Mol. Hum. Reprod. Advance Access originally published online on August 8, 2006
Molecular Human Reproduction 2006 12(10):647-652; doi:10.1093/molehr/gal069
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Multiple displacement amplification improves PGD for fragile X syndrome
1Faculté de Médecine, Université Paris-Descartes, Unité INSERM U781 Institut de Recherche Necker-Enfants Malades, Service de génétique médicale, Hôpital Necker-Enfants Malades (Assistance Publique-Hôpitaux de Paris), Paris, 2Service de Biologie et Génétique de la Reproduction and 3Service de Gynécologie-Obstétrique et Médecine de la Reproduction, UPRES 3538, Hôpital Antoine Béclère, Clamart, France
4 To whom correspondence should be addressed at: Service de génétique médicale, Hôpital Necker-Enfants Malades (Assistance Publique-Hôpitaux de Paris), 149 rue de Sèvres, 75743 Paris Cedex 15, France. E-mail: burlet{at}necker.fr
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Abstract |
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We report an improvement in the PGD test for fragile X syndrome (FXS). Recently, multiple displacement amplification (MDA) has been reported to yield large amounts of DNA from single cells. Taking into account this technique, we developed a new PGD test for FXS, enabling combined analysis of linked polymorphic markers with the study of the non-expanded CGG repeat. Single cell amplification efficiency was first assessed on single lymphocytes. Amplification rate of the different markers ranged from 85 to 95% with an allele drop-out (ADO) rate comprised between 7 and 34%. Using this test, eight PGD cycles were carried out for six couples, and 37 embryos were analysed after preliminary MDA. Amplification rate was increased by this technique from 41 to 66% so that embryos with no results were rarer (14 versus 45% without MDA). Reliability of the test was considerably improved by combining direct with indirect genetic analysis. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. Embryonic transfers were carried out in all but one PGD cycles. One biochemical and one clinical pregnancy resulted, and a healthy child was born. This single diagnosis procedure could be suitable to most patients carrying FXS.
Key words: fragile X syndrome/mental retardation/multiple displacement amplification/PGD
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Introduction |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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The fragile X syndrome (FXS, MIM 309550 [OMIM] ) is the most common monogenic cause of mental retardation. FXS is caused by mutations in the fragile X mental retardation-1 (FMR1) gene, >95% of which involve hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region (5'UTR) of the gene (Oberle et al., 1991
; Yu et al., 1991). In most affected patients, the CGG repeats are massively expanded to over 200 (full mutation), and the gene becomes methylated at CpG islands and is silenced. Individuals with CGG repeats in the premutation range of 55–200 repeats are clinically unaffected, but these repeats are likely to be unstable during transmission to the next generation (Fu et al., 1991). This instability depends on the size of the premutation allele and is much more pronounced through maternal transmission. Thus, females carrying a large premutation or a full mutation have a 50% risk of having a child carrying a full mutation. Furthermore, phenotypic expressivity of this disorder is variable, particularly in carrier females. A female fetus carrying a full mutation is indeed at risk of developing a mental retardation (two of three cases) but can be asymptomatic in one of three cases. Because there are no predictive criteria of mental retardation, the decision to terminate or continue the pregnancy for couples facing a prenatal diagnosis result of a female fetus carrying a full mutation is particularly difficult. Taken together, these data often lead couples at risk of transmitting FXS to their offspring to request PGD.
