MOLECULAR PATHOLOGY OF PRE-mRNA SPLICING. DIAGNOSTIC AND MECHANISTIC ASPECTS

It has been recently shown from the sequencing of human genome that more then one third of the human genes can produce several different proteins using the so called "alternative" splicing process, a strictly controlled basic mechanism involved in regulation of gene expression and genome diversity. Due to its intrinsic complexity the splicing process is susceptible to pathological derangement. In fact about 40 % of human genomic DNA mutations causing human hereditary diseases affect the process of RNA splicing. However the association of DNA mutations with splicing in several cases is not obvious, mainly because our knowledge of the basic mechanism controlling RNA splicing is limited. In particular in blanket genomic DNA screening of disease-associated genes several "orphan" splicing mutations are reported whose their exact role in disease is not clear. Frequently these mutations are found both in patients with the disease or in non affected individuals, or are found in patients with variable disease severity. In this proposal we intend to develop a system which permit the identification and the study of these "orphan" splicing mutations. The basic regulatory mechanism involved in some splicing mutations already identified in patients with Cystic Fibrosis, Ataxia Teleangectasia and Homocystinuria will be also evaluated in detail. The characterization and analysis of those genetic elements associated to pathological splicing is of utmost importance for the development of targeted therapeutic strategies.