8248 Med Genet 1991; 28: 824-829
Genotype prediction in the fragile X syndrome M C Hirst, Y Nakahori, S J L Knight, C Schwartz, S N Thibodeau, A Roche, T J Flint, M Connor, J-P Fryns, K E Davies
J
Abstract males are not retarded and either do not express the Fragile X positive, mentally retarded males fragile site or only express the fragile site at a low have been shown to have an insertion or level. This can make genetic counselling particularly amplification of DNA sequences at, or close to, difficult. Although closely linked genetic markers the site of expression of the fragile site. We have been developed,68 their use is sometimes show here the application of the detection of limited by the high recombination frequency, and in such changes to the diagnosis of affected males some cases the mode of inheritance (through the and female carriers and the identification of male or female line) is unclear. normal transmitting males. One fragile X The unusual inheritance pattern observed in the negative male with the clinical features of the fragile X syndrome has led to the hypothesis that the Martin-Bell syndrome also possesses an development of the full phenotype involves a two inserted/amplified DNA sequence. The im- step process.9" The first non-phenotypic, or preplications of these results for screening for the mutation, event is converted into the full mutation fragile X syndrome are discussed. only after passing through oogenesis. Several hypotheses have been proposed as to the nature of this second mutation event, including genomic The fragile X syndrome is the commonest inherited imprinting and amplification of DNA sequences at cause of mental impairment, affecting 1 in 1500 the fragile site. Recently evidence has accumulated males.' Fifty-six percent of female carriers of the for the possible role of both of these in the progresmutation are not affected while 20% of males are sion to the fragile X phenotype. We and others have phenotypically normal transmitters of the disorder shown that fragile X patients are hypermethylated at (so called normal transmitting males, NTMs)." The a CpG island close to the fragile site.'213 These CpG disease is associated with the expression of a folate dinucleotides are not generally methylated in normal sensitive fragile site at Xq27.3 although the level of transmitting males or in their daughters but do expression can vary from a few percent to as high as become methylated in the affected grandsons, sug50%.4 Typically, daughters of normal transmitting gesting that genomic imprinting could be occurring through methylation in early oogenesis. More recently several workers have reported the insertion/ Institute of Molecular Medicine, John Radcliffe amplification of DNA sequences adjacent to these Hospital, Headington, Oxford OX3 9DU. CpG residues in fragile X patients and normal M C Hirst, Y Nakahori, S J L Knight, A Roche, T J Flint, transmitting males. 146 This insertion/amplification K E Davies event can be readily observed on Southern blots by a Greenwood Genetic Center, Greenwood, South change of fragment size. In this paper we report the Carolina 29646, USA. use of this sequence change for the determination of C Schwartz genotype in fragile X positive families and in the diagnosis of fragile X negative males. Mayo Clinic, Rochester, Minnesota 55905, USA. S N Thibodeau
Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ. J M Connor Division of Human Genetics, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. J-P Fryns Correspondence to Dr Davies. Received for publication 3 July 1991. Revised version accepted for publication 6 August 1991.
Materials and methods SOUTHERN BLOT AND HYBRIDISATION ANALYSIS
DNA from the patients was restricted with the enzymes EcoRI, BglII, or HindIII, subjected to electrophoresis, and transferred onto membranes using standard methods. DNA probe Oxl.9 was isolated from a cosmid crossing the CpG region.'7 The 1 9 kb insert was radiolabelled by the random primed method as previously described."3 Filters were washed to a stringency of 1 x SSC and
825
Genotype prediction in the fragile X syndrome
Q
B Ea S N Bg
.1
I
E Bg
E
EH
Bm
H E
IIII-
I
->zzz"ROxl-9
Cen
Tel 1 kb
(i)
1
2
3
4
5
6
7
kb Figure I (A) Genomic restriction map of the DNA surrounding the hypermethylated HTF island associated with fragile X imprinting. The position of the DNA probe Oxl.9 is shown (solid bar) immediately next to the HTF island and the region exhibiting hypervariability (hatched box). The enzymes used to generate the map are BamHI (Bm), BglII (Bg), EcoRI (E), HindIII (H), BssHII (B), NruI (N), SacII (S), and EagI (Ea). (B) Hypervariation in the fragile X region detected by hybridisation of the DNA probe Oxl.9 to EcoRI digests of DNA from a family segregating for the fragile X syndrome and an unrelated affected male (lane 1). Normal males: lanes 2, 4, and 6; affected males: lanes 3 and 7; affected female: lane 5.
