Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies
Layal Antoury, Ningyan Hu, Leonora Balaj, Sudeshna Das, Sofia Georghiou, Basil Darras, Tim Clark, Xandra O Breakefield, Thurman M Wheeler, Layal Antoury, Ningyan Hu, Leonora Balaj, Sudeshna Das, Sofia Georghiou, Basil Darras, Tim Clark, Xandra O Breakefield, Thurman M Wheeler
Abstract
Urine contains extracellular RNA (exRNA) markers of urogenital cancers. However, the capacity of genetic material in urine to identify systemic diseases is unknown. Here we describe exRNA splice products in human urine as a source of biomarkers for the two most common forms of muscular dystrophies, myotonic dystrophy (DM) and Duchenne muscular dystrophy (DMD). Using a training set, RT-PCR, droplet digital PCR, and principal component regression, we identify ten transcripts that are spliced differently in urine exRNA from patients with DM type 1 (DM1) as compared to unaffected or disease controls, form a composite biomarker, and develop a predictive model that is 100% accurate in our independent validation set. Urine also contains mutation-specific DMD mRNAs that confirm exon-skipping activity of the antisense oligonucleotide drug eteplirsen. Our results establish that urine mRNA splice variants can be used to monitor systemic diseases with minimal or no clinical effect on the urinary tract.
Conflict of interest statement
Massachusetts General Hospital, T.M.W., X.O.B., and L.B. have filed a patent application on the use of urine exRNA to identify markers of muscular dystrophies. The remaining authors declare no competing interests.
Figures
References
- Scotti MM, Swanson MS. RNA mis-splicing in disease. Nat. Rev. Genet. 2016;17:19–32. doi: 10.1038/nrg.2015.3.
- Kanadia RN, et al. A muscleblind knockout model for myotonic dystrophy. Science. 2003;302:1978–1980. doi: 10.1126/science.1088583.
- Lin X, et al. Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Hum. Mol. Genet. 2006;15:2087–2097. doi: 10.1093/hmg/ddl132.
- Nakamori M, et al. Splicing biomarkers of disease severity in myotonic dystrophy. Ann. Neurol. 2013;74:862–872. doi: 10.1002/ana.23992.
- Wheeler TM, et al. Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA. Science. 2009;325:336–339. doi: 10.1126/science.1173110.
- Wheeler TM, et al. Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature. 2012;488:111–115. doi: 10.1038/nature11362.
- . (2016).
- Pandey SK, et al. Identification and characterization of modified antisense oligonucleotides targeting DMPK in mice and nonhuman primates for the treatment of myotonic dystrophy type 1. J. Pharmacol. Exp. Ther. 2015;355:329–340. doi: 10.1124/jpet.115.226969.
- Tkach M, Thery C. Communication by extracellular vesicles: where we are and where we need to go. Cell. 2016;164:1226–1232. doi: 10.1016/j.cell.2016.01.043.
- Skog J, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 2008;10:1470–1476. doi: 10.1038/ncb1800.
- Chen WW, et al. BEAMing and droplet digital PCR analysis of mutant IDH1 mRNA in glioma patient serum and cerebrospinal fluid extracellular vesicles. Mol. Ther. Nucleic Acids. 2013;2:e109. doi: 10.1038/mtna.2013.28.
- Nilsson J, et al. Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. Br. J. Cancer. 2009;100:1603–1607. doi: 10.1038/sj.bjc.6605058.
- San Lucas FA, et al. Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes. Ann. Oncol. 2016;27:635–641. doi: 10.1093/annonc/mdv604.
- Khan S, et al. Early diagnostic value of survivin and its alternative splice variants in breast cancer. BMC Cancer. 2014;14:176. doi: 10.1186/1471-2407-14-176.
- Neeb A, et al. Splice variant transcripts of the anterior gradient 2 gene as a marker of prostate cancer. Oncotarget. 2014;5:8681–8689. doi: 10.18632/oncotarget.2365.
- Romancino DP, et al. Identification and characterization of the nano-sized vesicles released by muscle cells. FEBS Lett. 2013;587:1379–1384. doi: 10.1016/j.febslet.2013.03.012.
- Forterre A, et al. Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation. Cell Cycle. 2014;13:78–89. doi: 10.4161/cc.26808.
- Hathout Y, et al. Clinical utility of serum biomarkers in Duchenne muscular dystrophy. Clin. Proteomics. 2016;13:9. doi: 10.1186/s12014-016-9109-x.
- Moeller MJ, Tenten V. Renal albumin filtration: alternative models to the standard physical barriers. Nat. Rev. Nephrol. 2013;9:266–277. doi: 10.1038/nrneph.2013.58.
- Noerholm M, et al. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC Cancer. 2012;12:22. doi: 10.1186/1471-2407-12-22.
- Miranda KC, et al. Massively parallel sequencing of human urinary exosome/microvesicle RNA reveals a predominance of non-coding RNA. PLoS ONE. 2014;9:e96094. doi: 10.1371/journal.pone.0096094.
- Erdbrugger U, Le TH. Extracellular vesicles in renal diseases: more than novel biomarkers? J. Am. Soc. Nephrol. 2016;27:12–26. doi: 10.1681/ASN.2015010074.
- Matsuzaki K, et al. MiR-21-5p in urinary extracellular vesicles is a novel biomarker of urothelial carcinoma. Oncotarget. 2017;8:24668–24678. doi: 10.18632/oncotarget.14969.
