Abstract
Purpose: Because allele dropout (ADO) is frequently observed in single-cell polymerase chain reaction analysis, it is important to develop a method for efficient detection of ADO, in order to avoid possible misdiagnosis in preimplantation diagnosis.
Methods: We introduced a simultaneous amplification of mutant genes and linked polymorphic markers, such as a 4-bp repeat (GATT) at the 3′ end of intron 6 in the cystic fibrosis (CF) gene and a short tandem repeat at the 5′ end of the β-globin gene. Three types of single heterozygous cells were studied for the amplification of both alleles, including 150 blastomeres, 1615 fibroblasts, and 170 first polar bodies, obtained from patients at risk for having children with cystic fibrosis (delta F-508 mutation) or sickle cell disease.
Results: ADO rates of as high as 33.3% for delta F-508 mutation and 22.8% for β-globin gene were observed in single blastomeres, compared to 7.1 and 7.7% in single fibroblasts and 5.9 and 9.6% in first polar bodies, respectively. The application of simultaneous amplification of the above linked polymorphic markers allowed detection of more than half of the cases of ADO in blastomeres (19.4% for cystic fibrosis and 12.3% for β-globin gene) and almost all ADOs in polar bodies, particularly when the two-step sequential analysis of the first and second polar body was applied in preimplantation diagnosis of single gene disorders.
Conclusions: Simultaneous amplification of linked polymorphic markers in single-cell DNA analysis of single-gene defects is an efficient method for avoiding the risk of misdiagnosis in preimplantation diagnosis.
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REFERENCES
Verlinsky Y, Kuliev A: Preimplantation Diagnosis of Genetic Diseases. A New Technique for Assisted Reproduction. New York: Wiley Liss, 1994
Handyside AH, Lesko JG, Tarin JJ, Winston RML, Hughes M: Birth of normal girl after in vitro fertilization and preimplantation diagnosis testing for cystic fibrosis. N Engl J Med 1992;327:905-909
Verlinsky Y, Munne S, Simpson JL, Kuliev A, Ao A, Ray P, et al.: Current status of preimplantation diagnosis. J Assist Reprod Genet 1997;14(2):71-75
Rechitsky S, Freidine M, Verlinsky Y, Strom C. Allele drop-out in sequential analysis of single cells by PCR and FISH. J Assist Reprod Genet 1996;13:115-124
Findlay I, Quirke P: Fluorescent polymerase chain reaction: Part I. A new method allowing genetic diagnosis allowing genetic diagnosis and DNA fingerprinting of single cells. Hum Reprod Update 1996;2(2):137-152
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
Verlinsky Y, Handyside A, Grifo J, Munne S, Liebaers I, Levison G, Arnheim N, Hughes M, Delhanty J, et al.: Preimplantation diagnosis of genetic and chromosomal Diseases. J Assist Reprod Genet 1994;11:236-243
Harper J. Preimplantation diagnosis of inherited disease by embryo biopsy. J Assist Reprod Genet 1996;13:90-95
Chebab D, Johnson J, Louie E, Goossens M, Kawasaki E, Erlich H: A diomorphic 4-bp repeat in the cystic fibrosis gene is an absolute linkage disequilibrium with a delta f508 mutation: Implications for prenatal diagnosis and mutation origin. J Hum Genet 1991;48:223-226
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
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
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
Sharma V, Litt M: Tetra nucleotide repeat polymorphism at the D21S11 locus. Hum Mol Genet 1992;1:67
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
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Rechitsky, S., Strom, C., Verlinsky, O. et al. Allele Dropout in Polar Bodies and Blastomeres. J Assist Reprod Genet 15, 253–257 (1998). https://doi.org/10.1023/A:1022532108472
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DOI: https://doi.org/10.1023/A:1022532108472