Abstract. Introduction. RBMOnline - Vol 7. No Reproductive BioMedicine Online; on web 18 June 2003

Transcription

1 RBMOnline - Vol 7. No Reproductive BioMedicine Online; on web 18 June 2003 Review Current status of preimplantation diagnosis for single gene disorders Dr Yury Verlinsky is a graduate, postgraduate and PhD of Kharkov University of the former USSR. His research interests include cytogenetics, embryology and prenatal and preimplantation genetics. He introduced polar body testing for preimplantation genetic diagnosis and developed the methods for karyotyping second polar body and individual blastomeres. He has published over 100 papers, as well as three books on preimplantation genetics. Dr Yury Verlinsky Dr Anver Kuliev received his PhD in Clinical Cytogenetics from Moscow Research Institute of Human Morphology in In 1979 he took the responsibility for the World Health Organization (WHO) s Hereditary Diseases Program in Geneva, where he developed the community-based programmes for prevention of genetic disorders and early approaches for prenatal diagnosis. He moved to the Reproductive Genetics Institute in 1990, where he heads the WHO Collaborating Center for Prevention of Genetic Disorders, and scientific research in prenatal and preimplantation genetics. He is an author on more than a hundred papers and nine books in the above areas, including three books in the field of Preimplantation Genetics. Dr Anver Kuliev Yury Verlinsky, Anver Kuliev 1 Reproductive Genetics Institute, 2825 North Halsted Street, Chicago, IL, USA 1 Correspondence: Tel: ; Fax: ; anverkuliev@hotmail.com or rgi@flash.net Abstract Preimplantation genetic diagnosis (PGD) has become an established procedure for avoiding the birth of affected children with single gene disorders. PGD is performed through polar body or blastomere biopsy, which has no deleterious effect on pre- and post-implantation development. This review describes the most recent developments and current changes in the spectrum of conditions for which PGD has been applied. The most recent applications of PGD include congenital malformations, blood group incompatibility and an increasing number of late onset disorders with genetic predisposition, all of which have not previously been diagnosed using PGD. Despite ethical concerns, PGD has also been used for preselection of unaffected and HLA matched embryos, and recently for preimplantation HLA matching without testing for the causative gene. This extends the practical value of PGD, with its utility being no longer limited to prevention of single gene disorders, by expanding it to treatment of siblings requiring stem cell transplantation. Keywords: blastomere biopsy, congenital malformations, ethics, HLA matching, late onset disorders, PGD Introduction Preimplantation genetic diagnosis (PGD) is a relatively new approach to detect genetic disorders before pregnancy, avoiding the need for prenatal diagnosis and abortion. It is based on testing of oocytes or embryos to pre-select and transfer only normal embryos back to the patient, achieving an unaffected pregnancy and the birth of a healthy child. PGD is performed either by testing single blastomeres removed from 8-cell preimplantation embryos, or by testing female gametes (Verlinsky and Kuliev, 2000). The latter is based on the removal and genetic analysis of the first and second polar bodies (PB1 and PB2), which represent the by-products of the first and second meiotic divisions. Because neither PB1 nor PB2 has any biological significance for embryo development, they may be removed and tested for their genetic content, in order to predict the resulting maternal contribution to the embryo. However, this approach cannot be applied to testing for paternally derived abnormalities and gender determination, which may be detected by genetic analysis of single blastomeres removed from cleavage stage embryos. Both methods, therefore, are complementary and may be applied 145

