Computational protocol: CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients

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Protocol publication

[…] The group of unrelated CF patients (N = 738) had been sequentially examined using IL tests: CFTR_19 and – if two mutations were not detected – CFTR_17TnUpdate. IL reverse dot blot kits rely on the DNA amplification and hybridization with a membrane-immobilized set of probes (Line Probe Assay). The probes correspond to the normal and mutated alleles, covering 36 most frequent European CFTR mutations. The procedure was performed as indicated by the manufacturer (InnoGenetics, Ghent, Belgium; see also ).To comprehensively determine the population frequency profile of CFTR mutations, a follow-up molecular screening was performed. All the patients with only one IL mutation (N = 109) and with no mutation found (N = 126) were analyzed using SSCP/HD (single strand conformational polymorphism and heteroduplex) technique as described in . Briefly, specific primer pairs were designed for each of the 27 CFTR exons, and the 5’ and 3’ UTR regions; to achieve the length of each amplicon <340 bp some exons were analyzed as overlapping fragments. For the SSCP/HD analysis, PCR-amplified segments were denatured and separated in 7 or 8% polyacrylamide (29∶1) in 0.5x or 1xTBE gels (optionally with ∼2 M urea and 10% glycerol) were run at 8–10 W for 20–40 h at RT or 4°C. Primer sequences, PCR conditions and detailed conditions used to separate each of the analyzed fragments are available from the authors upon request.Nucleotide changes underlying all the detected SSCP/HD migration variants were resolved by direct sequencing of the PCR products (BigDyeTerminator v3.1 on an ABI Prism 3130XL Analyzer, Applied Biosystems); trace files were checked and edited using FinchTV1.3.1. (Geospiza Inc.). Sequences were evaluated manually using Chromas 1.45 software and FASTA sequence comparison algorithm (, 2/3 of the samples, in which one or both mutations remained unidentified, were examined for possible intragenic rearrangements using MLPA technique with SALSA MLPA Kit P091-B1 CFTR (MRC-Holland). This P091-B1 CFTR probemix contains probes for each of the 24 CFTR exons and a second probe for exons 6, 14, 17 and 24. In addition, the probemix P091-B1 contains a mutation specific that detects the wildtype allele of the F508del mutation.The effect of amino acid changes on the protein stability was examined using SNPs3D online software (; the negative SVM value (<−0.5) was assumed to indicate a deleterious effect. […]

Pipeline specifications

Software tools FinchTV, SNPs3D
Application Sanger sequencing
Organisms Homo sapiens
Diseases Cystic Fibrosis, Huntington Disease