Cell, Vol. 64, 861-866,
February
22, 1991, Copyright
0 1991 by Cell Press
Physical Mapping across the Fragile X: Hypermethylation and Clinical Expression of the Fragile X Syndrome M. V. Bell,’ M. C. Hirst,l Y. Nakahori,’ R. N. MacKinnon,’ A. Roche,’ T. J. Flint,’ P A. Jacobs,2 N. Tommerup,3 L. Tranebjaerg4 U. Froster-lskenius,5 B. Kerr,‘j G. Turner,6 R. H. Lindenbaum,’ R. Winter,6 M. Pembrey,g S. Thibodeau,lO and K. E. Davies’ ‘Molecular Genetics Group Institute of Molecular Medicine John Radcliffe Hospital Headington, Oxford OX3 9DU, England 2Wessex Regional Genetics Laboratory General Hospital Salisbury, Wiltshire SP2 7SX, England 3Department of Medical Genetics Ulleval Hospital Blindern, 0315 Oslo 3, Norway 4Polar Institute of Medical Genetics Regional Hospital and University of Tromsoe 9012 Tromsoe, Norway 5Department of Gynaecology and Obstetrics Lubeck Medical University 2400 Lubeck, Germany 6Department of Medical Genetics Prince of Wales Children’s Hospital Randwick, New South Wales, Australia 2031 ‘Department of Medical Genetics The Churchill Hospital Headington, Oxford OX3 7LJ, England BThe Kennedy-Galton Centre North-West Thames Regional Genetics Service Clinical Research Centre, Northwick Park Hospital Harrow, Middlesex HA1 3UJ, England gMothercare Unit Institute of Child Health University of London London WCl, England lODepartment of Laboratory Medicine Mayo Clinic Rochester, Minnesota 55905
The most common genetic cause of mental retardation after Down’s syndrome, the fragile X syndrome, is associated with the occurrence of a fragile site at Xq27.3. This X-linked disease is intriguing because transmission can occur through phenotypically normal males. Theories to explain this unusual phenomenon include genomic rearrangements and methylation changes associated with a local block of reactivation of the X chromosome. Using microdissected markers close to the fragile site, we have been able to test these hypotheses. We present evidence for the association of methylation with the expression of the disease. However, there is no simple relationship between the degree of methylation and either the
level of expression of the fragile the clinical phenotype.
site or the severity
of
Introduction The fragile X syndrome (also called the Martin-Bell syndrome) is the most common genetic cause of mental retardation after Down’s syndrome. Population surveys have demonstrated that the prevalence in males is approximately 1 in 1250 (Gustavson et al., 1986; Webb et al., 1986). Unusually for an X-linked recessive disorder, about one-third of carrier females show some degree of mental impairment (Sherman et al., 1984). The overall prevalence of the mutation has been estimated to be 1 in 850, which suggests a very high new mutation rate for the disorder since affected individuals rarely reproduce. Affected males have characteristic long faces, large ears, and macro-orchidism (Martin and Bell, 1943; Turner et al., 1980; for review see Fryns, 1989). The phenotype is associated with the expression of a rare fragile site at Xq27.3 that can be induced in cultured cells under conditions of thymidine stress (Lubs, 1969; Sutherland, 1977; Harrison et al., 1983). However, the exact relationship between expression of the fragile X and the phenotype remains to be determined. There are, for example, cases where expression of the fragile X appears to segregate independently of the mutation (Voelckel et al., 1989). The fragile X syndrome is a puzzle for the geneticist because it shows an unusual segregation pattern (Sherman et al., 1984, 1985). Twenty percent of males with the mutation (normal transmitting males) appear to be phenotypitally normal and generally do not express the fragile site. The daughters of these males are almost never mentally retarded and usually do not express the fragile site. This segregation pattern has hampered genetic counseling of individuals at risk, but antenatal diagnosis and carrier detection have been much improved recently by the identification of closely linked polymorphic DNA segments (Oostra et al., 1990; Dahl et al., 1989; Suthers et al., 1989; Rousseau et al., 1991). However, DNA markers can be used only in those instances where the mode of transmission (either through the male or female line) is clear. Several hypotheses have been put forward to explain the observed inheritance pattern. Many of these involve a two-step process. The first is a premutation that in itself does not lead to the disease phenotype. As the premutated X chromosome passes through oogenesis, the second step occurs, resulting in the passing on of a fully mutated X chromosome. Conversion to the full mutation might involve a recombination event (Pembrey et al., 1985) amplification of sequences at or near the fragile site (Nussbaum et al., 1986), or the imprinting of DNA, perhaps via methylation, which would prevent the reactivation of genes near the mutation (Laird et al., 1987). We have recently identified DNA sequences that flank the fragile site (Hirst et al., 1991). This now provides an oppor-
Cell 662
Figure 1. Linkage and Long-Range Fragile Site
A MSSBBB NMNNMMNSS
MSSBBB NMNNMMNSSB
B
of Probes Restriction
VK21 and M749, Map across the
(A) Physical linkage of VK21 and M749. 48,XXXX lymphoblastoid cell line DNA was restricted with Not1 (N), Mlul (M), Sacll (S), and BssHll (B), singly and in double combinations. PFG conditions: 0.5x TEE, 0.8% agarose, pulse timeof 1200s 1.8Vlcm field strength, run time of 110 hr at 5%. The probes were hybridized to the same blot sequentially. Dashes at the side denote the positions of molecular weight standards: 250,1200,1600, and 3000 kb. (B) Long-range restriction map across the fragile site. The enzymes used were BssHll (E), Sacll (S), Notl (N), and MU (M). Numbers below the map refer to distances between Mlul sites in kb. Hatched bars indicate the partial digestion products observed in Figure 2. Parentheses indicate that a number is inferred. Numbers above the map indicate sizes for Sacll and BssHll fragments. The asterisk indicates the site of methylation occurring in patients (see later).
tunity to test these hypotheses directly. Here we present data supporting the concept that the expression of the fragile X phenotype is mediated via local inactivation through methylation. Results Physical Linkage across the Fragile Site We previously reported microdissection of the fragile X region (MacKinnon et al., 1990). Mapping on a somatic cell hybrid panel showed that two clones, M759 and M749 (defined by loci DXS532 and DXS533, respectively), flank the fragile site (Hirst et al., 1991). M749 maps in the same somatic cell hybrid interval as VK21 (DXS296), which lies at a recombination fraction of approximately 0.02-0.06 distal to the fragile X mutation. However, nothing was known about the position of M749 relative to VK21 and the fragile site. Pulsed-field gel (PFG) mapping studies show that M749 identifies BssHll and Sacll fragments different from VK21 but that these two loci can be linked on a single partial BssHll fragment of 1600 kb (solid triangle in Figure 1).
They lie on independent Not1 fragments: M749 identifies a fragment of approximately 3500 kb (not resolved on this gel), whereas VK21 identifies a 1000 kb Not1 fragment. Double digests with Sacll and BssHll give the same pattern as the single digests with both M749 and VK21, indicating that they are probably flanked by HTF islands (Lindsay and Bird, 1967). M759 identifies a Sacll fragment of 3000 kb that is not altered by BssHll digestion and identifies the same 3500 kb Not1 fragment as M749 (see later). The restriction map showing these sites is presented in Figure 1B. In Mlul digests of GM1416 (48,XxXx) DNA, VK21 identifies independent fragments of 350 kb and 1200 kb (running as a close doublet) and fragments of 3000 kb and 4500 kb that are also identified by M749 and M759 (Figure 2, solid triangles). M749 and M759 share an additional common fragment at 5000 kb (Figure 2, open triangle). U6.2 and Ml25 identify a common 4500 kb fragment detected by VK21, M749, and M759 (solid circle). The 5000 kb fragment common to M749 and M759 (open triangle) is very faintly visible for VK21 in some lanes but is not de-
Figure Site
r
-.
