Amino acid substitutions from human MTHFR orthologs tested for functional impact.

*Human-Macaque ancestral reconstruction and substitutions not found in any ortholog (P67V, R134C, R134F, D291N) are not listed.
**Boldface species are present in Figure 3.
***Ancestral reconstruction, >90% probability using PAML.
#The identities of these residues are conserved in the human lineage from the more recent of the two ancestors listed, at which point a longer-standing pattern of preservation (dating back to the more ancient of the two) was broken.
“Human Site Conserved in Clade” lists clades in which the identity of the human enzyme is seen multiple times. The sequence alignments for all positions interrogated in this study are in Table S2. “Human Lineage Preservation” is the most ancient MTHFR ancestor in which the identity of that site in the human enzyme has been preserved. “Substitution accepted in Ortholog”: MTHFR ortholog(s) in which the substitution can be found. Grantham score [5] estimates the physico-chemical dis-similarity between the parent and substituting amino acids. SIFT score [32] estimates whether the amino acid change can be tolerated based on the variability at that site in homologous proteins (see text for details). The order is presented as in Figure 2.