Somatic Mutations

We used all the somatic mutation calls from Level 2 (MAF format) data from the TCGA data portal for 517 samples. We merged calls from the different sequencing centers, removed duplicates and converted MAF to VCF using a custom script. We annotated the resulting comprehensive mutation list using ANNOVAR⁴² to obtain the resulting amino-acid changes.

In order to address the prevalence of specific mutations in HPV-HNSCC methylation subgroups, we performed an ANOVA on the presence or absence of mutation, excluding the HPV+ subgroup. In order to control for the mutation count per sample, we divided the presence of mutation (1) by the total amount of mutations, thus asking “Does the subgroup of a sample explain the variance observed in the fraction of mutations in a given gene”. Every gene was treated independently and p-values were corrected for multiple testing using Bonferroni.

To predict the impact of NSD1 missense mutations on the protein function, we used mutation assessor (http://mutationassessor.org) which evaluates the functional impact of an amino-acid substitution based on the evolutionary conservation of amino-acid residues in a protein family multiple sequence alignment, assuming that mutations that affect conserved residues are more likely to be functional.