Case report Pregnancy after preimplantation genetic diagnosis for brachydactyly type B

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1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 21 November 2008 Case report Pregnancy after preimplantation genetic diagnosis for brachydactyly type B Dr Ali Hellani graduated from the Lebanese University in 1994 with a Bachelor degree in molecular biology. He continued his post-doctoral studies in France where he finished his Master and PhD degrees in the molecular biology of male reproduction and preimplantation genetic diagnosis (PGD). He established the first PGD centre for single gene disorders in the Middle East in 1999 in the King Faisal Specialist Hospital in Riyadh. After serving at King Faisal for 6 years, he joined Saad Specialist Hospital in 2005 where he established the PGD and genetics facility. Dr Hellani was elected to the Board of the Human Genetics Society of Australia in Dr Ali Hellani Ali Hellani 1,4, Khaled Abu-Amero 2, Joseph Azouri 3, Hadeel Al-Sharif 1, Hamish Barblet 1, Siham El-Akoum 1 1 PGD Laboratory, Saad Specialist Hospital, Al-Khobar, 31952, KSA; 2 College of Medicine, King Saudi University, Riyadh, KSA; 3 A-Clinic, Mount Liban Hospital, Beirut, Lebanon 4 Correspondence: Tel: ; Fax: ; ahellani@gmail.com Abstract Brachydactyly type B (BDB) is an autosomal dominant disease caused by mutations in the ROR2 gene. Truncating mutations lead to the severe form of the disease, which is characterized by terminal deficiency of fingers and toes. Preimplantation genetic diagnosis (PGD) was carried out in a family suffering from severe BDB. The family was screened for mutations in exons 8 and 9 and found to harbour a known nonsense mutation (c.2265c A) in exon 9 of the ROR2 gene, which resulted in a premature stop-codon at residue 755. Three out of 10 linked markers tested were informative for this family and single cell work-up showed amplification efficiency in over 98% of the cells. Allele drop-out (ADO) was found in 0, 4.08 and 6.1% for D9S1803, D9S1842 and D9S280 respectively. The family underwent PGD using multiple displacement amplification, fluorescent polymerase chain reaction (informative short tandem repeat) and sequencing of exon 9. Two cells were taken from the three embryos generated in the PGD cycle and the diagnosis of both cells separately showed one normal embryo free of BDB abnormal allele. This embryo was transferred back to the mother and resulted in a singleton pregnancy. Postnatal DNA testing of the newborn confirmed the PGD result. Keywords: autosomal dominant, brachydactyly type B, MDA, preimplantation diagnosis, Introduction Autosomal dominant brachydactyly type B (BDB) is the most severe of the inherited brachydactylies. It is characterized by hypoplasia or complete absence of the distal and middle phalanges with variable degrees of distal and proximal symphalangism, often accompanied by nail dysplasia (Schwabe et al., 2000). BDB is caused by mutations in the ROR2 gene, which belongs to a small family of receptor tyrosine kinases. The gene is mapped to chromosome 9 and comprises nine exons. Preimplantation genetic diagnosis (PGD) has become an established alternative to prenatal diagnosis (PND) for couples carrying genetic conditions that may affect their offspring. The clear advantage of PGD over PND is that it allows diagnosis prior to implantation, thus avoiding the initiation of an affected pregnancy (Swanson et al., 2007; Kuliev et al., 2008). Despite the significant advantages provided by PGD, the setting up and testing of molecular diagnoses on single cell is work-intensive, technically challenging, expensive, and time-consuming. Labour-intensive development and validation of highly sensitive amplification strategies for single-cell diagnosis are required, usually using nested polymerase chain reaction (npcr), whole genome amplification (WGA), or fluorescent PCR methods. The main disadvantage of nested and fluorescent PCR is the difficulty in choosing primers for multiplex PCR (Van de Velde et al., 2004). On the other hand, the main disadvantages of WGA are the generation of non-specific amplification artefacts, incomplete coverage of loci, inefficiency of microsatellite amplification, and the generation of DNA less than 1 kb long (Dean et al., 2002). For those