Molecular Human Reproduction, Vol. 6, No. 5, 391-396,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis*
1 Department of Molecular Cell Biology & Genetics, Research Institute Grow and Development (GROW), Maastricht University, 6229 GR Maastricht, and 2 Department of Obstetrics & Gynaecology, Academic Hospital Maastricht, 6202 AZ Maastricht, The Netherlands
Abstract
Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)n repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0–2%) and single recombination (1.1–3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.
cystic fibrosis/multiplex marker PCR/preimplantation genetic diagnosis/single cell diagnosis
Introduction
Preimplantation genetic diagnosis (PGD) is a combination of IVF and the genetic diagnosis of embryos at the early cleavage stage. It allows the selection and transfer of unaffected preimplantation stage embryos to the uterus. For couples at risk of transferring a genetic disorder to their offspring, PGD offers an alternative to prenatal diagnosis. By choosing PGD, the difficult decision of pregnancy termination after genetic diagnosis by chorionic villus sampling or amniocentesis in the first and second trimesters of gestation can be avoided. In 1990, the first successful PGD was reported for a number of X-linked genetic disorders by sexing embryos using Y-specific DNA polymerase chain reaction (PCR) amplification (Handyside et al., 1990
). The first single gene defect for which PGD was successfully applied, was the triplet basepair 
F508 deletion in the cystic fibrosis transmembrane regulator (CFTR) gene, also by means of PCR (Handyside et al., 1992). Since then the number of disorders for which PGD can be performed is growing slowly, but steadily. Technically, PGD remains a challenge as only one or two blastomeres are available for analysis and this has to be completed within one day. The genetic analysis on one single blastomere has to meet high standards of PCR efficiency, allelic drop out (ADO) rate, reliability, and contamination control (Lissens et al. 1996). Therefore a PGD protocol is put through an extensive preclinical trial before it can be introduced into a clinic. In most PGD clinics, the cystic fibrosis (CF) F508 deletion is one of the single gene defects for which PGD is offered as an alternative for prenatal diagnosis.
CF is a common autosomal recessive genetic disorder with a prevalence of ~1 in 2500 live births and a carrier frequency of ~1 in 25 in the North Western European population (Findlay, 1997; Tsui and Durie, 1997). The disease phenotype is heterogeneous and depends on the specific mutation in the CFTR gene. Clinical characteristics of CF are accumulations of dehydrated mucus, resulting in chronic obstructive respiratory disease, pancreatic enzyme deficiency and obstruction of the small intestine. Also congenital bilateral absence of the vas deferens (CBAVD) is present in ~95% of the male CF patients, leading to male infertility (Chillón et al., 1995). Without treatment the disease usually causes early death from pulmonary infections. CF has a considerable impact on the quality of life and, although improved medical care often preserves life into adulthood, the median life expectancy is only 30 years (Tsui and Durie, 1997). Worldwide more than 700, mostly rare, CF-related mutations have been identified (Tsui and Durie, 1997). In the Netherlands, a phenylalanine deletion at amino acid position 508 (F508) is the most common mutation with a frequency of 77% of the CF chromosomes (Haigh and Kazazian, 1994). In 60% of CF couples, both partners carry the F508 mutation and a PGD protocol for this specific mutation is operational in our clinic (Liu et al., 1992). However, for the remaining 40% in which other CF mutations are involved no such test is available yet. Because the development of mutation-specific PGD protocols for all mutations other than the homozygous F508 mutation is impracticable, we developed a general marker-based protocol for PGD of CF.
Four closely linked highly polymorphic (CA)n repeat markers flanking the CFTR gene were used; D7S523 and D7S486 with heterozygote frequencies of 80 and 81%, located 1 and 0.2 cM proximal to the CFTR gene, and D7S480 and D7S490 with heterozygote frequencies of 86 and 78%, located 2 and 5 cM distal to the gene (Figure 1; de Vries et al., 1996). This protocol can extend the number of CF couples to whom PGD can be offered by nearly 2/3 of all the patients that request PGD for the CF F508 mutation. To perform a reliable marker-based CF diagnosis family studies are required to determine the wild-type and CF risk haplotype. At least two flanking markers must be informative. The aim of this study was to investigate the feasibility of a general PGD protocol using a multiplex PCR of the four markers at the single cell level.
