Exome sequencing identifies NBEAL2 as the causative gene for gray platelet syndrome

Abstract

Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.

Figure 1

Mutations in NBEAL2 in Gray Platelet Syndrome cases. (a) Electron micrographs showing discoid normal platelets with abundant α-granules in comparison with a selection of platelets from Gray Platelet Syndrome (GPS) cases A.II.3 and B.II.3. The GPS platelets have heterogeneous shapes and characteristically lack α-granules. They normally contain mitochondria (Mi); morphological abnormalities include the presence of membrane complexes (MC), occasional large vacuoles (V) and an overdeveloped Open Canalicular System (OCS). Bars represent 1μm. (b) Novel variants in neurobeachin-like 2 (NBEAL2) in 4 whole-exome sequenced index cases (A-D, Supplementary Table 1) explain GPS. Heterozygous mutations are shown in black and homozygous ones in red. InterPro protein domains are shown as colored bars above the transcript, where blue indicates the characteristic BEACH domain. Sequencing reads showed that the splice and S2268L variants occurred on the same chromosome. (c) Silencing of nbeal2 in zebrafish. To assess the function of nbeal2 in thrombopoiesis we investigated cd41 expression in caudal haematopoietic tissue (CHT) of Tg(cd41:EGFP) transgenic embryos, at 3 days post-fertilisation (dpf). nbeal2 morpholino (MO) knockdown resulted in a complete abrogation of thrombocytes (the zebrafish equivalent of human platelets) when compared to control (white arrowhead). White bars represent ~100μm. (d) Whole mount o-Dianisidine staining for mature erythrocytes at 3 dpf showed that the total number of mature erythrocytes was not affected in nbeal2-depleted embryos when compared to control (white arrow). (e) nbeal2 MO-injected embryos showed normal morphological development and vigorous circulation at 3 dpf, similar to control embryos. However, 41% of nbeal2 depleted embryos (N=78), and no control embryos (N=78) developed spontaneous bleedings visible within the tail of the embryo (left column). These spontaneous bleedings were confirmed with o-Dianisidine staining (right column, white arrows indicate the location of bleeding).