reasons, PGD requires a technique that would be able to amplify the single-cell Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 128 DNA with a high fidelity that suits the diagnosis of any known single-gene disorder by standard PCR techniques. Multiple displacement amplification (MDA) is an isothermal WGA technique based on the use of ø29 DNA polymerase and random primers. The ø29 polymerase combines high processivity with a strand displacement ability, leading to the synthesis of DNA fragments >10 kb and favouring uniform representation of sequences (Paez et al., 2004). MDA is a technique that is used in the amplification of minute DNA quantities in clinical samples, yielding a high quantity of good quality DNA (Dean et al., 2002). This report describes a successful pregnancy after application of PGD for an autosomal dominant single gene disorder. Materials and methods Patients A family approached the IVF centre for preconception counselling regarding their options in attempting a future pregnancy. The father was suffering from BDB and the family had one affected child who apparently inherited the disease from his father. Methods ROR2 mutation detection and PGD test design In order to assess the mutation causing BDB in the family, genomic DNA was extracted and exons 8 and 9 (a hotspot area for mutations which codes for the tyrosine kinase domain of the protein) of the ROR2 gene were screened for mutation(s). This was performed at the PGD laboratory of Saad Specialist Hospital. The primer sequences and the PCR conditions were as previously described (Schawbe et al., 2000). Briefly, ROR2 gene sequences were amplified from 100 to 200 ng of DNA using specific primers (5 mol/l), dntp (5 mmol/l), PCR buffer 10, and 1 unit of FastStart High Fidelity Taq polymerase (Roche, Germany). PCR products were purified (Qiagen, USA), then assessed by bio-analyser capillary electrophoresis using the DNA chip (Agilent, USA). The purified PCR product was sequenced on an ABI 3130xI Genetic Analyser (Applied Biosystems, USA) using forward and reverse primers. Due to the dominant mode of inheritance for BDB, PGD test design was based on mutation screening (c.2265c A) and linked marker detection for ascertaining the possible occurrence of allele drop-out (ADO). Polymorphic short tandem repeat (STR) markers D9S1796, D9S1781, D9S1842, D9S1815, D9S197, D9S1836, D9S1841, D9S1803, D9S196, D9S1689 were chosen, as they are known to be closely linked to the ROR2 gene (Gong et al., 1999; Oldridge et al., 2000). All 10 markers were tested in this family to determine which ones were informative. MDA protocol Single lymphocytes were isolated from peripheral blood of the affected child. Briefly, following Ficoll-Paque Plus centrifugation and dilution in sterile phosphate-buffered saline, 50 single lymphocytes were placed into 0.2 ml PCR tubes containing 3 μl alkaline lysis buffer (ALB; 0.2 mol/l KOH and 50 mmol/l dithiothreitol). Blank samples were also run in parallel with the tested samples in order to detect any contamination (if present). After 15 min incubation at 4 C, 3 l of neutralization buffer (900 mmol/l Tris-HCl, 300 mmol/l KCl, 200 mmol/l HCl) were added to the solution. Cell lysates were used directly for WGA using MDA (GE Healthcare, USA) by adding 20 μl of the master mix in a total volume of 30 μl. The mix was then incubated at 31ºC for 2 h, followed by heat inactivation at 65ºC for 10 min. MDA yield was quantified on a fluorometer using a picogreen quantification kit (Molecular Probes, Inc., Eugene, USA). Blanks did not show any amplification. A 5 l aliquot of the MDA product (diluted 10 ng/ l) was amplified using STR and exon 9 primers. PCR conditions were as follows: one cycle of denaturation at 95 C for 10 min, followed by 30 cycles of denaturation at 95 C for 30 s, annealing at 60 C for 30 s and extension at 72 C for 30 s. Final extension was performed at 72 C for 7 min. PCR products were analysed on 3130xl Genetic analyser for the labelled STR (Applied Biosystems, USA) or bioanalyser 2100 (Agilent, USA) for exon 9 before processing for sequencing. Ovarian stimulation protocol The flare protocol (Garcia et al., 1990) was followed for ovarian stimulation using gonadotrophin-releasing hormone analogue (GnRHa, Decapeptyl, 0.1 mg/day; Ipsen-Biotech, Paris, France) and human