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Materials and methods
Heterozygote frequencies of micro-satellite markers
To ascertain the heterozygote frequencies in the Dutch population of the (CA)n repeat markers, multiplex PCR was performed on 300 ng genomic DNA as described below, with a reduced number of 35 amplification cycles. DNA was obtained from 29 unrelated individuals of the Dutch population, and isolated from peripheral blood leukocytes according to standard protocols (Miller et al., 1988). The observed and published (de Vries et al., 1996) heterozygote frequency data were analysed using the 
2 test or Fisher's exact test where appropriate.
Collection of human leukocytes and blastomeres
Human leukocytes and blastomeres were used as single cells for testing the PCR method. Single leukocytes were collected in 2 µl Ca2+ and Mg2+-free phosphate-buffered saline solution (PBS) with 1% polyvinylpyrrolidone (PVP) molecular weight 360 kDa (Sigma, Aldrich Chemie BV, Zwijdrecht, The Netherlands) and 0.1 mg/ml Phenol Red (Sigma), with the help of a micromanipulator (ONO-121; Narishige, Paes Nederland BV) mounted on an inverted microscope (IX-70; Olympus, Zoeterwoude, The Netherlands). After transferring the cells to a 0.2 ml reaction tube, cellular DNase heat inactivation was accomplished by a 10 min incubation at 65°C. Cells were stored at –20°C until PCR was performed. Blastomeres were obtained from human embryos that were donated after IVF treatment. They were considered to be unsuitable for freezing. Seven surplus embryos after IVF were used from three different couples. Blastomeres were collected from these embryos after removing the zona pellucida by 3–5 min incubation in a 1/1 mixture of 500 IU/ml pronase (Sigma) and PBS. The blastomeres were separated from each other by gently flushing in a small pipette. Subsequently, they were rinsed in three droplets of PBS, with 1% PVP and Phenol Red (0.1 mg/ml), and transferred into a 0.2 ml reaction tube with the help of a dissection microscope. To obtain information on the maternal and paternal marker genotypes of the blastomeres, cumulus cells and spermatozoa served as PCR target material respectively. Cellular DNase inactivation and storage of the collected cells was performed as described previously. Blastomeres were only collected after couples had given consent that surplus embryos were used for these experiments. The protocol was approved by the local Ethical Committee.
PCR procedure
Prior to multiplex PCR, the alkaline lysis buffer and the PCR mix without primers or Taq/Pwo DNA polymerase (Roche Diagnostics, Nederland BV, Almere, The Netherlands), were decontaminated from DNA by UV-C irradiation for 1 h using an UV-C lamp type TUV 30W/G30T8 longlife (Philips, Eindhoven, The Netherlands). Blank samples were included in every PCR series to monitor DNA contamination. Cells were lysed by adding 2.5 µl of alkaline lysis buffer [50 mmol/l dithiothreitol (DTT; Pharmacia Biotech, Benelux, Roosendaal, The Netherlands)/200 mmol/l NaOH] followed by 10 min of incubation at 65°C. After the cell lysis, multiplex PCR was performed with the GeneAmp® PCR System 9700 (Perkin-Elmer Applied Biosystems, Nieuwerkerk a/d, Ijssel, The Netherlands) using the ExpandTM Long Template PCR System (Roche Diagnostics). The PCR reaction was performed in a total volume of 25 µl and contained 1x Buffer2ISOdia
