Differential Expression of Several miRNAs and the Host Genes AATK and DNM2 in Leukocytes of Sporadic ALS Patients

Abstract

Genetic studies have managed to explain many cases of familial amyotrophic lateral sclerosis (ALS) through mutations in several genes. However, the cause of a majority of sporadic cases remains unknown. Recently, epigenetics, especially miRNA studies, show some promising aspects. We aimed to evaluate the differential expression of 10 miRNAs, including miR-9, miR-338, miR-638, miR-663a, miR-124a, miR-143, miR-451a, miR-132, miR-206, and let-7b, for which some connection to ALS was shown previously in ALS culture cells, animal models or patients, and in three miRNA host genes, including C1orf61 (miR-9), AATK (miR-338), and DNM2 (miR-638), in leukocyte samples of 84 patients with sporadic ALS. We observed significant aberrant dysregulation across our patient cohort for miR-124a, miR-206, miR-9, let-7b, and miR-638. Since we did not use neurological controls we cannot rule out that the revealed differences in expression of investigated miRNAs are specific for ALS. Nevertheless, the group of these five miRNAs is worth of additional research in leukocytes of larger cohorts from different populations in order to verify their potential association to ALS disease. We also detected a significant up-regulation of the AAKT gene and down-regulation of the DNM2 gene, and thus, for the first time, we connected these with sporadic ALS cases. These findings open up new research toward miRNAs as diagnostic biomarkers and epigenetic processes involved in ALS. The detected significant deregulation of AAKT and DNM2 in sporadic ALS also represents an interesting finding. The DNM2 gene was previously found to be mutated in Charcot-Marie-Tooth neuropathy-type CMT2M and centronuclear myopathy (CNM). In addition, as recent studies connected AATK and frontotemporal dementia (FTD) and DNM2 and hereditary spastic paraplegia (HSP), these two genes together with our results genetically connect, at least in part, five diseases, including FTD, HSP, Charcot-Marie-Tooth (type CMT2M), CNM, and ALS, thus opening future research toward a better understanding of the cell biology involved in these partly overlapping pathologies.

Keywords: Slovenian population; amyotrophic lateral sclerosis; differential expression of miRNAs; down-regulation of AATK; leukocytes; sporadic ALS; up-regulation of DNM2.

Figure 1

(A) Relative expression of miR-124 in sALS patients compared to controls. (B) Relative expression of miR-132, miR-451a, and miR-663a, respectively, in sALS patients compared to controls. (C) Relative expression of miR-338 and its host gene AATK in sALS patients compared to controls. (D) Relative expression of miR-9, miR-206, and let-7b, respectively in sALS patients compared to controls. (E) Relative expression of miR-638 and its host gene DNM2 in sALS patients compared to controls. Ctrl, control group; ΔCt, delta Ct; ΔΔCt, delta delta Ct; sALS, sporadic amyotrophic lateral sclerosis; ^***p < 0.000. Horizontal lines represent standard deviation.

Figure 2

Heatmap of experimentally validated union pathways of investigated group of miRNAs using miRPath Database (Vlachos et al., 2015). In union pathways the enrichment analysis is performed and significance levels (p-values) are calculated between each miRNA and every pathway. Then, for each pathway a merged p-value is extracted using Fisher's meta-analysis method, which signify if a particular pathway is targeted by at least one miRNA out of the initially selected group (Vlachos et al., 2015). Different colors of cells mean different level of significance [log (p-value) in Color Key].