RNAi–Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function

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Genome-wide association studies (GWAS) are powerful tools to unravel genomic loci associated with common traits and complex human disease. However, GWAS only rarely reveal information on the exact genetic elements and pathogenic events underlying an association. In order to extract functional information from genomic data, strategies for systematic follow-up studies on a phenotypic level are required. Here we address these limitations by applying RNA interference (RNAi) to analyze 133 candidate genes within 56 loci identified by GWAS as associated with blood lipid levels, coronary artery disease, and/or myocardial infarction for a function in regulating cholesterol levels in cells. Knockdown of a surprisingly high number (41%) of trait-associated genes affected low-density lipoprotein (LDL) internalization and/or cellular levels of free cholesterol. Our data further show that individual GWAS loci may contain more than one gene with cholesterol-regulatory functions. Using a set of secondary assays we demonstrate for a number of genes without previously known lipid-regulatory roles (e.g. CXCL12, FAM174A, PAFAH1B1, SEZ6L, TBL2, WDR12) that knockdown correlates with altered LDL–receptor levels and/or that overexpression as GFP–tagged fusion proteins inversely modifies cellular cholesterol levels. By providing strong evidence for disease-relevant functions of lipid trait-associated genes, our study demonstrates that quantitative, cell-based RNAi is a scalable strategy for a systematic, unbiased detection of functional effectors within GWAS loci.

Vyšlo v časopise: RNAi–Based Functional Profiling of Loci from Blood Lipid Genome-Wide Association Studies Identifies Genes with Cholesterol-Regulatory Function. PLoS Genet 9(2): e32767. doi:10.1371/journal.pgen.1003338
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003338

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Zdroje

1. AulchenkoYS, RipattiS, LindqvistI, BoomsmaD, HeidIM, et al. (2009) Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat Genet 41: 47–55.

2. BurkhardtR, KennyEE, LoweJK, BirkelandA, JosowitzR, et al. (2008) Common SNPs in HMGCR in micronesians and whites associated with LDL-cholesterol levels affect alternative splicing of exon13. Arterioscler Thromb Vasc Biol 28: 2078–2084.

3. ChasmanDI, PareG, MoraS, HopewellJC, PelosoG, et al. (2009) Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterol content in genome-wide analysis. PLoS Genet 5: e1000730 doi:10.1371/journal.pgen.1000730..

4. ErdmannJ, GrosshennigA, BraundPS, KonigIR, HengstenbergC, et al. (2009) New susceptibility locus for coronary artery disease on chromosome 3q22.3. Nat Genet 41: 280–282.

5. HelgadottirA, ThorleifssonG, ManolescuA, GretarsdottirS, BlondalT, et al. (2007) A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science 316: 1491–1493.

6. KathiresanS, MelanderO, GuiducciC, SurtiA, BurttNP, et al. (2008) Six new loci associated with blood low-density lipoprotein cholesterol, high-density lipoprotein cholesterol or triglycerides in humans. Nat Genet 40: 189–197.

7. KathiresanS, WillerCJ, PelosoGM, DemissieS, MusunuruK, et al. (2009) Common variants at 30 loci contribute to polygenic dyslipidemia. Nat Genet 41: 56–65.

8. KoonerJS, ChambersJC, Aguilar-SalinasCA, HindsDA, HydeCL, et al. (2008) Genome-wide scan identifies variation in MLXIPL associated with plasma triglycerides. Nat Genet 40: 149–151.

9. McPhersonR, PertsemlidisA, KavaslarN, StewartA, RobertsR, et al. (2007) A common allele on chromosome 9 associated with coronary heart disease. Science 316: 1488–1491.

10. KathiresanS, VoightBF, PurcellS, MusunuruK, ArdissinoD, et al. (2009) Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nat Genet 41: 334–341.

11. PollinTI, DamcottCM, ShenH, OttSH, SheltonJ, et al. (2008) A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science 322: 1702–1705.

12. RidkerPM, PareG, ParkerAN, ZeeRY, MiletichJP, et al. (2009) Polymorphism in the CETP gene region, HDL cholesterol, and risk of future myocardial infarction: Genomewide analysis among 18 245 initially healthy women from the Women's Genome Health Study. Circ Cardiovasc Genet 2: 26–33.

13. SabattiC, ServiceSK, HartikainenAL, PoutaA, RipattiS, et al. (2009) Genome-wide association analysis of metabolic traits in a birth cohort from a founder population. Nat Genet 41: 35–46.

14. SamaniNJ, ErdmannJ, HallAS, HengstenbergC, ManginoM, et al. (2007) Genomewide association analysis of coronary artery disease. N Engl J Med 357: 443–453.

15. SandhuMS, WaterworthDM, DebenhamSL, WheelerE, PapadakisK, et al. (2008) LDL-cholesterol concentrations: a genome-wide association study. Lancet 371: 483–491.

16. SchunkertH, KonigIR, KathiresanS, ReillyMP, AssimesTL, et al. (2011) Large-scale association analysis identifies 13 new susceptibility loci for coronary artery disease. Nat Genet 43: 333–338.

17. TeslovichTM, MusunuruK, SmithAV, EdmondsonAC, StylianouIM, et al. (2010) Biological, clinical and population relevance of 95 loci for blood lipids. Nature 466: 707–713.

18. TregouetDA, KonigIR, ErdmannJ, MunteanuA, BraundPS, et al. (2009) Genome-wide haplotype association study identifies the SLC22A3-LPAL2-LPA gene cluster as a risk locus for coronary artery disease. Nat Genet 41: 283–285.

