Functional Studies of Polymorphisms

For most polymorphisms, functional information is not available. Therefore, it is important to predict whether a given polymorphism may result in a change in protein function, stability, or subcellular localization. One approach to understanding the functional effects of various types of genomic variations is to survey the mutations that have been associated with human Mendelian disease. DNA variations associated with diseases or traits most frequently are missense and nonsense mutations, followed by deletions. Of amino acid replacements associated with human disease, there is a high representation at residues that are most conserved evolutionarily. More radical changes in amino acids also are more likely to be associated with disease than more conservative changes; substitution of a charged amino acid (Arg) for a nonpolar, uncharged amino acid (Cys) is more likely to affect function than substitution of residues that are more chemically similar (e.g., Arg to Lys).

With an increasing number of SNPs being identified by large-scale SNP discovery projects, computational methods are needed to predict the functional consequences. To this end, predictive algorithms have been developed to identify potentially deleterious amino acid substitutions. These methods can be classified into two groups. The first group relies on sequence comparisons alone to identify and score substitutions according to their degree of conservation across multiple species. The second group of methods relies on mapping of SNPs onto protein structures, in addition to sequence comparisons.

For many proteins—including enzymes, transporters, and receptors—the mechanisms by which amino acid substitutions alter function have been characterized in kinetic studies. Figure 4-5 shows simulated curves depicting the rate of metabolism of a substrate by two amino acid variants of an enzyme and the most common genetic form of the enzyme. The metabolism of substrate by one variant enzyme, Variant A, is characterized by an increased Km. Such an effect can occur if the amino acid substitution alters the binding site of the enzyme leading to a decreased affinity for the substrate. An amino acid variant may also alter the maximum rate of substrate metabolism (V ) by the enzyme, as exemplified by Variant B. Such reductions in Vmax generally reflect reduced expression of the enzyme, which may occur because of decreased protein stability or changes in protein trafficking or recycling.

In contrast to studies with coding region SNPs, predicting the function of SNPs in noncoding regions of genes represents a major challenge in human genetics and pharmacogenetics. The principles of evolutionary conservation that have been validated in predicting the function of

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