and 2.5 IU of Taq/Pwo polymerase provided by the manufacturer (Roche Diagnostics), 0.2 mmol/l dNTP from each of the four deoxynucleotide triphosphates (dGTP/dATP/dCTP/dTTP; Pharmacia), 20 mmol/l Tricine pH 4.95 (Merck, Nederland BV, Amsterdam, The Netherlands) to neutralize the alkaline lysis buffer and the primersets of which the forward primer is fluorescently labelled (3 pmol 490R (AGC.TAC.TTG.CAG.TGT.AAC.AGC.ATT.T)/490F-TET (CCT.TGG.GCC. AAT.AAG.GTA.AG), 10 pmol 486R (GCC.C-AG.GTG.ATT.GAT.AGT.GC)/486F-HEX(AAA.GGC.CAA.TGG.T-AT.ATC.CC), 10 pmol 480R (AGC.TAC.CAT.AGG.GCT.GGA.GG)/480F-HEX(CTT.GGG. GAC.TGA.ACC.ATC. TT) and 20 pmol 523R (AAA.ACA.TTT.CCA.TTA.CCA.CTG)/523F-HEX(CTG.ATT.CA-T.AGC.AGC.ACT.TG) (Gyapay et al., 1994). PCR was started with an initial 5 min denaturation step at 95°C followed by 55 cycles of 30 s denaturation at 95°C, 60 s annealing at 55°C and 60 s elongation at 68°C with a final elongation step of 5 min at 68°C. PCR products were separated on a 8% Long Ranger (FMC BioProducts, Sanver TECH, Heerhugowaard, The Netherlands) denaturing polyacrylamide gel mounted on a ABI Prism 377 DNA Sequencer with automated fluorescent scanning detection and analysed using GeneScan Analysis Software version 2.1, to size the PCR fragments (PE Applied Biosystems). Within 10 h after sample preparation, the entire procedure of cell lysis, PCR amplification and PCR product analysis was completed.
Results
Heterozygote frequencies of micro-satellite markers
Heterozygote frequencies of the markers used were determined by genotyping 29 unrelated individuals from the Dutch population. The heterozygote frequencies of D7S523, D7S486, D7S480 and D7S490 were 81, 77, 87 and 80% respectively.
Optimizing single cell multiplex PCR
Initially 50 pg amounts of genomic DNA were used as target material, succeeded by single cells (leukocytes or blastomeres) in a later stage. The multiplex PCR of (CA)n repeat markers was optimized by looking for optimal but mutual PCR conditions of the individual markers, followed by combining the primer sets in one PCR reaction. However, combination of the primer sets in one PCR reaction required careful adjustment of the primer concentrations to obtain marker signals that could be scored within the same detection range. Furthermore, marker D7S490 is labelled with a different fluorescent label to distinguish it from background peaks from upstream markers. The electropherograms in Figure 2 show the single cell multiplex PCR results of blastomeres with the same marker genotypes, heterozygous for the four markers used. Electropherogram 1 demonstrates the complete genotype whereas in electropherogram 2 (ADO) and in electropherogram 3 the presence of an additional allele can be observed. Additional alleles are defined as alleles that cannot be deduced from the parents and are of non-paternal or maternal origin, either caused by contamination or PCR artefacts.
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Single cell testing
The optimized multiplex marker PCR was evaluated for PCR efficiency, ADO rate and the frequency of additional alleles. A total of 100 single leukocytes isolated from fresh blood obtained from four unrelated individuals, and 50 nucleated single blastomeres, obtained from seven surplus embryos from three couples, were analysed by multiplex PCR. All 100 leukocytes revealed positive PCR signals for each of the four markers tested whereas ADO rates determined in cells heterozygote for the concerning markers range from 1% for D7S490 to 6% for D7S480 as shown in Table I
. Of the 50 blastomeres, 49 gave positive PCR signals for each of the four markers tested and ADO rates varied from 0% for D7S523 to 7% for D7S486. The overall PCR efficiency of single cells tested was 99%, and the overall ADO rate varied from 2 to 5% (Table I). All 25 blanks included in the single cell test series were negative.
