Molecular Human Reproduction, Vol. 7, No. 5, 489-494,
May 2001
© 2001 European Society of Human Reproduction and Embryology
Reproductive genetic |
Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination
Département de génétique and INSERM U393, IRNEM, Hopital Necker Enfants Malades, 75743 Paris Cedex 15, France
Abstract
Large deletions in the dystrophin gene account for >60% of mutations responsible for Duchenne muscular dystrophy (DMD). We have developed a genetic test that can be used directly for the preimplantation genetic diagnosis (PGD) of a majority of couples at risk of transmitting DMD. The test, a double nested multiplex polymerase chain reaction assay for the amplification of exons 8, 19, 45, 47 and 51 allows the detection of over 70% of all DMD deletions. Amelogenin sequences on the X and the Y chromosomes were also co-amplified to provide a correlation between embryo gender and deletion status. The setting up of reliable single cell assays for preimplantation genetic diagnosis is delicate and time consuming. Assays have to be validated on a large number of single cells for each specific mutation to assess efficiency and accuracy before being applied clinically. The multiplex procedure permitted the validation of all tested loci in the same series of isolated lymphocytes rather than in separate series for each exon. One hundred single lymphocytes, 50 female and 50 male cells, were analysed with an overall amplification rate of 98% and an amplification failure of 2% per exon. We suggest that this test is reliable, easy to set up and much preferable to a mere sex determination with the selective transfer of female embryos.
Duchenne muscular dystrophy/dystrophin/gender determination/multiplex PCR/preimplantation genetic diagnosis
Introduction
Duchenne and Becker muscular dystrophy (B/DMD; OMIM accession number: 310200) are allelic X-linked recessive diseases responsible for early muscle degeneration and cardiac insufficiency in hemizygous boys (Boland et al., 1996
). The size of the disease-causing gene, dystrophin, probably accounts for the high frequency of this condition (1/3500 male births), and the high proportion of de-novo mutations. Deletions encompassing at least one of the 79 dystrophin exons account for two-thirds of all D/BMD mutations; the remaining mutations consist of large duplications and point mutations (Read et al., 1988; Tennyson et al., 1995). A number of multiplex polymerase chain reaction (PCR) protocols have been described, allowing the detection of an ever-increasing proportion of DMD deletions. This approach was pioneered by describing the co-amplification of six and, a year later, nine exons, allowing the detection of 80% of all DMD deletions (Chamberlain et al., 1988, 1990). Then an extra set of nine, then 11, primers were described, permitting the detection of 94 and 98% of all deletions respectively (Beggs et al., 1990; Abbs et al., 1991). The advantage of such a diagnostic test is obvious in terms of cost-effectiveness due to savings in both reagents and labour. Indeed, a single test allows the identification and the fairly accurate characterization of most deletions and can also be used directly for diagnostic purposes. For the diagnosis of duplications, however, Southern analysis is still usually required. Also, many point mutations remain uncharacterized and indirect diagnosis has to be carried out (Clemens et al., 1991).
The majority of preimplantation genetic diagnoses (PGD) for heterozygous female patients have so far consisted of the positive selection of female embryos, usually by fluorescent in-situ hybridization (FISH). The main disadvantage of this approach, however is that half of the discarded male embryos are unaffected. Allelic diagnoses, by either direct amplification of a deleted exon (Liu et al., 1995) or indirect identification of the mutant allele, have also been reported in the course of PGD (Lee et al., 1998). Others have described protocols for the amplification of several exons using whole genome amplification followed by the independent amplification of several exons and a Y-related sequence (Kristjansson et al., 1994). The preamplification of the whole genome by primed extension preamplification (PEP) provides a means of analysing up to 30 loci from a single cell (Zhang et al., 1992), but the initial amplification takes ~8 h and all loci to be analysed have to be subsequently amplified, usually individually by nested PCR. The time constraints associated with PGD and the scarcity of resources have probably prevented the clinical application of such an approach. More recently, a protocol for the duplex amplification of one of five DMD exons with a Y sequence for sex determination has been described (Hussey et al., 1999).
