Mol. Hum. Reprod. Advance Access originally published online on May 20, 2005
Molecular Human Reproduction 2005 11(6):463-468; doi:10.1093/molehr/gah176
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Multiple mutation analysis of the cystic fibrosis gene in single cells
1Unitat de Biologia Cel·lular i Genètica Mèdica, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra and 2Hospital Sant Joan de Deu, Barcelona, Spain
3 To whom correspondence should be addressed. Email: jsanchezg{at}servet.uab.es, Email: joaquima.navarro{at}uab.es
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Abstract |
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PGD is becoming an alternative to prenatal diagnosis. The combination of IVF techniques with the PCR technology allows for the detection of genetic abnormalities in first polar bodies from oocytes and blastomeres from cleavage-stage embryos. Dealing with a genetic disease with a heterogeneous spectrum of mutations like cystic fibrosis, one of the objectives of centres offering PGD is the application of simple and efficient protocols that allow for the detection of a wide range of mutations with a single procedure. In the present work, 29 normal loci and the 31 most frequent cystic fibrosis transmembrane conductance regulator (CFTR) mutations in Southern Europe could be detected at the same time in single cells applying a modified and improved primer extension preamplification-PCR. Two different Taq polymerases were tested in isolated buccal cells heterozygous for several mutations. The protocol that gave statistically significant better results was also successful in oocytes and their first polar bodies.
Key words: cystic fibrosis/embryo/first polar body/PEP-PCR/PGD
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Introduction |
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Cystic fibrosis is the most common autosomal-recessive disease which affects one in every 2500 newborns (Cutting, 1997
). Its main features are elevation of chloride in sweat, pulmonary disease and pancreatic failure. The gene responsible, cystic fibrosis transmembrane conductance regulator (CFTR), was cloned in 1989 (Riordan et al., 1989). The most frequent mutation in approximately 66% of all cases is a 3 bp deletion 
F508, but over 1000 different mutations have been described (http://www.genet.sickkids.on.ca/cftr-cgi-bin/FullTable). About 1 in 25–30 Caucasians is a carrier of a cystic fibrosis mutation. PGD is an alternative to prenatal diagnosis for the detection of chromosomal abnormalities or single gene defects and can prevent termination of pregnancy in the case of an affected fetus. PGD has been widely applied in cleavage-stage embryos by analysing one or two of their blastomeres (Sermon et al., 2005). PGD can also be performed by first polar body biopsy (PGD-1PB) (Strom et al., 1990). The 1PB is extruded during the first meiotic division and contains the complementary genotype to the oocyte arrested in metaphase II (MII). Its removal has no effect on the subsequent embryo since it is destined to degenerate and does not interfere with fertilization rates or the percentage of embryos entering cleavage (Verlinsky and Kuliev, 1992; Magli et al., 2004). In a female carrying a genetic disease, the absence or presence of the mutation can be detected indirectly in the oocyte by analysis of the 1PB. Only embryos resulting from genetically normal oocytes are transferred back to the mother. To date, PGD-1PB has been applied to several monogenic diseases (Wells and Sherlock, 1998) and its analysis must contemplate recombination events, which would lead to heterozygous gametes. The phenomenon of allele drop-out (ADO) also has to be considered in single-cell PCR (Findlay et al., 1995). It consists of the amplification of only one allele in a heterozygous single cell which could lead to misdiagnosis. The accuracy of the procedure has been improved by including techniques such as: more efficient lysis (El-Hashemite and Delhanty, 1997), multiplex PCR and fluorescent PCR (Findlay et al., 1995) which allows for more sensitive detection of PCR products. Recent studies in isolated cells and blastomeres are based on the identification of a single CFTR mutation (Ray et al., 2002; Vrettou et al., 2002) or on highly polymorphic short tandem repeats (STR) linked to the mutation (Dreesen et al., 2000; Eftedal et al., 2001; Goosens et al., 2003). The most recent alternatives for PGD of cystic fibrosis are sequencing and mini-sequencing methods, with which a wide range of mutations may be detected directly (Bermúdez et al., 2003; Fiorentino et al., 2003).
