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Alexandra Scott, Hanna M

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1 Functional analysis of synonymous substitutions predicted to affect splicing of the CFTR gene 
Alexandra Scott, Hanna M. Petrykowska, Timothy Hefferon, Valer Gotea, Laura Elnitski  Journal of Cystic Fibrosis  Volume 11, Issue 6, Pages (December 2012) DOI: /j.jcf Copyright © Terms and Conditions

2 Fig. 1 Summary of computational predictions for synonymous substitutions identified in CFTR. The Venn diagram depicts counts of mutations determined computationally to affect acceptor and donor SSs through SplicePort scoring (3′SS and 5′SS, respectively), or to affect the splicing regulatory landscape by Skippy (LOR>1.2). Mutations found to affect splicing are shown with a shaded background. Variant 4 (c.2604A>G) is not included here since it does not have a high LOR score, and does not lower scores of SSs in exon 15. Mutation c.1731C>T was not evaluated in this study, but was found to affect skipping by Pagani et al. [14]. ⁎The name for this mutation was determined from its position, but is not currently found in the CF Mutation Database. Journal of Cystic Fibrosis  , DOI: ( /j.jcf ) Copyright © Terms and Conditions

3 Fig. 2 Experimental assessment of splicing interference by five synonymous substitutions. A) Gel with splicing products collected following transient transfection into K562 cells. For each variant [1–5], constructs with the reference (R) and variant (V) alleles were analyzed separately. White arrows point to bands that indicate differential splicing; numbers correspond to band sizes. Results of quantitation with fluorescent labeled PCR primers (conducted in a separate experiment) are shown for variants 2 and 5, revealing different splicing patterns between reference and variant alleles. Exon+ and Exon− correspond to normal splicing (higher band) and cryptic splicing (lower band), respectively. B) Results for variants 2 and 5 were replicated in IB3-1 cells, a cell line more relevant for the phenotype of CF patients. Journal of Cystic Fibrosis  , DOI: ( /j.jcf ) Copyright © Terms and Conditions

4 Fig. 3 Schematic representation of splicing aberrations introduced by variants 2 and 5. A) Exon 8, in which variant 2 is located, is fully spliced with the reference allele (splicing pattern shown above by dashed lines). When the variant “T” allele is present at exon position 46, a total of 71bps are skipped from the 5′ end of the exon (splicing pattern shown below by dotted lines). B) In the case of variant 5 (c.3594G>T), 200bps are skipped from the 3′ end of exon 22. Flanking exons and introns are not shown to scale, whereas the middle exon is shown to scale. Numbers indicate size in bps of each section. The skipped region is shown with shaded background. Journal of Cystic Fibrosis  , DOI: ( /j.jcf ) Copyright © Terms and Conditions

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