H syndrome monocytes display altered subset distribution and elevated expression of inflammatory genes. (A) Representative flow cytometry analyses of healthy control (left) and patient with H syndrome (right) classical (CD14^high CD16⁻) and nonclassical (CD14^dim CD16⁺) monocyte subset distribution (the depicted cells are HLA-DR⁺ lineage [Lin]⁻ CD88⁺CD89⁺). Full gating strategy is depicted in supplemental Figure 3. (B) Dot plots depicting percentage of classical monocytes (left) and nonclassical monocytes (right) out of total monocytes, based on flow cytometry analysis (controls: n = 8; patients: n = 8). Statistical significance was determined using unpaired 2-tailed t tests; ∗∗∗P < .001. (C) Uniform manifold approximation and projection for dimension reduction (UMAP) visualization showing all cells included the single-cell RNA sequencing data sets, combining all healthy samples (n = 4, comprising a total of 13 028 single-cell transcriptome profiles) and all patient samples (n = 8, comprising a total of 34 375 single-cell transcriptome profiles). Monocyte fractions after enrichment were between 60% and 90% in different samples. (D) Principal component analysis (PCA) plot based on the top 1000 differentially expressed genes in monocytes (classical and nonclassical clusters); healthy controls (blue), H syndrome (red). (E) Volcano plots showing the differentially expressed genes in classical and nonclassical monocytes. Positive fold change (FC) represents genes that were upregulated in the H syndrome cohort and negative FC represents genes that were downregulated in the H syndrome cohort. FDR, false discovery rate; Not sig., not significant.