Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase. The disease is ...
Molecular Human Reproduction vol.5 no.7 pp. 691–696, 1999
Fluorescent PCR and automated fragment analysis in preimplantation genetic diagnosis for 21-hydroxylase deficiency in congenital adrenal hyperplasia H.Van de Velde1,3, K.Sermon2, A.De Vos1, W.Lissens2, H.Joris1, M.Vandervorst1, A.Van Steirteghem1 and I.Liebaers2 1Centre
for Reproductive Medicine and 2Centre for Medical Genetics, University Hospital and Medical School, Dutch-speaking Brussels Free University (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium
3To
whom correspondence should be addressed
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is most often caused by a deficiency in steroid 21-hydroxylase. The disease is characterized by a range of impaired adrenal cortisol and aldosterone synthesis combined with an increased androgen synthesis. These metabolic abnormalities lead to an inability to conserve sodium and virilization of females. The most common mutation causing the severe form of CAH is a conversion of an A or C at nucleotide (nt) 656 to a G in the second intron of the steroid 21-hydroxylase gene (CYP21) causing aberrant splicing of mRNA. A couple was referred to our centre for preimplantation genetic diagnosis (PGD) for 21-hydroxylase deficiency in CAH. A PGD was set up to detect the nt656 A/C→G mutation using fluorescent polymerase chain reaction (PCR) and subsequent restriction enzyme digestion and fragment analysis on an automated sequencer. Using DNA or single cells from the father, the normal allele could not be amplified. Non-amplification of the normal allele has been previously described in asymptomatic carriers, therefore the PCR was further developed using heterozygous lymphoblasts from the mother. The PCR was shown to be highly efficient (96% amplification), accurate (0% contamination) and reliable (0% allelic drop-out). The couple started PGD treatment and the second PGD cycle resulted in a twin pregnancy. The genotype of the fetuses was determined in our laboratory using chorionic villus sampling material using the method described here. Both fetuses were shown to be heterozygous carriers of the mutation, and two healthy girls were born. Key words: 21-hydroxylase deficiency/congenital adrenal hyperplasia (CAH)/fluorescent PCR analysis/preimplantation genetic diagnosis (PGD)
Introduction Congenital adrenal hyperplasia (CAH) is a common (1:10 000 to 1/18 000 live births) human genetic disease which in 90% of the cases are caused by a deficiency in the enzyme steroid 21-hydroxylase (Donohoue et al., 1995). This autosomal recessive disease is characterized by a wide range of impaired adrenal cortisol and aldosterone synthesis combined with an increased androgen synthesis. The defect in the cortisol and aldosterone synthesis leads to an inability to conserve sodium (‘salt wasting’), which can be fatal if left untreated. The adrenal overproduction of androgen precursors leads to prenatal masculinization (ambiguous genitalia) in females and postnatal virilization in both sexes. Three major disease phenotypes can be distinguished: (i) the classic salt-wasting form; (ii) the classic simple virilizing form; and (iii) the non-classic form. In the classic salt-wasting form, cortisol and aldosterone production is severely impaired and is accompanied by an overproduction of androgen precursors. In the classic simple virilizing form, cortisol synthesis is impaired but aldosterone synthesis is normal to elevated (non-salt wasting). These patients also undergo virilization. In the non-classic form, there is a subtle defect in cortisol synthesis and aldosterone production is normal. The androgen precursors are moderately © European Society of Human Reproduction and Embryology