_
M748
M769
* . .
.
:
”
.
2. Physical
Linkage
across
the Fragile
48,xXxX DNA was restricted with Mlul in the presence of ethigium bromide at (left to right) 0, 0.16, 0.33, 05~9.7, 0.8, and 1 ug/ml. PFG conditions: 0.25x TBE, 0.8% Fastlane agarose, pulse time of 3600 s, 1.5 V/cm field strength, run time of 72 hr at 5°C. The same blot was hybridized sequentially with the probes indicated. Positions of size markers are indicated on the left: 1200,1600,300& 4500, and 5700 kb. Symbols denote fragments depictedon the map in Figure 18.
Physical 863
Mapping
across
the Fragile
X
tected by U6.2 even in long exposures. Thus the 3000 kb and 5000 kb Mlul fragments identified by VK21 and shared by M749 and M759 extend proximally away from Ml25 and U6.2, whereas the 4500 kb fragment detected by all probes extends from M759 distally to U6.2. M749 and M759 thus lie proximal rather than distal to VK21, confirming the linear order given by hybrid mapping (Hirst et al., 1991). Mlul digestion of GM1416 DNA yields fragments of 350 kb and 1200 kb for VK21 (seen as a doublet in Figure 2). Ml25 detects fragments of 850 kb and 1200 kb (not shown) indicating that these two probes share a 1200 kb fragment but are separated by a partially cut Mlul site. The physical relationship between Mlul sites and the Notl, Sacll, and BssHll sites is determined by the slight reduction of the small Mlul fragment for VK21 by double digestion with Notl, Sacll, and BssHll. This is clearly visible in shortrange PFG experiments (not shown). A long-range restriction map encompassing M759, M749, and VK21 is given in Figure 18. This map must cross the region containing the fragile X mutation. The Mlul pattern in fragile X males is identical to that observed in normal males (data not shown). However, small changes of 100 kb would not be detected in gels of this range. A shift in the size of the BssHll and Sacll fragments was observed in fragile X-positive, mentally retarded patients. Analysis with M749 showed that these changes are associated with methylation of the BssHll and Sacll sites between M749 and M759 and are not due to a structural rearrangement (see below). Methylation in Mentally Retarded, Fragile X-Positive Males DNA isolated from mentally retarded, fragile X-positive males was digested with BssHll and analyzed using the probe 291L, which identifies the locus DXS533. (Probe 291L was derived from a YAC containing the original M749 sequence; see Experimental Procedures.) Hybridization with the probe U6.2, which identifies the distal locus DXS304, was used as the control and is seen as the lower band in all the digests. Figure 3A clearly demonstrates that the normal males give two bands, at 600 kb (291L) and 400 kb (U6.2). In contrast, mentally retarded males (solid squares, Figure 38) show no band, or a band of reduced intensity at 600 kb. Identical results are obtained from both fresh lymphocyte and transformed lymphocyte cell line DNA from an individual. Similar differences are also seen in the intensity of the 600 kb Sacll fragment. Figure 3C shows a similar analysis in two brothers, where both show reduced intensity of the band at 600 kb. Linkage analysis indicates that both brothers inherited the same X chromosome from their mother (Davies et al., 1985). The presence of a band of reduced intensity in some patients suggests that the presence or absence of the 600 kb BssHll band is not a deletion of a restriction enzyme site but an alteration of methylation at the site. The 1000 kb BssHll fragment identified by VK21 is unaltered in a patient who shows changes in the 600 kb M749 BssHll fragment (data not shown), indicating that the site being affected lies between the fragile site and
A
B 0
0
0
0
0
q 1
kb
...0.. 234
5
6
I
-600 -400 .‘.,I.
C
0
.
D
n
normal
B
:
s
N
patient
Figure 3. Methylation X-Positive Males
in Normal
and
Mentally
Retarded,
Fragile
(A-C) Analysis of BssHll fragments in fragile X Patients probed with 291L (600 kb) and U6.2 (400 kb). (A) Normal males (open squares). (B) Normal males (open squares) and fragile X-positive, mentally retarded males (filled squares). Lane 1, normal male; lane 2, patient 2532; lane 3, patient 1994; lane 4, patient 2252; lane 5, normal male; lane 6, patient 1523; lane 7, patient 1467. (C) Mother (circle) and her two fragile X-positive sons (filled squares). Left to right: mother, patient 129, pattent 130. PFG conditions: 0.25x TBE, 0.6% agarose. pulse time of 60 s, 5.6 V/cm field strength, run time of 16 hr at 5°C. (D) Fragments identified in the region of M759. N = Notl; S = Sacll; B = BssHII. Blots were probed with G9L. Gel conditions: 0.25x TBE, 0.6% Fastlane agarose, pulse time of 2700 s, 1.5 V/cm field strength, run time of 72 hr at 5%. Size markers are 3000 and 4500 kb.
M749 (see asterisk in Figure 1B). This conclusion is supported by the shift to higher molecular weight of the BssHll and Sacll fragments of M759 in a patient lacking the 600 kb BssHll fragment. Figure 3D shows that the Sacll and BssHll fragments migrate much closer to the Notl fragments in the fragile X male (patient in lane 6 of Figure 3B) than they do in the normal male control. We have analyzed 21 unrelated fragile X-positive, mentally retarded males. A summary of the results and the corresponding expression of the phenotype and fragile site is given in Table 1. The analysis of 32 normal male controls shows no reduction in signal of the 600 kb band. Two fragile X-positive males give patterns indistinguishable from those of the normal males (see Figure 38, lane 3, and Fig-
Cell 884
Table 1. Presence Mentally Retarded
Patient 129 130 1487 1498 1523 1525 1560 1806 (twin A) 1807 (twin 8) 1994 2046 2047 2049 2252 2306 2483 2532 2615 2804 2909 2910 2911 2912
of the Bssfill Males Fragile Site
37llOO 50/100 29/l 00 291100 30/l 50 6000 14150 371300 681300 71100 22/100 481100 6/I 00 10164 12150 11/60 15150 2%-9% 20%-40% 14/l 00 26/l 00 26/l 00 20/l 00
Site in Fragile
Retardation Phenotype
X-Positive,
800 kb SssHll
Sand
Reduced/faint Reduced/faint Absent Absent Absent Absent Absent Reduced Reduced Normal Absent Absent Absent Absent Absent/very faint Normal Faint Absent Absent Reduced Absent Reduced Absent
Severe Moderate Moderate Moderate Moderate Moderate Moderate Severe Mild Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Severe Moderate Moderate Moderate Moderate
Figure
ure 4A). In addition, we have tested one of two brothers who have the typical clinical features of the fragile X syndrome but who are fragile X negative. This male also gives a BssHll band of reduced intensity. Monozygotic twins who are discordant for the fragile X syndrome (Tommerup et al., 1987) were also analyzed. They both show a band at 600 kb of reduced intensity (Figure 48). Twin A is severely retarded whereas twin B is only mildly affected. Both express the fragile site at high levels. Methylation in Normal Transmitting We have investigated the methylation where transmission clearly occurs
B
0
A n
Males pattern in a family through a normal
B B
A
n
5. Methylation
in Normal
Transmitting
Males
(A) Analysis of BssHII fragments in a family showing normal male transmission, Probes U6.2 (400 kb) and 291L (600 kb) were used. Left to right: two control samples(C), affected female (filled circle), affected brother (filled square), carrier mother (circle), normal transmitting, fragile-X negative male (square). PFG conditions were as in Figures 3A-3C. (B) Analysis of a normal transmitting, fragile X-positive male (square) and a control sample (C).
