Transition from premutation to full mutation in fragile X syndrome is likely to be prezygotic
Transition from premutation to full mutation in fragile X syndrome is likely to be prezygoticCéline Moutou1,+, Marie-Claire Vincent1,+, Valérie Biancalana1 and Jean-Louis Mandel1,2,*
1Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine et CHRU, 67085 Strasbourg cedex, France and 2IGBMC, INSERM/CNRS/Université Louis Pasteur, 67404 Illkirch, France
Received January 9, 1997;Revised and Accepted April 1, 1997
In the fragile X syndrome, the transition from unmethylated moderate expansions of the CGG repeat (premutations) to methylated large expansions (full mutations) occurs only through maternal transmission. The risk of such transition is highly correlated with the size of the maternal premutation (PM), being very low for small PM alleles (~60 repeats), to 100% for alleles above 100 repeats. The timing of this transition was the object of much speculation. A postzygotic transition was proposed as a preferred model, based on the observation that males with full mutation (FM) have PM in sperm. Analysis of tissues from affected fetuses, including additional data reported here, indicate that such a putative postzygotic transition would have to occur very early in embryogenesis and most likely before determination of germ cell lineage. At least 15% of carriers of a FM show a significant proportion of white blood cells carrying a PM (mutation mosaics). We performed a simulation study showing that, if transition to FM is postzygotic, one should observe a much higher proportion of such mosaics in offspring of mothers with small PMs. This was compared with the actual pattern observed in 212 mutated offspring of 112 PM carrier mothers. We found no effect of maternal PM size on incidence of mosaicism in leucocytes. We propose that this is strong, albeit indirect evidence against a postzygotic transition to FM. A transition at an early morula stage (before day 3) cannot, however, be formally excluded.
The fragile X syndrome, the most common cause of inherited mental retardation, is caused by an unstable expansion of a CGG repeat located in the 5' untranslated region of the FMR1 gene (1 ,2 ). Three major types of alleles can be distinguished. Normal alleles have between six and ~50 CGG repeats, generally with one or two AGG interruptions. Premutations (PMs) are moderate expansions of the repeat (from 60 to ~200 units), that are unmethylated on an active X chromosome, and that do not affect FMR1 expression. They are thus found in clinically normal male or female carriers. Full mutations (FMs) are methylated larger expansions (>230 CGG repeats) that prevent transcription of the FMR1 gene and result in mental deficit in ~100% of males and in ~60% of heterozygous carrier females (3 ,4 ). The FM often shows somatic heterogeneity. In ~15% of carriers of a FM, a mixture of PM and FM can be detected by Southern blot analysis of leucocyte DNA. These have been called mutation mosaics (3 ). With more sensitive methods of detection, minor PM sized fragments can be detected in as many as 40% of the FM carriers (5 ).
The transition from PM to FM occurs exclusively by maternal transmission (3 ,6 ). It was shown that risk of expansion is highly correlated with the size of the maternal PM, being very low for PMs of ~60 repeats, and close to 100% for PMs larger than 90-100 repeats (7 -10 ). Even in the absence of such transition to FM, there is a tendency for increased length of PM upon maternal transmission, but not upon paternal transmission. These features of the transmission of the mutated FMR1 alleles account for the characteristics of the segregation of the fragile X syndrome (11 ).
Other diseases due to expansions of trinucleotide repeats (notably Huntington's disease and myotonic dystrophy), also show parental biases for the transmission of a more expanded allele, that account for the phenomenon of anticipation. In the case of Huntington's disease, a paternal bias for transmission of a larger pathologic allele can be explained by selective instability of the repeat in male gametogenesis (12 ,13 ).
