The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmission
The role of size, sequence and haplotype in the stability of FRAXA and FRAXE alleles during transmissionAnna Murray1,*, James N. Macpherson1, Michelle C. Pound1, Andrea Sharrock1, Sheila A. Youings1, Nick R. Dennis2, Nicky McKechnie2, Paul Linehan2, Newton E. Morton2 and Patricia A. Jacobs1,2
1Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury, Wiltshire SP2 8BJ, UK and 2Human Genetics, University of Southampton Medical School, Princess Anne Hospital, Southampton, Hampshire SO16 5YA, UK
Received October 4, 1996;Revised and Accepted November 15, 1996
Factors involved in the stability of trinucleotide repeats during transmission were studied in 139 families in which a full mutation, premutation or intermediate allele at either FRAXA or FRAXE was segregating. The transmission of alleles at FRAXA, FRAXE and four microsatellite loci were recorded for all individuals. Instability within the minimal and common ranges (0-40 repeats for FRAXA, 0-30 repeats for FRAXE) was extremely rare; only one example was observed, an increase in size at FRAXA from 29 to 39 repeats. Four FRAXA and three FRAXE alleles in the intermediate range (41-60 repeats for FRAXA, 31-60 repeats for FRAXE) were unstably transmitted. Instability was more frequent for FRAXA intermediate alleles that had a tract of pure CGG greater than 37 although instability only occurred in two of 13 such transmissions: the changes observed were limited to only one or two repeats. Premutation FRAXA alleles over 100 repeats expanded to a full mutation during female transmission in 100% of cases, in agreement with other published series. There was no clear correlation between haplotype and probability of expansion of FRAXA premutations. Instability at FRAXA or FRAXE was more often observed in conjunction with a second instability at an independent locus suggesting general genomic instability as a possible mechanism by which at least some FRAXA and FRAXE mutations arise.
A molecular mechanism for the non-Mendelian inheritance of the fragile X syndrome was found in 1991 with the discovery of the FMR1 gene and the CGG repetitive element in exon 1 (1 ,2 ). The CGG repeat, polymorphic in the general population, can expand during transmission such that full mutation carriers have between 200 and 2000 repeats (3 ). This gross expansion in repeat number coincides with hypermethylation of the repeat and the CpG island proximal to the gene (4 ). Methylation silences the FMR1 gene thus preventing protein production. The majority of mutations in FMR1 are expansions in CGG repeat number although a proportion, less than 5%, are due to deletions and point mutations (5 ,6 ). The phenotype of individuals with deletions and point mutations is indistinguishable from that of patients with an expansion and it therefore appears that absence of FMR1 protein causes the fragile X syndrome. The FRAXE gene is ~600 kb distal to the FRAXA gene and has a polymorphic triplet repeat of GCC at its 5' end (7 ,8 ). Expansion of this repeat is also associated with methylation of a CpG island resulting in affected individuals with a mild degree of learning disability (9 ,10 ).
Progression to a full mutation in both FRAXA and FRAXE occurs via a premutation allele which is unstably inherited and unmethylated and therefore generally considered to have no phenotypic effect (11 ,12 ). Transition from a minimal or common sized allele (<40 repeats for FRAXA, <30 repeats for FRAXE) to a premutation allele has never been observed. Rare examples of instability within the common range have been described, but have yet to be shown to progress to a full mutation (13 -15 ).
Numerous risk factors for the instability of premutation FRAXA alleles have been identified, the most important of which is the sex of the transmitting parent (16 ,17 ). It was appreciated in 1984 that expansion of fragile X alleles to a full mutation occurs exclusively during female transmission. Father to daughter transmission of a premutation is usually associated with small (<10 repeats) increases or decreases in size; male premutation carriers are therefore known as normal transmitting males (18 ).