PGD consists in the analysis of one or two blastomeres taken from an in vitro fertilized embryo at the third day stage. Only healthy embryos are transferred, avoiding the physical and psychic traumatism of the termination of pregnancy in the case of an affected fetus detected later by prenatal diagnosis. The development of PGD techniques has offered the possibility to select unaffected male or non-carrier female embryos for FXS (Sermon et al., 1999; Apessos et al., 2001; Platteau et al., 2002). However, carrying out PGD for FXS is difficult for multiple reasons. First, the large number of CGG repetitions in affected individuals precludes any attempt at sizing the mutant allele, leading to technical difficulties. Two alternative methods are therefore used for single blastomere analysis, i.e. the detection of the non-expanded CGG repeat allele (Sermon et al., 1999, 2001) or the use of linked polymorphic markers (Apessos et al., 2001; Harper et al., 2002). Direct detection of the normal allele is difficult because of the lack of PCR sensitivity despite specific PCR protocols (the expansion is refractory to PCR because of the high GC content of the repeat and surrounding sequences). Direct diagnosis exposes also to the risk of allele drop-out (ADO), a phenomenon by which only one allele present in a cell is amplified, leading to the consideration of a healthy embryo as affected. In addition, this approach is suitable for only 63% of couples, the heterozygosity of the repeat in the normal population (Fu et al., 1991). The main drawback of the indirect method is the risk of recombination and/or ADO, which may lead to misdiagnosis, and the non-informativity of the markers which required to establish a diagnostic test for each family. A second limitation to PGD for FXS is that women carrying premutations are at increased risk of premature ovarian failure (Murray et al., 1998; Marozzi et al., 2000). A reduced ovarian response to stimulation protocols often results in few embryos available to test (Platteau et al., 2002) and thus to transfer. Diagnosing each embryo is therefore a true challenge.
Recently, 
29 DNA polymerase and random exonuclease-resistant primers have been used to yield large amounts of DNA of high quality from single cells (Dean et al., 2001, 2002). This method [multiple displacement amplification (MDA)] is more accurate than whole genome amplification PCR-based methods previously described (Dean et al., 2002) and could provide a powerful test for PGD analysis of multiple loci (Handyside et al., 2004; Hellani et al., 2004, 2005). We have developed an alternative PGD diagnostic test for FXS using MDA as a preliminary step. Fluorescent PCR was secondly performed amplifying the non-expanded CGG repeats with markers linked to FMR1 and a sequence from the amelogenin gene, enabling embryonic gender determination simultaneously with the direct and indirect analyses of the FMR1 gene in preimplantation embryos. Using this test, we carried out eight PGD, resulting in genotyping of 37 embryos. Two pregnancies were obtained, one biochemical and one singleton, the latter one resulting in the birth of a healthy child.
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Patients and methods |
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Clinical PGD
Fourteen PGD cycles were performed for six couples at risk of transmitting FXS to their offspring. In four cases, women were carrying a full mutation (over 250 CGGs), and in two cases, a premutation was observed (70 and 100 CGGs). Three couples have experienced at least two termination of pregnancy for FXS-affected fetus, and two couples have affected children. Standard IVF protocols were realized. Embryo biopsies were performed in the morning of day 3 after insemination as described previously (Gigarel et al., 2004
). Usually two cells were removed from embryos with seven blastomeres or more and only one cell from the others, unless the first cell removed had lysed or had no visible nucleus. Under a binocular microscope, the blastomeres were rinsed in a drop of phosphate-buffered saline (PBS) supplemented with 0.1% of polyvinyl alcohol (Sigma Aldrich, Lyon, France) using a mouth-controlled, finely pulled glass pipette before being transferred into a transparent microcentrifuge tube containing 3 µl of lysis buffer (Cui et al., 1989). For each embryo, a small volume of rinsing medium was transferred similarly and used as a negative control. The cells were lysed by heating tubes for 15 min at 65°C. Seventy-seven embryos were biopsied, and 153 blastomeres were analysed. DNA from 37 embryos (75 blastomeres) was amplified using MDA. PCR results were retrospectively compared with those obtained without MDA (40 embryos and 78 blastomeres). When available, carrier embryos were reanalysed to confirm the results of the PGD test (n = 6).
Collection of human lymphocytes
Lymphocytes (n = 88) from controls and carrier individuals were used for validating the PCR method. Blood samples (10 ml unclotted with citrate-dextrose anticoagulant) were separated by centrifugation through Ficoll-paque (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s protocol. Cells were handled in a clean laboratory with a mouth-controlled, fine, heat-polished glass micropipette in drops of PBS (Sigma Aldrich) supplemented with 0.1% polyvinyl alcohol (Sigma Aldrich) and deposited in 3 µl of alkaline lysis buffer (Cui et al., 1989) under visual control using an inverted microscope. The cells were lysed by heating tubes for 15 min at 65°C.
