Am. J. Hum. Genet. 50:968-980, 1992
Fragile-X Syndrome: Unique Genetics of the Heritable Unstable Element S. Yu,* J. Mulley,* D. Loesch,t G. Turnert A. Donnelly,* A. Gedeon,* D. Hillen,* E. Kremer,* M. Lynch,* M. Pritchard* G. R. Sutherland,* and R. 1. Richards* *Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide; tDepartment of Psychology, La Trobe University, Bundoora, Australia; and tDepartment of Genetics, Prince of Wales Children's Hospital, Sydney
Summary The fragile site at Xq27.3 is an unstable microsatellite repeat, p(CCG)n. In fragile-X syndrome pedigrees, this sequence exhibits variable amplification, the length of which correlates with fragile-site expression. There is a direct relationship between increased p(CCG)n copy number and propensity for instability: individuals having large amplifications exhibit somatic variation due to increased instability. The instability of the p(CCG)n repeat, when transmitted through affected pedigrees, explains the unusual segregation patterns of fragile-X phenotype, referred to as the Sherman paradox. All individuals of fragile-X genotype were found (where testing was possible) to have a parent with amplified p(CCG)n repeat, indicating that few, if any, cases of fragile-X syndrome are not familial.
Introduction
Fragile-X syndrome, associated with a rare fragile site at Xq27.3, is the most common familial form of mental retardation (Sutherland 1985). Its segregation pattern is unique (Sherman et al. 1984, 1985); its cytogenetic detection can be difficult (Sutherland et al. 1985); and its molecular basis is only now beginning to be elucidated. Recently it has been demonstrated that fragile-X genotype is characterized by an unstable region of DNA (Oberle et al. 1991; Yu et al. 1991)-a trinucleotide repeat p(CCG)n of variable copy number (Kremer et al. 1991; Verkerk et al. 1991). In situ hybridization showed that the sequences flanking the p(CCG)n repeat also flank the fragile site (Kremer et al. 1991 ). The p(CCG)n repeat is embedded within the FMR-1 gene whose function is yet unknown (Verkerk et al. 1991). The FMR-1 gene is not expressed in fragile-X syndrome (Pieretti et al. 1991). Thus, all available experimental evidence leads to the conclusion Received November 4, 1991; revision received December 30, 1991.
Address for correspondence and reprints: Dr. Robert I. Richards, Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5005-0010$02.00
968
that the unstable p(CCG). repeat is the molecular basis of the fragile site, that cytogenetic expression of the fragile site is a reflection of increased repeat copy number, and that manifestation of fragile-X syndrome might be related to modulation of expression of a single gene. Fragile-X genotype is defined by the extent of amplification of the trinucleotide repeat, and we and others therefore hypothesized (Oberle et al. 1 991; Sutherland et al. 1991b; Yu et al. 1991) that the length of the unstable sequence might correlate with phenotype, which is known to be variable. DNA from several fragile-X individuals was previously reported to exhibit multiple bands when probed with pfxa3, possibly because of somatic instability of the unstable element (Yu et al. 1991). In addition, methylation of restriction-endonuclease recognition sites in the vicinity of the p(CCG)n repeat is associated with fragile-X phenotype (Bell et al. 1991; Vincent et al. 1991), although the relationship of methylation to genotype is not clear. In order to examine instability of the trinucleotide repeat during transmission from generation to generation and to assess possible correlations of the fragile-X genotype with phenotype, the genotypes of individuals from 49 fragile-X syndrome-affected pedigrees were determined.
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Fragile-X Syndrome Unstable Element Material and Methods Fragile X-affected Pedigrees
Fragile-X pedigrees assessed in this study have been described elsewhere (Richards et al. 1991a, 1991b). Index-case diagnosis was by cytogenetic detection of the fragile X. All consenting members of affected families were assessed clinically. The distinction between normal transmitting and affected males was made on the basis of assessment for mental retardation prior to DNA analysis of unstable-element band size. Chromosomal DNA was isolated by the method of Wyman and White (1980), from whole blood samples taken from members of affected families. Risk analysis was carried out using linked DNA markers according to a method described elsewhere (Mulley et al. 1987; Richards et al. 1991a, 1991b; Suthers et al. 1991a, 1991b). Fragile-X genotypes were confirmed by linkage analysis, with >95% probability, except for isolated cases where familial inheritance could only be demonstrated by using the pfxa3 probe. Detection of the Fragile-X Unstable Element with pfxa3 Chromosomal DNA was digested to completion with PstI. Samples were separated by electrophoresis on a .8% agarose gel and transferred to hybond N+
(Amersham) blotting membrane. The probe pfxa3 (Oncor) is a 536-bp sequence located immediately distal to the unstable element, p(CCG)n repeat, which is thought to constitute the fragile X (Kremer et al. 1991; Yu et al. 1991). The probe pS8 is an 800-bp PstI fragment from a yeast artificial chromosome isolated using VK21 (DXS296) as probe. pS8 was used as an internal positive control for hybridization with pfxa3. The probes were radioactively labeled with alpha32PdCTP by using a Multiprime labeling kit (Amersham) and were hybridized overnight at 421C in 5 x SSPE (pH 7.4), 1% SDS, 50% formamide, 10% dextran sulfate, and 100 jg salmon sperm DNA/ml. Membranes were washed in 0.1 % SDS, 0.1 x SSC at 70°C for at least 30 min prior to exposure to X-Omat, XK-1 film (Kodak) for 16-72 h at - 800C in the presence of two intensifying screens. The amount, in kilobase size, by which the unstable element was amplified relative to the normal 1.0-kb PstI fragment was measured as A, adopting the nomenclature of Oberle et al. (1991). For higher resolution of unstable-element band length, chromosomal DNA was digested to completion with Sau3AI prior to electrophoresis on 1.3% agarose gels and was probed with pfxa3 alone.
Cytogenetic Detection of the Fragile-X Site The fragile-X site was detected in lymphocytes by culture in folic acid-free medium or by induction with 300 mg thymidine/liter or 0.05 mg FUdR/liter (Sutherland and Baker 1990). Methylation at the Fragile-X Locus
Methylation at the fragile-X locus was detected by the resistance of the methylation-sensitive SacIl restriction site to digestion. To limit methylation detection to the single SacII site in the CpG island immediately proximal to the fragile X p(CCG)n repeat, the chromosomal DNA was also cleaved with EcoRI. The double-digested (SaclI and EcoRI) DNA was subjected to electrophoresis on 0.8% agarose gels and was probed with pfxa3 as described above. The increase in SacMI-Ecoll band length conferred by amplification of the fragile-X unstable element allowed distinction between the normal and fragile-X chromosomes in females. The percentage methylation was estimated by visual comparison of the relative intensity of the hybridization bands, approximated to the nearest 5% or 10%. PCR Across the p(CCG)n Repeat
PCR across the p(CCG)n repeat was accomplished using a modification of the reaction conditions of Kogan et al. (1987). Because of the exceptionally high GC content of this region, 7-deaza-dGTP was used in place of dGTP. The reactions were performed in a volume of 10 pl with 5 gCi of alpha-32PdCTP and were visualized by autoradiography for 72 h after electrophoresis on 6% polyacrylamide-urea gels. Thermal cycle conditions were as described elsewhere (Richards et al. 1991a, 1991b) and consisted of 10 cycles of 940C for 60 s, 60°C for 90 s, and 720C for 90 s, followed by 25 cycles of 940C for 60 s, 550C for 90 s, and 720C for 90 s and a final extension at 720C for 10 min. Primers utilized were 203 and 213 of Kremer et al. (1991). The electrophoretic mobility of the p(CCG)n-containing sequence (in certain instances of known length) was inconsistent with the mobility of DNA size markers on both acrylamide and agarose gels, presumably as a consequence of the unusual base composition. This is evident (in fig. 6B) by the large discrepancy between the dystrophin PCR markers (388 bp, 360 bp, and 331 bp) and the 203/213 PCR products when pfxa2 (Kremer et al. 1991) is used as a template (310 bp). Lengths of p(CCG)X-con-
.5(n 25
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A - Change in the Pst I fregment size detected by pfxa 3 (kb)
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Somatic variation at the fragile-X site. Southern Figure 4 blot analysis of PstI-digested chromosomal DNA from various cell types that was probed with pfxa3. The probe pS8, which hybridizes to a unique 800-bp PstI fragment on the X chromosome, was used as a positive control. A, DNAs from a fragile X-carrier female (KO from family 5). B, DNAs from a fragile X-affected male (AM from family 4).