We have developed a test which can directly be used for the diagnosis of the majority of DMD deletions. It consists of a 7-plex PCR allowing the amplification of five DMD exons as well as sequences located on the X amelogenin gene and its Y pseudogene. The multiplex PCR analysis was tested on 100 single lymphocytes, thus validating the diagnosis for all deletions covered by this panel and allowing the detection of >70% of all B/DMD deletions.
Materials and methods
Lymphocyte preparation
Lymphocytes were separated by centrifugation through Ficoll-paque (Pharmacia) from 10 ml of blood unclotted with citrate-dextrose anticoagulant (ACD) following the manufacturer's protocol. Lymphocytes were handled in a clean laboratory with a mouth-controlled fine heat-polished glass micropipette in drops of Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum. The cells were deposited in 3 µl of lysis buffer (200 mmol/l KOH, 50 mmol/l dithiothreitol), under visual control through an inverted microscope. The cells were then denatured at 65°C for 10 min and were kept frozen at –20°C until PCR analysis (Li et al., 1991).
Choice of exons and primers
The Leiden university medical centre compiles an extensive series of DMD-causing mutations (http://www.DMD.NL/deldup.html). From their pool of 305 B/DMD deletions we tried to find a combination of exons allowing the diagnosis of the majority of patients. A diagram representing this pool of deletions is shown in Figure 1. We calculated that 216/305 (71%) reported deletions could be detected by the co-amplification of exons 8, 19, 45, 47 and 51.
|
Primers used for the outer amplification were as previously described (Beggs et al., 1990
; Chamberlain et al., 1990). Inner primers and amelogenin outer and inner primers were chosen using the software Oligo 6.0, while trying to select primers with similar annealing temperatures. Sequences of the primers, melting temperatures, final working concentrations and sizes of the amplified fragments are reported in Table I.
|
Single cell PCR
A double nested multiplex PCR assay was used to co-amplify all the selected loci. The first round PCR contained the seven pairs of outer primers and the second round contained the seven pairs of inner primers with a target sequence located within the previously amplified sequences. Primer sequences and working concentrations are given in Table I
.
The outer PCR reaction contained 3 µl of lysis buffer, 3 µl of potassium-free PCR buffer (25 mmol/l MgCl2, gelatine 1 mg/ml, 100 mmol/l Tris–HCl, pH 8.3), 3 µl of neutralizing buffer (900 mmol/l Tris–HCl, pH 8.3, 300 mmol/l KCl, 200 mmol/l HCl), 0.1 mmol/l dNTP mix (Sigma), 3 µl of primer mix (Genset, see Table I), 1.5 U Amplitaq Gold (Perkin Elmer) and double-distilled water up to a final volume of 30 µl, covered with 30 µl of mineral oil. Three µl of the first amplification product were transferred to 27 µl of inner amplification mix which contained 3 µl of Perkin Elmer PCR buffer (500 mmol/l KCl, 100 mmol/l Tris–HCl, pH 8.3, 15 mmol/l MgCl2), 0.2 mmol/l dNTP mix, 3 µl of primer mix, and 1.5 U Amplitaq Gold.
Denaturation and annealing were carried out successively at 96°C for 25 s and 60°C for 45 s for both the outer and inner reactions followed by an extension phase at 72°C for 3 min during the outer and 2 min 30 s during the inner reaction. Twenty-two and 24 thermal cycles were carried out for the outer and inner reactions respectively. Both reactions were preceeded by a 10 min denaturation at 95°C (to activate the Ampligold) and terminated with a 7 min final extension at 72°C. Ten µl of amplified products were electrophoresed on 10% polyacrylamide mini gels for 40 min at 30 V/cm. The gels were then stained in 0.5 µg/ml ethidium bromide for 5 min, rinsed and exposed to 254 nm UV light for visualization (see Figure 2).
|
Results
Amplification efficiency
A total of 100 single normal lymphocytes were amplified, half male and half female cells. A summary of the amplification results is shown in Table II. All but two cells amplified at least five of the seven loci analysed (98% global amplification rate). Of these two cells, one female cell did not amplify any of the loci and one male only amplified exons X and 47. Overall, failure of amplification happened once for exon 47, twice for the Y-amelogenin sequence (4%), three times for exon 19 and 51 (3%), and four times for exons 8, 45 and the X-amelogenin sequence (4%). Out of the 49 positive male cells analysed, only one did not amplify the Y-amelogenin sequence and would have been mistyped as female (2%). Furthermore, two male cells did not amplify the X-amelogenin sequence while amplifying the Y-amelogenin. In this series the overall amplification failure amounted to the total number of failed loci (21) divided by the total number of loci which should have been amplified (50*7 + 50*6) = 2.8%, thus giving an overall amplification rate of 97.2%.
|
Contamination rate
Contamination of one or two loci was observed in 33 of the 100 negative controls analysed. The maximum number of loci amplified per negative control was two. Among the 100 control blanks analysed, amplification was observed once for the Y-amelogenin sequence (1%), four times for exon 8 (4%), five times for exon 45 (5%), six times for the X-amelogenin sequence (6%), seven times for exon 19 (7%), and eight times for exons 47 and 51 (8%). Exon 45 contamination can be seen in Figure 2
in the second negative control (Figure 2). From the 50 female lymphocytes studied, one wrongly amplified the Y locus and would have been mistyped as male. Overall, there were 39 instances of locus contamination from a total of 100x7 = 700 potential loci (5.6%). Discussion
We have described an efficient multiplex PCR protocol allowing the single cell diagnosis of >70% of all B/DMD deletions. Although the setting up of such a test is more labour intensive than that of a simpler mono or duplex reaction, we showed that once the correct PCR conditions have been defined, multiplex PCR is as efficient as for single locus PCR. The other advantage of such an approach is that the validation of all loci is performed in the same series of cells for all five exons and the sex-linked genes (here, the X and Y amelogenin). In one study, (Hussey et al., 1999), five exons were tested independently in conjunction with an SRY sequence. Amplification efficiency was assessed from 44 single lymphocytes and 22 negative controls for each duplex amplification, in a total of 220 cells and 110 negative controls. The mean amplification rate for the DMD exons obtained in their study was similar to the one observed here (97%). In that report, however, the contamination rate was lower (1%), although only 20 blanks were analysed per locus. With the multiplex procedure presented here, each exon was analysed in 100 reactions. If the same work had been carried out using a duplex reaction with one exon combined with a sexing procedure it would have been necessary to analyse 500 cells for each exon to be tested. The advantage in terms of cost is therefore clear, especially when taking labour time into consideration.
The most critical factor in achieving efficient multiplex amplification was found to be the primer sequence. For the outer reactions, primers previously described (Beggs et al., 1990; Chamberlain et al., 1990) were chosen, as they were shown to be reliable and compatible. An extra set of inner primers was designed for each of the five chosen exons and for the amplification of the amelogenin X and Y sequences. For these, a region of high homology between the X amelogenin gene and its Y pseudogene was chosen (Nakahori et al., 1991). The amplified sequence presented a 5 bp deletion on the X gene. Primers were chosen at location where 100% homology between the X and Y amelogenin sequences was observed. The size difference of 5 bp was thought to be ideal, as we wanted to minimize the problem of allele-specific amplification which is frequently encountered when allele sizes differ widely (Walsh et al., 1992), but wanted the size difference to be sufficient to allow easy band separation after standard gel electrophoresis.
Nested primers were tested for all five exons. Good amplification was achieved rapidly with inner primers 8, 19 and 51 but amplification with inner primers 45 and 47 was less satisfactory during the preliminary work (data not shown). At that point, since the two inner primers did not yield good amplification, two different hemi-nested combinations of primers were tested for each of these loci, i.e. outer forward primer with inner reverse and inner forward with outer reverse primer. Amplification was achieved with the first combination described for exon 45 and the second combination for exon 47 (see Table I). For many reactions, the intensity of amplification of the different loci was uneven: the amplification using the amelogenin gene primers was particularly strong while that using exon 45 primers was very weak. To circumvent that problem, various ratios of primers were tested, in particular the concentration of the weakly amplifying primers was increased in both outer and inner reactions and conversely with primers of high efficiency. Primer concentrations yielding even amplification of all loci were found and are listed in Table I. The concentrations used for exons 8, 19, 47 and 51 outer and inner primers ranged from 0.4 to 0.8 µmol/l and were slightly lower than what is currently used for most single locus PCR (i.e. 0.8–1 µmol/l). For the remaining two sets of primers, we adopted a more extreme primer concentration and we had a 28-fold difference between the outer primer concentration used respectively for the amelogenin and exon 45. The low amplification efficiency of exon 45 might be related to the larger size of the inner amplified product. The basis for the greater efficiency of the amelogenin primers remains unknown. It is interesting to note that the amplification failure rate per locus in male cells (4%) was almost twice that observed in female cells (2.3%). It is likely that this difference is due to the fact that there are two copies of each dystrophin allele in female cells as opposed to only one in male cells.
For the diagnosis of DMD as described here, when relying on the detection of deleted exons in affected hemizygous male cells, the contamination by a normal cell or by previously amplified PCR product of a deleted exon would result in a misdiagnosis. The mean contamination rate was 5.6% per exon. The fact that none of the 100 blanks amplified more than two exons clearly demonstrates that the contamination we observed was not cellular, as we would otherwise expect to have a majority of loci amplifying in the same control blank. Some of the contaminants could originate from free DNA released in the media from lysed lymphocytes. However, lymphocytes were carefully rinsed in Petri dishes which would only serve for a small number of lymphocytes. Presumably, the majority of contaminations came from air-borne amplified products generated in previous PCR reactions. These products are most likely to have been introduced into the PCR tube during single lymphocyte tubing or when adding the PCR mix. The preparation of PCR mixes was carried out in a clean class II laminar flow cabinet. We were, however, reassured to find no contamination in 20 negative controls prepared in our future clinical setting which is completely separated from the genetic laboratory where the analysis is carried out. This indicated that our PCR hood and reagents were `clean', and that the low level of contamination observed when working with single lymphocytes would probably not be observed during our clinical cases. Again, this contamination problem emphasizes the importance of physical separation between the biopsy area, dedicated to the clinical embryology work and the molecular biology area, where a separation between the pre-PCR set up and the post PCR analysis is also completely mandatory.
A number of strategies have been described to reduce carry over contamination. All aim at destroying or modifying the amplified products to prevent their subsequent amplification. Such strategies include the use of: (i) restriction enzymes prior to adding the template to the reaction mix; (ii) deoxyuracyl triphosphate (dUTP) instead of deoxythymidine triphosphate (dTTP) followed by the hydroxylation of dUTP containing amplified products with uracyl-DNA glycosylase (Pang et al., 1992; Thorton et al., 1992); (iii) primers containing a 3'-terminal ribose residue subsequently cleaved by ribonuclease or alkaline treatment (Walder et al., 1993); (iv) ipsoralen in the reaction and subsequent UV irradiation preventing reamplification (Cimino et al., 1991). Unfortunately, in our hands these strategies have proven to be either inefficient, impractical or interfering with the efficiency of the sensitive single cell protocols, and sometimes a combination of all three. Others propose to use a combination of polymorphic markers to detect contamination and to obtain confirmation of diagnosis (Findlay et al., 1995; Ray et al., 2000) and an indirect diagnosis has been used for DMD (Lee et al., 1998). Such strategies generally reduce the risks of misdiagnosis and should be encouraged but can in no way guarantee the accuracy of the diagnosis or the detection of contamination in the sample being analysed. The co-amplification of a contaminating cell will be detected as it will introduce extra alleles, but the results shown here demonstrate that `whole single cells' are rarely the source of contamination and therefore only an implausible allelic combination will be indicative of contamination. A greater accuracy of diagnosis would be achieved by analysing an increased number of polymorphic loci.
Here, when analyzing cells diagnosed as male the risk of misdiagnosis is directly related to the contamination rate. For cells diagnosed as female gender provides an internal control as hererozygous females are unaffected thus, if `whole cell contamination' is excluded, a misdiagnosis can only result from the concomitant failure of amplifying the amelogenin Y sequence and contamination with the exon of interest (probability = 0.05*0.02 = 0.1%). Consequently, until a larger series of negative controls prepared in our clinical setting has been analysed and found to be free of contamination, male undeleted embryos will only be considered for transfer when concordant results will have been obtained from two separate blastomeres. The chance of misdiagnosing a male cell taking into account our current rate of contamination would then be (0.056)2 = 0.3%. Also, since contamination was very seldom observed for more than one locus per blank, the risk of misdiagnosis is greatly reduced for patients with deletions encompassing more than one exon from the multiplex.
