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. 2022 Aug;269(8):4161-4173.
doi: 10.1007/s00415-022-11029-7. Epub 2022 Mar 2.

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis

Mridul Johari  1   2 , Anna Vihola  3   4   5 , Johanna Palmio  6 , Manu Jokela  5   7 , Per Harald Jonson  3   4 , Jaakko Sarparanta  3   4 , Sanna Huovinen  8 , Marco Savarese  3   4 , Peter Hackman  3   4 , Bjarne Udd  3   4   6   9
Affiliations

Comprehensive transcriptomic analysis shows disturbed calcium homeostasis and deregulation of T lymphocyte apoptosis in inclusion body myositis

Mridul Johari et al. J Neurol. 2022 Aug.

Abstract

Objective: Inclusion body myositis (IBM) has an unclear molecular etiology exhibiting both characteristic inflammatory T-cell activity and rimmed-vacuolar degeneration of muscle fibers. Using in-depth gene expression and splicing studies, we aimed at understanding the different components of the molecular pathomechanisms in IBM.

Methods: We performed RNA-seq on RNA extracted from skeletal muscle biopsies of clinically and histopathologically defined IBM (n = 24), tibial muscular dystrophy (n = 6), and histopathologically normal group (n = 9). In a comprehensive transcriptomics analysis, we analyzed the differential gene expression, differential splicing and exon usage, downstream pathway analysis, and the interplay between coding and non-coding RNAs (micro RNAs and long non-coding RNAs).

Results: We observe dysregulation of genes involved in calcium homeostasis, particularly affecting the T-cell activity and regulation, causing disturbed Ca2+-induced apoptotic pathways of T cells in IBM muscles. Additionally, LCK/p56, which is an essential gene in regulating the fate of T-cell apoptosis, shows increased expression and altered splicing usage in IBM muscles.

Interpretation: Our analysis provides a novel understanding of the molecular mechanisms in IBM by showing a detailed dysregulation of genes involved in calcium homeostasis and its effect on T-cell functioning in IBM muscles. Loss of T-cell regulation is hypothesized to be involved in the consistent observation of no response to immune therapies in IBM patients. Our results show that loss of apoptotic control of cytotoxic T cells could indeed be one component of their abnormal cytolytic activity in IBM muscles.

Keywords: Calcium; Differential expression; Differential splicing; Inclusion body myositis; T cells.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Fig. 1
Fig. 1
a Workflow and methodology used in this study. b Principal component analysis of gene expression results showing the pairwise comparison between different groups: IBM, TMD and Amputees. c IBM-specific differentially expressed genes were determined by comparing IBM cases with amputee and TMD groups. d Comparison between IBM-specific differentially expressed genes (cyan) and IBM-specific differentially spliced genes (magenta)
Fig. 2
Fig. 2
a Top 15 differentially expressed genes specific to IBM muscles. Log2 fold change (log2FC) of IBM versus amputees calculated by DEseq2 after shrinkage estimations. ' + '/'−' sign denotes the direction of change, i.e., positive log2FC values indicate overexpressed genes in IBM muscles, and negative log2FC values indicate underexpressed genes in IBM muscles. The p value of significance and adjusted p value using the Benjamini–Hochberg corrections and associated GO terms are shown for each gene. Genes marked with * are also observed as significantly dysregulated in Hamann et al. [13]. b Normalized gene expression in the different cohorts is presented as boxplots. Median and quartile values are shown, with whiskers reaching up to 1.5 times the interquartile range. Individual expression levels are shown as jitter points. The raincloud plots illustrate the distribution of data in each cohort. The scaled Y-axis shows log normalized counts
Fig. 3
Fig. 3
The calcium-induced T lymphocyte apoptosis pathway with gene expression changes observed in IBM compared to groups. Created with BioRender.com
Fig. 4
Fig. 4
Statistical over-representation tests were performed on a list of differentially spliced RNAs, using clusterProfiler for a Biological Processes, b Cellular component, and c Molecular function. d An UpSet plot is shown comparing six different sets, namely, IBM-specific differentially spliced (1271 genes), mobilization of Ca2+ (80 genes), calcium-induced T lymphocyte apoptosis (69 genes), the flux of Ca2+ (51 genes), quantity of Ca2+ (51 genes), and release of Ca2+ (33 genes). Dots and lines represent subsets of different lists. The horizontal bar graph (wine color) represents the size of each set, while the vertical histogram (black) represents the number of RNAs in each subset. The 10 RNAs that are both differentially expressed and differentially spliced are shown with a red circle with their gene names (black)
Fig. 5
Fig. 5
a Normalized LCK expression in the different cohorts (as explained in Fig. 2b). b Altered isoform expression of LCK using JunctionSeq showing estimated normalized mean read-pair count for each exon and splice junctions in the different cohorts (left) as well as for the whole LCK gene (right). The significantly alternatively spliced feature, E016 (pink), corresponds to chr1:32274818–32274992 (GRCh38). The alternative LCK transcripts used in the JunctionSeq analysis are shown below with their corresponding ENSEMBL identifiers
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