exposed against Kodak X-AR5 film five days.
at
-
70°C for
one to
Results We have isolated a genomic DNA fragment, Oxl.9, which lies immediately distal to the CpG island which is hypermethylated in fragile X positive, mentally retarded males (fig 1A). Analysis of patient DNA with restriction enzymes having recognition sites on either side of the CpG island showed alterations indicating the occurrence of hypervariation of fragment length in this region. This is shown in fig 1B with EcoRI digests of DNA from members of a family segregating for the fragile X syndrome together with an unrelated affected male. Normal subjects show an EcoRI fragment of 5-1 kb (lanes 2, 4, and 6) whereas affected males show either a faint smear of fragments or a discrete fragment of higher molecular weight (lanes 1, 3, and 7). The affected female (lane 5) in the family shows a 5-1 kb fragment corresponding to her normal X chromosome and additional fragments of higher molecular weight corresponding to the mutant X chromosome. This multi-fragment pattern of the fragile X chromosome is a common feature of the mutation and probably corresponds to somatic heterogeneity in the length of this fragment.
5s1
I
40
*MO
fkO
In some subjects the pattern seen with Oxl.9 is so heterogeneous that only a smear of hybridisation is seen. This allows diagnosis of the fragile X chromosome in males but can make the genotyping of females difficult. For example, in fig 2A the two affected males in lanes 2 and 3 clearly have the mutated X chromosome but the smear seen in their mother in lane 4 is only just visible. This female is cytogenetically positive and mildly affected. Samples in lanes 1 and 6 are from females predicted to be normal from linkage analysis. In fig 2B, an affected female (lane 2) clearly shows a fragment of increased size similar to her affected brother (lane 3). The clinically normal, cytogenetically negative mother in lane 4 shows no abnormal pattern. The occurrence of these fragment changes and their relationship to the expression of the disorder was studied in more detail through the analysis of an extended family segregating for the fragile X syndrome. Fig 3 shows the analysis of samples from such a family by HindIII digestion. Two phenotypically normal carrier females (lanes 3 and 11) show a larger sized fragment corresponding to the inheritance of the mutated X chromosome. Neither of these females is fragile X positive but they are obligate carriers because they have affected children. There are two additional equivalent female carriers in this pedigree, one of whom showed an altered fragment
826
Hirst, Nakahori, Knight, Schwartz, Thibodeau, Roche, Flint, Connor, Fryns, Davies
I
2
3
4
5
6
7
1
2
6
5
4
3
i... i,
0i
-
4.. W". ..
i..
Figure 2 (A) Hybridisation of DNA probe Oxl.9 to HindIII digests of DNA from normal males (lanes S and 7), normal females (lanes 1 and 6), affected males (lanes 2 and 3), and their mother (lane 4). (B) Hybridisation of the DNA probe Oxl.9 to HindIII digests of DNA from an affected male (lane 3) and his affected sister (lane 2) in a family segregating for the fragile X syndrome. Lane I is the normal sister of these subjects, lane 4 the mother, and lane 5 the father. Lane 6 is an unrelated normal male.
