MOLECULAR EPIDEMIOLOGY OF CYSTIC-FIBROSIS MUTATIONS AND HAPLOTYPES INSOUTHERN ITALY EVALUATED WITH AN IMPROVED SEMIAUTOMATED ROBOTIC PROCEDURE

We screened for 22 cystic fibrosis (CF) mutations in DNA from a first cohort of 69 CF patients from southern Italy using a semiautomated all ele specific oligonucleotide (ASO) dot blot procedure based on two mul tiplex PCR amplifications. Seven mutations (Delta F508, N1303K, G542X, 1717-1 G-->A, W1282X, 1148T, and R553X) identified 77.6% of CF chromo somes. Detection reached 79.8% with the 2183 AA-->G mutation analysed with the restriction generating PCR method. Thus, we included the 2183 AA-->G mutation in the ASO protocol and set up the conditions to ampl ify the gene regions that include the eight mutations in a single mult iplex PCR reaction. With this method we tested the DNA of the first co hort of 69 CF patients, a second cohort of 63 CF patients, and 300 car rier relatives; we also performed 12 prenatal diagnoses. The results f rom the 132 CF patients showed differences in the distribution of CF m utations between the south and north of Italy. The XV2c, KM19, and int ron 8 VNDR haplotypes suggested the presence, in CF chromosomes bearin g undetected mutations, of a limited number of unknown mutations typic al of southern Italy. Finally, for six of the eight mutations, we comp ared the ASO procedure with the methods based on restriction enzymes; the results obtained with the two procedures were identical for all th e 57 chromosomes compared.