Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis

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 deve lopment of mutation-specific PGD protocols is impracticable. The current st udy reports the development and evaluation of a general multiplex marker po lymerase chain reaction (PCR) protocol for PGD of CF Four closely linked hi ghly 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), mu ltiplex PCR results were obtained and the overall allelic drop out (ADO) ra te varied from 2 to 5%. After validation for the presence of ADO and additi onal alleles, 95% of the multiplex PCR results were accepted to construct t he marker genotypes. Depending on the genotype of the couple, and taking in to account the embryos lost for transfer due to validation criteria (5%), A DO (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 informativ e flanking markers and is <0.05%. Therefore, this polymorphic and multi-all elic marker system is a reliable and generally applicable alternative for m utation-directed PGD protocols. Furthermore, it provides a test for the ori gin of the detected genotype and also gives an indication of the chromosoma l ploidy status of the blastomere tested.