RBMOnline - Vol 1. No 2. 45-47; Reproductive BioMedicine Online webpaper 2000/014 on web 16/10/00
Article Preimplantation diagnosis for ornithine transcarbamylase deficiency 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.
DrYury V erlinsky
Y Verlinsky 1, S Rechitsky, O Verlinsky, C Strom, A Kuliev Reproductive Genetics Institute, Chicago, IL, USA 1Correspondence: 836 West Wellington avenue, Chicago, IL 60657, USA; Tel. 7732967095; Fax 773-8715221; e-mail:
[email protected]
Abstract Ornithine transcarbamylase (OTC) deficiency is a severe X-linked metabolic disorder leading to hyperammonaemia and death shortly after birth. Prenatal diagnosis for OTC deficiency is available, but may require termination of pregnancy if affected. Thus there is a need for an option for pre-pregnancy testing, to pre-select OTC deficiency-free embryos for transfer, thus avoiding prenatal diagnosis and pregnancy termination. Preimplantation genetic diagnosis (PGD) for OTC deficiency has been developed, using sequential first and second polar body analysis; it was applied in a woman carrying the R26Q mutation in the exon 1 of OTC gene. The first and second polar bodies were removed following maturation and fertilization of oocytes in a standard IVF protocol, and analysed using a multiplex nested PCR. R26Q mutation was tested simultaneously with linked markers in six zygotes, resulting in detection of the embryos with a mutation-free maternal contribution; these were transferred back to the patient, yielding pregnancy and birth of a healthy child. This is the first PGD for OTC deficiency resulting in the birth of an unaffected child.
Keywords: first and second polar bodies, linked polymorphic markers, multiplex nested PCR, ornithine transcarbamylase deficiency, preimplantation genetic diagnosis
Introduction
Materials and methods
Ornithine transcarbamylase (OTC) deficiency is a severe Xlinked metabolic disorder leading to hyperammonaemia and death of affected males shortly after birth. Heterozygous females have no signs or much milder phenotype, depending on skewed X-inactivation. Prenatal diagnosis for OTC deficiency is possible, but may lead to pregnancy termination of the male fetus. Pre-pregnancy testing, on the other hand, may allow preselection of the OTC deficiency-free embryos for transfer, avoiding prenatal diagnosis and pregnancy termination (Handyside et al., 1992; International Working Group on Preimplantation Genetics, 2001; Sermon et al., 1997; Verlinsky et al., 1999; Xu et al., 1999). This may be done by gender determination, but half of the male embryos in this case will be discarded despite being unaffected. We developed a preimplantation genetic diagnosis (PGD) test for OTC deficiency, which was applied in a patient carrying R26Q mutation in exon 1 of the OTC gene, yielding an unaffected pregnancy and birth of a healthy child.
A couple presented for PGD in connection with their first male offspring with OTC deficiency. The 30 year old mother was a carrier of R26Q mutation in exon 1 of the OTC gene due to single nucleotide substitution of G to A in codon 26. A map of the OTC gene with sites and locations of short tandem repeats (STR), dinucleotide repeats, restriction fragment length polymorphisms (RFLP) and R26Q mutation is shown in Figure 1. Following maturation of oocytes in a standard IVF protocol, the first polar bodies (PB1) were removed following maturation of oocytes using micromanipulation procedures described elsewhere (Verlinsky and Kuliev, 2000). Then oocytes were fertilized by intracytoplasmic sperm insertion (ICSI), and the second polar bodies (PB2) were removed by micromanipulation techniques mentioned above (Verlinsky and Kuliev, 2000). PB1 and PB2 were amplified by nested multiplex polymerase chain reaction (PCR) (Rechitsky et al.,
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Articles - Preimplantation diagnosis for ornithine transcarbamylase deficiency - Y Verlinsky et al.
Figure 1. PCR design for PGD for OTC mutation. Top: Human OTC gene showing sites and locations of short tandem repeats, dinucleotide repeats, restriction length polymorphisms, and R26Q mutation. Bottom: Primer sequences, primer melting temperatures and polymorphic restriction sites for loci shown in the map
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Figure 2. Results of PGD for R26Q mutation in OTC gene. Top: OTC gene exons, showing single nucleotide substitutions mutations, primer sets for nested PCR, and restriction sites for various enzymes. Middle: Restriction map for R26Q mutation analysis and linked polymorphism. Bottom: Mutation analysis (left) and linked marker analysis (right) of six oocytes by PB1 and PB2 (explanation in the text). Mutant allele lacks a DdeI site (- site), while the normal allele contains this site (+ site). Only oocyte no.5 is mutation-free, based on both mutation and linked marker analysis; it was replaced and resulted in pregnancy.
