Rebecca Wing-Yan Chan

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

Significance Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA, we obtained a bird’s eye view of the identities and contributions of these tissues to the circulating DNA pool. The tissue contributors and their relative proportions are identified by a bioinformatics deconvolution process that draws reference from DNA methylation signatures representative of each tissue type. We validated this approach in pregnant women, cancer patients, and transplant recipients. This method also allows one to identify the tissue of origin of genomic aberrations observed in plasma DNA. This approach has numerous research and diagnostic applications in prenatal testing, oncology, transplantation monitoring, and other fields. Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.

Intrarenal cytokine gene expression in lupus nephritis

Background: Lupus nephritis is characterised by intrarenal inflammation and lymphocyte activation. Aim: To examine the profile of cytokine gene expression in glomerulus and tubulointerstitium in patients with lupus nephritis. Methods: 36 consecutive patients with systemic lupus erythematosus having active renal disease were recruited, and they were required to undergo kidney biopsy. Glomerular and tubulointestitial cytokine expression of interleukin (IL)2, 4, 10, 12, 18, interferon γ (IFN)γ, T-bet (the Th1 transcription factor), GATA-3 (the Th2 transcription factor), transforming growth factorβ and monocyte chemoattractant protein (MCP)1 were studied by laser microdissection of the renal biopsy specimen, followed by real-time quantitative PCR. Results: There were 13 patients with World Health Organization class III nephritis, 14 patients with class IV nephritis and 9 patients with class V nephritis. There was a significant correlation between serum C3, C4 and anti-double strand DNA antibody level with glomerular expression of T-bet, IFNγ and IL2. There was a significant correlation between histological activity index and glomerular expression of IL12, IL18, IL10 and MCP1. In addition, the degree of glomerular leucocyte infiltration significantly correlated with glomerular expression of IFNγ, IL10, IL12 and IL18. By contrast, histological chronicity index correlated with the tubulointerstitial expression of IL2, MCP1 and GATA-3. Conclusions: Intraglomerular expression of certain target genes correlate with the severity of systemic as well as histological activity, whereas the tubulointerstitial expression of other target genes correlate with the degree of chronic kidney scarring. This result may shed light on the immunopathogenesis of lupus nephritis.

Plasma DNA aberrations in systemic lupus erythematosus revealed by genomic and methylomic sequencing

Significance Through the use of massively parallel sequencing, we have demonstrated a spectrum of plasma DNA abnormalities in patients with systemic lupus erythematosus. These abnormalities include aberrant measured genomic representations, hypomethylation, and DNA fragment size shortening. The binding of anti-double–stranded DNA antibody to plasma DNA appears to be an important factor associated with these abnormalities. These findings provide valuable insights into the biology of plasma DNA in an autoimmune disease and have potential implications for the development of new molecular markers for systemic lupus erythematosus. We performed a high-resolution analysis of the biological characteristics of plasma DNA in systemic lupus erythematosus (SLE) patients using massively parallel genomic and methylomic sequencing. A number of plasma DNA abnormalities were found. First, aberrations in measured genomic representations (MGRs) were identified in the plasma DNA of SLE patients. The extent of the aberrations in MGRs correlated with anti-double–stranded DNA (anti-dsDNA) antibody level. Second, the plasma DNA of active SLE patients exhibited skewed molecular size-distribution profiles with a significantly increased proportion of short DNA fragments. The extent of plasma DNA shortening in SLE patients correlated with the SLE disease activity index (SLEDAI) and anti-dsDNA antibody level. Third, the plasma DNA of active SLE patients showed decreased methylation densities. The extent of hypomethylation correlated with SLEDAI and anti-dsDNA antibody level. To explore the impact of anti-dsDNA antibody on plasma DNA in SLE, a column-based protein G capture approach was used to fractionate the IgG-bound and non–IgG-bound DNA in plasma. Compared with healthy individuals, SLE patients had higher concentrations of IgG-bound DNA in plasma. More IgG binding occurs at genomic locations showing increased MGRs. Furthermore, the IgG-bound plasma DNA was shorter in size and more hypomethylated than the non–IgG-bound plasma DNA. These observations have enhanced our understanding of the spectrum of plasma DNA aberrations in SLE and may provide new molecular markers for SLE. Our results also suggest that caution should be exercised when interpreting plasma DNA-based noninvasive prenatal testing and cancer testing conducted for SLE patients.

Messenger RNA expression of RANTES in the urinary sediment of patients with lupus nephritis

Background:  Lupus nephritis is characterized by intra‐renal inflammation. Patients with systemic lupus erythematosus (SLE) showed abnormal T‐cell expression of RANTES (regulated upon activation, normal T cell expressed) and its level in their serum. The authors studied the mRNA expression of RANTES in the urinary sediment of lupus patients.

Matrix metalloproteinase 9 mRNA: An early prognostic marker for patients with acute stroke

OBJECTIVES To investigate matrix metalloproteinase 9 (MMP9) mRNA as a prognostic marker in stroke. DESIGN AND METHODS MMP9 mRNA concentrations in 126 stroke patients were analyzed using quantitative reverse transcription-polymerase chain reaction. RESULTS The normalized MMP9 mRNA concentration was almost 3 times higher in non-survival patients compared to survival patients (P=0.0002); and 1.9-fold higher in patients with post-stroke modified Rankin score (mRS) >2 than patients with mRS≤2 (P<0.05). CONCLUSIONS MMP9 mRNA was a predictor of poor outcome and mortality in stroke.

Aberrant Concentrations of Liver-Derived Plasma Albumin mRNA in Liver Pathologies

BACKGROUND We hypothesized that liver-derived mRNA, such as ALB (albumin) mRNA, would be released into human plasma with liver cell death. METHODS We genotyped ALB mRNA molecules in samples of plasma and whole blood from liver and bone marrow transplant recipients by RNA single-nucleotide polymorphism analysis. Plasma and whole blood ALB mRNA genotypes were compared with the DNA genotypes of the recipients and donors. A reverse-transcription quantitative real-time PCR assay was used to measure plasma ALB mRNA concentrations in 107 patients [hepatocellular carcinoma (HCC), cirrhosis, or chronic hepatitis B (CHB)] and 207 healthy controls. RESULTS The RNA genotype data revealed ALB mRNA in plasma to be liver derived, whereas tissue compartments other than the liver also contributed to the ALB mRNA detected in whole blood. Statistically significant increases in plasma ALB mRNA concentrations were observed for HCC, cirrhosis, and active CHB, compared with controls. A cutoff of 835 copies/mL of plasma ALB mRNA identified by ROC curve analysis showed 85.5% diagnostic sensitivity and 92.8% diagnostic specificity for the detection of liver pathologies. Only 21.5% of patients with liver pathologies had increased alanine aminotransferase (ALT) activities, whereas 73.8% had increased plasma ALB mRNA concentrations. Only 48.6% of the HCC patients had increased serum alpha-fetoprotein concentrations, whereas 91.4% had increased plasma ALB mRNA concentrations. CONCLUSIONS ALB mRNA is liver specific in plasma, but not in whole blood. Plasma ALB mRNA is increased in some liver pathologies and may be more diagnostically sensitive than alpha-fetoprotein and ALT.

Multiple atherosclerosis-related biomarkers associated with short- and long-term mortality after stroke.

