Analysis of CFTR transcripts in nasal epithelial cells and lymphoblasts of a cystic fibrosis patient with 621 + 1G → T and 711 + 1G → T mutations

Abstract

We have analyzed the CFTR mRNA populations in a cystic fibrosis patient heterozygous for the 621 + 1G → T and 711 + 1G → T mutations. Total RNA isolated from the nasal epithelial cells and Epstein-Barr virus-transformed lymphoblasts derived from this patient was reversely transcribed and a region extending from exon 3 to exon 7 of the gene was amplified by the polymerase chain reaction and analyzed. Three abnormal products were identified, suggesting the presence of three aberrant transcripts, and their profiles were identical in both cell types. Two of the products were found to be missing either exon 4 or exon 5 as anticipated from the transcripts from the 621 + 1G → T or 711 + 1G → T alleles, respectively. The third product was apparently derived from an alternatively spliced mRNA species in the absence of the nominal splice site (in 621 + 1G → T) through the use of a cryptic splice donor sequence (TT 528/GTGAGG) within exon 4. Although reading frames appeared to be preserved in all three putative transcripts, significant portions of the presumed first and second transmembrane spans as well as the immediately following cytoplasmic domain would be deleted from the mutant CFTR polypeptides, if made. These observations are consistent with a loss of CFTR function in this cystic fibrosis patient.