Large-scale in vitro functional testing and novel variant scoring via protein modeling provide insights into alkaline phosphatase activity in hypophosphatasia.

Hypophosphatasia (HPP) is a rare metabolic disorder characterized by low tissue-nonspecific alkaline phosphatase (TNSALP) typically caused by ALPL gene mutations. HPP is heterogeneous, with clinical presentation correlating with residual TNSALP activity and/or dominant-negative effects (DNE). We measured residual activity and DNE for 155 ALPL variants by transient transfection and TNSALP enzymatic activity measurement. Ninety variants showed low residual activity and 24 showed DNE. These results encompass all missense variants with carrier frequencies above 1/25,000 from the Genome Aggregation Database. We used resulting data as a reference to develop a new computational algorithm that scores ALPL missense variants and predicts high/low TNSALP enzymatic activity. Our approach measures the effects of amino acid changes on TNSALP dimer stability with a physics-based implicit solvent energy model. We predict mutation deleteriousness with high specificity, achieving a true-positive rate of 0.63 with false-positive rate of 0, with an area under receiver operating curve (AUC) of 0.9, better than all in silico predictors tested. Combining this algorithm with other in silico approaches can further increase performance, reaching an AUC of 0.94. This study expands our understanding of HPP heterogeneity and genotype/phenotype relationships with the aim of improving clinical ALPL variant interpretation.