on her X chromosome and the other of whom showed a very small shift seen as a tight doublet (data not shown). These phenotypically normal females have a smaller increase in molecular weight of their fragments compared with the affected female in fig 2B. A sample from an affected female in this pedigree is shown in lane 1 where an extended smear of fragments is barely visible. The normal transmitting male in this pedigree (lane 5) shows a characteristic shift in molecular weight of the HindIII fragment. Normal transmitting males generally show a much smaller shift in fragment size compared to affected males (compare lanes 5 and 8). We have observed this insertion/amplification event in 56 out of 59 unrelated fragile X positive, mentally retarded males and in seven normal transmitting males. No insertion was observed in 43 normal X chromosomes. In the three cases that do not show an insertion, it is possible that these subjects are mosaics'617 or that they are cases of
misdiagnosis.
One mentally retarded subject who was fragile X negative on two occasions was a member of a family who had distant relatives suffering from the fragile X syndrome (see pedigree in fig 4, F1, subject 6). He has bilateral epicanthic folds, normal testes, and is in a special education class with an IQ of 58. BglII digests were analysed on this occasion as these were already available from previous RFLP typings with DXS15. The digest covers the same region of the fragile site as the EcoRI and HindIII digestion, but on a larger 12 kb fragment. All the fragile X positive males in this pedigree showed a characteristic smear of gel fragments similar to that in the affected male
(subject 3, fig 4) in family F2. The fragile X negative male also showed a small shift in the size of the BglII fragment (fig 4, lane 6). This suggests that his phenotype is indeed the result of a mutation at the fragile X locus even though he is fragile X negative. Why this affected male does not express the fragile site warrants further investigation. None of the other males in this branch of the pedigree shows
Genotype prediction in the fragile X syndrome
1
2
3
4
5
6
7
8
9
827
10
11
a0* -i 31
r
2
Figure 3 (A) Hybridisation of DNA probe Oxl.9 to HindIII digests of DNA from subjects from the pedigree illustrated in fig 3(B). The lane numbers correspond to the members of the pedigree.
abnormal fragments in BglII digests. They may, however, be normal male transmitters with smaller increases in the size of the fragment not detected in these digests. The analysis of subjects in another fragile X family is also shown in fig 4. The normal transmitting male in this pedigree shows an increase in size of the BglII fragment (F2, lane 5) whereas the affected males (lanes 3 and 9) show a smear of fragments. Small amplifications at the fragile X locus are difficult to detect in BglII digests. This may be the reason why carriers in fig 4 show a single band whereas carriers in fig 3 show doublets. However, even in other digests, female carriers do not always clearly show two bands (unpublished observations). Discussion We have shown that carriers of the fragile X syndrome and affected subjects possess an insertion/
amplification of DNA sequence adjacent to the CpG island which we previously showed to be abnormally methylated in patients."3 The data presented here, together with those already published," 1-7 suggest that this is a good test for the presence of the fragile X mutation. Although some affected males do show the normal sized band, owing to mosaicism'6 or molecular heterogeneity,'7 more than 95% show an altered fragment or smear of fragments. The size of the DNA fragment close to the fragile site increases even in normal transmitting males permitting this assay to be used to determine the mode of inheritance of the syndrome in many cases. This DNA analysis therefore provides information that cytogenetic analysis does not, since normal transmitting males are generally fragile X negative. As this insertion/amplification can be seen in BglII, EcoRI, and HindlIl digests of DNA samples, Ox1.9 may be used to analyse Southern blots previously used for RFLP typings of VK23 and DX13. This may prove useful for the analysis of subjects from whom DNA samples are limited. It has been suggested that larger fragment size changes are observed for affected subjects compared to phenotypically normal carriers and this may be useful for the diagnosis of the mental impairment of a carrier female.'6 Our analyses support this view although, as noted previously, the correlation is not absolute. The detection of changed fragments in females appears to be more variable. We find no obvious correlation between the fragment size observed and the degree of expression of the fragile site, nor is there an apparent association with inheritance of the mutant allele from an NTM or female carrier parent. It is now possible to screen for the fragile X mutation in males by assaying for an increase in fragment size in the region of the fragile site. This analysis is both simple and rapid. The increase in size of the BglII fragment in a fragile X negative, mentally retarded male suggests that this assay may be more sensitive than the cytogenetic expression of the fragile site. As a primary diagnosis, it may therefore be worth screening all fragile X negative males presenting with the Martin-Bell phenotype for fragment changes. The insertion/amplification event occurs immediately adjacent to the CpG island we have shown to be imprinted in fragile X males and thus may be influencing the change in methylation status of these CpG residues. As we can detect altered fragments in normal transmitting males, this indicates that the insertion/amplification event precedes the methylation changes. This order of events may therefore represent the two stages in the development of the Martin-Bell phenotype. More detailed analysis is in progress to define completely the genetic alterations detected in the fragile X population. A gene has
Hirst, Nakahori, Knight, Schwartz, Thibodeau, Roche, Flint, Connor, Fryns, Davies
828
F2
Fl
U
U
U
E
i* *e nk + 0tW..t.*h..
