Childhood Hypophosphatasia Associated with a Novel Biallelic ALPL Variant at the TNSALP Dimer Interface

Abstract

The goal of this study was to perform a clinical and molecular investigation in an eight-year-old female child diagnosed with hypophosphatasia (HPP). The proband and her family were evaluated by medical and dental histories, biochemical analyses, radiographic imaging, and genetic analysis of the tissue-nonspecific alkaline phosphatase (ALPL) gene. A bioinformatic analysis was performed to predict the structural and functional impact of the point mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) molecule and to define their potential contribution to the phenotype. We identified a novel combination of heterozygous ALPL missense variants in the proband, p.Ala33Val and p.Asn47His, compatible with an autosomal recessive mode of inheritance and resulting in skeletal and dental phenotypes. Computational modeling showed that the affected Asn47 residue is located in the coil structure close to the N-terminal α-helix, whereas the affected Ala33 residue is localized in the N-terminal α-helix. Both affected residues are located close to the homodimer interface, suggesting they may impair TNSALP dimer formation and stability. Clinical and biochemical follow-up revealed improvements after six years of ERT. Reporting this novel combination of ALPL variants in childhood HPP provides new insights into genotype-phenotype associations for HPP and specific sites within the TNSALP molecule potentially related to a childhood-onset HPP and skeletal and dental manifestations. Beneficial effects of ERT are implicated in skeletal and dental tissues.

Keywords: 3D modeling; alkaline phosphatase; genotype–phenotype association; hypophosphatasia; premature tooth loss.

Figure 1

Clinical presentation. Representative radiographs of the proband at 2 years of age show (A,B) abnormal heterogeneous trabecular bone in the ilium (yellow arrow in A), proximal femur, and the long bones at the knees, and radiolucent areas in the proximal metaphysis of the fibula (yellow arrow in B) and in the central metaphysis and epiphysis of the left distal femur. (C) The proband shows enlargement of the anterior costochondral junction (yellow arrows). (D) No abnormalities were observed for the long bones of the upper limbs. (E) Note a dolichocephalic skull and the presence of distractors after cranial vault decompression. (F–H) Asfotase alfa ERT was associated with injection site skin reactions including redness, swelling, and thickening (yellow arrows indicated affected regions) and (I) lipodystrophy, as shown in this photograph of the proband’s thigh. (J) The radiolucency and traverse fracture detected in the proximal right fibula (white arrows) were resolved after introduction of asfotase alfa ERT, with follow-up images at 3 years and 5 months and 4 years. (K) Panoramic oral radiograph of the proband at 8 years of age shows a mixed dentition. (L–N) Intraoral photos at 8 years of age show mild enamel hypoplastic lesions on the lower permanent incisors and upper deciduous left molar (yellow arrows).

Figure 2

Genetic and pedigree analysis. (A) Pedigree chart representing two generations of the family analyzed in this study. Segregation patterns of genotypes (p.Ala33Val and/or p.Asn47His) and phenotype (serum ALP levels) for the proband (filled circle). (B) Protein model of TNSALP showing the signal peptide (SP) and functional regions, including active site, ion binding regions, crown domain, and disulfide bonds. Amino acid substitutions identified in the proband (p.Ala33Val and p.Asn47His) are indicated in exon 3 with red and blue markers. (C) Residue conservation of orthologous ALPL sequences across 15 vertebrate species. The Ala33 and Asn47 native residues are indicated by a black and a blue arrow, respectively. Both Ala33 and As47 residues (indicated by arrows) are highly conserved. (D) The residue conservation is displayed by multiple sequence alignment in paralogous sequences of human ALPL, ALPP, ALPI, and ALPPL genes. (E) 3D model of TNSALP dimer showing the localization of affected residues by p.Ala33Val and p.Asn47His mutations in monomer 1 (cyan) and monomer 2 (orange). The Ala33 residue is located in the N-terminal alpha helix (α-helix), and Asn 47 residue is located in the coil structure close to the N-terminal (α-helix). The Ala33 residue in the α-helix structure is colored in pink and indicated by arrows, while the Asn47 is highlighted in the stick model and colored in red. Mg ions are colored green, and Zn ions colored yellow. The images were generated using the PyMol software. (F) Intramolecular interactions established by native Ala33 and mutant Val33 residues. (G) Intramolecular interactions established by native Asn47 and mutant His47 residues. A predicted new hydrogen bond between His47 and Thr48 is indicated by an asterisk. Hydrogen bonds (dashed green lines) and their distances are indicated. The images were generated using the Swiss-PdbViewer v4.1.