bridizing band of 2.8 kb. Fragile X-genotype chromosomal DNA gave fragments that increased from these sizes by the length of the amplification. These could be readily distinguished from the normal X chromosome in carrier females (fig. 3C). Figure 3A shows a clear correlation of methylation with length of the unstable element in males. The methylation status of the SacII sites on the normal and fragile-X chromosomes in carrier females was analyzed. As shown in figure 3B, the normal X chromosome usually exhibited 0.6 kb). This indicates again a correlation between methylation and unstable-element length. Somatic Variation
To test the hypothesis that the multiple bands frequently observed from fragile X-affected individuals
were due to somatic instability of the unstable element, DNA was isolated from lymphocytes and from cultured fibroblasts and/or Epstein-Barr virus-transformed lymphoblast cell lines from 23 individuals (including affected males, carrier females, and normals) in nine fragile X-syndrome pedigrees. Figure 4 demonstrates the results obtained for a female carrier and an affected male. In all 11 affected males, regardless of whether instability was observed in the lymphocyte DNA, one or more bands of different length were observed in cultured cell lines. The results of the analysis of the somatic variation between the different tissues are shown in table 1. No somatic variation was detected in eight normal individuals (four male and four female). Among four carrier females, the three carriers with small amplification (A = 0.1-0.2 kb) showed no somatic variation, while the carrier with a slightly larger amplification (A = 0.7 kb) exhibited obvious somatic variation between different tissues (table 1). All the individuals for whom somatic variation has been observed were positive for cytogenetic expression of the fragile X. Mutation
No evidence of new mutation was detected in the 42 initial pedigrees analyzed, which included three isolated cases, three pedigrees in which the earliest female carrier could have been a new mutant, and the four-generation pedigree shown in figure 5. In an attempt to identify instances of new mutation, the pedigrees of an additional seven apparently isolated cases of fragile X (G. Turner, unpublished data) were analyzed. In all 10 apparently isolated cases (eight affected males and two affected females), the pfxa3 probe demonstrated that the mother carried the fragile-X genotype; and, where DNA was available (in two families), it was found that one of the relevant grandparents was also a carrier. In another pedigree, several distant relatives of the affected girl were found to be carriers by pfxa3, indicating that one of the great-grandparents of the affected girl was an obligate carrier. All affected individuals were found to have both a parent who had a fragile-X genotype and (where testing was possible) a grandparent who had a fragile-X genotype. In all 14 affected pedigrees, in which it was possible to observe a new mutation (because of availability of material), no such new mutation was observed. It is clear that the mutation of the fragile X proceeds from a predisposed state, itself a minor amplification, which leads in subsequent generations to a major amplification. In different individuals, the relative size of
975
Fragile-X Syndrome Unstable Element Table I Somatic Variation (kb)
NAME (sex) REPEAT TYPE AND FAMILY
OF
INDIVIDUAL
% FRAX
Lymphocytes
Lymphoblasts
3
SL (M) ............
42
4
AM (M) ..........
36
1.0 2.3 2.1
4 4
BM (M) .......... GM (M) ..........
46 48
1.1 2.4 1.0 2.2 2.1 2.4
5
DO (M) ..........
6
JG (M) ...........
70
7 13 14
ME (M) .......... RM (M) .......... JB (M) ............ M F (M) ..........
30 22 38 42
27
SW (M) .
15
4
JM (F) ............
10
5 7 13
KO (F) ............ FE (F) ............. GM (F) ...........
0 0 0
.2 .3 .2
1.2 1.4 .2 .3 .2
CE (F) ............ SG (F) ............. DD (F) ............ AB (F) ............ CM (F) ........... SB (F) ............. AF (M) ........... GW (M) ..........
0 0 0 0 0 0 0 0
.0 .0 .0 .0 .0 .0 .0 .0
.0 .0 .0 .0 .0 .0 .0 .0
Fibroblasts
Affected:
13
1.1 2.3 3.4 .4 1.4 2.3 1.5 Smear 2.0 1.6 2.6 1.5 1.7
2.1 1.6 2.4 Smear
2.0 2.4 2.4 2.4
1.3
2.2 1.0 Smear 1.9 1.5 1.6
Carrier: 1.6 .2 .3
Normal: 4 6 7 9 13 13 14 27 b
Change in size of Pstl fragment detected by pfxa3. Less than half of dosage.
the unstable element detected by either Southern blot or PCR was identical (fig. 6), confirming the localization of the unstable region to the p(CCG). repeat and clearly demonstrating the wide range of lengths of this sequence in normal individuals. Each PCR product gave a cluster of four bands for each allele; while this is a common property for PCR products from microsatellite repeats, its molecular basis is not clear. While PCR was able to amplify across the repeat sequence of some fragile-X carriers, no products were obtained from carriers or from affected individuals with large
unstable-element sequences (data not shown). Furthermore, in carrier females there was preferential amplification of the normal (shorter) allele (fig. 6, lanes 4 and 7), indicating the limitations of PCR for fragile-X diagnosis. Application of PCR to diagnosis is further complicated by somatic variation, which may result in an affected individual having one or more bands in the normal range. The risk of false-negative results in such instances would appear substantial. The exact repeat length at which phenotype is affected is unknown, although males with an amplifi-
Yu et al.
976
longer p(CCG)n repeat to their offspring, and yet they would only be scored as carriers if this probability had been realized. Mutation in the fragile X would appear to be a continuous process rather than a simple oneor two-step process involving a premutation followed by a full mutation. Isolated affected individuals who have the amplified unstable element should therefore be considered as familial, rather than as possible new mutations. Discussion 3.60-
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=
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0.72-
-
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Inheritance of the fragile-X unstable element in a Figure 5 four-generation lineage from a large affected pedigree. Chromosomal DNA was digested with PstI and was probed with pfxa3. The control probe pS8 was included in the hybridization. Dot in square indicates normal carrier male; dot in circle indicates normal carrier female not expressing the fragile X; half-blackened circle indicates normal carrier female expressing the fragile X; blackened square indicates affected fragile X syndrome male expressing the fragile X; and unblackened circle indicates normal female. All carriers are obligate carriers.
