Deregulated megakaryocytic maturation, mitochondrial function and inflammatory signalling in <scp> <i>CYCS</i> (c.292T&gt;C) </scp> ‐related thrombocytopenia

The human CYCS gene encodes cytochrome c, a protein with important cellular functions related to cell survival, apoptosis and energy production. Several CYCS variants have been linked to CYCS-related thrombocytopenia, a non-syndromic autosomal dominant mild inherited thrombocytopenia (IT) characterized by reduced platelet counts (Introduction S1). This study has identified a novel CYCS variant (CYCS(NM_018947.6):c.292T>C, p.(Tyr98His), Accession RCV002287278.1) in a Greek family with thrombocytopenia (Figure S1; Table S1; Results S1) and examined the potential underlying mechanisms leading to thrombocytopenia. The novel variant was identified in patient IV1 using whole exome sequencing. Segregation analysis in family members revealed the presence of the variant at a heterozygous state in all members with thrombocytopenia, demonstrating a complete genotype–phenotype correlation (Results S1; Figure S1). This variant lies within a region already affected by other disease-causing variants1, 2 and is located in Tyr98, which contributes to protein folding.3 The substitution replaces the large hydrophobic tyrosine with a medium-sized hydrophilic histidine, thus compromising the protein's structural integrity.1 To assess its functional role, the variant was inserted in homozygous (C/C) and heterozygous (T/C) states into the megakaryocytic cell line Dami by CRISPR-Cas9 (Results S1; Figures S2 and S3; Table S2). As cytochrome c is involved in the intrinsic apoptotic pathway, the effect of the variant on apoptosis has been investigated. An increasing tendency in apoptosis in both C/C and T/C cells (Figure 1A) has been observed, in agreement with the augmented apoptotic activity reported in other models carrying CYCS variants.4, 5 Furthermore, the novel variant altered megakaryocytic maturation of Dami cells, potentially causing an impairment in thrombopoiesis (Results S1; Figure 1B). The observed decrease in CD42A and CD42B expression upon phorbol 12-myristate 13-acetate (PMA) treatment was unexpected and might be due to proteolytic cleavage of CD42 during culture, as previously reported.6 To validate this finding, mature megakaryocytes were examined in patients and found to be reduced (Results S1; Figure 1C–E). Notably, lower polyploidy and cell size were also detected (Results S1; Figure 1F). These findings support a potential mechanism impeding megakaryocytic maturation and leading to decreased platelet generation/numbers, and thus the thrombocytopenic phenotype. Collectively, a connection between thrombocytopenia and the reduced maturation in both Dami cells and patients is supported. Considering the role of cytochrome c as an electron transmitter in the mitochondrial respiratory chain, the adenosine triphosphate (ATP) production levels have been measured. In mitochondria, a decrease has been observed in T/C cells, while C/C exhibited only a slight reduction (Figure 1G). In cell lysates, a significant change in ATP levels was not apparent (Figure 1G), which can be at least partially attributed to ATP production through alternative pathways (Results S1). The reduced ATP levels detected here agree with the diminished respiratory activity observed in other CYCS variant models.2, 5, 7 To further profile the cellular effect of the variant, the superoxide oxidase activity of cytochrome c, a key indicator of its antioxidant function, has been assessed (Results S1). Lower levels of the reduced form of cytochrome c have been observed in C/C and T/C cells, correlating with decreased cytochrome c superoxide oxidase activity (Figure 1H; Results S1). The more severely affected T/C than C/C cells, regarding the levels of both ATP and the reduced form of cytochrome c, potentially indicate a dominant-negative function of the variant. To determine the transcriptomic changes evoked by the insertion of the novel variant, mRNA-Seq has been performed in wild-type (WT), C/C and T/C Dami cells (Results S1; Figure 2A,B; Figures S4 and S5; Tables S3 and S4). Functional annotation of the differentially expressed genes (DEGs) has indicated enrichment in apoptosis and oxidative phosphorylation pathways in both genotypes (Figure 2C), validating the findings mentioned above. Additionally, the metabolic pathway of oxidative phosphorylation, responsible for energy and reactive oxygen species (ROS) production, has been the most enriched in the heterozygous genotype (Figure 2C). This finding is noteworthy since the electron transport chain (ETC) is a crucial part of oxidative phosphorylation. Provided that the functional coupling between ROS and mitochondrial fission in megakaryocytes has been pivotal for the onset of thrombopoiesis,8 this further supports our hypothesis about the role of the novel variant in thrombocytopenia. Interestingly, the tumour necrosis factor Α (TNFΑ) signalling via the nuclear factor kappa B (NF-κB) pathway has been affected in both C/C and T/C conditions (Figure 2C), providing a potential mechanism through which the variant affects thrombopoiesis, given the previously established immunometabolic