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Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies

Abstract

No major predisposition gene for familial myeloproliferative neoplasms (MPN) has been identified. Here we demonstrate that the autosomal dominant transmission of a 700-kb duplication in four genetically related families predisposes to myeloid malignancies, including MPN, frequently progressing to leukemia. Using induced pluripotent stem cells and primary cells, we demonstrate that overexpression of ATG2B and GSKIP enhances hematopoietic progenitor differentiation, including of megakaryocytes, by increasing progenitor sensitivity to thrombopoietin (TPO). ATG2B and GSKIP cooperate with acquired JAK2, MPL and CALR mutations during MPN development. Thus, the germline duplication may change the fitness of cells harboring signaling pathway mutations and increases the probability of disease development.

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Figure 1: Pedigrees of the four families with MPN.
Figure 2: Characterization of the germline CNV.
Figure 3
Figure 4: ATG2B and GSKIP are overexpressed by the CNV.
Figure 5: Hematopoietic differentiation of iPSCs.
Figure 6: The CNV predisposition locus modifies the sensitivity to EPO and TPO in primary cells from patients.
Figure 7: ATG2B and GSKIP are involved in the CNV-induced phenotype.

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Acknowledgements

We greatly thank all the patients and family members involved in the study. We also thank O. Bawa and P. Opolon for the histopathological analysis of teratomas. We thank B. Benyahia for cytogenetic analysis. We thank M. Vestris for recruitment of patients. We greatly thank B. Job and the genomic platform for transcriptome and CGH analysis and also the iPSC platform of Institut Gustave Roussy. We thank S. Saker and T. Larmonier from the Généthon DNA and Cell Bank (Evry, France) for the establishment of B-lymphoblastoid cells from 'NMP' patients. We thank the cytometry platform of Institut Gustave Roussy (P. Rameau and Y. Lecluse). We thank C. Marzac for clinical data. We are grateful to S. Constantinescu and J. Feunteun for critical reading of the manuscript. We are also very grateful to E. Schwartz for proofreading the manuscript.

This work was supported by grants from Agence Nationale de la Recherche (ANR) (Blanc Megon 2009, Thrombocytosis 2011; ANR-13-JVSV1-GERMPN-01), Association pour la Recherche contre le Cancer (ARC) (Fondation ARC Libre 2012-SL220120605292), Groupe Information Santé (GIS)–Institute for Rare Diseases for High-Throughput Sequencing (AO9102LS), Association de Recherche sur la Moelle Osseuse (ARMO), regional Programme Hospitalier de Recherche Clinique (PHRC) AOR07014, Association Laurette Fugain and INCa-DGOS-INSERM 6043. Labex GR-Ex (I.P. and W.V.) is funded by the program 'Investissements d'Avenir'. G. Lenglet was supported by a postdoctoral fellowship from Ile-de-France Cancéropôle and ANR Molecular Medicine in Oncology (MMO) (funded by the program 'Investissements d'Avenir'). F.P. was supported by ARC. L.S. and J.S. were supported by doctoral grants from the Ile-de-France region (Cancéropôle and DIM Cellule Souche) and from Fondation pour la Recherche Médicale (FRM). C.M. was supported by ANR-13-JVSV1-GERMPN-01.

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Authors and Affiliations

Authors

Contributions

W.V., I.P. and C.B.-C. designed and performed research, analyzed data, prepared figures and wrote the manuscript. J.S. performed research on iPSCs and primary cells. G. Lenglet, A.D.S. and L.S. performed research on iPSCs. C.S.-M., N. Droin and G. Leroy performed pangenomic analysis. C.M. performed immunoblot and qRT-PCR analyses. A.P. purified primary cells from donors. E.M. performed colony assays. F.P., J.-C.M., C.D.-D., P.F., F.I. and N.C. were involved in the clinical aspect of the study. A.N. was involved in the clinical aspect of the study and initiated the familial study of MPN. S.G. and S.C. were involved in the generation of EBVCs and their study. B.K. performed cytogenetic analysis. M'b.D., J.M. and P.D. carried out bioinformatics study of exome and transcriptomic data. N. Debili and H.R. provided experimental and/or intellectual input on iPSC culture and hematopoietic differentiation. V.D.V. performed TCL1 mouse modeling. E.S. and O.A.B. contributed intellectual input. All authors contributed to writing and editing.