menopausal gonadotrophin (HMG, Menogon; Ferring Pharmaceuticals Ltd, USA). Human chorionic gonadotrophin (HCG-Pregnyl 10,000 IU; Organon, Netherlands) was administered when three follicles reached a diameter of 18 mm. Oocytes were recovered transvaginally under conscious sedation 35 h after HCG administration. Fertilization, embryo culture and biopsy Four oocytes were collected; three of which were mature (metaphase II) and were injected with the husband s spermatozoa using intracytoplasmic sperm injection (ICSI) (Van Steirteghem et al., 1993). ICSI was performed to eliminate contamination by spermatozoa when performing subsequent embryo biopsy. On day 3 of culture using ISM1,2 media (Medicult, Denmark) following oocyte retrieval, three embryos (eight cells grade 1, eight cells grade 1, six cells grade 2, with grade 1 being the best) underwent 2-cell biopsy. Embryos were incubated for 10 min in a calcium/ magnesium-free embryo biopsy medium (EBM, Medicult), prior to biopsy. The zona pellucida was pierced using the Saturn Laser System (Research Instrument, UK). Two blastomeres were gently aspirated through the piercing and each transferred to a 0.5 ml PCR tube containing the lysis buffer. The last wash drop served as a blank. Samples and blank PCR tubes were processed using the same protocol of PCR as previously described on single lymphocytes. Results Mutation screening of the ROR2 gene (exons 8 and 9) revealed the presence of a previously reported distal mutation (c.2265c A) in exon 9, which resulted in the replacement of tyrosine with a premature stop-codon at residue 755. This

3 mutation is known to be associated with the severe form of BDB (Oldridge et al., 2000). Initial analysis of the 10 polymorphic STR markers in the family revealed that three of the tested markers were informative (D9S1803, D9S1842 and D9S280). Genotype analysis of the three informative markers and the family pedigree are shown in Figure 1. The three informative markers were then tested on single cells (lymphocytes) to assess amplification efficiency and ADO rates (Table 1). MDA/PCR protocol applied on a single cell (lymphocyte) revealed the absence of contamination and a >98% amplification efficiency (49/50 cells were successfully amplified by MDA) for the three informative markers and exon 9. As for the ADO screening, the results showed the absence of ADO for D9S1803 (0%), 4.08% for D9S1842 and 6.1% for D9S280 marker (Table 1). Genotypes of the three biopsied embryos for the informative markers are shown in Table 1. One embryo was normal and two were abnormal, the result being confirmed on the three informative STR. ROR2 exon 9 confirmed the STR diagnosis in two embryos (one normal and one abnormal), while the third one showed a homozygous abnormal genotype as a result of ADO. Serum -HCG confirmed the pregnancy 2 weeks after the embryo was transferred. Transvaginal ultrasonography was performed 3 weeks after the transfer and revealed one intrauterine gestational sac. At the 7th week of gestation, fetal cardiac activity was confirmed. At 40 weeks of gestation, a live healthy male was delivered. Testing of the exon 9 amplification on the peripheral blood of the newborn corresponded with the PGD results originally performed on a single cell, and confirmed the absence of the c.2265 C A mutation. Discussion Heterozygous truncating mutations (nonsense, and frameshift) in the ROR2 gene were previously shown to cause BDB (Oldridge et al., 2000; Schwabe et al., 2000). This condition is the most severe of the brachydactylies and is characterized by terminal deficiency of fingers and toes. Mutations in the ROR2 gene can result in either of two distinct skeletal disorders, depending on the location and nature of the mutation; homozygous loss-of-function mutations spread throughout the gene and cause recessive Robinow syndrome (Patton and Afzal, 2002), whereas gain-of-function mutations cause BDB (Oldridge et al., 2000; Schwabe et al., 2000). To date, Figure 1. Pedigree of the affected family and the genotype results for the three informative markers. Table 1. Genotypes of the three-biopsied embryos for the informative markers. Embryo Marker Exon 9 Diagnosis D9S280 D9S1803 D9S Homozygous normal Normal Heterozygous Affected Homozygous affected a Affected a Amplification efficiency % (single >98 >98 >98 >98 lymphocytes, n = 50) ADO % (single lymphocytes, n = 50) a Homozygous affected status due to allele drop-out (ADO) as detected by sequencing exon 9 of the ROR2 gene. 129