0
transmitting male. We have reported the full pedigree of this family previously and shown that the affected grandson has inherited the grandpaternal alleles for markers flanking the fragile X (Patterson et al., 1988). A limited family tree is given above the autoradiograph in Figure 5A. The normal transmitting male (individual IV-9 in the original pedigree) gives a normal pattern as does his phenotypically normal daughter, who expresses the fragile site in only 1 in 200 cells. The mentally retarded grandson expresses the fragile site in 30/150 of his cells and in all samples tested always gave a pattern consistent with total methylation at this site. We also analyzed the DNA from a phenotypically normal transmitting male who expresses the fragile site in 34% of his cells. His grandson shows clinical features typical of the fragile X syndrome and expresses the fragile site in 26% of his cells. The 600 kb BssHll fragment is completely absent in this normal transmitting male (Figure 58). Discussion
Figure 4. Analysis of BssHll Fragments Monozygotic Twins Discordant for the Fragile
in a Family X Syndrome
and
in
(A) Analysis in a family. Left to right: patient 2483 (filled square), his normal sister (circle), the mother (circle). (B) Analysis in monozygotic twins discordant for the fragile X syndrome. Left to right: a normal male (open square), twin A (filled square), twin B (filled square), twin A (filled square; the sample block has been trimmed to give a more equivalent DNA load), the mother of the twins (circle). Probes were 291L (800 kb) and U6.2 (400 kb). PFG conditions were as in Figures 3A-3C, except that 0.8% Fastlane agarose was used.
We have demonstrated in this study that the fragile X syndrome is not associated with large structural rearrangements in Xq27.3 but is associated with methylation of DNA sequences distal to the fragile site. Significant differences are observed in the PFG patter&observed after BssHll digestion of DNA from fragile X-positive, mentally retarded individuals compared with normal male controls. In 19/21 affected males the normal 600 kb BssHll band is either absent or’of reduced intensity. The normal, fragile X-negative transmitting male gives a normal pattern whereas his mentally retarded, fragile‘X-positive grandson lacks the 600 kb BssHll f&merKThese observations suggest that the absence of the band in most patients is
Physical 885
Mapping
across
the Fragile
X
the result of the methylation of the BssHll site between the M749 locus and the fragile site rather than any sequence rearrangement in the DNA. The presence of a normal pattern in a normal, fragile X-negative transmitting male and the absence of the band in his affected grandson are consistent with Laird’s hypothesis of imprinting at this locus and are consistent with a two-step process for the expression of the disease. Laird et al. (1987) suggested that a normal transmitting male possesses a premutated X chromosome that he passes on to his phenotypically normal daughters. During early oogenesis, when both X chromosomes undergo reactivation, the mutated X chromosome is unable to reactivate in the region of the fragile site. This locally inactivated “imprinted” X chromosome is then inherited by the affected son, who shows complete methylation at this site (see Figure 5A). However, the total methylation of the BssHll site in the normal transmitting, fragile X-positive male (Figure 56) suggests that the situation is not as simple as this. The daughter of this transmitting male did not express the fragile site (O/100 cells), but her affected son expressed the fragile site in 26% of his cells. The methylation reported here is probably occurring in a CpG island. The methylation of CpG islands on the inactive X chromosome is well documented and has been shown to be stably maintained in tissues and cell lines (Wolf et al., 1984; Yen et al., 1984; Keith et al., 1986; Tonioloet al., 1988; Turker et al., 1989; Pfeifer et al., 1990).’ Partial methylation rather than total methylation of CpG sites can sometimes be seen on the inactive X chromosome, and thus partial methylation in some fragile X syndrome patients might be expected. The lack of expression of the gene associated with this CpG island could result in the disease phenotype. We observed a normal BssHll pattern in 2 out of the 21 unrelated affected males analyzed. These males are the only cases that we tested with no history of mental retardation in their families. We cannot exclude the possibility that the site was methylated to an extent not detectable in this assay. Direct sequencing across the site in these patients will be needed to analyze this further. Vincent et al. (1991) report similar methylation differences around the locus DXS465. We assume that we are detecting the same sites here, as the fragment lengths are identical and hybrid mapping places DXS465 in the same interval as M749 (Rousseau et al., 1991). However, these authors saw changes in all 21 patients and did not attempt to correlate the methylation patterns with clinical phenotype or fragile X expression. Our data show that there is no direct correlation between the degree of mental impairment or the level of fragile X expression and the degree of methylation at the BssHll site. It is of interest that one of two brothers with the typical clinical features of the fragile X syndrome but without fragile X expression also shows evidence of methylation at this site. We find no evidence for the presence of other CpG islands close to this region. The next proximal CpG island, detected by M759, lies 3000 kb away. The region around M759 may contain sequences responsible for fragile site expression that may affect the expression of genes lying
distal to it by causing a block in X chromosome reactivation. This hypothesis is consistent with analyses showing that a YAC containing M759 hybridizes across the fragile site whereas an adjacent YAC containing M749 hybridizes distal to the fragile site (Hirst, Rack, and Davies, unpublished data). The very high mutation rate observed in this disease (Sherman et al., 1984, 1985) indicates either that there is an unusual sequence at the fragile site or that the locus is very large. The fragile site might cover most of the 2000 kb interval that we have defined between DXS532 and DXS533. It should now be possible to introduce YACs from this region into animal cells and to test for expression of the fragile site. In summary, we have shown that methylation in the region of the fragile site is associated with the occurrence of the fragile X phenotype. However, the exact mechanism whereby this methylation occurs and its relationship to the fragile site and/or clinical expression of the disease remain obscure and must await the detailed analysis of clones covering the whole region. Experimental
Procedures
Cell Lines and Lymphocytes The lymphoblastoid cell line GM1416 (48,XxXx) was obtained from NIGMS. Cells were cast into agarose and DNA was prepared as described (Kenwrick et al., 1987). For direct DNA preparation from whole blood, an erythrocyte lysis buffer (155 mM NH&I, 10 mM NH,COs, 0.1 mM EDTA [pH 7.41) was used to purify white cells. Probes VK21 detects the locus DXS296 (Suthers et al., 1989). U6.2 detects the locus DXS304 (Dahl et al., 1989). M125, M749, and M759 are microdissected clones and define the loci DXS479, DXS533, and DXS532, respectively (Hirst et al., 1991). Probes 291L and G9L are derived from YACs containing M749 and M759, respectively (Hirst et al., unpublished data). Probes were labeled by the random priming method (Feinberg and Vogelstein, 1983) using T7 polymerase (Stratagene), or (for microdissected clones) by the polymerase chain reaction (Hirst et al., 1991). PFG Electrophoresis The equipment used was the Pharmacia-LKB Pulsaphor System. Exact running conditions are given for each figure. Restriction enzymes were obtained from BCL (Notl, Mlul, BssHII) and BRL (Sstll, an isoschizomer of Sacll). Digestions were performed as described (Kenwrick et al., 1987) sometimes in the presence of ethidium bromide (Barlow and Lehrach, 1990). The agarose used was BRL ultrapure or FMC Fastlane. Gels were depurinated in 0.25 M HCI (twice for 15 min) and transferred to charge-modified nylon membrane (Nytran [Schleicher and Schuell] or Zeta-probe [Bio-Rad]) in 1.5 M NaCI, 0.5 M NaOH. After neutralization in 50 mM sodium phosphate (pH 7.2) the membrane was baked at 8O“C for l-2 hr. For hybridization, filters were ftrst incubated for 2 hr in prehybridization buffer (0.5 M sodium phosphate [pH 7.21, 7% SDS, 10 mglml bovine serum albumin, 50 pg/ml salmon sperm DNA) before the addition of probe at 10 nglml in the same buffer without bovine serum albumin. Filters were washed in 3x SSC (for microdissection clones) or 0.1x SSC at 65°C. For sequential hybridizations, signal was removed from membranes by three treatments with boiling distilled water, followed by a check exposure on film overnight. Exposures of rehybridized membranes were always checked after one overnight exposure to ensure that any new signal detected was not merely residual. Acknowledgments We would
like to thank
the many
clinicians
and cytogeneticists
who
Cell 866
have contributed to the collection and typing of families. We thank Mark McKinley for PFG blocks of affected males and Bryan Bolton (PHLS, Porton Down) for cell lines. We are grateful to Grant Sutherland for probe VK.21 and Niklaus Dahl for U6.2. This research was funded by the Medical Research Council of Great Britain and Action Research for the Crippled Child. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.
