Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors

Abstract

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34 + cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34 + cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.

ETV6 in hematopoiesis and leukemia predisposition

“…Additional studies extended these findings to additional families with unique ETV6 germline mutations and predisposition to malignancy. (18) (19) (20) (21) (22). With one exception, all of the germline heterozygous ETV6 mutations identified in family studies cluster within the highly conserved ETS domain shared by all ETS family proteins and responsible for binding to DNA (Figure 1).…”

“…Examination of the bone marrow revealed frequent immature hypolobated megakaryocytes, mild dyserythropoiesis, and mild nuclear hypolobulation and hypogranulation of myeloid cells. (17) (18) (21) Increased numbers of circulating CD34+ cells were noted in patients harboring the P214L and Y401N mutations compared with healthy controls. (21)…”

Germline ETV6 mutations and predisposition to hematological malignancies

“…However, the platelets of carriers show an impaired ability to spread on fibrinogen [35]. Furthermore, patient-derived megakaryocytes reveal a defect in megakaryocytic maturation and formation of proplatelets both in terms of morphological and quantitative findings, demonstrated for P214L [33,38], Y401N [38], and R418G [33].…”

“…Interestingly, compared to the other germline variants, this mutation does not affect the ETS domain, but is located instead in a serine-proline phosphorylation motif present in the central regulatory domain and represses DNA binding by the ETS domain by approximately tenfold [19] and interacts with the corepressors mSin3A and SMRT [16]. Carriers of the P214L show a defect in proplatelet formation and megakaryocytic maturation, altered proplatelet spreading, reduced transcript levels encoding several cytoskeletal proteins, and modification of ETV6 subcellular localization [32][33][34]38]. There are three other amino acid residues that are affected by germline mutations in two families-the frameshift mutation N385Vfs, Y401N, and mutations of residue 369 (R369W, R369Q).…”

“…ETV6 interacts with several corepressors like SMRT, mSin3A, and N-CoR. For both A377T and Y401N, impaired interaction with these corepressors has been reported, which affects recruitment of the corepressors [38].…”