American Journal of Medical Genetics 4 3 1057- 1062 (1992)
DELAYED REPLICATION OF Xq27 IN INDIVIDUALS WITH THE FRAGILE X SYNDROME
Tessa Webb Department of Clinical Genetics, University of Birmingham Birmingham Maternity Hospital, Edgbaston, Birmingham, England, United Kingdom
The timing of late replicating bands on the X chromosome has been studied in individuals with the fragile X [fra (X)] syndrome. Compared to controls both affected individuals and symptomless carriers of the syndrome show delayed replication of the Xq27 region as shown by 2 different methods. The implications of this finding are discussed in relation to the proposal [Laird et al., 19871 that the fraX syndrome is associated with a failure to reactivate the Xq27 band correctly after it has been inactivated in a female. KEY WORDS: Replication, FRAXA, X-linked mental retardation, fragile X syndrome
INTRODUCTION The fragile X [fra(X)] syndrome is unusual in that it can be transmitted by unaffected carriers of either sex and that approximately one third of When an female heterozygotes are affected. unaffected male (known as a transmitting male TM) transmits the syndrome to his daughters they too are unaffected and rarely demonstrate the associated Xq27.3 fragility. However, passage of the X chromosome through a female can result in Received for publication January 10, 1992; revision received February 27, 1992. Address correspondence to Dr. Tessa Webb, Dept. of Clinical Genetics, West Midlands Regional Genetics Service, Birmingham Maternity Hospital, Edgbaston B15 2TG U.K.
0 1992 Wiley-Liss, Inc.
offspring of either sex who manifest both the syndrome and the cytogenetic abnormality. It was this requirement which prompted Pembrey et al  to propose their premutation theory. Several hypotheses have in fact been proposed to explain the unusual inheritance patterns shown by the fra(X) syndrome. One suggested that the mutation may be the result of a two-step process. The first step, designated a premutation, carried no phenotypic manifestation of the syndrome but if it occurred in a male it would be passed to his daughters. The second step which would occur during female meiosis would result in the phenotypic manifestation of the syndrome in a proportion of the children of these carrier but symptomless daughters [Pembrey et al., 19851. A second hypothesis, proposed by Nussbaum et al., , suggested that the mutation may be due to variation in a series of repeated pyrimidine nucleotides. This suggestion followed from the fact that in order for the fra(X) to become cytogenetically visible the normal balance of the nucleotide pool in the culture medium must be altered either by removing thymidine from the culture altogether or by adding a large excess. A further proposal was made by Laird et al.,  who suggested that the mutation could be
due to a failure of reactivation of a specific region of the inactive X- chromosome during oogenesis. Gene dosage compensation by random inactivation of one of the X chromosomes in females is thought to be linked to methylation of specific chromosomal sites [Riggs, 19751 and this epistatic effect must be removed during gametogenesis, when the chromosome becomes reactivated. If, due to retention of methylation, the fra(X) gene remained "inactive" or late replicating after passage from mother to child, then a failure in function, i.e. interference in transcription, could result in the abnormal phenotype. Laird et al.  proposed that the Xq27 region is methylated and has delayed replication in fra(X) chromosomes. Proof for any or all of these theories remained elusive until a series of papers began to delineate the possible structure and function of the fra(X) gene. An HTF island lying immediately upstream from the fragile site was found to be hypermethylated in affected individuals but not in normal or transmitting males [Vincent et al., 1991; Bell et al., 19911, while the finding of an unstable DNA sequence detectable as an amplification/insert which characterizes the fra(X) syndrome and permits an improved accuracy of diagnosis lent support to these theories [OberlC et al., 1991; Yu et al., 1991; Verkerk et al., 19911. If the insert remains at a size below 500 bp then the HTF island An appears to remain unmethylated. amplification/insertion of this type has been found in unaffected fra(X) gene carriers of either sex. This small insert has been designated the 'premutation' and it may on passage through a female, be unstable, as it can enlarge and the HTF island upstream become hypermethylated. It is then associated with manifestation of the fra(X) syndrome, in either sex. So mental retardation is associated with both an unstable insertion/amplification of DNA and with methylation. Specific unstable amplification of CGG repeat sequences has been detected in one of the exons of the FMR-1 gene [Verkerk et al.,1991]. If localised failure of reactivation at oogenesis is associated with the mutation, as Laird suggests, then it may be possible to recognise this as a delay in replication of the Xq27 region. Incorporation of
either 3HdTTP or BrdU into lymphocytes from fra(X) patients, carriers and controls and examination of the resulting replication patterns should demonstrate such a delay.