2
3
4
5
6
7
8
9
2
1
7
9
3
4
2 6 8
5
6
7
8
2
10
3
Figure 4 (A) Hybridisation of DNA probe Ox1.9 to BglII digests of DNA samples of subjects from fragile X families Fl (B) and F2 (C) including a fragile X negative, mentally retarded boy (Fl, 6), fragile X positive, mentally retarded males (F2, 3 and 9), and a fragile X negative, phenotypically normal male (F2, 5). The lane numbers correspond to the members of the pedigrees. Boxes indicate males discussed in the text. Filled boxes are affected subjects. Fragile site expression is scored as present ( + ) or absent (-) above key affected males (filled box) and an unaffected NTM (box) as discussed in the text.
Genotype prediction in the fragile X syndrome
recently been identified associated with this CpG island15 and the insertion/amplification event appears to occur near the 5' end of this gene. Hopefully, the mysteries of this fascinating disorder will soon begin to become unravelled. We are grateful to the many clinicians and cytogeneticists for patient samples and their analysis. We thank Helen Blaber for typing the manuscript. We are grateful to the Medical Research Council of Great Britain, Action Research for the Crippled Child, and the South Carolina Department of Mental Retardation for financial support. These studies were also funded in part by NIH grant No MH45916. 1
2
3 4 5
6
Fryns JP. X-linked mental retardation and the fragile X syndrome: a clinical approach. In: Davies KE, ed. The fragile X syndrome. Oxford: Oxford University Press, 1990:1-39. Sherman SL, Morton NE, Jacobs PA, Turner G. The marker (X) syndrome: a cytogenetic and genetic analysis. Ann Hum Genet 1984;48:21-37. Sherman SL, Jacobs PA, Morton NE, et al. Further segregation analysis of the fragile X syndrome with special reference to transmitting males. Hum Genet 1988;69:289-99. Lubs HA. A marker X-chromosome. Am Hum Genet 1969;21:231-44. Sutherland GR. Fragile sites on human chromosomes: demonstration of their dependence on the type of tissue culture medium. Science 1977;197:256-9. Rousseau F, Vincent A, Rivella S, et al. Four chromosomal breakpoints and four new probes mark out a 10 cM region Hum Genet encompassing the FRAXA locus. Am 1991;48:108-16.
