Novel missense and frameshift mutations in the tissue-nonspecific alkaline phosphatase gene in a Japanese patient with hypophosphatasia.
Novel missense and frameshift mutations in the tissue-nonspecific alkaline phosphatase gene in a Japanese patient with hypophosphatasia.
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日本低磷酸酯酶症患者的组织非特异性碱性磷酸酶基因出现新的错义和移码突变。
DOI：
10.1093/hmg/3.9.1683
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发表时间：
1994
影响因子：
3.5
通讯作者：
Tsuneo Hirayama
中科院分区：
文献类型：
--
作者：
Hideo Orimo;Zuisei Hayashi;Atsushi Watanabe;Tsunenori Hirayama;Tsuneo Hirayama
低磷症是一种罕见的常染色体隐性遗传病，其特征是由于组织非特异性碱性磷酸酶(TNSALP)缺乏导致骨骼矿化缺陷(1)。该病的临床严重程度变化很大，从宫内死亡到成年后的病理性骨折。TNSALP基因位于染色体Ip34-36.1(2，3)上，由12个外显子和11个内含子(4，5)组成。到目前为止，已经在患者的等位基因中发现了九种不同的错义突变。在这项研究中，我们分析了一名患有严重低磷症的日本患者的基因组TNSALP基因，发现了新的错义和移码突变。先证者是没有血缘关系的日本父母所生的第一个女婴，没有低磷血症的临床表现。患者表现出严重的骨骼矿化不足和呼吸窘迫，于5个月零27天死亡。患者血清碱性磷酸酶水平：父亲为4IU/L，父亲为92IU/L，母亲为36IU/L(正常范围65~205)。患者尿磷乙醇胺水平为3528.0 nmol/mg肌酐，父亲为54.5nmol/mg，母亲为70.4nmol/mg(正常范围：男性30-80，女性40-100)。从患者的尸检肝脏样本和她父母的外周白细胞中提取基因组DNA。根据已发表的TNSALP基因(4-8)的序列数据，设计了11对聚合酶链式反应(PCR)引物，用于扩增编码外显子(外显子2-12)。图1的图例中描述了用于PCR-单链构象多态(SSCP)和用于PCR-限制性片段长度多态(RFLP)的部分引物序列。其他引物的序列可根据要求提供。在对患者基因组DNA的PCR-SSCP分析中，包含外显子9、10和12的片段显示出异常的迁移率(图1A)。从SSCP凝胶中提取的异常条带的直接测序分析表明，外显子9的1068位核苷酸(G1068A)有G到A的转变，第10外显子的1190(C1190T)有C到T的转变(图2A，B)。G1068A突变导致密码子281(E281K)的氨基酸由谷氨酸变为赖氨酸，而C1190T突变在密码子321的组氨酸残基上无突变。将含有第12外显子的PCR扩增片段克隆到pUC19中并测序(图2C)。分析的七个克隆中有三个显示T在1735处缺失。这种删除导致在密码子503处的亮氨酸残基下游的帧移位，并且在508处的正常终止密码子将被消除。由于亚基终止密码子出现在突变DNA的第588密码子上，因此合成的蛋白质应该有80个额外的氨基酸。
Hypophosphatasia is a rare autosomal recessive disease which is characterized by the defect of skeletal mineralization due to tissue-nonspecific alkaline phosphatase (TNSALP) deficiency (1). Clinical severity of the disease is highly variable ranging from death in utero to pathological fracture in adulthood. The gene for TNSALP is located on chromosome Ip34—36.1 (2, 3) and consists of 12 exons and 11 introns (4, 5). To date, nine different missense mutations have been identified in patient's alleles. In this study, we analyzed the genomic TNSALP gene from a Japanese patient with a severe form of hypophosphatasia and found new missense and frameshift mutations. The proband was the first infant girl born to unrelated Japanese parents who had no clinical manifestations of hypophosphatasia. The affected patient exhibited profound skeletal hypomineralization and respiratory distress and died at age 5 month and 27 days. The serum levels of alkaline phosphatase were 4 IU/L in the patient, 92 IU/L in her father, and 36 IU/L in her mother (normal range 65—205). The urinary phosphoethanolamine level was 3528.0 nmol/mg of creatinine in the patient, 54.5 nmol/mg of creatinine in her father, and 70.4 nmol/mg of creatinine in her mother (normal range for male 30-80 and for female 40-100). Genomic DNA was extracted from the autopsy liver sample of the patient and from peripheral leukocytes of her parents. Based on the published sequence data of the TNSALP gene (4-8), eleven pairs of the polymerase chain reaction (PCR) primers were designed to amplify the coding exons (exons 2—12). Part of the sequences of the primers used for PCR-single strand conformation polymorphism (SSCP) and for PCR-restriction fragment length polymorphism (RFLP) are described in the legend for Figure 1. The sequences of the other primers are available upon request. In PCR-SSCP analysis of the patient's genomic DNA, the fragments containing exons 9, 10, and 12 revealed abnormal mobilities (Fig. 1A). Direct sequencing analysis of the abnormal bands extracted from the SSCP gel showed that a G to A transition at nucleotide position 1068 (G1068A) in exon 9 and a C to T transition at 1190 (C1190T) in exon 10 (Fig. 2A, B). G1068A mutation results in an amino acid change Glu to Lys at codon 281 (E281K), while Cl 190T mutation is silent at the His residue at codon 321. The PCR amplified fragment containing exon 12 was cloned in pUC19 and sequenced (Fig. 2C). Three out of seven clones analyzed showed a deletion of T at 1735. This deletion causes the frame shift downstream from the Leu residue at codon 503, and the normal termination codon at 508 would be eliminated. Since the inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein should have 80 additional amino acids.(a)