Preimplantation genetic diagnosis technologies in cystic fibrosis

Diagnosis should be set to all couples requiring preimplantation genetic diagnosis (PGD) for cystic fibrosis (CF). For clinical application, they must be optimized to minimize PCR failure, allelic drop-out (ADO) and contamination, while simultaneously detecting a wide spectrum of CF genotypes.

Different PCR configurations were tested, including duplex (IVS8CA/IVS17BTA, DeltaF508/ IVS8CA, or IVS17BTA and D7S486/D7S490), triplexes (F508, IVS8CA and IVS17bCA or I1CA and IVS17bTA) or quadruplex PCR, with co-amplification of intragenic polymorphic markers. Fluorescent or not multiplex PCR protocol allowing any combination amongst seven CFTR gene exons (4, 10, 11, 13 in two parts, 14b, 17b and 21) by nested PCR, also co-amplifying the DeltaF508 mutation and two CFTR intragenic polymorphic microsatellites (IVS8CA and IVS17bCA) and also 4 repeat markers D7S523, D7S486, D7S480 and D7S490, flanking the CF transmembrane regulator (CFTR) gene. Such an approach is justified since in 91% of the cases at least one partner of the couple carries the DeltaF508 mutation. Cases performed were at risk of transmitting mutations of DeltaF508 and R1162X, or R1066C, or 1341+1A. Cleavage-stage embryo biopsy resulted in transfer of one unaffected carrier embryo for each couple.

89 to 390 single lymphocytes from CF patients, heterozygotes and non-CF individual were used, the 97% of which a PCR signal was obtained and from 94% of blastomeres. PCR efficiency of the exons ranged from 90 to 100% for all experiments. A complete haplotype was achieved for 83.2 to 91% lymphoblasts for all experiments. Allele drop out (ADO) rate comprised between 0% and 11.6% for all experiments. These findings prove the concept that the DeltaF508 mutation of CF can be reliably and accurately diagnosed at the single cell level using microarray analysis.

cystic fibrosis - IVF - preimplantation genetic diagnosis