Date: Monday, October 22, 2018
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: The HMG-Cr form of Necrotizing Myopathy (NM) is a relatively newly recognized entity within the constellation of pediatric inflammatory myopathies. The purpose of this study is to assess peripheral blood mononuclear cells (PBMCs) from 2 children with NM, one with documented IgG antibody to HMG-Cr, for their pattern of gene dysregulation, compared with JDM and control PBMCs, using RNASeq.
Methods: After obtaining the appropriate informed consent, the following were enrolled in the CureJM Juvenile Myositis Registry: 4 girls with JDM (mean age 12.4±4.6 SD years), 2 girls with NM, (mean age 7.5± 8.8 years) and 4 controls, (mean age 8.5± 4.4 years); all participants were White/Hispanic. Sera for all current Myositis Specific Antibody as well as SRP and SAE antibody was tested by Oklahoma Research Labs. They were also tested for SAE and HMG-Cr antibody by RND labs (Culver, Ca.). RNASeq libraries were generated from PBMC RNA using the Clontech stranded high input ribosomal depletion total RNA kits. The samples were sequenced on an Illumina HiSeq2500 or HiSeq3000 in paired-end mode. Sequence reads were aligned to the reference genome GRCh38 by STAR. Read count normalization and differential expression analysis were performed by DESeq2.
Results: The MSAs of the 4 JDM were: p155/140=3; negative=1; the entire group was negative for SAE and SRP antibodies. Of the NM group, one was positive for IgG antibody to HMG-Cr. Cluster analysis of the RNAseq data suggests three distinct JDM subgroups (2 JDM each), with the two NM patients (one HMGCr +) which had very similar expression patterns. Overall, these 6 myositis transcriptome patterns were quite different from the 4 controls. One of the two groups of JDM was more similar to the controls (termed normal-like JDM). Using DESeq, we identified 209 genes that were differentially expressed in both HMGCr-normal-like JDM comparison and HMGCr-control comparison (False Discovery Rate (FDR) < 0.15). Pathway analysis by WebGestalt revealed several enriched pathways including neutrophil degranulation (29 genes, FDR< 0.00001); innate immune system (46 genes, FDR<1.6*E-8), Toll-like receptors cascades (11 genes, FDR=0.0023), complement and coagulation cascades (6 genes, FDR=0.048), which play a role in autoimmune disease. Of note, several genes with critical roles in T-cell receptor signaling (i.e., CD3D, CD8A, ICOS, ITK) were consistently down-regulated in the 2 NM children. Using weighted correlation network analysis, we identified 156 genes that were highly connected with the HMG-Cr gene. WebGestalt shows that this gene network is enriched in circadian clock/NAD metabolism and degradation of beta-catenin by the destruction complex.
Conclusion: Specific patterns of gene dysregulation determined by RNASeq appear to characterize PBMCs from these children with necrotizing myopathy. There is wide variation in gene dysregulation in PBMCs compared to JDM, revealing some shared modes of muscle destruction. When serological diagnosis is not established, characterization of the patient’s RNASeq pattern, using PBMCs, may be helpful in assessing necrotizing myopathy.
To cite this abstract in AMA style:Huang CC, Roberson EDO, Morgan GA, Huang H, Hans V, Pachman LM. Rnaseq of Peripheral Blood Mononuclear Cells from Juvenile Dermatomyositis, Necrotizing Myopathy and Controls Are an Aid in Diagnosis [abstract]. Arthritis Rheumatol. 2018; 70 (suppl 10). https://acrabstracts.org/abstract/rnaseq-of-peripheral-blood-mononuclear-cells-from-juvenile-dermatomyositis-necrotizing-myopathy-and-controls-are-an-aid-in-diagnosis/. Accessed August 3, 2020.
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