OBJECTIVES We investigated the relationships of biomarkers of various pathophysiologic pathways including high-sensitivity C-reactive protein (hs-CRP), lipocalin-2 (LCN2), myeloperoxidase (MPO) and matrix metalloproteinases 9 (MMP9) with mortality in stroke patients. DESIGN AND METHODS hs-CRP, LCN2 and MPO concentrations in 92 patients were determined using enzyme-linked immunosorbent assays. MMP9 mRNA concentrations were determined using real-time quantitative reverse transcription-polymerase chain reaction. RESULTS Twelve patients (13.0%) died at 6 months and 34 patients (37.0%) died at 5 years. The independent predictors for 6-month mortality were hs-CRP (adjusted OR=16.0) and LCN2 (adjusted OR=16.9), while for 5-year mortality was hs-CRP (adjusted OR=5.56). For patients with hs-CRP >3.4 mg/L, an increase in LCN2 was associated with 2.5-fold higher 6-month mortality, while an increase in normalized MMP9 mRNA was associated with 5.8-fold higher 6-month and 1.5-fold higher 5-year mortality. CONCLUSION hs-CRP was the most significant independent predictor of both short- and long-term mortality after stroke, with LCN2 and MMP9 mRNA each adding further to the risk stratification.

DNA of Erythroid Origin is Present in Human Plasma and Informs the Types of Anemia

BACKGROUND There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. METHODS Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. RESULTS Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. CONCLUSIONS Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.

The potential clinical utility of serial plasma albumin mRNA monitoring for the post-liver transplantation management.

OBJECTIVES Elevated albumin (ALB) mRNA concentration has been reported in the plasma of patients with liver diseases. The plasma ALB mRNA measurement was shown to be an effective indicator of liver pathologies with superior diagnostic sensitivity and specificity when compared with alanine transaminase (ALT). We hypothesized that serial plasma ALB mRNA analysis would be helpful in the early detection and monitoring of post-liver transplantation complications. DESIGN AND METHODS One hundred and five blood specimens were collected from 24 post-transplant recipients. Biochemical liver function test profiles and plasma ALB mRNA concentrations were assessed. RESULTS Over the study period, the health status of 14 recipients (58%) remained stable (Stable group). Their plasma ALB mRNA concentrations remained within a low-concentration range. In contrast, 10 recipients (42%) developed 14 episodes of hepatic complications (Unstable group). The median plasma ALB mRNA concentration of the Unstable group was 6.5-times higher than that of the Stable group. Plasma ALB mRNA concentration was elevated on 13/14 (93%) episodes of the hepatic complications while ALT was elevated only on 8/14 (57%) episodes. CONCLUSIONS The elevation of plasma ALB mRNA may allow sensitive detection of hepatic complications and monitoring of the clinical course in a dynamic fashion. Serial plasma ALB mRNA measurement is potentially useful for post-liver transplantation management.

Early Time-Dependent Dynamic Changes of TBET and GATA3 mRNA Expressions in Patients with Acute Coronary Syndrome

Background. T-box expressed in T cells (TBET) and guanine adenine thymine adenine sequence-binding protein 3 (GATA3) play important roles in the differentiation of Th1 and Th2 subsets, which contributes to the progression of acute coronary syndrome (ACS). Objective. This study aimed to investigate the temporal change of TBET/GATA3 mRNA ratio in ACS. Methods. Thirty-three patients suspected of ACS with symptom onset within 24 hours were recruited. Blood samples were taken after arrival at the emergency department and at hourly intervals until the 6th hour. The mRNA expressions of TBET and GATA3 were quantified by a real-time RT-qPCR. Results. The TBET/GATA3 mRNA ratio was elevated dramatically in patients with acute myocardial infarction (AMI) and exhibited biphasic M-shaped release kinetics with two distinct peaks. The ratio was elevated 2 hours after symptom onset, dropped to the lowest level at 10 hours, and rose to the second peak at 14 hours. A similar biphasic M-shaped curve was observed in AMI patients with blood samples taken prior to any intervention. Conclusions. The TBET/GATA3 mRNA ratio was elevated in AMI patients throughout most of the first 20 hours after symptom onset. The biphasic M-shaped release kinetics was more likely to reflect pathophysiological changes rather than treatment effects.