Influence of the duplication of CFTR exon 9 and its flanking sequences on diagnosis of cystic fibrosis mutations

Abstract

The DNA sequences of seven regions in the human genome were examined for sequence identity with exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is mutated in cystic fibrosis, and its intronic boundaries. These sequences were 95% to 96% homologous. Based on this nucleotide sequence similarity, PCR primers for CFTR exon 9 can potentially anneal with other homologous sequences in the human genome. Sequence alignment analysis of the CFTR exon 9 homologous sequences revealed that five registered mutations in the Cystic Fibrosis Mutation Database may be due to the undesired annealing of primers to a homologous sequence, resulting in inappropriate PCR amplification. For this reason, we propose that certain pseudomutations may result from the similarity between CFTR exon 9 (and its flanking introns) and related sequences in the human genome. Here we show that two mutations previously described in the CFTR database (c.1392 + 6insC; c.1392 + 12G>A) were inappropriately attributed to two individuals who sought carrier testing. A more detailed study by either direct sequencing or subcloning and sequencing of PCR products using specially designed primers revealed that these apparent mutations were not, in fact , present in CFTR. In addition, we present new PCR conditions that permit specific amplification of CFTR exon 9 and its flanking regions. Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology.