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Novel somatic and germline mutations in intracranial germ cell tumours


Intracranial germ cell tumours (IGCTs) are a group of rare heterogeneous brain tumours that are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographical and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically five- to eightfold greater in Japan and other East Asian countries than in Western countries1, with peak incidence near the time of puberty2. About half of the tumours are located in the pineal region. The male-to-female incidence ratio is approximately 3–4:1 overall, but is even higher for tumours located in the pineal region3. Owing to the scarcity of tumour specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next-generation sequencing, single nucleotide polymorphism array and expression array. We find the KIT/RAS signalling pathway frequently mutated in more than 50% of IGCTs, including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gains of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional co-repressor and tumour suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, which codes for a histone demethylase and is a coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway.

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Figure 1: Subgroup specificity of the recurrent genetic alterations identified in 62 IGCT patients.
Figure 2: Novel recurrent somatic and germline mutations in IGCT.
Figure 3: Frequent genetic alterations of the KIT/RAS and AKT/mTOR signalling pathways.
Figure 4: Enrichment of germline JMJD1C variants in IGCT.

Accession codes

Data deposits

All sequencing and genotyping data have been deposited in the NCBI database of Genotypes and Phenotypes (dbGaP, under accession number phs000725.v1.p1.


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This work was supported by research funding from the National Human Genome Research Institute (NHGRI, grant number 5U54HG003273) to D.A.W., the Children Brain Tumor Foundation, the Gillson Longenbaugh Foundation and Anderson Charitable Foundation to C.C.L., the CCBTP Fellowship from the Cancer Prevention & Research Institute of Texas (CPRIT grant RP101489) to S.Y., the St Baldrick’s Foundation to K.T. and the NLM predoctoral fellowships to M.D.B. (5T15 LM07093-18) and J.S. (5T15 LM07093-19). We thank H. H. Dinh and Y. Han for their technical support, J. G. Reid for Illumina sequence mapping.

Author information




L.W. conducted the bioinformatics analyses of the sequencing and single nucleotide polymorphism array data, integrated data from multiple platforms, wrote and revised the manuscript. S.Y. contributed to the conduct of the research. M.D.B., J.S., M.W. contributed to DNA copy number analysis. K.T. contributed to the coordination and conduct of the research. K.C. contributed to the mutation calling and annotation pipeline for AmpliSeq data. H.-K.N. and H.N. performed AKT1 immunohistochemistry assay. L.L. contributed to the construction of the AmpliSeq libraries. C.C.L., T.S., R.N., H.N., A.N., S.T., H.K.N., R.D., W.W. and A.A. collected tumour specimens, provided the histopathological confirmation and interpreted the clinical data. Y.Q., G.M. and L.W. contributed to clonality analysis. D.M.M. and H.D. managed the production pipeline. Z.H. and S.M.L. performed rare variant association tests for JMJD1C. R.A.G. contributed to the revision of the manuscript. D.A.W. and C.C.L. conceived the study, supervised the research, and contributed to the writing and revision of the manuscript.

Corresponding authors

Correspondence to David A. Wheeler or Ching C. Lau.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The performance of whole-exome sequencing.

a, The average read coverage across all samples in the discovery study. b, The base 20+ coverage. Base 20+ coverage, the percentage of targeted bases that were covered by at least 20 sequencing reads.

Extended Data Figure 2 The number and rate of validated somatic mutations.

a, The number of validated somatic nonsynonymous mutations across all tumours in the discovery study. b, The rate of somatic non-silent mutations across all tumours in the discovery study. c, The mutation rate of NGGCTs. Only validated non-silent somatic mutations were counted. Blue circles, mature teratomas; green circle, tumour defined as NGGCT but without detail information for subtype; brown circles, immature teratomas; red circles, yolk sac tumours. It is of note that M1 tumour is a mixture of germinoma and yolk sac tumour thus was included here. Mature teratomas, which are considered as histologically benign tumours, have the lowest mutation rate, followed by immature teratomas (0.1 per Mb). Yolk sac tumours have the highest mutation rates (0.6 per Mb).

Extended Data Figure 3 The mRNA expression levels of KIT.

The mRNA expression levels were determined by Affymetrix U133Plus2 human gene expression array for 37 out of the total 62 IGCT tumours with available RNA. Red dots, tumours with somatic mutation of KIT; green dot, tumour with somatic CBL mutation and was wild type for KIT; G, pure germinomas; mixed, mixed germ cell tumours with germinoma component; NGGCT, non-germinomatous germ cell tumours. P value was calculated using one-way ANOVA analysis.