PCR reactions and MDA
Primers
A panel of four (CA)n microsatellite markers located near or within the FMR1 gene (Figure 1) was chosen [i.e. DXS998 (Weissenbach et al., 1992), DXS548 (Riggins et al., 1992), FRAXAC1 (Richards et al., 1991) and DXS1215 (Gyapay et al., 1994)]. A method for gender determination of the embryos using the amelogenin sequence was included to enable distinction between ADO and hemizygosity (Nakahori et al., 1991). Primers were selected using the software Oligo 6.0 (Medprobe, Oslo, Norway) to have compatible primer sets with similar annealing temperatures (Table I).
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PCR reactions with no MDA
Blastomeres were denatured at 65°C for 10 min. PCR conditions were similar for the first (outer) and second (inner) amplifications: aliquots of 27 µl of PCR mix containing Expand Long Template Taq DNA Polymerase (1.5 U, Roche Diagnostics, Mannheim, Germany), x10 PCR buffer 2 (17.5 mM MgCl2) (3 µl), 0.5 µM of each primer (Table I) and 2 mM dNTP (deoxyribonucleoside triphosphates) mix (Roche Diagnostics) were added to the reaction tubes. The solution was covered with 30 µl of mineral oil to minimize the risks of contamination through aerosol production. ‘Inner’ amplification was performed by adding the first amplification product (3 µl) to five ‘inner’ amplification mixes (27 µl) containing ‘inner’ primers of DXS548, FRAXAC1, DXS998 and DXS1215 and the amelogenin sequence.
Denaturation was at 97°C for 3 min followed by 20 s of denaturation at 97°C, annealing at 60°C for 15 s and extension at 68°C for 1 min 15 s. Final extension is performed at 68°C for 10 min. The total number of cycles was 22 for the ‘outer’ and 30 for the ‘inner’ reactions. To reduce ADO, we performed denaturation of the first 10 cycles of the outer reaction at a higher temperature (98°C, Ray and Handyside, 1996).
PCR reactions after preliminary MDA
MDA was performed using the GenomiPhi kit (Amersham Biosciences) on lymphocytes or blastomeres. Nine microliters of sample buffer was added to the 3-µl lysis buffer containing the single cell, denatured at 95°C for 10 min and kept at 4°C. A second mixture containing 9 µl of reaction buffer and 1 µl of enzyme was added to the lysis sample and then incubated for at least 8 h at 30°C. The final step (65°C for 10 min) resulted in the inactivation of the 29 polymerase.
Linkage analysis was performed on the MDA product (1 µl) in two multiplexed PCRs, the first mix containing ‘inner’ primers of DXS548 and FRAXAC1 and the second mix containing ‘inner’ primers of DXS998, DXS1215 and the amelogenin sequence. PCR conditions were as described above: aliquots of 29 µl of PCR mix containing Expand Long Template Taq DNA Polymerase (1.5 U, Roche Diagnostics), x10 PCR buffer 2 0.5 µM of each primer (‘inner’ primers, Table I) and 2 mM of dNTP mix (Roche Diagnostics) were added to the reaction tubes.
Direct analysis of the non-expanded CGG repeat was simultaneously performed on the MDA product (1 µl), using primers FMR1-forward and FMR1-reverse Table I. Two mixes were prepared separately. Mix1 containing 500 µM dATP, dCTP, dTTP and 7-deaza-dGTP (Roche Diagnostics), 300 µM of each primer and 10% dimethylsulphoxide (DMSO) (Sigma Aldrich) was added to the MDA product (1 µl) in a final volume of 15 µl. Mix2 (10 µl), containing buffer 3 (27.5 mM MgCl2) and Expand Long Template Taq DNA Polymerase (1.7 units), was added to mix1 at a final volume of 25 µl. The PCR programme was run immediately in a GeneAmp 9700 thermocycler (Applied Biosystems, Courtaboeuf, France) and consisted of a first denaturation step of 95°C for 3 min, followed by 10 cycles (denaturation at 95°C for 30 s, annealing at 61°C for 30 s and extension at 68°C for 2 min), followed by 10 cycles using the same temperature and time profile, except that elongation was extended for 10 s after each cycle.