cation corresponding to more than "'240 copies of the repeat are affected, whereas those with fewer than this number were transmitting males. The exact length at which a random individual ought be considered an asymptomatic carrier is also not clear. The variation in length observed from high-resolution Southern blots in the normal population corresponds to 15-65 copies of the p(CCG)n repeat. The smallest amplification observed in apparently asymptomatic carriers (corresponding to "'70 copies of p(CCG)n) is very close to the biggest band observed in the normal population (fig. 6). Since the data suggest that instability is related to repeat length, individuals with longer repeats would be at greater risk of transmitting an even
The instability associated with the fragile X has been localized to a trinucleotide repeat p(CCG)n (Kremer et al. 1991; Yu et al. 1991), where in normal individuals n has been approximated to the range of 40 + 25 (by Southern blots; Kremer et al. 1991) or 30 + 24 (by PCR; Fu et al. 1991). The molecular basis for the instability could be either insertion or increase in repeat copy number (as instability is most frequently characterized by an increase in fragment length). The most parsimonious view of the molecular basis is variation in repeat copy number, since oligonucleotide repeat sequences of this type frequently exhibit such variation. Although the possibility of insertion of foreign sequences has not been excluded, such a mechanism is difficult to reconcile with the observed properties of the unstable sequence. Several reports have shown that differences in methylation in the vicinity of the fragile-X site exist between normal and affected members of fragile X-syndrome pedigrees (Bell et al. 1991; Oberle et al. 1991; Vincent et al. 1991). In addition, DNA from chorionic villi of an affected male fetus (Sutherland et al. 1991a) exhibited no SacII methylation, which was uncharacteristic of its 2.4-kb PstI fragment (A = 1.4 kb). Furthermore, no methylation was detected in this DNA at the NruI, BssHII, or EagI sites common to the SacII CpG island (data not shown). Tissues from the terminated fetus, other than fetal chorionic villi, exhibited both the 2.4-kb PstI fragment (A = 1.4 kb) and varying degrees of methylation of the SacII site. Methylation status does not provide additional information on fragile-X phenotype and may, in certain tissues such as chorionic villi, be misleading (Oberle et al. 1991; Sutherland et al. 1991a). Methylation is therefore likely to be a consequence of the fragile-X mutation (p(CCG)n amplification), and its progression through development may differ from one tissue to the next. Whether methylation has any causative role in fragile X-syndrome phenotype is yet to be established.
977
Fragile-X Syndrome Unstable Element
Laird (1987) has postulated a role for genetic imprinting in the transmission pattern of the fragile X. In this hypothesis, the passage of the X chromosomes
4'
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E
E
E Z~ ~ a
o
E qf
Kb A
_ 1.16 0.98
-
0.72
-
bp
B
-
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-
360
310 Variation in unstable-element length. A, Genomic Figure 6 DNA from normal and fragile-X individuals that was digested with Sau3AI and probed with pfxa3 to give accurate sizing and discrimination of unstable-element length. The normal male DNAs were chosen to represent the range including the maximum (lane 6), corresponding to ,.,60 copies of the p(CCG). repeat, and minimum (lane 1), corresponding to "'15 copies of the p(CCG)n repeat,
through a female predicts a different pattern of methylation on the inactive X compared with its active homologue. If the mutated X is methylated, the son of such a carrier female will be affected; if the mutated X is not methylated, then the son will be unaffected. Furthermore, according to Laird's X-inactivation imprinting model, once imprinted (methylation in fragile-X syndrome) the fragile-X chromosome remains stably imprinted when transmitted through the mother. However, four methylation-sensitive restriction-enzyme recognition sites in the vicinity of the fragile X-CpG island were unmethylated in chorionic villus, even though the SaclI site was methylated to different degrees in other tissues of the fetus tested (Sutherland et al. 1991a). In addition, in two affected males with multiple bands, methylation was detected in bands with A > 0.6 kb, whereas bands below this size were completely unmethylated (fig. 3B). These results fail to provide experimental evidence in support of the imprinting hypothesis. The correlation between the length ofthe unstable element and phenotype in males, and the observation that methylation correlates with length, suggest that methylation may be a consequence of amplification and that maternal X inactivation need not have a role in determining the phenotype of the offspring. Since the amplified sequence p(CCG). contains many additional targets for methylation, we propose that increased methylation is a function of amplification. Indeed, in fetal tissues, amplification has been observed in the absence of methylation (Oberle et al. 1991; Sutherland et al. 1991a). Methylation may have a role in the phenotypic manifestations of the disorder, but it does not appear to be involved in determining the transmission of the phenotype. In the normal population, the p(CCG). repeat is highly polymorphic although stable within pedigrees (Kremer et al. 1991). Variation in copy number of unstable-element lengths detected in >100 unrelated normal individuals tested. The three fragile-X carriers shown have the smallest fragile-X unstable elements (corresponding to "'70-80 copies of the p(CCG). repeat) detected in >200 fragile X-genotype individuals. Size markers are indicated in kilobases. B, PCR performed on the same chromosomal DNA samples as in A. Size markers were PCR products from the dystrophin locus (Chamberlain et al. 1988) and the 203/213 reaction using pfxa2 DNA (Kremer et al. 1991) as template. Arrows indicate the position of faint products of the affected allele in the two carrier females.
978
polymorphic oligonucleotide repeats is a common property of such sequences (Weber and May 1989). Extent of polymorphism is directly proportional to repeat length, suggesting that longer repeat sequences have a higher rate of new mutation. This rate is normally relatively low, as the majority of AC repeats (whose copy number is always 230), somatic variation is evident. The mutation rate of the repeat sequence is now so high as to result in variation between somatic cell lineages. In these instances, the further instability is often characterized by reduction in repeat copy number, giving rise to multiple bands in DNA samples from the one affected individual and even in DNA samples from a single tissue. This somatic variation does complicate the correlation between repeat length and phenotype, although all males who exhibit this variation are affected. It is possible that such instability may lead to deletion of the repeat -down to the range permissive of gene function in the tissues which produce the fragile-X phenotype and that the individual therefore would be unaffected. The mechanism of amplification is in itself puzzling. Linkage analysis using informative and closely linked flanking markers demonstrates that amplification does not occur as a result of meiotic recombination. The haplotype incorporating the affected allele is transmitted through affected pedigrees with rates of recombination consonant with map distances (Richards et al. 1991a, 1991 b; Suthers et al. 1991a, 1991 b). Amplifi-
Yu et al.
cation occurs in the pedigree in the absence ofrecombination between flanking markers. The trinucleotide p(CCG)n repeat has been found in several other locations in the human genome. Polymorphism of the p(CCG)n sequence in the ribosomal RNA genes on chromosome 21p has been reported (Gonzalez et al. 1985). This repeat is present at the breakpoint of a t(X;21) (p21;p12) translocation in a female with Duchenne muscular dystrophy (Bodrug et al. 1987) and in the 5' untranslated region of the BCR gene, which undergoes translocation with the ABL gene in the genesis of the t(9;22) Philadelphia chromosome (Zhu et al. 1990). The difficulties encountered in cloning the sequences containing the repeat, as well as the frequent deletion of these sequences during cloning, suggest that none of the reported DNA sequences, which have all been derived from cloned DNA, represent the true length of p(CCG). repeats in the human chromosome, although the sequences of 20 and 30 copies obtained by Oberle et al. (1991) and Verkerk et al. (1991), respectively, are in the normal range. PCR across these sequences, while successful in normal individuals and in carriers with small amplifications, has been problematic in affected individuals. Genomic sequencing, if technically feasible, may provide the only means of establishing the true in vivo length of p(CCG)n repeats and of confirming that amplification, rather than insertion, is involved in length variation in affected individuals. Mutation at the fragile X does not manifest at the molecular level in the same way we normally view insertions, substitutions, and deletions. The normal genotype consists of a range of copy numbers of the repeat, and an increase in copy number merely predisposes to further increases (or decreases). The correlation of repeat length and phenotype does imply a direct role for variation of the repeat copy number as the mechanism of mutation of the disease, although no functional data are yet available to support this. The 40 + 25 (or 30 + 24) copies of the repeat in normal individuals, which is uncommonly high for this type of repeat, suggests that this sequence may itself have a function. Index cases with rare autosomal folate-sensitive fragile sites (the same type of fragile site as the fragile X) always appear to inherit the fragile site from their mothers. In the study by Sherman and Sutherland (1986), 17 of 19 index cases had maternally derived fragile sites; the other two had an obligate carrier parent in whom the fragile site was not detected. In the same study it was shown that fragile sites were fully
Fragile-X Syndrome Unstable Element penetrant when transmitted by women but were only 50% penetrant when transmitted by men. Although the fragile X is the only one of these sites for which there is an associated phenotype, it should be noted that none of the other folate-sensitive fragile sites (which are all on autosomes) has ever been seen in a homozygous state. These segregation patterns suggest that a similar unstable element may be involved (Sutherland et al. 1991). Verkerk et al. (1991) have recently reported the partial sequence of a cDNA (FMR-1) from the fragile-X locus, which includes the p(CCG). repeat and sequences 3' to it. An open reading frame was observed, suggesting that the p(CCG)n repeat may be translated as well as transcribed. A consensus peptide sequence for nuclear translocation was identified, and the authors suggest that the FMR-1 gene might encode a nuclear protein. However, an ATG which conforms to the eukaryote consensus for initiation of translation is found 69 bases 3' to the p(CCG)n repeat, suggesting that the repeat may be located in the 5' untranslated region. Indeed, this methionine residue is followed by 13 of 20 hydrophobic amino acids, which is characteristic of an extracellular signal peptide. The compartmentalization and function of the FMR-1 protein product, as well as the translation of the p(CCG)n repeat, need to be clarified. It is clear that (a) the characterization of a gene, either FMR-1 or some other transcript, which is responsible for manifesting the fragile-X phenotype and (b) the characterization of other folate-sensitive fragile sites constitute important further approaches to understanding the molecular basis of fragile-X syndrome.