Summary of transmissions of FRAXA and FRAXE alleles by category
Category
FRAXA
FRAXE
Stable Male
Female
Unstable Male
Female
Stable Male
Female
Unstable Male
Female
Minimal
0-10
3
0
0
0
5
19
0
0
Common
A=11-40
83
245
1
0
105
394
0
0
E=11-30
Intermediate
A=41-60
21
80
0
4
10
17
1
2
E=31-60
Premutation
60-200
1
0
17
97
0
0
0
2
Full mutation
>200
0
0
0
15
0
0
1
6
Total
108
323
18
116
120
430
2
10
The size of the allele is also an important indicator of the likelihood of expansion. The larger the premutation the more likely it is to expand to a full mutation in one generation; alleles over ~100 repeats have never failed to expand to full mutations (3 ,19 -21 ). However, most premutation alleles have fewer than 100 repeats and these constitute the biggest problems for genetic counselling, particularly near the boundary of normal and premutation. FRAXA alleles as small as 56 repeats have been shown to expand to a full mutation (14 ), although not in one generation, while other alleles of similar size can be stably transmitted (22 ,23 ).
Recent studies have suggested that both the sequence of the triplet repeat and the flanking haplotype may be risk factors for expansion (24 ,25 ). The CGG repeat at FRAXA is not pure but is frequently interrupted by an AGG, usually at every 10th, 11th or 12th repeat within the tract (26 ,27 ). It has been postulated that the length of pure CGG repeats at the 3' end of a FRAXA allele is crucial in determining its stability; the threshold for instability is thought to be between 35 and 37 pure repeats (14 ). The expansion mechanism is still unclear but it is possible that AGG interruptions anchor the triplet repeat during replication and hence loss of AGGs leads to instability. Eichler et al. suggested that an individual allele could develop a long tract of pure CGG repeats by at least two mechanisms; gradual increase in repeat number from the 3' end of the repeat or sudden loss of an AGG interruption, giving more rapid progession to an unstable allele (24 ). The periodicity of AGG interruptions is correlated with the haplotype (24 ,25 ) and, if the distribution of AGGs predisposes an allele to lose an AGG repeat, this may explain why certain haplotypes are enriched on fragile X chromosomes. One particular haplotype (2-1-3) commonly associated with FRAXA full mutations is preferentially linked to alleles in the large normal or intermediate (41-60 repeats) size range. It is possible that alleles with this haplotype progress slowly to premutations by gradual increase in repeat number and hence this haplotype is over-represented in the intermediate class. Macpherson et al. suggest a third rapid mechanism of expansion involving genes responsible for general genomic instability, possibly analagous to mismatch repair genes (13 ,28 ). In this scenario, mutation would occur at a number of loci throughout the genome and could explain the presence of rare or unique haplotypes in ~20% of families with FRAXA full mutations.
Factors affecting the stability of FRAXE alleles have yet to be determined owing to the small number of affected families reported. However haplotype is clearly one factor as allele 3 at locus DXS1691 is significantly increased both in affected families and in conjunction with FRAXE alleles in the intermediate size range (23 ).
The aim of this study was to evaluate the stability of alleles of all sizes at the FRAXA and FRAXE loci in a large number of unrelated families, ascertained either from the diagnostic service in our region or via an ongoing population screening program (23 ). Families were selected because an allele of interest was segregating within them: such alleles included premutations, full mutations, intermediate alleles (41-60 repeats for FRAXA and 31-60 repeats for FRAXE), and minimal alleles with fewer than 11 repeats. We report our results on stability of transmission in 139 families. Four microsatellite markers flanking FRAXA and FRAXE were used to construct a haplotype and the sequence of the repeat region of FRAXA was determined wherever possible. Collation of data on FRAXA and FRAXE allele stability, haplotype and sequence was used to assess possible mechanisms of instability.
