Peripheral blood RNA expression profiling in illicit methcathinone users reveals effect on immune system

Abstract

Methcathinone (ephedrone) is relatively easily accessible for abuse. Its users develop an extrapyramidal syndrome and it is not known if this is caused by methcathinone itself, by side-ingredients (manganese), or both. In the present study we aimed to clarify molecular mechanisms underlying this condition. We used microarrays to analyze whole-genome gene expression patterns of peripheral blood from 20 methcathinone users and 20 matched controls. Gene expression profile data were analyzed by Bayesian modeling and functional annotation. Of 28,869 genes on the microarrays, 326 showed statistically significant differential expression with FDR adjusted p-values below 0.05. Quantitative real-time PCR confirmed differential expression for the most of the genes selected for validation. Functional annotation and network analysis indicated activation of a gene network that included immunological disease, cellular movement, and cardiovascular disease functions (enrichment score 42). As HIV and HCV infections were confounding factors, we performed additional stratification of subjects. A similar functional activation of the "immunological disease" category was evident when we compared subjects according to injection status (past versus current users, balanced for HIV and HCV infection). However, this difference was not large therefore the major effect was related to the HIV status of the subjects. Mn-methcathinone abusers have blood RNA expression patterns that mostly reflect their HIV and HCV infections.

Keywords: HIV; ephedrone; gene expression profiling; intravenous drug abuse; manganese; methcathinone.

Figure 1

Methcathinone users group distinctly on the hierarchical clustering heatmap of blood gene expression levels. The top 100 genes from the decreasing ordered list of moderated t-values were clustered according to similarity in their gene expression patterns. Signals are scaled to Z-scores of the rows. The colored bar above the heatmap indicates the grouping variable – goldenrod for methcathinone user, blue for controls. Column labels at the bottom combine HIV status (positive or negative) with the drug user status (current, past, never). Row labels are gene symbols and the asterisk denotes the subject without clinically relevant extrapyramidal syndrome. Note some overlap between the two groups at the middle of the heatmap.

Figure 2

Annotation enrichment analysis indicated that a network including “cell death, antigen presentation, neurological disease” gene functions is significantly enriched in the subjects with methcathinone abuse [score 47, score is −log (p-value)]. Red nodes designate upregulated genes and the number indicates log2 fold change (0 is for equal expression). Uncolored nodes are genes in this network that were not in our list of differentially expressed genes.