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M.M. Gorski et al.
abnormalities.15 Since STXBP5, a paralog of STXBP5L, pro- motes platelet secretion,34,35 perhaps STXBP5L may also play a role in this process. Another interesting candidate is the KCNMB3 gene that carries the p.P83H missense vari- ant. This gene encodes the Calcium-Activated Potassium Channel Subunit Beta-3 protein involved in a pathway activated in response to elevated platelet cytosolic Ca2+.
For patient C732, a gene defect was found in EXOC1, which is another candidate gene that influences platelet granule exocytosis. This gene encodes the Exocyst Complex Component 1 protein that functions as part of the exocyst complex and is required for targeting exocytic vesicles to specific docking sites on the plasma membrane.36
We also found a missense variant, p.A464P, in the RAP1GAP gene in patient C831. This variant has been classified as likely benign and for this reason, it was excluded from Table 2. Importantly, the Rap1GAP protein plays a regulatory role in platelet aggregation,37 suggesting that this missense variant may actually have a functional role.
As previously reported, PSD can be associated with pro- teins acting at different levels: signal transduction, platelet activation, degranulation, or exocytosis.4 Indeed, we found potential gene defects in proteins involved in all of these processes (Table 2). Importantly, several patients in our study had multiple defects in the above-mentioned genes and gene pathways, which may explain the com-
Table 2. Putative causal variants identified by whole-exome sequencing in 12/14 patients with primary platelet secretion defects according to the clas- sification of Leo et al.15 or by selecting singletons (Online Supplementary Figure S1). All variants were heterozygous.
ID Gene Nucleotide dbSNP Amino acid MAF MAF MAF SIFT change change 1000G ESP ExAC
C696 COL24A1 c.G4673A - p.G1558E - - - D C708 TTN c.G106955A rs200497615 p.R35652Q - 0.0007 0.0003 B
CSRNP1 c.C673T rs142034027 p.R225W - 0.0007 0.0001 D NRP1 c.G620A rs148308681 p.R207H - 0.0001 0.0001 D
C729 TTN c.C104564A - p.S34855Y - - - D ITGA2 c.G305A rs41392746 p.S102N - - 3.01E-05 B
Poly Mutation CADD Platelet
phen2 Taster Cscore expression (**)
Assess.
D D
B D D D D D
D D B B D D - -
D D
D D D D
D D B B D D D D D D D B D B - - - -
B D D D - D D D
D D
B D
B D D D
(*)
25 - VUS
25 + VUS 32 - VUS
MYO3A c.T1525C MUC2 c.G6931A
C732 EXOC1 c.G2009A C739 DIAPH1 c.T3227G
rs150793986 p.Y509H - 0.0003 0.0002 D rs200823008 p.V2311I - 0.0001 0.0008 -
rs35001804 p.G670E 0.003 0.0086 0.009 D
rs143763573 p.F1076C - - 0.0001 D - p.T1907M - - - D
20 20 27
33
32
26 33
22 24 38 27
24
15
12
- VUS
+ VUS + VUS - VUS - - VUS
ITPR3 c.C5720T
C740 TTN c.C72358T rs372309164 p.L24120F - 0.0002 0 B
+ VUS
+ VUS + VUS
+ VUS + VUS - VUS + VUS - VUS - VUS + VUS - VUS - - VUS
TTN c.G1895A rs150231219 p.G632D - 0.0002 0 D SLC2A7 c.C670T rs35776221 p.R224C 0.006 0.01 0.008 D STXBP5L c.G3430A rs139176240 p.D1120N - 0.0001 0.0001 B KCNMB3 c.C248A rs61734056 p.P83H - 1.50E-05 0.0001 D
18 19 27 25 27 23 23 -
LCN1 c.G298C PRKACG c.C280T MUC2 c.G2594A
MUC2 c.A5038G C749 LYST c.G8806A
rs117638349 p.G100R 0.006 0.008 0.004 D - p.R94C - - - D - p.S865N - - - -
rs371137719 p.T1680A 0.01 0.0024 0 - rs2753327 p.V2936I 0.001 0.0009 0.0009 B
- VUS + VUS - VUS + VUS
+ VUS
+ VUS
+ VUS + VUS + VUS + VUS
TTN c.G49413T rs202094100 p.W16471C - 0.0008 0.0006 D PHF14 c.G298T - p.E100X - - - - PTPN12 c.C1066T rs752211731 p.P356S - - 0 D
C797 TTN c.C17T rs201490999 p.P6L - - - D EGF c.G3073A - p.A1025T - - - B
C831 TTN c.T15768A rs138826545 p.H5256Q - 0.0002 0.0002 B EGF c.G1723A rs115396821 p.G575R 0.008 0.0024 0.0027 D TBXAS1 c.151_152del - p.V51fs - - - - VWF c.G8171A - p.C2724Y - - - D
C847 TTN c.C91384T rs373623340 p.R30462W - - 3.01E-05 D
26 - - -
D D
D D D D D D
B D
D D D D D D
26
+ VUS + VUS
PHACTR2 c.G1360C NOS3 c.C3385T
C1075 PRKCD c.A1043G C1107 PTPN7 c.G425A
- p.D454H - - - D
rs774447524 p.R1129C - - 2.31E-05 D
rs33911937 p.N348S - 0.0015 0.0016 B
26
26
34 - VUS
rs115136927 p.R142Q 0.003 0.0072 0.0062 B PRKCD c.G868T - p.A290S - - - B MMRN1 c.G3680T rs147451161 p.R1227L 0.003 0.0031 0.0036 D
15 + VUS
27 + VUS 25 + VUS 28 + VUS
dbSNP: Database of Single Nucleotide Polymorphisms v.138; MAF: minor allele frequency (MAF from European populations is shown); 1000G: the 1000 Genomes Project; ExAC: the Exome Aggregation Consortium; ESP: the Exome Sequencing Project; SIFT: Sorting Intolerant From Tolerant; PolyPhen2: Polymorphism Phenotyping v.2; Mutation Taster, prediction scores, D: dam- aging; B: benign; CADD C score: Combined Annotation Dependent Depletion score;41 VUS: variant of uncertain significance. (*) Platelet gene expression evaluated by the Human Proteome Map (HPM) (http://www.humanproteomemap.org);32 (**) Assess. – Assessment of variant pathogenicity assigned according to the American College of Medical Genetics and Genomics pathogenicity classification.31
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haematologica | 2019; 104(10)