Analysis of the PCR products
Amplified products (1 µl) were added to 15 µl of genetic analysis grade formamide (Applied Biosystems) and 0.3 µl of ROX 400HD (Applied Biosystems). After denaturation for 5 min at 95°C and fast cooling on ice, products were electrophoresed in an automated genetic analyser ABI 3130 (Applied Biosystems). Results were analysed with Genescan and Genotyper softwares (Applied Biosystems). For FMR1 analysis, the amplicon size corresponds to 62 bp + 3n, n being the number of trinucleotide repeats (62 bp off for primers and sequences surrounding the expansion).
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Results |
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Lymphocyte testing
Reliability of the protocol was first assessed on 88 single lymphocytes sampled from parents and control individuals. After preliminary MDA, three separate PCRs were performed: amplifying (CA)n microsatellite markers (DXS548, DXS1215 and DXS998), one intragenic microsatellite marker (FRAXAC1) with the amelogenin sequence and the non-expanded alleles of FMR1 alone. Amplification rate for five loci (i.e. DXS548, FRAXAC1, DXS1215, DXS998 and amelogenin sequence) was calculated (Table II). Amplification products were detected in 466/502 reactions (93%). Amplification failure for the (CA)n microsatellite markers was, respectively, 5, 8, 5, 7 and 7% for amelogenin (Table II). At heterozygous loci for these markers, ADO was detected in 59/284 (20%) reactions (Table II). Regarding the PCR amplifying the non-expanded CGG allele (FMR1), ADO rate was 25% (6/24 heterozygous cells), whereas amplification failure was 15% (8/62 cells) (Table II).
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Clinical PGD
Following the set-up on lymphocytes, the test was applied in eight clinical PGD for six couples at risk of transmitting FXS to their children. The six couples were shown to be informative for the non-expanded CGG alleles. Female carriers were informative for at least two of the four (CA)n microsatellite markers. Standard IVF protocols were performed, resulting in 37 embryos to test. A representative PGD result is shown in Figure 1. Results were retrospectively compared with the 40 embryos from six PGD cycles, analysed using multiplex-nested PCR without MDA amplification. MDA improved amplification yield which ranged from 61 to 74% (mean 66%) versus 31–52% (mean 41%) without MDA (Table II). Embryos with no results were less frequent (14 versus 45%, Table II). Among the 37 embryos tested after preliminary MDA, five embryos (12 cells) gave no PCR results at all, and interestingly, all originated from the same woman. In the remaining cases (32 embryos), a PCR result was available. As expected, half of the embryos were affected (19 embryos); only nine were healthy, and in four cases, the result was inconclusive, suggesting ADO, trisomy or recombination events. Six carrier embryos were reanalysed, and the results of the PGD test obtained from MDA products were confirmed (data not shown). None of the blank controls included in the test displayed amplification signals, indicating absence of contamination. Embryo transfers have been performed in all cases. One biochemical and one clinical pregnancy resulted. The latter patient refused to perform a prenatal test proposed as a PGD control, but diagnosis of a healthy embryo was confirmed on cord blood DNA analysis (data not shown).
To test whether multiplex PCR increases the risk of ADO and/or hampered PCR efficiency, we reanalysed blastomere using simplex PCRs on the same MDA product. Similar amplification efficiency and the same rate of ADO were obtained in all cases (data not shown).
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Discussion |
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TopAbstractIntroductionPatients and methodsResultsDiscussionReferences |
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We have applied PGD for FXS over a 2-year period using nested multiplex PCR of linked markers (DXS1215, FRAXAC1, DXS998 and DXS548) combined with the amelogenin sequence for sexing. There were several embryos without a diagnosis (45%, Table II), due in part to amplification failure [amplification efficiency was very low (41%), Table II] or recombination and/or ADO events, so that there were often no transferable embryos (half of the cases). This was particularly dramatic because in FXS, women are known to be at risk of premature ovarian failure. Furthermore, some couples were shown to be not informative, which resulted in the need of designing new diagnoses for PGD, a time-consuming procedure. Of six PGD cycles and 78 analysed blastomeres from 40 embryos, none of the patients were pregnant.