This assay was not tested on single blastomeres as the legislation in France is still unclear about using embryos for research purposes. Using the same cell lysis and PCR buffer system, we have demonstrated in the past that amplification rates were only marginally lower on single blastomeres, probably reflecting the higher number of abnormal or apoptotic cells in early development (Ray et al., 1996a,b, 1998). We are therefore confident that this test can be transposed reliably to single blastomeres.
In conclusion, the present work demonstrates that a single cell 7-plex PCR reaction can be as efficient as a mono or duplex PCR. Such an approach is efficient, as the validation of a single assay can be done once and then be used for the majority of patients. We believe that one is good at what he does most. Since the same assay can be used for many patients, it will be used more frequently and more reliably. When using a single cell test very rarely, as would be the case for single exon amplification, extensive series of lymphocytes would have to be assayed prior to each specific case. The wide applicability of our diagnostic test will increase the frequency of its use, making it more reliable while decreasing `the maintenance burden' of a whole battery of tests. We now are ready to use this diagnosis clinically and have four patients awaiting PGD with deletions of exons 6–8, 47, 47–52 and 46–47, all of which can be diagnosed using our multiplex assay.
Acknowledgements
This work was supported in part by the Association Franciaise contre les Myopathies and l'Assistance Publique – Hôpitaux de Paris.
Notes
1 To whom correspondence should be addressed. E-mail: ray{at}necker.fr 
References
Abbs, S., Yau, S.C., Clark, S. et al. (1991) A convenient multiplex PCR system for the detection of dystrophin gene deletions: a comparative analysis with cDNA hybridisation shows mistypings by both methods. J. Med. Genet., 28, 304–311.
Beggs, A.H., Koenig, M., Boyce, F.M. and Kunkel, L.M. (1990) Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum. Genet., 86, 45–48.[Web of Science][Medline]
Boland, B.J., Silbert, P.L., Groover, R.V. et al. (1996) Skeletal, cardiac, and smooth muscle failure in Duchenne muscular dystrophy. Pediat. Neurol., 14, 7–12.[Web of Science][Medline]
Chamberlain, J.S., Gibbs, R.A., Ranier, J.E. et al. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res., 9, 11141–11156.
Chamberlain, J. S., Gibbs, R. A., Ranier, J. E., and Caskey, C. T. (1990) In Innis, M., Gelfand, D. H., Sninsky, J. J. and White, T. J. (eds), PCR Protocols: A guide to Methods and Applications. Academic Press, San Diego, pp. 272–281.
Cimino, G.D., Metchette, K.C., Tessman, J.W. et al. (1991) Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res., 19, 99–107.
Clemens, P.R., Fenwick, R.G., Chamberlain, J.S., Gibbs, R.A. et al. (1991) Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms. Am. J. Hum. Genet., 49, 951–960.[Web of Science][Medline]
Findlay, I., Urquhart, A., Quirke, P. et al. (1995) Simultaneous DNA `fingerprinting', diagnosis of sex and single-gene defect status from single cells. Hum. Reprod., 10, 1005–1013.
Hussey, N.D., Donggui, H., Froiland, D.A. et al. (1999) Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells. Mol. Hum. Reprod., 5, 1089–1094.
Kristjansson, K., Chong, S.S., Van den Veyver, I.B. et al. (1994) Preimplantation single cell analyses of dystrophin gene deletions. Nat. Genet., 6, 19–23.[Web of Science][Medline]
Lee, S.H., Kwak, I.P., Cha, K.E. et al. (1998) Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis. Mol. Hum. Reprod., 4, 345–349.
Li, H., Cui, X. and Arnheim, N. (1991) In Albelson, J.N. and Simon, M.I., (eds), Methods. A Companion to Methods in Enzymology. Academic Press, New York, pp. 49–59.
Liu, J., Lissens, W., Van Broeckhoven, C. et al. (1995) Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion. Prenat. Diagn., 15, 351–358.[Web of Science][Medline]
Nakahori, Y., Takenaka, O. and Nakagome, Y. (1991) A human X-Y homologous region encodes `amelogenin'. Genomics, 9, 264–269.[Web of Science][Medline]
Pang, J., Modlin, J. and Yolken, R. (1992) Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. Mol. Cell. Probes, 6, 251–256.[Web of Science][Medline]
Ray P.F., Kaeda J.S., Bingham, J. et al. (1996a) Preimplantation genetic diagnosis of ß-thalassaemia major. Lancet, 347, 1696.