829 7 Suthers GK, Hyland VJ, Callen DF. Physical mapping of new probes near the fragile X (FRAXA) with a panel of cell lines. Am Hum Genet 1990;47:187-95. 8 Richards RI, Shen Y, Holman K, et al. Fragile X syndrome: diagnosis using highly polymorphic microsatellite markers. AmJ'Hum Genet 1991;48:1051-7. 9 Pembrey ME, Winter RM, Davies KE. A premutation that generates a defect at crossing over explains the inheritance of fragile X mental retardation. Am Hum Genet 1985; 21:709-17. 10 Nussbaum RL, Airhart SD, Ledbetter DH. Recombination and amplification of pyrimidine-rich sequences may be responsible for initiation and progression of the Xq27 fragile site: an hypothesis. Am Hum Genet 1986;23:715-22. 11 Laird C, Jaffe E, Karpen G, Lamb M, Nelson R. Fragile sites in human chromosomes as regions of late-replicating DNA. Trends Genet 1987;3:274-81. 12 Vincent A, Heitz D, Petit C, Kretz C, Oberle I, Mandel JL. Abnormal pattern detected in fragile X patients by pulsed field gel electrophoresis. Nature 1991;329:624-6. 13 Bell MV, Hirst MC, Nakahori Y, et al. Physical mapping across the fragile X: hypermethylation and clinical expression of the fragile X syndrome. Cell 1991;64:861-6. 14 Yu S, Pritchard M, Kremer E, et al. Fragile X genotype characterized by an unstable region of DNA. Science 1991;252:1 179-81. 15 Verkerk AJMH, Pieretti M, Sutcliffe JS, et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 1991;65:905-14. 16 Oberle I, Rousseau F, Heitz D, et al. Amazing instability of a 550bp DNA segment and abnormal methylation in fragile X syndrome. Science 1991;252:1097-102. 17 Nakahori Y, Knight SJL, Holland J, et al. Molecular heterogeneity of the fragile X syndrome. Nucleic Acids Res 1991;19:4355-9.
Genotype prediction in the fragile X syndrome M C Hirst, Y Nakahori, S J L Knight, C Schwartz, S N Thibodeau, A Roche, T J Flint, M Connor, J-P Fryns, K E Davies
J
Abstract males are not retarded and either do not express the Fragile X positive, mentally retarded males fragile site or only express the fragile site at a low have been shown to have an insertion or level. This can make genetic counselling particularly amplification of DNA sequences at, or close to, difficult. Although closely linked genetic markers the site of expression of the fragile site. We have been developed,68 their use is sometimes show here the application of the detection of limited by the high recombination frequency, and in such changes to the diagnosis of affected males some cases the mode of inheritance (through the and female carriers and the identification of male or female line) is unclear. normal transmitting males. One fragile X The unusual inheritance pattern observed in the negative male with the clinical features of the fragile X syndrome has led to the hypothesis that the Martin-Bell syndrome also possesses an development of the full phenotype involves a two inserted/amplified DNA sequence. The im- step process.9" The first non-phenotypic, or preplications of these results for screening for the mutation, event is converted into the full mutation fragile X syndrome are discussed. only after passing through oogenesis. Several hypotheses have been proposed as to the nature of this second mutation event, including genomic The fragile X syndrome is the commonest inherited imprinting and amplification of DNA sequences at cause of mental impairment, affecting 1 in 1500 the fragile site. Recently evidence has accumulated males.' Fifty-six percent of female carriers of the for the possible role of both of these in the progresmutation are not affected while 20% of males are sion to the fragile X phenotype. We and others have phenotypically normal transmitters of the disorder shown that fragile X patients are hypermethylated at (so called normal transmitting males, NTMs)." The a CpG island close to the fragile site.'213 These CpG disease is associated with the expression of a folate dinucleotides are not generally methylated in normal sensitive fragile site at Xq27.3 although the level of transmitting males or in their daughters but do expression can vary from a few percent to as high as become methylated in the affected grandsons, sug50%.4 Typically, daughters of normal transmitting gesting that genomic imprinting could be occurring through methylation in early oogenesis. More recently several workers have reported the insertion/ Institute of Molecular Medicine, John Radcliffe amplification of DNA sequences adjacent to these Hospital, Headington, Oxford OX3 9DU. CpG residues in fragile X patients and normal M C Hirst, Y Nakahori, S J L Knight, A Roche, T J Flint, transmitting males. 146 This insertion/amplification K E Davies event can be readily observed on Southern blots by a Greenwood Genetic Center, Greenwood, South change of fragment size. In this paper we report the Carolina 29646, USA. use of this sequence change for the determination of C Schwartz genotype in fragile X positive families and in the diagnosis of fragile X negative males. Mayo Clinic, Rochester, Minnesota 55905, USA. S N Thibodeau
Duncan Guthrie Institute of Medical Genetics, Yorkhill, Glasgow G3 8SJ. J M Connor Division of Human Genetics, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. J-P Fryns Correspondence to Dr Davies. Received for publication 3 July 1991. Revised version accepted for publication 6 August 1991.
