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Letters to the Editor |
1 University Women’s Hospital, University of Basel, Basel, Switzerland
2 Division of Paediatric Nephrology, University of the Witwatersrand, and Johannesburg Hospital, Johannesburg, South Africa
aAddress correspondence to this author at: Laboratory for Prenatal Medicine, University Women’s Hospital/Department of Research, Spitalstrasse 21, CH 4031 Basel, Switzerland. Fax 41-61-265-9399; e-mail shahn{at}unbs.ch.
To the Editor:
Several studies have reported that transplant-derived cell-free DNA (CF-DNA) can be detected in the urine of renal transplant recipients (1)(2)(3)(4)(5). These studies have indicated that the increased excretion of CF-DNA is associated with acute graft rejection but not with drug-induced kidney dysfunction (1)(2)(3)(5).
A limitation of these studies was that they frequently relied on the detection of Y-chromosome–specific sequences in sex-disparate donor–recipient pairs (1)(2)(5). Although the use of specific human lymphocyte antigens mismatches was examined, this approach is reliably useful only when donor and recipient are unrelated (5). We previously reported that the analysis of highly polymorphic short tandem repeat (STR) loci can be exploited for the detection of donor-specific urinary CF-DNA (4). Although we were able to detect such urinary CF-DNA in all of the cases studied, this approach as currently applied cannot reliably provide an accurate quantitative assessment of donor CF-DNA. For this reason, we are exploring alternative technologies.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been shown to permit the reliable detection of single-nucleotide polymorphisms (SNPs) (6), including fetal point mutations from fetal CF-DNA in maternal plasma (7). Such detection cannot be reliably performed by more-conventional PCR-based approaches (8). Using competitive PCR with oligonucleotides specific for the donor allele to be analyzed, MALDI-TOF MS allows quantitative allele-specific analyses (6).
We first sought to ascertain whether MS will permit the detection of donor-specific SNPs in urinary CF-DNA. For our small proof-of-concept study, we examined 20 SNP markers (G/C polymorphism; refer to Table 1
; kindly provided by Sequenom, Inc., San Diego, CA). These were tested on 4 cases involving related living-donor kidney transplants, which we have previously examined for the presence of STR loci (4). For our current analysis, urinary DNA was extracted from 1 mL of recipient urine by commercial column technology (Roche) (9) and eluted in 50 µL of elution buffer, of which 3 µL was used for the subsequent PCR reaction. This amount corresponds to more than 3000 genome-equivalents of CF-DNA/mL of urine in these samples, which had collected within 24 h of transplantation (2)(4).
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SNP loci were analyzed by the MassEXTEND MassARRAY® assay (Sequenom) (6)(7). Genotyping of the donor–recipient pairs, performed on 4 ng of genomic DNA, indicated that in only 16 instances was the donor SNP allele absent from the recipient genome (Table 1
). The pertinent donor SNP allele was detected in each of these 16 instances with allelic differences (Table 1
). Four representative traces are illustrated in the figure that accompanies the online version of this letter at http://www.clinchem.org/content/vol51/issue9/.
Our data therefore suggest that the analysis of such SNP markers by MS may provide a new alternative for the detection of donor-derived CF-DNA. The accuracy of this approach will, however, have to be examined in a larger scale study and include samples taken at later postoperative stages, when the concentrations of urinary CF-DNA are lower than those examined in this study (2). In addition, it remains to be determined whether this MS approach will permit the reliable quantification of donor SNPs in urinary CF-DNA. As donor-derived CF-DNA has been found in the plasma of other organ transplantation recipients (10), this approach may be useful for the monitoring of transplant rejection in these patients as well.
References
The following articles in journals at HighWire Press have cited this article:
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  Y. Li, W. Holzgreve, V. Kiefer, and S. Hahn MALDI-TOF Mass Spectrometry Compared With Real-Time PCR for Detection of Fetal Cell-Free DNA in Maternal Plasma Clin. Chem., December 1, 2006; 52(12): 2311 - 2312. [Full Text] [PDF]
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