Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders Sonia Giordano,

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Molecular and Functional Analysis of the Large 5′ Promoter Region of CFTR Gene Revealed Pathogenic Mutations in CF and CFTR-Related Disorders Sonia Giordano, Felice Amato, Ausilia Elce, Maria Monti, Carla Iannone, Pietro Pucci, Manuela Seia, Adriano Angioni, Federica Zarrilli, Giuseppe Castaldo, Rossella Tomaiuolo The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 331-340 (May 2013) DOI: 10.1016/j.jmoldx.2013.01.001 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Regulatory variants identified in CFTR promoter. Diagram of the chromosome 7q31.2 region containing CFTR and magnification of the sequenced region with annotation of the 23 mutations identified in the present study. The Journal of Molecular Diagnostics 2013 15, 331-340DOI: (10.1016/j.jmoldx.2013.01.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Relative quantification of CFTR mRNA expression in the HepG2, HeLa, PanC-1, and A549 cell lines. The values of CFTR mRNA are reported as means ± SD ratios to GAPDH housekeeping mRNA. The Journal of Molecular Diagnostics 2013 15, 331-340DOI: (10.1016/j.jmoldx.2013.01.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Relative promoter activity of mutations identified in the study. Plasmid constructs expressing firefly luciferase under control of WT or variant CFTR promoters were individually cotransfected with a Renilla luciferase reporter construct (pRL-CMV) in the HeLa, PanC-1, A549, and HepG2 cell lines. Firefly luciferase expression was measured and normalized to that of Renilla luciferase; then, Renilla-normalized promoter activities for mutated constructs were compared with those of the WT construct (indicated by the dashed horizontal line) to provide relative CFTR promoter activity of the variant. The assay was conducted at least in duplicate, and each set of transfections was repeated four times with independently purified plasmid DNA preparations and reaction mix. Data are given as means ± SD. ∗P < 0.001 compared with WT. The Journal of Molecular Diagnostics 2013 15, 331-340DOI: (10.1016/j.jmoldx.2013.01.001) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions