Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia
Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia
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DOI：
10.1359/jbmr.2001.16.12.2313
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发表时间：
2001-12-01
影响因子：
6.2
通讯作者：
Shimada, T
中科院分区：
文献类型：
--
作者：
Orimo, H;Girschick, HJ;Shimada, T
应用聚合酶链反应-单链构象多态性（PCR-SSCP）-直接测序法对5名德国儿童型低磷酸酶症（HOPS）家系成员的组织非特异性碱性磷酸酶（TNSALP）基因进行了分析。在相应的患者中检测到四种新的错义突变（T51 M，R54 S，L258 P和R374 H）和两种先前描述的错义突变（A160 T和R206 W）。在3例不同的患者中检测到突变A160 T，并且在1例患者和2例未将V505 A等位基因传递给先证者的父亲中检测到与L258 P突变相同的等位基因中的先前描述的多态性V505 A。2例患者未发现其他突变。突变蛋白在COS-1细胞中的瞬时表达表明，4个新突变和R206 W是严重等位基因，而A160 T是中度等位基因。酶活性和遗传传递模式分析证实V505 A是一个多态性。瞬时表达的蛋白质的免疫沉淀显示，80 kDa的成熟形式的酶的水平减少或不存在与严重的等位基因，相反，高分子量的二硫键连接的聚集体的水平增加。这些结果表明，在复合杂合子中，重度和中度等位基因的组合可能联合收割机导致儿童型HOPS中所见的轻度表型。
The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.