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The multiplex PCR results of the amplified leukocytes and blastomeres were validated with respect to the occurrence of ADO and additional alleles before they were accepted to construct the marker genotypes (Table II
). Multiplex PCR results were considered to be acceptable for marker genotype construction, when all markers gave amplification results and no more than one ADO event and/or one additional allele were detected. Although these criteria are somewhat arbitrary, the occurrence of ADO and the presence of additional alleles are an indication that the PCR results may not be reliable. No significant differences were found between leukocytes and blastomeres. The complete genotype without additional alleles or ADO was obtained in 73% of 149 single cells with a positive PCR result. In 22% of the tested single cells one additional allele and/or one ADO event was detected. In only 5% of the cases, more than one additional allele and/or more than one ADO event occurred.
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Furthermore, we demonstrated the inheritance of paternal and maternal marker haplotypes in 46 of 49 blastomeres which were accepted for genotyping according to the validation criteria. Paternal and maternal marker haplotypes were determined from the amplification of spermatozoa and cumulus cells respectively. Figure 3
shows the electropherograms obtained from the cumulus cells and sperm cells of one couple, electropherogram 1 and 2 respectively. Also the electropherograms (3, 4 and 5) of three single blastomeres collected from three different surplus embryos from this couple after IVF are shown. Blastomeres represented by electropherogram 3, 4 and 5 clearly demonstrated maternal and paternal alleles for all the four markers tested. In electropherogram 4, all markers revealed two different alleles demonstrating that this embryo was heterozygous for all the markers tested. The embryo represented by the blastomere analysed in electropherogram 3 was heterozygous for the markers D7S490 and D7S523 and homozygous for D7S486 and D7S480. Electropherogram 5 shows three different marker alleles (two maternal and one paternal allele) for the markers D7S490, D7S486 and D7S480. Marker D7S523 reveals only two different alleles. However it is likely that this embryo inherited both maternal 233 bp D7S523 alleles and that it was trisomic for the four markers amplified.
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Discussion
The heterozygote frequencies of the (CA)n repeat markers used in our population did not differ significantly from the heterozygote frequencies published by de Vries et al. (1996). Based on these heterozygote frequencies, we conclude that in ~87% of the couples at least one marker on each side of the CFTR gene is informative, which is a precondition for a reliable marker-based CF diagnosis. Although the confidence interval of this estimate is large, the vast majority of couples will be informative for this marker-based approach for PGD of CF. In single cell multiplex PCR, PCR efficiency, validation criteria, ADO and recombination events define the number of cells for which a reliable marker genotype can be constructed, and the number of embryos for which a marker-based CF diagnosis can be performed. Consequently these factors affect the number of embryos suitable for transfer in case of a PGD.
The PCR efficiency and the validation criteria affect the number of single cells for which a reliable genotype can be constructed, irrespective of the CF genotype of the single cells tested. In 99% of the single cells amplified, multiplex marker PCR data were obtained. After validating these data according to the criteria stated previously, we could reliably genotype 95% of the single cells for the markers used. This suggests that ~5% of the blastomeres obtained in a PGD cycle would consequently result in an embryo lost for transfer.
In the single cells for which a marker genotype could be constructed (95%), a fraction will result in embryos lost for transfer due to ADO and recombination events. The effect of ADO and single recombination events depend on the CF genotype. In cells containing both the maternal and paternal wild-type or CF-risk marker haplotypes, ADO or a single recombination event does not lead to embryos lost for transfer, or the transfer of an affected embryo. In these homozygous wild-type or CF-mutated cells one marker haplotype cannot be constructed, but the CF status can be ascertained by the other marker haplotype. However in carriers with two different marker haplotypes, the wild-type and CF risk haplotype, ADO and recombination events can prevent identification of the healthy marker haplotype. In case of PGD, these embryos are lost for transfer because only the risk haplotype can be constructed. The exact contribution of ADO and recombination events has to be derived from the parental genotypes prior to PGD and is predetermined by which markers and the number of markers that are informative. A thorough family study prior to PGD is essential to reliably construct the wild-type and CF risk marker haplotypes. If this is not the case, a single recombination event can lead to 50% of the embryos lost for transfer.
