Cell-free DNA levels of twins and sibling pairs indicate individuality and possible use as a personalized biomarker

Autoři: Lamyaa Alghofaili aff001;  Hannah Almubarak aff001;  Khawlah Gassem aff001;  Syed S. Islam aff001;  Serdar Coskun aff003;  Namik Kaya aff004;  Bedri Karakas aff001
Působiště autorů: Translational Cancer Research Section, Department of Molecular Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia aff001;  Alfaisal University Medical School, Riyadh, Saudi Arabia aff002;  Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia aff003;  Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia aff004
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223470


Cell-free DNA (cfDNA) in the human blood circulation has been under investigation since its initial observation in 1948. Plasma cfDNA is known to be significantly elevated in diseased people. Due to possible variation in the population, evaluating cfDNA as a non-invasive biomarker at disease onset alone may not be sensitive enough to accurately diagnose diseases, particularly early stage cancers on a personal level. To understand the factors that define the cfDNA levels on the personal level and for better use as a non-invasive biomarker, we isolated cfDNA from the plasma of healthy individuals with varying degrees of genetic and/or environmental similarities (monozygotic twins, dizygotic twins, sibling pairs, and unrelated individuals) as well as from patients with varying stages of breast and ovarian cancer undergoing treatment. Cell-free DNA levels were quantified by a fluorometer (ng/ml) and/or real-time PCR (copies/ml). The associations between individuals with various degrees of genetic and/or environmental similarities and their plasma cfDNA levels were evaluated. The ACE model (A = additive genetic, C = common environment, and E = specific environmental factors) was used to determine the proportion of each factor on the cfDNA levels. We found a high correlation (r = 0.77; p < 0.0001) in plasma cfDNA levels between monozygotic twins (n = 39). However, the correlation was gradually reduced to moderate (r = 0.47; p = 0.016) between dizygotic twins (n = 13) and low correlation (r = 0.28; p = 0.043) between sibling pairs (n = 26). The ACE model analysis showed that the plasma cfDNA level of a given healthy individual is influenced both by genetic and the environmental components in similar proportions (53% and 47%, respectively; A = 53%, C = 22.5%, E = 24.5%). Moreover, while age had no effect, gender significantly influenced the individual’s plasma cfDNA level. As expected, cfDNA levels were significantly higher in both breast (n = 26) (p<0.0001) and ovarian (n = 64) (p<0.0001) cancer patients compared to the healthy individuals. Our study demonstrated that both genome and environmental factors modulate the individual’s cfDNA level suggesting that its diagnostic sensitivity may be improved only if the person’s cfDNA level is known prior to disease presentation.

Klíčová slova:

Biomarkers – Blood plasma – Human genetics – Ovarian cancer – Polymerase chain reaction – Twins – Monozygotic twins – Dizygotic twins


1. Mandel P. Les acides nucleiques du plasma sanguin chez l'homme. CR Acad Sci Paris. 1948;142: 241–243.

2. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Research. 1977;37: 646–650. 837366

3. Steinman CR, Ackad A. Appearance of circulating DNA during hemodialysis. The American Journal of Medicine. 1977;62: 693–697. doi: 10.1016/0002-9343(77)90872-5 871126

4. Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiology Biomarkers & Prevention. 1994;3: 67–71.

5. Johnson PJ, Lo YMD. Plasma nucleic acids in the diagnosis and management of malignant disease. Clin Chem. 2002;48: 1186–1193. 12142371

6. Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. The Lancet. 1997;350: 485–487. doi: 10.1016/S0140-6736(97)02174-0

7. Li Y, Holzgreve W, Di Naro E, Vitucci A, Hahn S. Cell-Free DNA in Maternal Plasma: Is It All a Question of Size? Annals of the New York Academy of Sciences. 2006;1075: 81–87. doi: 10.1196/annals.1368.010 17108195

8. Ivanov M, Baranova A, Butler T, Spellman P, Mileyko V. Non-random fragmentation patterns in circulating cell-free DNA reflect epigenetic regulation. BMC Genomics. BioMed Central; 2015;16 Suppl 13: S1. doi: 10.1186/1471-2164-16-S13-S1 26693644

9. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999;64: 218–224. doi: 10.1086/302205 9915961

10. Jahr S, Hentze H, Englisch S, Hardt D, Fackelmayer FO, Hesch RD, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Research. 2001;61: 1659–1665. 11245480

11. Mouliere F, Chandrananda D, Piskorz AM, Moore EK, Morris J, Ahlborn LB, et al. Enhanced detection of circulating tumor DNA by fragment size analysis. Science Translational Medicine. American Association for the Advancement of Science; 2018;10: eaat4921. doi: 10.1126/scitranslmed.aat4921 30404863

12. Anker P, Stroun M, Maurice PA. Spontaneous extracellular synthesis of DNA released by human blood lymphocytes. Cancer Research. 1976;36: 2832–2839. 1277193

13. Ziegler A, Zangemeister-Wittke U, Stahel RA. Circulating DNA: a new diagnostic gold mine? Cancer Treat Rev. 2002;28: 255–271. doi: 10.1016/S0305-7372(02)00077-4 12435372

14. Schwarzenbach H, Hoon DSB, Pantel K. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer. Nature Publishing Group. Nature Publishing Group; 2011;11: 426–437. doi: 10.1038/nrc3066 21562580

15. Xue X, Teare MD, Holen I, Zhu YM, Woll PJ. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clinica Chimica Acta. 2009;404: 100–104. doi: 10.1016/j.cca.2009.02.018 19281804

16. Schmidt B, Weickmann S, Witt C, Fleischhacker M. Improved method for isolating cell-free DNA. Clin Chem. Clinical Chemistry; 2005;51: 1561–1563. doi: 10.1373/clinchem.2005.051003 16040863

17. Page K, Powles T, Slade MJ, De Bella MT, Walker RA, Coombes RC, et al. The Importance of Careful Blood Processing in Isolation of Cell-Free DNA. Annals of the New York Academy of Sciences. 2006;1075: 313–317. doi: 10.1196/annals.1368.042 17108226

18. Steinman CR. Free DNA in serum and plasma from normal adults. J Clin Invest. American Society for Clinical Investigation; 1975;56: 512–515. doi: 10.1172/JCI108118 1150882

19. Gal S, Fidler C, Lo YMD, Taylor M, Han C, Moore J, et al. Quantitation of circulating DNA in the serum of breast cancer patients by real-time PCR. Br J Cancer. 2004;90: 1211–1215. doi: 10.1038/sj.bjc.6601609 15026803

20. Yoon K-A, Park S, Lee SH, Kim JH, Lee JS. Comparison of circulating plasma DNA levels between lung cancer patients and healthy controls. J Mol Diagn. 2009;11: 182–185. doi: 10.2353/jmoldx.2009.080098 19324991

21. Sozzi G, Conte D, Mariani L, Vullo SL, Roz L, Lombardo C, et al. Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Research. AACR; 2001;61: 4675–4678.

22. Papadopoulou E, Davilas E, Sotiriou V, Georgakopoulos E, Georgakopoulou S, Koliopanos A, et al. Cell-free DNA and RNA in plasma as a new molecular marker for prostate and breast cancer. Annals of the New York Academy of Sciences. 2006;1075: 235–243. doi: 10.1196/annals.1368.032 17108217

23. Gormally E, Hainaut P, Caboux E, Airoldi L, Autrup H, Malaveille C, et al. Amount of DNA in plasma and cancer risk: A prospective study. Int J Cancer. Wiley Subscription Services, Inc., A Wiley Company; 2004;111: 746–749. doi: 10.1002/ijc.20327 15252845

24. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. Circulating mutant DNA to assess tumor dynamics Suppl Info. Nat Med. 2008;14: 985–990. doi: 10.1038/nm.1789 18670422

25. Murtaza M, Dawson S-J, Tsui DWY, Gale D, Forshew T, Piskorz AM, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 2013;497: 108–112. doi: 10.1038/nature12065 23563269

26. Karakas B. Allele-specific Emulsion PCR (asePCR) as a Liquid Biopsy Method for Residual Tumor Detection. Anatolian Clinic Journal of Medical Sciences. 2019;24: 47–52. doi: 10.21673/anadoluklin.441594

27. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. New England Journal of Medicine. 2012;366: 883–892. doi: 10.1056/NEJMoa1113205 22397650

28. Chan KCA, Jiang P, Zheng YWL, Liao GJW, Sun H, Wong J, et al. Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. Clin Chem. 2013;59: 211–224. doi: 10.1373/clinchem.2012.196014 23065472

29. Kleppe M, Levine RL. Tumor heterogeneity confounds and illuminates: assessing the implications. Nat Med. 2014;20: 342–344. doi: 10.1038/nm.3522 24710377

30. Heitzer E, Ulz P, Geigl JB. Circulating Tumor DNA as a Liquid Biopsy for Cancer. Clin Chem. 2014;61: 112–123. doi: 10.1373/clinchem.2014.222679 25388429

31. Karakas B, Qubbaj W, Al-Hassan S, Coskun S. Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer. Wanunu M, editor. PLoS ONE. 2015;10: e0126501–10 doi: 10.1371/journal.pone.0126501 25970589

32. Rago C, Huso DL, Diehl F, Karim B, Liu G, Papadopoulos N, et al. Serial assessment of human tumor burdens in mice by the analysis of circulating DNA. Cancer Research. 2007;67: 9364–9370. doi: 10.1158/0008-5472.CAN-07-0605 17909045

33. Schork NJ. Personalized medicine: Time for one-person trials. Nature. 2015;520: 609–611. doi: 10.1038/520609a 25925459

34. Sahin U, Türeci Ö. Personalized vaccines for cancer immunotherapy. Science. American Association for the Advancement of Science; 2018;359: 1355–1360 doi: 10.1126/science.aar7112 29567706

35. Rubin MA. Health: Make precision medicine work for cancer care. Nature. 2015;520: 290–291. doi: 10.1038/520290a 25877189

36. No JH, Kim K, Park KH, Kim Y-B. Cell-free DNA level as a prognostic biomarker for epithelial ovarian cancer. Anticancer Res. 2012;32: 3467–3471. 22843932

37. Skvortsova TE, Rykova EY, Tamkovich SN, Bryzgunova OE, Starikov AV, Kuznetsova NP, et al. Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation. Br J Cancer. 2006;94: 1492–1495. doi: 10.1038/sj.bjc.6603117 16641902

38. Boomsma D, Busjahn A, Peltonen L. Classical twin studies and beyond. Nat Rev Genet. 2002;3: 872–882. doi: 10.1038/nrg932 12415317

39. van Dongen J, Slagboom PE, Draisma HHM, Martin NG, Boomsma DI. The continuing value of twin studies in the omics era. Nat Rev Genet. 2012;13: 640–653. doi: 10.1038/nrg3243 22847273

40. Kong A, Thorleifsson G, Frigge ML, Vilhjalmsson BJ, Young AI, Thorgeirsson TE, et al. The nature of nurture: Effects of parental genotypes. Science. American Association for the Advancement of Science; 2018;359: 424–428. doi: 10.1126/science.aan6877 29371463

41. Al-Moghrabi N, Al-Showimi M, Al-Yousef N, Al-Shahrani B, Karakas B, Alghofaili L, et al. Methylation of BRCA1 and MGMT genes in white blood cells are transmitted from mothers to daughters. Clin Epigenet. 2018;10: 1861–10. doi: 10.1186/s13148-018-0529-5 30049288

42. Kamat AA, Sood AK, Dang D, Gershenson DM, Simpson JL, Bischoff FZ. Quantification of Total Plasma Cell-Free DNA in Ovarian Cancer Using Real-Time PCR. Annals of the New York Academy of Sciences. 2006;1075: 230–234. doi: 10.1196/annals.1368.031 17108216

43. Teo YV, Capri M, Morsiani C, Pizza G, Faria AMC, Franceschi C, et al. Cell-free DNA as a biomarker of aging. Aging Cell. 2018;18: e12890–14. doi: 10.1111/acel.12890 30575273

Článek vyšel v časopise


2019 Číslo 10