When the maternal bias in expansion of the CGG repeat was found, it was initially proposed to occur during oogenesis (14 ). However, Reyniers et al. (15 ) showed that three male carriers of a FM (and a fourth with a mosaic PM-FM pattern) had a PM in sperm. Furthermore, a small number of affected males with a FM who have procreated, were found to have premutated daughters (16 ,17 ). This led to the suggestion that transition to FM is a postzygotic event that spares the male germ line, and occurs early in embryogenesis (15 ). Indeed several observations have suggested that somatic instability of fragile X mutations is linked to an early period of embryo development (18 ,19 ). A postzygotic expansion occurring only for a PM carried on the maternal X chromosome would, however, require an imprinting mechanism distinguishing it from a PM carried on the paternal X, that would not expand (15 ,20 ). The alternate hypothesis of prezygotic expansion to FM, followed by postzygotic reversion and selection against FM (or in favour of expression of the FMR1 protein) was thought unlikely (15 ), and this appeared confirmed by observations in knockout mice that are defective for FMRP expression, but do not show male sterility (21 ), and in a family with a paternally-derived deletion in the FMR1 gene (22 ). We pointed out, however, that selection may still be present, even in cells that do not appear to depend on FMR1 expression (1 ,23 ). More recently, an elaborate model has been proposed that features limited expansion of the PM during oogenesis and spermatogenesis, and a postzygotic expansion to FM limited to maternal X chromosomes (24 ).
Here we present data that further indicates that if expansion is postzygotic, it must occur very early during embryogenesis. Furthermore, we show that if the strong dependence of the risk of transition to FM upon size of the maternal PM reflects an effect at the individual cell level, one would predict that PM-FM mosaics would be more likely to have mothers with small PMs. Systematic analysis in 112 premutated mothers and in their offspring shows that this prediction is not fulfilled. We thus conclude that a postzygotic expansion to FM is very unlikely.
We have examined the heterogeneity of the FM in leucocytes of patients and in tissues of two affected fetuses, in order to estimate a window for a putative postzygotic transition. One hundred FMs were examined in EcoRI and/or EcoRI+EagI blots where the size of fragment is 5.2 kb plus the size of expansion (3 ). In 24 of them, a single narrow band was observed, and in another 20 cases two such bands were detected. Half of the patients showed a more heterogeneous pattern (Fig. 1 ). The simplicity of the pattern observed in half of the patients suggests that the transition to FM cannot occur after the time of commitment of white blood cell precursors.
The probability of transition from PM in the mother to FM in child is strikingly dependent on the size of the maternal PM. In particular, the studies of Heitz et al. (9 ), Vaisanen et al. (26 ) and Fisch et al. (10 ) have corrected for ascertainment bias in favour of affected males (carrying a FM), and thus better reflect the effect of PM on the probabilities of transition than the earlier studies (7 ,8 ). Under the hypothesis that this transition is postzygotic, one has to assume that the observed effect reflects a probability of transition at the individual cell level. Intuitively, one would expect that if the zygote carries a small maternal PM, the probability that all cells undergo transition would be lower than if the zygote carries a large PM, with a 100% transition probability at the cell level. One would thus predict that mosaics for PM and FM are more likely to be children of mothers with relatively small PMs.
We wished however to check by simulation analysis that this expectation would lead to measurable differences in the nature (mosaic or `pure' FM) of the offspring as a function of the maternal PM size, under various hypotheses concerning the timing of the transition or the shape of the transition probability curve for an individual cell. We assumed that, if postzygotic, such a transition should occur very early in the embryo. If the transition occurred when a large number of cells are present, that are precursors for various tissues, one would expect that very heterogeneous and dissimilar patterns would be found in different tissues. Analysis of several affected fetuses has shown that this is not the case (see above).
As diagnosis of FM, PM or mosaic status, is based on analysis of white blood cells, we had to take into account for our simulations the number of cells in the embryo proper when the transition occurs, and the number of committed precursors from which white blood cells are derived. We tested transition at the 4, 8 or 16 cell stages (assuming that a later transition would not account for the similarity in mutation patterns in various fetal tissues). We assumed that the number of white blood cells precursors was between 8 and 32. This is based on the observation that ~10% of normal women show strongly biased inactivation in leucocytes (9 ,27 ). This rules out both a very small number of precursors ( <= 4) that would imply a much larger proportion of biased inactivation, or a larger number ( >= 32) that would be incompatible with the observed proportion of women with biased inactivation. A rather small number of precursors is also consistent with the very simple mutation pattern observed in white blood cells in about half of the patients tested (Fig. 1 ).
We first tested various models for transition probabilities at the single cell level assuming that the probability was 0 for 50 CGGs and 100% for 100 CGGs. We assumed that a child diagnosed with a FM must have at least 50% of his cells carrying a FM. This is justified by the fact that the penetrance of mental retardation in males with a FM is very close to 100% while it is only 60% in carrier females, who have on average 50% of all cells without a functional FMR1 gene (3 ,4 ,28 ). Quadratic or cubic functions fitted better with published observations (10 ), than a linear function for a transition at the 4 or 8 cell stages (not shown). We then used such functions to model the observations in white blood cells. We assumed that in a standard Southern blot experiment, individuals with >= 90% FM and <= 10% PM will be detected as FM only. Similarly, individuals with less than 25% FM in leucocytes will appear as PM only (as FM is often heterogeneous, a small proportion will be harder to detect than for a PM). Examples of simulations of the proportion of PMs, mosaics or FMs that would be observed in leucocytes from offspring as a function of maternal PM size, are shown in Figure 3 . In the various cases tested, there was always a strong effect of maternal PM size on the proportion of observed mosaics, that will be highest for the intermediate sizes. This prediction was then compared with the experimental observations.
We have tested by PCR analysis PM size in 112 carrier women who had at least one child carrying an expanded allele. These mothers had a total of 212 children with a PM, a FM or a mosaic pattern, and had been ascertained through our diagnostic laboratory using Southern blot analysis (EcoRI+EagI digest) (3 ). The only selection was availability of DNA for study. Selection through a diagnostic laboratory will lead to under-representation of small PMs with a low risk of transition to FM, but will not affect our conclusions that are based on relative risk of outcome for each PM size class. We also re-tested 185 of the 212 children by PCR analysis in order to detect and size PM-sized expansions (there was no DNA left for the 27 remaining children) (Fig. 4 ). In order to reliably amplify and size the PM, we tested various methods of PCR amplification. We found it was critical to use NaOH denaturation prior to amplification and to omit KCl from the reaction buffer (following a personal communication from Dr W. Block, Applied Biosystems).
Since the discovery of the maternal only transition from PM to FM, the question of timing of such an event has been debated. When it was found that FM males have a PM in sperm, and that the few known to have children had premutated daughters, it was proposed that such observations fitted best with a postzygotic transition model, where transition would not occur in the male germ line (15 ,16 ). The alternative prezygotic expansion model necessitates postzygotic contraction events followed by a selection for PM sized alleles in the male germ line (20 ). It was proposed that such a selection is unlikely, based on the observation of a single family where an apparently clinically normal male (of unknown mutation pattern), transmitted an FMR1 inactivating deletion to his three daughters (22 ), and on the fact that FMR1 deficient knockout male mice are fertile (21 ). These observations suggested that the FMR1 protein is not required for spermatogenesis, despite its presence in early spermatogonia (29 ,30 ). The postzygotic expansion model requires, however, an equally unproven imprinting that would distinguish, in the fertilized zygotes, between a PM carried on the paternal chromosome (that would escape transition to FM), and a PM carried on the maternal chromosome, that would undergo such transition with up to 100% probability (1 ,15 ,20 ). Furthermore, one would need to assume that transition occurs separately in trophectoderm and, only after determination of the male germ line, in the rest of the embryonic cells (20 ). The observation that in an affected fetus various tissues show a very similar mutation pattern renders the latter timing unlikely (18 ,19 ,25 ; Fig. 2 ). In fact, the presence, in several fetuses examined, of a major band common to chorionic villi and other embryonic tissues (18 ; Fig. 2 ), suggests that expansion must have occurred before separation of the trophectoderm and inner cell mass. The sparing of the male germ line would have thus to result from reversion and selection. The higher heterogeneity of mutation in chorionic villi samples may be due to a higher instability of unmethylated large expansions, as methylation was proposed to stabilize long repeats (25 ).
