Establishment of Alkaline Phosphatase deficient odontogenous cell lines to analyze dental aspects of hypophosphatasia

Introduction Hypophosphatasia (HPP) is a rare inherited disorder caused by compound- or dominant negative heterozygous loss-of-function mutations in the ALPL gene encoding the Tissue- Nonspecific Alkaline Phosphatase (TNAP). Besides skeletal symptoms, some patients also present dental abnormalities, like for example the premature loss of deciduous teeth without prior root resorption.

Methods Here we generated and subsequently characterized five different TNAP-deficient immortalized hTERT (human telomerase reverse transcriptase) periodontal ligament (PDL) derived cell lines using CRISPR-Cas9.

Results The mutations varied from a moderate heterozygous point mutation to a severe homozygous deletion leading to a premature stop codon. Analysis of the ALPL expression (qPCRs), TNAP activity measurements (CSPD Assays), and TNAP stainings revealed a significant decrease in all TNAP-deficient cell lines, with a strong connection between the residual activity and the extend of the mutation resembling the complex genotype-phenotype correlation in HPP. The osteogenic differentiation capacity of hTERT PDL cells was completely abolished in the homozygously mutated cell line.

Discussion Putative key mechanisms for the premature exfoliation in HPP are the restricted formation and mineralization of the cementum and the impaired insertion of elastic dental fibers. The newly generated TNAP-deficient cell lines provide a promising and donor independent in vitro model to gain better understanding of the molecular mechanisms leading to dental problems in HPP.

Keywords Hypophosphatasia, Tissue-nonspecific alkaline phoshatase, CRISPR-Cas9, Periodontal Ligament

Korrespondenzadresse Sofia Paulus, IZKF Group Tissue Regeneration in Musculoskeletal Diseases, Bernhard-Heine Zentrum für Bewegungsfors, Universitätsklinikum Würzburg, Röntgenring 11, 97070 Würzburg, Deutschland

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Publication History

Article published online:
05 March 2021