A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals

Given q as the global frequency of the alleles causing a disease, any allel e with a frequency higher than q minus the cumulative frequency of the prev iously known disease-causing mutations (threshold) cannot be the cause of t hat disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whe ther they are the cause of cystic fibrosis. A total of 191 DNA samples fl-o m random individuals from Italy, France, and Spain were investigated by DGG E (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE wer e identified by sequencing. The sample size was sufficient to select essent ially all mutations with a frequency of at least 0.01. A total of 46 mutati ons was detected, 20 of which were missense mutations. Four new mutations w ere identified: 1341+28 C/T, 2082 C/T, L1096R, and I1131V. Thirteen mutatio ns (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2 693 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classi fied as non-CF-causing alleles on the basis of their frequency. The remaini ng mutations have a cumulative frequency far exceeding q; therefore, most o f them cannot be CF-causing mutations. This is the first random survey capa ble of detecting all the polymorphisms of the coding sequence of a gene.