Abstract

Background: Single ventricle (SV) and hypoplastic left heart syndrome (HLHS) present significant sex differences in terms of surgical outcomes and long-term prognosis between male and female patients. Peripheral blood mononuclear cells (PBMCs) drive tissue damage and cardiac dysfunction, contributing to the sex-specific clinical outcomes. Aims: We used single-cell RNA sequencing (scRNA-seq) to assess gene expression profiles in PBMCs, aiming to gain insights into sex effects and identify new biomarkers for assessing disease prognosis. Methods: We studied PBMCs from 32 cases with SV/HLHS. The experiments were conducted in 3 independent batches (9 females/11 males; 2 females/6 males; 2 females/2 males), using 10X Chromium Single Cell Gene Expression assay (10x Genomics, Single Cell 3' v3). Sequencing was performed using the Illumina NovaSeq6000. The data analysis was conducted using the Seurat R package, employing SCTransform for data normalizing and scaling (Satija, Farrell et al. 2015, Butler, Hoffman et al. 2018). To mitigate batch effects, the 3 batches were analyzed separately. Results: 42 genes were consistently identified of differential expression (DE) (|log 2 FC|≥0.25, P adj<0.05) in each of the 3 batches, including 38 upregulated (6 on ChrX) and 4 downregulated genes in females. Notably, 3 autosome genes were also identified of DE in SV/HLHS cases compared to controls: PPP1R15A upregulated in cases has lower expression in females; EVL and CLEC2D downregulated in cases have higher expression in females. The expression of these 3 genes is more significant in naïve CD4 + T cells, CD14 + Monocytes, NK cells, and naïve B cells. Conclusion: PPP1R15A mediates cellular stress responses and apoptosis (Blais, Filipenko et al. 2004). EVL negatively regulates cell migration (Lambrechts, Kwiatkowski et al. 2000). CLEC2D mediates immune sensing of cell death through the recognition of histone sequences (Lai, Cruz et al. 2020). The expression changes in females may mitigate inflammation and immune dysregulation in SV/HLHS patients. The significance of these genes as prognostic markers and potential targets for therapeutic intervention in SV/HLHS management warrants further research.