Myalgic encephalomyelitis/chronic fatigue syndrome from current evidence to new diagnostic perspectives through skeletal muscle and metabolic disturbances.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a demanding medical condition for patients and society. It has raised much more public awareness after the COVID-19 pandemic since ME/CFS and long-COVID patients share many clinical symptoms such as debilitating chronic fatigue. However, unlike long COVID, the etiopathology of ME/CFS remains a mystery despite several decades' research. This review moves from pathophysiology of ME/CFS through the compelling evidence and most interesting hypotheses. It focuses on the pathophysiology of skeletal muscle by proposing the hypothesis that skeletal muscle tissue offers novel opportunities for diagnosis and treatment of this syndrome and that new evidence can help resolve the long-standing debate on terminology.

Secreted Metabolome of ALS-Related hSOD1(G93A) Primary Cultures of Myocytes and Implications for Myogenesis.

Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease associated with progressive muscle atrophy, paralysis, and eventually death. Growing evidence demonstrates that the pathological process leading to ALS is the result of multiple altered mechanisms occurring not only in MNs but also in other cell types inside and outside the central nervous system. In this context, the involvement of skeletal muscle has been the subject of a few studies on patients and ALS animal models. In this work, by using primary myocytes derived from the ALS transgenic hSOD1(G93A) mouse model, we observed that the myogenic capability of such cells was defective compared to cells derived from control mice expressing the nonpathogenic hSOD1(WT) isoform. The correct in vitro myogenesis of hSOD1(G93A) primary skeletal muscle cells was rescued by the addition of a conditioned medium from healthy hSOD1(WT) myocytes, suggesting the existence of an in trans activity of secreted factors. To define a dataset of molecules participating in such safeguard action, we conducted comparative metabolomic profiling of a culture medium collected from hSOD1(G93A) and hSOD1(WT) primary myocytes and report here an altered secretion of amino acids and lipid-based signaling molecules. These findings support the urgency of better understanding the role of the skeletal muscle secretome in the regulation of the myogenic program and mechanisms of ALS pathogenesis and progression.

Acute myeloid leukemia: from NGS, through scRNA-seq, to CAR-T. dissect cancer heterogeneity and tailor the treatment.

Acute myeloid leukemia (AML) is a malignant blood cancer with marked cellular heterogeneity due to altered maturation and differentiation of myeloid blasts, the possible causes of which are transcriptional or epigenetic alterations, impaired apoptosis, and excessive cell proliferation. This neoplasm has a high rate of resistance to anticancer therapies and thus a high risk of relapse and mortality because of both the biological diversity of the patient and intratumoral heterogeneity due to the acquisition of new somatic changes. For more than 40 years, the old gold standard "one size fits all" treatment approach included intensive chemotherapy treatment with anthracyclines and cytarabine.The manuscript first traces the evolution of the understanding of the pathology from the 1970s to the present. The enormous strides made in its categorization prove to be crucial for risk stratification, enabling an increasingly personalized diagnosis and treatment approach.Subsequently, we highlight how, over the past 15 years, technological advances enabling single cell RNA sequencing and T-cell modification based on the genomic tools are affecting the classification and treatment of AML. At the dawn of the new millennium, the advent of high-throughput next-generation sequencing technologies has enabled the profiling of patients evidencing different facets of the same disease, stratifying risk, and identifying new possible therapeutic targets that have subsequently been validated. Currently, the possibility of investigating tumor heterogeneity at the single cell level, profiling the tumor at the time of diagnosis or after treatments exist. This would allow the identification of underrepresented cellular subclones or clones resistant to therapeutic approaches and thus responsible for post-treatment relapse that would otherwise be difficult to detect with bulk investigations on the tumor biopsy. Single-cell investigation will then allow even greater personalization of therapy to the genetic and transcriptional profile of the tumor, saving valuable time and dangerous side effects. The era of personalized medicine will take a huge step forward through the disclosure of each individual piece of the complex puzzle that is cancer pathology, to implement a "tailored" therapeutic approach based also on engineered CAR-T cells.

Transcriptome-wide selection and validation of a solid set of reference genes for gene expression studies in the cephalopod mollusk Octopus vulgaris.

Octopus vulgaris is a cephalopod mollusk and an active marine predator that has been at the center of a number of studies focused on the understanding of neural and biological plasticity. Studies on the machinery involved in e.g., learning and memory, regeneration, and neuromodulation are required to shed light on the conserved and/or unique mechanisms that these animals have evolved. Analysis of gene expression is one of the most essential means to expand our understanding of biological machinery, and the selection of an appropriate set of reference genes is the prerequisite for the quantitative real-time polymerase chain reaction (qRT-PCR). Here we selected 77 candidate reference genes (RGs) from a pool of stable and relatively high-expressed transcripts identified from the full-length transcriptome of O. vulgaris, and we evaluated their expression stabilities in different tissues through geNorm, NormFinder, Bestkeeper, Delta-CT method, and RefFinder. Although various algorithms provided different assemblages of the most stable reference genes for the different kinds of tissues tested here, a comprehensive ranking revealed RGs specific to the nervous system (Ov-RNF7 and Ov-RIOK2) and Ov-EIF2A and Ov-CUL1 across all considered tissues. Furthermore, we validated RGs by assessing the expression profiles of nine target genes (Ov-Naa15, Ov-Ltv1, Ov-CG9286, Ov-EIF3M, Ov-NOB1, Ov-CSDE1, Ov-Abi2, Ov-Homer2, and Ov-Snx20) in different areas of the octopus nervous system (gastric ganglion, as control). Our study allowed us to identify the most extensive set of stable reference genes currently available for the nervous system and appendages of adult O. vulgaris.

Comparison among Neuroblastoma Stages Suggests the Involvement of Mitochondria in Tumor Progression.

Neuroblastoma (NB) is the most common extracranial tumor of early childhood and accounts for 15% of all pediatric cancer mortalities. However, the precise pathways and genes underlying its progression are unknown. Therefore, we performed a differential gene expression analysis of neuroblastoma stage 1 and stage 4 + 4S to discover biological processes associated with NB progression. From this preliminary analysis, we found that NB samples (stage 4 + 4S) are characterized by altered expression of some proteins involved in mitochondria function and mitochondria-ER contact sites (MERCS). Although further analyses remain necessary, this review may provide new hints to better understand NB molecular etiopathogenesis, by suggesting that MERCS alterations could be involved in the progression of NB.

Monoamine Oxidase-Dependent Pro-Survival Signaling in Diabetic Hearts Is Mediated by miRNAs.

Diabetes leads to cardiomyopathy and heart failure, the leading cause of death for diabetic patients. Monoamine oxidase (MAO) inhibition in diabetic cardiomyopathy prevents oxidative stress, mitochondrial and endoplasmic reticulum stress and the development of diastolic dysfunction. However, it is unclear whether, in addition to the direct effects exerted on the mitochondria, MAO activity is able to post-transcriptionally regulate cardiomyocyte function and survival in diabetes. To this aim, we performed gene and miRNA expression profiling in cardiac tissue from streptozotocin-treated mice (model of type 1 diabetes (T1D)), administered with either vehicle or MAOs inhibitor pargyline for 12 weeks. We found that inhibition of MAO activity in T1D hearts leads to profound transcriptomic changes, affecting autophagy and pro-survival pathways activation. MAO activity in T1D hearts increased miR-133a-3p, -193a-3p and -27a-3p expression. These miRNAs target insulin-like growth factor receptor 1 (Igf1r), growth factor receptor bound protein 10 and inositol polyphosphate 4 phosphatase type 1A, respectively, all components of the IGF1R/PI3K/AKT signaling pathway. Indeed, AKT activation was significantly downregulated in T1D hearts, whereas MAO inhibition restored the activation of this pro-survival pathway. The present study provides an important link between MAO activity, transcriptomic changes and activation of pro-survival signaling and autophagy in diabetic cardiomyopathy.

The Interplay of Microtubules with Mitochondria-ER Contact Sites (MERCs) in Glioblastoma.