elevated in these patients, but this is clinically important only in pubertal or adult females. The treatment of patients with the simple virilizing form includes glucocorticoid therapy and surgical correction of the ambiguous genitalia of the females. Patients having the saltwasting form of the disease require an additional treatment with mineralcorticoids. Adult male patients with CAH appear to be fertile, on the basis of their sperm counts and reproductive histories (Urban et al., 1978). A high number of adult female patients lack sexual activity (Federman, 1987). Those who have had surgical correction and have an adequate introı¨tus show a low fertility rate: 7% in the salt-wasting form and 60% in the virilizing form (Mulaikal et al, 1987). The gene encoding 21-hydroxylase (CYP21) is located on 6p21.3. The molecular genetic basis of the disease has been thoroughly investigated (White, 1989; Strachan et al., 1990). The mutations can be divided into different groups according to severity, which makes it possible to predict the clinical outcome in affected subjects on the basis of genotyping (Speiser et al., 1992; Wedell, 1998). The majority of the mutations causing the deficiency are the result of recombination processes between CYP21 and a highly homologous closely linked pseudogene CYP21P. In fact, most of these mutations 691
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are gene conversions between CYP21P and CYP21, so that the mutation(s) that normally inactivate(s) the pseudogene are instead found in the normally expressed or active gene (Higashi et al., 1986; White et al., 1986). The most common conversion is a mutation in the second intron at nucleotide (nt)656, in which an A or C in CYP21 is replaced by a G from CYP21P (Higashi et al., 1991), resulting in aberrant splicing of mRNA. The nt656 A/C→G mutation is associated with both the severe salt-wasting form and the simple virilizing non-salt-wasting form of the disease (Speiser et al., 1992). A previously described problem when amplifying the nt656 region is the absence (non-amplification) of the normal allele in healthy carriers resulting in an affected genotype (Day et al., 1996). Polymerase chain reaction (PCR)-based genotyping has facilitated genetic counselling of families with CAH. A couple with a child diagnosed for 21-hydroxylase deficiency was referred to our centre for preimplantation genetic diagnosis (PGD). Both parents carry the mutation at nt656. Here we describe the development of a single cell PCR for the detection of the nt656 splice site mutation in the CYP21 gene causing 21-hydroxylase deficiency in CAH. The second PGD cycle led to a twin pregnancy of two carrier babies which was confirmed at prenatal diagnosis using the method developed for PGD. Two healthy girls were born.
Materials and methods Patient description A young non-consanguinous couple (woman 34 years old, man 39 years old) was referred to our centres for PGD because of a risk of 21-hydroxylase deficiency (25%). She was Gravitas8Partus2Abortus6. Their first child, a boy, had been born and was shown to be affected by the disease. Seven pregnancies followed, three miscarried spontaneously, three were terminated after prenatal diagnosis and one healthy boy was born. Both parents carry the most common mutation of CYP21 in the second intron at nt656. In addition, the father and the affected boy carry the Val281Leu mutation in exon 7 of the same allele. Collection of single lymphoblasts Peripheral blood lymphocytes from both partners and the affected child were transformed with Epstein Bar Virus. The lymphoblasts were cultured according to standard procedures (Ventura et al., 1988). Single lymphoblasts were collected as described (Sermon et al., 1998). Briefly, a few colonies were aspirated and transferred to a 1.5 ml Eppendorf tube. The cells were washed three times with phosphatebuffered saline (PBS). Finally, they were resuspended in 50 µl PBS and kept at 4°C. Single lymphoblasts were washed three times in 3 µl droplets of Ca21-and Mg21-free medium (136.8 mM NaCl, 2.7 mM KCl, 0.4 mM NaH2PO4, 5.6 mM glucose, 3.5 mM EDTA, 11.9 mM NaHCO3, 0.01% w/v Phenol Red) supplemented with 15 mg/ml bovine serum albuminin (BSA) in a Petri dish using fine hand-drawn microcapillaries (30 µl; Drummond Scientific Company, USA). They were transferred blindly by mouth controlled pipetting to 2.5 µl alkaline lysis buffer (ALB) (200 mM KOH, 50 mM dithiothreitol) in 200 µl PCR tubes. An aliquot from the last washing droplet was transferred to a PCR tube containing ALB to serve as a blank per five cells collected. The samples were stored (not longer than 1 week) at –80°C until further processing.
