Abstract
Although urine-based liquid biopsy has received considerable attention, there is a lack of a simple model to optimize assay parameters, including cell-free DNA (cfDNA) extraction, bisulfite modification, and bis-DNA recovery after conversion for methylation analysis in urine. The primary aim of this work was to establish a practical model by developing a quantitative methylation-sensitive PCR (qMS-PCR) assay for PAX2 based on hypermethylated PAX2 cfDNA that could be detected in healthy human urine. We first studied the methylation status of PAX2 in kidney tissues and whole blood, followed by an assessment of commercial kits for bisulfite conversion and bis-DNA recovery. Furthermore, we investigated the influence of urine storage and collection conditions on the preservation of methylated PAX2 in urine samples by qMS-PCR. As expected, PAX2 methylation was identified in urine but not in blood. Two commercial kits (CellCook and Zymo Research) had similar conversion efficiency and bis-DNA recovery. Urine storage for up to 5 days did not change PAX2 methylation estimates. Overall, cold storage of urine samples and the CellCook urine container maintained higher levels of methylated PAX2 compared to urine kept at room temperature and the conventional tubes, respectively. These findings highlight the importance of using the correct approaches/kits and optimizing experimental conditions as a diagnostic tool in the clinical setting. Our study provides insights on the development of urine-based liquid biopsy with DNA methylation as a universal biomarker.
Similar content being viewed by others
References
Heitzer, E., Ulz, P., & Geigl, J. B. (2015). Circulating tumor DNA as a liquid biopsy for cancer. Clinical Chemistry, 61(1), 112–123.
Frenel, J. S., Carreira, S., Goodall, J., Roda, D., Perez-Lopez, R., Tunariu, N., Riisnaes, R., Miranda, S., Figueiredo, I., Nava-Rodrigues, D., et al. (2015). Serial next-generation sequencing of circulating cell-free DNA evaluating tumor clone response to molecularly targeted drug administration. Clinical Cancer Research, 21(20), 4586–4596.
Salvi, S., Gurioli, G., De Giorgi, U., Conteduca, V., Tedaldi, G., Calistri, D., & Casadio, V. (2016). Cell-free DNA as a diagnostic marker for cancer: Current insights. Oncotargets and Therapy, 9, 6549–6559.
Garcia-Murillas, I., Schiavon, G., Weigelt, B., Ng, C., Hrebien, S., Cutts, R. J., Cheang, M., Osin, P., Nerurkar, A., Kozarewa, I., et al. (2015). Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer. Sci Transl Med, 7(302), 302ra133.
Roy, D., & Tiirikainen, M. (2020). Diagnostic power of DNA methylation classifiers for early detection of cancer. Trends Cancer, 6(2), 78–81.
Constancio, V., Nunes, S. P., Henrique, R., & Jeronimo, C. (2020). DNA Methylation-Based Testing in Liquid Biopsies as Detection and Prognostic Biomarkers for the Four Major Cancer Types. Cells, 9(3), 624.
Fleischhacker, M., & Schmidt, B. (2007). Circulating nucleic acids (CNAs) and cancer–a survey. Biochimica et Biophysica Acta, 1775(1), 181–232.
Holmes, E. E., Jung, M., Meller, S., Leisse, A., Sailer, V., Zech, J., Mengdehl, M., Garbe, L. A., Uhl, B., Kristiansen, G., et al. (2014). Performance evaluation of kits for bisulfite-conversion of DNA from tissues, cell lines, FFPE tissues, aspirates, lavages, effusions, plasma, serum, and urine. PLoS ONE, 9(4), 93933.
Izzi, B., Binder, A. M., & Michels, K. B. (2014). Pyrosequencing evaluation of widely available bisulfite conversion methods: Considerations for application. Medical Epigenet, 2(1), 28–36.
Liu, X., Ren, J., Luo, N., Guo, H., Zheng, Y., Li, J., Tang, F., Wen, L., & Peng, J. (2019). Comprehensive DNA methylation analysis of tissue of origin of plasma cell-free DNA by methylated CpG tandem amplification and sequencing (MCTA-Seq). Clinical Epigenetics, 11(1), 93.
Zhou, Y., Xiong, M., Niu, J., Sun, Q., Su, W., Zen, K., Dai, C., & Yang, J. (2014). Secreted fibroblast-derived miR-34a induces tubular cell apoptosis in fibrotic kidney. Journal of Cell Science, 127(Pt 20), 4494–4506.
Merchant, M. L., Rood, I. M., Deegens, J. K. J., & Klein, J. B. (2017). Isolation and characterization of urinary extracellular vesicles: Implications for biomarker discovery. Nature Reviews. Nephrology, 13(12), 731–749.
Priante, G., Gianesello, L., Ceol, M., Del Prete, D., & Anglani, F. (2019). Cell death in the kidney. International Journal of Molecular Sciences, 20(14), 3598.
Su, Y. H., Wang, M., Brenner, D. E., Ng, A., Melkonyan, H., Umansky, S., Syngal, S., & Block, T. M. (2004). Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. The Journal of Molecular Diagnostics, 6(2), 101–107.
