Transcript analysis of CFTR frameshift mutations in lymphocytes using the reverse transcription-polymerase chain...

mRNA transcripts of the cystic fibrosis transmembrane conductance regulator (CFTR) gene were analyzed from lymphocytes of two cystic fibrosis compound heterozygotes (394delTT/3195del6 and 1215delG/ 2423delG), of five related carriers heterozygous for one of these mutations, and of five normal individuals. After reverse transcription of total RNA and amplification by the polymerase chain reaction, fragments were investigated by sequencing and by the protein truncation test (PTT). The three frameshift mutants were correctly detected by PTT, as they introduced a premature termination codon resulting in shortened protein products. The PTT approach thus provides a simple and reliable alternative method for detecting frameshift, nonsense, or splice site mutations, and for ascertaining their putative effect on the reading frame of the mRNA. In addition, we have identified 6 alternatively spliced forms of CFTR mRNA, two of which (transcripts lacking 4 + 5 or 17B) have not been described previously.