Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies


Autoři: Anya T. Joynt aff001;  Taylor A. Evans aff001;  Matthew J. Pellicore aff001;  Emily F. Davis-Marcisak aff001;  Melis A. Aksit aff001;  Alice C. Eastman aff001;  Shivani U. Patel aff002;  Kathleen C. Paul aff001;  Derek L. Osorio aff001;  Alyssa D. Bowling aff001;  Calvin U. Cotton aff003;  Karen S. Raraigh aff001;  Natalie E. West aff002;  Christian A. Merlo aff002;  Garry R. Cutting aff001;  Neeraj Sharma aff001
Působiště autorů: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America aff001;  Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins Hospital, Baltimore, Maryland, United States of America aff002;  Departments of Pediatrics, Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, United States of America aff003
Vyšlo v časopise: Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies. PLoS Genet 16(10): e1009100. doi:10.1371/journal.pgen.1009100
Kategorie: Research Article
doi: 10.1371/journal.pgen.1009100

Souhrn

Elucidating the functional consequence of molecular defects underlying genetic diseases enables appropriate design of therapeutic options. Treatment of cystic fibrosis (CF) is an exemplar of this paradigm as the development of CFTR modulator therapies has allowed for targeted and effective treatment of individuals harboring specific genetic variants. However, the mechanism of these drugs limits effectiveness to particular classes of variants that allow production of CFTR protein. Thus, assessment of the molecular mechanism of individual variants is imperative for proper assignment of these precision therapies. This is particularly important when considering variants that affect pre-mRNA splicing, thus limiting success of the existing protein-targeted therapies. Variants affecting splicing can occur throughout exons and introns and the complexity of the process of splicing lends itself to a variety of outcomes, both at the RNA and protein levels, further complicating assessment of disease liability and modulator response. To investigate the scope of this challenge, we evaluated splicing and downstream effects of 52 naturally occurring CFTR variants (exonic = 15, intronic = 37). Expression of constructs containing select CFTR intronic sequences and complete CFTR exonic sequences in cell line models allowed for assessment of RNA and protein-level effects on an allele by allele basis. Characterization of primary nasal epithelial cells obtained from individuals harboring splice variants corroborated in vitro data. Notably, we identified exonic variants that result in complete missplicing and thus a lack of modulator response (e.g. c.2908G>A, c.523A>G), as well as intronic variants that respond to modulators due to the presence of residual normally spliced transcript (e.g. c.4242+2T>C, c.3717+40A>G). Overall, our data reveals diverse molecular outcomes amongst both exonic and intronic variants emphasizing the need to delineate RNA, protein, and functional effects of each variant in order to accurately assign precision therapies.

Klíčová slova:

Amino acid substitution – Electromyography – Chlorides – Introns – Messenger RNA – Nucleotides – Reverse transcriptase-polymerase chain reaction – RNA splicing


Zdroje

1. Shapiro MB, Senapathy P. RNA splice junctions of different classes of eukaryotes:sequence statistics and functional implications in gene expression. Nucleic Acids Res. 1987;15:7155–7175. doi: 10.1093/nar/15.17.7155 3658675

2. Watakabe A, Tanaka K, Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993;7(3):407–418. doi: 10.1101/gad.7.3.407 8449402.

3. Caputi M, Casari G, Guenzi S, Tagliabue R, Sidoli A, Melo CA, et al. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon. Nucleic Acids Res. 1994;22(6):1018–1022. doi: 10.1093/nar/22.6.1018 8152907; PubMed Central PMCID: PMC307924.

4. Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, et al. Deciphering the splicing code. Nature. 2010;465(7294):53–59. doi: 10.1038/nature09000 20445623.

5. Rivas MA, Pirinen M, Conrad DF, Lek M, Tsang EK, Karczewski KJ, et al. Human genomics. Effect of predicted protein-truncating genetic variants on the human transcriptome. Science. 2015;348(6235):666–669. doi: 10.1126/science.1261877 25954003; PubMed Central PMCID: PMC4537935.

6. Zhang S, Samocha KE, Rivas MA, Karczewski KJ, Daly E, Schmandt B, et al. Base-specific mutational intolerance near splice sites clarifies the role of nonessential splice nucleotides. Genome Res. 2018;28(7):968–974. doi: 10.1101/gr.231902.117 29858273; PubMed Central PMCID: PMC6028136.

7. Wieringa B, Meyer F, Reiser J, Weissmann C. Unusual splice sites revealed by mutagenic inactivation of an authentic splice site of the rabbit beta-globin gene. Nature. 1983;301(5895):38–43. doi: 10.1038/301038a0 6296682.

