Functional and In Silico Characterization of ALPL Gene Variants Reveals Genotype–Phenotype Correlations in Italian Hypophosphatasia Patients

TL;DR

This study analyzed 21 ALPL gene variants in Italian hypophosphatasia patients to understand how genetic changes affect protein function and disease severity.

Contribution

The study provides a multifaceted genotype-phenotype correlation framework using functional, clinical, and in silico data for hypophosphatasia.

Findings

Most ALPL variants were downregulated or not expressed, and all showed impaired enzymatic activity in vitro.

In silico predictions aligned with functional data, enabling preliminary variant categorization based on protein stability and structure.

Over 70% of variants showed coherence among bioinformatics, experimental, and clinical data.

Abstract

What are the main findings?

First bullet: functional characterization of 21 ALPL genetic variants identified in well clinical ascertained HPP patients that provided novel insights about the protein stability and residual enzymatic activity of the mutants.

Second bullet: a multifaceted comparison among functional, genotypic, clinical/biochemical and in silico data that might represent a higher and more accurate level of a basic genotype-phenotype correlation.

What are the implications of the main findings?

First bullet: a greater comprehension of the presumed effect of an ALPL genetic variant on the whole TNAP protein function.

Second bullet: the multi-data comparison might represent for the clinician an innovative tool for the follow up and management of a HPP patient.

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.

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