2 146 depending on the PGD objectives in each patient. More that 5000 PGD cycles have been performed, resulting in the birth of well over 1000 children (Verlinsky et al., 2003a). The overall experience of using PGD for single gene disorders presently exceeds 1000 cases, showing that it is an established alternative to traditional prenatal diagnosis, and may be reliably used as an integral part of genetic practices (IWGPG, 2001; Kuliev and Verlinsky, 2002; ESHRE PGD Consortium, 2002). Changing spectrum of PGD application The list of disorders, presently comprising more than 50 different conditions, for which PGD has been applied is being extended beyond the indications for prenatal diagnosis, although the most frequent ones are still cystic fibrosis and haemoglobin disorders (IWGPG, 2001; ESHRE PGD Consortium, 2002). Of approximately 400 PGD cycles for single gene disorders, almost half were performed for cystic fibrosis and haemoglobin disorders, followed by myotonic dystrophy and X-linked mental retardation (XMR1) (Verlinsky et al., unpublished data), similar to experience in other active centres (ESHRE PGD Consortium, 2002). As there have been many previous reports on PGD for different single gene disorders, as well as extensive reviews on the subject (Handyside et al., 1992; Sermon et al., 1997, 1999, 2000; Kuliev et al., 1998, 1999; Xu et al., 1998; Kanavakis et al., 1999; De Vos et al., 2000; Goossens et al., 2000; Carvalho et al., 2001; Daniels et al., 2001; Georgiou et al., 2001; Harper et al., 2002; Hellani et al., 2002; Stern et al., 2002), this review will be limited to the most recent developments and current changes in the spectrum of conditions for which PGD has been applied. Specific diagnosis for X-linked diseases More than half of PGD cases for single gene disorders, from the very beginning, have involved gender determination for X- linked conditions, using either polymerase chain reaction (PCR) or fluorescence in-situ hybridization (FISH) (IWGPG, 2001; ESHRE PGD Consortium, 2002). This was not only because the sequence information was not always available, but also because it was technically easier to identify female embryos by DNA analysis or FISH, despite the obvious cost of discarding 50% healthy male embryos. On the other hand, testing for X-linked genetic disorders may be entirely limited to oocytes, because the mutations involved are fully maternally derived. Therefore, testing of oocytes for maternally derived specific mutations makes it possible to avoid further testing of the resulting embryos, which may be transferred irrespective of gender or any contribution from the father. Initially, the technique was applied for ornithine transcarbamylase deficiency (OTC) (Verlinsky et al., 2000). It has since been extended to the specific diagnosis of other X- linked disorders (Verlinsky et al., 2002a), and currently comprises experience of specific diagnosis in 35 cycles performed for OTC, XMR1, myotubular myotonic dystrophy and X-linked hydrocephalus. This resulted in the transfer of 68 mutation-free embryos in 33 cycles, with 12 unaffected clinical pregnancies. Specific diagnosis for X-linked mutations has also been performed at the cleavage stage (Ray et al., 1999, 2000). The data demonstrate the clinical usefulness of specific polar body or blastomere testing for X-linked disorders as an alternative to PGD by gender determination. Couples with homozygous affected partner PGD has also been successfully used in couples with one homozygous or compound-heterozygous affected partner with thalassaemia or phenylketonuria (PKU) (Verlinsky and Kuliev, 2000; Verlinsky et al., 2001a). Although the risk of producing an affected child in such couples is 50%, irrespective of maternal or paternal affected status, the strategy of PGD in such cases will depend on whether the father or mother is affected. In one such case when the father was affected, PGD was concentrated on the preselection of mutation free oocytes, while in the other, with the mother affected, a cleavage stage PGD was required to identify those few embryos containing the normal gene. Testing is particularly complicated if the parents are carrying different mutations. In one such case performed for thalassaemia, the affected mother was double heterozygous (IVS I-110; IVS I-6), while the male partner was a heterozygous carrier of the third mutation (IVS II-745) (Verlinsky and Kuliev, 2000). This required a complex PGD design to exclude preferential amplification of each of the three alleles tested in biopsied blastomeres, as described also in PGD for PKU (Verlinsky et al., 2001a). In this case, the affected father was compound heterozygous for R408 and Y414C mutations in exon 12 of the phenylalanine hydroxylase (PAH) gene, and the carrier mother heterozygous for the R408W mutation in the same exon. PGD strategy was based on the pre-selection of the mutation-free oocytes using sequential PB1 and PB2 DNA analysis. Based on multiplex hemi-nested PCR analysis, four embryos resulting from the