Materials and methods SOUTHERN BLOT AND HYBRIDISATION ANALYSIS
DNA from the patients was restricted with the enzymes EcoRI, BglII, or HindIII, subjected to electrophoresis, and transferred onto membranes using standard methods. DNA probe Oxl.9 was isolated from a cosmid crossing the CpG region.'7 The 1 9 kb insert was radiolabelled by the random primed method as previously described."3 Filters were washed to a stringency of 1 x SSC and
825
Genotype prediction in the fragile X syndrome
Q
B Ea S N Bg
.1
I
E Bg
E
EH
Bm
H E
IIII-
I
->zzz"ROxl-9
Cen
Tel 1 kb
(i)
1
2
3
4
5
6
7
kb Figure I (A) Genomic restriction map of the DNA surrounding the hypermethylated HTF island associated with fragile X imprinting. The position of the DNA probe Oxl.9 is shown (solid bar) immediately next to the HTF island and the region exhibiting hypervariability (hatched box). The enzymes used to generate the map are BamHI (Bm), BglII (Bg), EcoRI (E), HindIII (H), BssHII (B), NruI (N), SacII (S), and EagI (Ea). (B) Hypervariation in the fragile X region detected by hybridisation of the DNA probe Oxl.9 to EcoRI digests of DNA from a family segregating for the fragile X syndrome and an unrelated affected male (lane 1). Normal males: lanes 2, 4, and 6; affected males: lanes 3 and 7; affected female: lane 5.
exposed against Kodak X-AR5 film five days.
at
-
70°C for
one to
Results We have isolated a genomic DNA fragment, Oxl.9, which lies immediately distal to the CpG island which is hypermethylated in fragile X positive, mentally retarded males (fig 1A). Analysis of patient DNA with restriction enzymes having recognition sites on either side of the CpG island showed alterations indicating the occurrence of hypervariation of fragment length in this region. This is shown in fig 1B with EcoRI digests of DNA from members of a family segregating for the fragile X syndrome together with an unrelated affected male. Normal subjects show an EcoRI fragment of 5-1 kb (lanes 2, 4, and 6) whereas affected males show either a faint smear of fragments or a discrete fragment of higher molecular weight (lanes 1, 3, and 7). The affected female (lane 5) in the family shows a 5-1 kb fragment corresponding to her normal X chromosome and additional fragments of higher molecular weight corresponding to the mutant X chromosome. This multi-fragment pattern of the fragile X chromosome is a common feature of the mutation and probably corresponds to somatic heterogeneity in the length of this fragment.