8. Lin JH, Tang XY, Boulling A, Zou WB, Masson E, Fichou Y, et al. First estimate of the scale of canonical 5' splice site GT>GC variants capable of generating wild-type transcripts. Hum Mutat. 2019;40(10):1856–1873. doi: 10.1002/humu.23821 31131953.

9. Lin JH, Masson E, Boulling A, Hayden M, Cooper DN, Ferec C, et al. 5' splice site GC>GT and GT>GC variants differ markedly in terms of their functionality and pathogenicity. Hum Mutat. 2020. doi: 10.1002/humu.24029 32369867.

10. Teraoka SN, Telatar M, Becker-Catania S, Liang T, Onengut S, Tolun A, et al. Splicing defects in the ataxia-telangiectasia gene, ATM: underlying mutations and consequences. Am J Hum Genet. 1999;64(6):1617–1631. doi: 10.1086/302418 10330348; PubMed Central PMCID: PMC1377904.

11. Ars E, Kruyer H, Morell M, Pros E, Serra E, Ravella A, et al. Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet. 2003;40(6):e82. doi: 10.1136/jmg.40.6.e82 12807981; PubMed Central PMCID: PMC1735494.

12. Krawczak M, Thomas NS, Hundrieser B, Mort M, Wittig M, Hampe J, et al. Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat. 2007;28(2):150–158. doi: 10.1002/humu.20400 17001642

13. Orkin SH, Kazazian HH Jr., The mutation and polymorphism of the human beta-globin gene and its surrounding DNA. Annu Rev Genet. 1984;18:131–171. doi: 10.1146/annurev.ge.18.120184.001023 6084979.

14. Marvit J, DiLella AG, Brayton K, Ledley FD, Robson KJ, Woo SL. GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria. Nucleic Acids Res. 1987;15(14):5613–5628. doi: 10.1093/nar/15.14.5613 3615198; PubMed Central PMCID: PMC306010.

15. Takeshima Y, Yagi M, Okizuka Y, Awano H, Zhang Z, Yamauchi Y, et al. Mutation spectrum of the dystrophin gene in 442 Duchenne/Becker muscular dystrophy cases from one Japanese referral center. J Hum Genet. 2010;55(6):379–388. doi: 10.1038/jhg.2010.49 20485447.

16. Eriksson M, Brown WT, Gordon LB, Glynn MW, Singer J, Scott L, et al. Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature. 2003;423(6937):293–298. doi: 10.1038/nature01629 12714972.

17. Chu C-S, Trapnell BC, Murtagh JJ, Moss J, Dalemans W, Jallat S, et al. Variable deletion of exon 9 coding sequences in cystic fibrosis transmembrane conductance regulator gene mRNA transcripts in normal bronchial epithelium. EMBO J. 1991;10:1355–1363. 1709095

18. Highsmith WE, Burch LH, Zhou Z, Olsen JC, Boat TE, Spock A, et al. A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations. N Engl J Med. 1994;331(15):974–980. doi: 10.1056/NEJM199410133311503 7521937

19. Chillon M, Dork T, Casals T, Gimenez J, Fonknechten N, Will K, et al. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA—>G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype. Am J Hum Genet. 1995;56(3):623–629. 7534040

20. Highsmith WE Jr., Burch LH, Zhou Z, Olsen JC, Strong TV, Smith T, et al. Identification of a splice site mutation (2789 +5 G > A) associated with small amounts of normal CFTR mRNA and mild cystic fibrosis. Hum Mutat. 1997;9(4):332–338. doi: 10.1002/(SICI)1098-1004(1997)9:4<332::AID-HUMU5>3.0.CO;2-7 9101293.

21. Cutting GR. Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet. 2015;16(1):45–56. Epub 2014/11/18. doi: 10.1038/nrg3849 25404111; PubMed Central PMCID: PMC4364438.

22. McCague AF, Raraigh KS, Pellicore MJ, Davis-Marcisak EF, Evans TA, Han ST, et al. Correlating Cystic Fibrosis Transmembrane Conductance Regulator Function with Clinical Features to Inform Precision Treatment of Cystic Fibrosis. Am J Respir Crit Care Med. 2019;199(9):1116–1126. doi: 10.1164/rccm.201901-0145OC 30888834; PubMed Central PMCID: PMC6515867.

