We and others recently described families with autosomal dominant thrombocytopenia, variable platelet function defects, and a predisposition to leukemia caused by heterozygous germline mutations in ETV6 . One of these mutations in ETV6, encoding an amino acid change at position 214 (P214L), was described in five independent families. The P214L mutation, located in the central domain of ETV6, was shown to disrupt nuclear localization of the ETV6 protein and to block megakaryocyte maturation of human cells overexpressing the mutant protein in vitro . Additionally, RNA seq of platelets from patients with the ETV6-P214L mutation showed downregulation of cytoskeletal transcripts and transcripts involved in platelet production and function. To further study the effect of the P214L mutation on ETV6 and on platelet production and function, we have generated a CRISPR-Cas9 knock-in mouse with the equivalent mutation to the human P214L in the mouse Etv6 ortholog (in the corresponding amino acid P216 in mice). CRISPR-Cas9 knock-in mice were generated by designing short guide RNAs specific to the site of Etv6 encoding the proline to be mutated. Additionally, a ssDNA oligo was designed encoding the mutation (cca>cta, P>L) and flanking homologous sequence to provide a template for recombination. Heterozygous and homozygous mice were viable. Heterozygous by heterozygous crosses yielded ratios not significantly different from expected Mendelian ratios: wild type (WT) 18.0%, heterozygous 63.0%, and homozygous 19.1% (p=0.18, Chi-squared test). Complete blood counts were obtained by retro-orbital bleeds and showed a mild but significant decrease in platelet counts in homozygous mice (WT 967.3 x 103/uL, heterozygous 840 x 103/uL, homozygous 807.5 x 103/uL, p=0.03). Western blot indicated normal expression of Etv6 protein in mouse bone marrow. Immunofluorescence and confocal imaging of megakaryocytes from these mice showed an increased percentage of small, immature megakaryocytes in homozygous mice compared to WT mice. Flow cytometry analysis suggested normal distribution of the hematopoietic stem cell compartment. These data are consistent with the patient phenotype and in vitro data, which showed that the P214L mutation resulted in variable thrombocytopenia, immature megakaryocytes, and normal ETV6 protein expression.Considering the downregulation of transcripts related to platelet function in patients with the P214L mutation and the variable platelet function defects reported in patients with germline ETV6 mutations, we characterized the platelet function of Etv6 mutant mice. Platelets from homozygous mice showed decreased platelet activation upon high (10 ug/ml) and low (2 ug/ml) collagen stimulation as measured by light transmission aggregometry (p=0.041 and 0.019, respectively). Additionally, homozygous mice had decreased markers of platelet activation (thrombin and convulxin) including granule secretion (P-selectin exposure) and aIIbb3 activation (JONA binding).In conclusion, we have generated a CRISPR-Cas9 knock-in mouse with the orthologous mutation to the human P214L recurrent germline mutation. Mice homozygous for this mutation recapitulate the human phenotype of decreased platelet counts and impaired megakaryocyte maturation. Additionally, mice homozygous for this mutation show a significant defect in platelet activation, further supporting that Etv6 is important for platelet function. Although complete knock-outs of Etv6 are embryonic lethal and Etv6 is expressed in the hematopoietic stem cell, we did not find a significant hematopoietic stem cell defect in our mutant mice. This finding suggests that the most important function of Etv6 is further downstream in hematopoiesis. The Etv6 P214L mouse represents a reliable model for studying the role of Etv6 in megakaryocyte biology, platelet function, and potentially future studies in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.
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Jun 1, 2022
Janaína Ribeiro Oliveira + 4
Open Access