Gliomas are heterogeneous neoplasms, classified into grade I to IV according to their malignancy and the presence of specific histological/molecular hallmarks. The higher grade of glioma is known as glioblastoma (GB). Although progress has been made in surgical and radiation treatments, its clinical outcome is still unfavorable. The invasive properties of GB cells and glioma aggressiveness are linked to the reshaping of the cytoskeleton. Recent works suggest that the different susceptibility of GB cells to antitumor immune response is also associated with the extent and function of mitochondria-ER contact sites (MERCs). The presence of MERCs alterations could also explain the mitochondrial defects observed in GB models, including abnormalities of energy metabolism and disruption of apoptotic and calcium signaling. Based on this evidence, the question arises as to whether a MERCs-cytoskeleton crosstalk exists, and whether GB progression is linked to an altered cytoskeleton-MERCs interaction. To address this possibility, in this review we performed a meta-analysis to compare grade I and grade IV GB patients. From this preliminary analysis, we found that GB samples (grade IV) are characterized by altered expression of cytoskeletal and MERCs related genes. Among them, the cytoskeleton-associated protein 4 (CKAP4 or CLIMP-63) appears particularly interesting as it encodes a MERCs protein controlling the ER anchoring to microtubules (MTs). Although further in-depth analyses remain necessary, this perspective review may provide new hints to better understand GB molecular etiopathogenesis, by suggesting that cytoskeletal and MERCs alterations cooperate to exacerbate the cellular phenotype of high-grade GB and that MERCs players can be exploited as novel biomarkers/targets to enhance the current therapy for GB.

Regulation of Endoplasmic Reticulum-Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3ß.

Mitochondria-ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER-mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3ß, previously associated with MERCs disruption.

Expression and Functional Analyses of Nymphaea caerulea MADS-Box Genes Contribute to Clarify the Complex Flower Patterning of Water Lilies.

Nymphaeaceae are early diverging angiosperms with large flowers characterized by showy petals and stamens not clearly whorled but presenting a gradual morphological transition from the outer elements to the inner stamens. Such flower structure makes these plant species relevant for studying flower evolution. MADS-domain transcription factors are crucial components of the molecular network that controls flower development. We therefore isolated and characterized MADS-box genes from the water lily Nymphaea caerulea. RNA-seq experiments on floral buds have been performed to obtain the transcript sequences of floral organ identity MADS-box genes. Maximum Likelihood phylogenetic analyses confirmed their belonging to specific MADS-box gene subfamilies. Their expression was quantified by RT-qPCR in all floral organs at two stages of development. Protein interactions among these transcription factors were investigated by yeast-two-hybrid assays. We found especially interesting the involvement of two different AGAMOUS-like genes (NycAG1 and NycAG2) in the water lily floral components. They were therefore functionally characterized by complementing Arabidopsis ag and shp1 shp2 mutants. The expression analysis of MADS-box genes across flower development in N. caerulea described a complex scenario made of numerous genes in numerous floral components. Their expression profiles in some cases were in line with what was expected from the ABC model of flower development and its extensions, while in other cases presented new and interesting gene expression patterns, as for instance the involvement of NycAGL6 and NycFL. Although sharing a high level of sequence similarity, the two AGAMOUS-like genes NycAG1 and NycAG2 could have undergone subfunctionalization or neofunctionalization, as only one of them could partially restore the euAG function in Arabidopsis ag-3 mutants. The hereby illustrated N. caerulea MADS-box gene expression pattern might mirror the morphological transition from the outer to the inner floral organs, and the presence of transition organs such as the petaloid stamens. This study is intended to broaden knowledge on the role and evolution of floral organ identity genes and the genetic mechanisms causing biodiversity in angiosperm flowers.

MyoData: An expression knowledgebase at single cell/nucleus level for the discovery of coding-noncoding RNA functional interactions in skeletal muscle.

Non-coding RNAs represent the largest part of transcribed mammalian genomes and prevalently exert regulatory functions. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) can modulate the activity of each other. Skeletal muscle is the most abundant tissue in mammals. It is composed of different cell types with myofibers that represent the smallest complete contractile system. Considering that lncRNAs and miRNAs are more cell type-specific than coding RNAs, to understand their function it is imperative to evaluate their expression and action within single myofibers. In this database, we collected gene expression data for coding and non-coding genes in single myofibers and used them to produce interaction networks based on expression correlations. Since biological pathways are more informative than networks based on gene expression correlation, to understand how altered genes participate in the studied phenotype, we integrated KEGG pathways with miRNAs and lncRNAs. The database also integrates single nucleus gene expression data on skeletal muscle in different patho-physiological conditions. We demonstrated that these networks can serve as a framework from which to dissect new miRNA and lncRNA functions to experimentally validate. Some interactions included in the database have been previously experimentally validated using high throughput methods. These can be the basis for further functional studies. Using database information, we demonstrate the involvement of miR-149, -214 and let-7e in mitochondria shaping; the ability of the lncRNA Pvt1 to mitigate the action of miR-27a via sponging; and the regulatory activity of miR-214 on Sox6 and Slc16a3. The MyoData is available at https://myodata.bio.unipd.it.

