Impact of the ENPP1 mutation on bone mineralization and ectopic calcification: evidence from in vitro and in vivo models

Abstract

Background: Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) plays a key role in mineralization processes, and mutations in this gene are associated with various severe diseases. Clinical case reports have implicated the ENPP1 Y451C mutation in diffuse idiopathic skeletal hyperostosis patients, but its precise impact on bone mineralization and ectopic calcification remains unclear.

Methods: We used bioinformatics tools and in vitro functional assays to assess the impact of the ENPP1 Y451C mutation on protein structure and enzymatic activity. Furthermore, we generated a knock-in mouse model (Enpp1^Y433C ) to evaluate microarchitecture or signs of ectopic calcification by Micro-CT.

Results: Bioinformatics analysis and in vitro assays showed that the Y451C mutation affects the ENPP1 protein's structure, reducing enzymatic activity by approximately 50%. We successfully generated the Enpp1^Y433C knock-in mouse model. However, no significant differences were observed in body phenotype or biochemical markers in Enpp1^Y433C mice at 3, 5, and 10 months, compared to wild-type controls. Similarly, no significant changes were observed in bone microarchitecture or signs of ectopic calcification.

Conclusion: The ENPP1 Y451C mutation significantly reduces enzymatic activity in vitro, yet the Enpp1^Y433C knock-in mouse model shows no significant abnormalities in mineralization, providing additional evidence for the pathogenicity assessment of ENPP1 Y451C variant. Given that these results are from mouse models, further studies are required to clarify its pathogenicity in humans.

Keywords: bone mineralization; diffuse idiopathic skeletal hyperostosis; ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1); ectopic calcification; ossification of the posterior longitudinal ligament.

Figure 1

Structure prediction of ENPP1 Y451C mutation. (A, B) ENPP1 structure and the location of the Y451C variants.

Figure 2

Functional analysis of ENPP1 Y451C mutation in vitro. (A) Western blotting and quantification of ENPP1 protein expression in HEK293T cells transfected with ENPP1 wild-type (WT) and Y451C plasmids. (B) Immunofluorescence imaging showing the expression and localization of ENPP1 in MC3T3-E1 cells. (C) Enzymatic activity in HEK293T cells. Data are expressed as mean ± standard deviation (n=4-6). Statistical analysis was performed using unpaired student's t-tests for comparisons between two groups, *** p<0.001.

Figure 3

Generation of the Enpp1^Y433C mutant mouse. (A) Design methods of Enpp1^Y433C knock-in mice were performed by CRISPR-Cas9. (B) Genotyping of Enpp1^Y433C via sequencing, the red box highlights the mutation site (A>G). (C-E) Whole-body photographs, body weight and nose-tail length measurements of 10-month-old male mice. (F) Western blotting and quantification of ENPP1 protein expression in tibial tissue from 10-month-old male mice. ENPP1 protein levels were normalized to ACTIN. Data are expressed as mean ± standard deviation(n=3-6). Statistical analysis was performed using one-way ANOVA for comparisons among three groups.

Figure 4

Biochemistry analysis in 5- and 10- months-old male mice. (A-E) Biochemistry features of phosphate (P), calcium (Ca), magnesium (Mg), alkaline phosphatase (ALP) and FGF23 levels in 5- month-old mice. (F-J) Biochemistry features of phosphate (P), calcium (Ca), magnesium (Mg), alkaline phosphatase (ALP) and FGF23 levels in 10-month-old mice. Data are expressed as mean ± standard deviation(n=5-7). Statistical analysis was performed using one-way ANOVA for comparisons among three groups.

Figure 5

Ectopic calcification analysis in 5- and 10- months old male mice. (A, B) X-ray images of Enpp1^Y433C mice in both anterior-posterior and lateral views. (C, D) 3D reconstruction of whole-body and paw imaging.

Figure 6

Bone microarchitecture and biomechanics of 10-month-old male mice. (A) Three-dimensional reconstructed images of femoral trabecular and cortical bone. (B) Quantification of trabecular BV/TV, trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp). (C) Quantification of cortical BV/TV, cortical thickness (Ct.Th), and cortical density (Ct. BMD). (D) Femur length. (E) Quantification of femur biomechanical properties (maximum load, stiffness). Data are expressed as mean ± standard deviation(n=5-6). Statistical analysis was performed using one-way ANOVA for comparisons among three groups.