N1303K (c.3909C>G) Mutation and Splicing: Implication of Its c.[744-33GATT(6); 869+11C>T] Complex Allele in CFTR Exon 7 Aberrant Splicing

Figure 1

Identified complex allele in the Lebanese population and constructions of four hybrid minigenes. (a) The identified CFTR complex allele combining the 744-33GATT(6) polymorphism (intron 6), c.869+11C>T polymorphism (intron 7), and the c.3909C>G mutation (exon 24). (b) The pTBNdeI plasmid used in the hybrid minigene approach. This plasmid contains a reporter gene used to study the mRNA splicing. The reporter gene contains, at the 5′ end, a promoter/enhancer sequence indicated by the arrow. This is followed by α-globin (G1, G2, G3, and G4) and fibronectin (F1 and F2) exons separated by intronic sequences. The fibronectin intronic region, located between F1 and F2, contains a unique NdeI restriction site. Fragments of interest can be inserted in this site. (c) The four inserts used in this study. The impact of the c.[744-33GATT(6); 869+11C>T] complex allele on splicing was evaluated by the use of insert 1 (c.[744-33GATT(7); 869+11C]) and insert 2 (c.[744-33GATT(6); 869+11C>T]). Inserts 1 and 2 contain a part of intron 6 (335 bp), exon 7 (126 bp), and a part of intron 7 (326 bp). The impact of the c.3909C>G mutation on splicing was assessed using inserts 3 (WT) and 4 (c.3909C>G). Inserts 3 and 4 contain intron 23 (100 bp), exon 24 (96 bp), and intron 24 (100 bp). Inserts 1, 2, and 3 are obtained from patients and were inserted in the pTBNdeI plasmid. Plasmid containing insert 4 was obtained by directed mutagenesis realized on the plasmids containing insert 1.

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