Theoretically, using our prediction formulae, scores approximately equal to zero indicate that there is no significant change in the underlying amino acid probabilities whereas scores less than zero indicate that an unfavorable substitution has been observed, that is, the mutant residue is less likely to be observed than the wild-type residue, and scores greater than zero indicate that a favorable substitution has been observed, that is, the mutant residue is more likely to be observed than the wild-type residue. However, in practice, FATHMM is sensitive to small fluctuations in the amino acid probabilities modeled within the HMMs. For example, the slightest reduction in amino acid probabilities would yield a pathogenic prediction in our unweighted/species-independent algorithm. Therefore, to eliminate the effects of these fluctuations, we plotted the distribution of the predicted magnitude of effect for both disease-associated and functionally neutral AASs within the SwissVar dataset (Fig. 1). From this, we calculated prediction thresholds for our unweighted and weighted methods at which the specificity and sensitivity were both maximized (−3.0 and −1.5, respectively). Using our unweighted method, we noted that the majority