METHODS In order to demonstrate FRAXA, lymphocytes from fra(X) positive individuals were cultured in FXl medium containing neither folic acid nor thymidine. Areas of late replication were studied by different methods. 1) Three or four hours prior to harvest 5pC/ml of 3HdTTP were added to each culture. Harvesting and slide making were by standard methods and the cells were GTG banded. Individual mitoses were identified, and the positions of the X chromosomes (both normal and FRAXA positive) within each cell noted for future reference. The slides were destained, dipped in Ilford K2 emulsion and exposed at 4O C for about 14 days. Developing and fixing of the emulsion layer was followed by restaining of the slides which permitted relocation of each X-chromosome. The areas of late replication were identified by the intensity of silver grains in the emulsion above them. When there were definite "bands" of activity in the chromosome the positions and intensities of these were recorded. Cells with activity throughout the X chromosome and those with only very few grains were rejected. 2) As BrdU is an analogue of thymidine, addition to the culture medium of levels recommended for visualising the late replicating X chromosome markedly reduced the level of detectable fra(X). In order to overcome this, a maximum of 10 pg/ml was added to each culture to facilitate R banding. Well-banded cells were selected for study. The X chromosome had light staining late-replicating bands at Xp21 and Xq21 and in females, the active and inactive X chromosomes were easily distinguishable. The frequency of occurrence of each pale band on the X chromosome was recorded for males and on the early- replicating or active X chromosome for females. The order of replication of the bands can be ascertained by counting the frequency with which they occur on the single X chromosome in males
Delayed Replication in FRA(X)
and the early replicating or active X chromosome in females. A consensus order has been established previously with only minor individual variation being observed [Latt et a1.,1981; Reddy et al.,1988]. Xq21 is the latest and Xp21 the penultimate band to replicate in normal human X chromosomes. So it is expected that the Xq21 band would be paler or late replicating more often than the Xq27 band.
RESULTS When 3HdTTP is incorporated during the final phase of replication dark grains become visible in the emulsion layer above the latest replicating bands. Figure 1 shows a mitotic cell from an affected Fra(X) positive male which has heavy labelling at Xq27-28 only, indicating late replication of this band.
Figure 1. Dividing cell from an affected male with heavy 3HdTTP labelling at Xq27-28 but not elsewhere in the cell.
Figure 2 Active chromosomes from carrier females, all showing grains at Xq27-28.
Several X chromosomes are shown in Figure 2, demonstrating late labelling of Xq27-28 in the active X in female carriers both affected and unaffected. The number of times Xq27- 28 had incorporated 3HdTTP during the final period of replication is recorded in Table I. For the females, only the active X chromosome is considered. Comparisons made between different individuals indicate that carriers of the fra(X) syndrome, whether affected or not, tend to have the Xq27-28 region of the X chromosome later replicating than the controls. This difference is significant at the 0.1 > p > 0.05 level for both males and females. TABLE I: Late Replication (LR) at Xq27-28 of Active X Chromosomes Only. Subjects
ActiveTotal % Cells Cells +ve 2 37 5.4
FRAXA 4 Male FRAXA +ve 1 TM.
When the individual Xchromosomes were further divided into those which demonstrated FRAXA and those which did not, it was found that the FRAXA positive chromosomes from normal carriers incorporated 3HdTTP only at a level comparable with that of the control X chromosomes (Table 11) whereas early replicating FRAXA positive cells from affected individuals of either sex showed a marked increase in 3HdTTP incorporation indicating a shift to later replication.