Corresponding authors

Correspondence to William Vainchenker, Christine Bellanné-Chantelot or Isabelle Plo.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 A common CNV at 14q32 segregated with MPN in all families.

(a) A common haplotype at 14q32 segregated with MPN in both families. The haplotype of interest is depicted as a black bar. Sixteen microsatellite markers were genotyped: D14S81, D14S749, D14S1054, D14S1434, D14S1030, D14S265, D14S996, D14S987, D14S605, D14S131, D14S51, D14S979, D14S611, D14S1067, D14S65 and D14S267. Recombinations occurred in family F1 (patient F1:II.7) downstream of D14S265 (first marker excluded from the common haplotype) and in family F2 between D14S611 and D14S1067. (b) Gel electrophoresis showing the amplification of a 962-bp junction fragment in patients (F1:III-4, F2:III-10, F3:II-2 and F4:II-3) and its absence in non-carrier controls (F1:III-6, F2:IV-8 and F4:II-5). The primers used for PCR were Chr14_B2-C2.1F and Chr14_B1-C2.3R.

Supplementary Figure 2 Molecular characterization of P3-CNV-VF-TET2.

(a) Electropherograms of TET2 DNA sequences. (b) Measurements of the percentage of 5hmC levels by ELISA in P3-CNV-VF-TET2 versus P3-CNV-VF (one experiment). (c) Measurements of 5hmC/1000mC by liquid chromatography–mass spectrometry in erythroblasts from F2:II-4 compared to controls (results are the mean of three independent experiments ± s.e.m. for controls and one experiment for the patient).

Supplementary Figure 3 Molecular characterization of iPSCs.

(a) CGH array analysis is represented by hierarchical clustering with Pearson distance on 14 samples including iPSCs (a or b) and their respective CD34+ progenitor cells from control, P2, P3 and P4. Dots correspond to amplifications (in green, log2 (ratio) > 1.5) and deletions (in red, log2 (ratio) < –1.5), and lines correspond to gains (in blue, 0 < log2 (ratio) < 1.5) and losses (in red, 0 > log2 (ratio) > –1.5). Note that all iPSCs showed a CNV at the 14q locus, indicated by an arrow. As expected, the CGH array analysis showed that the 14q32.13-q32.2 duplication was present in P4-CNV, P3-CNV-VF and P3-CNV-VF-TET2 clones but did not identify significant acquired chromosome abnormalities in the iPSCs compared to the starting CD34+ progenitor cells. (b) Karyotypes of iPSCs from control, P4-CNV, P3-CNV-VF and P3-CNV-VF-TET2. iPSCs showed a normal karyotype except for a constitutional chromosomal abnormality inv(2)(p24.1q14.1) also observed in the primary cells of several patients from family F2 but absent from affected patients from families F1 and F4. Whole-exome sequencing analysis of iPSC clones compared to CD34+ starting cells identified a number of additional mutations compatible with previous reports, but none of these affected genes involved in hematological malignancies.

Supplementary Figure 4 Biological characterization of iPSCs.

