Introduction: Hypophosphatasia (HPP) characterized by reduced mineralization results from mutations in the tissue non-specific alkaline phosphatase (ALPL) gene. HPP is clinically variable with extensive allelic heterogeneity in the ALPL gene. We report the findings of in vitro functional studies following site-directed mutagenesis in bi-allelic mutations causing extreme clinical phenotypes; severe perinatal and asymptomatic HPP.
Objectives: Elucidate genotype-phenotype correlation using in vitrofunctional studies and 3 dimensional (3D) ALP modelling.
Methods: Clinical, biochemical and radiological features were recorded in two subjects (S) with extreme HPP phenotypes: S1: Perinatal HPP with compound heterozygous mutations (c.110T>C; c.532T>C); S2: Asymptomatic with homozygous missense mutation (c.715G>T). S2's affected siblings (3 homozygous, 1 heterozygous) were also studied.
Plasmids created for mutants 1 c.110T>C (L37P), 2 c.532T>C (Y178H) and 3 c.715G>T(D239Y) using in vitro mutagenesis were transfected into human osteosarcoma (U2OS) cells and compared to wild type (WT) and mock cDNA. ALP activity was measured using enzyme kinetics with p-nitrophenylphosphate. Mineral deposition was evaluated photometrically with Alizarin Red S staining after culture with beta-glycerophosphate. Western blot analysis was performed to identify the mature type protein expression (80 kDa). Mutations were located on a 3D ALP model.
Results: Phenotype: S1 had extremely under-mineralized bones and pulmonary hypoplasia, typical of perinatal HPP. S2, diagnosed incidentally at 4 years, had normal growth, dentition and radiology similar to the siblings. All subjects had typical biochemical features of HPP (low ALP, high serum pyridoxal-5'-phosphate and urinary phosphoethanolamine) except heterozygous sibling (normal ALP).
Functional assay: Mutants 1 and 2 demonstrated negligible ALP activity and mineralization (7.9% and 9.3% of WT, respectively). Mutant 3 demonstrated 50% ALP activity and 15.5% mineralization of WT. Western blot analysis detected mutants 1 and 2 as faint bands indicating reduced expression and mutant 3 as mature form protein (50% of WT expression). Mutant 1 was located near the Glycosylphosphatidylinositol anchor, 2 at the core structure and 3 at the periphery of the ALP protein structure.
Conclusion: Our findings expand the current knowledge of functional effect of individual mutations and the importance of their location in the ALP structure. c.715G>T homozygous mutation showed no clinical variability enabling phenotype prediction in offsprings and genetic counselling.
19 - 21 Sep 2019
European Society for Paediatric Endocrinology