Abstract
Lately, much effort has been made to find mRNA biomarkers for tuberculosis (TB) disease/infection with microarray-based approaches. In a pilot investigation, through RNA sequencing technology, we observed a prominent modulation of DOCK9, EPHA4, and NPC2 mRNA abundance in the blood of TB patients. To corroborate these findings, independent validations were performed in cohorts from different areas. Gene expression levels in blood were evaluated by quantitative real-time PCR (Brazil, n = 129) or reanalysis of public microarray data (UK: n = 96; South Africa: n = 51; Germany: n = 26; and UK/France: n = 63). In the Brazilian cohort, significant modulation of all target-genes was observed comparing TB vs. healthy recent close TB contacts (rCt). With a 92% specificity, NPC2 mRNA high expression (NPC2high) showed the highest sensitivity (85%, 95% CI 65%–96%; area under the ROC curve [AUROC] = 0.88), followed by EPHA4 (53%, 95% CI 33%–73%, AUROC = 0.73) and DOCK9 (19%, 95% CI 7%–40%; AUROC = 0.66). All the other reanalyzed cohorts corroborated the potential of NPC2high as a biomarker for TB (sensitivity: 82–100%; specificity: 94–97%). An NPC2high profile was also observed in 60% (29/48) of the tuberculin skin test positive rCt, and additional follow-up evaluation revealed changes in the expression levels of NPC2 during the different stages of Mycobacterium tuberculosis infection, suggesting that further studies are needed to evaluate modulation of this gene during latent TB and/or progression to active disease. Considering its high specificity, our data indicate, for the first time, that NPC2high might serve as an accurate single-gene biomarker for TB.
Highlights
Mycobacterium tuberculosis bacilli spread through the air, and the lung parenchyma is the main site of infection
In an exploratory investigation, a small set of recent close TB contacts exposed to a bacillary index case, and TB patients, were submitted to whole blood transcriptomic profiling via RNA sequencing (RNAseq); the raw transcriptome data can be found at the NCBI Gene Expression Omnibus (GEO) under the accession code GSE84076
Individual expression of DOCK9, EPHA4, and NPC2 mRNAs was obtained as crossing point (Cp) values (Guerreiro et al, 2013), which were subsequently normalized to RPL13A mRNA expression, using the qPCR library (Ritz and Spiess, 2008) for the R statistical package 3.2.3 (R Development Core Team, 2014), resulting in relative expression data for the three target genes
Summary
Mycobacterium tuberculosis bacilli spread through the air, and the lung parenchyma is the main site of infection. The aim of the present study was to identify new mRNA biomarkers for TB in whole blood specimens For this purpose, in an exploratory investigation, a small set of recent close TB contacts (rCt) exposed to a bacillary index case, and TB patients, were submitted to whole blood transcriptomic profiling via RNAseq (unpublished data); the raw transcriptome data can be found at the NCBI Gene Expression Omnibus (GEO) under the accession code GSE84076. In an exploratory investigation, a small set of recent close TB contacts (rCt) exposed to a bacillary index case, and TB patients, were submitted to whole blood transcriptomic profiling via RNAseq (unpublished data); the raw transcriptome data can be found at the NCBI Gene Expression Omnibus (GEO) under the accession code GSE84076 Mining these data, highly prominent regulation of DOCK9, EPHA4, and NPC2 mRNA expression was observed in the TB samples. Following a first validation by quantitative realtime PCR (RT-qPCR) in a cohort of rCt and TB cases from Rio de Janeiro/Brazil, expression data from previous microarray studies (Berry et al, 2010; Maertzdorf et al, 2012; Bloom et al, 2013) were used for an independent validation of these potential mRNA biomarkers in populations with different levels of TB incidence and diverse genetic backgrounds
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