Characterization of Genetic Variants of Uncertain Significance for the ALPL Gene in Patients With Adult Hypophosphatasia

Abstract

Hypophosphatasia (HPP) a rare disease caused by mutations in the ALPL gene encoding for the tissue-nonspecific alkaline phosphatase protein (TNSALP), has been identified as a potentially under-diagnosed condition worldwide which may have higher prevalence than currently established. This is largely due to the overlapping of its symptomatology with that of other more frequent pathologies. Although HPP is usually associated with deficient bone mineralization, the high genetic variability of ALPL results in high clinical heterogeneity, which makes it difficult to establish a specific HPP symptomatology. In the present study, three variants of ALPL gene with uncertain significance and no previously described (p.Del Glu23_Lys24, p.Pro292Leu and p.His379Asn) were identified in heterozygosis in patients diagnosed with HPP. These variants were characterized at phenotypic, functional and structural levels. All genetic variants showed significantly lower in vitro ALP activity than the wild-type (WT) genotype (p-value <0.001). Structurally, p.His379Asn variant resulted in the loss of two Zn²⁺ binding sites in the protein dimer which may greatly affect ALP activity. In summary, we identified three novel ALPL gene mutations associated with adult HPP. The correct identification and characterization of new variants and the subsequent study of their phenotype will allow the establishment of genotype-phenotype relationships that facilitate the management of the disease as well as making it possible to individualize treatment for each specific patient. This would allow the therapeutic approach to HPP to be personalized according to the unique genetic characteristics and clinical manifestations of each patient.

Keywords: alkaline phosphatase; bone; enzymatic activity; genetic variant; hypophosphatasia; mineralization; pyridoxal 5´ phosphate.

Figure 1

(A) TNSALP is attached to the cell membrane, where it degrades inorganic pyrophosphate (PPi) to inorganic phosphate (Pi), needed for hydroxyapatite (HA) crystal formation and further bone mineralization. TNSALP hydrolyses PLP (active form of B6) to pyridoxal (PL) in order to cross the cell membranes, after which is, then intracellularly rephosphorylated into PLP. (B) PPi is not degraded to Pi. Excess of PPi inhibits HA crystal formation with further inhibition of bone mineralization. PLP is not hydrolyzed to pyridoxal (PL), and accumulates extracellularly. The low activity of ALP causes the accumulation of PPi and PLP, causing symptoms in various organs of the human body (the symptoms related to PPi accumulation appears in orange boxes while symptoms related to PLP accumulation as vitamin B6 deficiency are indicated in yellow box). Figure created with BioRender.com.

Figure 2

ALP activity in HEK293T cells transfected with pcDNA 3.1 or pcDNA 3.1+ insert. Quantitative results of the ALP assay expressed in U/mg are shown. Results are expressed as mean ± SEM of TNSALP activity. ANOVA was used for comparisons between groups. *p < 0.001 vs WT; ^# p < 0.001 vs all groups.

Figure 3

Total mRNA expression. The results are expressed as the percentage of expression of the Cts ± SEM normalized with the expression values of the constitutive gene RPL13. ANOVA corrected by Tukey’s test was used for comparisons between groups. to empty pcDNA3.1 are indicated with an *p < 0.05 vs all groups.

Figure 4

Viability cell assay graphs. ANOVA corrected by Tukey’s test was used for comparisons between groups Statistically significant differences was set at a p-value <0.05. (A) Percentage of cell viability. (B) Percentage of apoptotic cells.

Figure 5

Three-dimensional representation of the TNSALP protein in the form of ribbons. (A) TNSALP WT. Zn²⁺ atoms bound to the corresponding Zn²⁺ binding sites are highlighted in green. The arrows indicate the location of the different mutations identified in this study. (B) TNSALP with the genetic variant p.Del Glu23_Lys24. The deletion is marked in yellow and the major structural changes are highlighted with a yellow circle. (C) TNSALP with the genetic variant p.Pro292Leu. The amino acid substitution is marked in yellow and the major structural changes are highlighted with a yellow circle. (D) TNSALP with the genetic variant p.His379Asn. The loss of 2 of the 4 Zn²⁺ binding sites in the dimeric protein is highlighted with a yellow circle in one of the protein monomers.

Figure 6

Three-dimensional representation of the TNSALP protein according to its hydrophobicity. Hydrophobicity is represented on the Kyte and Doolittle hydrophobicity scale (Kd scale); from the most hydrophilic amino acids in light blue to the most hydrophobic in red. Changes to more hydrophilic and hydrophobic amino acids are marked in yellow and black circles respectively. (A) TNSALP WT. (B) TNSALP with the genetic variant p.Del Glu23_Lys24. (C) TNSALP with the genetic variant p.Pro292Leu. (D) TNSALP with the genetic variant p.His379Asn.