zygotes predicted to contain no mutant allele of the PAH gene were transferred, resulting in an unaffected twin pregnancy and the birth of the healthy twins. With improvements in treatment and extended life expectancy for many genetic disorders, an increasing number of couples with affected maternal or paternal partners may require PGD in order to have unaffected children. Cancer predisposition Cancer predisposition has not traditionally been considered as an indication for prenatal diagnosis, as this would lead to pregnancy termination, which is scarcely justifiable on the basis of genetic predisposition alone. On the other hand, the possibility of choosing embryos free of genetic predisposition for transfer would obviate the need for considering pregnancy termination, as only potentially normal pregnancies would be established. PGD for such conditions appears acceptable on ethical grounds, because only a limited number of the embryos available from hyperstimulation are selected for transfer. The first PGD for inherited cancer predisposition was performed for couples carrying p53 tumour suppressor gene

3 mutations, known to determine a strong predisposition to the majority of cancers. It resulted in a singleton pregnancy and the birth of a healthy child free from the mutation predisposing to Li Fraumeni syndrome (Verlinsky et al., 2001b). The couple presented with the paternally derived missense mutation due to transversion of G to A in exon 5 of the p53 tumour suppressor gene. The carrier was a 38-year-old proband with Li Fraumeni syndrome (LFS), diagnosed with rhabdomyosarcoma of the right shoulder at the age of 2 years, followed by right upper extremity amputation. At the age of 31 years, he was also diagnosed with a high-grade leiomyosarcoma of the bladder and underwent a radical cystoprostatectomy. His mother was diagnosed with leimyosarcoma at age 37 years. At present, PGD is also being used for other cancers, including familial adenomatous polyposis coli, Von Hippel Lindau syndrome, retinoblastoma, neurofibromatosis type I and II and familial posterior fossa brain tumour (Rechitsky et al., 2002). Overall, 20 PGD cycles have been performed for 10 couples, resulting in preselection and transfer of 40 mutation-free embryos, which yielded five unaffected clinical pregnancies and four healthy children born to date. Despite the controversy surrounding PGD use for late onset disorders, the data demonstrate the usefulness of this approach as the only acceptable option for at-risk couples to avoid the birth of children with inherited predisposition to cancer, and to have a healthy child. Other late-onset disorders with genetic predisposition One of the first experiences of PGD for late-onset disorders was for genetic predisposition to one of the forms of Alzheimers disease (AD) (Verlinsky et al., 2002b). This condition is caused by an autosomal dominant familial predisposition to a presenile form of dementia, determined by a nearly completely penetrant autosomal dominant mutation in the amyloid precursor protein (APP) gene, for which no treatment is available despite a possible predictive diagnosis. The 30-year-old woman had no signs of AD, but was a carrier of the V717L mutation, resulting from a G to C substitution in exon 17 of the APP gene. Predictive testing in the patient was performed because of early onset AD in her sister, who carried this mutation and developed symptoms of AD at the age of 38 years. This sister is still alive, but her cognitive problems progressed to the point that she was placed in an assisted living facility. Her father had died at age 42 years and had also a history of psychological difficulties and marked memory problems. V717L mutation was also detected in one of her brothers, who experienced mild short-term memory problems as early as 35 years of age, with a moderate decline in memory, new learning and sequential tracking in the next 2 3 years. The other family members, including one brother and two sisters, were asymptomatic, although predictive testing was performed only in the sisters, who appeared to be free from a mutation in the APP gene. PGD was performed by DNA analysis of PB1 and PB2, resulting in a singleton clinical pregnancy and birth of an unaffected child. It may be argued that PGD should not be provided for a patient who might not be able to raise her child into adulthood, but it was the patient s decision to undertake PGD, and it is arguably a better option than to reproduce without testing, with