Instability at FRAXA was observed infrequently in the minimal, common and intermediate ranges (Table 1 and 2 ). There were no cases of instability in the minimal range and one increase in size from 29 to 39 repeats in the common range, the paternal 29 allele being transmitted stably to a second daughter (Table 2 , #34). This family is unique as the instability at FRAXA is associated with several other anomalies (Table 2 , #13) which are described in more detail below and have been partially reported previously (13 ). There were four instabilities of FRAXA alleles in the intermediate range (Table 2 , #1, #6, #17 and #37), three of which were changes of just one triplet repeat. A change from 50 to 51 repeats was in a survey family and the proband boy inherited the 51 allele from his mother in a stable fashion but her mother had only 50 repeats (#17). This is one of only two examples in our population of alleles that have been transmitted both stably and unstably. A change from 55 to 56 repeats (#6) was in a family in which a FRAXA full mutation was segregating, on an independent chromosome (#5). Thirdly there was a single repeat increase from 53 to 54 repeats (#1) reported previously in a family ascertained in the screening survey (23 ). Finally, one allele fell into our intermediate category at 60 repeats, but was shown to expand first to 68 repeats and then to a full mutation (#37). All four instabilities of intermediate FRAXA alleles occurred during female transmission, but this is likely to be due to the excess of female transmissions studied; 84 female versus 21 male transmissions (Table 1 ).
Instabilities at FRAXA by haplotype and sex of parent and offspring; families are separated within each haplotype
Haplotype
Ch no.
Female -> Male
Female -> Female
Male -> Female
Parental size
Offspring size
Parental size
Offspring size
Parental size
Offspring size
213
1
53
54
2
F (690)
F* (430)
3
P (130)
F (660)
P (130)
F (600)
4
P (?)
F (360)
5
F (280)
F (1630)
91
F (280)
6
55
56
7
80
159
80
P (107)
P (107)
F (830)
80
100
100
F (760)
100
F (290)
8
F (560)
F (1000)
F (560)
F (1530)
644
9
78
F (460)
67
78
79
87
78
F (530)
78
F (240)
67
104
10
88
F (490)
88
F (460)
F (460)
F (560)
F (460)
F (560)
11
95
P (147)
95
F (430)
95
F (330)
95
F (230)
12
82
F (180)
74
82
79
81
74
79
82
F (280)
79
82
79
73*
79
71*
13
86
F (480)
86
F (160)
14
71
74
15
92
P (97)
P (87)*
16
78
F (560)
645
17
50
51
18
97
F (890)
P (157)
F (?)
97
F (350)
19
70
F (280)
70
F (660)
20
82
F (?)
87
82*
21
100
F (260)
100
F (730)
22
71
F (660)
71
71
23
84
F (1560)
24
P (113)
F (?)
P (113)
F (290)
P (80)
P (113)
25
F (360)
F (330)*
734+
26
65
71
67
79
65
67
27
P (90)
F (360)
28
80
F (760)
74
80
61
74
74
96
Table 2.Continued
74
65*
29
101
F (270)
72
101
80
F (310)
69
72
80
F (220)
69
80
30
F (400)
F (460)
77
F (400)
77
F (800)
F (400)
F (760)
31
74
F (600)
32
P (130)
F (?)
P (87)
P (130)
P (93)
P (170)
33
F (700)
F (250)*
F (700)
F (830)
734
34
29
39
35
100
F (260)
83
F (350)
36
P (123)
F (1160)
P (123)
F (1860)
79
P (123)
37
68
F (?)
60
68
38
F (460)
F (1130)
834+
39
76
F (?)
71
69*
76
83
71
75
40
88
F (?)
88
F (830)
41
P (167)
F (700)
74
P (167)
P (167)
F (830)
42
76
F (500)
713
43
104
F (1200)
87
104
44
89
124
45
83
F (180)
724
46
F (800)
F (500)*
84
F (800)
47
70
F (1200)
735
48
F (180)
F (800)
P (70)
F (180)
49
F (900)
F (210)*
113
50
P (210)
F (760)
212
51
80
F (560)
74
80
234
52
P (130)
F (400)
64
P (130)
P (130)
F (560)
P (130)
F (860)
445
53
P (187)
F (560)
89
P (187)
613
54
P (200)
F (300)
82
P (200)
634
55
P (117)
F (500)
P (117)
F (313)
F (313)
F (660)
634+
56
97
P (107)
646+
57
95
F (?)