Articles - Preimplantation diagnosis for ornithine transcarbamylase deficiency - Y Verlinsky et al. 1999), using a primer design and primer melting temperatures shown in Figure 1. Restriction digestion of PCR product was done using enzyme A1wNI for mutation and Ddel for polymorphic marker (restriction maps for both genes are shown in Figure 2). Based on sequential PB1 and PB2 mutation and linked marker analysis (Verlinsky et al., 1997), the embryos resulting from mutation-free oocytes were transferred back to patients, while those predicted affected were further amplified to confirm PB diagnosis. The diagnosis was also confirmed in a resulting pregnancy by chorionic villus sampling (CVS).
Results and discussion
References
Of six oocytes available for testing, the results for both PB1 and PB2 were obtained in five of them, allowing reliable diagnosis. In one of the oocytes (oocyte no.1) with the results only in PB1, the resulting genotype could not be predicted, because both normal and mutant alleles were present. Of five oocytes with both PB1 and PB2 data, four were predicted affected (oocytes 2, 3, 4, and 6), based on heterozygous PB1, containing both normal and mutant alleles, and normal PB2, suggesting that the resulting oocytes should have contained a mutant allele (Figure 2). Only one oocyte (oocyte no.5) was predicted to contain a normal allele, based on the presence of a mutant allele in PB1 and the normal allele in PB2 (Figure 2). These results were in agreement with the linked marker analysis, showing that the homozygous status of PB1 in oocyte no.5 was not due to allele drop out (ADO) of the normal allele, otherwise the normal status of PB2 would mean that the resulting oocyte was abnormal and could not have been transferred. As seen from Figure 2, the marker analysis also confirmed the mutant status of oocyte nos 2, 3, 4, and 6, which showed heterozygous status of PB1 and hemizygous normal PB2. The embryos resulting from these embryos were followed up by mutation and marker analysis, confirming the presence of a mutant allele in all of them.
Handyside AH, Lesko JG, Tarin JJ et al. 1992 Birth of a normal girl after in vitro fertilization and preimplantation diagnosis testing for cystic fibrosis. New England Journal of Medicine 327, 905-909. International Working Group on Preimplantation Genetics 2001 10th Anniversary of Preimplantation Genetic Diagnosis. Report of the 10th Annual Meeting International Working Group on Preimplantation Genetics, in conjunction with 3rd International Symposium on Preimplantation Genetics, Bologna, June 23, 2000. Journal of Assisted Reproduction and Genetics 18, 66-72 Rechitsky S, Strom C, Verlinsky O et al. 1999 Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. Journal of Assisted Reproduction and Genetics 16, 192-198. Sermon K, Lissens W, Joris H. et al. 1997 Clinical application of preimplantation diagnosis for myotonic dystrophy. Prenatal Diagnosis 17, 925-932. Verlinsky Y, Kuliev A 2000 Atlas of Preimplantation Genetic Diagnosis. Parthenon, NewYork and London. Verlinsky Y, Rechitsky S, Cieslak J et al. 1997 Preimplantation diagnosis of single gene disorders by two-step oocyte analysis using first and second polar pody. Biochemical and Molecular Medicine 62,182-187. Verlinsky Y, Rechitsky S, Verlinsky O et al. 1999 Prepregnancy testing for single-gene disorders by polar body analysis. Genetic Test 3, 185-190. Xu K, Shi ZM, Veeck LL et al. 1999 First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell disease. Journal of the American Medical Association 281, 1701-1706.
The embryo resulting from oocyte no.5 was transferred back to the patient, yielding a clinical pregnancy, confirmed by CVS to be an unaffected male. A healthy child was born, who is currently one year old with normal pattern of growth. The high accuracy of the PGD strategy has been demonstrated by the follow-up study of affected embryos, which showed complete concordance with the results of sequential PB1 and PB2 analysis. Overall, 750 PGD cycles have so far been performed for at least two dozen different single gene disorders worldwide, resulting in ~150 clinical pregnancies and >100 healthy children born up to the present time (International Working Group on Preimplantation Genetics, 2001). To our knowledge, this is the first PGD for OTC deficiency resulting in the birth of an unaffected child, demonstrating the usefulness of PGD for preselection of mutation-free oocytes in couples at risk for producing a child with severe X-linked disorders.
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