The number of possible and observed non-synonymous SNV.

The total number of possible non-synonymous SNVs, the number of which are observed in the MSA, and the proportion of resistance mutations that are observed in the MSA are shown. For example, if no evolutionary constraints whatsoever had been in effect, 679 non-synonymous SNVs would have been possible in exons 20 and 21 of EGFR. In reality, only 438 are observed. The other 241 non-synonymous SNVs presumably interfere with the biological activity of the enzyme and are selected against. When examining only the residues that are linked to resistance mutations (Table S1), none of the variations falls outside of the MSA. Overall, only 5% of the non-synonymous SNVs that lead to resistance mutations fall within 38% of the SNVs that are possible but not observed in the MSA, which indicates that the resistance mutations are subject to evolutionary constraints.