a high risk of having an affected child. PGD therefore provides a further option for patients who may wish to avoid transmission of the mutant gene predisposing to late onset disorders in their potential children. Because such diseases present beyond early childhood, and even later may not be expressed in 100% of cases, the application of PGD for this group of disorders is still controversial. However, for diseases with no current prospect for treatment, arising despite pre-symptomatic diagnosis and follow-up, PGD may be offered as the only solution for at-risk couples. One example of the application of PGD in a late onset disorder is that of Huntington disease (HD). Non-disclosure PGD has been considered for at-risk couples, and involves the transfer of disease-free embryos, while the prospective parents do not learn their own status (Stern et al., 2002). Parents receive no information about number of oocytes obtained after hormonal stimulation, number of embryos developed, and the number of embryos available for transfer. However, there might be no unaffected embryos for transfer, or no affected embryos can be found, suggesting that the parent is genetically normal. The other alternative in HD is exclusion-pgd testing, but embryos with a detected grandparental allele would be excluded from transfer, notwithstanding that only half of these embryos would contain the affected allele. However, some European centres prefer performing direct testing of embryos from known disease gene carriers (Sermon et al., 2001). Blood group incompatibility The first PGD for materno fetal incompatibility resulting in a healthy pregnancy was performed for Kell (K1) genotype, which is one the major antigenic systems in human red blood cells. It is comparable in importance to RhD, causing materno fetal incompatibility leading to severe haemolytic disease of the newborn (HDN) in sensitized mothers (Verlinsky et al., 2003c). The K1 allele is present in 9% of populations, in contrast to its highly prevalent allelic variant K2. The gene is located on chromosome 7 (7q33), consisting of 19 exons, with the only C to T base substitution in exon 6 in K1 compared with K2 antigen. In a pregnancy with a K1 fetus in a K2 mother, antibodies to K1 may develop, leading to materno fetal incompatibility causing severe HDN. Although prenatal diagnosis is available for identification of pregnancies at risk for HDN, this may not always prevent potential complications for the fetus, stillbirth or neonatal death. PGD is therefore a possible option for preventing both Kell and Rhesus haemolytic diseases. PGD for Kell disease was performed for two at-risk couples with a history of neonatal death in previous pregnancies due to HDN. The preselection and transfer of embryos free from the K1 allele of the KEL gene was possible in each case, yielding a clinical pregnancy and the birth of healthy twins, confirmed to be free of the K1 allele. A number of attempts have also been undertaken to perform PGD for Rhesus disease, although they have not yet resulted in 147

4 a clinical pregnancy (Van Den Veyver, 2000). Both Kell disease and Rhesus incompatibility are quite prevalent (approximately 15% frequency for RhD and 9% for KEL antigen), presenting a risk for alloimmunization that may lead to HDN in some at-risk couples. Therefore, PGD may be a useful option for these couples to avoid the establishment of a RhD or K1 pregnancy in sensitized mothers. Although at-risk pregnancies detected by prenatal diagnosis may be treated by intrauterine transfusion, potential complications for the fetus cannot be completely excluded even after this procedure. Pregnancy termination in such cases will also be unacceptable, as the antibodies to K1, for example, are developed in only 5% of persons exposed to incompatible blood. On the other hand, some at-risk couples have had such an unfortunate experience of HDN, resulting in neonatal death, that they regard PGD as their only option to plan another pregnancy. This makes PGD attractive for patients at risk for alloimmunization, although such conditions have rarely been an indication for prenatal diagnosis. Congenital malformations Congenital malformations are highly prevalent (29.3/1000 live births) and are usually sporadic. However, with progress of the Human Genome Project, an increasing number of inherited forms are being described, which, therefore, may be avoided through PGD. For example, Sonic Hedgehog (SHH) gene mutation, for which the first PGD was recently performed (Verlinsky et al., 2003b), causes failure of the cerebral hemispheres to separate into distinct left and right halves and leads to holoprosencephaly (HPE), which is one of the most common developmental anomalies of forebrain and mid-face. Although the majority of HPE are sporadic, familial cases are not rare, with clear autosomal dominant inheritance. Significant intrafamilial clinical variability of HPE, from alobar HPE and cyclopia, to cleft lip and palate, microcephaly, ocular hypertelorism, and even normal phenotype, suggests interaction of the SHH gene with other genes expressed during craniofacial development and the possible involvement of environmental factors. This may explain the fact that almost one-third of carriers of SHH mutations may be clinically unaffected. Therefore, even in familial cases, the detection of SHH mutations in prenatal diagnosis might not justify pregnancy termination, making PGD a more attractive option for couples at risk for producing children with HPE. In the case described (Verlinsky et al., 2003b), the couple presented for PGD because of the previous birth of two children with clinical signs of HPE. One, a female with severe HPE and cleft lip and palate, died shortly after birth. Both the child and the parents were chromosomally normal, but DNA analysis in the child s autopsy material demonstrated the presence of SHH nonsense mutation due to GAG TAG sequence change, leading to premature termination of the protein at position 256 (Glu256 stop). The same mutation was found in their 5-year-old son, who was born after a fullterm normal pregnancy. This child had less severe facial dysmorphisms, which included microcephaly, Rathke s pouch 148 Figure 1. PGD for Sonic Hedgehog (SHH) mutation: family pedigree. Upper panel: the father (I-1) has a gonadal mosaicism for SHH mutation, which is linked to 156 bp dinucleotide C-A repeat allele of D7S550 polymorphic marker, while the mother (I-2) is normal, with one normal allele linked to 158 bp repeat and the other to 138 bp repeat alleles. Lower panel: reproductive outcomes of this couple, including three previous pregnancies, one resulting in the birth of affected child with holoprosencephaly, carrying the mutant gene (lower left) (II-1), one in perinatal death (II-2), also carrying the mutant gene [lower middle (circle)], and one in a spontaneously aborted fetus with Turner syndrome (II-3), free from SHH mutation [lower middle (triangle)]. The lower right open circle (II-4) represents the outcome of preimplantation diagnosis, resulting in an unaffected clinical pregnancy and birth of a healthy child, following confirmation of mutation-free status by amniocentesis. Reprinted with permission, from Verlinsky et al. (2003b).

5 cyst, single central incisor and choanal stenosis. There was also clinodactyly of the fifth fingers and incurved fourth toes bilaterally. The child s growth was slow in the first 2 years, but since then, he has been maintaining reasonably good growth. PGD was performed by blastomere biopsy and multiplex nested PCR analysis, involving specific mutation testing simultaneously with linked marker analysis. Of nine tested embryos, four were free of the mutant gene, of which two were transferred back to the patient, resulting in a singleton pregnancy and birth of a healthy child following confirmation of the mutation free status by amniocentesis (Figure 1). A similar approach has been used for PGD of Crouson syndrome (Abou-Sleiman et al., 2002). The data suggest the clinical usefulness of PGD for familial cases of congenital malformations. Because of the high prevalence of congenital anomalies, the approach may have practical implications for at-risk couples as a preventive measure to be employed in genetic practice. Preimplantation HLA matching combined with causative gene test Preimplantation HLA matching was first introduced in combination with mutation analysis for Fanconi anaemia, with the objective of establishing an unaffected pregnancy yielding a potential donor progeny for transplantation in an affected sibling (Verlinsky et al., 2001c). This resulted in a clinical pregnancy and birth of an unaffected child, whose cord blood was transplanted to the affected sibling, saving her life. This strategy is unlikely to be clinically acceptable through traditional prenatal genetic diagnosis, because of possible clinical pregnancy termination after HLA matching. However, PGD for such purposes should be acceptable, because