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 DeltaF508 as a model mutation. To this end, oligonucleotide probes representing both the normal and DeltaF508 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 DeltaF508 genotypes assigned from the fluorescence bound to each allelic probe. The performance of the array was evaluated by its ability to detect DeltaF508 mutations in target DNA. Strong binding of the target to the probes was observed, allowing the expected DeltaF508 genotypes to be assigned. The reliability and accuracy of the microarray diagnosis for DeltaF508 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 DeltaF508 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 DeltaF508 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.

Effect of growth factors on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in Rat-1 fibroblasts.

The activation of glycolytic flux is a biochemical characteristic of growing cells. Several reports have demonstrated the role of fructose 2,6-bisphosphate in this process. In this paper we show that the levels of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (6PF2K/Fru-2,6-P2ase) mRNA are modulated in response to serum and growth factors and this effect is due to regulation of its transcription rate. The modulation of the expression of this enzyme by growth factors differs according their mitogenic effect; both lysophosphatidic acid and epidermal growth factor, when added alone, increased the mRNA levels, but endothelin had no effect. Furthermore, cAMP, which acts as an antimitogenic signal in Rat-1 fibroblasts, produced a decrease in 6PF2K/Fru-2, 6-P2ase mRNA and inhibited the effects of lysophosphatidic acid and epidermal growth factor on 6PF2K/Fru-2,6-P2ase expression. PD 098059, a specific inhibitor of the activation of the mitogen-activated protein kinase, was able to prevent the effect of EGF on 6PF2K/Fru-2, 6-P2ase gene expression. These results imply that activation of mitogen-activated protein kinase is required for the stimulation of the transcription of 6PF2K/Fru-2,6-P2ase by EGF.

Hepatocyte growth factor and transforming growth factor beta regulate 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression in rat hepatocyte primary cultures.

Hepatocyte growth factor (HGF) and transforming growth factor beta (TGF-beta) are believed to be of major importance for hepatic regeneration after liver damage. We have studied the effect of these growth factors on fructose 2,6-bisphosphate (Fru-2,6-P2) levels and the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-BPase) in rat hepatocyte primary cultures. Our results demonstrate that HGF activates the expression of the 6PF2K/Fru-2,6-BPase gene by increasing the levels of its mRNA. As a consequence of this activation, the amount of 6PF2K/Fru-2,6-BPase protein and 6-phosphofructo-2-kinase activity increased, which was reflected by a rise in Fru-2,6-P2 levels. In contrast, TGF-beta decreased the levels of 6PF2K/Fru-2,6-BPase mRNA, which led to a decrease in the amount of 6PF2K/Fru-2,6-BPase protein and Fru-2,6-P2. The different actions of HGF and TGF-beta on 6PF2K/Fru-2,6-BPase gene expression are concomitant with their effect on cell proliferation. Here we show that, in the absence of hormones, primary cultures of hepatocytes express the F-type isoenzyme. In addition, HGF increases the expression of this isoenzyme, and dexamethasone activates the L-type isoform. HGF and TGF-beta were able to inhibit this activation.