In the optimal situation where all four markers are informative, ADO does not result in carrier embryos lost for transfer. After ADO (Table I
) of one marker allele of the healthy haplotype, there are still markers left flanking the CFTR gene, making construction of the healthy marker haplotype possible. However, if three markers are informative, the chance that a CF carrier embryo cannot be transferred due to ADO, is determined by half the ADO rate of the sole distal or proximal marker. In the ADO affected carrier embryos, the incidence of the risk haplotype being lost is equal to that of the healthy wild-type marker haplotype being lost. Therefore, the chance that only the risk haplotype is ascertained is 1–2.5%. If only two markers are informative, one on each site of the CFTR gene, this chance is determined by the sum of half the ADO rates of the two flanking informative markers and varies from 2 to 4%. Because carrier embryos are 50% of the total embryo population, the overall contribution of ADO to embryos lost for transfer varies from 0 to 2%, depending on the number and type of informative markers.
Recombination occurs in 2.2–6% of the paternal and maternal haplotypes depending on which flanking markers are informative. Therefore the total change on recombination varies from 4.4 to 12%. However, recombination events, similar to ADO events, only lead to embryos being lost for transfer in carrier embryos. Of the heterozygous embryos affected by recombination 50% still can be transferred because the wild-type marker haplotype can be ascertained. Moreover, of the total embryo population, 50% are CF carriers. This means that due to recombination events, and depending on the genetic distances of the informative markers, 1.1–3% of the embryos are lost for transfer.
ADO and single recombination events do not result in erroneous transfer of an affected embryo provided that the marker haplotype unaffected by ADO and recombination can be reliably ascertained. On the contrary, double recombination between flanking heterozygous markers of the paternal or maternal CF risk marker haplotype may lead to a marker-based CF misdiagnosis in a CF compound heterozygous or homozygous embryo. The double recombination event of such an embryo can result in the detection of a wild-type marker haplotype although the CF mutation is present. Hence the embryo displays a marker genotype of a carrier embryo although it is compound heterozygous or homozygous for the CF mutation. However, the risk of transferring an affected embryo with a double recombination between flanking markers is <0.05%.
An additional source of misdiagnosis can be caused by contamination with foreign DNA. Precautions taken to avoid contamination as described in Materials and methods appear to be sufficient. This can be concluded from the 25 blank samples included in the single cell test series to monitor contamination, which were all negative. Furthermore this multiplex PCR marker system provides a test for contamination. Prior to PGD the maternal and paternal marker haplotypes are determined and the expected marker alleles for testing a single cell are known. Therefore the use of this highly polymorphic multiplex PCR system provides also a control for contamination. The four dinucleotide repeat marker alleles identify the origin of the amplified DNA. They confirm the amplification of blastomeric template DNA or expose contamination by foreign DNA as has been shown previously (Pickering et al., 1994; Findlay et al., 1995). Pickering used a dinucleotide repeat sequence to obtain a crude DNA fingerprint whereas Findlay used six tetranucleotide micro-satellite sequences to determine a DNA fingerprint from single cells. Deduced from the allele frequencies of the individual four markers used (data not shown), the observed genotype has a <1 in 24 000 chance of not being from the amplified cell.
An additional advantage of this highly polymorphic multi allelic marker PCR system is demonstrated in Figure 3. Although the markers only give information about the number of chromosomes 7, e.g. trisomy 7 (Figure 3; embryo 3), an indication about the ploidy status of the tested embryos is obtained as well. This prevents triploid embryos from being transferred. The use of fluorescent PCR of polymorphic small tandem repeats for determining chromosomal trisomies have been reported for application in prenatal diagnosis and PGD (Findlay et al., 1998; Verma et al., 1998; Blake et al., 1999).