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Stimulation and oocyte preparation Ovarian stimulation was carried out by a desensitizing protocol using the gonadotrophin-releasing hormone (GnRH) agonist, buserelin (Suprefact, Hoechst, Brussels, Belgium) combined with human menopausal gonadotrophin (HMG, Humegon; Organon, Oss, The Netherlands) and human chorionic gonadotrophin (HCG, Pregnyl, Organon; or Profasi; Serono, Brussels, Belgium). Intravaginally administered progesterone (Utrogestan; Piette, Brussels, Belgium) was used for luteal phase supplementation. The details of this stimulation protocol have already been reported (Ubaldi et al., 1995). Oocytes were retrieved by vaginal ultrasound-guided puncture of ovarian follicles 36 h after HCG administration. Oocyte denudation was performed as described using enzymatic and mechanical procedures (Van de Velde et al., 1997). Semen preparation The partner’s semen was collected in a sterile container after masturbation. Sperm concentration and motility were evaluated according to the recommendations of the World Health Organization (WHO, 1992). Sperm morphology was determined after Shorr staining (WHO, 1992) of a smear using the strict Tygerberg criteria (Kruger et al., 1986). The freshly ejaculated semen was prepared by washing only (De Vos et al., 1997). ICSI procedure Intracytoplasmic sperm injection (ICSI) was used for fertilization to prevent contamination with sperm cells (Lissens et al., 1997). The preparation of the holding and injection pipettes has been described in detail elsewhere (Van Steirteghem et al., 1995; Joris et al., 1997). Only metaphase II oocytes were injected. The ICSI procedure has been described in detail elsewhere (Van Steirteghem et al., 1995). Oocytes were held by the holding pipette with the polar body at the 6 o’clock position and the injection pipette was inserted into the oocyte at the 3 o’clock position (Nagy et al., 1995). After injection, the oocytes were washed and stored in 25 µl microdrops of B2 medium under oil in a Petri dish. They were kept in an incubator at 37°C in an atmosphere of 5% CO2, 5% O2 and 90% N2. Assessment of survival, fertilization and embryo development The morning after injection (16–22 h later), the oocytes were checked for survival and fertilization (Nagy et al, 1994). The numbers and aspects of polar bodies and pronuclei were recorded. The criteria for normal fertilization were the presence of two individualized or fragmented polar bodies together with two clearly visible pronuclei. Embryo cleavage and quality were evaluated 2 and 3 days after ICSI as described: type A (0% anucleate fragmentation), type B (1–20% fragmentation), type C (21–50% fragmentation) and type D (.50% fragmentation) (Van de Velde et al., 1997). On day 3, only transferable embryos (0–50% anucleate fragmentation) were considered for biopsy. Embryo biopsy Biopsy was performed on day 3 in the morning as described (De Vos et al., 1998). The embryo was fixed by the holding pipette and a hole was made in the zona pellucida by a stream of acidic Tyrode’s (pH 2.3) using a fine needle (5–7 µm inner diameter). Most of the embryos were already compacted and were, therefore, preincubated in Ca21- and Mg21-free medium for 5 min. If the embryo had six or fewer cells, only one blastomere was aspirated. If the embryo had seven or more cells, two blastomeres were removed. The blastomeres were gently aspirated through the hole using a pipette with an inner diameter of 40–45 µm and were released next to the embryo. They were checked for the presence of a nucleus. Single blastomeres were
PGD for 21-hydroxylase deficiency washed three times in 3 µl droplets of Ca21- and Mg21-free medium supplemented with 4 mg/ml BSA in a Petri-dish using a fine handdrawn Pasteur pipette. Finally, they were transferred to a 200 µl PCR tube containing 2.5 µl ALB under visual control using a stereomicroscope. For each collected blastomere, an aliquot from the last washing droplet was transferred to a PCR tube containing ALB to serve as a blank. The samples were kept at –80°C for at least 30 min.
Cell lysis and PCR procedure The cells were lysed by incubation at 65°C for 10 min. The ALB was neutralized with 2.5 µl neutralization buffer (NB), containing 900 µM Tris–HCl pH 8.3, 300 mM KCl and 200 mM HCl. The reaction mix for the fluorescent PCR was decontaminated with the restriction enzyme AluI (New England Biolabs Inc, Westburg, Leusden, The Netherlands) by incubation for at least 3 h at 37°C. The enzyme was inactivated by incubation for at least 30 min at 65°C. The primer set used was: the fluorescently labelled forward primer CAH3F 59-CAGGTCCCTGGAAGCTCTTG-39 and the reverse primer CAH2 59-CCAGAGCAGGGAGTAGTCTC-39 (Eurogentec, Seraing, Belgium). The reaction mix was added to the lysed cells to a final volume of 25 µl and final concentrations of 50 mM KCl, 10 mM Tris–HCl pH 8.3, 2 mM MgCl2, 0.1 mg/ml gelatin, 0.2 mM dNTP (Pharmacia, Gent, Belgium), 0.4 µM primers and 1.25 IU Taq polymerase (Perkin Elmer, Brussels, Belgium). The PCR programme was as follows: 5 min at 96°C; 10 cycles of 96°C for 30 s, 61°C for 30 s and 72°C for 30 s; followed by 37 cycles of 94°C for 30 s, 61°C for 30 s and 72°C for 30 s; followed by 7 min at 72°C. Purification and digestion The PCR product was purified using the High Pure PCR Product Purification Kit (Boehringer Mannheim, Germany) using the manufacturer’s protocol, with some small modifications. Briefly, 125 µl binding buffer