83
95
83
87
845
58
P (?)
F ( 360)
744
59
F (310)
F (660)
P, premutation; F, full mutation; *decrease in size.Numbers in parentheses are an estimate of repeat number from Southern blot.Entries in bold type are associated with a second instability on the same chromosome.
At least one transmission of the mutated allele was studied in 55 families in which a FRAXA full or premutation was segregating. The relationship between the size of a premutation allele and its likelihood of expanding to a full mutation during female transmission is shown in Figure 1 . Probands in each family were excluded to correct for ascertainment bias. Premutation sizes were calculated from PCR band sizes or were estimated from Southern blots; premutation/full mutation mosaics being treated as full mutations. Premutations over 100 repeats expanded to full mutations in 100% of cases.
Analysis of the FRAXA repeat by the MnlI digestion technique (14 ) was performed on all alleles in males that were in the intermediate and premutation ranges and visible by PCR. The distribution of AGG triplets within the CGG repeat was determined in 54 males and compared to the stability of the allele in the family and its haplotype (Table 3 ). With the exception of one common and one intermediate allele, individual alleles in families were either always stably inherited or always unstably inherited, although for many alleles only one or two transmissions were studied. All premutations contained either a pure CGG tract or had a single AGG interspersion at the 5' end of the gene. Thirty five meioses, 15 male and 20 female, involving the sequenced premutation alleles were studied and all were unstably transmitted. There was no obvious correlation between haplotype and the presence or absence of an interspersion in the premutations.
One of the two unstable alleles from the intermediate range was found to have no AGG interruptions. The allele increased by only one repeat from 53 to 54 during female to male transmission (Table 2 , #1). This allele was on a 2-1-3 haplotype, which is usually associated with at least two AGG interruptions (24 ). In our sample there were 19 stably transmitted alleles occurring on the 2-1-3 haplotype and 18 of these had two or more interruptions. The other intermediate FRAXA allele was both stably and unstably inherited (Table 2 , #17) and had a single AGG interspersion with 41 pure CGGs at the 3' end, which is above the predicted threshold for instability of 35-37 repeats (14 ). In addition to these two cases, there were six independent alleles (#60, #61, #62, #63, #64 and #65) with pure CGG tracts greater than 35, that were transmitted a total of 10 times, all stably. One of these (#62) was stably transmitted during four meioses despite having a 44 pure CGG repeat tract. This allele was on a 7-2-4 haplotype, which is enriched on fragile X chromosomes; it represents 6.6% of fragile X haplotypes but only 0.1% of controls (28 ). Thirty six of the 42 intermediate alleles stably transmitted had tracts of pure CGGs less than 35 and were stably transmitted through 76 meioses.
The correlation between haplotype and repeat sequence was similar to previously published series, i.e. all 7-3-4+ haplotypes had 10 or 11 CGGs at the 5' end of the repeat compared to nine CGGs on most other haplotypes. The 43 repeat allele in our study on a 6-4-4 haplotype had a 9+10+12+9 interspersion pattern (#99), confirming that the middle CGG repeat tracts are usually longer than the flanking tracts on this haplotype (24 ).
Pattern of AGG interspersion for all intermediate and premutation FRAXA alleles, in males, compared to numbers of stable and unstable transmissions, number of CGG repeats and haplotype
. Instabilities at FRAXE, grouped by sex of parent and offspring
DXS1691
Ch no.
