Duplex, triplex and quadruplex PCR for the preimplantation genetic diagnosis (PGD) of cystic fibrosis (CF), an exhaustive approach

Abstract

Most of cystic fibrosis (CF) pre-implantation genetic diagnosis (PGD) cases described to date are limited to the detection of DeltaF508. Beside this predominant mutation, over 1000 mutations have been identified, rendering the development of a mutation-based PGD protocol impracticable. This is the reason why we, as well as the others, have developed PGD strategies on the basis of the identification of the pathogenic haplotype instead of the mutation(s). In a previous article, we reported the conditions for the co-amplification of two intragenic polymorphic markers and the F508 locus. Here we describe an improved protocol allowing the additional amplification of two new intragenic markers, intron 1 CA repeat (I1CA) and IVS17bTA. This new protocol should, theoretically, allow us to provide a diagnosis to all couples requiring PGD for CF. Using single lymphoblasts, we have tested four different PCR configurations, including one duplex, two triplexes and one quadruplex PCR. All of them gave results compatible with a clinical application. The number of single lymphoblasts tested in each series varied from 89 to 155. PCR efficiency ranged from 95.4 to 100%. A complete haplotype was achieved for 83.2 to 90.7% of the tested cells, with an allele drop out (ADO) rate comprised between 6.0 and 11.6%. We present here three cases that we performed either with the former test (one case using the triplex PCR combining F508, IVS8CA and IVS17bCA) or with the new one (one case using the triplex combining F508, I1CA and IVS17bTA and one case using a quadruplex test). We obtained two single pregnancies.

Five years’ experience of preimplantation genetic diagnosis in the Parisian Center: outcome of the first 441 started cycles

“…To reduce the risk of allele dropout and amplification failure, PCR conditions had to be optimized. In the future, the incorporation of linked polymorphic microsatellite markers, the use of multiplex PCR (50,51), and the improvement of the accuracy of FISH probes are probably better ways to control misdiagnoses and avoid the removal of two cells. Other new methods for diagnosis are being developed at a fast rate; at the single cell level, real-time PCR, microarrays, and comparative genomic hybridization (CGH) are promising (52).…”

Preimplantation Genetic Diagnosis

“…Following IVF PGD is performed either on polar body biopsy 3 or on the blastomere or blastomeres removed from embryos usually on day 3 after fertilization when the embryos are on the 6–8 cell stage 4 . The following techniques are used for the identification of defects on the embryo: polymerase chain reaction (PCR) 5 and fluorescent in situ hybridization (FISH) 6 …”

Misdiagnosing cystic fibrosis in the era of gene analysis: Case reports

“…was found to carry the exact two CF mutations as her sister, 3849 þ 10 kbc > T and l148T. However, according to the more advanced knowledge of CF genetics in 2003, [8][9][10][11][12] the l148T mutation without 3199-del6 was no longer categorized as CFdisease-contributing.…”

“…was treated as a CF patient for 5 years on the basis of her prenatal diagnosis made in 1999. Only because of her sibling's more advanced gene analysis in 2003, using new technology and knowledge which were not available in 1999, [8][9][10][11][12] did it become clear that A.P. did not have CF.…”