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In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

CFTR transcripts have been qualitatively and quantitatively analysed in nasal epithelial and vas deferens cells by means of reverse transcription PCR. Alternative splicing of exon 9, which is known to occur in nasal epithelial cells, also occurred in vas deferens cells. The extent of this alternative splicing was determined by the allele present at the Tn locus at the end of intron 8 of the CFTR gene. However, the proportion of transcripts lacking exon 9 sequences was increased in vas deferens cells compared with nasal epithelial cells, independent of the Tn genotype. We postulate that this tissue specific difference in the proportion of CFTR transcripts lacking exon 9 sequences could contribute to the tissue specific disease phenotype observed in individuals with congenital bilateral absence of the vas deferens.

Ten polymorphic loci, located in a 1 Mb interval across the cystic fibrosis locus, were analyzed on normal and mutant CFTR genes. A different distribution of haplotype backgrounds among normal and mutant CFTR genes was observed. With exception of the D7S8 locus, the three most common mutations, delta F508, G542X and N1303K, were found on an identical haplotype background. In agreement with the observed linkage equilibrium between the Q1463Q and D7S8 loci, both alleles at the D7S8 locus were found on delta F508 CFTR genes. However, the G542X and N1303K mutations, which have been estimated to be at least 35000 years old, were found to be associated with a single allele at the D7S8 locus. Absence of recombination between the D7S8 and Q1463Q loci was also observed on normal CFTR genes with this haplotype background. At the Tn locus in intron 8, allele 9 known to result in very efficient splicing was associated with the most frequent mutations. At the M470V locus, located in a conserved region of the first nucleotide binding fold, the amino acid methionine was found to be associated with the frequent mutations, in particular with mutations located in one of the two nucleotide binding folds which are generally known as severe mutations with regard to exocrine pancreatic function. On mutant CFTR gene, this locus was in complete association with the centromeric D9 locus, in the absence of a complete association with the intervening loci.(ABSTRACT TRUNCATED AT 250 WORDS)