Conclusions
Multiplex PCR is a reliable and generally applicable alternative for mutation-directed PGD protocols, irrespective of the CF mutations involved. The protocol expands the number of CF couples to whom PGD can be offered, by nearly 2/3 of all the patients that request PGD for the CF F508 mutation. It makes the development of individual mutation-directed PGD protocols redundant, provided that the couple at risk is informative for the markers used. However, the multiplex marker-based CF diagnosis requires a thorough family study to ascertain the CF risk and wild-type marker haplotype. The exact number of family members to be analysed to construct haplotypes depends on the family constitution. The majority of couples who are carriers of a recessive genetic disorder have affected offspring, limiting the number of family members to be analysed for the markers used, to four: the couple at risk; their affected child; and an unaffected sibling. If no unaffected brothers or sisters are available, paternal and maternal grandparents of the index patient may be analysed to rule out recombination, and accurately determine the CF risk and wild-type marker haplotype. Additional information on the paternal haplotypes can be obtained by analysing single sperm cells. This family study is not necessary for mutation-directed protocol for which only information about the mutations involved is required. Nevertheless, for single cell analyses the marker-based approach clearly is advantageous over the direct mutation approach. Unlike the marker-based CF diagnosis, ADO in mutation-directed protocols can lead to the transfer of affected embryos in PGD of compound heterozygotes. If (due to ADO) one mutant allele is not detected and only the wild-type allele is observed, the embryo can be genotyped as a carrier of the only detected mutation. Hence transfer of an affected embryo can occur. Furthermore, the polymorphic and multi-allelic character of this marker system provides a test for the origin of the detected genotype and gives an indication about the chromosomal ploidy of the blastomere tested. This is not possible with the mutation-directed protocols, in which information is obtained of the mutant and wild-type alleles only. Depending on the genotype of the couple, and taking into account the embryos lost for transfer by the validation criteria (5%), the ADO event (0–2%) and the single recombination event (1.1–3%), >90% of the embryos can be reliably genotyped on a single blastomere. Moreover, the risk of misdiagnosis equals the chance of a double recombination between flanking markers and is <0.05%. Therefore this approach will enable PGD as a choice for virtually all CF carriers provided that at least two flanking markers are informative.
Notes
3 To whom correspondence should be addressed at: Department of Molecular Cell Biology & Genetics, POB 1475, 6201 BL Maastricht, The Netherlands 
* This work is based in part on the Established Scientists Award Winning paper presented at the 15th Annual Meeting of ESHRE, June 27–30, 1999, Tours, France
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Submitted on November 15, 1999; accepted on February 8, 2000.
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 E Kanavakis and J Traeger-Synodinos Preimplantation genetic diagnosis in clinical practice J. Med. Genet., January 1, 2002; 39(1): 6 - 11. [Abstract] [Full Text] [PDF]
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I. Eftedal, M. Schwartz, H. Bendtsen, A.N. Andersen, and S. Ziebe Single intragenic microsatellite preimplantation genetic diagnosis for cystic fibrosis provides positive allele identification of all CFTR genotypes for informative couples Mol. Hum. Reprod., March 1, 2001; 7(3): 307 - 312. [Abstract] [Full Text] [PDF]
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ESHRE PGD Consortium Steering Committee ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: data collection II (May 2000) Hum. Reprod., December 1, 2000; 15(12): 2673 - 2683. [Abstract] [Full Text] [PDF]
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 A. Makrigiannakis, K. Amin, G. Coukos, J. L. Tilly, and C. Coutifaris Regulated Expression and Potential Roles of p53 and Wilms' Tumor Suppressor Gene (WT1) during Follicular Development in the Human Ovary J. Clin. Endocrinol. Metab., January 1, 2000; 85(1): 449 - 459. [Abstract] [Full Text]
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 A. Makrigiannakis, G. Coukos, M. Christofidou-Solomidou, B. J. Gour, G. L. Radice, O. Blaschuk, and C. Coutifaris N-Cadherin-Mediated Human Granulosa Cell Adhesion Prevents Apoptosis : A Role in Follicular Atresia and Luteolysis? Am. J. Pathol., May 1, 1999; 154(5): 1391 - 1406. [Abstract] [Full Text] [PDF]
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