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M.M. Gorski et al.
secretion was impaired with two stimuli in 4/14 patients, with three stimuli in 4/14, and with more than three stim- uli in 6/14 (Table 1). These findings confirmed the diagno- sis of primary PSD in all patients.
The concentrations of total serotonin, ADP, and ATP were normal in all patients as was the ATP/ADP ratio, which is considered a diagnostic hallmark for δ-storage pool deficiency (Online Supplementary Table S1). Similarly, fibrinogen from platelet α-granules was normal. All together, these data excluded that the secretion defect of these patients was attributable to the presence of α- or δ- storage pool deficiency.
Exome sequencing and candidate gene discovery
NGS data analysis revealed 101,562 variants that passed quality control and were sequenced with an average read depth of 51 over each site. Of those, 96,432 were single SNV and 5,130 were indels. The number of singletons, defined as private variants occurring exclusively in a single individual, was 11,430 (mean, 762) in PSD cases and 23,564 (mean, 1,473) in controls. In addition, we identified 30,973 rare variants with a minor allele frequency (MAF) ≤1% and 11,187 of these variants were considered novel, i.e., not listed in the Database of Single Nucleotide Polymorphisms (dbSNP) or any other variant database.
Table 1. Clinical and biological characteristics of 14 unrelated patients with platelet secretion defects.
ID Sex Age BSS Parents consang.
C696 F 34 4 no
C708 F 36 5 no C729 F 3 2 no C732F2515 no C739 M 5 0 no
First-grade family Bleeding history
No
Mother(mildPSD) No
No Mother
Father (mild PSD), Sister (mild bleeding diathesis without PSD)
Motherandsister with thrombocytopenia
Mother No Mother Mother No Brother
No
Platelet secretion stimulus
ADP ADP Collagen U46619 TRAP
4μM 20μM 2μg/mL 1μM 10μM
+ -
+ + + + + + + +
+ +
+ +
+ +
+ +
+ -
+ +
+ +
+ +
+ +
- +-
+ + - - - n/p - +- - - n/p
C740
C749
C783
C797
C831
C847
C862
C1075
C1107
M 19 10 no
F 55 9 no
F 63 13 no
F 31 5 no
F 20 7 no
F 19 7 no
F3 7 no
F 52 15 no
F 60 8 no
+
+
-
+ +
- +
- - + - - + + +
+ n/p + -+
- - + - -
+ +
ID: patient identity; BSS: bleeding severity score; U46619: a defect; (+) indicates defective platelet secretion response
thromboxane A2 analog; TRAP: thrombin receptor activator peptide. F: female; M: male; PSD: platelet secretion to the stimulus; (-) indicates response within the normal range; n/p: data not present.
AB
Figure 1. Platelet aggregation and secretion of 14 unrelated patients with platelet secretion defects. Dot plots of platelet (A) aggregation and (B) secretion. Boxes indicate our internal range of normality (5th-95th percentiles). U46619: thromboxane A2 analog; TRAP: thrombin receptor activator peptide.
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haematologica | 2019; 104(10)