Bone Abstracts (2014) 3 PP369 | DOI: 10.1530/boneabs.3.PP369

A homozygous 20 bp intronic deletion in front of exon 8 of the ALPL-gene causes infantile hypophosphatasia: a functional characterization

Birgit Mentrup1, Hermann Girschick2, Franz Jakob1 & Christine Hofmann3

1Orthopedic Department, Orthopedic Center for Musculoskeletal Research, University of Wuerzburg, Wuerzburg, Germany; 2Children’s Hospital, Vivantes Hospital im Friedrichshain, Berlin, Germany; 3Pediatric
Rheumatology and Osteology, Children’s hospital, University of
Wuerzburg, Wuerzburg, Germany.

Mutations of the ALPL-gene are closely related to hypophosphatasia (HPP), an inherited disorder of bone and mineral metabolism with clinically heterogeneous symptoms. To date 278 different mutations have been described, leading to reduction or completely loss of enzymatic activity of the tissue nonspecific alkaline phosphatase (TNAP).

We present the case of a 6-year-old boy with clinical features and laboratory results consistent with infantile HPP, but without any mutation in the coding region of the ALPL-gene. Intensified genomic DNA analyses, performed with informed consent provided by the parents, revealed a homozygous 20 bp-deletion in front of exon 8: c.793del-14_33. Both parents, who are consanguine, are heterozygous. Examination of mRNA transcripts resulted in three different splice variants; the main transcript shows a deletion of exon 8, a second, weaker transcript has a deletion of exons 7 and 8, and only a very weak signal referred to the full length transcript. Deletion of exon 8 or both exons leads to a shift of the reading frame resulting in a stop codon in exon 9. Since the main transcript misses exon 8 and we detected a protein of ~ 40 kDa in PBMC and serum from the patient by western- blotting with a TNAP-specific antibody, we generated an expression construct for the deduced C-terminally truncated TNAP protein of 275 aa and investigated intracellular localization by immunocytochemistry and enzymatic activity in transfection/co-transfection studies with not mutated TNAP expression plasmid.

The protein cannot be localized in the cell-membrane due to the loss of its C-terminal membrane-anchor. Furthermore it has no basal enzymatic activity and it does not seem to affect an unimpaired dimerization partner.

This intronic deletion can explain the reduced alkaline phosphatase activity and clinical symptoms of HPP, which cannot be compensated by an unaffected allele like in the case of the healthy parents.

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