Molecular Human Reproduction, Vol. 6, No. 5, 391-396,
May 2000
© 2000 European Society of Human Reproduction and Embryology
Genetic diagnosis |
Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis*
1 Department of Molecular Cell Biology & Genetics, Research Institute Grow and Development (GROW), Maastricht University, 6229 GR Maastricht, and 2 Department of Obstetrics & Gynaecology, Academic Hospital Maastricht, 6202 AZ Maastricht, The Netherlands
Abstract
Cystic fibrosis (CF) is the first monogenic disorder for which single cell preimplantation genetic diagnosis (PGD) has been successfully applied. The spectrum of mutations in CF is extremely heterogeneous, and hence, the development of mutation-specific PGD protocols is impracticable. The current study reports the development and evaluation of a general multiplex marker polymerase chain reaction (PCR) protocol for PGD of CF. Four closely linked highly polymorphic (CA)n repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the cystic fibrosis transmembrane regulator (CFTR) gene, were used. In 99% of the single cells tested (100 leukocytes and 50 blastomeres), multiplex PCR results were obtained and the overall allelic drop out (ADO) rate varied from 2 to 5%. After validation for the presence of ADO and additional alleles, 95% of the multiplex PCR results were accepted to construct the marker genotypes. Depending on the genotype of the couple, and taking into account the embryos lost for transfer due to validation criteria (5%), ADO (0–2%) and single recombination (1.1–3%), in general >90% of the embryos could be reliably genotyped by PGD using a single blastomere. The risk of misdiagnosis equals the chance of a double recombination between informative flanking markers and is <0.05%. Therefore, this polymorphic and multi-allelic marker system is a reliable and generally applicable alternative for mutation-directed PGD protocols. Furthermore, it provides a test for the origin of the detected genotype and also gives an indication of the chromosomal ploidy status of the blastomere tested.
cystic fibrosis/multiplex marker PCR/preimplantation genetic diagnosis/single cell diagnosis
Notes
3 To whom correspondence should be addressed at: Department of Molecular Cell Biology & Genetics, POB 1475, 6201 BL Maastricht, The Netherlands
* This work is based in part on the Established Scientists Award Winning paper presented at the 15th Annual Meeting of ESHRE, June 27–30, 1999, Tours, France
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