Table 11: Late Replication (LR) at Xq27-28. Active FRAXA Positive Chromosomes Only. Subjects No. No. Total % Cells Cells +ve
Table IV: Order of Appearance of Late-Replicating 3HdTTP Positive Bands in Early X Chromosomes From Female Carriers of the Fragile-X Syndrome.
Band order in: Controls
Previous studies of the dynamics of replication of the X chromosome have resulted in'the consensus that Xq21 is the latest band to replicate, preceded by Xp21 and then either Xq27 or Xq25, the latter bands showing greater variation in replication time than the former [Reddy et al.,1988]. Table I11 shows that in 3/4 affected males, band Xq27-28 has become later replicating
Table 111: Order of Appearance of Late -Replicating 3HdTTP Positive Bands in FRAXA Subjects Band order in controls Band order in affected Males
1 2 3 4
p21 q27 p21 p21
q21 p21 927 q27
q27 q21 q21 q21
Conclusion: Band q27 is later replicating in FRAXA positive males than in control males.
When the replication patterns of the active X chromosome in carrier females is considered [Table IV] then band Xq27 again moves its position in the replication order in both of the two affected females. The order remains the same as that found in normal individuals for both of the unaffected carriers.
Conclusion: Band Xq27 is later replicating in the early X chromosome from affected carriers, but not from carriers of normal IQ.
In order to try to quantify this effect it was reasoned that if Xq21 is the last band to replicate then the ratio of appearance of any other band to that of Xq21 should be less than unity. It can be seen from Table V that this is as expected for controls, both male and female, and for the normal fra(X) positive female carriers. Affected individuals of either sex all had Xq27/Xq21 band frequency ratios around unity indicating that the Xq27 band had become as late replicating as the Xq2 1 band.
Table V: Ratio of Xq27 to Xq21 Band Frequency as Shown by Incorporation of 3HdTTP. Active X Chromosomes Only. Subjects
Xq27/Total Bands 0.20
FRAXA Male 4
Delayed Replication in FRA(X)
R-banding of cells from 5 affected male subjects and one affected female showed that the Xq27 region almost invariably demonstrated a large pale area characteristic of late replication in cells carrying the fra(X) gene. This occurred in both FRAXA positive and negative cells and was always larger than the comparable pale area in controls. The large pale late replicating R-band at Xq27 in two affected males is shown in Figure 3.
Figure 3. Two R-banded fragile-X chromosomes from affected males showing a large pale latereplicating band atXq27.
patterns of methylation in expressing tissues and hypomethylation of the same genes in nonexpressing tissues [Bird, 19861. The pattern of methylation which influences this level of expression is contained within HTF islands which lie in regions critical for the regulation of genes. These islands contain clusters of CpG dinucleotides which tend to be unmethylated in active genes. Methylation inhibits gene expression suggesting a role as a control element Wolf and Migeon,1985]. So if methylation of both autosomal and X- linked genes is implicated in gene control and ra(X) positive individuals demonstrate hypermethylation of an HTF island upstream of the FMR-1 gene [Verkerk et al.,1991], then it is likely that this hypermethylation is involved in reduced gene expression and that one of its manifestations is in the delayed replication of the region. Such hypermethylation could have been achieved via an error in the reactivation of a previously inactivated FMR-1 gene. It remains to be seen whether the hypermethylation of the HTF island delays replication and interferes with transcription by similar methods such as in affecting the binding of specific factors [Bird, 1986, Dynan, 19891.
DISCUSSION REFERENCES As shown by incorporation of 3HdTTP or BrdU into mitotic cells during the final stages of S-phase, fra(X) positive individuals tend to defer the timing of replication of the Xq27 region when compared to controls. If only those cells which are demonstrably FRAXA positive are considered then affected individuals of either sex show later replication of the Xq27 region than do female carriers of normal intelligence. Thus, affected individuals with the fra(X) syndrome do have delayed replication of band Xq27. These results are in agreement with those of Yu et al. [19901 who studied late replication of band Xq27 in lymphoblastoid cell lines from fra(X) positive individuals. R-banding of mitotic cells after incorporation of BrdU during late Sphase showed late Xq terminal replication when compared to controls. Beside the apparent association between methylation and X-inactivation in females, many autosomal and X- linked genes also show precise
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