for methionine has been taken to mean that amino acid metabolism or one-carbon transfer via S-adenosyl-methionine is most important in fra(X) expression. The inhibitory effect of folic acid can be overcome, as we have shown, by the addition of 5'-fluorodeoxyuridine (FUdR) to the culture medium, suggesting that depletion of dTMP available for DNA synthesis is most important in expression. We tested the combined effects of FUdR addition and methionine depletion on the fra(X). We found expression of the fra(X), indicating that methionine per se is not necessary for expression. Our work supports the suggestion that expression of the fra(X) is due to specific limitation of the dTMP pool available for DNA synthesis.     

Frequency of fragile X chromosomes,fra(X), in lymphocytes in relation to blood storage time and culture techniques

Summary The fra(X) frequencies in metaphases of lymphocytes from seven male patients with X-linked mental retardation and macroorchidism were scored after use of different culture techniques and different times of blood storage. No statistically significant differences were found between the fra(X) rates of lymphocytes grown either in folic acid deficient medium (TC 199) or in medium containing folic acid as well as a folic acid antagonist (methotrexate: MTX, aminopterine: AP). With respect to the effect of the time interval between blood sampling and culture set-up, a statistically significant decrease in the fra(X) frequency was observed in all culture types after 4 or 7 days of blood storage.     

Auditory brain-stem responses in the fragile X syndrome.

Auditory brain-stem responses (ABRs) were recorded from a group of 12 mentally retarded males with the fragile X (fra[X]). The responses were analyzed in terms of ABR thresholds, absolute latencies, and interpeak latencies. One patient had increased ABR thresholds, indicating hearing impairment. Five fra(X) subjects had prolonged I-V interpeak latencies. Comparisons between the fra(X) group (excluding one possible hard-of-hearing subject) and a control group of age-matched males with normal intelligence showed that the fra(X) group's interpeak latencies were significantly prolonged for the III-V and I-V but not for the I-III. This pattern of prolongation of interpeak latencies suggests that central, as opposed to peripheral, nervous-system dysfunction predominates in many patients having this syndrome. In addition, frequently observed prolongation of the transmission time may indicate that brain-stem white-matter functioning is also apt to be involved in this syndrome.