The aim of the present work was to develop a sensitive and reliable method for the identification of a large number of different mutations in a single procedure. Isolated cells were subjected to a modified and improved primer extension preamplification PCR (I-PEP-PCR) (Dietmaier et al., 1999). The products obtained were used in a multiplex PCR and a fluorescent oligonucleotide ligation assay (OLA) for the identification of normal and mutant loci of the 31 most frequent mutations in Southern Europe (including the 25 most common worldwide). In our hands, the first protocol of PEP-PCR (Zhang et al., 1992) was successful in small clumps of cells (5–30) and in 20 pg of genomic DNA. In order to improve the performance of whole genome amplification in single cells, two different Taq polymerases were tested.
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Materials and methods |
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Strategy
To develop a unique PGD system to detect diverse mutations, whole genome amplification was applied to isolated cells, enabling the use of a commercial kit which allows for fluorescent detection of 31 cystic fibrosis mutations. Two different Taq polymerases were tested to improve the performance of the preamplification step. The validation of both procedures was based on the height of the alleles detected in the electropherogram. Moreover, the method which yielded the best results was tested in oocytes.
Cell samples
Isolated buccal cells were collected by mouthwashes from three normal individuals, three patients affected by mutations F508, F508/1078delT and F508/3849+10kbC>T and five heterozygous carriers for CFTR mutations F508, N1303K, G542X, R347P and 2183AA>G.
Ten matured oocytes remaining unfertilized after IVF, donated by assisted reproductive centres, were available for analysis. Both MII oocytes and 1PBs were studied separately.
All procedures were approved by the Ethics Committees of the participating centres.
Cell isolation
Stringent precautions were followed to guard against contamination during the entire process of isolation, lysis and amplification (Wells and Sherlock, 1998; Thornhill and Snow, 2002). The work was performed in a restricted area in a flow hood with no DNA or PCR product access. The technician wore a gown, surgical mask and gloves, and dedicated pipettes and filter tips were used in the procedure. The reagents used were subjected to filtration, sterilization and UV exposure.
Manipulation was performed using UV-sterilized pulled-glass micropipettes. Buccal cell suspension was diluted in several droplets of sterile phosphate-buffered saline (PBS) with 0.1% polyvinyl alcohol (PVA) to prevent cell sticking, until a single cell was obtained. Buccal cells were checked under stereomicroscope for the presence of a visible nucleus. MII oocytes and 1PBs were separated by digestion of the zona pellucida with either 30 mg/ml trypsin in PBS, pH 7.2, for 2–3 min at 37°C, or with Tyrode's acid solution (pH 2.5). Cells were washed three times in PBS/0.1% PVA and transferred to a 0.2 ml PCR tube. The presence of the cell inside the tube was confirmed.
Cell lysis
One microlitre of sodium dodecyl sulphate 17 µM (Sigma, USA) and 2 µl of PCR-grade proteinase K (125 mg/ml) (Roche, Switzerland) were added to each tube and covered with one drop of light mineral oil (Sigma, USA). The lysis was performed by incubation for 1 h at 37°C, followed by 15 min at 95°C to inactivate the enzyme.
Whole genome amplification in single cells
The lysed cells were subjected to I-PEP-PCR whole genome amplification (Dietmaier et al., 1999) with several modifications: (1) The reaction was performed in 40 µl with five units of Taq polymerase, 4 µl of 10x Taq buffer, 3.3 µl of a 400 µM solution of totally degenerated 15-mer primers (Zhang et al., 1992) and 2 µl of a mixture of the four dNTPs (2 mM each); (2) Two Taq polymerases were used: SuperTaq polymerase (Taq A) (HT Biotechnology, UK), specially recommended in single-cell PCR and SuperTaq Plus polymerase (Taq B) (Ambion, USA), a mixture of thermostable DNA polymerases with proofreading activity, enabling a high yield of PCR products up to 20 kb. Twenty single cells were amplified with Taq A and a further 30 isolated buccal cells and 10 1PB-MII pairs were amplified with Taq B; (3) Hotstart was performed keeping the tubes in ice and placing them in a Tgradient (Biometra, Germany) thermal cycler when it reached the required temperature of 80°C; (4) The initial denaturation was performed at 94°C for 7 min and (5) Modified I-PEP-PCR consisted of 50 cycles of 1 min at 94°C, 2 min at 31.5°C, a programmed ramp of 0.1°C per second, 55°C for 4 min and a final step at 68°C for 30 s. Tubes were then cooled down to 4°C. Blank controls of the isolation solutions and PCR reagents were included to check contamination. Tubes were kept at –80°C if necessary.
