ESPE Abstracts (2019) 92 RFC6.5

Evaluating Genotype-Phenotype Correlation using an in vitro Mutagenesis Model in Bi-Allelic Mutations Resulting in Extreme Hypophosphatasia Clinical Phenotypes

Suma Uday1,2, Tomohiro Matsumura3, Vrinda Saraff1, Shiho Saito3, Hideo Orimo3, Wolfgang Högler4,2


1Birmingham Women's and Children's Hospital, Birmingham, United Kingdom. 2University of Birmingham, Birmingham, United Kingdom. 3Nippon Medical School, Tokyo, Japan. 4Johannes Kepler University, Linz, Austria


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.

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