Female -> Male
Female -> Female
Male -> Female
allele
Parental size
Offspring size
Parental size
Offspring size
Parental size
Offspring size
3
55
41
42
3
102
40
41
3
103
37
27/37
3
104
66
87
3
105
P (98)
F (350)
F (680)
P (98)*
3
106
F (550)
F (480)*
F (380)
F (550)
F (380)
F (365)*
F (380)
F (615)
F (350)
F (330)*
F (550)
F (480)*
P, premutation; F, full mutation; *decrease in size.Numbers in parentheses are an estimate of repeat number from Southern blot.Entries in bold type are associated with a second instability on the same chromosome.
At FRAXE no instabilities were detected in the minimal or common ranges but there were three instabilities in the intermediate range (Table 4 ). One, 41-42 repeats (#55), was found in conjunction with an expansion at FRAXA from a premutation to a full mutation. The second, 40-41 repeats (#102), was in a family in which a FRAXA full mutation was segregating on an independent chromosome (#18). Finally we found an unstable transmission of a 37 repeat allele which gave a mosaic pattern in the offspring of 27 and 37 repeats (#103) (23 ).
We have three families with FRAXE pre- or full mutations who have been reported previously (9 ,23 ,29 ); the unstable transmissions in these families are summarised in Table 4 . One family, ascertained as a result of the fragile X screening survey, is a rare example of a FRAXE premutation and in this case unique as it was ascertained in the absence of a full mutation carrier (#104). A second family is also notable because a male with a full mutation has reproduced and had a daughter with a premutation allele (#105).
Four microsatellite markers spanning ~1 Mb were tested. In more than 100 male and 400 female meioses studied no changes in repeat number during transmission were detected at three of the markers, FRAXAC1, FRAXAC2 and DXS1691, (Table 5 ). At the most proximal marker, DXS548, five apparent mutations were detected; however we have evidence to suggest that four of these can be accounted for by a PCR artifact, possibly due to a rare polymorphism at one of the PCR primer binding sites. This is suggested by the haplotypes of the individual chromosomes on which these `mutations' occured (Table 6 ); all except #104 have an identical 4-29-6+ FRAXAC1-FRAXA-FRAXAC2 haplotype. Females with this haplotype appear homozygous at DXS548. However if this rare polymorphism causes inefficient primer annealing, there may be preferential amplification of the common allele, independent of its size, and a heterozygote would appear homozygous.
Table 5 Numbers of stable and unstable transmissions for the microsatellite markers
Stable meioses
Unstable meioses (mutation)
Male
Female
Male
Female
DXS548
121
432
1
4
FRAXAC1
122
437
0
0
FRAXAC2
121
434
0
0
DXS1691
122
436
0
0
There is, therefore, only a single genuine mutation at one of the microsatellites, DXS548 (#104). The mother's allele was seven but the son had a mosaic pattern, with apparently three alleles (3/4+/4) and is of particular interest because it occurred in conjunction with a second mutation on this chromosome, an instability at FRAXE in the premutation range.
In four transmissions a single cross-over event occurred and in one transmission there appeared to be two exchanges (Fig. 2 and Fig. 3 ). As expected from the physical map, the most common region for recombination was between FRAXAC2 and FRAXE, a distance of ~600 kb (29 ,30 ). Two definite cross-over events occurred in this region (Families 4 and 5) but a third, (Family 3) was not completely informative occurring either between FRAXA and FRAXAC2 or between FRAXAC2 and FRAXE. Recombination in Family 3 was also associated with a second instability on one of the chromosomes involved in the cross-over (Table 2 , #9), a FRAXA premutation of 79 repeats in a male, which we assume was inherited unstably, although we were unable to obtain a sample from his mother. Nevertheless, we are confident of the maternal genotype because we have tested several of his sibs (Fig. 3 ). The cross-over in Family 2 occurred between FRAXAC1 and FRAXA, a physical distance of ~7 kb (31 ).
