The Genom of Homo sapiens.pdf

474 WALLACE, RUIZ-PESINI, AND MISHMARFigure 4. Geographic variation in mtDNA polypeptides. Thehaplogroups included in the “Tropical” category are L0–L3;“Temperate” are H, V, U, J, T, X, N1b, and W; and “arctic” areA, C, D, G, X, Y, and Z. The distribution of the Ka/(Ks + constant)values is given by the vertical line. The colored dot is themean; the square encompasses the 25–75% range. The statisticaldifference of distribution of Ka/Ks values of the temperateand arctic zones, relative to the tropical zone, are presentedabove the distributions. (Adapted from Mishmar et al. 2003b.)joining, phylogeny (Fig. 2). This phylogeny was thenused to position all nucleotide substitutions in the tree.Mutations that were shared by a number of relatedmtDNA haplotypes were placed at the internal branch oftheir common ancestor. Mutations that were found inonly one mtDNA were placed at the ends of the branches.Therefore, the position of each mutation within the tree isindicative of its relative age.Three different categories of nucleotide variants wouldbe expected. The first is neutral mutations. These mutationscould be either synonymous or nonsynonymous, yethave minimal phenotypic effect. Neutral variants wouldaccumulate in the mtDNA phylogeny by chance at a relativelyconstant rate and be uniformly distributedthroughout the tree. Hence, the number of synonymousmutations in a segment of the tree would be proportionalto that segment’s age. The second class of mutationswould be deleterious replacement substations. These mutationswould be rapidly eliminated by purifying selection,experienced at the individual level as mitochondrialdisease. The third class of mutations would be advantageousreplacements. These would become enriched in apopulation by adaptive selection. Such adaptive mutationswould be rare, but they would tend to foundbranches of the tree that were successful in new environments.Thus, adaptive mutations would be common in internalbranches but rare at the terminal branches (Templeton1996).By this logic, we can distinguish between deleteriousand adaptive mutations by their distribution in themtDNA tree. If only purifying selection has been actingon a region-specific branch of the tree, the frequency ofreplacement mutations will be low at the internalbranches (Nodes). Consequently, the ratio of the replacementmutation frequencies (NS/S) of the Nodes dividedby Terminal Branches (Tips) will be low. However, ifadaptive selection has acted, then the frequency of replacementmutations at the Nodes will be increased andthe Nodes/Tips replacement mutation frequency will alsobe increased. Purifying selection should predominate instable environments, whereas adaptive selection shouldbecome more important in changing environments.Since neutral mutations would be dispersed throughoutthe phylogeny, we had to distinguish between the neutraland the adaptive replacement mutations. This was accomplishedby analyzing the inter-specific conservation of themutant amino acid, since functionally important aminoacids would tend to be conserved through organismal evolution.To determine whether a mutation altered a highlyconserved amino acid, we examined the mtDNA proteinsequences from 39 vertebrates listed in our MITOMAPdatabase (www.mitomap.org). The number of thesespecies that have the ancestral human mtDNA sequence isdefined as that amino acid’s Conservation Index.We then calculated the mean Conservation Index of theTip and Node replacement mutations and found that theConservation Index of the Tips was greater than that ofthe Nodes (Fig. 5). This is probably due to the fact thatdeleterious mutations can alter any amino acid, but adaptivemutations can only modify a function, not eliminateit. Hence, adaptive mutations are more constrained.To determine the level of Conservation Index thatwould result in a deleterious phenotype, we turned to theMITOMAP collection of all known human pathogenicmissense mutations. The Conservation Indices of all 22reported pathogenic replacement mutations were thencalculated. The average of these values proved to be36.4 ± 5.2 (93 ± 13%), the maximum value being 39.Figure 5. Global amino acid Conservation Indices forpathogenic, nodal, and terminal replacement mutations. “Path”equals the mean Conservation Index for 22 known pathogenicmutations, “Tips” and “Nodes” equal Conservation Indices foramino acid substitutions at internal branches versus terminalbranches, “Node (NC)” and “Node (CO)” represent the averageconservation of the nodal replacement mutations that are outsideand within two standard deviations from the pathogenic mutationsmean.