We thus developed a more efficient and reliable new PGD test enabling PGD for most FXS couples. We used MDA as a first step because this technique was shown to produce good quality DNA compared with classical whole genome amplification protocols previously described. This technique is a reliable method for single-cell analysis, different reports showing high-amplification rates (88–95%) with an ADO rate between 7 and 31% (Handyside et al., 2004; Hellani et al., 2004, 2005). Furthermore, after MDA amplification, a single round of PCR is needed, which is appropriate with the time available for a PGD test. Using this test, we carried out eight PGD. PCR efficiency was increased, with a better amplification rate (66%), and reliability of the test was also considerably improved by combining both direct and indirect analyses of the cells, the use of several polymorphic markers providing independent diagnostic confirmations. The risk of misdiagnosis due to ADO at one locus was indeed avoided by extrapolating the information obtained from other loci, and the use of the amelogenin sequence, a method for gender determination, enabled distinction between ADO and hemizygosity. Furthermore, in cases of fully expanded alleles too large to be amplified by PCR, this test gives an internal amplification control. It can also detect contaminations by showing exogenous alleles in linkage analysis and give some information on the ploidy of the embryos. Non-conclusive or aberrant results led to no transfer of the embryos (five embryos in our series). Finally, this new test enables a successful diagnosis in 86% of the embryos, and a uterine transfer of healthy embryos was possible in all cases but one, leading to one biochemical and one clinical pregnancy. A healthy child was born.
Overall, our data for PGD of FXS are worse when compared with other disorders, with a lesser amplification efficiency. Interestingly, this is due to five embryos from the same patient, who gave no PCR results. This can be due to unknown technical events, even if other embryos and controls gave results. This could also be explained by poor embryonic quality associated with DNA-strand breakage. Avoiding heat denaturation by using gentle alkaline denaturation, provided by other MDA commercially available kit, may be a mean to protect poor-quality DNA from breakage. Amplification of the CGG-containing sequence in the non-expanded FMR1 alleles in lymphocytes gave lower amplification rates (85 versus 94%), and higher ADO rates in our series (25 versus 1.9%) than previously reported (Sermon et al., 1999). This can be due to MDA artefact; however, the gain of accuracy of the assay and the possibility to offer this diagnosis to semi- or non-informative FMR1 couples make this test a valuable tool for PGD of FXS. Futhermore, additional markers (SNP (single nucleotide polymorphism), microsatellites, etc.) can easily be added to the PCR reaction in case of ‘non-informative’ couples.
Our data demonstrate the successful strategy of using multiplex fluorescent PCR to simultaneously amplify the mutation site and polymorphic loci after MDA in FXS. MDA improves both sensitivity and reliability of our PGD results, and this method should be appropriate to most patients carrying fragile X.
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Acknowledgements |
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We thank Dr H. Etchevers for critical reading of the manuscript and Dr F. Rousseau and R. Gesny for technical advice. This work was supported by grants from AP-HP (Assistance Publique-Hôpitaux de Paris: PHRC HUS-3173). J.S. is supported by Association Française contre les Myopathies (grant number 10554).
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References |
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Apessos A, Abou-Sleiman PM, Harper JC and Delhanty JD (2001) Preimplantation genetic diagnosis of the fragile X syndrome by use of linked polymorphic markers. Prenat Diagn 21,504–511.[CrossRef][ISI][Medline]
Cui XF, Li HH, Goradia TM, Lange K, Kazazian HH Jr, Galas D and Arnheim N (1989) Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc Natl Acad Sci USA 86,9389–9393.
Dean FB, Nelson JR, Giesler TL and Lasken RS (2001) Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11,1095–1099.
Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P, Sun Z, Zong Q, Du Y, Du J et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 99,5261–5266.
Fu YH, Kuhl DP, Pizzuti A, Pieretti M, Sutcliffe JS, Richards S, Verkerk AJ, Holden JJ, Fenwick RG Jr, Warren ST et al. (1991) Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell 67,1047–1058.[CrossRef][ISI][Medline]
Gigarel N, Frydman N, Burlet P, Kerbrat V, Steffann J, Frydman R, Munnich A and Ray PF (2004) Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti loci on Xq28. Hum Genet 114,298–305.[CrossRef][ISI][Medline]
Gyapay G, Morissette J, Vignal A, Dib C, Fizames C, Millasseau P, Marc S, Bernardi G, Lathrop M and Weissenbach J (1994) The 1993–94 Genethon human genetic linkage map. Nat Genet 7,246–339.[CrossRef][ISI][Medline]
Handyside AH, Robinson MD, Simpson RJ, Omar MB, Shaw MA, Grudzinskas JG and Rutherford A (2004) Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Mol Hum Reprod 10,767–772.