was added to the 25 µl PCR product and mixed thoroughly. The mixture was centrifuged for 30 s at 14 000 g on a High Pure filter tube combined with a collection tube. The flowthrough was discarded and the filter was washed twice with 500 and 200 µl respectively of wash buffer by centrifugation for 30 s at 14 000 g. Finally, the filter tube was placed in a new Eppendorf tube and the DNA was eluted with 50 µl sterile distilled H2O by centrifugation for 30 s at 14 000 g. The sample was digested using the restriction enzyme MwoI (New England Biolabs Inc), which cuts into a 59...GCN6GC...39 site. Purified PCR product (10 µl) was incubated for 1 h at 60°C in a final volume of 20 µl restriction buffer containing 150 mM NaCl, 50 mM Tris– HCl, 10 mM MgCl2, 1 mM dithiothrietol and 100 µg/ml BSA. Blanks were not purified and digested, they were simply applied onto the gel. Electrophoresis Restricted PCR product (6 µl) was mixed with 6 µl loading buffer (10 mM Tris–HCl pH 7.6 and 1 mM EDTA and 5 mg/ml Dextran Blue 2000 in formamide) and loaded on the gel (6% acrylamide/bisacrylamide (19:1), 7 M urea, Tris Borate Electrophoresis buffer, Sequencing gel; Immunosource, Zoersel, Belgium). The electrophoresis unit used was an ALF Automated DNA Sequencer from Pharmacia Biotech, Gent, Belgium. The results were processed using the Fragment Analyser software provided by the manufacturer.
Results In a first step, the genotype of the family members was confirmed. The PCR was set up using a primer set consisting of a fluorescently labelled forward primer and a reverse primer
overlapping the 8 bp deletion region in the pseudogene CYP21P avoiding amplification of the pseudogene. The total PCR product is 185 bp. The mutation at nt656 is recognized by restriction enzyme digestion using MwoI which cuts the mutated allele into two fragments: 65 bp (which is not fluorescently labelled and which would coincide with the primer peak because it is too small) and 120 bp. DNA (100 pg) was amplified and digested. The normal control DNA showed one 185 bp peak after digestion indicating that the pseudogene CYP21P, which contains the mutation, is not amplified by this primer set (Figure 1a, lane 5). Both parents were shown to have the 120 bp affected peak and the normal 185 bp peak and were thus confirmed as carriers of the mutation (Figure 1a, lanes 3 and 4). Unexpectedly, their affected child was also shown to be heterozygous for the mutation (Figure 1a, lane 2). Based on the idea that some PCR reagents might interfere with the digestion reaction, the DNA was purified before digestion using a DNA purification kit as described above. The normal control DNA showed one 185 bp peak after purification and digestion (Figure 1b, lane 5), whereas the affected child now appeared to have only the affected 120 bp peak (Figure 1b, lane 2). The mother was confirmed to be heterozygous (Figure 1b, lane 4), however the healthy father appeared to be homozygous for the affected gene although he has no clinical signs of the disease (Figure 1b, lane 3). Although non-amplification of the normal allele at nt 656 is a well known phenomenon, we have tried to investigate this technical problem by using more DNA (1 µg), AmpliTaq Gold polymerase instead of AmpliTaq polymerase and a different primer set. We have not been able to obtain the heterozygous genotype of the father. Using single cell PCR on lymphoblasts, similar results were obtained (Table I). The overall amplification rate was 96% (72/75 lymphoblasts) and contamination was not observed (0/25 blanks). To determine the allelic drop-out (ADO), 30 single lymphoblasts from the mother were analysed. Of these cells were amplified, 29 (97%) and none of them showed ADO (0%) (Table II). Single cells (n 5 15) from the father were also analysed, 15 cells were amplified (100%) and they all showed the phenomenon of non-amplification of the normal allele. Despite the above technical problem with regard to the father’s genotype, the couple started PGD treatment because the preferential amplification of the affected allele would lead only to misdiagnosis of a healthy heterozygous embryo, which would appear affected and would not be transferred. In the first PGD cycle (Table II) 10 cumulus–oocyte complexes (COCs) were retrieved which were mature and those were all injected. The semen characteristics were normal (76% progressive motility and 18% normal morphology). The next morning, seven oocytes showed normal fertilization. On the third day, seven blastomeres were retrieved from six embryos (two embryos had reached the 8 cell-stage, two embryos had reached the 6-cell stage and two embryos were only at the 4-cell stage). Six out of seven cells were amplified (86%). Two embryos were homozygous normal, two were heterozygous and one was homozygous affected. In one embryo no result was obtained, but it later turned out to be heterozygous. The 693
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Figure 1. Results of the polymerase chain reaction (PCR)-based genotyping of DNA from the family after fluorescent PCR, (a) without and (b) with DNA purification, before restriction enzyme digestion and fragment analysis on an automated sequencer: lane 2, the patient; lane 3, the father; lane 4 the mother; lane 5, an unrelated normal control. Lane 6 represents undigested PCR product from the patient, and lane 1 represents a molecular weight standard showing 100, 150, 200 and 250 bp peaks. The x axis shows the running time in minutes.
