Towards preimplantation diagnosis of cystic fibrosis using microarrays

Cystic fibrosis (CF) is a common indication for preimplantation genetic diagnosis (PGD). A 3-bp deletion (DeltaF508) in the cftr gene, which accounts for approximately 80% of all CF mutations in the Caucasian population, is normally diagnosed in IVF embryos using fluorescent PCR (FL-PCR) and allelic sizing. In PGD, the possibility of using microarrays for genetic diagnosis is largely unexplored. Therefore, the aim of this study was to prove the diagnostic capability of microarrays for PGD. using AF508 as a model mutation. To this end, oligonucleotide probes representing both the normal and AF508 disease alleles were used to construct a single microarray platform. Target DNA, which was generated by PCR and labelled with the fluorescent dye Cy3, was hybridized to the array and the AF508 genotypes assigned from the fluorescence bound to each allelic probe. The performance of the array was evaluated by its ability to detect AF508 mutations in target DNA. Strong binding of the target to the probes was observed, allowing the expected AF508 genotypes to be assigned. The reliability and accuracy of the microarray diagnosis for AF508 was blindly assessed on 10 samples with either a homozygous normal, homozygous affected or heterozygous genotype. All samples were correctly genotyped. In addition, PCR products from a previous PGD case involving AF508 were re-evaluated on the array, with results in complete concordance with allelic sizing methods used to make the original diagnosis. Together, these findings prove the concept that the AF508 mutation of CF can be reliably and accurately diagnosed at the single cell level using microarray analysis. The availability of more cost-effective array platforms comprising mutation probes for common single-gene disorders and a reliable method of whole genome amplification (WGA) would allow PGD to be offered to the majority of PGD patients with minimal or no change to methodology.