5s1
I
40
*MO
fkO
In some subjects the pattern seen with Oxl.9 is so heterogeneous that only a smear of hybridisation is seen. This allows diagnosis of the fragile X chromosome in males but can make the genotyping of females difficult. For example, in fig 2A the two affected males in lanes 2 and 3 clearly have the mutated X chromosome but the smear seen in their mother in lane 4 is only just visible. This female is cytogenetically positive and mildly affected. Samples in lanes 1 and 6 are from females predicted to be normal from linkage analysis. In fig 2B, an affected female (lane 2) clearly shows a fragment of increased size similar to her affected brother (lane 3). The clinically normal, cytogenetically negative mother in lane 4 shows no abnormal pattern. The occurrence of these fragment changes and their relationship to the expression of the disorder was studied in more detail through the analysis of an extended family segregating for the fragile X syndrome. Fig 3 shows the analysis of samples from such a family by HindIII digestion. Two phenotypically normal carrier females (lanes 3 and 11) show a larger sized fragment corresponding to the inheritance of the mutated X chromosome. Neither of these females is fragile X positive but they are obligate carriers because they have affected children. There are two additional equivalent female carriers in this pedigree, one of whom showed an altered fragment
826
Hirst, Nakahori, Knight, Schwartz, Thibodeau, Roche, Flint, Connor, Fryns, Davies
I
2
3
4
5
6
7
1
2
6
5
4
3
i... i,
0i
-
4.. W". ..
i..
Figure 2 (A) Hybridisation of DNA probe Oxl.9 to HindIII digests of DNA from normal males (lanes S and 7), normal females (lanes 1 and 6), affected males (lanes 2 and 3), and their mother (lane 4). (B) Hybridisation of the DNA probe Oxl.9 to HindIII digests of DNA from an affected male (lane 3) and his affected sister (lane 2) in a family segregating for the fragile X syndrome. Lane I is the normal sister of these subjects, lane 4 the mother, and lane 5 the father. Lane 6 is an unrelated normal male.
on her X chromosome and the other of whom showed a very small shift seen as a tight doublet (data not shown). These phenotypically normal females have a smaller increase in molecular weight of their fragments compared with the affected female in fig 2B. A sample from an affected female in this pedigree is shown in lane 1 where an extended smear of fragments is barely visible. The normal transmitting male in this pedigree (lane 5) shows a characteristic shift in molecular weight of the HindIII fragment. Normal transmitting males generally show a much smaller shift in fragment size compared to affected males (compare lanes 5 and 8). We have observed this insertion/amplification event in 56 out of 59 unrelated fragile X positive, mentally retarded males and in seven normal transmitting males. No insertion was observed in 43 normal X chromosomes. In the three cases that do not show an insertion, it is possible that these subjects are mosaics'617 or that they are cases of
misdiagnosis.
One mentally retarded subject who was fragile X negative on two occasions was a member of a family who had distant relatives suffering from the fragile X syndrome (see pedigree in fig 4, F1, subject 6). He has bilateral epicanthic folds, normal testes, and is in a special education class with an IQ of 58. BglII digests were analysed on this occasion as these were already available from previous RFLP typings with DXS15. The digest covers the same region of the fragile site as the EcoRI and HindIII digestion, but on a larger 12 kb fragment. All the fragile X positive males in this pedigree showed a characteristic smear of gel fragments similar to that in the affected male
(subject 3, fig 4) in family F2. The fragile X negative male also showed a small shift in the size of the BglII fragment (fig 4, lane 6). This suggests that his phenotype is indeed the result of a mutation at the fragile X locus even though he is fragile X negative. Why this affected male does not express the fragile site warrants further investigation. None of the other males in this branch of the pedigree shows
Genotype prediction in the fragile X syndrome
1
2
3
4
5
6
7
8
9
827
10
11
a0* -i 31
r
2
Figure 3 (A) Hybridisation of DNA probe Oxl.9 to HindIII digests of DNA from subjects from the pedigree illustrated in fig 3(B). The lane numbers correspond to the members of the pedigree.