23. Ramsey BW, Davies J, McElvaney NG, Tullis E, Bell SC, Drevinek P, et al. A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N Engl J Med. 2011;365(18):1663–1672. doi: 10.1056/NEJMoa1105185 22047557

24. Rowe SM, Daines C, Ringshausen FC, Kerem E, Wilson J, Tullis E, et al. Tezacaftor-Ivacaftor in Residual-Function Heterozygotes with Cystic Fibrosis. N Engl J Med. 2017;377(21):2024–2035. Epub 2017/11/03. doi: 10.1056/NEJMoa1709847 29099333.

25. Durmowicz T, Pacanowski M. Novel Approach Allows Expansion of Indication for Cystic Fibrosis Drug https://www.fda.gov2017 [updated May 18, 2017; cited 2017 June 29].

26. Middleton PG, Mall MA, Drevinek P, Lands LC, McKone EF, Polineni D, et al. Elexacaftor-Tezacaftor-Ivacaftor for Cystic Fibrosis with a Single Phe508del Allele. N Engl J Med. 2019;381(19):1809–1819. doi: 10.1056/NEJMoa1908639 31697873.

27. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, et al. Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A. 2009;106(44):18825–18830. doi: 10.1073/pnas.0904709106 19846789

28. Yu H, Burton B, Huang CJ, Worley J, Cao D, Johnson JP Jr., et al. Ivacaftor potentiation of multiple CFTR channels with gating mutations. J Cyst Fibros. 2012;11(3):237–245. S1569-1993(11)00251-7 [pii]; doi: 10.1016/j.jcf.2011.12.005 22293084

29. Van Goor F, Yu H, Burton B, Hoffman BJ. Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function. J Cyst Fibros. 2014;13(1):29–36. S1569-1993(13)00113-6 [pii]; doi: 10.1016/j.jcf.2013.06.008 23891399

30. Raraigh KS, Han ST, Davis E, Evans TA, Pellicore MJ, McCague AF, et al. Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity. Am J Hum Genet. 2018. Epub 2018/05/17. doi: 10.1016/j.ajhg.2018.04.003 29805046.

31. Scott A, Petrykowska HM, Hefferon T, Gotea V, Elnitski L. Functional analysis of synonymous substitutions predicted to affect splicing of the CFTR gene. J Cyst Fibros. 2012;11(6):511–517. doi: 10.1016/j.jcf.2012.04.009 22591852; PubMed Central PMCID: PMC3440543.

32. Aissat A, de Becdelievre A, Golmard L, Vasseur C, Costa C, Chaoui A, et al. Combined computational-experimental analyses of CFTR exon strength uncover predictability of exon-skipping level. Hum Mutat. 2013;34(6):873–881. doi: 10.1002/humu.22300 23420618

33. Raynal C, Baux D, Theze C, Bareil C, Taulan M, Roux AF, et al. A Classification Model Relative to Splicing for Variants of Unknown Clinical Significance: Application to the CFTR Gene. Hum Mutat. 2013. doi: 10.1002/humu.22291 23381846

34. Sosnay PR, Siklosi KR, Van Goor F, Kaniecki K, Yu H, Sharma N, et al. Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene. Nat Genet. 2013;45(10):1160–1167. Epub 2013/08/25. doi: 10.1038/ng.2745 23974870; PubMed Central PMCID: PMC3874936.

35. Masvidal L, Igreja S, Ramos MD, Alvarez A, De Gracia J, Ramalho A, et al. Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis. Eur J Hum Genet. 2013. ejhg2013238 [pii]; doi: 10.1038/ejhg.2013.238 24129438

36. Sharma N, Sosnay PR, Ramalho AS, Douville C, Franca A, Gottschalk LB, et al. Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions. Hum Mutat. 2014;35(10):1249–1259. doi: 10.1002/humu.22624 25066652

37. Lee M, Roos P, Sharma N, Atalar M, Evans TA, Pellicore MJ, et al. Systematic Computational Identification of Variants That Activate Exonic and Intronic Cryptic Splice Sites. Am J Hum Genet. 2017;100(5):751–765. doi: 10.1016/j.ajhg.2017.04.001 28475858; PubMed Central PMCID: PMC5420354.

38. Sharma N, Evans TA, Pellicore MJ, Davis E, Aksit MA, McCague AF, et al. Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis. PLoS Genet. 2018;14(11):e1007723. doi: 10.1371/journal.pgen.1007723 30444886.

