Bone Abstracts

Introduction: Hypophosphatasia (HPP) characterized by reduced mineralization occurs from mutations in the tissue non-specific alkaline phosphatase (ALPL) gene. Individuals harbouring bi-allelic mutations are generally reported to be severely affected. 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 vitro functional studies and 3 dimensional (3D) ALP modelling.

Methods: Clinical, biochemical and radiological features were recorded in two patients (P) with extreme HPP phenotypes: P1: perinatal HPP with compound heterozygous mutations (c.110T>C; c.532T>C); P2: asymptomatic with homozygous missense mutation (c.715G>T). P2’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 cells and compared to wildtype (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: P1 had extremely under-mineralized bones and pulmonary hypoplasia, typical of perinatal HPP. P2, diagnosed incidentally at 4 years, had normal growth and radiology similar to the siblings. All had typical biochemical features of HPP (low ALP, high serum pyridoxal-5’-phosphate) 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. Unlike the high intrafamilial variability reported with compound heterozygous mutations, homozygous mutations showed no variability enabling phenotype prediction in offsprings and genetic counselling.

Disclosure: Wolfang Högler has been an investigator in Alexion-sponsored clinical trials, is a board member of the global HPP registry and has received honoraria and consulting fees from Alexion.

Funding: This investigator-initiated study was financially supported by Alexion Pharma Ltd. Alexion had no role in the conduct of the study, analyses or writing of the abstract. Alexion provided courtesy medical review. The authors made the final decision on content and submission of the abstract.