PURPOSE: Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. METHODS: We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. RESULTS: Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. CONCLUSION: Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.
PURPOSE: Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. METHODS: We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. RESULTS: Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. CONCLUSION: Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.
Authors: William Brockman; Pablo Alvarez; Sarah Young; Manuel Garber; Georgia Giannoukos; William L Lee; Carsten Russ; Eric S Lander; Chad Nusbaum; David B Jaffe Journal: Genome Res Date: 2008-01-22 Impact factor: 9.043
Authors: Neeraj Sharma; Patrick R Sosnay; Anabela S Ramalho; Christopher Douville; Arianna Franca; Laura B Gottschalk; Jeenah Park; Melissa Lee; Briana Vecchio-Pagan; Karen S Raraigh; Margarida D Amaral; Rachel Karchin; Garry R Cutting Journal: Hum Mutat Date: 2014-09-10 Impact factor: 4.878
Authors: Ahmad N Abou Tayoun; Christopher D Tunkey; Trevor J Pugh; Tristen Ross; Minita Shah; Clarence C Lee; Timothy T Harkins; Wendy A Wells; Laura J Tafe; Christopher I Amos; Gregory J Tsongalis Journal: Clin Chem Date: 2013-06-17 Impact factor: 8.327
Authors: Fred A Wright; Lisa J Strug; Vishal K Doshi; Clayton W Commander; Scott M Blackman; Lei Sun; Yves Berthiaume; David Cutler; Andreea Cojocaru; J Michael Collaco; Mary Corey; Ruslan Dorfman; Katrina Goddard; Deanna Green; Jack W Kent; Ethan M Lange; Seunggeun Lee; Weili Li; Jingchun Luo; Gregory M Mayhew; Kathleen M Naughton; Rhonda G Pace; Peter Paré; Johanna M Rommens; Andrew Sandford; Jaclyn R Stonebraker; Wei Sun; Chelsea Taylor; Lori L Vanscoy; Fei Zou; John Blangero; Julian Zielenski; Wanda K O'Neal; Mitchell L Drumm; Peter R Durie; Michael R Knowles; Garry R Cutting Journal: Nat Genet Date: 2011-05-22 Impact factor: 38.330
Authors: YuanYuan Gu; Isaac T W Harley; Lindsay B Henderson; Bruce J Aronow; Ilja Vietor; Lukas A Huber; John B Harley; Jeffrey R Kilpatrick; Carl D Langefeld; Adrienne H Williams; Anil G Jegga; Jing Chen; Marsha Wills-Karp; S Hasan Arshad; Susan L Ewart; Chloe L Thio; Leah M Flick; Marie-Dominique Filippi; H Leighton Grimes; Mitchell L Drumm; Garry R Cutting; Michael R Knowles; Christopher L Karp Journal: Nature Date: 2009-02-25 Impact factor: 49.962
Authors: Melissa Lee; Patrick Roos; Neeraj Sharma; Melis Atalar; Taylor A Evans; Matthew J Pellicore; Emily Davis; Anh-Thu N Lam; Susan E Stanley; Sara E Khalil; George M Solomon; Doug Walker; Karen S Raraigh; Briana Vecchio-Pagan; Mary Armanios; Garry R Cutting Journal: Am J Hum Genet Date: 2017-05-04 Impact factor: 11.025
Authors: Martina I Lefterova; Peidong Shen; Justin I Odegaard; Eula Fung; Tsoyu Chiang; Gang Peng; Ronald W Davis; Wenyi Wang; Martin Kharrazi; Iris Schrijver; Curt Scharfe Journal: J Mol Diagn Date: 2016-02-01 Impact factor: 5.568