Multilevel analysis of patients with hypophosphatasemia: genomic, epigenomic and isoenzyme activity profiles

Background Hypophosphatasemia is the biochemical hallmark of (HPP), a rare genetic condition caused by deficient activity of tissue-nonspecific alkaline phosphatase (ALP) due to pathogenic variants in the ALPL gene. However, a substantial number of patients with persistently low serum levels of ALP (hypophosphatasemia) lack identifiable mutations, complicating diagnosis and management. Aim To investigate the molecular basis of low ALP activity in adult patients without detectable ALPL mutations using an integrative multi-omics approach and isoenzyme profiling. Methods We studied 50 adult patients with low serum ALP and 20 healthy controls. Genetic analysis included targeted sequencing of ALPL exons and promoter, and whole-exome sequencing in mutation-negative cases. Promoter methylation was explored by MassARRAY, and ALPL mRNA expression was determined by RT-qPCR. Total and bone-specific ALP, and bone turnover markers were determined. Isoenzyme fractions were analyzed by agarose gel electrophoresis. Results Pathogenic ALPL variants were identified in 24 patients, whereas the remaining 26 were non-carriers. Non-carriers did not have identifiable variants in the ALPL promoter or in other genes related to bone and mineral homeostasis. Likewise, ALPL expression levels, and promoter methylation were similar in all groups. PINP and β-CTX levels were similarly reduced in carriers and non-carriers, in comparison with controls. In contrast, mutation carriers exhibited a marked reduction in the hepatic ALP isoenzyme, while mutation-negative individuals showed relatively preserved liver ALP activity. Moreover, in non-carriers, we did not find the expected correlation between total and bone-specific ALP in serum. Conclusions Taken together, these findings, along the normal serum levels of pyridoxal-phosphate, suggest that in patients with persistent hypophosphatasemia without ALPL mutations, low serum ALP levels may reflect an enzymatic defect affecting bone more than liver. Longitudinal follow-up of these patients will be essential to determine whether low ALP levels are associated with long-term musculoskeletal consequences.