California Institute for Quantitative Biosciences, Department of Molecular and Cellular Biology, University of California Berkeley, Berkeley, California, United States of America.
PLoS Genet. 2010 May 27;6(5):e1000968. doi: 10.1371/journal.pgen.1000968.
Computational predictions of the functional impact of genetic variation play a critical role in human genetics research. For nonsynonymous coding variants, most prediction algorithms make use of patterns of amino acid substitutions observed among homologous proteins at a given site. In particular, substitutions observed in orthologous proteins from other species are often assumed to be tolerated in the human protein as well. We examined this assumption by evaluating a panel of nonsynonymous mutants of a prototypical human enzyme, methylenetetrahydrofolate reductase (MTHFR), in a yeast cell-based functional assay. As expected, substitutions in human MTHFR at sites that are well-conserved across distant orthologs result in an impaired enzyme, while substitutions present in recently diverged sequences (including a 9-site mutant that "resurrects" the human-macaque ancestor) result in a functional enzyme. We also interrogated 30 sites with varying degrees of conservation by creating substitutions in the human enzyme that are accepted in at least one ortholog of MTHFR. Quite surprisingly, most of these substitutions were deleterious to the human enzyme. The results suggest that selective constraints vary between phylogenetic lineages such that inclusion of distant orthologs to infer selective pressures on the human enzyme may be misleading. We propose that homologous proteins are best used to reconstruct ancestral sequences and infer amino acid conservation among only direct lineal ancestors of a particular protein. We show that such an "ancestral site preservation" measure outperforms other prediction methods, not only in our selected set for MTHFR, but also in an exhaustive set of E. coli LacI mutants.
计算预测遗传变异的功能影响在人类遗传学研究中起着关键作用。对于非同义编码变异，大多数预测算法都利用给定位置同源蛋白中观察到的氨基酸替换模式。特别是，其他物种的同源蛋白中观察到的替换通常假定在人类蛋白中也是耐受的。我们通过在酵母细胞功能测定中评估一组典型人类酶甲基四氢叶酸还原酶（MTHFR）的非同义突变体来检验这一假设。正如预期的那样，在人类 MTHFR 中在跨越远缘同源物的保守位点发生的替换导致酶功能受损，而在最近分化的序列中发生的替换（包括“复活”人类-猕猴祖先的 9 位点突变体）导致功能酶。我们还通过在人类酶中创建至少一个 MTHFR 同源物接受的替换来研究 30 个具有不同保守程度的位点。令人惊讶的是，这些替换中的大多数对人类酶都是有害的。结果表明，选择压力在进化枝之间存在差异，因此将远缘同源物纳入推断人类酶的选择压力可能会产生误导。我们提出，同源蛋白最适合用于重建祖先序列，并推断特定蛋白质的直系祖先之间的氨基酸保守性。我们表明，这种“祖先位点保存”度量标准不仅在我们选择的 MTHFR 组中，而且在大肠杆菌 LacI 突变体的详尽组中都优于其他预测方法。