Cystic fibrosis assay in single cells
Six microlitres of the I-PEP-PCR products were used in an adaptation of the ‘Cystic Fibrosis Assay’ (Applied Biosystems, USA), a commercially available test for the screening of the most common CFTR mutations. It consists of a multiplex PCR in which 15 pairs of primers amplify the regions of the gene which may contain the mutations. Then, a multiplex, fluorescent OLA detects normal and mutant alleles with a complementary probe for each sequence which are ligated with a common probe for every locus labelled with 6-FAM (6-Carboxyfluorescein), TET (tetrachlorafluorescein) or HEX (hexachlorofluorescein). The normal and mutant oligonucleotides have different lengths of pentaethylene oxide tails, with subsequent electrophoretic separation. The mutations assayed are: F508, I507, Q493X, V520F, 1717-1G>A, G542X, G551D, R560T, S459R, S459N and R553X labelled with FAM (blue), 3849+10kbC>T, 3849+4A>G, R1162X, 3659delC, W1282X, 3905insT, N1303K, 1078delT, R347P, R347H and R334W labelled with TET (green) and A455E, 1898+1G>A, 2183AA>G, 2789+5G>A, G85E, 621+1G>T, R117H, Y122X and 711+1G>T labelled with HEX (yellow). The 10 µl of the OLA products of a single cell were additionally ethanol-precipitated for 1 h at –80°C and resuspended in 1 µl of nuclease-free water and a TAMRA (Carboxytetramethylrhodamine) size standard was added. Single cells produced a very small amount of PCR products, so it was necessary to ethanol-precipitate the OLA reaction to concentrate the fragments obtained. The samples were run in an ABI Prism 377 (Applied Biosystems, USA) automated gel electrophoresis. Subsequent analysis was performed with GeneScan and Genotyper softwares (Applied Biosystems, USA). Each allele was identified by its name, fragment size in base pairs and fluorescence intensity in relative fluorescence units (RFU).
Scoring criteria
Detection of an allele has been considered when a single peak was equal to or above 50 RFU in height. A rigorous classification of three categories, based on allele height, was established for evaluating the success and quality of both procedures: peaks below 50 RFU were considered not detected, when the peak ranged from 50 to 250 RFU the detection was considered as low and peaks over 250 RFU were classified as high. Statistical analysis was performed with SPSS software, version 12.5, to compare the results obtained with both Taq A and B.
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Results |
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Identification of the normal and mutant alleles of the CFTR gene was achieved with both polymerases tested in preamplified DNA of single cells. Since the mutations to be analysed are known, the rest of the peaks of the normal loci can be used as a control of the amplification. No additional unknown mutations were observed after whole genome amplification. Thus the genotype of the cells was determined correctly. Figure 1A shows an electropherogram of a buccal cell carrying mutations
F508 and 3849+10kbC>T.
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Twenty single buccal cells were analysed using Taq A for amplification with PEP-PCR. Preamplification with Taq B was performed in 30 single buccal cells. Table I shows the number of cells analysed for the indicated allele, the percentage of cells in each category of classification and amplification rates for both polymerases. From a total of 30 alleles, a mean of 22.3 alleles was identified per cell using Taq A, ranging from 15 to 30. In the case of Taq B, an average of 26.4 alleles was identified correctly, ranging from 19 to 31, from a total of 31 alleles. The median values for efficiencies of amplification were 80% with Taq A and 90% with Taq B, both showing statistically significant differences (P<0.0001) in a Wilcoxon test for non-parametric data. Peaks included in the high category (>250 RFU) were used for identifying which Taq achieved better results. Taq A revealed a median value of 15% of high peaks, while in the case of Taq B it was 40%. The difference between both polymerases was also statistically significant (P<0.0001) with the same test.
