A molecular approach to dominance in hypophosphatasia

Hypophosphatasia is an inherited disorder characterized by defective bone m ineralization and a deficiency of tissue-nonspecific alkaline phosphatase ( TNSALP) activity. The disease is highly variable in its clinical expression , because of various mutations in the TNSALP gene. In approximately 14% of the patients tested in our laboratory, only one TNSALP gene mutation was fo und, despite exhaustive sequencing of the gene, suggesting that missing mut ations are harbored in intron or regulatory sequences or that the disease i s dominantly transmitted. The distinction between these two situations is o f importance, especially in terms of genetic counseling, but dominance is s ometimes difficult to conclusively determine by using familial analysis sin ce expression of the disease may be highly variable, with parents of even s everely affected children showing no or extremely mild symptoms of the dise ase. We report here the study of eight point mutations (G46 V, A99T, S164L, R167 W, R206 W, G232 V, N461I, 1473F) found in patients with no other dete ctable mutation. Three of these mutations, G46 V, S164L, and 1473F, have no t previously been described. Pedigree and/or serum alkaline phosphatase dat a suggested possible dominant transmission in families with A99T, R167 W, a nd G232 V. By means of site-directed mutagenesis, transfections in COS-1 ce lls, and three-dimensional (3D) modeling, we evaluated the possible dominan t effect of these eight mutations. The results showed that four of these mu tations (G46 V, A99T, R167 W, and N461I) exhibited a negative dominant effe ct by inhibiting the enzymatic activity of the heterodimer, whereas the fou r others did not show such inhibition. Strong inhibition resulted in severe hypophosphatasia, whereas partial inhibition resulted in milder forms of t he disease. Analysis of the 3D model of the enzyme showed that mutations ex hibiting a dominant effect were clustered in two regions, viz., the active site and an area probably interacting with a region having a particular bio logical function such as dimerization, tetramerization, or membrane anchori ng.