With rapid advances in sequencing technologies, tens of millions of DNA variants have now been discovered in the human population. However, there are currently no experimental methods available for examining the impact of DNA variants in a high-throughput fashion. As a result, we have no functional data on the vast majority of these variants, which is a major roadblock to generating novel biological insights and developing new disease prevention therapeutic strategies. To address this issue, we have successfully developed the first massively-parallel site-directed mutagenesis approach, Clone-seq, to leverage the power of next-generation sequencing to generate a large number of mutant alleles in a fast and cost-effective manner. In conjunction with Clone-seq, we established a high-throughput comparative interactome-scanning pipeline to experimentally elucidate the effect of variants on protein stability and interactions. Additionally, Clone-seq can be used to generate clones for all DNA variants, including those in non-coding regions.
Vyšlo v časopise: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations. PLoS Genet 10(12): e32767. doi:10.1371/journal.pgen.1004819 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004819
1. StensonPD, MortM, BallEV, HowellsK, PhillipsAD, et al. (2009) The Human Gene Mutation Database: 2008 update. Genome Med 1 : 13.
2. ConsortiumTGP (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491 : 56–65.
3. FuW, O'ConnorTD, JunG, KangHM, AbecasisG, et al. (2013) Analysis of 6,515 exomes reveals the recent origin of most human protein-coding variants. Nature 493 : 216–220.
4. HindorffLA, SethupathyP, JunkinsHA, RamosEM, MehtaJP, et al. (2009) Potential etiologic and functional implications of genome-wide association loci for human diseases and traits. Proc Natl Acad Sci U S A 106 : 9362–9367.
5. VidalM, CusickME, BarabasiAL (2011) Interactome networks and human disease. Cell 144 : 986–998.
6. ZhongQ, SimonisN, LiQR, CharloteauxB, HeuzeF, et al. (2009) Edgetic perturbation models of human inherited disorders. Mol Syst Biol 5 : 321.
7. WangX, WeiX, ThijssenB, DasJ, LipkinSM, et al. (2012) Three-dimensional reconstruction of protein networks provides insight into human genetic disease. Nat Biotechnol 30 : 159–164.
8. BermanHM, WestbrookJ, FengZ, GillilandG, BhatTN, et al. (2000) The Protein Data Bank. Nucleic Acids Res 28 : 235–242.
9. YuH, BraunP, YildirimMA, LemmensI, VenkatesanK, et al. (2008) High-quality binary protein interaction map of the yeast interactome network. Science 322 : 104–110.
10. BraunP, TasanM, DrezeM, Barrios-RodilesM, LemmensI, et al. (2009) An experimentally derived confidence score for binary protein-protein interactions. Nat Methods 6 : 91–97.
11. RualJF, VenkatesanK, HaoT, Hirozane-KishikawaT, DricotA, et al. (2005) Towards a proteome-scale map of the human protein-protein interaction network. Nature 437 : 1173–1178.
12. VenkatesanK, RualJF, VazquezA, StelzlU, LemmensI, et al. (2009) An empirical framework for binary interactome mapping. Nat Methods 6 : 83–90.
13. YuH, TardivoL, TamS, WeinerE, GebreabF, et al. (2011) Next-generation sequencing to generate interactome datasets. Nat Methods 8 : 478–480.
14. HI2012 (2012) http://interactomedfciharvardedu/indexphp?page=login&lg=/H_sapiens/indexphp?page=newrelease.
15. SuzukiY, KagawaN, FujinoT, SumiyaT, AndohT, et al. (2005) A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system. Nucleic Acids Res 33: e109.
16. Salehi-AshtianiK, YangX, DertiA, TianW, HaoT, et al. (2008) Isoform discovery by targeted cloning, ‘deep-well’ pooling and parallel sequencing. Nat Methods 5 : 597–600.
17. DasJ, LeeHR, SagarA, FragozaR, LiangJ, et al. (2014) Elucidating common structural features of human pathogenic variations using large-scale atomic-resolution protein networks. Hum Mutat 35 : 585–593.