We reasoned that another prediction of a postzygotic model would be that for small maternal PMs in the 60-90 range, that give rise either to PM or FM offspring, with a ratio that strongly depends on size of repeat, one would expect that such dependence reflects a probability of transition inferior to 100% in individual postzygotic cells. Intuitively, one would thus expect that mosaic children, who carry both PMs and FM-sized fragments should arise preferentially from mothers with small PMs. We tested this assumption under simulation models that state postzygotic transition at a very early stage (4-16 cells in the inner cell mass) and various size dependent transition probabilities. Under the various models tested, there was always a strong prediction that offspring showing detectable FM and PM mosaicism in leucocytes would arise preferentially from mothers with intermediate sized mutations (60-100 CGGs). We did not test the recent model of Ashley and Sherman (24 ), that assumes a moderate expansion during gametogenesis, followed by a postzygotic transition to FM. In this model, the size of PM in the fertilized zygote will be different than in the mother, but the probability to enter this meiotic expansion process was stated to depend on maternal PM size. Thus, the predicted effect on mosaicism should be similar.
Analysis of PM size in 112 mothers who had 212 children with expanded alleles failed to show any dependence of mosaicism upon maternal PM size. We propose that this is strong, albeit indirect, evidence against a postzygotic transition model. We cannot, however, formally exclude a transition occurring at a very early morula stage (before the 8 cell stage at day 3, that would correspond to about four precursors of the embryo proper). The main element in support of the postzygotic model, the apparent sparing of the male germ cell lineage, that would require a transition after day 5-6 (15 ,30 ) cannot be accounted for by our results. One has thus to assume that the prezygotic model with selection against FM in the male germ line is more likely, and that mosaicism arises in a random (stochastic) manner as a postzygotic retraction of FMs. This would also account for the very good correlation between clinical status, likely to reflect mutation pattern in brain, and the mutation pattern observed in leucocytes (100% penetrance in males diagnosed with a methylated FM; only very rare cases of mental retardation in males diagnosed as PM carrier). If expansion was postzygotic and mosaicism created by lack of expansion in some cells, one would expect large differences between the ratio of mutated/premutated cells in various tissues, and a frequent lack of correlation between clinical status and mutation pattern in leucocytes. On the contrary, if mosaicism is due to random retractions in a minority of cells of a fetus with FM, this would fit with the observation that mosaic patients show, in general, a minor proportion of PM, and with the phenotype/genotype correlation (almost 100% penetrance of mental retardation in males with a FM in leucocytes, background level incidence of mental retardation in males with a PM).
Selection for PM carrying cells during male gametogenesis could occur at the level of DNA replication, favouring smaller repeat size. It has been shown that the FM is responsible for a late replication of the FMR1 gene during the cell cycle (31 ), and the induction of the fragile site indicates that, under certain conditions, this may lead to abnormal chromatin structure and thus be detrimental. The large number of replication cycles linked to spermatogenesis may enhance such a selection. Alternatively, despite the observations suggesting that the protein FMRP is not absolutely required for spermatogenesis (see above), it may still provide a selective advantage to spermatogonia in which it is expressed. Such a selective advantage conferred by FMR1 expression has indeed been found in leucocytes. We have previously shown that selection operates in females carrying a FM that results, in adults carriers, in a strongly biased X-inactivation pattern, favouring the presence of cells with the normal allele on the active X chromosome (23 ). This selection occurs despite the absence of any detectable dysfunction of white blood cells in males carrying a FM. Indeed, immunohistochemical analysis of FMRP expression in testes from two fetuses with a FM has very recently suggested that a selection mechanism may indeed occur during development of the testis (32 ). The finding, in the same study, of a FM in oocytes of a 16 week old female fetus carrying the same mutation in all other tissues tested, is not formal proof in favour of a prezygotic transition (the expansion could have occurred, in a postzygotic model, after determination of the female gametic lineage), and the authors still propose the two alternate models. A definitive conclusion would require the analysis of oocytes in ovaries from an embryo carrying, in somatic cells, a PM of at least 90-100 CGGs. Such a study is not possible (unless the fetus is affected by another serious genetic problem or malformation) for obvious ethical reasons.
