Preclinical work-up

Before clinical application, extensive preclinical trials have to be performed on hundreds of single cells isolated under an inverted microscope (lymphocytes, lymphoblasts or fibroblasts), including the development of specific PCR protocols as well as techniques chosen to identify mutations and polymorphic markers. Otherwise, the aim of these preclinical experiments is to evaluate amplification efficiencies and ADO rates for all the DNA sequences to be studied in the procedure.

For most of PGD indications, the development, optimization and validation procedures can take several weeks to several months, according to the genes studied and techniques used. Work-up results on single cells are considered satisfactory for clinical application when they fall within the limits set in the ESHRE PGD consortium’s guidelines : amplification efficiencies may be above 90% and ADO rates may be lower than 10%. However, single cell amplification efficiencies and ADO rates can vary greatly with different cell types, especially in blastomeres from poor embryo quality that frequently exhibit inconclusive results.

ADO is a chance phenomenon, and whatever its cause, all PCR-based genotyping methods should include internal monitoring to detect it. An efficient strategy is to combine the application of direct diagnosis (study of the causative mutation) and indirect genetic diagnosis (study of the haplotypes linked to the mutant and normal alleles). The multiplex PCR technique allows the co-amplification of the disease-specific allele with at least two informative-linked markers, as close as possible to the disease causing mutation, providing back-up diagnostic information.

Because of the large spectrum of molecular defects identified in some genes, the development of mutation-specific PGD protocols is impracticable. Furthermore, some mutations as large rearrangements can not be studied at the single cell level. Therefore, indirect genetic diagnosis based upon the analysis of linked microsatellite markers, may be suitable for all couples requesting PGD for the same indication, whatever the mutations involved, provided that the couples are informative for the different markers.

In the preclinical work up, fresh blood samples from the couple and affected relatives are requested in order to check the previously identified causative mutation(s). Therefore, a familial segregation analysis is performed using the microsatellite markers that have been set up on single cells. Sometimes, some couples ar not informative for the markers tested ; therefore, other polymorphic markers may be tested and multiplex PCR protocols developed on hundreds of single cells.