39. Aksit MA, Bowling AD, Evans TA, Joynt AT, Osorio D, Patel S, et al. Decreased mRNA and protein stability of W1282X limits response to modulator therapy. J Cyst Fibros. 2019. doi: 10.1016/j.jcf.2019.02.009 30803905.

40. Pandey KR, Maden N, Poudel B, Pradhananga S, Sharma AK. The curation of genetic variants: difficulties and possible solutions. Genomics Proteomics Bioinformatics. 2012;10(6):317–325. doi: 10.1016/j.gpb.2012.06.006 23317699; PubMed Central PMCID: PMC5054708.

41. Bareil C, Bergougnoux A. CFTR gene variants, epidemiology and molecular pathology. Arch Pediatr. 2020;27 Suppl 1:eS8–eS12. doi: 10.1016/S0929-693X(20)30044-0 32172939.

42. den Dunnen JT, Dalgleish R, Maglott DR, Hart RK, Greenblatt MS, McGowan-Jordan J, et al. HGVS Recommendations for the Description of Sequence Variants: 2016 Update. Human Mutation. 2016;37(6):564–569. doi: 10.1002/humu.22981 26931183

43. Jaganathan K, Kyriazopoulou Panagiotopoulou S, McRae JF, Darbandi SF, Knowles D, Li YI, et al. Predicting Splicing from Primary Sequence with Deep Learning. Cell. 2019;176(3):535–548 e524. doi: 10.1016/j.cell.2018.12.015 30661751.

44. Molinski SV, Gonska T, Huan LJ, Baskin B, Janahi IA, Ray PN, et al. Genetic, cell biological, and clinical interrogation of the CFTR mutation c.3700 A>G (p.Ile1234Val) informs strategies for future medical intervention. Genet Med. 2014;16(8):625–632. doi: 10.1038/gim.2014.4 24556927.

45. De Boeck K, Munck A, Walker S, Faro A, Hiatt P, Gilmartin G, et al. Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation. J Cyst Fibros. 2014;13(6):674–680. S1569-1993(14)00216-1 [pii]; doi: 10.1016/j.jcf.2014.09.005 25266159

46. Amato F, Scudieri P, Musante I, Tomati V, Caci E, Comegna M, et al. Two CFTR mutations within codon 970 differently impact on the chloride channel functionality. Hum Mutat. 2019;40(6):742–748. doi: 10.1002/humu.23741 30851139.

47. Gottschalk LB, Vecchio-Pagan B, Sharma N, Han ST, Franca A, Wohler ES, et al. Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants. J Cyst Fibros. 2016;15(3):285–294. doi: 10.1016/j.jcf.2015.11.010 26694805; PubMed Central PMCID: PMC4879073.

48. Romey MC, Desgeorges M, Malzac P, Sarles J, Demaille J, Claustres M. Homozygosity for a novel missense mutation (I175V) in exon 5 of the CFTR gene in a family of Armenian descent. Hum Mol Genet. 1994;3(4):661–662. doi: 10.1093/hmg/3.4.661 7520799.

49. Caputo A, Hinzpeter A, Caci E, Pedemonte N, Arous N, Di Duca M, et al. Mutation-specific potency and efficacy of cystic fibrosis transmembrane conductance regulator chloride channel potentiators. J Pharmacol Exp Ther. 2009;330(3):783–791. doi: 10.1124/jpet.109.154146 19491324; PubMed Central PMCID: PMC2729795.

50. Jones CT, McIntosh I, Keston M, Ferguson A, Brock DJH. Three novel mutations in the cystic fibrosis gene detected by chemical cleavage: Analysis of variant splicing and a nonsense mutation. Hum Mol Genet. 1992;1:11–17. doi: 10.1093/hmg/1.1.11 1284466

51. Scotti MM, Swanson MS. RNA mis-splicing in disease. Nat Rev Genet. 2016;17(1):19–32. doi: 10.1038/nrg.2015.3 26593421.

52. Wang Z, Xiao X, Van Nostrand E, Burge CB. General and specific functions of exonic splicing silencers in splicing control. Mol Cell. 2006;23(1):61–70. doi: 10.1016/j.molcel.2006.05.018 16797197; PubMed Central PMCID: PMC1839040.

53. Caceres EF, Hurst LD. The evolution, impact and properties of exonic splice enhancers. Genome Biol. 2013;14(12):R143. doi: 10.1186/gb-2013-14-12-r143 24359918; PubMed Central PMCID: PMC4054783.