Several assumptions were made for our purpose: (i) the transition from PM to FM is post-zygotic; (ii) this transition occurs at an early stage in embryonic development; (iii) as mosaicism is experimentally tested in white blood cells, we assumed that these derive from a small number of hematopoietic precursors sampled after the transition occurred; (iv) there is no selection in favour of cells carrying a PM (versus those with a FM), i.e. division rates and probability to differentiate towards white blood cells are equivalent for PM and FM cells.
The simulation estimates the proportion of premutated, full mutated and mosaic children according to the size of the maternal PM. The functions tested for the transition probability at the single cell level assumed that the probability is 0 for 50 repeats and 100% for 100 repeats. Linear, quadratic or cubic functions are thus:
Pcell = 1/100 (2r - 100);
Pcell = 1/100 [(r2/75) - (100/3)];
Pcell = 1/100 [(r3/8750) - (100/7)], respectively, with Pcell corresponding to the transition probability and r corresponding to the size of the maternal PM.
The probability that n among b white blood cell precursors carry a FM, if transition occurs when t cells are present in the embryo (transition stage), is given by the equation:
P (n mutated precursors) ={sum from {i = 0} to t} {C sup {t from i}} cdot {{P sub {{roman {c e l l}}}} sup i} cdot ( 1 - {P sub {{roman {c e l l}}}} {) sup {t - i}} {fwd 29 {{{italic C} sup {b from n}} cdot ( i / {{t )} sup n} cdot [ ( t - i ) / t {] sup {b - n}}}}(1)
The proportion of premutated children is defined as: Prop(PM) = P(less than 25% of full mutated precursors).
The proportion of mutated children is defined as: Prop(M) = P(at least 90% of full mutated precursors).
Finally, the proportion of mosaic children is defined as: Prop(MoMP) = 1 - Prop(PM) - Prop(M).
All DNA samples were derived from peripheral blood leucocytes. Prior to PCR, 200 ng of genomic DNA was chemically denatured using 0.4 M NaOH and 0.4 mM EDTA in a total volume of 50 [mu]l for 10 min at room temperature. It was then neutralized with 3 M NH3OAc (pH 5.2) and precipitated with cold absolute ethanol (33 ).
The denatured DNA was amplified using the following procedure: 25 [mu]l amplification reaction contained 25 pmol of each primer [21 first base pairs of primer A (34 ), and primer E (35 )], 2.5 [mu]l PCR buffer (Stratagene mix without KCl), 200 [mu]M C7deaza dGTP, 0.2 mM of dATP, dCTP and dTTP, 15% DMSO, and 1. 25 U polymerase exo Pfu (Stratagene). Cycling conditions were 94oC for 2 min as an initial step followed by 30 cycles consisting of denaturation at 94oC for 10 s, annealing at 61oC for 1 min, and elongation at 72oC for 2 min (Thermal Cycler Perkin Elmer), and a final elongation at 72oC for 10 min.
