Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. 
However, DNA copy number and gene expression signatures indicate differences 
between adult and paediatric cases. To explore the genetic events underlying 
this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic 
mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 
44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the 
replication-independent histone 3 variant H3.3, were observed in 31% of tumours, 
and led to amino acid substitutions at two critical positions within the histone 
tail (K27M, G34R/G34V) involved in key regulatory post-translational 
modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome 
X-linked) and DAXX (death-domain associated protein), encoding two subunits of a 
chromatin remodelling complex required for H3.3 incorporation at pericentric 
heterochromatin and telomeres, were identified in 31% of samples overall, and in 
100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations 
were identified in 54% of all cases, and in 86% of samples with H3F3A and/or 
ATRX mutations. Screening of a large cohort of gliomas of various grades and 
histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly 
prevalent in children and young adults. Furthermore, the presence of 
H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative 
lengthening of telomeres and specific gene expression profiles. This is, to our 
knowledge, the first report to highlight recurrent mutations in a regulatory 
histone in humans, and our data suggest that defects of the chromatin 
architecture underlie paediatric and young adult GBM pathogenesis.