CD34 expression and granule contents of RUNX1- and GFI1B-mutated platelets. (A) Pedigree of the family harboring a heterozygous RUNX1 p.Q154fs mutation (indicated by ±). Thrombocytopenia is indicated with black shapes. Also the deceased mother of case I:1 had a history of thrombocytopenia (data not shown), suggesting autosomal dominant inheritance of the disease. Four family members were screened for the mutation (all ±). □: male, ○: female. Pedigrees from the RUNX1 tandem duplication exons 2-6 and GFI1B p.Q287* families were published before.1,5 (B) MGG-stained blood film of proband I:1 (RUNX1 p.Q154fs) showing normal, hypogranular, and agranular (gray) platelets (arrows). Original magnification ×40, VisionTek Sakura, VisionTek Live 2.6. (C) Sequencing of RUNX1 revealed a heterozygous mutation (c.460del NM_001754.4, p.Q154fs) in all tested affected family members. Results from the proband I:1 are shown. (D) Geometric MFI of CD34 expression on CD41-positive platelets measured by flow cytometry showing that thrombocytopenic individuals from RUNX1-mutated families (p.Q154fs cases I:1, II:1-II:3, and tandem duplication exons 2-6 cases II:3 and II:6) have higher platelet CD34 expression than healthy controls. The line indicates the median (left). Geometric MFI of CD62p on CD42a-positive (middle) and CD63 on CD42b-positive (right) platelets (thrombin-stimulated minus unstimulated). On average, individuals from RUNX1-mutated families (p.Q154fs cases I:1, II:2, II:3, and tandem duplication exons 2-6 cases II:3 and II:6) and the GFI1B p.Q287*-mutated family (cases II:6, II:10, and the last case from Table 1, plus cases II:3 and II:8 from Monteferrario et al1 ) had decreased CD62p and CD63 expression compared with healthy controls. (E) Whole mount EM on platelets from proband I:1. All δ-granules are indicated with arrows. The lower of the two platelets is devoid of δ-granules. Original magnification ×1200, Jeol JEM 1400, Gatan digital micrograph software. (F) Quantification of δ-granules using whole mount EM. The number of δ-granules was determined for 40 or 50 platelets (counters were blinded to diagnosis). As reference, the left panel shows the percentage of platelets harboring 0 to 9 or ≥10 δ-granules for healthy controls (n = 10, each bar with whisker shows mean + 99.55% CI). The middle and right panels show that 2 of 3 thrombocytopenic individuals from RUNX1-mutated families and 3 of 3 from GFI1B p.Q287*-mutated families had an increased percentage of platelets harboring no δ-granules (affected: 12% to 40%; healthy controls: 0% to 4.4% with a 99.55% CI). The median number of δ-granules per platelet was significantly decreased for GFI1B p.Q287* cases compared with controls (P = .013). Because results from the 3 individuals from RUNX1-mutated families were not consistent, the median number of δ-granules per platelet was not significantly decreased for these cases compared with controls (P = .08). One of 3 and 2 of 3 individuals from RUNX1- and GFI1B-mutated families, respectively, had increased percentages of platelets containing ≥10 δ-granules (affected: 10% to 22%; healthy controls: 0% to 5.2% with a 99.55% CI). ‡These individuals were thrombocytopenic, but not tested for the indicated mutation. †This case is a son of case II:10 and nephew of II:6, but was not included in the previous publication.1 All P values were obtained using the 2-sided Mann-Whitney U test. CI, confidence interval; MFI, mean fluorescence intensity; TD, tandem duplication.
