Functional and In Silico Characterization of ALPL Gene Variants Reveals Genotype-Phenotype Correlations in Italian...

Background. Hypophosphatasia (HPP) is a rare genetic disorder caused by impaired tissue non-specific alkaline phosphatase (ALPL/TNSALP) activity that impacts the musculoskeletal and neurological systems. It is extremely variable, with up to six forms of increasing severity. The large phenotypic variability and the still remaining high number of variants of uncertain significance (VUS) in the ALPL gene represent a conundrum for clinicians dealing with people suspected to be suffering from HPP. Methods. We applied a multi-faceted bench-based and high-throughput bioinformatics analysis to investigate the effect of 21 ALPL variants (18 deleterious-pathogenic or likely pathogenic-and 3 VUS) on the structure and function of the mutated encoded protein. The results were compared with available clinical and biochemical data. Results. Most variants were downregulated or not expressed by Western blot analysis. Impairment of the enzymatic activity was confirmed in vitro for all variants by a specific colorimetric enzymatic assay. In silico prediction was in line with functional data and allowed for preliminary categorization of variants based on their impact on both the overall stability of the protein complex and local structural alterations. Coherence among bioinformatics, experimental and clinical data was documented for more than 70% of the variants. Conclusions. Functional and in silico characterizations of ALPL variants in people with a suspicion of HPP offer integrative strategies to genotyping in assisting clinicians for diagnosis confirmation in doubtful cases.