Perturbations of the Proteome and of Secreted Metabolites in Primary Astrocytes from the hSOD1(G93A) ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins-and of secreted metabolites-involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome-lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte-MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.

Insights into how environment shapes post-mortem RNA transcription in mouse brain.

Most biological features that occur on the body after death were already deciphered by traditional medicine. However, the molecular mechanisms triggered in the cellular microenvironment are not fully comprehended yet. Previous studies reported gene expression alterations in the post-mortem condition, but little is known about how the environment could influence RNA degradation and transcriptional regulation. In this work, we analysed the transcriptome of mouse brain after death under three concealment simulations (air exposed, buried, and submerged). Our analyses identified 2,103 genes differentially expressed in all tested groups 48 h after death. Moreover, we identified 111 commonly upregulated and 497 commonly downregulated genes in mice from the concealment simulations. The gene functions shared by the individuals from the tested environments were associated with RNA homeostasis, inflammation, developmental processes, cell communication, cell proliferation, and lipid metabolism. Regarding the altered biological processes, we identified that the macroautophagy process was enriched in the upregulated genes and lipid metabolism was enriched in the downregulated genes. On the other hand, we also described a list of biomarkers associated with the submerged and buried groups, indicating that these environments can influence the post-mortem RNA abundance in its particular way.

Single-Cell RNAseq Analysis of lncRNAs.

Mammalian genomes are pervasively transcribed and a small fraction of RNAs produced codify for proteins. The importance of noncoding RNAs for the maintenance of cell functions is well known (e.g., rRNAs, tRNAs), but only recently it was first demonstrated the involvement of microRNAs (miRNAs) in posttranscriptional regulation and then the activity of long noncoding RNAs (lncRNAs) in the regulation of miRNAs, DNA structure and protein function. LncRNAs have an expression more cell specific than other RNAs and basing on their subcellular localization exert different functions. In this book chapter we consider different protocols to evaluate the expression of lncRNAs at the single cell level using genome-wide approaches. We considered the skeletal muscle as example because the most abundant tissue in mammals involved in the regulation of metabolism and body movement. We firstly described how to isolate the smallest complete contractile system responsible for muscle metabolic and contractile traits (myofibers). We considered how to separate long and short RNAs to allow the sequencing of the full-length transcript using the SMART technique for the retrotranscription. Because of myofibers are multinucleated cells and because of it is better to perform single cell sequencing on fresh tissues we described the single-nucleus sequencing that can be applied to frozen tissues. The chapter concludes with a description of bioinformatics approaches to evaluate differential expression from single-cell or single-nucleus RNA sequencing.

Neurocognitive assessment and DNA sequencing expand the phenotype and genotype spectrum of Alström syndrome.

Alström syndrome (OMIM#203800) is an ultra-rare autosomal recessive monogenic disease presenting pathogenic variants in ALMS1 (chromosome 2p13). It is characterized by early onset of blindness, hearing loss and systemic comorbidities, with delayed development without cognitive impairment. We aimed to investigate the cognitive functions and describe new pathogenic variants in Alström syndrome patients. Nineteen patients (13 adults, 6 children) underwent a thorough clinical, genetic, laboratory, instrumental, and neurocognitive assessment. Six new pathogenic variants in ALMS1 including the first described in exon 6 were identified. Four patients displayed a "mild phenotype" characterized by slow disease onset or absence of complications, including childhood obesity and association with at least one pathogenic variant in exon 5 or 6. At neurocognitive testing, a significant proportion of patients had deficits in three neurocognitive domains: similarities, phonological memory, and apraxia. In particular, 53% of patients showed difficulties in the auditory working memory test. We found ideomotor and buccofacial apraxia in 74% of patients. "Mild phenotype" patients performed better on auditory working memory and ideomotor apraxia test than "typical phenotype" ones (91.9 + 16.3% vs. 41.7 + 34.5% of correct answers, Z = 64.5, p < .01 and 92.5 + 9.6 vs. 61.7 + 26.3, Z = 61, p < .05, respectively). Deficits in auditory working memory, ideomotor, and buccofacial apraxia were found in these patients and fewer neuropsychological deficits were found in the "mild" phenotype group. Furthermore, in the "mild" phenotype group, it was found that all pathogenic variants are localized before exon 8.