Oostra, B. A.. Hupkes, ker, E., Halley, D. J. J., B., and van Oost, 8. A. the fragile site FRAXA.
Received
Pfeifer, G. f?, Tanguay, R. L., Steigerwald, S. D., and Riggs, A. D. (1990). In ViVo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev. 4, 1277-1287.
January
15, 1991; revised
February
4, 1991.
References Barlow, D. P., and Lehrach, H. (1990). Partial Notl digests, generated by low enzyme concentration or the presence of ethidium bromide, can be used to extend the range of pulsed field gel mapping. Technique 2, 79-87.
F? E., Perdon, L. F., van Bennekom, C. A., BakSchmidt, M., Du Sart, D., Smits, A., Wieringa, (1990). New polymorphic DNA marker close to Genomics 6, 129-132.
Patterson, M., Bell, M., Kress, W., Davies, K. E., and Froster-lskenius, U. (1968). Linkage studies in a large fragile X family. Am. J. Hum. Genet. 43, 664-688. Pembrey, M. E., Winter, Ft. M., and Davies, K. E. (1985). A premutation that generates a defect at crossing over explains the inheritance of fragile X mental retardation. Am. J. Med. Genet. 27, 709-717.
Rousseau, F., Vincent, A., Rivella, S., Heitz, D., Tribioli, C., Maestrini, E., Warren, S. T., Suthers, G. K., Goodfellow, P., Mandel, J.-L., Toniolo, D., and Oberle, I. (1991). Four chromosomal breakpoints and four new probes mark out a 1OcM region encompassing the FRAXA locus. Am. J. Hum. Genet. 48, 108-118.
Dahl, N., Goonewardena, P., Malmgren, H., Gustavson, K. H., Holmgren, G., Seemanova, E., Anneren, G., Flood, A., and Pettersson, U. (1989). Linkage analysis of families with fragile-X mental retardation using a novel RFLP marker (DXS304). Am. J. Hum. Genet. 45, 304-309.
Sherman, S. L., Morton, N. E., Jacobs, The marker (X) syndrome: a cytogenetic Hum. Genet. 48, 21-37.
Davies, K. E., Mattei, M. G., Mattei, J. F., Veenama, H., McGlade, S., Harper, K.. Tommerup, N., Nielsen, K. B., Beighton, P., Drayna, D., White, R., and Pembrey, M. E. (1985). Linkagestudiesof X-linked mental retardation: high frequency of recombination in the telomeric region of the human X chromosome. Hum. Genet. 70, 249-255.
Sherman, S. L., Jacobs, l? A., Morton, N. E., Froster-lskenius, U., Howard-Peebles, P. N., Nielsen, K. B., Partington, M. W., Sutherland, G. R., Turner, G., and Watson, M. (1985). Further segregation analysis of the fragile X syndrome with special reference to transmitting males. Hum. Genet. 69, 3289-3299.
Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 732, 6-13.
Sutherland, G. R. (1977). Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium. Science 797, 256-266.
Fryns, J.-P (1989). X-linked mental retardation and the drome: a clinical approach. In The Fragile X Syndrome, ed. (Oxford: Oxford University Press), pp. l-39. Gustavson, G., Bloomquist, H. K., and Holmgren, G. lence of the fragile-X syndrome in mentally retarded Swedish county. Am. J. Med. Genet. 23, 581-587.
Suthers, G. K., Callen, D. F., Hyland, V. J., Kozman, H. M., Baker, E.. Eyre, H., Harper, P. S., Roberts,’ S. H., Hors-Cayla, M. C., Davies, K. E., Bell, M. V., and Sutherland, G. R. (1989). A new DNA marker tightly linked to the fragile X locus (FRAXA). Science 246, 1298-1300.
fragile X synK. E. Davies, (1986). Prevachildren in a
Harrison, C. J., Jack, E. M., Allen, T. D.. and Harris, R. (1983). Thefragile X: a scanning electron microscope study. J. Med. Genet. 20, 280-285. Hirst, M. C., Roche, A., Flint, T. J., MacKinnon, R. N., Bassett, J. H. D., Nakahori, Y., Watson, J. E. V., Bell, M. V., Patterson, M. N., Boyd, Y., Thomas, N. S. T., Knight, S. J. L.. Warren, S. T., Hors-Cayla, M., Schmidt, M., and Davies, K. E. (1991). Linear order of new and established DNA markers around the fragile site at Xq27.3. Genomics, in press.
P A., and Turner, G. (1984). and genetic analysis. Ann.
Tommerup, N., Tranebjaerg, L., Tonnesen, T., Kastern, W., Hansen, H., and Dissing, J. (1987). Identical expression of the fragile X but discordant clinical affection in two monozygotic twins with Martin-Bell syndrome. Abstracts, Third International Workshop on the Fragile X and X-Linked Mental Retardation, Troina, 13-16 September. Toniolo, D., Martini, G., Migeon, 8. R., and Dono, R. (1988). Expression of the GGPD locus on the human X chromosome is associated with demethylation of three CpG islands within 100kb of DNA. EMBO J. 7, 401-406. Turker, M. S., Swisshelm, K., Smith, A. C., and Martin, G. M. (1989). A partial methylation profile for a CpG site is stably maintained in mammalian tissues and cultured cell lines. J. Biol. Chem. 264, 11632-l 1636.
Keith, D. H., Singer-Sam, J., and Riggs, A. D. (1986). Active X chromosome DNA is unmethylated at eight CCGG sites clustered in a guanosine-plus-cytosine-rich island at the 5’end of the gene for phosphoglycerate kinase. Mol. Cell. Biol. 6, 4122-4125.
Turner, G., Daniel, A., and Forst, M. (1980). X-linked mental retardation, macroorchidism and the Xq27 fragile site. J. Pediatr. 96, 837-841.
Kenwrick, S., Patterson, M., Speer, A., Fischbeck, F., and Davies, K. E. (1987). Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis. Cell 48, 351-357.
Vincent, A., Heitz, D., Petit, C., Kretz, C., Oberle, I., and Mandel, J.-L. (1991). Abnormal pattern detected in fragile X patients by pulsed field gel electrophoresis. Nature, in press.
Laird, C., Jaffe, E., Karpen, G., Lamb, M., and Nelson, R. (1987). Fragile sites in human chromosomes as regions of late-replicating DNA. Trends Genet. 3, 274-281.
Voelckel, M. A., Philip, N., Piquet, C., Pellissier, M. C., Oberle, I., Birg. F, Mattei, M. G., and Mattei, J. F. (1989).%dyof a family with a fragile site of the X chromosome at Xq27-28 without mental retardation. Hum. Genet. 87, 353-357.
Lindsay, potential
S., and Bird, A. I? (1987). Use of restriction enzymes to detect gene sequences in mammalian DNA. Nature 327, 336-338.
Lubs, H. A. (1969). A marker 231-244.
X-chromosome.
Am. J. Hum. Genet.
27,
Webb, T., Bundey, S., Thake, of the fragile X chromosome Med. Genet. 23, 398-399.
J., and Todd, A. (1986). The frequency among schoolchlrdren in Coventry. J.
