Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosis

Abstract

One major limitation of pre-implantation genetic diagnosis (PGD) practice comes from the need to develop single cell PCR protocols. For a disease such as cystic fibrosis (CF), for which almost 1000 mutations have been identified, the development of a mutation based PGD protocol is impracticable. An elegant way to overcome this problem is to set up an indirect diagnosis using polymorphic markers allowing the identification of the pathogenic haplotype instead of the mutation. We present here a new PGD protocol for CF. Our strategy is based on a multiplex fluorescent PCR co-amplifying the DF508 mutation and two CFTR intragenic polymorphic microsatellites (IVS8CA and IVS17bCA). Such an approach is justified since in 91% of the cases at least one partner of the couple carries the DF508 mutation. The use of intragenic markers reduces the risk of misdiagnosis due to meiotic recombination. In 97% of the single lymphoblasts (151/155) tested a PCR signal was obtained. A complete haplotyping was achieved in 137/151 (91%) lymphoblasts and a 6% rate of allele drop out (ADO) was observed. Three cases were performed. Case one was at risk of transmitting mutations DF508 and R1162X, case 2 DF508 and R1066C and case 3 DF508 and 1341+1A. Considering these three cases and the re-analysis of the affected embryos, we have analysed 62 blastomeres from which we had PCR signal for 58 (94%) and a complete haplotype for 49 (84%). With the degree of polymorphism of the markers used in this work (48 and 39%) and the fact that we co-amplified the F508 locus our test should be suitable for nearly 80% of the couples requesting PGD for CF. This fluorescent multiplex PCR indirect diagnosis provides also a safer test since it allows the confirmation of the diagnosis, the detection of contamination and could give an indication on the ploidy of the embryos tested.

New tools for preimplantation genetic diagnosis of Huntington's disease and their clinical applications

“…Tubing and embryo biopsy procedures were performed as previously described. 10,11 PCR reactions Cell treatments were as described previously. 10 PCR primer sequences are given in Table 1.…”

“…10,11 PCR reactions Cell treatments were as described previously. 10 PCR primer sequences are given in Table 1. One primer of each pair was fluorescently labelled with the 6-fam, ned or hex fluorochrome (Applied Biosystems, Warrington, UK).…”

“…12 CAG repeats for couples with known genetic status and haplotype analysis for the different microsatellites were performed using procedures previously described. 10 For group I patients, we used a simplex PCR amplifying the CAG repeats. 5 The drawback of this test comes from the difficulty of amplifying the long repeats, meaning that the couple has to carry nonpathological CAG repeats of different sizes.…”

Improving preimplantation genetic diagnosis for Fragile X syndrome: two new powerful single-round multiplex indirect and direct tests

“…Patient ovarian stimulation, oocyte pick up, ICSI and biopsy procedures were carried out as previously described. 10 Two blastomeres were analysed per embryo, except for one cycle (one blastomere).…”

“…Single cells (lymphoblasts or biopsied blastomeres) were handled as previously described 10 and lysed in 2.5 μl lysis buffer (LB: 200 mM KOH, 50 mM DTT) for 10 min at 65°C.…”