Using an independent test set of 238 mutations, we confirmed our results in a comparison with related work.
Of these nonsense mutations, we confirmed 15 of 16 selected loci within the Kaga, HNI, and Nilan strains by targeted PCR and sequencing.
The pattern of base substitutions in the JH4 intronic regions derived from NBS patients (386 mutations) was again different from that in controls (797 mutations) and we confirmed that, as in the VH3-23 therecripts, there were significantly more transversions occurring at G residues (18.9% vs. 13.4%, χ2 test, p<0.01) and G mutations altogether (38.1% vs. 28.9%, χ2 test, p<0.01).
The lamD395 allele was generated by excision of the P-element insertion responsible for the lamP mutation [19], and we confirmed it is a null allele by showing the lamD395 deletion removes exon II containing the translation start codon (Fig. 2).
Among novel mutations observed in this study, we confirmed the mutation to be of germline origin in three out of four patients from whom WBC DNA was available.
Unlike the remarkable intra-tumoral heterogeneity of driver mutations in adult HGG, we confirmed the presence of heterozygous K27M mutations in H3F3A or HIST1H3B in 100%% of tumor cells across all disease compartments in four and three DIPGs, respectively.
Two relatively early-onset patients from the same family carried a H137L mutation in RP9, which we confirmed by both genomic and cDNA sequencing (Fig. S2).
ZIC4 and PLCB1 were the genes with the lowest methylation frequency in HNSCC, exhibiting also low mutation frequency, and yet, we confirmed ZIC4 and PLCB1 promoter methylation differences between normal and cancer samples in the Validation cohort.
We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products.