February
22, 1991, Copyright
0 1991 by Cell Press
Physical Mapping across the Fragile X: Hypermethylation and Clinical Expression of the Fragile X Syndrome M. V. Bell,’ M. C. Hirst,l Y. Nakahori,’ R. N. MacKinnon,’ A. Roche,’ T. J. Flint,’ P A. Jacobs,2 N. Tommerup,3 L. Tranebjaerg4 U. Froster-lskenius,5 B. Kerr,‘j G. Turner,6 R. H. Lindenbaum,’ R. Winter,6 M. Pembrey,g S. Thibodeau,lO and K. E. Davies’ ‘Molecular Genetics Group Institute of Molecular Medicine John Radcliffe Hospital Headington, Oxford OX3 9DU, England 2Wessex Regional Genetics Laboratory General Hospital Salisbury, Wiltshire SP2 7SX, England 3Department of Medical Genetics Ulleval Hospital Blindern, 0315 Oslo 3, Norway 4Polar Institute of Medical Genetics Regional Hospital and University of Tromsoe 9012 Tromsoe, Norway 5Department of Gynaecology and Obstetrics Lubeck Medical University 2400 Lubeck, Germany 6Department of Medical Genetics Prince of Wales Children’s Hospital Randwick, New South Wales, Australia 2031 ‘Department of Medical Genetics The Churchill Hospital Headington, Oxford OX3 7LJ, England BThe Kennedy-Galton Centre North-West Thames Regional Genetics Service Clinical Research Centre, Northwick Park Hospital Harrow, Middlesex HA1 3UJ, England gMothercare Unit Institute of Child Health University of London London WCl, England lODepartment of Laboratory Medicine Mayo Clinic Rochester, Minnesota 55905
The most common genetic cause of mental retardation after Down’s syndrome, the fragile X syndrome, is associated with the occurrence of a fragile site at Xq27.3. This X-linked disease is intriguing because transmission can occur through phenotypically normal males. Theories to explain this unusual phenomenon include genomic rearrangements and methylation changes associated with a local block of reactivation of the X chromosome. Using microdissected markers close to the fragile site, we have been able to test these hypotheses. We present evidence for the association of methylation with the expression of the disease. However, there is no simple relationship between the degree of methylation and either the
level of expression of the fragile the clinical phenotype.
site or the severity
of
Introduction The fragile X syndrome (also called the Martin-Bell syndrome) is the most common genetic cause of mental retardation after Down’s syndrome. Population surveys have demonstrated that the prevalence in males is approximately 1 in 1250 (Gustavson et al., 1986; Webb et al., 1986). Unusually for an X-linked recessive disorder, about one-third of carrier females show some degree of mental impairment (Sherman et al., 1984). The overall prevalence of the mutation has been estimated to be 1 in 850, which suggests a very high new mutation rate for the disorder since affected individuals rarely reproduce. Affected males have characteristic long faces, large ears, and macro-orchidism (Martin and Bell, 1943; Turner et al., 1980; for review see Fryns, 1989). The phenotype is associated with the expression of a rare fragile site at Xq27.3 that can be induced in cultured cells under conditions of thymidine stress (Lubs, 1969; Sutherland, 1977; Harrison et al., 1983). However, the exact relationship between expression of the fragile X and the phenotype remains to be determined. There are, for example, cases where expression of the fragile X appears to segregate independently of the mutation (Voelckel et al., 1989). The fragile X syndrome is a puzzle for the geneticist because it shows an unusual segregation pattern (Sherman et al., 1984, 1985). Twenty percent of males with the mutation (normal transmitting males) appear to be phenotypitally normal and generally do not express the fragile site. The daughters of these males are almost never mentally retarded and usually do not express the fragile site. This segregation pattern has hampered genetic counseling of individuals at risk, but antenatal diagnosis and carrier detection have been much improved recently by the identification of closely linked polymorphic DNA segments (Oostra et al., 1990; Dahl et al., 1989; Suthers et al., 1989; Rousseau et al., 1991). However, DNA markers can be used only in those instances where the mode of transmission (either through the male or female line) is clear. Several hypotheses have been put forward to explain the observed inheritance pattern. Many of these involve a two-step process. The first is a premutation that in itself does not lead to the disease phenotype. As the premutated X chromosome passes through oogenesis, the second step occurs, resulting in the passing on of a fully mutated X chromosome. Conversion to the full mutation might involve a recombination event (Pembrey et al., 1985) amplification of sequences at or near the fragile site (Nussbaum et al., 1986), or the imprinting of DNA, perhaps via methylation, which would prevent the reactivation of genes near the mutation (Laird et al., 1987). We have recently identified DNA sequences that flank the fragile site (Hirst et al., 1991). This now provides an oppor-
Cell 662
Figure 1. Linkage and Long-Range Fragile Site
A MSSBBB NMNNMMNSS
MSSBBB NMNNMMNSSB
B
of Probes Restriction
VK21 and M749, Map across the
(A) Physical linkage of VK21 and M749. 48,XXXX lymphoblastoid cell line DNA was restricted with Not1 (N), Mlul (M), Sacll (S), and BssHll (B), singly and in double combinations. PFG conditions: 0.5x TEE, 0.8% agarose, pulse timeof 1200s 1.8Vlcm field strength, run time of 110 hr at 5%. The probes were hybridized to the same blot sequentially. Dashes at the side denote the positions of molecular weight standards: 250,1200,1600, and 3000 kb. (B) Long-range restriction map across the fragile site. The enzymes used were BssHll (E), Sacll (S), Notl (N), and MU (M). Numbers below the map refer to distances between Mlul sites in kb. Hatched bars indicate the partial digestion products observed in Figure 2. Parentheses indicate that a number is inferred. Numbers above the map indicate sizes for Sacll and BssHll fragments. The asterisk indicates the site of methylation occurring in patients (see later).
tunity to test these hypotheses directly. Here we present data supporting the concept that the expression of the fragile X phenotype is mediated via local inactivation through methylation. Results Physical Linkage across the Fragile Site We previously reported microdissection of the fragile X region (MacKinnon et al., 1990). Mapping on a somatic cell hybrid panel showed that two clones, M759 and M749 (defined by loci DXS532 and DXS533, respectively), flank the fragile site (Hirst et al., 1991). M749 maps in the same somatic cell hybrid interval as VK21 (DXS296), which lies at a recombination fraction of approximately 0.02-0.06 distal to the fragile X mutation. However, nothing was known about the position of M749 relative to VK21 and the fragile site. Pulsed-field gel (PFG) mapping studies show that M749 identifies BssHll and Sacll fragments different from VK21 but that these two loci can be linked on a single partial BssHll fragment of 1600 kb (solid triangle in Figure 1).
They lie on independent Not1 fragments: M749 identifies a fragment of approximately 3500 kb (not resolved on this gel), whereas VK21 identifies a 1000 kb Not1 fragment. Double digests with Sacll and BssHll give the same pattern as the single digests with both M749 and VK21, indicating that they are probably flanked by HTF islands (Lindsay and Bird, 1967). M759 identifies a Sacll fragment of 3000 kb that is not altered by BssHll digestion and identifies the same 3500 kb Not1 fragment as M749 (see later). The restriction map showing these sites is presented in Figure 1B. In Mlul digests of GM1416 (48,XxXx) DNA, VK21 identifies independent fragments of 350 kb and 1200 kb (running as a close doublet) and fragments of 3000 kb and 4500 kb that are also identified by M749 and M759 (Figure 2, solid triangles). M749 and M759 share an additional common fragment at 5000 kb (Figure 2, open triangle). U6.2 and Ml25 identify a common 4500 kb fragment detected by VK21, M749, and M759 (solid circle). The 5000 kb fragment common to M749 and M759 (open triangle) is very faintly visible for VK21 in some lanes but is not de-
Figure Site
r
-.
