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Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas


Gliomas arising in the brainstem and thalamus are devastating tumors that are difficult to surgically resect. To determine the genetic and epigenetic landscape of these tumors, we performed exomic sequencing of 14 brainstem gliomas (BSGs) and 12 thalamic gliomas. We also performed targeted mutational analysis of an additional 24 such tumors and genome-wide methylation profiling of 45 gliomas. This study led to the discovery of tumor-specific mutations in PPM1D, encoding wild-type p53–induced protein phosphatase 1D (WIP1), in 37.5% of the BSGs that harbored hallmark H3F3A mutations encoding p.Lys27Met substitutions. PPM1D mutations were mutually exclusive with TP53 mutations in BSG and attenuated p53 activation in vitro. PPM1D mutations were truncating alterations in exon 6 that enhanced the ability of PPM1D to suppress the activation of the DNA damage response checkpoint protein CHK2. These results define PPM1D as a frequent target of somatic mutation and as a potential therapeutic target in brainstem gliomas.

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Figure 1: Exome sequencing results for BSGs and thalamic gliomas.
Figure 2: Mutation status of TP53, PPM1D, H3F3A and IDH1 in the extended series.
Figure 3: Landscape of PPM1D alterations and immunohistochemical staining for PPM1D in tumor samples.
Figure 4: Heat map of variant methylation probes in 45 glioma samples, with genetic information.
Figure 5: Effect of cancer-derived truncated PPM1D on CHK2, p53 and H2AX activation and cell growth.

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This study was partly supported by National Cancer Institute grant R01CA140316 (H.Y.), the American Cancer Society (RSG-10-126-01-CCE) (H.Y.), a Pediatric Brain Tumor Foundation Institute grant (D.D.B.), a Voices Against Brain Cancer Foundation grant (D.D.B.), a James S. McDonnell Foundation grant (H.Y.), The V Foundation (H.Y.), an Accelerate Brain Cancer Cure Foundation grant (H.Y.), a Pediatric Brain Tumor Foundation Institute at Duke Early Career Award (Z.J.R.) and Natural Science Foundation of China grants 30772237 and 81241078 (L.Z.).

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Authors and Affiliations



L.Z., H.Y., Z.J.R. and L.H.C. designed research. L.H.C., Z.J.R., J.B.W. and H.Y. wrote the manuscript. L.Z., L.H.C., H.W., W.Z., J.F., S.J., R.Y., P.J.K., J.Z., Z. Wu, S.H., Y.W. and J.B.W. performed the experiments. Z. Wang and H.Z. contributed to the biological validation of the functions of mutant PPM1D. H.S.F. and A.H.F. contributed to the inspection of clinical data. L.H.C., S.Y., S.J., S.W., G.L., R.E.M., Y.H., Z.J.R., D.D.B. and H.Y. analyzed the data.

Corresponding authors

Correspondence to Zachary J Reitman or Hai Yan.

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Competing interests

Under agreements between Duke University, Agios Pharmaceuticals, Blueprint Medicines and Personal Genome Diagnostics, Inc., H.Y. and D.D.B. are entitled to a share of the royalties received by Duke University on the sales of products related to genes described in this manuscript. S.W. and H.Y. are co-founders and own stocks of Beijing Pangenomics Technology, Co. Ltd.

Integrated supplementary information

Supplementary Figure 1 Kaplan-Meier survival curves of patients with brainstem tumors.

(a) Comparison of patients with PPM1D mutation versus wild-type PPM1D. (b) Comparison of patients with PPM1D mutation versus TP53 mutation. (c) Comparison of patients with PPM1D/H3F3A mutations versus TP53/H3F3A mutations.

Supplementary Figure 2 Consensus clustering by methylation β value and Lorenz curve.

Our three groups could be mapped almost perfectly to IDH1 and H3F3A mutation status. We also noted that samples with hemizygous IDH1 mutation could be differentiated from samples with heterozygous IDH1 mutation.

Supplementary Figure 3 Principal-component analysis for our 45 samples.

Tumor samples with H3F3A mutation (HIST), IDH1 mutation (IDH) and neither mutation (WT) could be differentiated by DNA methylation levels.

Supplementary Figure 4 Box plot of DNA methylation levels for sample averages.

The group with IDH1 mutation shows a hypermethylated pattern, whereas the group with H3F3A mutation shows a hypomethylated pattern.

Supplementary Figure 5 Density plot of probes for DNA methylation levels (β values) in different groups.

The group with IDH1 mutation (red) shows a hypermethylated pattern, and the group with H3F3A (K27) mutation (orange) shows a hypomethylated pattern.

Supplementary Figure 6 Electropherograms of HCT116 parental, repaired 1 and repaired 2 lines.

Electropherograms demonstrate repair of the heterozygous frameshift mutation to a wild-type PPM1D sequence in these lines.

Supplementary Figure 7 Colony formation assay for the HCT116 parental, repaired 1 and repaired 2 lines.

Colony formation was assessed in the absence of ionizing radiation and under 2 Gy or 4 Gy of ionizing radiation. Representative stained plates with colony formation are shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1, 2, 5–7 and 9 (PDF 1543 kb)

Supplementary Tables 3, 4 and 8

Supplementary Tables 3, 4 and 8 (XLSX 108 kb)

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Zhang, L., Chen, L., Wan, H. et al. Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas. Nat Genet 46, 726–730 (2014).

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