(a) Analysis of alkaline phosphatase (AP) activity in iPSC colonies from P2-VF, P3-CNV-VF, P3-CNV-VF-TET2, P4-CNV or control. (b) Flow cytometry analysis of the TRA-1-81 and SSEA-4 pluripotency markers using specific antibodies or control immunoglobulin. (c) qRT-PCR performed for expression of the exogenous transgenes TgSOX2, TgOCT4, TgKLF4 and TgC-MYC in undifferentiated iPSCs using specific primers. PPIA was used as a housekeeping gene. Positive controls correspond to 293EBNA cells transduced with the retroviral vectors. (d) Fold change in exogenous transgenes (TgSOX2, TgOCT4, TgKLF4 and TgC-MYC) levels were compared to positive controls in iPSC-derived GPA+ cells by qRT-PCR (one experiment in duplicate). (e) Fold changes in endogenous pluripotent transcription factors (NANOG, POU5F1 and SOX2) in undifferentiated iPSCs were compared to ES cells by qRT-PCR. (f) The spontaneous differentiation of iPSCs and ES cells was induced by embryoid body formation. The presence of the three germ layers was assessed at day 5 by qRT-PCR performed on BRACHYURY (T), FOXA2 and PAX6. PPIA was used as a housekeeping gene. Results are expressed as fold change compared to the undifferentiated state (mean ± s.e.m., n = 4). (g) IPSCs were injected into immunodeficient NSG mice, and teratomas were analyzed after hematoxylin-eosin-saffron (HES) staining.

Supplementary Figure 5 Duplication modifies the sensitivity to EPO and TPO in iPSCs.

(a) GPA+CD41+ cells from P2-VF, P3-CNV-VF, P3-CNV-VF-TET2, P4-CNV or control were plated in methylcellulose in the presence of SCF and increasing concentrations of EPO. Erythroid progenitor colonies were counted 12 d later. (b) The percentage of large erythroid progenitor colonies (>50 cells per colony) was also calculated. Results are the mean ± s.e.m. of two experiments in duplicate. (c) Pictures of erythroid progenitors. (d) GPA+ cells were deprived of cytokines overnight in serum-free medium and seeded in IMDM alone for 4 h. Cells were stimulated or not with 10 U/ml EPO and analyzed by immunoblot. (e) CD41+ cells from P2-VF, P3-CNV-VF, P3-CNV-VF-TET2, P4-CNV or control were plated in plasma clots without or with increasing concentrations of TPO. CFU-MK colonies were counted at day 10 after indirect staining for CD41a. Results are the mean ± s.e.m. of two experiments in duplicate. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (f) CD41+ cells were sorted and grown, and the percentage of hyperploid cells (>8N) was calculated after propidium iodide labeling and flow cytometry analysis (mean ± s.e.m., n = 5; *P < 0.05, **P < 0.01). (g) Pictures of megakaryocyte cells in culture.

Supplementary Figure 6 Duplication is associated with autophagy.

(a) Platelets from healthy donors, patients or asymptomatic carriers were submitted to immunoblot analysis using LC3-I/II antibody. ß-actin was used as a loading control. Fold increases were calculated compared to control 1. (b) Relative fold change in WIPI1 expression in control EBV cell lines (n = 3 in triplicate) compared to patient cell lines (n = 5 in triplicate). (***P < 0.001.)

Supplementary Figure 7 Model of predisposition.

(a) In sporadic cases, essential thrombocythemia developed with a low frequency at a median age of 67 years as a result of the selection of a first hit (i.e., signaling mutation) either by aging or additional genetic or environmental events. Essential thrombocythemia (ET) can progress to myelofibrosis (MF). (b) In familial cases, the hematological malignancies or essential thrombocythemia developed with a high frequency at a median age of 41 years as a result of the CNV predisposition locus whose molecules cooperate with signaling mutations (JAK2 V617F) to change fitness and induce a growth advantage at the HSC/progenitor level. The progression is also more active and acute and is correlated with TET2 and/or IDH2 mutations.

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Saliba, J., Saint-Martin, C., Di Stefano, A. et al. Germline duplication of ATG2B and GSKIP predisposes to familial myeloid malignancies. Nat Genet 47, 1131–1140 (2015). https://doi.org/10.1038/ng.3380

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