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In the
F508 locus, a 30% increase in peaks over 250 RFU was found in both normal and mutant alleles by changing Taq A for the proofreading Taq, while the amplification rate was similar. In most cases, PEP-PCR with Taq B achieved better results and amplification rates than Taq A. About 10% heterozygous F508 cells amplified with Taq B displayed alleles below 50 RFU, but most of them could be properly identified. A similar result was seen in heterozygous cells for G542X and 3849+10kbC>T. Heterozygous cells for the R347P mutation successfully amplified both alleles with the proofreading Taq. Locus A455 showed the highest values in all cells tested. Other loci with high detection were V520, I507, R334 and 1078. On the other hand, amplification in locus 2183AA was extremely low (25% in Protocol A and 33.3% in B), and mutation 2183AA>G was barely detected in only one of seven cases (14.3%). The ADO rate for the assay with Taq A was 16.6% because 3 of 18 heterozygous samples generated only one signal. The ADO rate in Protocol B was 13.2% (5/38). Ten oocytes were also analysed with Taq B (Table II), the average of amplification rate of oocytes was 74.5% and in 1PBs 44.5%. The total number of alleles identified in the 10 pairs of 1PBs and MII oocytes is shown in Table III. On average, the alleles detected in 1PBs were 12.9, while in MII oocytes it was 21.7, from a total of 29 normal alleles. Figure 1B and C show electropherograms obtained in the analysis of MII and 1PB pair of Oocyte 2 in Table III, respectively.
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The contamination rate was 2.7% (2 of 70 blanks showed amplification).
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Discussion |
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The present work describes an improved whole genome amplification in isolated cells which allows for performing standard laboratory methods for single gene diagnosis with no extensive modifications. PEP-PCR is a reliable method of preamplification because the expected genotype in isolated cells is found and artefacts after PEP were not observed with either of the polymerases chosen. Considering that an increase in denaturing time has beneficial effects on the ADO rate (Piyamongkol et al., 2003
), a long initial denaturation step at 94°C for 7 min was performed for ensuring dissociated strands. This option was preferred, instead of increasing the temperature, for avoiding DNA damage. A step of 30 s at 68°C was added (Dietmaier et al., 1999) because it is the optimal temperature of polymerization for both polymerases (according to manufacturers) and longer genetic fragments are obtained.
Amplification rates were calculated considering only peaks over 50 RFU. The protocol with Taq B showed a better amplification rate (90%) over Taq A (80%), and Taq B yielded more results over 250 RFU than Taq A (median values of 40 versus 15%). This may be explained by the fact that proofreading activity repairs mistakes during elongation in PCR, permitting high yield as well as longer DNA fragments. The critical choice of the polymerase has been previously reported (Thornhill et al., 2005). Locus R347P reached a 100% amplification rate, while most of the heterozygous loci displayed efficiencies barely over 90%, suggesting that as PCR primers have to be shared between the two alleles, heterozygous samples seem to have less amplification than homozygotes. The ADO rate was evaluated considering heterozygous samples in which one of the alleles was undetected. Taq B showed an acceptable ADO rate of 13.2%, similar to other recent reports (Bermúdez et al., 2003; Handyside et al., 2004). Whole genome amplification does not reduce the ADO rate (Wells and Sherlock, 1998), but PEP-PCR was necessary to have a considerable amount of template because of the variety of mutations detected simultaneously with the reactions of multiplex PCR and OLA. OLA detection works with short fragments, which at first should be less inclined to experience ADO (Piyamongkol et al., 2003).
Only in the case of allele 2183AA and its mutation 2183AA>G, as a result of the failures of amplification accumulated in this locus, would PGD of this mutation not be advisable. The loci with amplification rates below 85% (3849+10kbC>T, N1303K, 2789+5G>A and 711+1G>T) should be studied more in-depth, preferably analysing fresh oocytes. The rest of the loci showed efficiencies of amplification suitable for use in clinical application. The ‘Cystic Fibrosis Assay’ (Applied Biosystems, USA) commercial kit is used in molecular diagnostic centres for the screening of CFTR mutations in cystic fibrosis patients and for carrier identification. Genomic DNA from peripheral blood showed low fluctuations in the height of the alleles due to differences in the efficiencies of amplification for each locus and these variations were increased in preamplified single cells. The possibility of optimization of the kit is rather limited, but the efficiencies of amplification obtained in single cells were very satisfactory in the great majority of alleles.