Aliquots of PCR product (4 [mu]l) plus 8 [mu]l of loading buffer (95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 20 mM EDTA) were electrophoresed in a 6% denaturing polyacrylamide gel (0.75 mm thick) at 1000 V for 2 h with TBE buffer. An end-labelled MspI digest of plasmid pBR322 was used as length standard. PCR products were transferred by electroblotting (3 mA/cm2 for 30 min) to a positively charged nylon membrane (Amersham). The membrane was hybridized with a 32P-end-labelled (CGG)10 oligonucleotide in a hybridization buffer (25% formamide, 2 mM EDTA, 5 mM NaCl, 50 mM phosphate buffer, 2* polyvinylpyrrolidone, 2* ficoll, 0.1 mg/ml salmon sperm, 20% SDS) and washed twice in 0.5* SSPE at 65oC. After a 30-60 min exposure period at 4oC with X-ray film (Kodak), repeat length was determined using the length standard.
We thank our clinical colleagues for referring fragile X families and J.-C. Bouix for providing blots of the families. This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National pour la Recherche Scientifique (CNRS), the Centre Hospitalier Universitaire (CHU) of Strasbourg, and a grant from the Groupement de Recherches et d'Etudes sur les Génomes (GREG).
1 Imbert,G. and Mandel,J-L. (1996) The fragile X mutation. Mental Retard. Dev. Disabil. Res. Rev., 1, 251-262.
2 Warren,S.T. and Ashley,C.T.,Jr. (1995) Triplet repeat expansion mutations: the example of fragile X syndrome. Annu. Rev. Neurosci., 18, 77-99.MEDLINE Abstract
3 Rousseau,F., Heitz,D., Biancalana,V., Blumenfeld,S., Kretz,C., Boué,J., Tommerup,N., Van der Hagen,C., DeLozier-Blanchet,C., Croquette,M.-F., Gilgenkrantz,S., Jalbert,P., Voelckel,M.A., Oberlé,I. and Mandel,J.L. (1991) Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation. N. Engl. J. Med., 325, 1673-1681.
4 Hagerman,R.J. (1995) Molecular and clinical correlations in fragile X syndrome. Mental Retard. Dev. Disabil. Res. Rev., 1, 276-280.
5 Nolin,S.L., Glicksman,A., Houck,G.E., Brown,W.T. and Dobkin,C.S. (1994) Mosaicism in fragile X affected males. Am. J. Med. Genet., 51, 509-512.MEDLINE Abstract
6 Oberlé,I., Rousseau,F., Heitz,D., Kretz,C., Devys,D., Hanauer,A., Boue,J., Bertheas,M.F. and Mandel,J.L. (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science, 252, 1079-1102.
7 Fu,Y.H., Kuhl,D.P.A., Pizzuti,A., Pieretti,M., Sutcliffe,J.S., Richards,S., Verkerk,A.J.M.H., Holden,J.J.A., Fenwick Jr.,R.G., Warren,S.T., Oostra,B.A., Nelson,D.L. and Caskey,C.T. (1991) Variation of the CGG repeat at the Fragile X site results in genetic instability: resolution of the Sherman paradox. Cell, 67, 1047-1058.
8 Yu,S., Mulley,J., Loesch,D., Turner,G., Donnelly,A., Gedeon,A., Hillen,D., Kremer,E., Lynch,M., Pritchard,M., Sutherland,G.R. and Richards,R.I. (1992) Fragile-X syndrome: unique genetics of the heritable unstable element. Am. J. Hum. Genet., 50, 968-980.
9 Heitz,D., Devys,D., Imbert,G., Kretz,C. and Mandel,J.L. (1992) Inheritance of the fragile X syndrome: size of the fragile X premutation is a major determinant to full mutation. J. Med. Genet., 29, 794-801.
10 Fisch,G.S., Snow,K., Thibodeau,S.N., Chalifaux,M., Holden,J.J.A., Nelson,D.L., Howard-Peebles,P.N. and Maddalena,A. (1995) The fragile X premutation in carriers and its effect on mutation size in offspring. Am. J. Hum. Genet., 56, 1147-1155.