Partial F8 gene duplication (factor VIII Padua) associated with high factor VIII levels and familial thrombophilia.

High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.

The immune receptor CD300e negatively regulates T cell activation by impairing the STAT1-dependent antigen presentation.

CD300e is a surface receptor, expressed by myeloid cells, involved in the tuning of immune responses. CD300e engagement was reported to provide the cells with survival signals, to trigger the expression of activation markers and the release of pro-inflammatory cytokines. Hence, CD300e is considered an immune activating receptor. In this study, we demonstrate that the ligation of CD300e in monocytes hampers the expression of the human leukocyte antigen (HLA) class II, affecting its synthesis. This effect, which is associated with the transcription impairment of the signal transducer and activator of transcription 1 (STAT1), overcomes the capacity of interferon gamma (IFN-γ) to promote the expression of the antigen-presenting molecules. Importantly, the decreased expression of HLA-II on the surface of CD300e-activated monocytes negatively impacts their capacity to activate T cells in an antigen-specific manner. Notably, unlike in vitro- differentiated macrophages which do not express CD300e, the immune receptor is expressed by tissue macrophages. Taken together, our findings argue against the possibility that this molecule should be considered an activating immune receptor sensu stricto. Moreover, our results support the notion that CD300e might be a new player in the regulation of the expansion of T cell-mediated responses.

A Single Cell but Many Different Transcripts: A Journey into the World of Long Non-Coding RNAs.

In late 2012 it was evidenced that most of the human genome is transcribed but only a small percentage of the transcripts are translated. This observation supported the importance of non-coding RNAs and it was confirmed in several organisms. The most abundant non-translated transcripts are long non-coding RNAs (lncRNAs). In contrast to protein-coding RNAs, they show a more cell-specific expression. To understand the function of lncRNAs, it is fundamental to investigate in which cells they are preferentially expressed and to detect their subcellular localization. Recent improvements of techniques that localize single RNA molecules in tissues like single-cell RNA sequencing and fluorescence amplification methods have given a considerable boost in the knowledge of the lncRNA functions. In recent years, single-cell transcription variability was associated with non-coding RNA expression, revealing this class of RNAs as important transcripts in the cell lineage specification. The purpose of this review is to collect updated information about lncRNA classification and new findings on their function derived from single-cell analysis. We also retained useful for all researchers to describe the methods available for single-cell analysis and the databases collecting single-cell and lncRNA data. Tables are included to schematize, describe, and compare exposed concepts.

SAMHD1-deficient fibroblasts from Aicardi-Goutières Syndrome patients can escape senescence and accumulate mutations.

In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.

RNA-sequencing reveals that STRN, ZNF484 and WNK1 add to the value of mitochondrial MT-COI and COX10 as markers of unstable coronary artery disease.

Markers in monocytes, precursors of macrophages, which are related to CAD, are largely unknown. Therefore, we aimed to identify genes in monocytes predictive of a new ischemic event in patients with CAD and/or discriminate between stable CAD and acute coronary syndrome. We included 66 patients with stable CAD, of which 24 developed a new ischemic event, and 19 patients with ACS. Circulating CD14+ monocytes were isolated with magnetic beads. RNA sequencing analysis in monocytes of patients with (n = 13) versus without (n = 11) ischemic event at follow-up and in patients with ACS (n = 12) was validated with qPCR (n = 85). MT-COI, STRN and COX10 predicted new ischemic events in CAD patients (power for separation at 1% error rate of 0.97, 0.90 and 0.77 respectively). Low MT-COI and high STRN were also related to shorter time between blood sampling and event. COX10 and ZNF484 together with MT-COI, STRN and WNK1 separated ACS completely from stable CAD patients. RNA expressions in monocytes of MT-COI, COX10, STRN, WNK1 and ZNF484 were independent of cholesterol lowering and antiplatelet treatment. They were independent of troponin T, a marker of myocardial injury. But, COX10 and ZNF484 in human plaques correlated to plaque markers of M1 macrophage polarization, reflecting vascular injury. Expression of MT-COI, COX10, STRN and WNK1, but not that of ZNF484, PBMCs paired with that in monocytes. The prospective study of relation of MT-COI, COX10, STRN, WNK1 and ZNF484 with unstable CAD is warranted.