Yang Haiou, Wang Lili, Geng Juan, Yu Tingting, Yao Ru-En, Shen Yongnian, Yin Lei, Ying Daming, Huang Rongkui, Zhou Yunfang, Chen Huijin, Liu Lanbo, Mo Xi, Shen Yiping, Fu Qihua, Yu Yongguo
Department of Laboratory Medicine, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Cell Physiol Biochem. 2013;32(3):635-44. doi: 10.1159/000354467. Epub 2013 Sep 10.
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.
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.
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.
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.
低磷酸酯酶症是一种罕见的遗传性疾病，其特征为骨骼和牙齿矿化缺陷，由碱性磷酸酶（TNSALP）组织非特异性同工酶的各种突变引起。我们的目的是确定三名被诊断为低磷酸酯酶症的中国儿童TNSALP基因的突变情况。
从患者及其父母的全血样本中提取基因组DNA。然后对TNSALP编码区进行测序。构建表达野生型或各种突变体的质粒，并进行体外研究，以确定这些氨基酸替换是否会影响TNSALP酶活性。
从三个独立家系中鉴定出六个错义突变。在这六个错义突变中，四个是新发现的，两个先前已有报道。与野生型（WT）TNSALP相比，Y28D、A111T和T389N突变体在体外仅表现出可忽略不计的碱性磷酸酶（ALP）活性。缺陷主要是由于66 KD未成熟形式的蛋白质表达显著降低，以及80 KD成熟形式的蛋白质表达几乎检测不到。此外，当与TNSALP（WT）共转染时，所有三个突变体对WT蛋白都有显性负效应。M219V和R136L突变体均表现出部分酶活性，这与两种形式的TNSALP中蛋白质表达降低一致，进一步表现出中度显性负效应。此外，Y388H突变体表现出较弱的ALP活性。蛋白质印迹分析表明，TNSALP成熟形式信号的极度降低可能是酶活性降低的主要原因，因为在未成熟形式中观察到强信号。
在三个血清碱性磷酸酶活性低于正常水平的中国低磷酸酯酶症家系中鉴定出六个错义突变。我们的结果表明，这三名患者血清碱性磷酸酶活性低主要是由于突变体蛋白质表达缺陷。这可能是这些患者低磷酸酯酶症的潜在分子机制。
