To predict the potential functional impact of amino-acid substitutions we used two different methods—Sorting Intolerant From Tolerant (SIFT)41 and Polymorphism Phenotyping (PolyPhen).42 SIFT uses sequence homology to predict whether a substitution is likely to affect protein function, and PolyPhen predicts protein function on the basis of 3D structure and multiple alignments of homologous sequences.