- Nielsen M, Qaseem A. Hematuria as a marker of occult urinary tract cancer: advice for high-value care from the American College of Physicians. Ann. Intern. Med. 2016;164:488–497. doi: 10.7326/M15-1496.
- Wise GJ, Schlegel PN. Sterile pyuria. N. Engl. J. Med. 2015;372:1048–1054. doi: 10.1056/NEJMra1410052.
- Dorrenhaus A, et al. Cultures of exfoliated epithelial cells from different locations of the human urinary tract and the renal tubular system. Arch. Toxicol. 2000;74:618–626. doi: 10.1007/s002040000173.
- Bharadwaj S, et al. Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology. Stem Cells. 2013;31:1840–1856. doi: 10.1002/stem.1424.
- CDER. Guidance for Industry and FDA Staff: Qualification Process for Drug Development Tools. 1–32 (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Silver Spring, 2014).
- Ihaka R, Gentleman R. R: a language for data analysis and graphics. J. Comput. Graph Stat. 1996;5:299–314.
- Raychaudhuri, S., Stuart, J.M. & Altman, R.B. Principal components analysis to summarize microarray experiments: application to sporulation time series. Pac. Symp. Biocomput. 455–466 (2000).
- Ringner M. What is principal component analysis? Nat. Biotechnol. 2008;26:303–304. doi: 10.1038/nbt0308-303.
- Wagner SD, et al. Dose-dependent regulation of alternative splicing by MBNL proteins reveals biomarkers for myotonic dystrophy. PLoS Genet. 2016;12:e1006316. doi: 10.1371/journal.pgen.1006316.
- Mevik BH, Wehrens R. The pls package: principal component and partial least squares regression in R. J. Stat. Softw. 2007;18:1–24. doi: 10.18637/jss.v018.i02.
- Spitali P, et al. Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy. Lab. Invest. 2010;90:1396–1402. doi: 10.1038/labinvest.2010.98.
- Verheul RC, van Deutekom JC, Datson NA. Digital droplet PCR for the absolute quantification of exon skipping induced by antisense oligonucleotides in (pre-)clinical development for duchenne muscular dystrophy. PLoS ONE. 2016;11:e0162467. doi: 10.1371/journal.pone.0162467.
- Liquori CL, et al. Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science. 2001;293:864–867. doi: 10.1126/science.1062125.
- Suominen T, et al. Population frequency of myotonic dystrophy: higher than expected frequency of myotonic dystrophy type 2 (DM2) mutation in Finland. Eur. J. Hum. Genet. 2011;19:776–782. doi: 10.1038/ejhg.2011.23.
- Wheeler TM, Lueck JD, Swanson MS, Dirksen RT, Thornton CA. Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy. J. Clin. Invest. 2007;117:3952–3957.
- Groh WJ, et al. Electrocardiographic abnormalities and sudden death in myotonic dystrophy type 1. N. Engl. J. Med. 2008;358:2688–2697. doi: 10.1056/NEJMoa062800.
- Motamedinia P, et al. Urine exosomes for non-invasive assessment of gene expression and mutations of prostate cancer. PLoS ONE. 2016;11:e0154507. doi: 10.1371/journal.pone.0154507.
- Urquidi V, et al. Urinary mRNA biomarker panel for the detection of urothelial carcinoma. Oncotarget. 2016;7:38731–38740.
- Du H, et al. Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy. Nat. Struct. Mol. Biol. 2010;17:187–193. doi: 10.1038/nsmb.1720.
- Mankodi A, et al. Myotonic dystrophy in transgenic mice expressing an expanded CUG repeat. Science. 2000;289:1769–1773. doi: 10.1126/science.289.5485.1769.
- Cirak S, et al. Exon skipping and dystrophin restoration in patients with Duchenne muscular dystrophy after systemic phosphorodiamidate morpholino oligomer treatment: an open-label, phase 2, dose-escalation study. Lancet. 2011;378:595–605. doi: 10.1016/S0140-6736(11)60756-3.
- Aartsma-Rus A, Krieg AM. FDA approves eteplirsen for duchenne muscular dystrophy: the next chapter in the eteplirsen saga. Nucleic Acid. Ther. 2017;27:1–3. doi: 10.1089/nat.2016.0657.
- Bouge AL, et al. Targeted RNA-seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle. Sci. Rep. 2017;7:39094. doi: 10.1038/srep39094.
- Davis BM, McCurrach ME, Taneja KL, Singer RH, Housman DE. Expansion of a CUG trinucleotide repeat in the 3’ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts. Proc. Natl Acad. Sci. USA. 1997;94:7388–7393. doi: 10.1073/pnas.94.14.7388.
- Martinez-Fernandez M, Paramio JM, Duenas M. RNA detection in urine: from RNA extraction to good normalizer molecules. J. Mol. Diagn. 2016;18:15–22. doi: 10.1016/j.jmoldx.2015.07.008.
- Imbeaud S, et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 2005;33:e56. doi: 10.1093/nar/gni054.
- Savkur RS, Philips AV, Cooper TA. Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat. Genet. 2001;29:40–47. doi: 10.1038/ng704.
- Koressaar T, Remm M. Enhancements and modifications of primer design program Primer3. Bioinformatics. 2007;23:1289–1291. doi: 10.1093/bioinformatics/btm091.
- Untergasser A, et al. Primer3--new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115. doi: 10.1093/nar/gks596.
Source: PubMed