Characterization of Six Missense Mutations in the Tissue-Nonspecific Alkaline Phosphatase (TNSALP) Gene in Chinese Children with Hypophosphatasia

Abstract

Aims: Hypophosphatasia, a rare inherited disease characterized by defective mineralization of bone and teeth, is caused by various mutations in the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) gene. Our aim was to determine the mutations on TNSALP gene in three Chinese children diagnosed as having hypophosphatasia. Methods: Genomic DNA was extracted from whole blood samples of patients and their parents. The TNSALP coding regions were then sequenced. Plasmids expressing wild-type or various mutants were built and in vitro studies were performed in order to determine whether these amino acid replacements could affect the TNSALP enzymatic activity. Results: Six missense mutations were identified from three independent pedigrees. Of the six missense mutations, four were novel and two had been previously reported. The Y28D, A111T and T389N mutants displayed only negligible ALP activity in vitro compared to the wild-type (WT) TNSALP. The defect was mainly due to the significantly decreased protein expression in the 66 KD immature forms and the nearly undetectable protein expression in the 80 KD mature forms. Moreover, all three mutants had a dominant negative effect on the WT protein when co-transfected with TNSALP (WT). M219V and R136L mutants both exhibited partial enzymatic activities which were consistent with reduced protein expression in both forms of TNSALP which further exhibited moderate dominant-negative effect. In addition, Y388H mutant showed weak ALP activity. Western blot analysis indicated that the extreme reduction in signal from the mature forms of TNSALP could be the main cause of decreased enzymatic activity, since a strong signal was observed in the immature forms. Conclusion: Six missense mutations were identified in three Chinese hypophosphatasia pedigrees with subnormal serum ALP activity. Our results show that the low activity of serum ALP in the three patients is due mainly to a defect in the protein expression of the mutants. This may be the underling molecular mechanism for hypophosphatasia in these patients.

Molecular phenotype of tissue-nonspecific alkaline phosphatase with a proline (108) to leucine substitution associated with dominant odontohypophosphatasia

“…Attempts have been made to understand the molecular mechanism by which loss-of-function mutations in the ALPL gene culminate in the development of HPP by expressing and examining various TNSALP mutant proteins produced in vitro in cultured cells [7][8][9][10][11][12][13][14][15][16][17]. However, even though there are an increasing number of dominantly inherited TNSALP mutations, especially in milder HPP [6,14,18], their molecular study has been limited to a small number of cases to date [19][20][21][22].…”

“…However, even though there are an increasing number of dominantly inherited TNSALP mutations, especially in milder HPP [6,14,18], their molecular study has been limited to a small number of cases to date [19][20][21][22]. In this study, we characterized TNSALP (P108L) associated with dominant odonto-HPP both in transiently transfected COS-1 cells and in an established cell line.…”

“…In theory, when the transcription of the ALPL gene is not impaired, these loss-of-function mutations could affect any biosynthetic step of TNSALP from the ER to the cell membrane, resulting in diminished and/or altered cell surface ALP activity. Studies in vitro in cultured cells have indicated that the cell surface localization of TNSALP mutant proteins is retarded to various degrees due to improper folding and resultant perturbed protein trafficking [7][8][9][10][11][12][13][14]. Some TNSALP mutant proteins are retained in the ER or cis-Golgi and eventually subjected to ER-associated degradation [10,11,13].…”

“…Of interest is a frame-shift mutation that converts TNSALP to a GPI-anchorless form; this frame-shift mutant protein no longer resides on the cell membrane, but is largely degraded within the cell [17]. An increasing number of TNSALP mutations associated with dominant HPP have been identified [6,14,18]; however, the molecular characterization of these TNSALP mutant proteins has remained limited [19][20][21][22]. TNSALP (P108L) was first reported in a 9-year-old boy with dominantly transmitted odonto-HPP [23].…”

A novel missense mutation in the ALPL gene causes dysfunction of the protein

“…The cells were lysed with 0.2 ml lysis buffer (Beyotime Institute of Biotechnology). A protease inhibitor mixture without any phosphatase inhibitor was added to the cell lysate as described previously (14). The protease inhibitor mixture, which consisted of phenylmethylsulfonyl fluoride (100 mM), aprotinin (15 µM), leupeptin (100 µM), bestatin (100 µM), pepstatin A (100 µM) and E-64 (80 µM), was purchased from Shanghaibocai (Shanghai, China).…”