_
M748
M769
* . .
.
:
”
.
2. Physical
Linkage
across
the Fragile
48,xXxX DNA was restricted with Mlul in the presence of ethigium bromide at (left to right) 0, 0.16, 0.33, 05~9.7, 0.8, and 1 ug/ml. PFG conditions: 0.25x TBE, 0.8% Fastlane agarose, pulse time of 3600 s, 1.5 V/cm field strength, run time of 72 hr at 5°C. The same blot was hybridized sequentially with the probes indicated. Positions of size markers are indicated on the left: 1200,1600,300& 4500, and 5700 kb. Symbols denote fragments depictedon the map in Figure 18.
Physical 863
Mapping
across
the Fragile
X
tected by U6.2 even in long exposures. Thus the 3000 kb and 5000 kb Mlul fragments identified by VK21 and shared by M749 and M759 extend proximally away from Ml25 and U6.2, whereas the 4500 kb fragment detected by all probes extends from M759 distally to U6.2. M749 and M759 thus lie proximal rather than distal to VK21, confirming the linear order given by hybrid mapping (Hirst et al., 1991). Mlul digestion of GM1416 DNA yields fragments of 350 kb and 1200 kb for VK21 (seen as a doublet in Figure 2). Ml25 detects fragments of 850 kb and 1200 kb (not shown) indicating that these two probes share a 1200 kb fragment but are separated by a partially cut Mlul site. The physical relationship between Mlul sites and the Notl, Sacll, and BssHll sites is determined by the slight reduction of the small Mlul fragment for VK21 by double digestion with Notl, Sacll, and BssHll. This is clearly visible in shortrange PFG experiments (not shown). A long-range restriction map encompassing M759, M749, and VK21 is given in Figure 18. This map must cross the region containing the fragile X mutation. The Mlul pattern in fragile X males is identical to that observed in normal males (data not shown). However, small changes of 100 kb would not be detected in gels of this range. A shift in the size of the BssHll and Sacll fragments was observed in fragile X-positive, mentally retarded patients. Analysis with M749 showed that these changes are associated with methylation of the BssHll and Sacll sites between M749 and M759 and are not due to a structural rearrangement (see below). Methylation in Mentally Retarded, Fragile X-Positive Males DNA isolated from mentally retarded, fragile X-positive males was digested with BssHll and analyzed using the probe 291L, which identifies the locus DXS533. (Probe 291L was derived from a YAC containing the original M749 sequence; see Experimental Procedures.) Hybridization with the probe U6.2, which identifies the distal locus DXS304, was used as the control and is seen as the lower band in all the digests. Figure 3A clearly demonstrates that the normal males give two bands, at 600 kb (291L) and 400 kb (U6.2). In contrast, mentally retarded males (solid squares, Figure 38) show no band, or a band of reduced intensity at 600 kb. Identical results are obtained from both fresh lymphocyte and transformed lymphocyte cell line DNA from an individual. Similar differences are also seen in the intensity of the 600 kb Sacll fragment. Figure 3C shows a similar analysis in two brothers, where both show reduced intensity of the band at 600 kb. Linkage analysis indicates that both brothers inherited the same X chromosome from their mother (Davies et al., 1985). The presence of a band of reduced intensity in some patients suggests that the presence or absence of the 600 kb BssHll band is not a deletion of a restriction enzyme site but an alteration of methylation at the site. The 1000 kb BssHll fragment identified by VK21 is unaltered in a patient who shows changes in the 600 kb M749 BssHll fragment (data not shown), indicating that the site being affected lies between the fragile site and
A
B 0
0
0
0
0
q 1
kb
...0.. 234
5
6
I
-600 -400 .‘.,I.
C
0
.
D
n
normal
B
:
s
N
patient
Figure 3. Methylation X-Positive Males
in Normal
and
Mentally
Retarded,
Fragile
(A-C) Analysis of BssHll fragments in fragile X Patients probed with 291L (600 kb) and U6.2 (400 kb). (A) Normal males (open squares). (B) Normal males (open squares) and fragile X-positive, mentally retarded males (filled squares). Lane 1, normal male; lane 2, patient 2532; lane 3, patient 1994; lane 4, patient 2252; lane 5, normal male; lane 6, patient 1523; lane 7, patient 1467. (C) Mother (circle) and her two fragile X-positive sons (filled squares). Left to right: mother, patient 129, pattent 130. PFG conditions: 0.25x TBE, 0.6% agarose. pulse time of 60 s, 5.6 V/cm field strength, run time of 16 hr at 5°C. (D) Fragments identified in the region of M759. N = Notl; S = Sacll; B = BssHII. Blots were probed with G9L. Gel conditions: 0.25x TBE, 0.6% Fastlane agarose, pulse time of 2700 s, 1.5 V/cm field strength, run time of 72 hr at 5%. Size markers are 3000 and 4500 kb.
M749 (see asterisk in Figure 1B). This conclusion is supported by the shift to higher molecular weight of the BssHll and Sacll fragments of M759 in a patient lacking the 600 kb BssHll fragment. Figure 3D shows that the Sacll and BssHll fragments migrate much closer to the Notl fragments in the fragile X male (patient in lane 6 of Figure 3B) than they do in the normal male control. We have analyzed 21 unrelated fragile X-positive, mentally retarded males. A summary of the results and the corresponding expression of the phenotype and fragile site is given in Table 1. The analysis of 32 normal male controls shows no reduction in signal of the 600 kb band. Two fragile X-positive males give patterns indistinguishable from those of the normal males (see Figure 38, lane 3, and Fig-
Cell 884
Table 1. Presence Mentally Retarded
Patient 129 130 1487 1498 1523 1525 1560 1806 (twin A) 1807 (twin 8) 1994 2046 2047 2049 2252 2306 2483 2532 2615 2804 2909 2910 2911 2912
of the Bssfill Males Fragile Site
37llOO 50/100 29/l 00 291100 30/l 50 6000 14150 371300 681300 71100 22/100 481100 6/I 00 10164 12150 11/60 15150 2%-9% 20%-40% 14/l 00 26/l 00 26/l 00 20/l 00
Site in Fragile
Retardation Phenotype
X-Positive,
800 kb SssHll
Sand
Reduced/faint Reduced/faint Absent Absent Absent Absent Absent Reduced Reduced Normal Absent Absent Absent Absent Absent/very faint Normal Faint Absent Absent Reduced Absent Reduced Absent
Severe Moderate Moderate Moderate Moderate Moderate Moderate Severe Mild Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Severe Moderate Moderate Moderate Moderate
Figure
ure 4A). In addition, we have tested one of two brothers who have the typical clinical features of the fragile X syndrome but who are fragile X negative. This male also gives a BssHll band of reduced intensity. Monozygotic twins who are discordant for the fragile X syndrome (Tommerup et al., 1987) were also analyzed. They both show a band at 600 kb of reduced intensity (Figure 48). Twin A is severely retarded whereas twin B is only mildly affected. Both express the fragile site at high levels. Methylation in Normal Transmitting We have investigated the methylation where transmission clearly occurs
B
0
A n
Males pattern in a family through a normal
B B
A
n
5. Methylation
in Normal
Transmitting
Males
(A) Analysis of BssHII fragments in a family showing normal male transmission, Probes U6.2 (400 kb) and 291L (600 kb) were used. Left to right: two control samples(C), affected female (filled circle), affected brother (filled square), carrier mother (circle), normal transmitting, fragile-X negative male (square). PFG conditions were as in Figures 3A-3C. (B) Analysis of a normal transmitting, fragile X-positive male (square) and a control sample (C).
