5’UTR point substitutions and N-terminal truncating mutations of ANKRD26 in acute myeloid leukemia
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Thrombocytopenia 2 (THC2) is an inherited disorder caused by monoallelic single nucleotide substitutions in the 5’UTR of the ANKRD26 gene. Patients have thrombocytopenia and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). Given the association of variants in the ANKRD26 5’UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5’UTR. We found variants in four patients. One patient had the c.−125T>G substitution in the 5’UTR, while three patients carried two different variants in the 5’ end of the ANKRD26 coding region (c.3G>A or c.105C>G). Review of medical history showed that the patient carrying the c.−125T>G was actually affected by typical but unrecognized THC2, highlighting that some apparently sporadic AML cases represent the evolution of a well-characterized familial predisposition disorder. As regards the c.3G>A and the c.105C>G, we found that both variants result in the synthesis of N-terminal truncated ANKRD26 isoforms, which are stable and functional in cells, in particular, have a strong ability to activate the MAPK/ERK signaling pathway. Moreover, investigation of one patient with the c.3G>A showed that mutation was associated with strong ANKRD26 overexpression in vivo, which is the proposed mechanism for predisposition to AML in THC2 patients. These data provide evidence that N-terminal ANKRD26 truncating mutations play a potential pathogenetic role in AML. Recognition of AML patients with germline ANKRD26 pathogenetic variants is mandatory for selection of donors for bone marrow transplantation.
KeywordsANKRD26 gene Acute myeloid leukemia Inherited predisposition to leukemia Inherited thrombocytopenia
Acute myeloid leukemia
Hematopoietic stem cell
Thrombocytopenia 2 (THC2, MIM 188000) is an autosomal dominant disorder caused by monoallelic single nucleotide substitutions in the 5’UTR of the ANKRD26 gene [1, 2]. Patients have mild to moderate thrombocytopenia, mild or no bleeding tendency, and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). The analysis of 222 consecutive THC2 patients showed that the incidence of AML, myelodysplastic syndromes, and chronic myelogenous leukemia was significantly higher than expected, with an estimated risk of AML 24-fold increased with respect to the general population . The role of ANKRD26 in hematopoiesis is poorly understood. A recent investigation indicated that thrombocytopenia of THC2 patients is caused by ANKRD26 overexpression in megakaryocytes due to defective downregulation by RUNX1 and FLI1, which, in turn, derives from impaired binding of these transcription factors to the mutated 5’UTR .
A growing body of evidence indicates that a significant proportion of apparently sporadic, adult-onset AML cases originate from a germline predisposition, which often is not recognized [5, 6]. Given the association of variants in the ANKRD26 5’UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5’UTR. Genomic DNA was obtained from peripheral blood at the time of diagnosis.
We found three different variants in four patients, whose clinical features are reported in Additional file 1: Table S1. One patient carried the c.−125T>G substitution in the 5’UTR that was previously reported as responsible for THC2 . Review of personal and family history disclosed that this subject had thrombocytopenia since childhood, and one sister and her son had independently received the diagnosis of THC2 due to the same mutation. We could confirm that the c.−125T>G had a germinal origin (Additional file 1: Table S1). Therefore, this AML case represented the evolution of a typical but unrecognized THC2.
Two patients carried the c.3G>A variant of ANKRD26 that is predicted to cause the loss of the physiologic start codon (p.Met1?). In both patients, we could analyze the DNA from different tissues (urinary epithelium, saliva, and blood collected in complete remission), which demonstrated the germinal origin of the variant. Finally, one patient had the c.105C>G substitution resulting in the generation of a stop codon at position 35 (p.Tyr35*). We thus investigated the effects of these two variants in the 5’ end of the ANKRD26 coding region.
To investigate the stability of the mutant proteins in cells, we blocked the protein synthesis by adding cycloheximide to HeLa cultures 24 h after transfection and measured the kinetics of the subsequent reduction of the amounts of transfected proteins. These experiments showed that both variants presented a similar stability as the WT protein (Additional file 1: Figure S1).