Extended Data Figure 4 The somatic mutations identified in KRAS and NRAS.

a, The distribution of somatic mutations identified in KRAS and NRAS. The positions and amino acid changes were indicated for each mutation. b, KRAS/NRAS mutations are mutually exclusive with mutations in KIT. Two-by-two table showing that mutations in KIT and KRAS/NRAS were mutually exclusive. Left-tailed Fisher’s exact test was applied to calculate the P value. P = 0.018.

Extended Data Figure 5 The clonal and subclonal loss of heterozygosity events on chromosome 11q (11qLOH).

a, Topographic maps showing regions of clonal or subclonal 11qLOH spanning the CBL locus in individual patients. b, The clonal and subclonal 11qLOH in two representative cases G4 and G11. The red rectangle indicates the CBL locus. BAF, B allele frequency; LRR, Log2 R ratio; CN, copy number.

Extended Data Figure 6 Recurrent DNA copy number gains at 14q32 identified in the discovery study.

a, Recurrent focal amplification of 14q32.33 spanning the AKT1 locus. Regions of absolute DNA copy number are plotted for 14q (top panel) and 14q32.33 spanning the AKT1 locus (bottom panel). Each row represents an individual tumour. Tumours were sorted in descending order by their absolute copy number within the boundaries of AKT1 (indicated to the right). The mean copy number across chromosomes 1–22 (ploidy) is indicated alongside the tumour IDs on the left. The x axis shows chromosome 14 genomic locations in megabase pairs (Mb). CN, copy number inferred by Omni2.5 SNP array. b, The correlation between the DNA copy number status and levels of mRNA expression of two representative genes, XRCC3 and CDCA4, presented in focal amplified region 14q32.33. The P value across all groups was calculated by Spearman’s rank-order correlation analysis. Rho, the Spearman’s correlation coefficient.

Extended Data Figure 7 The workflow for filtering of germline variants.

Germline variants were filtered step by step to pick up the potentially interesting candidates. First, select the non-silent variants including missense, nonsense, frameshift, and splice-site variants. Second, select the high-confidence variants that meet the following criteria: (1) variant allele fraction in both tumour and normal equal to or greater than 0.20; (2) variant calling was supported by at least 4 sequencing reads for both tumour and normal samples. Third, select novel variants that have not been reported in dbSNP database (dbSNP135). Then, select genes with COSMIC evidence, that is, genes for which mutations have been reported in COSMIC database in at least 100 times. After that, for all 1,876 genes left in the above list, calculate the fold of enrichment of the germline variants in Japanese IGCT patients by comparing its frequency to that of Japanese patients in the control cohort and performed Fisher’s exact test to calculate the P values. Then, select potentially interesting genes based on the IGCT frequency bias (≥ 4) and significant P values (<0.05).

Extended Data Figure 8 The mRNA expression levels of JMJD1C and AR in IGCT.

a, The mRNA expression levels of JMJD1C. b, The mRNA expression levels of AR. The mRNA expression levels were determined by Affymetrix U133Plus2 human gene expression array for 37 out of the total 62 tumours with available RNA. Left, the expression level of JMJD1C or AR in all IGCT tumours analysed; middle and right, the expression level of JMJD1C or AR comparing to other known genes in representative tumours. Selected genes were highlighted in different colours and the remaining genes were coloured in grey. The red dashed lines indicate median values of expression.

Extended Data Figure 9 Subgroup specificity of LOH and chromosomal imbalance in IGCT.

Summary of the gross chromosomal alterations based on genome-wide Illumina Omni2.5 SNP array. Ploidy was predicted by Genome Alteration Print (GAP) algorithm30. Chromosomal imbalances are represented by the change of B-allele frequency (BAF) pattern with or without the loss of heterozygosity (LOH). G, germinoma; NGGCT, non-germinomatous germ cell tumour; M, mixed GCTs with germinoma component.

Extended Data Figure 10 An overview of the subclonal signatures across all tumours in the discovery study.

Those cases without SNP array data or without detectable copy number changes were excluded for clonality analysis. Each peak in the plot indicates a subclone. The x axis indicates mBAF and the y axis indicate the number of heterozygous SNPs. Those cases with single peak are monoclonal and those with multiple peaks are polyclonal. Some subclones were highlighted, such as the subclone in G4, for a better visualization. The amplitude of the peaks in the plot has nothing to do with the fractions of cells that are harbouring each event.

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Wang, L., Yamaguchi, S., Burstein, M. et al. Novel somatic and germline mutations in intracranial germ cell tumours. Nature 511, 241–245 (2014).

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