Acknowledgments We wish to thank Sharon Lane for growing cell lines, Elizabeth Baker for providing data on frequency of fragilesite expression, Drs. J.-L. Mandel and D. L. Nelson for discussion on PCR conditions, and Weiping Gai for artwork. R.I.R. thanks Shelley Richards for support and encouragement, particularly during the preparation of the manuscript. This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Adelaide Children's Hospital Research Foundation.
979 across the fragile X: hypermethylation and clinical expression of the fragile X syndrome. Cell 64:861-866 Bodrug SE, Ray PN, Gonzalez IL, Schmickel RD, Sylvester JE, Worton RG (1987) Molecular analysis of a constitutional X-autosome translocation in a female with muscular dystrophy. Science 237:1620-1624 Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multplex DNA amplification. Nucleic Acids Res 16:11141-11156 Dracopoli NC, O'Connell P, Elsner TI, Lalouel J-M, White RL, Buetow KH, Nishimura DY, et al (1991) The CEPH consortium linkage map of human chromosome 1. Genomics 9:686-700 Fu Y-H, Kuhl DPA, Pizzuti A, Pieretti M, SutcliffeJS, Richards S, Verkerk AJMH, et al (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67:1-20 Gonzalez IL, Gorski JL, Campen TJ, Dorney DJ, Erickson JM, Sylvester JE, Schmickel RD (1985) Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci USA 82:7666-7670 Kogan SC, Doherty BS, Gitshier J (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 317:985990 Kremer EJ, Pritchard M, Lynch M, Yu S, Holman K, Baker E, Warren ST, et al (1991) Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252:1711-1714 Laird CD (1987) Proposed mechanism of inheritance and expression of the human fragile-X syndrome of mental retardation. Genetics 117:587-599 Mulley JC, Gedeon AK, Thorn KA, Bates LJ, Sutherland GR (1987) Linkage and genetic counselling for the fragile X using DNA probes 52A, F9, DX13 and St14. AmJ Med Genet 27:435-448 Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boue I, et al (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252:1087-1102 Pieretti M, Zhang F, Fu Y-H, Warren ST, Oostra BA, Caskey CT, Nelson DL (1991) Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 66:817-822 Richards RI, Holman K, Kozman HJ, Kremer E, Lynch M, Pritchard M, Yu S, et al (1991a) Fragile X syndrome: genetic localization by linkage mapping of two microsatellite repeats FRAXAC1 and FRAXAC2 which immediately flank the fragile site. J Med Genet 28:818-823 Richards RI, Shen Y, Holman K, Kozman H, Hyland VJ, Mulley JC, Sutherland GR (1991b) Fragile X syndrome:
diagnosis using highly polymorphic microsatellite mark-
References Bell MV, Hirst MC, Nakahori Y, MacKinnon RN, Roche A, Flint TJ, Jacobs PA, et al (1991) Physical mapping
ers. Am J Hum Genet 48:1051-1057
Sherman SL, Jacobs PA, Morton NE, Froster-Iskenius U, Howard-Peebles PN, Nielsen KB, Partingon MW, et al (1985) Further segregation analysis of the fragile X syn-
980 drome with special reference to transmitting males. Hum Genet 69:289-299 Sherman SL, Morton NE, Jacobs PA, Turner G (1984) The marker (X) syndrome: a cytogenetic and genetic analysis. Ann Hum Genet 48:21-37 Sherman SL, Sutherland GR (1986) Segregation analysis of rare autosomal fragile sites. Hum Genet 72:123-128 Sutherland GR (1985) The enigma of the fragile X chromosome. Trends Genet 1:108-112 Sutherland GR, Baker E (1990) The common fragile site in band q27 of the human X chromosome is not coincident with the fragile X. Clin Genet 37:167-172 Sutherland GR, Gedeon A, Kornman L, Donnelly A, Byard RW, MulleyJC, Kremer E, et al (199la) Prenatal diagnosis of fragile X syndrome by direct detection of the characteristic unstable DNA sequence. N Engl J Med 325: 17201722 Sutherland GR, Haan EA, Kremer E, Lynch M, Pritchard M, Yu S. Richards RI (1991b) Hereditary unstable DNA: a new explanation for some old genetic questions? Lancet 338:289-292 Sutherland GR, Hecht F, Mulley JC, Glover TW, Hecht BK (1985) Fragile sites on human chromosomes. Oxford University Press, New York Suthers GK, Mulley JC, Voelckel MA, Dahl N, Vaisanen ML, Steinbach P, Glass IA, et al (1991a) Genetic mapping
Yu
et
al.
of new DNA probes at Xq27 defines a strategy for DNA studies in the fragile X syndrome. Am J Hum Genet 48: 460-467 Suthers GK, Oberle I, NancarrowJ, MulleyJC, Hyland VJ, Wilson PJ, McCure J, et al (1991b) Genetic mapping of new RFLPs at Xq27-q28. Genomics 9:37-43 Verkerk AJMH, Pieretti M, SutcliffeJS, Fu Y-H, Kuhl DPA, Pizzuti A, Reiner 0, et al (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65:905-914 Vincent A, Heitz D, Petit C, Kretz C, Oberle I, Mandel J-L (1991) Abnormal pattern detected in fragile X patients by pulsed-field gel electrophoresis. Nature 349:624-626 Weber JL, May PE (1989) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388-396 Wyman AR, White R (1980) A highly polymorphic locus in human DNA. Proc Natl Acad Sci USA 77:6754-6758 Yu S, Pritchard M, Kremer E, Lynch M, NancarrowJ, Baker E, Holman K, et al (1991) Fragile X genotype characterized by an unstable region of DNA. Science 252:11791181 Zhu Q-S, Heisterkamp N, Groffen J (1990) Unique organization of the human BCR gene promoter. Nucleic Acids Res 18:7119-7125
Fragile-X Syndrome: Unique Genetics of the Heritable Unstable Element S. Yu,* J. Mulley,* D. Loesch,t G. Turnert A. Donnelly,* A. Gedeon,* D. Hillen,* E. Kremer,* M. Lynch,* M. Pritchard* G. R. Sutherland,* and R. 1. Richards* *Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide; tDepartment of Psychology, La Trobe University, Bundoora, Australia; and tDepartment of Genetics, Prince of Wales Children's Hospital, Sydney
Summary The fragile site at Xq27.3 is an unstable microsatellite repeat, p(CCG)n. In fragile-X syndrome pedigrees, this sequence exhibits variable amplification, the length of which correlates with fragile-site expression. There is a direct relationship between increased p(CCG)n copy number and propensity for instability: individuals having large amplifications exhibit somatic variation due to increased instability. The instability of the p(CCG)n repeat, when transmitted through affected pedigrees, explains the unusual segregation patterns of fragile-X phenotype, referred to as the Sherman paradox. All individuals of fragile-X genotype were found (where testing was possible) to have a parent with amplified p(CCG)n repeat, indicating that few, if any, cases of fragile-X syndrome are not familial.
