Clustering analysis (with heatmap) of variants identified in 26 Italian severe hemophilia A patients by WES. Unsupervised hierarchical clustering was performed using 1364 statistically significant variants (P ≤ .05) by Cochran-Armitage (trend model) and Fisher’s exact (dominant model) t tests. Two main, distinct patterns of homozygous variants were revealed that are characteristic of inhibitor-positive (risk) and inhibitor-negative (protective) patients. Differences in modes of inheritance between all brothers are emphasized through pattern differences in the heatmap: concordant brothers E287-E1147 (inhibitor negative; green rectangle) and E1361-E502 (inhibitor positive; orange rectangle) exhibit greater similarity across homo- and heterozygous variants, whereas the discordant brothers E835-E1309 and E172-E1355 (blue rectangles) exhibit greater differences across these variant types. Red bars under the heatmaps underline inhibitor-positive and black bars underline inhibitor-negative patients. Heatmap: red, homozygous variants; green, heterozygous variants; black, reference calls. Unsupervised hierarchical clustering was performed using R.³¹