role of the TNFA in platelets and megakaryocytes.9, 10 Furthermore, gene set enrichment analysis has determined a positive enrichment in the TNFA signalling via the NF-κB gene sets in both genotypes (Figure 2D), implicating TNFΑ in CYCS-related thrombocytopenia. Transcriptional changes among significant DEGs within the TNFA signalling via the NF-κB gene set have revealed distinct expression patterns (Figure 2E). Additionally, negative enrichment in gene sets involved in ETC (Figure 2D) and complex IV (Results S1; Figure S6) has been revealed, supporting our hypothesis about the defective role of the variant in ETC, leading to reduced energy production and ATP levels. Moreover, several gene sets involved in differentiation have been negatively enriched (Figure 2D; Figure S6), and stemness and stem cell-associated gene sets have been positively enriched (Results S1; Figure S6), supporting the findings of reduced differentiation of mature megakaryocytes in Dami cells and patients, and corroborating that the novel variant affects megakaryocytic maturation and thus thrombopoiesis. Our findings support a potential link between the identified CYCS variant and the deregulation of the TNFA via NF-κB pathway, which may contribute to the development of the thrombocytopenia phenotype observed in the affected members of the Greek family. Megakaryocytes express TNF receptors,11 activated by TNF binding, leading to NF-κB activation, which has been significant in platelet function, being present in both megakaryocytes and platelets.12 The increased apoptosis observed in C/C and T/C Dami cells, although small, might occur via the extrinsic TNF-induced apoptosis pathway. Furthermore, decreased ATP levels and cytochrome c superoxide oxidase activity associated with the novel variant also agree with the deregulation of genes involved in the TNFA via NF-κB pathway, as mitochondrial ETC, along with ROS production, are vital for NF-κB activation.13 Collectively, transcriptomic data in the megakaryocytic cell line Dami connect the novel variant with thrombocytopenia, driven by increased apoptosis and reduced bioenergetics and differentiation, potentially mediated by deregulated TNF-mediated signalling in megakaryocytes. The deregulation of TNF-mediated signalling was validated in patient-derived mature megakaryocytes using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for selected targets associated with TNFA via NF-κB signalling, identified through RNA-seq in Dami cells. Specifically, the expression of the CXCL1, TNFAIP2, and ZFP36 genes was examined and found to be significantly increased in patient samples compared to controls (Figure 2F; Table S5), in agreement with the RNA-seq data. Upregulation of these targets indicates sustained TNFA via NF-κB deregulation, which drives inflammatory signalling. In haematopoietic contexts, chronic NF-κB-driven inflammation and altered survival/apoptosis signalling can impair megakaryocytic differentiation and platelet release, offering a link between the CYCS variant and thrombocytopenia.14 In summary, this study identified a novel CYCS variant and validated its pleiotropic functional effects in apoptosis, cellular bioenergetics, differentiation/maturation and TNFA via NF-κB signalling of megakaryocytes, contributing to thrombocytopenia. The deregulation of the TNFA via NFK-κB signalling, determined in this study, is proposed as a mediator in CYCS-related thrombocytopenia, creating a potential link between inflammatory signalling and abnormal thrombopoiesis. This study, by reporting an association between inflammatory signalling and CYCS-related thrombocytopenia, highlights important aspects of cytochrome c function related to IT and provides potential novel pathways/targets for future investigation. KG performed experiments, analysed data and drafted the manuscript; ESF performed experiments, analysed RNA-seq data and wrote the manuscript; SG performed sanger sequencing and segregation analysis; AB and AK managed the patients; FK performed haematological/biochemical tests for patient management; AA performed cell sorting experiments; NMM performed WES; GV performed RNA-seq; VP managed the patients, designed and supervised the project; EK designed, supervised the project and wrote the manuscript. All authors have corrected and approved the final version of the manuscript. We thank Dr. Laura Gutiérrez (Platelet Research Laboratory, ISPA, Oviedo, Spain) for providing the Dami cell line, and Drs. Ekati Drakopoulou, Dimitra Kerdidani and Ioanna Galani (Biomedical Research Foundation, Academy of Athens) for helpful discussions on FACS analysis. The publication of this article in OA mode was financially supported by HEAL-Link. This work has been supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813091 to EK. The authors declare no conflict of interest. The study has been conducted under all ethical standards. All patients have provided written informed consent. The authors make their original data and protocols available to other investigators upon request without restrictions. RNA-Seq data have been deposited in GEO (accession GSE290319). Data S1. Table S3. Table S4. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.