0
transmitting male. We have reported the full pedigree of this family previously and shown that the affected grandson has inherited the grandpaternal alleles for markers flanking the fragile X (Patterson et al., 1988). A limited family tree is given above the autoradiograph in Figure 5A. The normal transmitting male (individual IV-9 in the original pedigree) gives a normal pattern as does his phenotypically normal daughter, who expresses the fragile site in only 1 in 200 cells. The mentally retarded grandson expresses the fragile site in 30/150 of his cells and in all samples tested always gave a pattern consistent with total methylation at this site. We also analyzed the DNA from a phenotypically normal transmitting male who expresses the fragile site in 34% of his cells. His grandson shows clinical features typical of the fragile X syndrome and expresses the fragile site in 26% of his cells. The 600 kb BssHll fragment is completely absent in this normal transmitting male (Figure 58). Discussion
Figure 4. Analysis of BssHll Fragments Monozygotic Twins Discordant for the Fragile
in a Family X Syndrome
and
in
(A) Analysis in a family. Left to right: patient 2483 (filled square), his normal sister (circle), the mother (circle). (B) Analysis in monozygotic twins discordant for the fragile X syndrome. Left to right: a normal male (open square), twin A (filled square), twin B (filled square), twin A (filled square; the sample block has been trimmed to give a more equivalent DNA load), the mother of the twins (circle). Probes were 291L (800 kb) and U6.2 (400 kb). PFG conditions were as in Figures 3A-3C, except that 0.8% Fastlane agarose was used.
We have demonstrated in this study that the fragile X syndrome is not associated with large structural rearrangements in Xq27.3 but is associated with methylation of DNA sequences distal to the fragile site. Significant differences are observed in the PFG patter&observed after BssHll digestion of DNA from fragile X-positive, mentally retarded individuals compared with normal male controls. In 19/21 affected males the normal 600 kb BssHll band is either absent or’of reduced intensity. The normal, fragile X-negative transmitting male gives a normal pattern whereas his mentally retarded, fragile‘X-positive grandson lacks the 600 kb BssHll f&merKThese observations suggest that the absence of the band in most patients is
Physical 885
Mapping
across
the Fragile
X
the result of the methylation of the BssHll site between the M749 locus and the fragile site rather than any sequence rearrangement in the DNA. The presence of a normal pattern in a normal, fragile X-negative transmitting male and the absence of the band in his affected grandson are consistent with Laird’s hypothesis of imprinting at this locus and are consistent with a two-step process for the expression of the disease. Laird et al. (1987) suggested that a normal transmitting male possesses a premutated X chromosome that he passes on to his phenotypically normal daughters. During early oogenesis, when both X chromosomes undergo reactivation, the mutated X chromosome is unable to reactivate in the region of the fragile site. This locally inactivated “imprinted” X chromosome is then inherited by the affected son, who shows complete methylation at this site (see Figure 5A). However, the total methylation of the BssHll site in the normal transmitting, fragile X-positive male (Figure 56) suggests that the situation is not as simple as this. The daughter of this transmitting male did not express the fragile site (O/100 cells), but her affected son expressed the fragile site in 26% of his cells. The methylation reported here is probably occurring in a CpG island. The methylation of CpG islands on the inactive X chromosome is well documented and has been shown to be stably maintained in tissues and cell lines (Wolf et al., 1984; Yen et al., 1984; Keith et al., 1986; Tonioloet al., 1988; Turker et al., 1989; Pfeifer et al., 1990).’ Partial methylation rather than total methylation of CpG sites can sometimes be seen on the inactive X chromosome, and thus partial methylation in some fragile X syndrome patients might be expected. The lack of expression of the gene associated with this CpG island could result in the disease phenotype. We observed a normal BssHll pattern in 2 out of the 21 unrelated affected males analyzed. These males are the only cases that we tested with no history of mental retardation in their families. We cannot exclude the possibility that the site was methylated to an extent not detectable in this assay. Direct sequencing across the site in these patients will be needed to analyze this further. Vincent et al. (1991) report similar methylation differences around the locus DXS465. We assume that we are detecting the same sites here, as the fragment lengths are identical and hybrid mapping places DXS465 in the same interval as M749 (Rousseau et al., 1991). However, these authors saw changes in all 21 patients and did not attempt to correlate the methylation patterns with clinical phenotype or fragile X expression. Our data show that there is no direct correlation between the degree of mental impairment or the level of fragile X expression and the degree of methylation at the BssHll site. It is of interest that one of two brothers with the typical clinical features of the fragile X syndrome but without fragile X expression also shows evidence of methylation at this site. We find no evidence for the presence of other CpG islands close to this region. The next proximal CpG island, detected by M759, lies 3000 kb away. The region around M759 may contain sequences responsible for fragile site expression that may affect the expression of genes lying
distal to it by causing a block in X chromosome reactivation. This hypothesis is consistent with analyses showing that a YAC containing M759 hybridizes across the fragile site whereas an adjacent YAC containing M749 hybridizes distal to the fragile site (Hirst, Rack, and Davies, unpublished data). The very high mutation rate observed in this disease (Sherman et al., 1984, 1985) indicates either that there is an unusual sequence at the fragile site or that the locus is very large. The fragile site might cover most of the 2000 kb interval that we have defined between DXS532 and DXS533. It should now be possible to introduce YACs from this region into animal cells and to test for expression of the fragile site. In summary, we have shown that methylation in the region of the fragile site is associated with the occurrence of the fragile X phenotype. However, the exact mechanism whereby this methylation occurs and its relationship to the fragile site and/or clinical expression of the disease remain obscure and must await the detailed analysis of clones covering the whole region. Experimental
Procedures
Cell Lines and Lymphocytes The lymphoblastoid cell line GM1416 (48,XxXx) was obtained from NIGMS. Cells were cast into agarose and DNA was prepared as described (Kenwrick et al., 1987). For direct DNA preparation from whole blood, an erythrocyte lysis buffer (155 mM NH&I, 10 mM NH,COs, 0.1 mM EDTA [pH 7.41) was used to purify white cells. Probes VK21 detects the locus DXS296 (Suthers et al., 1989). U6.2 detects the locus DXS304 (Dahl et al., 1989). M125, M749, and M759 are microdissected clones and define the loci DXS479, DXS533, and DXS532, respectively (Hirst et al., 1991). Probes 291L and G9L are derived from YACs containing M749 and M759, respectively (Hirst et al., unpublished data). Probes were labeled by the random priming method (Feinberg and Vogelstein, 1983) using T7 polymerase (Stratagene), or (for microdissected clones) by the polymerase chain reaction (Hirst et al., 1991). PFG Electrophoresis The equipment used was the Pharmacia-LKB Pulsaphor System. Exact running conditions are given for each figure. Restriction enzymes were obtained from BCL (Notl, Mlul, BssHII) and BRL (Sstll, an isoschizomer of Sacll). Digestions were performed as described (Kenwrick et al., 1987) sometimes in the presence of ethidium bromide (Barlow and Lehrach, 1990). The agarose used was BRL ultrapure or FMC Fastlane. Gels were depurinated in 0.25 M HCI (twice for 15 min) and transferred to charge-modified nylon membrane (Nytran [Schleicher and Schuell] or Zeta-probe [Bio-Rad]) in 1.5 M NaCI, 0.5 M NaOH. After neutralization in 50 mM sodium phosphate (pH 7.2) the membrane was baked at 8O“C for l-2 hr. For hybridization, filters were ftrst incubated for 2 hr in prehybridization buffer (0.5 M sodium phosphate [pH 7.21, 7% SDS, 10 mglml bovine serum albumin, 50 pg/ml salmon sperm DNA) before the addition of probe at 10 nglml in the same buffer without bovine serum albumin. Filters were washed in 3x SSC (for microdissection clones) or 0.1x SSC at 65°C. For sequential hybridizations, signal was removed from membranes by three treatments with boiling distilled water, followed by a check exposure on film overnight. Exposures of rehybridized membranes were always checked after one overnight exposure to ensure that any new signal detected was not merely residual. Acknowledgments We would
like to thank
the many
clinicians
and cytogeneticists
who
Cell 866
have contributed to the collection and typing of families. We thank Mark McKinley for PFG blocks of affected males and Bryan Bolton (PHLS, Porton Down) for cell lines. We are grateful to Grant Sutherland for probe VK.21 and Niklaus Dahl for U6.2. This research was funded by the Medical Research Council of Great Britain and Action Research for the Crippled Child. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact.
