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.
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.