Introduction
Fragile-X syndrome, associated with a rare fragile site at Xq27.3, is the most common familial form of mental retardation (Sutherland 1985). Its segregation pattern is unique (Sherman et al. 1984, 1985); its cytogenetic detection can be difficult (Sutherland et al. 1985); and its molecular basis is only now beginning to be elucidated. Recently it has been demonstrated that fragile-X genotype is characterized by an unstable region of DNA (Oberle et al. 1991; Yu et al. 1991)-a trinucleotide repeat p(CCG)n of variable copy number (Kremer et al. 1991; Verkerk et al. 1991). In situ hybridization showed that the sequences flanking the p(CCG)n repeat also flank the fragile site (Kremer et al. 1991 ). The p(CCG)n repeat is embedded within the FMR-1 gene whose function is yet unknown (Verkerk et al. 1991). The FMR-1 gene is not expressed in fragile-X syndrome (Pieretti et al. 1991). Thus, all available experimental evidence leads to the conclusion Received November 4, 1991; revision received December 30, 1991.
Address for correspondence and reprints: Dr. Robert I. Richards, Department of Cytogenetics and Molecular Genetics, Adelaide Children's Hospital, North Adelaide, SA 5006, Australia. i 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5005-0010$02.00
968
that the unstable p(CCG). repeat is the molecular basis of the fragile site, that cytogenetic expression of the fragile site is a reflection of increased repeat copy number, and that manifestation of fragile-X syndrome might be related to modulation of expression of a single gene. Fragile-X genotype is defined by the extent of amplification of the trinucleotide repeat, and we and others therefore hypothesized (Oberle et al. 1 991; Sutherland et al. 1991b; Yu et al. 1991) that the length of the unstable sequence might correlate with phenotype, which is known to be variable. DNA from several fragile-X individuals was previously reported to exhibit multiple bands when probed with pfxa3, possibly because of somatic instability of the unstable element (Yu et al. 1991). In addition, methylation of restriction-endonuclease recognition sites in the vicinity of the p(CCG)n repeat is associated with fragile-X phenotype (Bell et al. 1991; Vincent et al. 1991), although the relationship of methylation to genotype is not clear. In order to examine instability of the trinucleotide repeat during transmission from generation to generation and to assess possible correlations of the fragile-X genotype with phenotype, the genotypes of individuals from 49 fragile-X syndrome-affected pedigrees were determined.
969
Fragile-X Syndrome Unstable Element Material and Methods Fragile X-affected Pedigrees
Fragile-X pedigrees assessed in this study have been described elsewhere (Richards et al. 1991a, 1991b). Index-case diagnosis was by cytogenetic detection of the fragile X. All consenting members of affected families were assessed clinically. The distinction between normal transmitting and affected males was made on the basis of assessment for mental retardation prior to DNA analysis of unstable-element band size. Chromosomal DNA was isolated by the method of Wyman and White (1980), from whole blood samples taken from members of affected families. Risk analysis was carried out using linked DNA markers according to a method described elsewhere (Mulley et al. 1987; Richards et al. 1991a, 1991b; Suthers et al. 1991a, 1991b). Fragile-X genotypes were confirmed by linkage analysis, with >95% probability, except for isolated cases where familial inheritance could only be demonstrated by using the pfxa3 probe. Detection of the Fragile-X Unstable Element with pfxa3 Chromosomal DNA was digested to completion with PstI. Samples were separated by electrophoresis on a .8% agarose gel and transferred to hybond N+
(Amersham) blotting membrane. The probe pfxa3 (Oncor) is a 536-bp sequence located immediately distal to the unstable element, p(CCG)n repeat, which is thought to constitute the fragile X (Kremer et al. 1991; Yu et al. 1991). The probe pS8 is an 800-bp PstI fragment from a yeast artificial chromosome isolated using VK21 (DXS296) as probe. pS8 was used as an internal positive control for hybridization with pfxa3. The probes were radioactively labeled with alpha32PdCTP by using a Multiprime labeling kit (Amersham) and were hybridized overnight at 421C in 5 x SSPE (pH 7.4), 1% SDS, 50% formamide, 10% dextran sulfate, and 100 jg salmon sperm DNA/ml. Membranes were washed in 0.1 % SDS, 0.1 x SSC at 70°C for at least 30 min prior to exposure to X-Omat, XK-1 film (Kodak) for 16-72 h at - 800C in the presence of two intensifying screens. The amount, in kilobase size, by which the unstable element was amplified relative to the normal 1.0-kb PstI fragment was measured as A, adopting the nomenclature of Oberle et al. (1991). For higher resolution of unstable-element band length, chromosomal DNA was digested to completion with Sau3AI prior to electrophoresis on 1.3% agarose gels and was probed with pfxa3 alone.
Cytogenetic Detection of the Fragile-X Site The fragile-X site was detected in lymphocytes by culture in folic acid-free medium or by induction with 300 mg thymidine/liter or 0.05 mg FUdR/liter (Sutherland and Baker 1990). Methylation at the Fragile-X Locus
Methylation at the fragile-X locus was detected by the resistance of the methylation-sensitive SacIl restriction site to digestion. To limit methylation detection to the single SacII site in the CpG island immediately proximal to the fragile X p(CCG)n repeat, the chromosomal DNA was also cleaved with EcoRI. The double-digested (SaclI and EcoRI) DNA was subjected to electrophoresis on 0.8% agarose gels and was probed with pfxa3 as described above. The increase in SacMI-Ecoll band length conferred by amplification of the fragile-X unstable element allowed distinction between the normal and fragile-X chromosomes in females. The percentage methylation was estimated by visual comparison of the relative intensity of the hybridization bands, approximated to the nearest 5% or 10%. PCR Across the p(CCG)n Repeat
PCR across the p(CCG)n repeat was accomplished using a modification of the reaction conditions of Kogan et al. (1987). Because of the exceptionally high GC content of this region, 7-deaza-dGTP was used in place of dGTP. The reactions were performed in a volume of 10 pl with 5 gCi of alpha-32PdCTP and were visualized by autoradiography for 72 h after electrophoresis on 6% polyacrylamide-urea gels. Thermal cycle conditions were as described elsewhere (Richards et al. 1991a, 1991b) and consisted of 10 cycles of 940C for 60 s, 60°C for 90 s, and 720C for 90 s, followed by 25 cycles of 940C for 60 s, 550C for 90 s, and 720C for 90 s and a final extension at 720C for 10 min. Primers utilized were 203 and 213 of Kremer et al. (1991). The electrophoretic mobility of the p(CCG)n-containing sequence (in certain instances of known length) was inconsistent with the mobility of DNA size markers on both acrylamide and agarose gels, presumably as a consequence of the unusual base composition. This is evident (in fig. 6B) by the large discrepancy between the dystrophin PCR markers (388 bp, 360 bp, and 331 bp) and the 203/213 PCR products when pfxa2 (Kremer et al. 1991) is used as a template (310 bp). Lengths of p(CCG)X-con-
.5(n 25
3 > 20
°4
15
5
10
-0
,0
z 5
0
1.2
0.8
OA
240
1.6
28
2.4
3.6
3.2
2 bands multiple bands
A - Change in the Pst I fregment size detected by pfxa 3 (kb)
x
Im CdS
80r aO.691pp-
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LL
-J
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-J
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0
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-
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4.84 4.84 3.59 2.81 1.95
-
3.59 2.81
-
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-
1.16
-
-
1.51 1.39 -
1:9I--
0. 98 -i
pS8
A
B
Somatic variation at the fragile-X site. Southern Figure 4 blot analysis of PstI-digested chromosomal DNA from various cell types that was probed with pfxa3. The probe pS8, which hybridizes to a unique 800-bp PstI fragment on the X chromosome, was used as a positive control. A, DNAs from a fragile X-carrier female (KO from family 5). B, DNAs from a fragile X-affected male (AM from family 4).