Harper JC, Wells D, Piyamongkol W, Abou-Sleiman P, Apessos A, Ioulianos A, Davis M, Doshi A, Serhal P, Ranieri M et al. (2002) Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders. Prenat Diagn 22,525–533.[CrossRef][ISI][Medline]
Hellani A, Coskun S, Benkhalifa M, Tbakhi A, Sakati N, Al-Odaib A and Ozand P (2004) Multiple displacement amplification on single cell and possible PGD applications. Mol Hum Reprod 10,847–852.
Hellani A, Coskun S, Tbakhi A and Al-Hassan S (2005) Clinical application of multiple displacement amplification in preimplantation genetic diagnosis. Reprod Biomed Online 10,376–380.[ISI][Medline]
Marozzi A, Vegetti W, Manfredini E, Tibiletti MG, Testa G, Crosignani PG, Ginelli E, Meneveri R and Dalpra L (2000) Association between idiopathic premature ovarian failure and fragile X premutation. Hum Reprod 15,197–202.
Murray A, Webb J, Grimley S, Conway G and Jacobs P (1998) Studies of FRAXA and FRAXE in women with premature ovarian failure. J Med Genet 35,637–640.[Abstract]
Nakahori Y, Takenaka O and Nakagome Y (1991) A human X-Y homologous region encodes "amelogenin". Genomics 9,264–269.[CrossRef][ISI][Medline]
Oberle I, Rousseau F, Heitz D, Kretz C, Devys D, Hanauer A, Boue J, Bertheas MF and Mandel JL (1991) Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 252,1097–1020.
Platteau P, Sermon K, Seneca S, Van Steirteghem A, Devroey P and Liebaers I (2002) Preimplantation genetic diagnosis for fragile Xa syndrome: difficult but not impossible. Hum Reprod 17,2807–2812.
Ray PF and Handyside AH (1996) Increasing the denaturation temperature during the first cycles of amplification reduces allele dropout from single cells for preimplantation genetic diagnosis. Mol Hum Reprod 2,213–218.
Richards RI, Holman K, Kozman H, Kremer E, Lynch M, Pritchard M, Yu S, Mulley J and Sutherland GR (1991) Fragile X syndrome: genetic localisation by linkage mapping of two microsatellite repeats FRAXAC1 and FRAXAC2 which immediately flank the fragile site. J Med Genet 28,818–823.[Abstract]
Riggins GJ, Sherman SL, Oostra BA, Sutcliffe JS, Feitell D, Nelson DL, van Oost BA, Smits AP, Ramos FJ, Pfendner E et al. (1992) Characterization of a highly polymorphic dinucleotide repeat 150 KB proximal to the fragile X site. Am J Med Genet 43,237–243.[CrossRef][ISI][Medline]
Sermon K, Seneca S, Vanderfaeillie A, Lissens W, Joris H, Vandervorst M, Van Steirteghem A and Liebaers I (1999) Preimplantation diagnosis for fragile X syndrome based on the detection of the non-expanded paternal and maternal CGG. Prenat Diagn 19,1223–1230.[CrossRef][ISI][Medline]
Sermon K, Seneca S, De Rycke M, Goossens V, Van de Velde H, De Vos A, Platteau P, Lissens W, Van Steirteghem A and Liebaers I (2001) PGD in the lab for triplet repeat diseases – myotonic dystrophy, Huntington’s disease and Fragile-X syndrome. Mol Cell Endocrinol 183,S77–S85.
Weissenbach J, Gyapay G, Dib C, Vignal A, Morissette J, Millasseau P, Vaysseix G and Lathrop M (1992) A second-generation linkage map of the human genome. Nature 359,794–801.[CrossRef][Medline]
Yu S, Pritchard M, Kremer E, Lynch M, Nancarrow J, Baker E, Holman K, Mulley JC, Warren ST, Schlessinger D et al. (1991) Fragile X genotype characterized by an unstable region of DNA. Science 252,1179–1181.
Submitted on April 10, 2006; resubmitted on July 4, 2006; accepted on July 8, 2006.
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