Hypophosphatasia (HPP) is a rare disorder of the bone metabolism, characterized by genetically determined low alkaline phosphatase (ALP) activity. Low ALP may also be observed in some common causes of bone fragility, such as in osteoporosis treated with antiresorptive drugs. This study aimed to verify whether differences in bone turnover markers (BTMs) could help differentiate adult patients with HPP from those with osteoporosis undergoing antiresorptive treatment. In this multicenter study, we enrolled 23 adult patients with a diagnosis of HPP and compared them with 46 osteoporotic subjects previously treated with zoledronic acid or denosumab. BTMs such as CTX, N-terminal propeptide of type I procollagen (P1NP), total ALP, and bone ALP (bALP) were measured, and ratios between BTMs were also calculated. Considering that the control group included only females, in the primary analysis we compared their characteristics with that of the 16 female patients with HPP. Both individual BTMs (CTX and P1NP) and 4 BTM ratios (ALP/P1NP, bALP/P1NP, ALP/CTX, and bALP/CTX) showed satisfactory discriminatory power, outperforming ALP alone. P1NP, in particular, had an area under the curve (AUC) of 0.962 with a cut-off of 32 μg/L, while as for the BTMs ratios, the ALP/P1NP ratio had an AUC of 0.964 with a cut-off of 1.114. Similar results were confirmed when including male HPP patients, when adjusting for age and sex, and finally when performing a sensitivity analysis only in patients with ALP less than or equal to 32 U/L (ie, the median of the distribution of the entire population). In cases of low ALP and bone fragility, BTM and their ratios could help distinguish HPP patients from osteoporotic individuals treated with antiresorptive drugs, aiding in accurate diagnosis and reducing the risk of inappropriate treatment.

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.