54. Fairbrother WG, Chasin LA. Human genomic sequences that inhibit splicing. Mol Cell Biol. 2000;20(18):6816–6825. doi: 10.1128/mcb.20.18.6816-6825.2000 10958678; PubMed Central PMCID: PMC86212.

55. Wang Y, Ma M, Xiao X, Wang Z. Intronic splicing enhancers, cognate splicing factors and context-dependent regulation rules. Nat Struct Mol Biol. 2012;19(10):1044–1052. doi: 10.1038/nsmb.2377 22983564; PubMed Central PMCID: PMC3753194.

56. Skalska L, Beltran-Nebot M, Ule J, Jenner RG. Regulatory feedback from nascent RNA to chromatin and transcription. Nat Rev Mol Cell Biol. 2017;18(5):331–337. doi: 10.1038/nrm.2017.12 28270684.

57. Anna A, Monika G. Splicing mutations in human genetic disorders: examples, detection, and confirmation. J Appl Genet. 2018;59(3):253–268. doi: 10.1007/s13353-018-0444-7 29680930; PubMed Central PMCID: PMC6060985.

58. Becirovic E, Bohm S, Nguyen ON, Riedmayr LM, Koch MA, Schulze E, et al. In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors. PLoS Genet. 2016;12(1):e1005811. doi: 10.1371/journal.pgen.1005811 26796962; PubMed Central PMCID: PMC4722987.

59. Khan AO, Becirovic E, Betz C, Neuhaus C, Altmuller J, Maria Riedmayr L, et al. A deep intronic CLRN1 (USH3A) founder mutation generates an aberrant exon and underlies severe Usher syndrome on the Arabian Peninsula. Sci Rep. 2017;7(1):1411. doi: 10.1038/s41598-017-01577-8 28469144; PubMed Central PMCID: PMC5431179.

60. Petersen-Jones SM, Occelli LM, Winkler PA, Lee W, Sparrow JR, Tsukikawa M, et al. Patients and animal models of CNGbeta1-deficient retinitis pigmentosa support gene augmentation approach. J Clin Invest. 2018;128(1):190–206. doi: 10.1172/JCI95161 29202463; PubMed Central PMCID: PMC5749539.

61. Cooper TA. Use of minigene systems to dissect alternative splicing elements. Methods. 2005;37(4):331–340. S1046-2023(05)00173-8 [pii]; doi: 10.1016/j.ymeth.2005.07.015 16314262

62. Yeo GW, Van Nostrand EL, Liang TY. Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements. PLoS Genet. 2007;3(5):e85. 06-PLGE-RA-0548R2 [pii]; doi: 10.1371/journal.pgen.0030085 17530930

63. Pagani F, Stuani C, Tzetis M, Kanavakis E, Efthymiadou A, Doudounakis S, et al. New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12. Hum Mol Genet. 2003;12(10):1111–1120. doi: 10.1093/hmg/ddg131 12719375

64. Valley HC, Bukis KM, Bell A, Cheng Y, Wong E, Jordan NJ, et al. Isogenic cell models of cystic fibrosis-causing variants in natively expressing pulmonary epithelial cells. J Cyst Fibros. 2019;18(4):476–483. doi: 10.1016/j.jcf.2018.12.001 30563749.

65. Jennings S, Ng HP, Wang G. Establishment of a DeltaF508-CF promyelocytic cell line for cystic fibrosis research and drug screening. J Cyst Fibros. 2019;18(1):44–53. doi: 10.1016/j.jcf.2018.06.007 30670178.

66. Tzetis M, Efthymiadou A, Doudounakis S, Kanavakis E. Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A→G, 2751+2T→A, 296+1G→C, 1717–9T→C-D565G) and one nonsense mutation (E822X) in the CFTR gene. Human Genetics. 2001;109(6):592–601. doi: 10.1007/s00439-001-0631-0 11810271

67. Hinzpeter A, Aissat A, Sondo E, Costa C, Arous N, Gameiro C, et al. Alternative splicing at a NAGNAG acceptor site as a novel phenotype modifier. PLoS Genet. 2010;6(10).

68. Faa V, Coiana A, Incani F, Costantino L, Cao A, Rosatelli MC. A synonymous mutation in the CFTR gene causes aberrant splicing in an italian patient affected by a mild form of cystic fibrosis. J Mol Diagn. 2010;12(3):380–383. doi: 10.2353/jmoldx.2010.090126 20190016; PubMed Central PMCID: PMC2860476.