Ray, P.F. and Handyside, A.H. (1996b) Increasing the denaturation temperature during the first cycles of nested amplification reduces allele dropout from single cells for preimplantation genetic diagnosis. Mol. Hum. Reprod., 2, 213–218.
Ray P.F., Gigarel, N., Bonnefont, J.P. et al. (2000) First specific preimplantation genetic diagnosis (PGD) for ornithine transcarbamylase deficiency. Prenat. Diagn., 20, 1048–1054.[Web of Science][Medline]
Ray, P.F., Winston, R.M.L.and Handyside, A.H. (1998) Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the 
508 deletion causing cystic fibrosis in a clinical practice. Prenat. Diagn., 18, 1402–1412.[Web of Science][Medline]
Read, A.P., Mountford, R.C., Forrest, S.M. et al. (1988) Patterns of exon deletions in Duchenne and Becker muscular dystrophy. Hum. Genet., 80, 152–156.[Web of Science][Medline]
Tennyson, C.N., Klamut, H.J. and Worton, R.G. (1995) The human dystrophin gene requires 16 h to be transcribed and is cotranscriptionally spliced. Nature Genet., 9, 184–190.[Web of Science][Medline]
Thornton, C.G., Hartley, J.L. and Rashtchian, A. (1992) Utilizing uracil DNA glycosylase to control carryover contamination in PCR: characterization of residual UDG activity following thermal cycling. Biotechniques, 13, 180–184.[Web of Science][Medline]
Walder, R.Y., Hayes, J.R. and Walder, J.A. (1993) Use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences. Nucleic Acids Res., 21, 4339–4343.
Walsh, P.S., Erlich, H.A. and Higuchi, R. (1992) Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Appl., 1, 241–250.[Medline]
Zhang, L., Cui, X., Schmitt, K. et al. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc. Natl Acad. Sci. USA, 89, 5847–5851.
Submitted on October 27, 2000; accepted on February 28, 2001.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Malmgren, I. White, S. Johansson, L. Levkov, E. Iwarsson, M. Fridstrom, and E. Blennow PGD for dystrophin gene deletions using fluorescence in situ hybridization Mol. Hum. Reprod., May 1, 2006; 12(5): 353 - 356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Fiorentino, A. Biricik, A. Nuccitelli, R. De Palma, S. Kahraman, M. Iacobelli, V. Trengia, D. Caserta, M.A. Bonu, A. Borini, et al. Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders Hum. Reprod., March 1, 2006; 21(3): 670 - 684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Feyereisen, A. Amar, V. Kerbrat, J. Steffann, A. Munnich, M. Vekemans, R. Frydman, and N. Frydman Myotonic dystrophy: does it affect ovarian follicular status and responsiveness to controlled ovarian stimulation? Hum. Reprod., January 1, 2006; 21(1): 175 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Girardet, S. Hamamah, H. Dechaud, T. Anahory, C. Coubes, B. Hedon, J. Demaille, and M. Claustres Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy Mol. Hum. Reprod., July 1, 2003; 9(7): 421 - 427. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. D. Hussey, T. Davis, J. R. Hall, M. F. Barry, R. Draper, R. J. Norman, and Z. Rudzki Preimplantation genetic diagnosis for {beta}-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci Mol. Hum. Reprod., December 1, 2002; 8(12): 1136 - 1143. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Tachdjian, N. Frydman, F. Audibert, P. Ray, V. Kerbrat, P. Ernault, R. Frydman, and J.-M. Costa Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre Hum. Reprod., August 1, 2002; 17(8): 2183 - 2186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. F. Ray, N. Frydman, T. Attie, S. Hamamah, V. Kerbrat, G. Tachdjian, S. Romana, M. Vekemans, R. Frydman, and A. Munnich Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination Mol. Hum. Reprod., July 1, 2002; 8(7): 688 - 694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Thornhill and K. Snow Molecular Diagnostics in Preimplantation Genetic Diagnosis J. Mol. Diagn., February 1, 2002; 4(1): 11 - 29. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||