title: Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain.
abstract: Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed 'Digital Deletion Detection' (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10(-8) . 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.
authors.affiliation: Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave, Seattle, WA, 98109, USA.
authors.forename: Sean D
authors.initials: SD
authors.lastname: Taylor
authors.forename: Nolan G
authors.initials: NG
authors.lastname: Ericson
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Burton
authors.forename: Tomas A
authors.initials: TA
authors.lastname: Prolla
authors.forename: John R
authors.initials: JR
authors.lastname: Silber
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.forename: Jason H
authors.initials: JH
authors.lastname: Bielas
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000235:genetics; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000235:genetics*; D018740:Genetic Heterogeneity; D006801:Humans; D008875:Middle Aged; D008938:Mitosis; D017384:Sequence Deletion; D055815:Young Adult
chemical_list: D004272:DNA, Mitochondrial
keywords: aging; genome instability; mitochondrial DNA; mitochondrial disease; next-generation sequencing; rare deletion detection
references.citation: Clin Chem. 2003 Aug;49(8):1309-17
references.pmid: 12881447
references.citation: Rejuvenation Res. 2009 Jun;12(3):217-9
references.pmid: 19594330
references.citation: Nat Genet. 2011 Jun 26;43(8):806-10
references.pmid: 21706004
references.citation: Trends Genet. 2009 Feb;25(2):91-8
references.pmid: 19110336
references.citation: Anal Chem. 2012 Jan 17;84(2):1003-11
references.pmid: 22122760
references.citation: J Pathol. 2012 Jan;226(2):274-86
references.pmid: 21989606
references.citation: Expert Rev Mol Diagn. 2005 Sep;5(5):809-15
references.pmid: 16149882
references.citation: Nucleic Acids Res. 1990 Dec 11;18(23):6927-33
references.pmid: 2263455
references.citation: Hum Mol Genet. 2009 Mar 15;18(6):1028-36
references.pmid: 19095717
references.citation: Cell Mol Life Sci. 2004 Nov;61(22):2799-811
references.pmid: 15558210
references.citation: Methods. 2008 Dec;46(4):263-8
references.pmid: 18948200
references.citation: Ageing Res Rev. 2010 Nov;9 Suppl 1:S47-58
references.pmid: 20816876
references.citation: Trends Genet. 1989 Jan;5(1):9-13
references.pmid: 2652392
references.citation: Ann Neurol. 1998 Feb;43(2):217-23
references.pmid: 9485063
references.citation: Nat Genet. 2008 Mar;40(3):275-9
references.pmid: 18305478
references.citation: Am J Hum Genet. 2001 Mar;68(3):802-6
references.pmid: 11179029
references.citation: Ann N Y Acad Sci. 2004 Jun;1019:240-4
references.pmid: 15247022
references.citation: Nat Genet. 2008 Apr;40(4):392-4
references.pmid: 18311139
references.citation: Neurosci Res. 2011 Apr;69(4):331-6
references.pmid: 21185889
references.citation: Mutat Res. 2003 Jan 28;522(1-2):13-9
references.pmid: 12517407
references.citation: Exp Neurol. 2009 Aug;218(2):316-9
references.pmid: 19426731
references.citation: J Biol Chem. 2000 Aug 11;275(32):24818-28
references.pmid: 10827171
references.citation: Z Gerontol Geriatr. 2007 Oct;40(5):325-33
references.pmid: 17943235
references.citation: Biochim Biophys Acta. 2010 Jun-Jul;1797(6-7):1159-62
references.pmid: 20388490
references.citation: PLoS Comput Biol. 2011 Nov;7(11):e1002287
references.pmid: 22125488
references.citation: Exp Gerontol. 2008 Jul;43(7):645-52
references.pmid: 18439778
references.citation: Nat Methods. 2005 Apr;2(4):285-90
references.pmid: 15782221
references.citation: Front Genet. 2012 Jun 20;3:103
references.pmid: 22723804
references.citation: Neuromuscul Disord. 2006 Jun;16(6):381-6
references.pmid: 16684599
references.citation: PLoS Genet. 2011 Mar;7(3):e1002028
references.pmid: 21455489
references.citation: Nat Genet. 2011 Jul 27;43(8):726-7
references.pmid: 21792237
references.citation: Nat Genet. 2001 Jun;28(2):147-50
references.pmid: 11381261
references.citation: Nucleic Acids Res. 2002 Jul 15;30(14):e68
references.pmid: 12136116
references.citation: Methods. 2008 Dec;46(4):269-73
references.pmid: 18955147
references.citation: Anal Chem. 2011 Nov 15;83(22):8604-10
references.pmid: 22035192
references.citation: Mutat Res. 2001 Apr 18;475(1-2):169-83
references.pmid: 11295162
references.citation: Nat Genet. 2012 Nov;44(11):1282-5
references.pmid: 23042113
references.citation: J Biomed Biotechnol. 2012;2012:251364
references.pmid: 22829749