11 Sherman,S.L., Jacobs,P.A., Morton,N.E., Froster-Iskenius,U., Howard-Peebles,P.N., Brondum-Nielsen,K., Partington,M.W., Sutherland,G.R., Turner,G. and Watson,M. (1985) Further segregation analysis of the Fragile X syndrome with special reference to transmitting males. Hum. Genet., 71, 184-186.
12 Mac Donald,M.E., Barnes,G., Srinidhi,J., Duyao,M.P., Ambrose,C.M., Myers,R.H., Gray,J., Conneally,P.M., Young,A., Penney,J., Shoulson,I., Hollingswoth,Z., Koroshetz,W., Bird,E., Vonstattel,J.P., Bonilla,E., Moscowitz,C., Penchaszadeh,G., Brzustowicz,L., Alvir,J., Bickham Conde,J., Cha,J.-H., Dure,L., Gomez,F., Ramos-Arroyo,M., Sanchez-Ramos,J., Snodgrass,S.R., de Young,M., Wexler,N.S., MacFarlane,H., Anderson,M.A., Jenkins,B. and Gusella,J.F. (1993) Gametic but not somatic instability of CAG repeat length in Huntington's disease. J. Med. Genet., 30, 982-986.
13 Telenius,H., Kremer,B., Goldberg,Y.P., Theilmann,J., Andrew,S.E., Zeisler,J., Adam,S., Greeberg,C., Ives,E.J., Clarke,L.A. and Hayden,M.R. (1994) Somatic and gonodal mosaicism of the Huntington disease gene CAG repeat in brain and sperm. Nature Genet., 6, 409-414.
14 Morton,N.E. and Macpherson,J.N. (1992) Population genetics of the fragile-X syndrome: multiallelic model for the FMR1 locus. Proc. Natl. Acad. Sci. USA, 89, 4215-4217.MEDLINE Abstract
15 Reyniers,E., Vits,L., De Boulle,K., Van Roy,B., Van Velzen,D., de Graaff,E., Verkerk,A.J., Jorens,H.Z., Darby,J.K., Oostra,B. and Willems,P.J. (1993) The full mutation in the FMR-1 gene of male fragile X patients is absent in their sperm. Nature Genet., 4, 143-146.
16 Willems,P.J., Van Roy,B., De Boulle,K., Vits,L., Reyniers,E., Beck,O., Dumon,J.E., Verkerk,A. and Oostra,B. (1992) Segregation of the fragile X mutation from an affected male to his normal daughter. Hum. Mol. Genet., 1, 511-515.
17 Rousseau,F., Robb,L.J., Rouillard,P. and Der Kaloustian,V.M. (1994) No mental retardation in a man with 40% abnormal methylation at the FMR-1 locus and transmission of sperm cell mutations as premutations. Hum. Mol. Genet., 3, 927-930.
18 Devys,D., Biancalana,V., Rousseau,F., Boué,J., Mandel,J.L. and Oberlé,I. (1992) Analysis of full fragile X mutations in fetal tissues and monozygotic twins indicate that abnormal methylation and somatic heterogeneity are established early in development. Am. J. Med. Genet., 43, 208-216.
19 Wöhrle,D., Hennig,I., Vogel,W. and Steinbach,P. (1993) Mitotic stability of fragile X mutations in differentiated cells indicates early post-conceptional trinucleotide repeat expansion. Nature Genet., 4, 140-142.
20 Trottier,Y., Devys,D. and Mandel,J-L. (1993) An expanding story. Curr. Biol., 3, 783-786.
21 The Dutch-Belgian fragile X consortium, (1994) Fmr1 knockout mice: a model to study fragile X mental retardation. Cell, 78, 23-33.
