The Gene Contains Hotspots for L1 Endonuclease-Dependent Insertion
Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the ∼100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as ∼0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA–based mutation analysis protocols, resulting in improved detection compared to gDNA–based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)–23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.
Vyšlo v časopise:
The Gene Contains Hotspots for L1 Endonuclease-Dependent Insertion. PLoS Genet 7(11): e32767. doi:10.1371/journal.pgen.1002371
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002371
Souhrn
Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the ∼100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as ∼0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA–based mutation analysis protocols, resulting in improved detection compared to gDNA–based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)–23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.
Zdroje
1. LanderESLintonLMBirrenBNusbaumCZodyMC 2001 Initial sequencing and analysis of the human genome. Nature 409 860 921
2. BrouhaBSchustakJBadgeRMLutz-PriggeSFarleyAH 2003 Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A 100 5280 5285
3. DewannieuxMEsnaultCHeidmannT 2003 LINE-mediated retrotransposition of marked Alu sequences. Nat Genet 35 41 48
4. CordauxRHedgesDJBatzerMA 2004 Retrotransposition of Alu elements: how many sources? Trends Genet 20 464 467
5. DeiningerPLBatzerMAHutchisonCA3rdEdgellMH 1992 Master genes in mammalian repetitive DNA amplification. Trends Genet 8 307 311
6. DanielsGRDeiningerPL 1991 Characterization of a third major SINE family of repetitive sequences in the galago genome. Nucleic acids research 19 1649 1656
7. SwergoldGD 1990 Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol 10 6718 6729
8. DeiningerPLBatzerMA 2002 Mammalian retroelements. Genome research 12 1455 1465
9. DombroskiBAMathiasSLNanthakumarEScottAFKazazianHHJr 1991 Isolation of an active human transposable element. Science 254 1805 1808
10. ScottAFSchmeckpeperBJAbdelrazikMComeyCTO'HaraB 1987 Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence. Genomics 1 113 125
11. FengQMoranJVKazazianHHJrBoekeJD 1996 Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87 905 916
12. MathiasSLScottAFKazazianHHJrBoekeJDGabrielA 1991 Reverse transcriptase encoded by a human transposable element. Science 254 1808 1810
13. JurkaJ 1997 Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons. Proc Natl Acad Sci U S A 94 1872 1877
14. LuanDDKormanMHJakubczakJLEickbushTH 1993 Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72 595 605
15. KazazianHHJr 2004 Mobile elements: drivers of genome evolution. Science 303 1626 1632
16. BatzerMADeiningerPL 2002 Alu repeats and human genomic diversity. Nat Rev Genet 3 370 379
17. BeckCRCollierPMacfarlaneCMaligMKiddJM 2010 LINE-1 retrotransposition activity in human genomes. Cell 141 1159 1170
18. HuangCRSchneiderAMLuYNiranjanTShenP 2010 Mobile interspersed repeats are major structural variants in the human genome. Cell 141 1171 1182
19. IskowRCMcCabeMTMillsREToreneSPittardWS 2010 Natural mutagenesis of human genomes by endogenous retrotransposons. Cell 141 1253 1261
20. WitherspoonDJXingJZhangYWatkinsWSBatzerMA 2010 Mobile element scanning (ME-Scan) by targeted high-throughput sequencing. BMC Genomics 11 410
21. KazazianHHJrWongCYoussoufianHScottAFPhillipsDG 1988 Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature 332 164 166
22. BelancioVPHedgesDJDeiningerP 2008 Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. Genome research 18 343 358
23. GoodierJLKazazianHHJr 2008 Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell 135 23 35
24. ChenJMStensonPDCooperDNFerecC 2005 A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease. Hum Genet 117 411 427
25. WallaceMRAndersenLBSaulinoAMGregoryPEGloverTW 1991 A de novo Alu insertion results in neurofibromatosis type 1. Nature 353 864 866
26. KazazianHHJrMoranJV 1998 The impact of L1 retrotransposons on the human genome. Nat Genet 19 19 24
27. CallinanPABatzerMA 2006 Retrotransposable elements and human disease. Genome Dyn 1 104 115
28. ChenJMFerecCCooperDN 2006 LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease: mutation detection bias and multiple mechanisms of target gene disruption. J Biomed Biotechnol 2006 56182