bridizing band of 2.8 kb. Fragile X-genotype chromosomal DNA gave fragments that increased from these sizes by the length of the amplification. These could be readily distinguished from the normal X chromosome in carrier females (fig. 3C). Figure 3A shows a clear correlation of methylation with length of the unstable element in males. The methylation status of the SacII sites on the normal and fragile-X chromosomes in carrier females was analyzed. As shown in figure 3B, the normal X chromosome usually exhibited 0.6 kb). This indicates again a correlation between methylation and unstable-element length. Somatic Variation
To test the hypothesis that the multiple bands frequently observed from fragile X-affected individuals
were due to somatic instability of the unstable element, DNA was isolated from lymphocytes and from cultured fibroblasts and/or Epstein-Barr virus-transformed lymphoblast cell lines from 23 individuals (including affected males, carrier females, and normals) in nine fragile X-syndrome pedigrees. Figure 4 demonstrates the results obtained for a female carrier and an affected male. In all 11 affected males, regardless of whether instability was observed in the lymphocyte DNA, one or more bands of different length were observed in cultured cell lines. The results of the analysis of the somatic variation between the different tissues are shown in table 1. No somatic variation was detected in eight normal individuals (four male and four female). Among four carrier females, the three carriers with small amplification (A = 0.1-0.2 kb) showed no somatic variation, while the carrier with a slightly larger amplification (A = 0.7 kb) exhibited obvious somatic variation between different tissues (table 1). All the individuals for whom somatic variation has been observed were positive for cytogenetic expression of the fragile X. Mutation
No evidence of new mutation was detected in the 42 initial pedigrees analyzed, which included three isolated cases, three pedigrees in which the earliest female carrier could have been a new mutant, and the four-generation pedigree shown in figure 5. In an attempt to identify instances of new mutation, the pedigrees of an additional seven apparently isolated cases of fragile X (G. Turner, unpublished data) were analyzed. In all 10 apparently isolated cases (eight affected males and two affected females), the pfxa3 probe demonstrated that the mother carried the fragile-X genotype; and, where DNA was available (in two families), it was found that one of the relevant grandparents was also a carrier. In another pedigree, several distant relatives of the affected girl were found to be carriers by pfxa3, indicating that one of the great-grandparents of the affected girl was an obligate carrier. All affected individuals were found to have both a parent who had a fragile-X genotype and (where testing was possible) a grandparent who had a fragile-X genotype. In all 14 affected pedigrees, in which it was possible to observe a new mutation (because of availability of material), no such new mutation was observed. It is clear that the mutation of the fragile X proceeds from a predisposed state, itself a minor amplification, which leads in subsequent generations to a major amplification. In different individuals, the relative size of
975
Fragile-X Syndrome Unstable Element Table I Somatic Variation (kb)
NAME (sex) REPEAT TYPE AND FAMILY
OF
INDIVIDUAL
% FRAX
Lymphocytes
Lymphoblasts
3
SL (M) ............
42
4
AM (M) ..........
36
1.0 2.3 2.1
4 4
BM (M) .......... GM (M) ..........
46 48
1.1 2.4 1.0 2.2 2.1 2.4
5
DO (M) ..........
6
JG (M) ...........
70
7 13 14
ME (M) .......... RM (M) .......... JB (M) ............ M F (M) ..........
30 22 38 42
27
SW (M) .
15
4
JM (F) ............
10
5 7 13
KO (F) ............ FE (F) ............. GM (F) ...........
0 0 0
.2 .3 .2
1.2 1.4 .2 .3 .2
CE (F) ............ SG (F) ............. DD (F) ............ AB (F) ............ CM (F) ........... SB (F) ............. AF (M) ........... GW (M) ..........
0 0 0 0 0 0 0 0
.0 .0 .0 .0 .0 .0 .0 .0
.0 .0 .0 .0 .0 .0 .0 .0
Fibroblasts
Affected:
13
1.1 2.3 3.4 .4 1.4 2.3 1.5 Smear 2.0 1.6 2.6 1.5 1.7
2.1 1.6 2.4 Smear
2.0 2.4 2.4 2.4
1.3
2.2 1.0 Smear 1.9 1.5 1.6
Carrier: 1.6 .2 .3
Normal: 4 6 7 9 13 13 14 27 b
Change in size of Pstl fragment detected by pfxa3. Less than half of dosage.
the unstable element detected by either Southern blot or PCR was identical (fig. 6), confirming the localization of the unstable region to the p(CCG). repeat and clearly demonstrating the wide range of lengths of this sequence in normal individuals. Each PCR product gave a cluster of four bands for each allele; while this is a common property for PCR products from microsatellite repeats, its molecular basis is not clear. While PCR was able to amplify across the repeat sequence of some fragile-X carriers, no products were obtained from carriers or from affected individuals with large
unstable-element sequences (data not shown). Furthermore, in carrier females there was preferential amplification of the normal (shorter) allele (fig. 6, lanes 4 and 7), indicating the limitations of PCR for fragile-X diagnosis. Application of PCR to diagnosis is further complicated by somatic variation, which may result in an affected individual having one or more bands in the normal range. The risk of false-negative results in such instances would appear substantial. The exact repeat length at which phenotype is affected is unknown, although males with an amplifi-
Yu et al.
976
longer p(CCG)n repeat to their offspring, and yet they would only be scored as carriers if this probability had been realized. Mutation in the fragile X would appear to be a continuous process rather than a simple oneor two-step process involving a premutation followed by a full mutation. Isolated affected individuals who have the amplified unstable element should therefore be considered as familial, rather than as possible new mutations. Discussion 3.60-
2.80 l.§S legs
=
1U9 1.1
-"
0.72-
-
1E D-pS8
Inheritance of the fragile-X unstable element in a Figure 5 four-generation lineage from a large affected pedigree. Chromosomal DNA was digested with PstI and was probed with pfxa3. The control probe pS8 was included in the hybridization. Dot in square indicates normal carrier male; dot in circle indicates normal carrier female not expressing the fragile X; half-blackened circle indicates normal carrier female expressing the fragile X; blackened square indicates affected fragile X syndrome male expressing the fragile X; and unblackened circle indicates normal female. All carriers are obligate carriers.