19. WallaceC, NewhouseSJ, BraundP, ZhangF, TobinM, et al. (2008) Genome-wide association study identifies genes for biomarkers of cardiovascular disease: serum urate and dyslipidemia. Am J Hum Genet 82: 139–149.

20. WillerCJ, SannaS, JacksonAU, ScuteriA, BonnycastleLL, et al. (2008) Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nat Genet 40: 161–169.

21. WaterworthDM, RickettsSL, SongK, ChenL, ZhaoJH, et al. (2010) Genetic variants influencing circulating lipid levels and risk of coronary artery disease. Arterioscler Thromb Vasc Biol 30: 2264–2276.

22. The Wellcome Trust Case Control Consortium (2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447: 661–678.

23. SaxenaR, VoightBF, LyssenkoV, BurttNP, de BakkerPI, et al. (2007) Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 316: 1331–1336.

24. MusunuruK, StrongA, Frank-KamenetskyM, LeeNE, AhfeldtT, et al. (2010) From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature 466: 714–719.

25. ManolioTA (2010) Genomewide association studies and assessment of the risk of disease. N Engl J Med 363: 166–176.

26. LanderES (2011) Initial impact of the sequencing of the human genome. Nature 470: 187–197.

27. AltshulerD, DalyMJ, LanderES (2008) Genetic mapping in human disease. Science 322: 881–888.

28. ManolioTA, CollinsFS, CoxNJ, GoldsteinDB, HindorffLA, et al. (2009) Finding the missing heritability of complex diseases. Nature 461: 747–753.

29. MohrS, BakalC, PerrimonN (2010) Genomic screening with RNAi: results and challenges. Annu Rev Biochem 79: 37–64.

30. BartzF, KernL, ErzD, ZhuM, GilbertD, et al. (2009) Identification of cholesterol-regulating genes by targeted RNAi screening. Cell Metab 10: 63–75.

31. ErfleH, NeumannB, LiebelU, RogersP, HeldM, et al. (2007) Reverse transfection on cell arrays for high content screening microscopy. Nat Protoc 2: 392–399.

32. NeumannB, WalterT, HericheJK, BulkescherJ, ErfleH, et al. (2010) Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature 464: 721–727.

33. MoreauD, KumarP, WangSC, ChaumetA, ChewSY, et al. (2011) Genome-Wide RNAi Screens Identify Genes Required for Ricin and PE Intoxications. Dev Cell 21: 231–244.

34. BrassAL, DykxhoornDM, BenitaY, YanN, EngelmanA, et al. (2008) Identification of host proteins required for HIV infection through a functional genomic screen. Science 319: 921–926.

35. ZhouH, XuM, HuangQ, GatesAT, ZhangXD, et al. (2008) Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe 4: 495–504.

36. KonigR, ZhouY, EllederD, DiamondTL, BonamyGM, et al. (2008) Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 135: 49–60.

37. BechlerME, DoodyAM, HaKD, JudsonBL, ChenI, et al. (2011) The phospholipase A2 enzyme complex PAFAH Ib mediates endosomal membrane tubule formation and trafficking. Mol Biol Cell 22: 2348–2359.

38. GoldsteinJL, BrownMS (2009) The LDL receptor. Arterioscler Thromb Vasc Biol 29: 431–438.

39. GoldsteinJL, DeBose-BoydRA, BrownMS (2006) Protein sensors for membrane sterols. Cell 124: 35–46.

40. MoonYA, LiangG, XieX, Frank-KamenetskyM, FitzgeraldK, et al. (2012) The Scap/SREBP pathway is essential for developing diabetic fatty liver and carbohydrate-induced hypertriglyceridemia in animals. Cell Metab 15: 240–246.

41. MehtaN, LiM, WilliamD, KheraA, DerOhannessianS, et al. (2011) The novel atherosclerosis locus at 10q11 regulates plasma CXCL12 levels. European heart journal 32: 963–971.

42. FredericksWJ, McGarveyT, WangH, LalP, PuthiyaveettilR, et al. (2011) The bladder tumor suppressor protein TERE1 (UBIAD1) modulates cell cholesterol: implications for tumor progression. DNA Cell Biol 30: 851–864.

43. UebiT, ItohY, HatanoO, KumagaiA, SanosakaM, et al. (2012) Involvement of SIK3 in Glucose and Lipid Homeostasis in Mice. PLoS ONE 7: e37803 doi:10.1371/journal.pone.0037803..

44. GoldsteinJL, RawsonRB, BrownMS (2002) Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Archives of biochemistry and biophysics 397: 139–148.

45. CollinetC, StoterM, BradshawCR, SamusikN, RinkJC, et al. (2010) Systems survey of endocytosis by multiparametric image analysis. Nature 464: 243–249.

46. ErfleH, NeumannB, RogersP, BulkescherJ, EllenbergJ, et al. (2008) Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates. J Biomol Screen 13: 575–580.

47. CarpenterAE, JonesTR, LamprechtMR, ClarkeC, KangIH, et al. (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7: R100.

48. ArthurJR, HeineckeKA, SeyfriedTN (2011) Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain. J Lipid Res 52: 1345–1351.

49. SimpsonJC, JoggerstB, LaketaV, VerissimoF, CetinC, et al. (2012) Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway. Nature cell biology 14: 764–774.