Page 250 - Haematologica Vol. 107 - September 2022

LETTER TO THE EDITOR
ETV6-related thrombocytopenia: dominant negative effect of mutations as common pathogenic mechanism
Inherited thrombocytopenias are a group of rare diseases characterized by low platelet count and variable bleeding tendency. In some forms, patients might develop addi- tional phenotypes during life, such as myeloid neoplasms as in ETV6-related thrombocytopenia (ETV6-RT). ETV6-RT is caused by germline heterozygous mutations of ETV6, a gene encoding a master hematopoietic transcriptional re- pressor structured in three functional domains: the N-ter- minal pointed (PNT), the central regulatory domain (CRD) and C-terminal DNA-binding (ETS).1
To our knowledge, at least 15 different ETV6 variants, mainly amino acid substitutions, have been associated with thrombocytopenia,2–9 though their pathogenic role has not always been clarified. Moreover, whereas ETV6 is well known as a tumor suppressor in chromosomal trans- locations associated to childhood leukemia,10 the molecu- lar mechanism responsible for ETV6-RT remains to be elucidated.2,3 Understanding the molecular mechanisms involved in ETV6-RT pathogenesis is important in order to clarify the role of ETV6 in megakaryopoiesis and leukemia, to identify a possible therapeutic approach able to correct platelet biogenesis and to prevent the onset of leukemia. For these reasons we have studied seven ETV6 missense variants identified in patients with thrombocytopenia, demonstrating that five of them reduce the repression ac- tivity of ETV6 preventing its localization into the nucleus, as confirmed by inhibition of the nuclear export by lep- tomycin B. Moreover, we have demonstrated that the mu- tations act through a dominant negative effect, which results in accumulation of the wild-type (WT) protein in the cytoplasm likely due to formation of WT-mutant dimer of ETV6.
Individuals with suspicion of inherited thrombocytopenia due to their low platelet count were refereed to our Insti- tution for molecular diagnostic purpose. A next generation sequencing (NGS) approach allowed us to identify seven missense variants in ETV6 gene, some of which were pre- viously reported in patients with thrombocytopenia or leukemia (Online Supplementary Table S1).5–7,9
All the variants are clustered within the ETS domain ex- cept for the S22N, that hits the PNT domain, and the H224Q, which is located in the CRD as the control muta- tion P214L. The ETS domain is a critical site for the binding of ETV6 to the DNA, suggesting that its impairment can lead to a loss or an alteration in transcriptional repression. Accordingly, different bioinformatic tools suggest a higher impact on the protein functions for the five variants that reside in the ETS domain, while H224Q and S22N were
predicted as likely benign substitutions. The pathogenic role of these five variants clustered within the ETS domain is supported also by structural analysis revealing a poten- tial effect not only of R369W and W380R6 but also of the novel Q347P and R396G variants on protein stability and folding (Online Supplementary Figure S1A). Moreover, since structural analysis of ETS bound to a specific DNA se- quence revealed that R396 and R399 are involved in elec- trostatic interactions with its DNA cognate (Online Supplementary Figure S1B), R396G and R399H substitu- tions are likely to prevent the binding of the mutant forms of ETV6 to its targets.
Notably, two germline disease causing mutations, c.1106G>A and c.1195C>T involving R369 and R399, re- spectively, but with a different amino acid substitution have been previously reported.3,9
In order to verify the predictions obtained from in silico analyses, we tested the activity of reporter luciferase gene under the control of the Stromelysin-1 (MMP3) promoter, a validated target of ETV6.11 In HEK293T cells, over- expression of the WT form of ETV6 resulted in a re- pression of luciferase activity to 44.3±8.6% compared to the empty vector. On the contrary, transfecting the Q347P, R369W, W380R, R396G and R399H mutant (M-ETV6) forms, as well as P214L used as control,2,3 we observed significantly higher luciferase activity (Online Supplemen- tary Figure S2).
These results highlight the loss of the repression ability of these ETV6 variants on the MMP3 promoter, confirming their pathogenicity. On the contrary, the repressive activity was maintained by S22N and H224Q fully comparable to the WT, suggesting the absence of any pathogenic role, according to the in silico predictions.
In order to determine whether the loss of repression ac- tivity of the M-ETV6 forms could be explained also with a reduced nuclear localization of the protein, as observed for other mutations,2,3 we performed immunofluorescence assays in HeLa cells. Whereas the WT, S22N and H224Q forms are mainly detected in the nucleus, the other mu- tant proteins are prevalently cytoplasmatic (Figure 1A and B). The aberrant localization of the Q347P, R369W, W380R, R396G and R399H mutants was confirmed by western blot assays of cellular fractions. While the WT protein de- tected in the nucleus is 69.9±16.5%, the nuclear fractions of mutant proteins account for 5,7±4,1%, 28,3±9.7%, 7.7±5.8%, 30.8±15.3% and 23.2±8.8%, respectively (Figure 1C and D).
We next investigated the mechanisms retaining the M-
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