Instabilities outside the premutation and full mutation ranges have been rarely described, possibly because transmissions of alleles in this range are infrequently studied owing to the presumed lack of clinical significance of these alleles. It is therefore difficult to draw conclusions from reports of instability with no indication of how many stable transmissions were studied. We have selected families with intermediate, premutation and full mutation alleles at FRAXA or FRAXE and compared the incidence of instability at these loci to both sequence and haplotype. One hundred and thirty nine families were analysed and a range of repeat alleles were represented, albeit enriched for the larger size ranges. Change in repeat number within the minimal and common ranges was rare, only one such example being found in a common FRAXA allele. This family is a special case and will be discussed in detail later. We studied 87 male and 245 female meioses of FRAXA alleles and 110 male and 413 female transmissions of FRAXE alleles in the minimal and common ranges, and no other instabilities were detected. In our recent survey of males with learning difficulties, stability of alleles during female transmission was studied and we found no instances of instability in the minimal and common ranges in 726 transmissions (23 ). We are aware of only two cases, other than ours, in which a FRAXA allele with fewer than 40 repeats or a FRAXE allele with fewer than 30 repeats has changed during transmission: a 34 repeat FRAXA allele that was both stably and unstably transmitted in two separate transmissions (14 ) and a 29 repeat allele that reduced in size to 21 repeats (15 ). Mutation at FRAXA and FRAXE therefore appears to be rare for alleles in the common and minimal ranges. Obviously cryptic mutations may occur, such as conversion of an AGG to a CGG, which would go undetected by our screening protocol, but it has been shown that such mutations account for very little of the heterozygosity seen at FRAXA and therefore we may infer that this is a rarer form of FRAXA mutation than simple gain or loss of triplet repeats (32 ).
We observed seven changes in repeat number during transmission of intermediate alleles (four at FRAXA and three at FRAXE). The likelihood of expansion of an individual allele is thought to correlate with the length of pure CGG repeats rather than total repeat number; we therefore sequenced FRAXA intermediate and premutation alleles. FRAXE alleles were not sequenced as a convenient technique was not available and also it has been shown that the GCC repeat is uninterrupted in 21 independent alleles (33 ); we have therefore assumed our FRAXE alleles to be pure.
Only two of the four FRAXA intermediate alleles which increased in size during transmission could be sequenced as a male carrier is required for the test. One had a pure sequence of 54 repeats and increased in size by only one triplet during female transmission. A similar allele of 54 pure repeats (14 ) was originally described by Fu et al. (3 ) and was unstably inherited during all five transmissions studied, ranging from 52 to 60 repeats in the offspring of the 54 repeat carrier female. In both families the 54 repeat alleles were on a 2-1-3 haplotype (Table 3 ; 24 ) and neither had a full mutation proband. A third example of a completely pure intermediate sized allele was reported by Eichler and Nelson (32 ): a paternal 42 repeat allele was transmitted stably to one daughter but expanded by one triplet to a second daughter. These limited data certainly suggest that pure intermediate alleles are unstable, although not in 100% of transmissions, but the change appears limited to fewer than five repeats. Whether these pure intermediate alleles should be regarded as incipient premutations is debatable but it is important that the outcome of future transmissions be monitored so that accurate risk assessments can be made. FRAXE alleles appear to be pure and therefore any allele over ~35 repeats should be regarded with the same degree of caution.
The second sequenced intermediate allele in our series shown to be unstable had 51 repeats and although the index male had inherited an unchanged allele his maternal grandmother had only 50 repeats. This allele had only one AGG interspersion leaving 41 pure CGGs at the 3' end of the repeat, in excess of the predicted threshold for instability of ~35 repeats. However it is clear from this case and others in the literature (24 ,34 ) that instability is not certain at each transmission for intermediate alleles of this kind. In our series there were six alleles with >35 pure repeats that were always stably transmitted. Premutations usually have one or no AGGs in contrast to the modal pattern of two AGG interspersions (26 ,35 ). Thus loss of AGG interruptions may be a frequent event in the generation of an unstable premutation (14 ) but it may be necessary for this loss to coincide with a second event, possibly an expansion in repeat number, before an allele becomes committed to the full mutation expansion.