Controls against contamination are critical when using whole genome amplification, since contaminating cells or amplicons can be over-amplified rather than the assayed cell and thus lead to misdiagnosis. It is a matter that can be dealt with by using severe measures to prevent it and by the application of techniques which detect it (i.e. STRs). Up to six aliquots of each PEP-PCR product can be analysed simultaneously and this technique allows for additional amplification of STRs of the CFTR gene (data not shown) for the detection of contamination and ADO. According to our experience, the phenomenon of slippage, reported in the amplification of preamplified repetitive sequences (Foucault et al., 1996; Wells et al., 1999) did not occur when using the Taq with proofreading activity (Taq B), as mistakes are corrected when using this polymerase.
The present study has been performed under standard electrophoretic conditions for genomic DNA in an ABI Prism 377 sequencer. The step of precipitation should be avoided as varying results were obtained in different aliquots assayed from the same PEP product. Results could be improved by using more sensitive apparatus, such as an ABI Prism 3100. Variations in injection time or voltage could make detection of certain fragment sizes and weaker fluorescent dyes better. Additionally, electrophoresis is performed simultaneously in 16 capillaries, and all cells tested can be analysed in a single run.
Buccal cells are not completely representative, but they are easier to obtain and can be selected from carriers of mutations in the gene under study. Spare embryos and oocytes from finished IVF cycles are in limited availability and have variable, often suboptimal genetic quality. Oocytes were processed at least one day after retrieval, which could explain the better results of MII compared to 1PB, where degeneration by apoptosis may have started earlier than in the oocyte (Gitlin et al., 2003). Several 1PBs were extensively degraded and completely failed to amplify (1PB numbers 5 and 10 in Table III) and in only one case (1PB number 2) were all normal alleles detected. This oocyte ageing could also explain the difference between the oocytes and the buccal cells. Ageing produces nicks in the DNA with detrimental amplification results.
Handyside et al. (2004) have recently described isothermal amplification and its possible application in PGD. Their technique needs overnight incubation (about 16 h), while our method of preamplification lasts 6 h less, in which molecular diagnosis could be performed. Saving time is crucial in the case of PGD in blastomeres, when only 32 h are available for diagnosis before transfer. The entire process (lysis, I-PEP-PCR, multiplex PCR, OLA, electrophoresis and analysis) takes about 30 h, so it would be compatible with 1PB analysis and Day +2 embryo replacement in the case of single gene defects maternally inherited. Blastomere analysis should also be possible since embryo transfer can be carried out the third or fourth day after oocyte retrieval (Durban et al., 2001).
This work represents the first description of the adaptation for PGD of a commercial test for the screening of cystic fibrosis mutations with fluorescent detection. The use of the commercial assay allows for the use of a single procedure for the identification of the 31 most common mutations, avoiding the need for having a different protocol for every mutation tested. Therefore, it could be a very useful strategy for its application in centres offering PGD. The method described here directly detects the mutation as in mini-sequencing strategies (Bermúdez et al., 2003; Fiorentino et al., 2003). However, it is important to note that these procedures are not exempt from primer design for every different mutation tested. This procedure would be applicable to 80% of couples worldwide carrying a CFTR mutation. This data is close to the 87% described by Dreesen et al. (2000) in the Dutch population with their microsatellite approach. A global strategy including the present technique and STR analysis for additional indirect diagnosis (when informative) and detection of contamination and ADO would be a very powerful approach.
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Acknowledgements |
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The authors wish to thank Dr Francisco Vidal for critical reading of the manuscript and Drs Mari Carme Pons and Mark Grossmann (Centro Médico Teknon), Josep Maria Calafell (Hospital Clínic i Provincial) and Carmen Márquez (Hospital Vall d'Hebron) for the provision of oocytes. This work received financial support from the Fundació La Marató de TV3, Project 98/1510, 2001-SGR-00201 and the Fondo de Investigaciones Sanitarias (FIS), Project PI-020168.
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Submitted on March 3, 2005; accepted on April 12, 2005.
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