abnormal fragments in BglII digests. They may, however, be normal male transmitters with smaller increases in the size of the fragment not detected in these digests. The analysis of subjects in another fragile X family is also shown in fig 4. The normal transmitting male in this pedigree shows an increase in size of the BglII fragment (F2, lane 5) whereas the affected males (lanes 3 and 9) show a smear of fragments. Small amplifications at the fragile X locus are difficult to detect in BglII digests. This may be the reason why carriers in fig 4 show a single band whereas carriers in fig 3 show doublets. However, even in other digests, female carriers do not always clearly show two bands (unpublished observations). Discussion We have shown that carriers of the fragile X syndrome and affected subjects possess an insertion/
amplification of DNA sequence adjacent to the CpG island which we previously showed to be abnormally methylated in patients."3 The data presented here, together with those already published," 1-7 suggest that this is a good test for the presence of the fragile X mutation. Although some affected males do show the normal sized band, owing to mosaicism'6 or molecular heterogeneity,'7 more than 95% show an altered fragment or smear of fragments. The size of the DNA fragment close to the fragile site increases even in normal transmitting males permitting this assay to be used to determine the mode of inheritance of the syndrome in many cases. This DNA analysis therefore provides information that cytogenetic analysis does not, since normal transmitting males are generally fragile X negative. As this insertion/amplification can be seen in BglII, EcoRI, and HindlIl digests of DNA samples, Ox1.9 may be used to analyse Southern blots previously used for RFLP typings of VK23 and DX13. This may prove useful for the analysis of subjects from whom DNA samples are limited. It has been suggested that larger fragment size changes are observed for affected subjects compared to phenotypically normal carriers and this may be useful for the diagnosis of the mental impairment of a carrier female.'6 Our analyses support this view although, as noted previously, the correlation is not absolute. The detection of changed fragments in females appears to be more variable. We find no obvious correlation between the fragment size observed and the degree of expression of the fragile site, nor is there an apparent association with inheritance of the mutant allele from an NTM or female carrier parent. It is now possible to screen for the fragile X mutation in males by assaying for an increase in fragment size in the region of the fragile site. This analysis is both simple and rapid. The increase in size of the BglII fragment in a fragile X negative, mentally retarded male suggests that this assay may be more sensitive than the cytogenetic expression of the fragile site. As a primary diagnosis, it may therefore be worth screening all fragile X negative males presenting with the Martin-Bell phenotype for fragment changes. The insertion/amplification event occurs immediately adjacent to the CpG island we have shown to be imprinted in fragile X males and thus may be influencing the change in methylation status of these CpG residues. As we can detect altered fragments in normal transmitting males, this indicates that the insertion/amplification event precedes the methylation changes. This order of events may therefore represent the two stages in the development of the Martin-Bell phenotype. More detailed analysis is in progress to define completely the genetic alterations detected in the fragile X population. A gene has
Hirst, Nakahori, Knight, Schwartz, Thibodeau, Roche, Flint, Connor, Fryns, Davies
828
F2
Fl
U
U
U
E
i* *e nk + 0tW..t.*h..
2
3
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5
6
7
8
9
2
1
7
9
3
4
2 6 8
5
6
7
8
2
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Figure 4 (A) Hybridisation of DNA probe Ox1.9 to BglII digests of DNA samples of subjects from fragile X families Fl (B) and F2 (C) including a fragile X negative, mentally retarded boy (Fl, 6), fragile X positive, mentally retarded males (F2, 3 and 9), and a fragile X negative, phenotypically normal male (F2, 5). The lane numbers correspond to the members of the pedigrees. Boxes indicate males discussed in the text. Filled boxes are affected subjects. Fragile site expression is scored as present ( + ) or absent (-) above key affected males (filled box) and an unaffected NTM (box) as discussed in the text.
Genotype prediction in the fragile X syndrome
recently been identified associated with this CpG island15 and the insertion/amplification event appears to occur near the 5' end of this gene. Hopefully, the mysteries of this fascinating disorder will soon begin to become unravelled. We are grateful to the many clinicians and cytogeneticists for patient samples and their analysis. We thank Helen Blaber for typing the manuscript. We are grateful to the Medical Research Council of Great Britain, Action Research for the Crippled Child, and the South Carolina Department of Mental Retardation for financial support. These studies were also funded in part by NIH grant No MH45916. 1
2
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