Where not shown, errors bars are smaller than symbol size
Where not shown, errors bars are smaller than symbol size. of internal initiation codons. The N-truncated CFTR generated by this mutation has decreased stability, very low processing efficiency, and drastically reduced function. Analysis of mutants of four methionine codons downstream to M1 (M82, M150, M152, M156) revealed that each of the codons M150/M152/M156 (exon 4) can mediate CFTR alternative translation. Conclusions: The CFTR N-terminus has an important role in avoiding CFTR turnover and in rendering effective its plasma membrane traffic. These data correlate well with the severe clinical phenotype of CF patients bearing the c.120del23 mutation. Key Words:CFTR, Cystic Fibrosis, Mutation, Truncated proteins, Alternative translation, N-terminus == Introduction == Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, MIM# 602421) gene cause Cystic Fibrosis (CF, MIM# 219700) the most common life-threatening autosomal recessive disease among Caucasians [1]. The major CF symptoms are progressive pulmonary dysfunction, pancreatic insufficiency and elevated sweat electrolyte concentration [1]. CFTR protein, a Cl-channel in the apical membrane of epithelial cells comprises several domains, namely: two membrane-spanning domains (MSD1/2), two nucleotide binding domains (NBD1/2) and a regulatory domain (RD), besides the N- and C-termini which are both also functionally relevant [2,3]. Although 1,600 variants of theCFTRgene have been identified [4], F508del, the most frequent mutation (70% of CF chromosomes worldwide) is associated with a severe clinical phenotype. Most F508del-CFTR is retained and degraded at the level of Acemetacin (Emflex) the endoplasmic reticulum (ER) [5]. In Southern Europe the incidence of F508del is lower, in Portugal it only accounts for 60% of CF chromosomes [6]. Hence, there is a higher prevalence of non-F508del alleles among Portuguese CF patients and this necessitates close assessment of other predicted mutations to determine their functional consequences at the cellular and physiological levels. The functional characterization of such naturally occurring CFTR mutations is of high relevance for genetic counselling and clinical prognosis, not just for such gene variants, but also for mutations in other disease-associated genes which perturb the same molecular mechanism. Here, we describe a novel CFTR mutation in exon 1, c.120del23, which we reported previously [4], that was found in two CF patients from the Azores islands, both with F508del in the other allele. As deletion of these 23 nucleotides (120-142) removes the translation initiation codon (133-135), we asked whether this mutation still allows for synthesis of CFTR protein through usage of alternative internal initiation codon(s), MEKK as shown for other CFTR variants [7]. We demonstrate that truncated CFTR proteins are produced and show that M150/M152/M156 (but not M82) can mediate this alternative translation initiation. By functionally characterizing these N-truncated protein(s) we show that at least one of these reaches the cell membrane, but it is unstable and has drastically reduced Cl-channel activity. These findings correlate well with the severe clinical phenotype of the two patients bearing this mutation. == Materials and Methods == == DNA analysis == The 27 CFTR exons, intronic flanking regions, 5 and 3 UTRs and [TG]mTnwere analysed by sequencing as described elsewhere [8]. Acemetacin (Emflex) == Transcript analysis == Following approval of this study by the hospital ethical committee and written informed consent from the parents, nasal epithelial cells from one of the CF patients with the F508del/ c.120del23 genotype were obtained. RNA extraction and cDNA synthesis were performed as described before [9]. Briefly, quantitative (log-phase) RT-PCR analysis of CFTR transcripts from nasal epithelial cells was performed in the region of exons 8-10, using a FAM-labelled primer [10]. After separation in the automatic sequencer, quantitative analysis of RT-PCR products was performed with the GeneScan software as before [10]. == In silico analysis of the potential alternative in-frame initiation codons == The CFTR mRNA sequence (GenBank:M28668, position 1-4575) was assessed for in-frame AUG codons as possible alternative initiation codons using the AUG Acemetacin (Emflex) evaluator and Open-Reading-Frame-Finder online software [11]. == Constructs == By site-directed mutagenesis (QuikChange, Stratagene, La Jolla, CA, USA) c.120del23 was Acemetacin (Emflex) introduced into the wt-CFTR-cDNA in the pNUT mammalian expression vector [12,13] using as primer the patient’s RT-PCR product (positions Acemetacin (Emflex) 96-265). The exon-1-lacking-CFTR-cDNA-construct forin vitrotranslation resulted from cloning (CFTR-cDNA, exons 2-24) into the pSP73 vector. Other CFTR mutants were produced by mutagenesis on c.120del23-CFTR-cDNA-pNUT or on the exon2-24-CFTR-cDNA-pSP73 (primers shown in Table1). == Table 1. == Primers used for site-directed mutagenesis. Primers used to mutagenize the CFTR methionines in positions 82, 150, 152 and 156 into valines in the 120del23-CFTR-pNUT and in the pSP73CFTRex2-24 constructs. == Stable CFTR-expressing BHK cells == The c.120del23-CFTR cDNA pNUT plasmid and its methionine mutants were used (2 g) to.