only a limited number of the embryos are usually pre-selected for transfer, which in this case will represent unaffected embryos with a perfect match for affected siblings in need of a transplant. Since the multiplex single cell PCR used in PGD presents the opportunity for combined PGD and HLA testing, it has become a useful way to pre-select an embryo which may be an HLA match to the affected sibling requiring stem cell transplantation. The method has currently been applied for HLA genotyping in two dozen cycles in combination with PGD for thalassaemia, Fanconi anaemia, hyper-immunoglobulin M syndrome, X- linked adrenoleukodystrophy and Wiscott Aldrich syndrome. The results confirm the usefulness of preimplantation HLA matching as part of PGD, with the prospect of applying this approach to other inherited conditions, also requiring an HLA compatible donor for bone marrow transplantation (Verlinsky et al., unpublished data). This provides a realistic option for couples desiring to avoid the birth of an affected child, together with the establishment of a healthy pregnancy, potentially providing an HLA match progeny for treatment of an affected sibling. Ethical issues The increasing use of PGD for late onset disorders with genetic predisposition, and preimplantation HLA typing to produce an HLA compatible donor to treat a family member requiring stem cell transplantation, raises important ethical issues (see Edwards et al., 2003). Although there is no actual difference in the application of PGD for the latter conditions, the controversy can be explained by the fact that in traditional prenatal diagnosis, if the fetus is found to carry the gene predisposing to late-onset disorder or to be HLA unmatched, the couple would have to make an extremely difficult decision regarding pregnancy termination, which could hardly be justified by such findings. Alternatively, PGD technology allows genetic testing of human eggs and embryos before pregnancy is established, making it completely realistic to establish only HLA matched or potentially normal pregnancies without genetic predisposition to late onset disorders. In any case, as seen from the above review, PGD is now becoming an established clinical option in reproductive medicine and is applied using separate consent forms and the research protocols approved by institutional ethics committees. The number of apparently healthy children born after PGD has passed its first thousand, showing that there is no evidence of any incurred adverse effect. However, these protocols would still require confirmatory chorionic villous sampling or amniocentesis and follow-up monitoring of safety and accuracy. Although PGD will help solve some longstanding ethical problems, such as the abortion issue (which will be avoided as a result of this new approach), others could become a serious obstacle, particularly those related to designer babies. These considerations are highly relevant to the subject of PGD, as well as to any other new methods, as further development of appropriate technology for controlling genetic disability takes place. References Abou-Sleiman PM, Apessos A, Harper JC et al Pregnancy following preimplantation genetic diagnosis for Crouson syndrome. Molecular and Human Reproduction 8, Carvalho F, Sousa M, Fernandes S et al Preimplantation genetic diagnosis for familial amyloidotic plyneuropathy (FAP). Prenatal Diagnosis 21, Daniels G, Pettigrew R, Thornhill A et al Six unaffected livebirths following preimplantation diagnosis for spinal muscular atrophy. Molecular and Human Reproduction 7, De Vos A, Sermon K, Van de Velde H et al Two pregnancies after preimplantation genetic diagnosis of ostegenesis imperfecta type I and type IV. Human Genetics 106, Edwards RG 2003 Ethics of preimplantation diagnosis: recordings from the Fourth International Symposium on Preimplantation Genetics. Reproductive BioMedicine Online 6, ESHRE Preimplantation Genetic Diagnosis Consortium 2002 Data Collection III, May Human Reproduction, 17, Georgiou I, Sermon K, Lissens W et al Preimplantation genetic diagnsis for spinal and bulbar muscular atrophy (SBMA). Human Genetics 108, Goossens V, Sermon K, Lissens W. et al Clinical application of preimplantation genetic diagnosis for cystic fibrosis. Prenatal Diagnosis 20, Handyside AH, Lesko JG, Tarin JJ et al Birth of a normal girl after in vitro fertilization and preimplantation diagnosis testing for cystic fibrosis. New England Journal of Medicine 327, Harper J, Wells D, Piyamongkol W et al Preimplantation genetic diagnosis for single gene disorders: experience with five single gene disorders. Prenatal Diagnosis 22, Hellani A, Lauge A, Ozand P et al Pregancy after preimplantation genetic diagnosis for ataxia telangectasia. 149

