High sensivity and specificity of denaturing high performance liquid chromatography (DHPLC) for mutation analysis of the FBN1 gene in patients with Marfan syndrome.

High sensivity and specificity of denaturing high performance liquid chromatography (DHPLC) for mutation analysis of the FBN1 gene in patients with Marfan syndrome. A. Mori 1 , S. Ansaldi 1 , M. Grasso 1 , A. Pilotto 1 , C. Lucchelli 1 , L. Lanzarini 2 , M. Diegoli 3 , L. Tavazzi 2 , E. Arbustini 1 ; 1 Cardiovascular Pathol. and Molec. Diagn. - Res.Transplantation Lab. , IRCCS Policlinico S.Matteo, Pavia, Italy, 2 Cardiology Division, IRCCS Policlinico S.Matteo, Pavia, Italy, 3 Department of Pathology - University of Pavia, Pavia, Italy. Marfan syndrome is an autosomal dominant inherited disorder of the connective tissue that principally involves the cardiovascular,ocular and skeletal systems. The incidence is estimated to be 1:5000, with 25% sporadic cases. The leading cause of death is related to the cardiovascular involvement, in particular aortic root dilatation and rupture. The disease is caused by alteration in FBN1 gene (65 exons, located at 15q15-q21.1). Causal mutations are scattered throughout the gene and are largely unique to individual families. The FBN1 gene was analyzed in 29 unrelated patients suspected to be affected by Marfan syndrome. To develop an efficient and faster method capable of identify all possible mutations in this gene, we introduced DHPLC technology in the analysis of 25 exons in which mutations recur. We first analysed the FBN1 exons and exon- flanking non coding regions gene coding regions with automated sequencing of all 65 exons (ABI PE- 373 DNA Sequencer) to identify mutations and polymorphisms. Then, DHPLC analysis was carried out on the WaveTM DNA Fragment Analysis System (Transgenomic, Cheshire, UK). DNA fragment elution profiles were displayed using the Transgenomic WAVEMAKER-TM software. Chromatograms were analysed and amplified fragments showing alterations were re-confirmed by automated sequencing. Overall, by direct sequencing we indentify 19 variants (14 in coding regions and 5 in intronic sequences). A corresponding number of heteroduplex 297 profiles was detected with DHPLC with 100% correspondence to the variant-containing regions previously identified by direct sequencing. Our results confirms that DHPLC is a highly sensitive and specific technology for DNA sequence variant detection.