We then investigated whether these mutant proteins maintain their function. The best known functional activity of ANKRD26 is the modulation of different kinase signaling pathways [4, 7], especially the MAPK/ERK pathway. ANKRD26 regulates ERK phosphorylation in mouse embryonic fibroblasts . Hyperactivation of ERK in human megakaryocytes is the mechanism of thrombocytopenia in THC2 and increased ERK signaling at the level of the myeloid progenitors could contribute to predisposition to myeloid malignancies . In HeLa cells, transfection of either WT or mutant ANKRD26 (but not of the empty vector) resulted in a marked phosphorylation of ERK, while it had no effects on some other signal transduction kinases such as AKT or p38MAPK (Fig. 2a, b). The efficiency of exogenous ANKRD26 in phosphorylating ERK was measured as the p-ERK/ERK ratio weighted for FLAG: this value was 2.7- to 3.3-fold higher for the mutants compared with WT ANKRD26 (Fig. 2c). We concluded that the N-truncated ANKRD26 variants do maintain the ability to activate the ERK pathway of the WT protein and could be even more potent ERK activators than the WT ANKRD26.
Interestingly, the c.3G>A and the c.105C>G variants were not present in an in-house cohort of 510 consecutive control individuals of the same geographic origin (Additional file 1: Methods) and resulted in a significantly higher frequency in our cohort of AML patients in comparison to the non-The Cancer Genome Atlas subset of the Exome Aggregation Consortium (exac.broadinstitute.org), with p values of 0.012 and 0.032 for the c.3G>A and c.105C>G, respectively.
In summary, the analysis of a large case series showed that variants in the ANKRD26 5’UTR are infrequent among non-familial AML patients. However, some apparently sporadic, adult-onset AML cases represent the evolution of an unrecognized THC2. Identification of these cases is imperative especially in patients who are candidates for hematopoietic stem cell (HSC) transplantation from a family donor, in order to avoid the use of HSC from a donor affected by the same inherited disorder. In fact, several reports indicate that the use of HSC from donors with germline mutations predisposing to hematological malignancies resulted in the development of donor-derived leukemia in the recipient and/or poor transplant engraftment [5, 8, 9, 10]. Of note, the sister of patient 1 with THC2 developed chronic myelomonocytic leukemia 2 years after the onset of AML in the proband.
Moreover, we observed that mutations in the ANKRD26 coding sequence resulting in the truncation of the protein N-terminus also have a regulatory effect, causing ANKRD26 overexpression and thus playing a potential role in AML. In fact, ANKRD26 overexpression is the proposed pathogenetic mechanism for both thrombocytopenia and predisposition to AML in THC2 patients . Since none of the patients 2–4 presented thrombocytopenia before AML, we suggest that, unlike THC2 mutations, the coding variants described here induce ANKRD26 overexpression though a mechanism independent of RUNX1/FLI1 interaction with the 5’UTR of the gene and possibly due to increased mRNA stability. In this way, the transcription factors are still able to bind the 5’UTR and downregulate ANKRD26 in megakaryocytes, thus avoiding thrombocytopenia. Whatever the mechanisms of ANKRD26 upregulation, we showed that these N-truncated isoforms are stable in cells and have a strong ability to activate the MAPK/ERK pathway. Although further investigation is required, the present data strongly suggest that N-terminal truncating mutations of ANKRD26 have a potential pathogenetic role in apparently sporadic AML. Since our investigation was restricted to non-familial AML cases, prevalence of ANKRD26 pathogenetic variants in AML could be greater than we found.
This work was supported in part by grants from the Telethon Foundation, Italy (GGP10089), the Cariplo Foundation, Italy (2012–0529, 2010–0807), the Italian Ministry of Health (RF-2010-2310098), and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
Availability of data and materials
All data generated and analyzed during the current study are included in the submitted article and its supplementary information file. Materials as well as additional information are available from the corresponding author on reasonable request.
CM, MS, and AP designed the research, interpreted the data, and wrote the manuscript. GSi, GM, and GSa designed the research, enrolled the patients, and interpreted the data. MT and IP designed the research and interpreted the data. IC, VB, TP, FM, and SA performed the experiments and interpreted the data. All the authors critically revised the manuscript and approved the final version.
The authors declare that they have no competing interests.
Consent for publication
The four individuals whose ANKRD26 mutations and clinical data are reported in the paper provided written informed consent for publication in an anonymous form.
Ethics approval and consent to participate
The study was approved by the Institutional Review Board of the Institute of Hematology “L. and A. Seràgnoli”, University of Bologna, Bologna, Italy, and the Institutional Review Board of the San Luigi Hospital, University of Turin, Orbassano, Turin, Italy—the two institutions that enrolled the patients. All patients provided written informed consent in accordance with the Declaration of Helsinki.
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