Hotel Estoril Eden, Monte Estoril,
5-8 October 2005



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enomic and Proteomics Approaches to Study the Genetic Disease Cystic Fibrosis

Margarida D. Amaral
University of Lisbon,  Dept of Chemistry and Biochemistry; Centre of Human Genetics, National Institute of Health, Portugal

Cystic Fibrosis (CF), which is the most common lethal genetic disease with a recessive and autossomic pattern among Caucasians, is caused by mutations on CF transmembrane conductance regulator (CFTR) protein. CFTR is an ATP-binding cassette (ABC) transporter expressed at the apical membrane of epithelial cells where it mainly functions as a chloride (Cl-) channel.

Our aim is to identify marker genes showing robust differential expression in human Cystic Fibrosis (CF) vs normal cell lines and native tissues.

To identify genes that are differentially expressed at the level of messenger RNA (mRNA) we used human 40K microarrays (MWG-Biotech) containing approximately 40,000 probes covering essentially the entire human genome. To identify differential gene expression at the protein level, we analysed samples from nasal cells from CF patients and non-CF controls by two-dimensional (2D) gels.

Results from microarrays experiments with two pairs of human CF-related cell lines (CFBE-41o- / 16HBE-14o- and 6CFSMEo- / Calu-3 ) revealed a small number of significantly regulated genes for each CF cell line (approximately 15 upregulated and 55 downregulated), only very few of which have a known role in CF pathology. The 40K array experiment was also performed on pooled nasal epithelial cells obtained by nasal brushing from two groups of CF patients and age/sex-matched non-CF controls, for in vivo validation of data obtained in cell lines.

Comparison of all data sets (microarrays in cell lines and tissues and proteomics) is currently being undertaken with the aim of generating a short-list (5-10) of very strongly regulated CF marker genes, with priority given to the genes regulated in vivo.

Work supported by EU Contract # QLK3-CT-2001-01982 (CF-CHIP) and FCT 2001 research grants: POCTI/1999/MGI/35737 and POCTI/MGI/40848/.