SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects

Abstract

Inherited thrombocytopenias are a group of disorders that are characterized by a low platelet count and are sometimes associated with excessive bleeding that ranges from mild to severe. We evaluated 36 unrelated patients and 17 family members displaying thrombocytopenia that were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study. All patients had a history of excessive bleeding of unknown etiology. We performed platelet phenotyping and whole-exome sequencing (WES) on all patients and identified mutations in schlafen 14 (SLFN14) in 12 patients from 3 unrelated families. Patients harboring SLFN14 mutations displayed an analogous phenotype that consisted of moderate thrombocytopenia, enlarged platelets, decreased ATP secretion, and a dominant inheritance pattern. Three heterozygous missense mutations were identified in affected family members and predicted to encode substitutions (K218E, K219N, and V220D) within an ATPase-AAA-4, GTP/ATP-binding region of SLFN14. Endogenous SLFN14 expression was reduced in platelets from all patients, and mutant SLFN14 expression was markedly decreased compared with that of WT SLFN14 when overexpressed in transfected cells. Electron microscopy revealed a reduced number of dense granules in affected patients platelets, correlating with a decreased ATP secretion observed in lumiaggregometry studies. These results identify SLFN14 mutations as cause for an inherited thrombocytopenia with excessive bleeding, outlining a fundamental role for SLFN14 in platelet formation and function.

Figure 3. Functional characterization of SLFN14 in patient platelets.

(A) Representative Western blot analysis of healthy volunteer and patient platelet lysates, demonstrating decreased SLFN14 protein levels in patient platelets when compared with healthy individuals. (B) Densitometry quantification of 4 independent Western blots (A) performed using a single platelet sample obtained from each patient or healthy volunteer. **P ≤ 0.005, ***P ≤ 0.001, compared with control, Student’s t test. (C) Whole-mount electron microscopy images showing reduced dense granules (δ) in patient platelets in comparison to healthy volunteer platelets. Scale bar: 2 μm.(D) Quantification of data in C. *P ≤ 0.05, ***P ≤ 0.001, compared with control, Student’s t test. 40 platelets were analyzed per patient/healthy volunteer from a single platelet sample. All values are mean ± SEM.

Figure 2. Platelet phenotyping of patients from the 3 families with SLFN14 mutations.

(A) Representative impaired aggregation traces in platelet-rich plasma from patients from the 3 families (family A, IV:4; family B, I:2; family C, II:2; and representative control), following lumiaggregometry performed on undiluted platelet-rich plasma to assess the percentage aggregation after stimulation with ADP (10 μM), collagen (3 μg/ml), and PAR-1 peptide (100 μM). (B) Reduced ATP secretion from dense granules in representative patients from each of the 3 families using Chronolume after stimulation with PAR-1 peptide (100 μM). Lumiaggregometry studies shown in A and B were repeated once and performed on single platelet samples from multiple affected individuals (family A, n = 5; family B, n = 1; and family C, n = 1). (C) Flow cytometric assessment of platelet function in platelet-rich plasma from patient III.2 from family A (n = 1 from one experiment). Agonist responses are determined using anti-CD62P. IgGk1, isotype control. Data for healthy volunteers are shown as mean ± SD (n = 9). Data for healthy volunteers (1:3 dilution) (with PBS) are shown to demonstrate the effect of moderate thrombocytopenia alone on this assay.

Figure 1. Identification of SLFN14 mutations in 3 unrelated families with a dominant form of thrombocytopenia.

(A) Pedigrees from 3 families with moderate thrombocytopenia. Affected individuals are shaded; question marks denote that platelet count is unknown; asterisks indicate those patients whose exomes were sequenced; and arrows indicate nucleotide change. Representative Sanger electropherograms confirming SLFN14 mutations in patients are shown below the relevant families. (B) Linear domain organization of SLFN14 protein, showing the amino acid position of the 3 different missense SLFN14 mutations (K218E, K219N, and V220D) located in the ATPase-AAA-4 domain and conservation of the protein in higher order species.