S2 1. Genetics 5 CFTR mutations detection in patients with cystic fibrosis from Romania using Elucigene CF29 kit L. Tamas 1 , I. Popa 2 , L. Pop 2 , I.M. Ciuca Popa 2 , Z. Popa 3 , A. Anghel 1 , M. Motoc 1 . 1 Biochemistry Department, University of Medicine and Pharmacy Victor Babes, Timisoara, Romania; 2 Pediatric IInd Department, University of Medicine and Pharmacy Victor Babes, Timisoara, Romania; 3 National Cystic Fibrosis Center, Clinical County Hospital, Timisoara, Romania Introduction: Cystic fibrosis is the most common autosomal recessive disease in Caucasian populations. The aim of this study was to perform genetic analysis and to identify CFTR mutations in patients from the National Center of Cystic Fibrosis from Timisoara. Material and Methods: Based on clinical findings and sweat test, 46 patients (92 alleles) were selected for performing genetic testing. 29 mutations were investi- gated and the detection was performed using the Elucigene CF29 kit which detects point mutations or small deletions in deoxyribonucleic acid using a method based on ARMS (amplification refractory mutation system) allele specific amplification technology. Genomic DNA isolated from peripheral blood samples was amplified by PCR and the PCR products were visualized on a UV transiluminator after electrophoresis on agarose gel and staining with ethidium bromide. Results: 5 mutations were identified: DF508, G542X, N1303K, I148T and 621 + 1G>T. 39 alleles (42.4%) remained unidentified. The frequency of CFTR mutations was calculated. Conclusions: In Romanian population the most frequent CFTR mutations are DF508 (46.8%) and G542X (6.52%), these two mutations having the highest frequency in Europe. Many mutations remain unidentified by investigating only the 29 mutations panel from Elucigene CF29 kit. The great number of mutations and polymorphisms (25) identified up to date by this study and previous studies, reflects the genetic heterogeneity of Romanian population. 6* Large CFTR gene rearrangements in Italian population V. Paracchini 1 , L. Porcaro 1 , L. Costantino 1 , P. Capasso 1 , D. Degiorgio 1 , D. Coviello 1 , C. Colombo 2 , L. Claut 2 , R. Padoan 3 , M. Seia 1 . 1 Molecular Genetics, IRCCS Fondazione Policlinico, Mangiagalli, Regina Elena, Milan, Italy; 2 Cystic Fibrosis Center, IRCCS Fondazione Policlinico, Mangiagalli, Regina Elena, Milan, Italy; 3 Cystic Fibrosis Support Center, A.O. Spedali Civili, Brescia, Italy Cystic fibrosis (CF) is mainly caused by small molecular lesions of the CFTR gene, but a number of CF patients still remain uncharacterized using PCR-techniques. The CF mutation database (www.genet.sickkids.on.ca) lists more than 25 large deletions encompassing one or more exons, and a single case of duplication. We report here the results of our screening for CTFR gene rearrangements, performed on Italian CF patients. A sample of 689 unrelated Italian patients (for a total of 1378 alleles), followed at CF Center of Regione Lombardia (Milan, Italy), was collected. All patients had a classical form of CF with typical pulmonary and gastrointestinal symptoms, and positive sweat test. The Innogenetics assay and the DHPLC screening showed a mutation detection rate of 93%. After these analyses, 100 alleles still remain unidentified (7%). In order to detect the rearrangements in the CFTR gene, the 27 exons were screened in these patients with the MLPA assay based on commercial Kit “SALSA P091 CFTR MALPA Kit”. The MLPA assay was performed for 38 patients (76 alleles). We characterized 10 different deletions for a total of 19 deleted alleles. Taking into account the total number of CF alleles, 25% (19/76) alleles had a large gene deletion. The deletion of exons 22-23 (6/76) is the most frequent in our population. In conclusion, analysis of CF patients performed with MLPA techniques have made possible to reach a 94.4% detection rate, improving the process of carrier detection and genetic counselling in Italian population. Supported by: Ministry of Health and Regione Lombardia. 7 GT repetition on 5T allele M. Maschio, Z. Cannioto, M. Morgutti, F. Poli. Department of Reproduction and Development Sciences, University of Trieste-IRCCS Burlo Garofolo, Trieste, Italy 5T allele over the introne 8 of CFTR gene represents the second most frequent mutation of CFTR gene and it seems to be expressed on 10% of general population. It is characterised by a variable penetrance and its clinical expression is related to be in trans position with a mayor CFTR mutation. In this kind of genetic pattern some clinical features such as different agenesis, nasal poliposis, recurrent pancreatitis, bronchiectasie and neonatal ipertripsinemia seems to be more frequent leading to a typical form of Cystic Fibrosis. We confirm this kind of clinical pattern in two familiar cases; one in a child of 14 years old with an important story of recurrent sinusitis and endoscopic features of nasal poliposis. His sweet test resulted border line (Na 59 mEq/l, Cl 57 mEq/l) with a genetic evaluation demonstrating dele2,3 (21Kb)(GT)11-(GT)12T5 with polyT 5/7 genotype. His father resulted (GT)10T7-(GT)12T5 and appeared symp- tomatic while his mother, whose genetic characterisation is dele2,3 (21Kb)(GT)11- (GT)11T5, presented with an autoimmune disorder such as pernicious anaemia. Surely a bigger sample of data will be necessary to investigate the hypothesis of a genotype-phenotype correlation but the genetic pattern of this family seems to suggest that bad clinical features are related to more GT repetition on 5T allele. (Sequence evaluation of other 5 patients with 5T allele is on stand). 8 DHPLC in Cystic Fibrosis diagnostics in the German population U. Froster, M. Skrzypczak, M. Mierzejewski. Institute of Human Genetics, University of Leipzig, Leipzig, Germany. Cystic Fibrosis is a common autosomal recessive disorder that primarily affects the epithelial cells in the intestine, respiratory system, pancreas, gall bladder and sweat glands. The frequency of CF in the German population is 1:3300. Over 1400 mutations and more than 200 polymorphisms have currently been identified in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that are associated with CF disease. Many without recognised effects on CFTR, the molecular diagnosis can be troublesome. The CF phenotype is highly heteroge- neous among individual patients, even between siblings carrying identical CFTR mutations. The aim of our study was to develop a highly sensitive, automated and economical molecular pre-screening method based on denaturing high performance liquid chromatography (DHPLC) for analyse the complete coding sequence of the CFTR gene (24 exons). Complete automated sequencing, the standard procedure, however, is time consuming, laborious and expensive. We have tested 108 patients from the CF-DNA bank from the Institute of Human Genetics, University of Leipzig (including 21 cases with one molecular genetic confirmed CF mutation as positive control). Following PCR and heteroduplexformation, all 24 exons of the CFTR gene were screened for mutations or polymorphisms using DHPLC. We found 37 nucleotide changes including mutations (8) on 11 alleles and polymorphisms (29, including 6 novel variants) on 430 alleles. DHPLC has proofed again to be important for large gene mutation screening. With this technology efficient and fast screening for point mutations can be applied.