69. Wang C, Zhou W, Huang Y, Yin H, Jin Y, Jia Z, et al. Presumed missense and synonymous mutations in ATP7B gene cause exon skipping in Wilson disease. Liver Int. 2018;38(8):1504–1513. doi: 10.1111/liv.13754 29637721.

70. Linde L, Boelz S, Nissim-Rafinia M, Oren YS, Wilschanski M, Yaacov Y, et al. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin. J Clin Invest. 2007;117(3):683–692. doi: 10.1172/JCI28523 17290305

71. Haggie PM, Phuan PW, Tan JA, Xu H, Avramescu RG, Perdomo D, et al. Correctors and Potentiators Rescue Function of the Truncated W1282X-CFTR Translation Product. J Biol Chem. 2016. doi: 10.1074/jbc.M116.764720 27895116.

72. Peabody Lever JE, Mutyam V, Hathorne HY, Peng N, Sharma J, Edwards LJ, et al. Ataluren/ivacaftor combination therapy: Two N-of-1 trials in cystic fibrosis patients with nonsense mutations. Pediatr Pulmonol. 2020;55(7):1838–1842. doi: 10.1002/ppul.24764 32281737.

73. Fernandez Alanis E, Pinotti M, Dal Mas A, Balestra D, Cavallari N, Rogalska ME, et al. An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. Hum Mol Genet. 2012;21(11):2389–2398. doi: 10.1093/hmg/dds045 22362925; PubMed Central PMCID: PMC3349419.

74. Igreja S, Clarke LA, Botelho HM, Marques L, Amaral MD. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides. Hum Mutat. 2016;37(2):209–215. doi: 10.1002/humu.22931 26553470.

75. Michaels WE, Bridges RJ, Hastings ML. Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells. Nucleic Acids Res. 2020;48(13):7454–7467. doi: 10.1093/nar/gkaa490 32520327; PubMed Central PMCID: PMC7367209.

76. Sanz DJ, Hollywood JA, Scallan MF, Harrison PT. Cas9/gRNA targeted excision of cystic fibrosis-causing deep-intronic splicing mutations restores normal splicing of CFTR mRNA. PLoS One. 2017;12(9):e0184009. doi: 10.1371/journal.pone.0184009 28863137; PubMed Central PMCID: PMC5581164.

77. Maule G, Casini A, Montagna C, Ramalho AS, De Boeck K, Debyser Z, et al. Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing. Nat Commun. 2019;10(1):3556. doi: 10.1038/s41467-019-11454-9 31391465; PubMed Central PMCID: PMC6685978.

78. Han ST, Rab A, Pellicore MJ, Davis EF, McCague AF, Evans TA, et al. Residual function of cystic fibrosis mutants predicts response to small molecule CFTR modulators. JCI Insight. 2018;3(14). Epub 2018/07/25. doi: 10.1172/jci.insight.121159 30046002; PubMed Central PMCID: PMC6124440.

79. Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, et al. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol. 2012;180(2):599–607. doi: 10.1016/j.ajpath.2011.10.036 22189618; PubMed Central PMCID: PMC3349876.

80. Liu X, Krawczyk E, Suprynowicz FA, Palechor-Ceron N, Yuan H, Dakic A, et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc. 2017;12(2):439–451. doi: 10.1038/nprot.2016.174 28125105.

81. Gentzsch M, Boyles SE, Cheluvaraju C, Chaudhry IG, Quinney NL, Cho C, et al. Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells. Am J Respir Cell Mol Biol. 2017;56(5):568–574. doi: 10.1165/rcmb.2016-0276MA 27983869.

82. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–359. nmeth.1923 [pii]; doi: 10.1038/nmeth.1923 22388286

83. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25(9):1105–1111. btp120 [pii]; doi: 10.1093/bioinformatics/btp120 19289445

Štítky
Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics


2020 Číslo 10

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Kurzy

Zvyšte si kvalifikaci online z pohodlí domova

Antiseptika a prevence ve stomatologii
nový kurz
Autoři: MUDr. Ladislav Korábek, CSc., MBA

Diagnostika a léčba deprese pro ambulantní praxi
Autoři: MUDr. Jan Hubeňák, Ph.D

Snímatelné zubní náhrady a fixační krémy
Autoři: doc. MUDr. Hana Hubálková, Ph.D.

Nová éra v léčbě migrény
Autoři: MUDr. Eva Medová, MUDr. Tomáš Nežádal, Ph.D.

Význam nutraceutik u kardiovaskulárních onemocnění
Autoři:

Všechny kurzy
Přihlášení
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.

Přihlášení

Nemáte účet?  Registrujte se