Abstract
Purpose: Preimplantation genetic diagnosis (PGD) is an important option for couples at risk of having children with β-globin mutations to avoid selective abortions of affected fetuses following prenatal diagnosis.
Methods: We performed PGD for thalassemia in 12 clinical cycles (IVS1-110, and IVS-745 mutations) using biopsy of the first and second polar bodies (PBs) extruded from oocytes during maturation and fertilization, coupled with nested polymerase chain reaction analysis and restriction digestion.
Results: A total of 118 oocytes was obtained, of which 78 had results for both the first and the second PBs. This resulted in the selection and transfer of 30 unaffected embryos (2.5 embryos per cycle). To avoid a possible misdiagnosis due to allele dropout (ADO), we have also introduced simultaneous detection of two highly polymorphic linked markers, a short tandem repeat immediately at the 5′ end of the globin gene and HUMTH01 which is a syntenic short tandem repeat. The application of multiplex polymerase chain reaction of the β-globin gene and linked polymorphic markers enabled detection of ADO in five first PBs, thus avoiding the transfer of potentially affected embryos resulting from their corresponding oocytes.
Conclusions: Confirmation studies of the embryos resulting from the oocytes predicted to contain an affected gene confirmed the diagnosis in 98% of the cases, thus demonstrating the accuracy and reliability of PB PGD of thalassemia mutations. The application of PB analysis in six patients resulted in two ongoing pregnancies with a thalassemia-free fetus already confirmed in both of them by prenatal diagnosis.
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REFERENCES
Verlinsky Y, Kuliev A: Progress in preimplantation diagnosis. J Assist Reprod Genet 1998;15:9-11
Modell B, Kuliev A, Wagner M (eds): Community Genetics Services in Europe. Copenhagen, World Health Organization, Regional Office for Europe, Copenhagen, 1991, p 137
Verlinsky Y, Rechitsky S, Cieslak J, Ivakhnenko V, Wolf G, Lifchez A, Kaplan B, Moise J, Walle J, White M, Ginsberg N, Strom C, Kuliev A: Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body. Biochem Mol Med 1997;62:182-187
Rechitsky S, Freidine M, Verlinsky Y, Strom C: Allele dropout in sequential analysis of single cells by PCR and FISH. J Assist Reprod Genet 1996;13:115-124
Varawala NY, Dokras A, Old JM, Sargent IL, Barlow DH: An approach to preimplantation diagnosis of beta-thalassaemia. Prenat Diagn 1991;11:775-785
Lindeman R, Hu SP, Volpata F, Trent RJ: Polymerase chain reaction (PCR) mutagenesis enabling rapid nonradioctive diagnosis of common beta-thalassemia mutation in Mediterraneans. Br J Hematol 1991;78:100-104
Sheardown S, Findlay I, Turner A, Greaves D, Bolton V, Mitchell M, Layton DM, Muggleton-Harris AL: Preimplantation diagnosis for an Hbs transgene in biopsied trophectoderm cells and blasomeres of the mouse embryo. Hum Reprod 1992;7:1290-1303
Findlay I, Ray P, Rutherford A, Lilford R. Allele drop-out and preferential amplification in single cells and human blastomeres: Implications for preimplantation diagnosis of sex and cystic fibrosis. Hum Reprod 1995;10:1609-1618
Ray P, Winston R, Handyside A: Reduced allele drop-out in single cell analysis for preimplantation genetic diagnosis of cystic fibrosis. J Assist Reprod Genet 1996;13:104-106
Loudianos G, Cao A, Pirastu M. Feasibility of prenatal diagnosis of beta-thalassemia using two highly polymorphic microsatellites 5′ to the beta-globin gene. Haematologica 1992;77:361-362
Puers C, Hammond HA, Caskey T, Lins AM, Sprecher CJ, Brinkman B, Schumm JW: Allelic ladder characterization of the short tandem repeat polymorphism located in the 5′ flanking region to the human coagulation factor A subunit gene. Genomics 1994;23:260-264
Peake IR, Bowen D, Bignell P, Liddel MB, Ladler GE, Standen G, Bloom AL: Family studies and prenatal diagnosis in severe Von Willebrand disease by polymerase chain reaction amplification of variable tandem repeat region of the Von Willebrand factor gene. Blood 1990;76:555-561
Sharma V, Litt M: Tetranucleotide repeat polymorphism at the D21S11 locus. Hum Mol Genet 1992;1:67
Li H, Cui X, Arnheim N. Analysis of DNA sequences variation in single cells. Methods Comp Methods Enzymol 1991;2:49-59
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Kuliev, A., Rechitsky, S., Verlinsky, O. et al. Preimplantation Diagnosis of Thalassemias. J Assist Reprod Genet 15, 219–225 (1998). https://doi.org/10.1023/A:1022571822585
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DOI: https://doi.org/10.1023/A:1022571822585