We have described 55 families with an instability at FRAXA and three with an instability at FRAXE, in the pre- or full mutation ranges. At FRAXA the likelihood of expansion of a premutation to a full mutation was correlated with size of the maternal premutation, with 100% of premutations greater than 100 repeats expanding to a full mutation in one generation. This agrees well with other published data (3 ,19 -21 ). We corrected for ascertainment bias in the same way as Heitz et al. (19 ), by removal of affected probands. Decreases in repeat number were seen in 10 transmissions, however the female transmissions all involved full mutation alleles and review of the Southern blots revealed that the smears representing the expansions extended into the size ranges seen in the offspring and therefore may not be true examples of decreases. In contrast the male transmissions with decreases in repeat number showed no such overlap with offspring sizes and must be considered genuine. Therefore, size reductions appear more common in male transmissions and it has been suggested that this may be due to lack of expansion rather than contraction in repeat number, as males may have smaller FRAXA alleles in sperm than in somatic tissue (21 ). Thus in some male transmissions post-zygotic expansion may not exceed the somatic expansion that occurred in the father and the allele would appear to have reduced in size.
At FRAXE the number of families with full and premutations is too small to make accurate risk assessments and these families have been discussed in previous publications (9 ,29 ).
The haplotypes of all unstably transmitted chromosomes are shown in Table 2 and 2 and those tested for AGG interspersions in Table 3 . Apart from the observation reported previously by us (28 ) and others (36 -38 ) that there is a different distribution of haplotypes in FRAXA full mutation carriers compared to normals, no clear correlation between haplotype and stability of the repeat was found. There appeared to be no difference in the size of expansions at each transmission between families grouped by haplotype but after removal of probands the numbers in each class were too small for statistical comparison. This implies, however, that although haplotype is a major risk determinant for fragile X it probably acts at the level of the initial mutation event, which commits an allele to progress to a full mutation, and once that threshold has been reached premutations on any haplotype adopt a similar expansion course.
We observed no mutations at three of the four microsatellite markers tested, FRAXAC1, FRAXAC2 and DXS1691. This is perhaps not unexpected as the estimated mutation rate for microsatellites is ~0.1% (39 ,40 ) and we have only studied ~500 transmissions. However, Zhong et al. reported an unexpectedly high mutation rate for FRAXAC2 of 3.3% and described FRAXAC2 as a highly mutable polymorphism (41 ). In reply to Zhong et al. three groups were unable to substantiate these findings (42 ) and commented that the four mutations detected by Zhong et al. originated from two families, in the first family the result could be explained by recombination and in the second, mutations to three sibs suggests that this family is not representative. Our data agree with those of Richards et al. and Mornet et al. (42 ); despite being a complex repeat, FRAXAC2 is no more mutable than other dinucleotide repeats. We did detect a mutation at DXS548, where the parental allele was seven but the offspring had a mosaic pattern that did not include the parental seven allele indicating mitotic instability of this allele. This case was particularly interesting as the individual had a premutation for FRAXE, inherited unstably from his mother.
Five transmissions were seen in which a crossover event was detected and, as expected from the physical distances involved, were most frequently in the FRAXAC2/FRAXE interval, although one was seen between FRAXAC1 and FRAXA, a distance of only ~7 kb apart (31 ); however this family was otherwise unremarkable. Recombination between DXS548 and FRAXA is not rare, several having been described including one in our series (43 ).