Crisafulli, G., Mussolin, B., Cassingena, A., Montone, M., Bartolini, A., Barault, L., Martinetti, A., Morano, F., Pietrantonio, F., Sartore-Bianchi, A., et al. (2019). Whole exome sequencing analysis of urine trans-renal tumour DNA in metastatic colorectal cancer patients. ESMO Open, 4(6), e000572.
Salvi, S., Gurioli, G., Martignano, F., Foca, F., Gunelli, R., Cicchetti, G., De Giorgi, U., Zoli, W., Calistri, D., & Casadio, V. (2015). Urine Cell-Free DNA integrity analysis for early detection of prostate cancer patients. Disease Markers, 2015, 574120.
Casadio, V., Salvi, S., Martignano, F., Gunelli, R., Ravaioli, S., & Calistri, D. (2017). Cell-Free DNA integrity analysis in urine samples. Journal of Visualized Experiments. https://doi.org/10.3791/55049
Smith, C. G., Moser, T., Mouliere, F., Field-Rayner, J., Eldridge, M., Riediger, A. L., Chandrananda, D., Heider, K., Wan, J. C. M., Warren, A. Y., et al. (2020). Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors. Genome Med, 12(1), 23.
Hong, S. R., & Shin, K. J. (2021). Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion. Frontiers in Genetics, 12, 618955.
Grunau, C., Clark, S. J., & Rosenthal, A. (2001). Bisulfite genomic sequencing: Systematic investigation of critical experimental parameters. Nucleic Acids Research, 29(13), E65-65.
Leontiou, C. A., Hadjidaniel, M. D., Mina, P., Antoniou, P., Ioannides, M., & Patsalis, P. C. (2015). Bisulfite conversion of DNA: Performance comparison of different kits and methylation quantitation of epigenetic biomarkers that have the potential to be used in non-invasive prenatal testing. PLoS ONE, 10(8), e0135058.
Kint, S., De Spiegelaere, W., De Kesel, J., Vandekerckhove, L., & Van Criekinge, W. (2018). Evaluation of bisulfite kits for DNA methylation profiling in terms of DNA fragmentation and DNA recovery using digital PCR. PLoS ONE, 13(6), e0199091.
Wu, H., Chen, Y., Liang, J., Shi, B., Wu, G., Zhang, Y., Wang, D., Li, R., Yi, X., Zhang, H., et al. (2005). Hypomethylation-linked activation of PAX2 mediates tamoxifen-stimulated endometrial carcinogenesis. Nature, 438(7070), 981–987.
Patricio, P., Ramalho-Carvalho, J., Costa-Pinheiro, P., Almeida, M., Barros-Silva, J. D., Vieira, J., Dias, P. C., Lobo, F., Oliveira, J., Teixeira, M. R., et al. (2013). Deregulation of PAX2 expression in renal cell tumours: Mechanisms and potential use in differential diagnosis. Journal of Cellular and Molecular Medicine, 17(8), 1048–1058.
Chen, X., Zhang, J., Ruan, W., Huang, M., Wang, C., Wang, H., Jiang, Z., Wang, S., Liu, Z., Liu, C., et al. (2020). Urine DNA methylation assay enables early detection and recurrence monitoring for bladder cancer. The Journal of Clinical Investigation, 130(12), 6278–6289.
Su, Y. H., Wang, M., Brenner, D. E., Norton, P. A., & Block, T. M. (2008). Detection of mutated K-ras DNA in urine, plasma, and serum of patients with colorectal carcinoma or adenomatous polyps. Annals of the New York Academy of Sciences, 1137, 197–206.
Bach, S., Paulis, I., Sluiter, N. R., Tibbesma, M., Martin, I., van de Wiel, M. A., Tuynman, J. B., Bahce, I., Kazemier, G., & Steenbergen, R. D. M. (2021). Detection of colorectal cancer in urine using DNA methylation analysis. Science and Reports, 11(1), 2363.
Oshi, M., Murthy, V., Takahashi, H., Huyser, M., Okano, M., Tokumaru, Y., Rashid, O. M., Matsuyama, R., Endo, I., & Takabe, K. (2021). Urine as a source of liquid biopsy for Cancer. Cancers (Basel), 13(11), 2652.
Liu, B., Ricarte Filho, J., Mallisetty, A., Villani, C., Kottorou, A., Rodgers, K., Chen, C., Ito, T., Holmes, K., Gastala, N., et al. (2020). Detection of promoter DNA methylation in urine and plasma aids the detection of non-small cell lung cancer. Clinical Cancer Research, 26(16), 4339–4348.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Zhou, Xc., Chen, Qp., Yuan, Jj. et al. Kidney-Derived Methylated PAX2 Sequences in the Urine of Healthy Subjects as a Convenient Model for Optimizing Methylation-Based Liquid biopsy. Mol Biotechnol 64, 1088–1094 (2022). https://doi.org/10.1007/s12033-022-00481-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-022-00481-5