Table I. Results of the polymerase chain reaction (PCR)-based genotyping of lymphoblasts from the family and an unrelated normal control after fluorescent PCR
Patient Father Mother Control
Phenotype
Genotype
Amplification (%) Contamination (%) ADO (%)
homozygous affected heterozygous heterozygous homozygous normal
homozygous affected homozygous affected heterozygous homozygous normal
14/15 15/15 29/30 14/15
(93) (100) (97) (93)
0/5 (0) 0/5 (0) 0/10 (0) 0/5 (0)
NA NA 0/29 (0) NA
ADO 5 allelic drop-out; NA 5 not applicable.
diagnosis of the affected embryo was confirmed later. No ADO was found in this cycle. Two homozygous normal embryos and one heterozygous embryo were transferred, and the remaining heterozygous embryo was frozen. At the moment of transfer, only the heterozygous embryo had cleaved further. The patient did not become pregnant in this cycle. In the second cycle (Table II), 12 COCs were obtained. Mature oocytes (n 5 10) were injected (normal semen parameters with 67% progressive motility and 22% normal morphology), and six were normally fertilized the next morning. On day 3, six embryos were available for biopsy (all but one had reached the 7–8-cell stage). All the cells (11 blastomeres) were amplified (100%) so that diagnosis was obtained for all embryos: one normal, three heterozygous and 694
two affected. In this cycle there was no ADO. In one embryo, two blastomeres from the embryo were heterozygous while one of the two blanks was contaminated. The genotype of this contaminating peak could not be tested later. This embryo was not transferred because the presence of a contaminating normal allele might have led to the transfer of an affected embryo. The embryo was frozen. The diagnosis of the affected embryos was confirmed later. The homozygous normal embryo was transferred together with two heterozygous embryos. None of these embryos had cleaved further between the time of biopsy and the moment of transfer. This PGD cycle resulted in a twin pregnancy. The diagnosis was confirmed in our laboratory after chorionic villus sampling and genotype analysis using the method described here to detect the mutation. The two
PGD for 21-hydroxylase deficiency
Table II. Results of two preimplanation genetic diagnosis (PGD) cycles for 21-hydroxylase deficiency
No. of COCs No. of MII oocytes No. of survived oocytes (%)a No. of 2PN (%)a No. of embryos biopsied (type A,B and C) (%)b No. of blastomeres analysed amplification (%) ADO (%)* contamination (%) Resulting embryos: homozygous normal heterozygous homozygous affected no diagnosis No. of embryos transferred No. of embryos frozen No. of embryos implanted
Cycle 1
Cycle 2
10 10 8 (80) 7 (70) 6 (85) 7 6/7 (86) 0/2 (0) 0/7 (0)
12 10 9 (90) 6 (60) 6 (100) 11 11/11 (100) 0/6 (0) 1/11 (9)
2 2 1 1 3 1 0
1 3 2 0 3 1 2
COCs 5 cumulus–oocyte complexes; MII 5 metaphase II; 2PN 5 two pronuclear; ADO 5 allelic drop-out. aPercentage of injected MII oocytes. bPercentage of 2PN *Non-amplification of the normal allele cannot be seen.
implanted embryos were healthy carriers of the nt656 mutation and were dizygous. Two healthy girls were born after 36 weeks of pregnancy.
