A noninvasive assay for monitoring renal allograft status
Transplant rejection is a serious complication, sometimes threatening life of the patient. Although recent development of the new generation of immunosuppressive drugs reduced the incidence of acute rejection in kidney transplantation, the absence of noninvasive biomarkers of the rejection does not allow often the optimization of a prompt antirejection therapy. Serum creatinine is the most widely used marker for allograft function, however, it is not sensitive and specific enough to detect acute rejection. Other biomarkers are even less valuable for this purpose. Histological examination of renal allograft biopsy still remains the golden standard for diagnosing acute renal allograft rejection. Therefore, there is a high demand for reliable biomarkers for noninvasive monitoring of renal allograft status. Examination of urine in renal transplant recipients provides a logical and readily accessible approach for this monitoring. The high potency biomarkers for kidney allograft monitoring are fragments of DNA in recipient urine that originated from renal allograft cells. Because of the difference in the genetic origin these DNA can be distinguished from recipient DNA. Quantitative analysis of donor’s DNA, derived from cells of renal allograft, in recipient’s urine might be a reliable predictive tool for the kidney transplant rejection. We developed an assay to quantitate donor DNA content in recipient urine. Application of the technique—coamplification at lower denaturation temperature-PCR (COLD-PCR) increased the abundance of donor DNA that usually presents in recipient urine in quantities that are out of the detection range. This assay has a potential for routine application in clinical practice after statistical validation and additional modifications.
1. Muthukumar T, Dadhania D, Ding R, Snopkowski C, Naqvi R, et al. (2005) Messenger RNA for FOXP3 in the urine of renal-allograft recipients. N Engl J Med 353: 2342-2351.
2. Alachkar N (2012) Serum and urinary biomarkers in acute kidney transplant rejection. Nephrol Ther 8: 13-19.
3. Garcia Moreira V, Prieto Garcia B, Baltar Martin JM, Ortega Suarez F, Alvarez FV (2009) Cell-free DNA as a noninvasive acute rejection marker in renal transplantation. Clin Chem 55: 1958-1966.
4. Ting YT, Coates PT, Walker RJ, McLellan AD (2012) Urinary tubular biomarkers as potential early predictors of renal allograft rejection. Nephrology (Carlton) 17: 11-16.
5. Alachkar N, Rabb H, Jaar BG (2011) Urinary biomarkers in acute kidney transplant dysfunction. Nephron Clin Pract 118: c173-181; discussion c181.
6. Li Y, Hahn D, Zhong XY, Thomson PD, Holzgreve W, et al. (2003) Detection of donor-specific DNA polymorphisms in the urine of renal transplant recipients. Clin Chem 49: 655-658.
7. Zhong XY, Hahn D, Troeger C, Klemm A, Stein G, et al. (2001) Cell-free DNA in urine: a marker for kidney graft rejection, but not for prenatal diagnosis? Ann N Y Acad Sci 945: 250-257.
8. Zhang Z, Ohkohchi N, Sakurada M, Mizuno Y, Miyagi T, et al. (2001) Diagnosis of acute rejection by analysis of urinary DNA of donor origin in renal transplant recipients. Transplant Proc 33: 380-381.
9. Sigdel TK, Vitalone MJ, Tran TQ, Dai H, Hsieh SC, et al. (2013) A rapid noninvasive assay for the detection of renal transplant injury. Transplantation 96: 97-101.
10. Snyder TM, Khush KK, Valantine HA, Quake SR (2011) Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci U S A 108: 6229-6234.
11. Kang L, Zhang X, Liu K, Zhao J (2009) [Sequence polymorphisms of the mitochondrial DNA HVR I and HVR II regions in the Deng populations from Tibet in China]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 26: 690-695.
12. Kohnemann S, Sibbing U, Pfeiffer H, Hohoff C (2008) A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians. Int J Legal Med 122: 517-523.
13. Li J, Wang L, Mamon H, Kulke MH, Berbeco R, et al. (2008) Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 14: 579-584.
14. Castellanos-Rizaldos E, Milbury CA, Guha M, Makrigiorgos GM (2014) COLD-PCR enriches low-level variant DNA sequences and increases the sensitivity of genetic testing. Methods Mol Biol 1102: 623-639.
15. Milbury CA, Li J, Makrigiorgos GM (2011) Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic Acids Res 39: e2.
16. Castellanos-Rizaldos E, Liu P, Milbury CA, Guha M, Brisci A, et al. (2012) Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment. Clin Chem 58: 1130-1138.
17. Milbury CA, Li J, Makrigiorgos GM (2009) COLD-PCR-enhanced high-resolution melting enables rapid and selective identification of low-level unknown mutations. Clin Chem 55: 2130-2143.
18. Dwight Z, Palais R, Wittwer CT (2011) uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics 27: 1019-1020.
19. Cao P, Wang QJ, Zhu XT, Zhou H, Li R, et al. (2011) Quantitative determination of allele frequency in pooled DNA by using sequencing method. J Chromatogr B Analyt Technol Biomed Life Sci 879: 527-532.
20. Li J, Wang L, Janne PA, Makrigiorgos GM (2009) Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR. Clin Chem 55: 748-756.