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Letters to the Editor |
1 Accident and Emergency Medicine, Academic Unit
2 Department of Chemical Pathology
3 Li Ka Shing Institute of Health, Sciences and
4 Department of Paediatrics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR
aAddress correspondence to this author at: Department of Chemical Pathology, Rm. 38061, 1/F, Clinical Sciences Bldg., Prince of Wales Hospital, 30-32 Ngan Shing St., Shatin, Hong Kong SAR. Fax 852-2636-5090; e-mail loym{at}cuhk.edu.hk.
To the Editor:
The analysis of circulating RNA in plasma opens up new possibilities for the noninvasive monitoring of a variety of physiological and pathological conditions (1). In this study, we demonstrate the usefulness of a bone marrow transplantation (BMT) model system for ascertaining the tissue origin of plasma RNA species. These data were generated as part of a project to develop plasma nucleic acid markers for brain injury. Because brain injury, such as stroke, involves cell death and disruption of the blood-brain barrier, we hypothesized that brain-derived nucleic acids may be released into the circulation. Hence, the measurement of brain-derived RNA markers in plasma may reflect the degree of brain tissue damage.
Numerous reports indicate that S100 calcium binding protein B (S100B) is released after brain injury and its concentrations in serum are significantly correlated with injury severity and outcome (2). To investigate whether S100B mRNA might be detectable in the plasma and might serve as a marker for brain injury, we used a real-time reverse transcriptase PCR assay to measure plasma S100B mRNA concentrations in 67 stroke patients and 16 healthy age- and sex-matched individuals. Among the stroke patients, stroke was diagnosed as ischemic in 48 patients and hemorrhagic in 6 patients, and 13 patients had no acute changes on computed tomography or magnetic resonance neuroimaging. Median patient scores for the National Institutes of Health Stroke Scale (NIHSS) and Glasgow Coma Scale (GCS) were 5 (range 0–34) and 15 (range 4–15), respectively. The median time from symptom onset to blood sampling was 11 h (range 1–23.8 h). Eight stroke patients died during hospitalization or within 30 days after discharge. Plasma S100B mRNA concentrations, however, showed no statistically significant differences in stroke patients compared with controls (P = 0.38, Mann–Whitney test), and plasma S100B mRNA concentrations in stroke patients showed no statistically significant correlation with NIHSS or GCS scores. These unexpected results prompted us to investigate whether the detected plasma S100B mRNA was indeed derived from the brain. Previous data from a BMT model have demonstrated that hematopoietic cells are important contributors of plasma DNA. It is possible that hematopoietic cells might also contribute to the detected plasma S100B mRNA through illegitimate transcription(3).
We thus proceeded to use the BMT model to test whether the hematopoietic system might indeed contribute significantly to plasma S100B mRNA. Genotypes of S100B mRNA molecules in plasma of recipients after BMT were compared with those before BMT as well as with the donor genotypes. Contribution of plasma S100B mRNA by blood cells could be inferred if the transcripts in plasma of recipients bore the donor genotypes among the informative donor-recipient pairs, in which the donor and recipient had different S100B genotypes before BMT.
The local clinical research ethics committee approved the study. Twenty-three myeloablative BMT patients who attended the Department of Paediatrics of the Prince of Wales Hospital, Hong Kong, were recruited, and informed consent was obtained from either the patients or responsible guardians. Archived pre-BMT peripheral blood mononuclear cells (PBMCs) of the recipients were retrieved for DNA extraction. A post-BMT blood sample (5 mL EDTA) was withdrawn from each patient and then centrifuged at 100g for 10 min at 4 °C. Plasma and buffy coat samples were then collected separately. Plasma samples were filtered with a 5-µm filter to remove any residual blood cells. Buffy coat samples were centrifuged at 230g for 5 min at 4 °C, and any residual plasma was removed.
The methods of nucleic acid extraction and single nucleotide polymorphism (SNP) analysis of RNA molecules, i.e., RNA-SNP, were as previously described (4)(5). In brief, the analysis was based on the detection by mass spectrometry of an SNP within an RNA transcript after a primer extension reaction. The extension products for each allele demonstrated distinct masses that could be resolved by MALDI-TOF mass spectrometry.
A highly polymorphic C/T SNP, rs9722 (minor allele frequency 0.43), located within the coding region of S100B was selected from the Sequenom RealSNPTM Assay Database. Post-BMT plasma S100B genotypes of recipients were determined from the RNA specimens that were extracted from the recipients’ post-BMT plasma samples. Pre-BMT S100B genotypes of the recipients were determined from the DNA specimens that were extracted from the archived pre-BMT PBMC samples of the recipients. The S100B genotypes of the bone marrow donors were determined from both DNA and RNA specimens that were extracted from the post-BMT buffy coat samples of the corresponding recipients.
As shown in Table 1
, 8 of 23 donor-recipient pairs were informative. The S100B mRNA genotypes in the plasma of these recipients were altered after BMT and became identical to those of the corresponding donors.
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Our study has clearly demonstrated that after BMT the S100B mRNA in recipient plasma switched to the donor’s genotype. Thus, plasma S100B was predominantly of hematopoietic origin and could not be used as a brain-specific plasma marker. Hence, caution is required in the interpretation of the presumed tissue origin of plasma RNA markers. The BMT model described here can provide a valuable confirmation step for showing or excluding the hematopoietic contribution of a plasma RNA marker.
Acknowledgments
Grant/funding support: This work was supported by funding from the Li Ka Shing Foundation and the Chinese University of Hong Kong research direct grant account 4450148. Y.M.D.L. is supported by the Chair Professorship Scheme of the Li Ka Shing Foundation.
Financial disclosures: T.H.R., N.Y.L.L., R.W.K.C., and Y.M.D.L. have filed patent applications on aspects of plasma nucleic acid analysis.
References
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