Oostra, B. A.. Hupkes, ker, E., Halley, D. J. J., B., and van Oost, 8. A. the fragile site FRAXA.
Received
Pfeifer, G. f?, Tanguay, R. L., Steigerwald, S. D., and Riggs, A. D. (1990). In ViVo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev. 4, 1277-1287.
January
15, 1991; revised
February
4, 1991.
References Barlow, D. P., and Lehrach, H. (1990). Partial Notl digests, generated by low enzyme concentration or the presence of ethidium bromide, can be used to extend the range of pulsed field gel mapping. Technique 2, 79-87.
F? E., Perdon, L. F., van Bennekom, C. A., BakSchmidt, M., Du Sart, D., Smits, A., Wieringa, (1990). New polymorphic DNA marker close to Genomics 6, 129-132.
Patterson, M., Bell, M., Kress, W., Davies, K. E., and Froster-lskenius, U. (1968). Linkage studies in a large fragile X family. Am. J. Hum. Genet. 43, 664-688. Pembrey, M. E., Winter, Ft. M., and Davies, K. E. (1985). A premutation that generates a defect at crossing over explains the inheritance of fragile X mental retardation. Am. J. Med. Genet. 27, 709-717.
Rousseau, F., Vincent, A., Rivella, S., Heitz, D., Tribioli, C., Maestrini, E., Warren, S. T., Suthers, G. K., Goodfellow, P., Mandel, J.-L., Toniolo, D., and Oberle, I. (1991). Four chromosomal breakpoints and four new probes mark out a 1OcM region encompassing the FRAXA locus. Am. J. Hum. Genet. 48, 108-118.
Dahl, N., Goonewardena, P., Malmgren, H., Gustavson, K. H., Holmgren, G., Seemanova, E., Anneren, G., Flood, A., and Pettersson, U. (1989). Linkage analysis of families with fragile-X mental retardation using a novel RFLP marker (DXS304). Am. J. Hum. Genet. 45, 304-309.
Sherman, S. L., Morton, N. E., Jacobs, The marker (X) syndrome: a cytogenetic Hum. Genet. 48, 21-37.
Davies, K. E., Mattei, M. G., Mattei, J. F., Veenama, H., McGlade, S., Harper, K.. Tommerup, N., Nielsen, K. B., Beighton, P., Drayna, D., White, R., and Pembrey, M. E. (1985). Linkagestudiesof X-linked mental retardation: high frequency of recombination in the telomeric region of the human X chromosome. Hum. Genet. 70, 249-255.
Sherman, S. L., Jacobs, l? A., Morton, N. E., Froster-lskenius, U., Howard-Peebles, P. N., Nielsen, K. B., Partington, M. W., Sutherland, G. R., Turner, G., and Watson, M. (1985). Further segregation analysis of the fragile X syndrome with special reference to transmitting males. Hum. Genet. 69, 3289-3299.
Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 732, 6-13.
Sutherland, G. R. (1977). Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium. Science 797, 256-266.
Fryns, J.-P (1989). X-linked mental retardation and the drome: a clinical approach. In The Fragile X Syndrome, ed. (Oxford: Oxford University Press), pp. l-39. Gustavson, G., Bloomquist, H. K., and Holmgren, G. lence of the fragile-X syndrome in mentally retarded Swedish county. Am. J. Med. Genet. 23, 581-587.
Suthers, G. K., Callen, D. F., Hyland, V. J., Kozman, H. M., Baker, E.. Eyre, H., Harper, P. S., Roberts,’ S. H., Hors-Cayla, M. C., Davies, K. E., Bell, M. V., and Sutherland, G. R. (1989). A new DNA marker tightly linked to the fragile X locus (FRAXA). Science 246, 1298-1300.
fragile X synK. E. Davies, (1986). Prevachildren in a
Harrison, C. J., Jack, E. M., Allen, T. D.. and Harris, R. (1983). Thefragile X: a scanning electron microscope study. J. Med. Genet. 20, 280-285. Hirst, M. C., Roche, A., Flint, T. J., MacKinnon, R. N., Bassett, J. H. D., Nakahori, Y., Watson, J. E. V., Bell, M. V., Patterson, M. N., Boyd, Y., Thomas, N. S. T., Knight, S. J. L.. Warren, S. T., Hors-Cayla, M., Schmidt, M., and Davies, K. E. (1991). Linear order of new and established DNA markers around the fragile site at Xq27.3. Genomics, in press.
P A., and Turner, G. (1984). and genetic analysis. Ann.
Tommerup, N., Tranebjaerg, L., Tonnesen, T., Kastern, W., Hansen, H., and Dissing, J. (1987). Identical expression of the fragile X but discordant clinical affection in two monozygotic twins with Martin-Bell syndrome. Abstracts, Third International Workshop on the Fragile X and X-Linked Mental Retardation, Troina, 13-16 September. Toniolo, D., Martini, G., Migeon, 8. R., and Dono, R. (1988). Expression of the GGPD locus on the human X chromosome is associated with demethylation of three CpG islands within 100kb of DNA. EMBO J. 7, 401-406. Turker, M. S., Swisshelm, K., Smith, A. C., and Martin, G. M. (1989). A partial methylation profile for a CpG site is stably maintained in mammalian tissues and cultured cell lines. J. Biol. Chem. 264, 11632-l 1636.
Keith, D. H., Singer-Sam, J., and Riggs, A. D. (1986). Active X chromosome DNA is unmethylated at eight CCGG sites clustered in a guanosine-plus-cytosine-rich island at the 5’end of the gene for phosphoglycerate kinase. Mol. Cell. Biol. 6, 4122-4125.
Turner, G., Daniel, A., and Forst, M. (1980). X-linked mental retardation, macroorchidism and the Xq27 fragile site. J. Pediatr. 96, 837-841.
Kenwrick, S., Patterson, M., Speer, A., Fischbeck, F., and Davies, K. E. (1987). Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis. Cell 48, 351-357.
Vincent, A., Heitz, D., Petit, C., Kretz, C., Oberle, I., and Mandel, J.-L. (1991). Abnormal pattern detected in fragile X patients by pulsed field gel electrophoresis. Nature, in press.
Laird, C., Jaffe, E., Karpen, G., Lamb, M., and Nelson, R. (1987). Fragile sites in human chromosomes as regions of late-replicating DNA. Trends Genet. 3, 274-281.
Voelckel, M. A., Philip, N., Piquet, C., Pellissier, M. C., Oberle, I., Birg. F, Mattei, M. G., and Mattei, J. F. (1989).%dyof a family with a fragile site of the X chromosome at Xq27-28 without mental retardation. Hum. Genet. 87, 353-357.
Lindsay, potential
S., and Bird, A. I? (1987). Use of restriction enzymes to detect gene sequences in mammalian DNA. Nature 327, 336-338.
Lubs, H. A. (1969). A marker 231-244.
X-chromosome.
Am. J. Hum. Genet.
27,
Webb, T., Bundey, S., Thake, of the fragile X chromosome Med. Genet. 23, 398-399.
J., and Todd, A. (1986). The frequency among schoolchlrdren in Coventry. J.