cation corresponding to more than "'240 copies of the repeat are affected, whereas those with fewer than this number were transmitting males. The exact length at which a random individual ought be considered an asymptomatic carrier is also not clear. The variation in length observed from high-resolution Southern blots in the normal population corresponds to 15-65 copies of the p(CCG)n repeat. The smallest amplification observed in apparently asymptomatic carriers (corresponding to "'70 copies of p(CCG)n) is very close to the biggest band observed in the normal population (fig. 6). Since the data suggest that instability is related to repeat length, individuals with longer repeats would be at greater risk of transmitting an even
The instability associated with the fragile X has been localized to a trinucleotide repeat p(CCG)n (Kremer et al. 1991; Yu et al. 1991), where in normal individuals n has been approximated to the range of 40 + 25 (by Southern blots; Kremer et al. 1991) or 30 + 24 (by PCR; Fu et al. 1991). The molecular basis for the instability could be either insertion or increase in repeat copy number (as instability is most frequently characterized by an increase in fragment length). The most parsimonious view of the molecular basis is variation in repeat copy number, since oligonucleotide repeat sequences of this type frequently exhibit such variation. Although the possibility of insertion of foreign sequences has not been excluded, such a mechanism is difficult to reconcile with the observed properties of the unstable sequence. Several reports have shown that differences in methylation in the vicinity of the fragile-X site exist between normal and affected members of fragile X-syndrome pedigrees (Bell et al. 1991; Oberle et al. 1991; Vincent et al. 1991). In addition, DNA from chorionic villi of an affected male fetus (Sutherland et al. 1991a) exhibited no SacII methylation, which was uncharacteristic of its 2.4-kb PstI fragment (A = 1.4 kb). Furthermore, no methylation was detected in this DNA at the NruI, BssHII, or EagI sites common to the SacII CpG island (data not shown). Tissues from the terminated fetus, other than fetal chorionic villi, exhibited both the 2.4-kb PstI fragment (A = 1.4 kb) and varying degrees of methylation of the SacII site. Methylation status does not provide additional information on fragile-X phenotype and may, in certain tissues such as chorionic villi, be misleading (Oberle et al. 1991; Sutherland et al. 1991a). Methylation is therefore likely to be a consequence of the fragile-X mutation (p(CCG)n amplification), and its progression through development may differ from one tissue to the next. Whether methylation has any causative role in fragile X-syndrome phenotype is yet to be established.
977
Fragile-X Syndrome Unstable Element
Laird (1987) has postulated a role for genetic imprinting in the transmission pattern of the fragile X. In this hypothesis, the passage of the X chromosomes
4'
aa-
E
E
E Z~ ~ a
o
E qf
Kb A
_ 1.16 0.98
-
0.72
-
bp
B
-
3#1
-
360
310 Variation in unstable-element length. A, Genomic Figure 6 DNA from normal and fragile-X individuals that was digested with Sau3AI and probed with pfxa3 to give accurate sizing and discrimination of unstable-element length. The normal male DNAs were chosen to represent the range including the maximum (lane 6), corresponding to ,.,60 copies of the p(CCG). repeat, and minimum (lane 1), corresponding to "'15 copies of the p(CCG)n repeat,
through a female predicts a different pattern of methylation on the inactive X compared with its active homologue. If the mutated X is methylated, the son of such a carrier female will be affected; if the mutated X is not methylated, then the son will be unaffected. Furthermore, according to Laird's X-inactivation imprinting model, once imprinted (methylation in fragile-X syndrome) the fragile-X chromosome remains stably imprinted when transmitted through the mother. However, four methylation-sensitive restriction-enzyme recognition sites in the vicinity of the fragile X-CpG island were unmethylated in chorionic villus, even though the SaclI site was methylated to different degrees in other tissues of the fetus tested (Sutherland et al. 1991a). In addition, in two affected males with multiple bands, methylation was detected in bands with A > 0.6 kb, whereas bands below this size were completely unmethylated (fig. 3B). These results fail to provide experimental evidence in support of the imprinting hypothesis. The correlation between the length ofthe unstable element and phenotype in males, and the observation that methylation correlates with length, suggest that methylation may be a consequence of amplification and that maternal X inactivation need not have a role in determining the phenotype of the offspring. Since the amplified sequence p(CCG). contains many additional targets for methylation, we propose that increased methylation is a function of amplification. Indeed, in fetal tissues, amplification has been observed in the absence of methylation (Oberle et al. 1991; Sutherland et al. 1991a). Methylation may have a role in the phenotypic manifestations of the disorder, but it does not appear to be involved in determining the transmission of the phenotype. In the normal population, the p(CCG). repeat is highly polymorphic although stable within pedigrees (Kremer et al. 1991). Variation in copy number of unstable-element lengths detected in >100 unrelated normal individuals tested. The three fragile-X carriers shown have the smallest fragile-X unstable elements (corresponding to "'70-80 copies of the p(CCG). repeat) detected in >200 fragile X-genotype individuals. Size markers are indicated in kilobases. B, PCR performed on the same chromosomal DNA samples as in A. Size markers were PCR products from the dystrophin locus (Chamberlain et al. 1988) and the 203/213 reaction using pfxa2 DNA (Kremer et al. 1991) as template. Arrows indicate the position of faint products of the affected allele in the two carrier females.
978
polymorphic oligonucleotide repeats is a common property of such sequences (Weber and May 1989). Extent of polymorphism is directly proportional to repeat length, suggesting that longer repeat sequences have a higher rate of new mutation. This rate is normally relatively low, as the majority of AC repeats (whose copy number is always 230), somatic variation is evident. The mutation rate of the repeat sequence is now so high as to result in variation between somatic cell lineages. In these instances, the further instability is often characterized by reduction in repeat copy number, giving rise to multiple bands in DNA samples from the one affected individual and even in DNA samples from a single tissue. This somatic variation does complicate the correlation between repeat length and phenotype, although all males who exhibit this variation are affected. It is possible that such instability may lead to deletion of the repeat -down to the range permissive of gene function in the tissues which produce the fragile-X phenotype and that the individual therefore would be unaffected. The mechanism of amplification is in itself puzzling. Linkage analysis using informative and closely linked flanking markers demonstrates that amplification does not occur as a result of meiotic recombination. The haplotype incorporating the affected allele is transmitted through affected pedigrees with rates of recombination consonant with map distances (Richards et al. 1991a, 1991 b; Suthers et al. 1991a, 1991 b). Amplifi-
Yu et al.
cation occurs in the pedigree in the absence ofrecombination between flanking markers. The trinucleotide p(CCG)n repeat has been found in several other locations in the human genome. Polymorphism of the p(CCG)n sequence in the ribosomal RNA genes on chromosome 21p has been reported (Gonzalez et al. 1985). This repeat is present at the breakpoint of a t(X;21) (p21;p12) translocation in a female with Duchenne muscular dystrophy (Bodrug et al. 1987) and in the 5' untranslated region of the BCR gene, which undergoes translocation with the ABL gene in the genesis of the t(9;22) Philadelphia chromosome (Zhu et al. 1990). The difficulties encountered in cloning the sequences containing the repeat, as well as the frequent deletion of these sequences during cloning, suggest that none of the reported DNA sequences, which have all been derived from cloned DNA, represent the true length of p(CCG). repeats in the human chromosome, although the sequences of 20 and 30 copies obtained by Oberle et al. (1991) and Verkerk et al. (1991), respectively, are in the normal range. PCR across these sequences, while successful in normal individuals and in carriers with small amplifications, has been problematic in affected individuals. Genomic sequencing, if technically feasible, may provide the only means of establishing the true in vivo length of p(CCG)n repeats and of confirming that amplification, rather than insertion, is involved in length variation in affected individuals. Mutation at the fragile X does not manifest at the molecular level in the same way we normally view insertions, substitutions, and deletions. The normal genotype consists of a range of copy numbers of the repeat, and an increase in copy number merely predisposes to further increases (or decreases). The correlation of repeat length and phenotype does imply a direct role for variation of the repeat copy number as the mechanism of mutation of the disease, although no functional data are yet available to support this. The 40 + 25 (or 30 + 24) copies of the repeat in normal individuals, which is uncommonly high for this type of repeat, suggests that this sequence may itself have a function. Index cases with rare autosomal folate-sensitive fragile sites (the same type of fragile site as the fragile X) always appear to inherit the fragile site from their mothers. In the study by Sherman and Sutherland (1986), 17 of 19 index cases had maternally derived fragile sites; the other two had an obligate carrier parent in whom the fragile site was not detected. In the same study it was shown that fragile sites were fully
Fragile-X Syndrome Unstable Element penetrant when transmitted by women but were only 50% penetrant when transmitted by men. Although the fragile X is the only one of these sites for which there is an associated phenotype, it should be noted that none of the other folate-sensitive fragile sites (which are all on autosomes) has ever been seen in a homozygous state. These segregation patterns suggest that a similar unstable element may be involved (Sutherland et al. 1991). Verkerk et al. (1991) have recently reported the partial sequence of a cDNA (FMR-1) from the fragile-X locus, which includes the p(CCG). repeat and sequences 3' to it. An open reading frame was observed, suggesting that the p(CCG)n repeat may be translated as well as transcribed. A consensus peptide sequence for nuclear translocation was identified, and the authors suggest that the FMR-1 gene might encode a nuclear protein. However, an ATG which conforms to the eukaryote consensus for initiation of translation is found 69 bases 3' to the p(CCG)n repeat, suggesting that the repeat may be located in the 5' untranslated region. Indeed, this methionine residue is followed by 13 of 20 hydrophobic amino acids, which is characteristic of an extracellular signal peptide. The compartmentalization and function of the FMR-1 protein product, as well as the translation of the p(CCG)n repeat, need to be clarified. It is clear that (a) the characterization of a gene, either FMR-1 or some other transcript, which is responsible for manifesting the fragile-X phenotype and (b) the characterization of other folate-sensitive fragile sites constitute important further approaches to understanding the molecular basis of fragile-X syndrome.
