Study Finds Distinct Enzyme Patterns in Low ALP Patients

Researchers at the Hospital Universitario Marqués de Valdecilla in Santander, Spain, have identified distinct biochemical patterns in adult patients with persistently low serum alkaline phosphatase (ALP) who lack identifiable mutations in the ALPL gene. The study, published in Frontiers in Endocrinology, utilized an integrative multi-omics approach to investigate the molecular basis of hypophosphatasemia in these individuals. By combining genetic, epigenetic, transcriptomic, and isoenzyme profiling, the team sought to clarify why a substantial number of patients with low ALP activity do not carry pathogenic variants in the ALPL gene, a condition that complicates diagnosis and clinical management.

The investigation included 50 adult patients with persistently low serum ALP levels and 20 healthy controls matched by age and sex. The patients were referred for evaluation after showing ALP levels below 40 U/L on at least two separate occasions more than six months apart. Secondary causes of hypophosphatasemia, such as medication use or underlying systemic diseases, were systematically excluded through detailed medical history, physical examination, and laboratory tests. This rigorous screening ensured that the observed low ALP levels were not attributable to external factors, allowing the researchers to focus on intrinsic biological mechanisms.

Genetic analysis revealed that 24 of the 50 patients carried pathogenic or likely pathogenic variants in the ALPL gene, while the remaining 26 were non-carriers. The identified variants were predominantly heterozygous missense substitutions, which are expected to affect the catalytic activity, folding, or stability of the tissue-nonspecific alkaline phosphatase enzyme. Among the mutation carriers, 65% exhibited high serum levels of pyridoxal-5′-phosphate (PLP), a major circulating form of vitamin B6 that accumulates when ALP activity is deficient. In contrast, none of the mutation-negative individuals had serum PLP levels above the reference range, suggesting a fundamental difference in the underlying enzymatic defect between the two groups.

To explore potential regulatory mechanisms in the mutation-negative group, the researchers performed targeted sequencing of the ALPL promoter region and analyzed DNA methylation patterns. Sanger sequencing of the proximal promoter region did not identify any sequence variants that could account for the reduced ALP activity. Furthermore, DNA methylation analysis using the MassARRAY system revealed uniformly low methylation levels across all groups, consistent with a hypomethylated state that is permissive for gene transcription. These findings argue against transcriptional silencing via CpG hypermethylation as the primary driver of low ALP activity in non-carriers.

Whole-exome sequencing was conducted in the mutation-negative patients to identify variants in other genes related to bone and mineral homeostasis. Phenotype-driven analysis using Human Phenotype Ontology terms did not identify pathogenic variants in genes associated with inherited disorders of bone metabolism or other Mendelian conditions that could explain the persistent hypophosphatasemia. Additionally, RT-qPCR analysis of peripheral blood cells showed no statistically significant differences in ALPL mRNA expression levels between mutation carriers, mutation-negative patients, and controls. This suggests that genomic and epigenetic factors in peripheral blood are unlikely to be the primary determinants of the reduced ALP activity observed in this group.

Given the lack of explanatory genetic or epigenetic findings, the study focused on the biochemical characterization of circulating ALP isoenzymes. Total serum ALP activity was significantly lower in ALPL mutation carriers than in mutation-negative patients, with mean values of 20 ± 5 U/L and 32 ± 6 U/L, respectively. Both patient groups had markedly lower total ALP levels than the healthy controls, who had a mean of 56 ± 16 U/L. Bone-specific ALP activity was also modestly but significantly lower in mutation carriers compared to mutation-negative patients, indicating a broader impact of the genetic variants on enzyme activity across different tissue isoforms.

Isoenzyme electrophoresis provided critical insights into the tissue-specific distribution of ALP activity. The relative abundance of the major hepatic isoform (H1) was comparable between controls and mutation-negative patients, at 40.4 ± 15.3% and 38.5 ± 18.5%, respectively. In contrast, the H1 fraction was significantly reduced in mutation carriers, at 26.9 ± 20.2%. When absolute activities were calculated, total hepatic ALP activity was markedly lower in mutation carriers than in non-carriers. This preservation of liver ALP activity in mutation-negative individuals suggests that their low total ALP levels may reflect a defect primarily affecting bone-derived enzyme activity rather than a global reduction in ALP function.

Another key finding was the dissociation between total and bone-specific ALP activity in mutation-negative patients. In healthy controls and mutation carriers, total ALP activity showed a significant positive correlation with bone-specific ALP levels. However, no significant correlation was observed in mutation-negative individuals with low total ALP activity. This lack of correlation suggests a tissue-specific difference in enzyme regulation or distribution in this group. Bone turnover markers, including procollagen type I N-terminal propeptide (P1NP) and β-C-terminal telopeptide of type I collagen (β-CTX), were similarly reduced in both carriers and non-carriers compared to controls, indicating that both groups exhibit altered bone metabolism despite their different genetic backgrounds.

The study highlights the importance of isoenzyme profiling in distinguishing between different mechanisms of ALP deficiency. The preservation of liver ALP activity in mutation-negative patients, combined with the normal levels of pyridoxal-5′-phosphate, suggests that these individuals may have a distinct biochemical phenotype. The authors propose that these patients should be considered as having “hypophosphatasemia of unknown significance,” a term that acknowledges the biochemical abnormality while recognizing the lack of clear genetic etiology. This classification is important for clinical follow-up, as it distinguishes these individuals from those with confirmed hypophosphatasia due to ALPL mutations.

The researchers acknowledge several limitations of the study, including the sample size and the assessment of methylation in peripheral blood, which may not fully capture the epigenetic landscape of bone or liver tissues. DNA methylation patterns are cell- and tissue-specific, and the absence of liver and bone samples precluded a more detailed analysis of tissue-specific regulatory mechanisms. Additionally, the isoenzyme analysis relied on chromatographic separation rather than direct estimation of enzymatic activity for each form. Despite these limitations, the study provides valuable insights into the molecular basis of low ALP activity in mutation-negative patients and underscores the need for longitudinal follow-up to determine the long-term musculoskeletal consequences of this biochemical profile.

The findings suggest that the absence of detectable ALPL mutations does not exclude abnormal bone turnover or a hypophosphatasia-like biochemical phenotype. Future studies incorporating bone and liver tissue analysis will be critical to elucidate the mechanisms driving these abnormalities and to refine diagnostic criteria for mutation-negative cases of low ALP activity. The study contributes to the growing understanding of hypophosphatasemia as a complex condition with diverse molecular underpinnings, emphasizing the need for a nuanced approach to diagnosis and management in clinical practice.

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

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

Article References: Del Real, Á., Álvarez Villalobos, S., Ortiz Espejo, M., Sañudo, C., Vega, A. I., Riancho-Zarrabeitia, L., Amigo, T., García-Unzueta, M. T., Puente Ruiz, N., Garcia Tellado, A., Gutiérrez Saiz, O., Lopez Montiel, J., Ansede-Bermejo, J., Fernández-Luna, J. L., González-Lamuño, D., Carracedo, A., & Riancho, J. A. (2026). Multilevel analysis of patients with hypophosphatasemia: genomic, epigenomic and isoenzyme activity profiles. Frontiers in Endocrinology, 17, Article 1910750. https://doi.org/10.3389/fendo.2026.1910750

Image Credits: AI Generated

DOI: 10.3389/fendo.2026.1910750

Keywords: Multilevel, analysis, patients, hypophosphatasemia, genomic, epigenomic, isoenzyme, activity, profiles, scientific research