4 130 a total of 16 mutations associated with BDB phenotype have been reported in the ROR2 gene. Seven are nonsense, six are missense mutations, two are small deletions and one is a small insertion (human gene mutation database available at accessed November 2008). A genotype phenotype correlation between the distal and the proximal mutations in the ROR2 gene was detected, with distal gene mutations being less variable and more severe. The clinical picture seen here, in the proband, is of the severe type, and is consistent with the clinical features of previously reported patients with the distal (Y755X) mutation (Oldridge et al., 2000; Schwabe et al., 2000). Single-cell PCR was the first technique developed for the analysis of DNA from single cells (Ao et al., 1996). Setting up a new diagnosis utilizing the PGD technique is a time consuming process because: (i) primers have to be designed to amplify all linked markers in a single nested multiplex PCR reaction; and (ii) PCR conditions have to be optimized in such a way to reduce the risk of ADO and amplification failure. Since the first application of MDA on a single cell (Handyside et al., 2004; Hellani et al., 2004), many publications using this technique in routine PGD and on diverse inherited diseases have emerged (Burlet et al., 2006; Lledó et al., 2007; Ren et al., 2007). So far, the average rate of ADO and failure of amplification varies from 10 to 34% and 0 to 5% respectively. ADO variation would be due to the quality of blastomere DNA, the region of DNA amplified and less probably to the PCR primers (Burlet et al., 2006). Interestingly, Ren et al. (2007) reported ADO variation between lymphocytes (9%) and blastomeres (25%). Such an observation favours the adoption of intensive measures, such as informative linked markers and 2-cell biopsy. This will lead to the detection of ADO, even though its rate on lymphocytes is low. The current report shows the application of MDA in a PGD cycle for a dominant single gene disorder as has also been reported for Marfan syndrome (Lledó et al., 2006). Therefore, precautions to avoid ADO should be extremely strict in order to exclude any risk of misdiagnosis. In accordance with this hypothesis, this PGD strategy was developed for the BDB affected family. Three out of the 10 tested STRs markers were informative. Analysis of 50 single cells showed 0, 4, 6 and 20% ADO occurrence. Such results favour the use of fluorescence PCR over sequencing method when a single cell is amplified by MDA. In theory, the occurrence of ADO should be 1% if the three linked markers applied simultaneously. Such a theory is valid on the set of 50 lymphocytes diagnosed (every cell was diagnosed by at least one linked marker). Based on these results, the PGD test was effective in diagnosing single cells for BDB. As an extra vigilance step, a 2-cell biopsy process was adopted where each cell was diagnosed separately. Theoretically, cells can be removed for diagnosis at any stage between the 2-cell stage embryo and the blastocyst. In human embryos, the eight-cell stage is ideal because the cells are still totipotent (each cell can replace another cell). Although up to a quarter of cells from a human embryo can be removed without impairing its in-vitro development (Hardy et al., 1990), the random removal of two cells could interfere with the embryo s early differentiation (Edwards and Beard, 1997; Cohen et al., 2007). However, routine 2-cell biopsy (Van de Velde et al., 2000; Burlet et al., 2006; Goossens et al., 2008) has resulted in pregnancies and live birth rates comparable with one-cell biopsy. The routine in the PGD protocol is to take one cell for FISH and two cells for array comparative genomic hybridization and PCR analysis. Preliminary investigations (Hellani et al., unpublished data) suggest that the pregnancy rate resulting from 2-cell biopsy PCR cycles (20/44) is comparable to the world-wide known one-cell biopsy PCR cycles (Goossens et al., 2008), although the number of patients in the investigation was low. Interestingly, one report (Hellani et al., 2008) shows that five out of six patients (recurrent IVF failure) who