18. KhuranaE, FuY, ColonnaV, MuXJ, KangHM, et al. (2013) Integrative annotation of variants from 1092 humans: application to cancer genomics. Science 342 : 1235587.
19. VandenbrouckeI, Van MarckH, VerhasseltP, ThysK, MostmansW, et al. (2011) Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications. Biotechniques 51 : 167–177.
20. ForbesSA, BindalN, BamfordS, ColeC, KokCY, et al. (2011) COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res 39: D945–950.
21. McKennaA, HannaM, BanksE, SivachenkoA, CibulskisK, et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20 : 1297–1303.
22. HanksS, ColemanK, ReidS, PlajaA, FirthH, et al. (2004) Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B. Nat Genet 36 : 1159–1161.
23. SuijkerbuijkSJ, van OschMH, BosFL, HanksS, RahmanN, et al. (2010) Molecular causes for BUBR1 dysfunction in the human cancer predisposition syndrome mosaic variegated aneuploidy. Cancer Res 70 : 4891–4900.
24. MatsuuraS, MatsumotoY, MorishimaK, IzumiH, MatsumotoH, et al. (2006) Monoallelic BUB1B mutations and defective mitotic-spindle checkpoint in seven families with premature chromatid separation (PCS) syndrome. Am J Med Genet A 140 : 358–367.
25. RothS, SistonenP, SalovaaraR, HemminkiA, LoukolaA, et al. (1999) SMAD genes in juvenile polyposis. Genes Chromosomes Cancer 26 : 54–61.
26. HoulstonR, BevanS, WilliamsA, YoungJ, DunlopM, et al. (1998) Mutations in DPC4 (SMAD4) cause juvenile polyposis syndrome, but only account for a minority of cases. Hum Mol Genet 7 : 1907–1912.
27. MassagueJ (2008) TGFbeta in Cancer. Cell 134 : 215–230.
28. NasimMT, OgoT, AhmedM, RandallR, ChowdhuryHM, et al. (2011) Molecular genetic characterization of SMAD signaling molecules in pulmonary arterial hypertension. Hum Mutat 32 : 1385–1389.
29. MachadoRD (2012) The molecular genetics and cellular mechanisms underlying pulmonary arterial hypertension. Scientifica (Cairo) 2012 : 106576.
30. TuncbagN, GursoyA, NussinovR, KeskinO (2011) Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM. Nat Protoc 6 : 1341–1354.
31. ZhangQC, PetreyD, DengL, QiangL, ShiY, et al. (2012) Structure-based prediction of protein-protein interactions on a genome-wide scale. Nature 490 : 556–560.
32. MeyerMJ, DasJ, WangX, YuH (2013) INstruct: a database of high-quality 3D structurally resolved protein interactome networks. Bioinformatics 29 : 1577–1579.
33. MoscaR, CeolA, AloyP (2013) Interactome3D: adding structural details to protein networks. Nat Methods 10 : 47–53.
34. DasJ, FragozaR, LeeHR, CorderoNA, GuoY, et al. (2014) Exploring mechanisms of human disease through structurally resolved protein interactome networks. Mol Biosyst 10 : 9–17.
35. PeltomakiP, VasenHF (1997) Mutations predisposing to hereditary nonpolyposis colorectal cancer: database and results of a collaborative study. The International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer. Gastroenterology 113 : 1146–1158.
36. KondoE, SuzukiH, HoriiA, FukushigeS (2003) A yeast two-hybrid assay provides a simple way to evaluate the vast majority of hMLH1 germ-line mutations. Cancer Res 63 : 3302–3308.
37. OngSE, BlagoevB, KratchmarovaI, KristensenDB, SteenH, et al. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics 1 : 376–386.
38. OngSE, MannM (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1 : 2650–2660.
39. OhouoPY, Bastos de OliveiraFM, AlmeidaBS, SmolkaMB (2010) DNA damage signaling recruits the Rtt107-Slx4 scaffolds via Dpb11 to mediate replication stress response. Mol Cell 39 : 300–306.
40. LeungWK, KimJJ, WuL, SepulvedaJL, SepulvedaAR (2000) Identification of a second MutL DNA mismatch repair complex (hPMS1 and hMLH1) in human epithelial cells. J Biol Chem 275 : 15728–15732.
