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. 2015 Jun 23;11(6):e1005262.
doi: 10.1371/journal.pgen.1005262. eCollection 2015 Jun.

Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

Sabine Topka  1 , Joseph Vijai  1 , Michael F Walsh  2 , Lauren Jacobs  3 , Ann Maria  3 , Danylo Villano  3 , Pragna Gaddam  3 , Gang Wu  2 , Rose B McGee  2 , Emily Quinn  2 , Hiroto Inaba  2 , Christine Hartford  2 , Ching-Hon Pui  2 , Alberto Pappo  2 , Michael Edmonson  2 , Michael Y Zhang  4 , Polina Stepensky  5 , Peter Steinherz  3 , Kasmintan Schrader  6 , Anne Lincoln  3 , James Bussel  7 , Steve M Lipkin  7 , Yehuda Goldgur  8 , Mira Harit  5 , Zsofia K Stadler  3 , Charles Mullighan  2 , Michael Weintraub  5 , Akiko Shimamura  9 , Jinghui Zhang  2 , James R Downing  2 , Kim E Nichols  2 , Kenneth Offit  10
Affiliations

Germline ETV6 Mutations Confer Susceptibility to Acute Lymphoblastic Leukemia and Thrombocytopenia

Sabine Topka et al. PLoS Genet. .

Abstract

Somatic mutations affecting ETV6 often occur in acute lymphoblastic leukemia (ALL), the most common childhood malignancy. The genetic factors that predispose to ALL remain poorly understood. Here we identify a novel germline ETV6 p. L349P mutation in a kindred affected by thrombocytopenia and ALL. A second ETV6 p. N385fs mutation was identified in an unrelated kindred characterized by thrombocytopenia, ALL and secondary myelodysplasia/acute myeloid leukemia. Leukemic cells from the proband in the second kindred showed deletion of wild type ETV6 with retention of the ETV6 p. N385fs. Enforced expression of the ETV6 mutants revealed normal transcript and protein levels, but impaired nuclear localization. Accordingly, these mutants exhibited significantly reduced ability to regulate the transcription of ETV6 target genes. Our findings highlight a novel role for ETV6 in leukemia predisposition.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Identification of germline mutations in ETV6 in 2 unrelated kindreds.
(a) In Kindred 1, targeted sequencing identified a germline ETV6 L349P mutation. Sequencing was performed on 9 individuals including the proband (arrow) affected with thrombocytopenia and/or ALL and 7 unaffected individuals as noted in Table 1. (b) In Kindred 2, clinical whole exome sequencing was performed on the proband (arrow) with ALL, MDS and AML, the mother with thrombocytopenia as well as the unaffected father. An ETV6 N385fs mutation was identified. In both kindreds, the ETV6 mutations segregated with disease.
Fig 2
Fig 2. Location of somatic and germline ETV6 mutations and structural modeling.
(a) Schematic depicting the germline ETV6 mutations detected in the MSKCC and SJCRH kindred, reported separately or somatic mutations detected as part of the Pediatric Cancer Genome Project. Somatic and germline mutations are indicated by separate green and purple lines, respectively. (b) Structural modeling of ETV6 with the germline ETV6 L349P and N385fs mutations. The ETV6 L349P amino acid substitution is located on an α-helix within the DNA binding domain and causes extensive kinking of the protein structure. The ETV6 N385fs mutation results in truncation of the DNA binding domain.
Fig 3
Fig 3. Effect of germline ETV6 mutations on transcription.
(a) The effects of germline mutations on ETV6 function were examined using a Dual Luciferase Reporter Assay. Each of the mutants tested exhibited significantly (****P ≤0.0001) impaired transcriptional repression from the PF4 and MMP3 promoter constructs when contrasted with the WT ETV6 in the co-transfection experiment. The experiment was performed with 6 replicates for each condition and repeated 3 times. Statistical analysis was done using an unpaired t-test, the error bars show the Standard Error of Mean (SEM). (b) The effects of V37M and R181H germline mutations on ETV6 function were examined using a Dual Luciferase Reporter Assay. The experiment was performed with 6 replicates for each condition and repeated twice. Statistical analysis was done using an unpaired t-test, the error bars show the Standard Deviation (SD). (c) Quantitative PCR of ETV6 transcriptional targets EGR1 and TRAF1 showed reduced transcriptional abundance in the mutants when contrasted with the WT. The effect was most pronounced in the frameshift mutant. The experiment was performed in triplicate for each condition and repeated three times. Statistical analysis was done using an unpaired t-test, the error bars show the Standard Error of Mean (SEM).
Fig 4
Fig 4. Germline ETV6 mutations impair localization of the ETV6 protein.
Western blots of HeLa cell fractions probed for ETV6 protein show (a) presence of ETV6 within the cells transiently overexpressing ETV6 WT and the P214L, R369Q and R339C mutants within the nucleus. Both the L349P and N385fs mutant were not detected in the nuclear fraction. (b) Presence of ETV6 protein is abundant in the cytoplasmic fraction. The frameshift mutant N385fs showed a protein product that was smaller (45kDa) than the full-length protein (53kDa). (c) Densitometric analysis of the western blots shows that the WT localization is predominantly nuclear, while the L349P and N385fs are cytoplasmic. Other mutants P214L, R369Q and R339C show localization to a lesser extent in the nucleus.
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