had embryo transfer after 2-cell biopsy in a comparative genomic hybridization cycle became pregnant. This report shows the first delivery of a normal baby in a family with BDB. The success of MDA/fluorescent PCR and fragment analysis in the current report in terms of low ADO ( 1%) and failure of amplification ( 2%) gives more reason to use this technique in the field of PGD (dominant and recessive inheritance). Most of the reported MDA cases use linked markers in addition to mutation detection. Such a strategy improves the rate of successful diagnosis through decreasing the ADO rate. References Ao A, Ray P, Harper J et al Brachydactyly type B1: report of a family with de novo ROR2 mutation. Prenatal Diagnosis 70, Burlet P, Frydman N, Gigarel N et al Multiple displacement amplification improves PGD for fragile X syndrome. Molecular Human Reproduction 12, Cohen J, Wells D, Munné S 2007 Removal of 2 cells from cleavage stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates. Fertility and Sterility 87, Dean FB, Hosono S, Fang L et al Comprehensive human genome amplification using multiple displacement amplification. Proceedings of the National Academy of Sciences of the USA 99, Edwards RG, Beard HK 1997 Oocyte polarity and cell determination in early mammalian embryos. Molecular Human Reproduction 3, Garcia J, Padilla S, Bargati J et al Follicular phase gonadotropin-releasing hormone agonist and human gonadotropins: a better alternative for in vitro fertilization. Fertility and Sterility 53, Gong Y, Chitayat D, Kerr B et al Brachydactyly type B: clinical description, genetic mapping to chromosome 9q, and evidence for a shared ancestral mutation. American Journal of Human Genetic 64, Goossens V, De Rycke M, De Vos A et al Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Human Reproduction 23, Handyside AH, Robinson MD, Simpson RJ et al Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Molecular Human Reproduction 10, Hardy K, Martin KL, Leese HJ et al Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage. Human Reproduction 5, Hellani A, Abu-Amero KK, Akoum S, Azouri J 2008 Successful pregnancies after application of array-comparative genomic hybridization in PGS aneuploidy screening. Reproductive BioMedicine Online, e-pub ahead of print 30 October. Hellani A, Coskun S, Benkhalifa M et al Multiple displacement amplification on single cell and possible PGD applications.

5 Molecular Human Reproduction 10, Kuliev A, Verlinsky Y 2008 Preimplantation genetic diagnosis: technological advances to improve accuracy and range of applications. Reproductive BioMedicine Online 16, Lledó B, Bernabeu R, Ten J et al Preimplantation genetic diagnosis of X-linked adrenoleukodystrophy with gender determination using multiple displacement amplification. Fertility and Sterility 88, Lledó B, Ten J, Galán FM, Bernabeu R 2006 Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification. Fertility and Sterility 86, Oldridge M, Fortuna AM, Maringa M et al Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B. Nature Genetic 24, Paez JG, Lin M, Beroukhim R et al Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acid Research 32, e71. Patton MA, Afzal AR 2002 Robinow syndrome. Journal of Medical Genetics 39, Ren Z, Zhou C, Xu Y et al Mutation and haplotype analysis of Duchenne muscular dystrophy by single multiple displacement amplification. Molecular Human Reproduction 13, Schwabe GC, Tinschert S, Buschow C et al Distinct mutations in the receptor tyrosine kinase gene ROR2 cause brachydactyly type B. American Journal of Human Genetics 67, Swanson A, Strawn E, Lau E et al Preimplantation genetic diagnosis: technology and clinical applications. Wisconsin Medical Journal 106, Van de Velde H, Georgiou I, De Rycke M et al Novel universal approach for preimplantation genetic diagnosis of betathalassaemia in combination with HLA matching of embryos. Human Reproduction 19, Van de Velde H, De Vos A, Sermon K et al Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis. Prenatal Diagnosis 20, Van Steirteghem AC, Nagy Z, Joris H et al High fertilization and implantation rate after intracytoplasmic sperm injection. Human Reproduction 8, Declaration: The authors report no financial or commercial conflicts of interest. Received 31 March 2008; refereed 14 May 2008; accepted 14 August