41. PengM, LitmanR, XieJ, SharmaS, BroshRMJr, et al. (2007) The FANCJ/MutLalpha interaction is required for correction of the cross-link response in FA-J cells. EMBO J 26 : 3238–3249.
42. GoldfarbSB, KashlanOB, WatkinsJN, SuaudL, YanW, et al. (2006) Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels. Proc Natl Acad Sci U S A 103 : 5817–5822.
43. DeLuca-FlahertyC, McKayDB, ParhamP, HillBL (1990) Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LCa to stimulate ATP hydrolysis. Cell 62 : 875–887.
44. KauferS, CoffeyCM, ParkerJS (2012) The cellular chaperone hsc70 is specifically recruited to reovirus viral factories independently of its chaperone function. J Virol 86 : 1079–1089.
45. FowlerDM, ArayaCL, FleishmanSJ, KelloggEH, StephanyJJ, et al. (2010) High-resolution mapping of protein sequence-function relationships. Nat Methods 7 : 741–746.
46. StaritaLM, PrunedaJN, LoRS, FowlerDM, KimHJ, et al. (2013) Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis. Proc Natl Acad Sci U S A 110: E1263–1272.
47. ArayaCL, FowlerDM, ChenW, MuniezI, KellyJW, et al. (2012) A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc Natl Acad Sci U S A 109 : 16858–16863.
48. PittJN, Ferre-D'AmareAR (2010) Rapid construction of empirical RNA fitness landscapes. Science 330 : 376–379.
49. StranskyN, EgloffAM, TwardAD, KosticAD, CibulskisK, et al. (2011) The mutational landscape of head and neck squamous cell carcinoma. Science 333 : 1157–1160.
50. AtlasTCG (2012) Comprehensive molecular characterization of human colon and rectal cancer. Nature 487 : 330–337.
51. CunninghamBC, WellsJA (1989) High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244 : 1081–1085.
52. ConsortiumAIM (2011) Evidence for network evolution in an Arabidopsis interactome map. Science 333 : 601–607.
53. DasJ, VoTV, WeiX, MellorJC, TongV, et al. (2013) Cross-species protein interactome mapping reveals species-specific wiring of stress response pathways. Sci Signal 6: ra38.
54. Reece-HoyesJS, BarutcuAR, McCordRP, JeongJS, JiangL, et al. (2011) Yeast one-hybrid assays for gene-centered human gene regulatory network mapping. Nat Methods 8 : 1050–1052.
55. BergerMF, PhilippakisAA, QureshiAM, HeFS, EstepPW3rd, et al. (2006) Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat Biotechnol 24 : 1429–1435.
56. YakhninAV, YakhninH, BabitzkeP (2012) Gel mobility shift assays to detect protein-RNA interactions. Methods Mol Biol 905 : 201–211.
57. BandyopadhyayA, SaxenaK, KasturiaN, DalalV, BhattN, et al. (2012) Chemical chaperones assist intracellular folding to buffer mutational variations. Nat Chem Biol 8 : 238–245.
58. FranzosaEA, XiaY (2011) Structural principles within the human-virus protein-protein interaction network. Proc Natl Acad Sci U S A 108 : 10538–10543.
59. SteinA, CeolA, AloyP (2011) 3did: identification and classification of domain-based interactions of known three-dimensional structure. Nucleic Acids Res 39: D718–723.
60. FinnRD, MarshallM, BatemanA (2005) iPfam: visualization of protein-protein interactions in PDB at domain and amino acid resolutions. Bioinformatics 21 : 410–412.
61. YangX, BoehmJS, Salehi-AshtianiK, HaoT, ShenY, et al. (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods 8 : 659–661.
62. LiH, DurbinR (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 : 1754–1760.
63. LiH, HandsakerB, WysokerA, FennellT, RuanJ, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25 : 2078–2079.
64. WalhoutAJ, VidalM (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24 : 297–306.
65. BehrendsC, SowaME, GygiSP, HarperJW (2010) Network organization of the human autophagy system. Nature 466 : 68–76.
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