Discussion Steroid 21-hydroxylase deficiency causing congenital adrenal hyperplasia is an autosomal recessive disease that is characterized by salt wasting and virilization of female fetuses. The molecular basis of the disease is diverse, the most common mutation is a conversion of an A or C at nt656 to a G in the second intron of the CYP21 gene encoding 21-hydroxylase causing impaired mRNA splicing. We describe here an efficient (amplification was 96% using lymphoblasts and 96% during PGD) and accurate (contamination was 0% using lymphoblasts and 6% during PGD) PCR for the PGD of the nt656G/G mutation in the CYP21 gene by using fluorescent PCR, DNA purification, restriction enzyme digestion and fragment analysis on an automated sequencer. In our experience, it is unusual to insert a DNA purification step between the PCR and the restriction enzyme digestion. The MwoI enzyme is probably inhibited by reagents from the PRC mix. An additional problem was the finding that the husband was genotyped as homozygous affected for the nt656 mutation although he showed no clinical signs of the disease. The problem was also not solved by using a different primer set, excluding the possibility that the non-amplification had been due to polymorphism of one of the PCR priming sites. The normal allele was also not amplified using a hot-start PCR system with AmpliTaq Gold polymerase. Most probably, the normal allele is not amplified (i.e. is ‘dropped-out’) during the reaction, leading to an ‘asymptomatic’ affected result from an individual carrying a normal allele that remains undetectable because of preferential amplification of the allele carrying the
nt656 splicing mutation. This phenomenon has already been described (Day et al., 1996), and it was found that in pedigrees of 150 patients with 21-hydroxylase deficiency caused by the nt656 splicing mutation, 13 first-degree family members were genotyped as affected but showed no clinical signs of CAH. ADO is a major problem of single-cell PCR (Findlay et al., 1995), but we found that the normal allele also failed to be amplified after PCR using 1 µg of DNA. The non-amplification of the normal allele could be investigated further, by use of microsatellite haplotyping (Day et al., 1996), but this has not been done because it would not be of any use for the PGD. Absence of the normal allele would never lead to transfer of an affected embryo but would result in the misdiagnosis of a healthy heterozygous embryo that would appear affected and consequently would not be transferred. In addition, the undetectable normal allele is not necessarily absent in the offspring of the ‘asymptomatic’ affected parent because it can appear again in the next generation (Day et al., 1996). Consequently, the ‘ADO’ obtained using lymphoblasts from the father was 100%. We assume that non-amplification of his normal allele is due to a technical problem specific for his DNA and, therefore, this number does not represent the dropout caused by the PCR technique. Therefore, we prefer to use the term ‘non-amplification’ when discussing results obtained using cells or DNA from the father. The real ADO was determined using heterozygous lymphoblasts from the mother and was shown to be 0%. This finding makes the test reliable because the probability of transferring an affected embryo after misdiagnosis by ADO on two blastomeres is low. For these reasons, the PGD treatment was started. In the first cycle, two homozygous normal embryos and one heterozygous embryo were transferred, but the patient did not become pregnant. In the second cycle, one homozygous normal embryo and two embryos carrying the nt656 mutation were transferred. In all, nine embryos were analysed: three were homozygous normal, six were heterozygous and three were affected (expected ratio 25/50/25). There was no ADO in these PGD cycles, but the group of the affected embryos might contain one or more heterozygous embryo(s) with an undetectable normal allele that was not amplified. The second cycle resulted in a twin pregnancy of two carrier fetuses. The diagnosis was confirmed in our laboratory by applying the PGD strategy on chorionic villus sampling at 10 weeks of pregnancy. Two healthy girls were born. In conclusion, we developed a PGD for the detection of 21hydroxylase deficiency in CAH caused by the nt656A/C〈〈G splicing mutation in the CYP21 gene using fluorescent PCR, DNA purification, restriction enzyme digestion and fragment analysis on an automated sequencer. Non-amplification of the normal allele can occur, resulting in the misdiagnosis and subsequent loss of healthy heterozygous embryos that appear affected for the nt656 mutation.
Acknowledgements The authors wish to thank the clinical, scientific, nursing and technical staff of the Centre for Reproductive Medicine and Centre for
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H.Van de Velde et al. Medical Genetics, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel-VUB, Belgium). We would like to thank Prof. Rodesch (Universite´ Libre de Bruxelles-ULB, Brussels, Belgium) for referring the patient and Dr. Morel (Hospices Civils de Lyon, Lyon, France) for giving technical advice. Furthermore we are grateful to Mr. Winter of the Language Education Centre of our University for proofreading the English in this manuscript. This work has been supported by grants from the Fund for Medical ResearchFlanders and the University Research Council.
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