The β-thalassemia (thal) and sickle cell syndromes, and cystic fibrosis (CF) are the most common monogenic disorders in Greece, with a carrier incidence of approxi­mately 10 and 5%, respectively. Prevention programs involving prenatal diagnosis (PND) are well established for both diseases, but for some couples pre-implantation genetic diagnosis (PGD) may be a more appropriate op­tion. Pre-implantation genetic diagnosis represents an alternative to PND which avoids terminating affected on-going pregnancies through the selection of unaffected in vitro fertilized (IVF) embryos for transfer. Embryo biopsy and genetic analysis is most commonly performed on blas­tomeres from cleavage stage embryos, and PGD for mono­genic disorders is based on the polymerase chain reaction (PCR). More than a decade of experience has highlighted several inherent pitfalls associated with single-cell DNA amplification, including potential sample contamination, total PCR failure, and allelic drop-out (ADO), all of which should be minimized for any PGD PCR protocol prior to clinical application. In addition, the chosen method must reliably and accurately characterize the ge­notype of the embryo relative to the disorder under inves­tigation. In Greece, both β-thal and CF are caused by a wide range of mutations.
Thus, for each disease, we chose to develop PGD analytical protocols applicable to a wide range of affected genotypes, rather than having to develop case-specific protocols each time. The methods of choice included de­naturing gradient gel electrophoresis (DGGE) and more recently, real-time PCR. The experience accumulated following more than 5 years of clinical application is sum­marized, highlighting approaches for improvement, pitfalls and overall limitations when applying PGD within the context of a preventive genetic service for common reces­sive diseases.
Key words: Cystic fibrosis (CF); Pre-implantation genetic diagnosis (PDG); Thalassemia syndromes; Sickle cell syndromes; Single cell genotyping.