One family in our series is remarkable and although described elsewhere (13 ) an additional rare event was observed as a result of the current study. The mother underwent prenatal testing because she was a FRAXA premutation carrier, her fetus did not carry the premutation but the liveborn child subsequently died from DiGeorge syndrome due to a 22q deletion. Haplotype analysis of both parents and the child showed a paternal expansion at FRAXA within the normal range (29 repeats increased to 39) and also a recombination between DXS548 and FRAXAC1 on the maternal chromosome. More extensive haplotype analysis in the present study demonstrated a second recombination event on the maternal chromosome between FRAXAC2 and FRAXE. Such a double cross-over is not considered possible over this short distance (<750 kb) due to physical restrictions on chiasma formation and our observation probably represents a more complex scenario such as mutation plus recombination, or gene conversion. Thus, at least three rare genetic changes have occurred in this one individual. The parents subsequently had a second child which inherited the mother's normal FRAXA allele and is clinically and genetically normal. A similar complex event involving multiple exchanges was reported by Ouweland et al. (44 ); in this family the event involved a maternal X carrying a FRAXA premutation but, as in our example, the resulting recombined chromosome excluded the expanded allele.
These unusual families led us to test the hypothesis that a mutation and/or recombination event occurs more frequently on chromosomes that have also undergone a change at FRAXA or FRAXE, compared to the normal frequency of mutation and recombination.We studied 145 transmissions with a change at either the FRAXA or FRAXE locus and looked at how often there was a second change; either a mutation at one of the other five microsatellites in that haplotype or a recombination involving the chromosome of interest (Table 7 ). Of these 145 transmissions two chromosomes had an instability at a second locus, one at FRAXE, and one at DXS548; a third chromosome was involved in a recombination event. The original family with the FRAXA expansion plus several other genetic changes was also included in this group. In the 430 transmissions in which there was no change at FRAXA or FRAXE (group 2) there were no mutations at other loci and three recombinations. When mutation and recombination were considered together the difference in the incidence of a second change between the two groups was suggestive but did not reach statistical significance by Fishers exact test, one tailed (P = 0.071). When only mutations and the `double recombinant' are included the difference is significant by Fishers exact test, one-tailed (P = 0.016); single recombinants appear independent of mutation. The small number of events observed makes any conclusions drawn from these data tentative but preliminary evidence supports the hypothesis that some chromosomes or perhaps individuals are predisposed to mutational changes at more than one locus. Such effects may be regulated by genes in cis or trans and act either randomly across the whole genome or more locally over a restricted area. This hypothesis could explain the occurrence of rare or unique haplotypes in families with FRAXA full mutations. If expansion at FRAXA were caused by such an instability gene it is plausible that other loci could be affected by the same gene thereby giving rise to new alleles and hence rare or unique haplotypes; this would also be seen as an increase in heterozygosity for these loci as reported by Zhong et al. (37 ).
Individuals studied were from families that were ascertained for one of two reasons; either families in which a fragile X full mutation was segregating, collected as part of the diagnostic service in the Wessex region over the last 15 years, or from our fragile X screening survey (23 ) (Table 8 ). All individuals from the survey shown to have a FRAXA or FRAXE allele outwith the common range (11-40 for FRAXA, 11-30 for FRAXE) were visited, a family pedigree taken and samples requested from as many family members as possible in order to determine the stability of the alleles of interest during transmission. A few families fell into both categories, either because they were from fragile X families with a male child who satisfied our criteria for inclusion in the survey or because they were families of boys with a full mutation ascertained during the survey. Transmissions of all alleles within the families were studied, not just those of particular interest.
DNA from blood or buccal cells was used to determine the number of repeats at both FRAXA and FRAXE, and a haplotype was constructed using four polymorphic makers, DXS548, FRAXAC1, FRAXAC2 and DXS1691, flanking the two triplet repeats using fluorescent sequencer technology described previously (23 ). All individuals from families in which a full mutation was segregating were screened by Southern blot to determine the size of the expansion (45 ). The sequence of 54 FRAXA alleles was determined using an MnlI digestion technique (14 ), the recognition site for this enzyme is GAGG and therefore it detects AGG repeats interrupting the CGG repeat. The test can only be done on males, and all males with either an intermediate or premutation FRAXA allele, visible by PCR, were analysed.
We would like to thank Evan Eichler for assistance with the MnlI digestion technique. This work was supported by a programme grant from the Wellcome Trust.
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