22 Meijer,H., de Graaff,E., Merckx,D.M., Jongbloed,R.J., de Die-Smulders,C.E., Engelen,J.J., Fryns,J.P., Curfs,P.M. and Oostra,B.A. (1994) A deletion of 1.6 kb proximal to the CGG repeat of the FMR1 gene causes the clinical phenotype of the fragile X syndrome. Hum. Mol. Genet, 3, 615-620.MEDLINE Abstract
23 Rousseau,F., Heitz,D., Oberlé,I. and Mandel,J.L. (1991) Selection in blood cells from female carriers of the fragile X syndrome: inverse correlation between age and proportion of active X chromosomes carrying the full mutation. J. Med. Genet., 28, 830-836.
24 Ashley,A.E. and Sherman,S.L. (1995) Population dynamics of a meiotic/mitotic expansion model for the fragile X syndrome. Am. J. Hum. Genet., 57, 1414-1425.MEDLINE Abstract
25 Wohrle,D., Kennerknecht,I., Wolf,M., Enders,H., Schwemmle,S. and Steinbach,P. (1995) Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a casual involvement of methyl-directed DNA mismatch repair in triplet repeat stability. Hum. Mol. Genet., 4, 1147-1153.
26 Vaisanen,M.L., Kahkonen,M. and Leisti,J. (1994) Diagnosis of fragile X syndrome by direct mutation analysis. Hum. Genet., 93, 143-147.
27 Naumova,A.K., Plenge,R.M., Bird,L.M., Leppert,M., Morgan,K., Willard,H.F. and Sapienza,C. (1996) Heritability of X chromosome- inactivation phenotype in a large family. Am. J. Hum. Genet., 58, 1111-1119.
28 Taylor,A.K., Safanda,J.F., Fall,M.Z., Quince,C., Lang,K.A., Hull,C.E., Carpenter,I., Staley,L.W. and Hagerman,R.J. (1994) Molecular predictors of cognitive involvement in female carriers of fragile X syndrome. J. Am. Med. Assoc., 271, 507-514.
29 Devys,D., Lutz,Y., Rouyer,N., Bellocq,J.P. and Mandel,J.L. (1993) The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation. Nature Genet., 4, 335-340.
30 Bachner,D., Manca,A., Steinbach,P., Wohrle,D., Just,W., Vogel,W., Hameister,H. and Poustka,A. (1993) Enhanced expression of the murine FMR1 gene during germ cell proliferation suggests a special function in both the male and the female gonad. Hum. Mol. Genet., 2, 2043-2050.
31 Hansen,R.S., Canfield,T.K., Lamb,M.M., Gartler,S.M. and Laird,C.D. (1993) Association of fragile X syndrome with delayed replication of the FMR1 gene. Cell, 73, 1403-1409.
32 Malter,H.E., Iber,J.C., Willemsen,R., de Graaff,E., Tarleton,J.C., Leisti, J., Warren,S.T. and Oostra,B.A. (1997) Characterization of the full fragile X syndrome mutation in fetal gametes. Nature Genet., 15, 165-169.
33 Agarwal,R.K. and Perl,A. (1993) PCR amplification of highly GC-rich DNA template after denaturation by NaOH. Nucleic Acids Res., 21, 5283-5284.MEDLINE Abstract
34 Eichler,E.E., Holden,J.J.A., Popovich,B.W., Reiss,A.L., Snow,K., Thibodeau,S.N., Richards,C.S., Ward,P.A. and Nelson,D.L. (1994) Length of uninterrupted CGG repeats determines instability in the FMR1 gene. Nature Genet., 8, 88-94.
35 Wang,Q., Green,E., Bobrow,M. and Mathew,C.G. (1995) A rapid, non-radioactive screening test for fragile X mutations at the FRAXA and FRAXE loci. J. Med. Genet., 32, 170-173.
*To whom correspondence should be addressed at: IGBMC, BP 163, 67404 Illkirch, C.U. de Strasbourg, France. Tel: +33 3 88653244; Fax: +33 3 88653246; Email:mandeljl{at}igbmc.u-strasbg.fr
+Both authors contributed equally to this work
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