29. JurkaJ 2000 Repbase update: a database and an electronic journal of repetitive elements. Trends Genet 16 418 420
30. BoissinotSChevretPFuranoAV 2000 L1 (LINE-1) retrotransposon evolution and amplification in recent human history. Mol Biol Evol 17 915 928
31. MyersJSVincentBJUdallHWatkinsWSMorrishTA 2002 A comprehensive analysis of recently integrated human Ta L1 elements. Am J Hum Genet 71 312 326
32. GilbertNLutzSMorrishTAMoranJV 2005 Multiple fates of L1 retrotransposition intermediates in cultured human cells. Mol Cell Biol 25 7780 7795
33. GilbertNLutz-PriggeSMoranJV 2002 Genomic deletions created upon LINE-1 retrotransposition. Cell 110 315 325
34. SymerDEConnellyCSzakSTCaputoEMCostGJ 2002 Human l1 retrotransposition is associated with genetic instability in vivo. Cell 110 327 338
35. SkowronskiJFanningTGSingerMF 1988 Unit-length line-1 transcripts in human teratocarcinoma cells. Mol Cell Biol 8 1385 1397
36. KazazianHHJr 1999 An estimated frequency of endogenous insertional mutations in humans. Nat Genet 22 130
37. MessiaenLWimmerK 2011 Mutation analysis of the NF1 gene by cDNA-based sequencing of the coding region Soares Gonçalves CunhaKGellerM Advances in Neurofibromatosis Research Nova Science Publishers
38. TeugelsEDe BrakeleerSGoelenGLissensWSermijnE 2005 De novo Alu element insertions targeted to a sequence common to the BRCA1 and BRCA2 genes. Hum Mutat 26 284
39. ConleyMEPartainJDNorlandSMShurtleffSAKazazianHHJr 2005 Two independent retrotransposon insertions at the same site within the coding region of BTK. Hum Mutat 25 324 325
40. CordauxRHedgesDJHerkeSWBatzerMA 2006 Estimating the retrotransposition rate of human Alu elements. Gene 373 134 137
41. MorrishTAGilbertNMyersJSVincentBJStamatoTD 2002 DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat Genet 31 159 165
42. ChenJMFerecCCooperDN 2007 Mechanism of Alu integration into the human genome. Genomic Med 1 9 17
43. MorrishTAGarcia-PerezJLStamatoTDTaccioliGESekiguchiJ 2007 Endonuclease-independent LINE-1 retrotransposition at mammalian telomeres. Nature 446 208 212
44. KojimaKK 2010 Different integration site structures between L1 protein-mediated retrotransposition in cis and retrotransposition in trans. Mob DNA 1 17
45. OstertagEMKazazianHHJr 2001 Twin priming: a proposed mechanism for the creation of inversions in L1 retrotransposition. Genome research 11 2059 2065
46. WimmerKYaoSClaesKKehrer-SawatzkiHTinschertS 2006 Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients. Genes Chromosomes Cancer 45 265 276
47. BergetSM 1995 Exon recognition in vertebrate splicing. J Biol Chem 270 2411 2414
48. WimmerKRocaXBeiglbockHCallensTEtzlerJ 2007 Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5′ splice-site disruption. Hum Mutat 28 599 612
49. KralovicovaJVorechovskyI 2007 Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition. Nucleic Acids Res 35 6399 6413
50. MeischlCBoerMAhlinARoosD 2000 A new exon created by intronic insertion of a rearranged LINE-1 element as the cause of chronic granulomatous disease. Eur J Hum Genet 8 697 703
51. MessiaenLMCallensTMortierGBeysenDVandenbrouckeI 2000 Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum Mutat 15 541 555
52. KobayashiKNakahoriYMiyakeMMatsumuraKKondo-IidaE 1998 An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature 394 388 392
53. YoshidaKNakamuraAYazakiMIkedaSTakedaS 1998 Insertional mutation by transposable element, L1, in the DMD gene results in X-linked dilated cardiomyopathy. Hum Mol Genet 7 1129 1132
54. HanJSSzakSTBoekeJD 2004 Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature 429 268 274
55. Perepelitsa-BelancioVDeiningerP 2003 RNA truncation by premature polyadenylation attenuates human mobile element activity. Nat Genet 35 363 366
56. VandenbrouckeIvan DoornRCallensTCobbenJMStarinkTM 2004 Genetic and clinical mosaicism in a patient with neurofibromatosis type 1. Hum Genet 114 284 290
57. WimmerKEckartMRehderHFonatschC 2000 Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients. Hum Genet 106 311 313
58. ThompsonJDHigginsDGGibsonTJ 1994 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 4673 4680
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2011 Číslo 11
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