6 Molecular and Human Reproduction 8, International Working Group on Preimplantation Genetics (IWGPG) 2001 Preimplantation genetic diagnosis experience of three thousand clinical cycles. Report of the 11th Annual Meeting International Working Group on Preimlantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 15, Reproductive BioMedicine Online 3, Kanavakis E, Vrettou C, Palmer G et al Preimplantation genetic diagnosis in 10 couples at risk for transmitting betathalassemia major: clinical experience including the initiation of six singleton pregnancies. Prenatal Diagnosis 19, Kuliev A, Verlinsky Y 2002 Current features of preimplantation genetic diagnosis. Reproductive BioMedicine Online 5, Kuliev A, Rechitsky S, Verlinsky O et al Preimplantation diagnosis of thalassemias. Journal of Assisted Reproduction and Genetics 15, Kuliev A, Rechitsky S, Verlinsky O et al Birth of healthy children after reimplantation diagnosis of thalassemias. Journal of Assisted Reproduction and Genetics 16, Ray P, Harper J, Ao A et al Successful preimplantation genetic diagnosis for sex linked Lesch Nyhan syndrome using specific diagnosis. Prenatal Diagnosis 19, Ray PF, Gigarel N, Bonnefont JP et al First specific preimplantation genetic diagnosis for ornithine transcarbamylase deficiency. Prenatal Diagnosis 20, Rechitsky S, Verlinsky O, Chistokina A et al Preimplantation genetic diagnosis for cancer predisposition. Reproductive BioMedicine Online 5, Sermon K, Lissens W, Joris H et al Clinical application of preimplantation diagnosis for myotonic dystrophy. Prenatal Diagnosis 17, Sermon K, Lissens W, Messiaen L et al Preimplantation genetic diagnosis of Marfan syndrome with the use of fluorescent polymerase chain reaction and automated laser fluorescence DNA sequencer. Fertility and Sterility 71, Sermon K, Henderix P, Lissens W et al Preimplantation genetic diagnosis for medium-chain acyl-coa dehydrogenase (MCAD) deficiency. Molecular and Human Reproduction 16, Sermon K, Seneca S, De Rycke M et al PGD in the lab for triplet diseases myotonic dystrophy, Huntington s disease and Fragile-X syndrome. Molecular and Cellular Endocrinology 183, S77 S85. Stern HJ, Harton GL, Sisson ME et al Non-disclosing preimplantation genetic diagnosis for Huntington disease. Prenatal Diagnosis 22, Van Den Veyver IB 2000 Prenatal and preimplantation genetic diagnosis for rhd alloimmunization. 10th International Conference on Prenatal Diagnosis and Therapy. Barcelona (Spain), June 19 21, University of Barcelona, pp Verlinsky Y, Kuliev A 2000 Atlas of Preimplantation Genetic Diagnosis. Parthenon Publishing Group, London, New York. Verlinsky Y, Rechitsky S, Verlinsky O et al Preimplantation diagnosis for ornithine transcarbamylase deficiency. Reproductive BioMedicine Online 1, Verlinsky Y, Rechitsky S, Verlinsky O et al. 2001a Preimplantation diagnosis for PKU. Fertility and Sterility 76, Verlinsky Y, Rechitsky S, Verlinsky O et al. 2001b Preimplantation diagnosis for p53 tumor suppressor gene mutations. Reproductive BioMedicine Online 2, Verlinsky Y, Rechitsky S, Verlinsky O et al. 2001c Preimplantation diagnosis for Fanconi anemia combined with HLA matching. Journal of the American Medical Association 285, Verlinsky Y, Rechitsky S, Verlinsky O et al. 2002a Polar body based preimplantation diagnosis for X-linked genetic disorders. Reproductive BioMedicine Online 4, Verlinsky Y, Rechitsky S, Verlinsky O et al. 2002b Preimplantation diagnosis for early onset Alzheimer disease caused by V717L mutation. Journal of the American Medical Association 287, Verlinsky Y, Cohen J, Munne S et al. 2003a The first thousand babies born after preimplantation genetic diagnosis. Fertility and Sterility, in press. Verlinsky Y, Rechitsky S, Verlinsky O et al. 2003b Preimplantation diagnosis for Sonic Hedgehog mutation causing familial holoprosencephaly. New England Journal of Medicine 348, Verlinsky Y, Rechitsky S, Verlinsky O et al. 2003c Preimplantation diagnosis for Kell genotype. Fertility and Sterility, in press. Xu K, Shi ZM, Veeck LL, Hughes MR, Rosenwaks Z 1999 First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell disease. Journal of the American Medical Association 281, Received 4 April 2003; refereed 22 April 2003 ; accepted 25 April