Acknowledgments We wish to thank Sharon Lane for growing cell lines, Elizabeth Baker for providing data on frequency of fragilesite expression, Drs. J.-L. Mandel and D. L. Nelson for discussion on PCR conditions, and Weiping Gai for artwork. R.I.R. thanks Shelley Richards for support and encouragement, particularly during the preparation of the manuscript. This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Adelaide Children's Hospital Research Foundation.
979 across the fragile X: hypermethylation and clinical expression of the fragile X syndrome. Cell 64:861-866 Bodrug SE, Ray PN, Gonzalez IL, Schmickel RD, Sylvester JE, Worton RG (1987) Molecular analysis of a constitutional X-autosome translocation in a female with muscular dystrophy. Science 237:1620-1624 Chamberlain JS, Gibbs RA, Ranier JE, Nguyen PN, Caskey CT (1988) Deletion screening of the Duchenne muscular dystrophy locus via multplex DNA amplification. Nucleic Acids Res 16:11141-11156 Dracopoli NC, O'Connell P, Elsner TI, Lalouel J-M, White RL, Buetow KH, Nishimura DY, et al (1991) The CEPH consortium linkage map of human chromosome 1. Genomics 9:686-700 Fu Y-H, Kuhl DPA, Pizzuti A, Pieretti M, SutcliffeJS, Richards S, Verkerk AJMH, et al (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67:1-20 Gonzalez IL, Gorski JL, Campen TJ, Dorney DJ, Erickson JM, Sylvester JE, Schmickel RD (1985) Variation among human 28S ribosomal RNA genes. Proc Natl Acad Sci USA 82:7666-7670 Kogan SC, Doherty BS, Gitshier J (1987) An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. N Engl J Med 317:985990 Kremer EJ, Pritchard M, Lynch M, Yu S, Holman K, Baker E, Warren ST, et al (1991) Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252:1711-1714 Laird CD (1987) Proposed mechanism of inheritance and expression of the human fragile-X syndrome of mental retardation. Genetics 117:587-599 Mulley JC, Gedeon AK, Thorn KA, Bates LJ, Sutherland GR (1987) Linkage and genetic counselling for the fragile X using DNA probes 52A, F9, DX13 and St14. AmJ Med Genet 27:435-448 Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boue I, et al (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252:1087-1102 Pieretti M, Zhang F, Fu Y-H, Warren ST, Oostra BA, Caskey CT, Nelson DL (1991) Absence of expression of the FMR-1 gene in fragile X syndrome. Cell 66:817-822 Richards RI, Holman K, Kozman HJ, Kremer E, Lynch M, Pritchard M, Yu S, et al (1991a) Fragile X syndrome: genetic localization by linkage mapping of two microsatellite repeats FRAXAC1 and FRAXAC2 which immediately flank the fragile site. J Med Genet 28:818-823 Richards RI, Shen Y, Holman K, Kozman H, Hyland VJ, Mulley JC, Sutherland GR (1991b) Fragile X syndrome:
diagnosis using highly polymorphic microsatellite mark-
References Bell MV, Hirst MC, Nakahori Y, MacKinnon RN, Roche A, Flint TJ, Jacobs PA, et al (1991) Physical mapping
ers. Am J Hum Genet 48:1051-1057
Sherman SL, Jacobs PA, Morton NE, Froster-Iskenius U, Howard-Peebles PN, Nielsen KB, Partingon MW, et al (1985) Further segregation analysis of the fragile X syn-
980 drome with special reference to transmitting males. Hum Genet 69:289-299 Sherman SL, Morton NE, Jacobs PA, Turner G (1984) The marker (X) syndrome: a cytogenetic and genetic analysis. Ann Hum Genet 48:21-37 Sherman SL, Sutherland GR (1986) Segregation analysis of rare autosomal fragile sites. Hum Genet 72:123-128 Sutherland GR (1985) The enigma of the fragile X chromosome. Trends Genet 1:108-112 Sutherland GR, Baker E (1990) The common fragile site in band q27 of the human X chromosome is not coincident with the fragile X. Clin Genet 37:167-172 Sutherland GR, Gedeon A, Kornman L, Donnelly A, Byard RW, MulleyJC, Kremer E, et al (199la) Prenatal diagnosis of fragile X syndrome by direct detection of the characteristic unstable DNA sequence. N Engl J Med 325: 17201722 Sutherland GR, Haan EA, Kremer E, Lynch M, Pritchard M, Yu S. Richards RI (1991b) Hereditary unstable DNA: a new explanation for some old genetic questions? Lancet 338:289-292 Sutherland GR, Hecht F, Mulley JC, Glover TW, Hecht BK (1985) Fragile sites on human chromosomes. Oxford University Press, New York Suthers GK, Mulley JC, Voelckel MA, Dahl N, Vaisanen ML, Steinbach P, Glass IA, et al (1991a) Genetic mapping
Yu
et
al.
of new DNA probes at Xq27 defines a strategy for DNA studies in the fragile X syndrome. Am J Hum Genet 48: 460-467 Suthers GK, Oberle I, NancarrowJ, MulleyJC, Hyland VJ, Wilson PJ, McCure J, et al (1991b) Genetic mapping of new RFLPs at Xq27-q28. Genomics 9:37-43 Verkerk AJMH, Pieretti M, SutcliffeJS, Fu Y-H, Kuhl DPA, Pizzuti A, Reiner 0, et al (1991) Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65:905-914 Vincent A, Heitz D, Petit C, Kretz C, Oberle I, Mandel J-L (1991) Abnormal pattern detected in fragile X patients by pulsed-field gel electrophoresis. Nature 349:624-626 Weber JL, May PE (1989) Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet 44:388-396 Wyman AR, White R (1980) A highly polymorphic locus in human DNA. Proc Natl Acad Sci USA 77:6754-6758 Yu S, Pritchard M, Kremer E, Lynch M, NancarrowJ, Baker E, Holman K, et al (1991) Fragile X genotype characterized by an unstable region of DNA. Science 252:11791181 Zhu Q-S, Heisterkamp N, Groffen J (1990) Unique organization of the human BCR gene promoter. Nucleic Acids Res 18:7119-7125
