Watanabe H, Hashimoto-Uoshima M, Goseki-Sone M, Orimo H, Ishikawa I
Department of Hard Tissue Engineering (Periodontology), Graduate School, Tokyo Medical and Dental University, Yushima, Japan.
Oral Dis. 2001 Nov;7(6):331-5. doi: 10.1034/j.1601-0825.2001.00740.x.
Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineralization, deficiency of tissue-non-specific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In this study we analysed the genomic TNSALP gene from a patient with HOPS, her family, and unrelated normal controls.
The proband was a 52-year-old Japanese woman with adult onset HOPS. The patient showed deficiency in alkaline phosphatase (ALP) activity, increased urinary excretion of phosphoethanolamine and severe periodontal disease. Genomic DNA was extracted from the peripheral leukocytes of the subjects. Based on published sequence data in the TNSALP gene, 11 pairs of polymerase chain reaction (PCR) primers were used to amplify the coding exons. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis.
By PCR-SSCP analysis of the patient's genomic DNA, fragments containing exon 5 revealed abnormal mobility. This abnormal mobility (exon 5) was also found in the genomic DNA in her mother's sister, but were not detected in her father, brothers or sisters, and unrelated normal controls. Sequencing analysis of the abnormal band extracted from the SSCP gel revealed a C to T transition at nucleotide position 571 (C571T) in exon 5. This mutation resulted in a substitution of Ala-115 with a Val in the mature TNSALP polypeptide. PCR-ASO analysis also confirmed this missense point mutation. The result of this study showed that the pro-band has inherited the C571T mutation in exon 5 from her mother alone and the disease in this family was inherited as an autosomal dominant trait from the pedigree.
The C571T mutation is a new missense point mutation and appears to cause significant changes in the structure and function of TNSALP because Ala-115 is highly conserved in rat TNSALP and human tissue-non-specific, intestinal and placental ALPs.
低磷酸酯酶症（HOPS）是一种遗传性疾病，其特征为骨骼矿化缺陷、组织非特异性碱性磷酸酶（TNSALP）活性缺乏以及乳牙过早脱落。TNSALP基因位于1号染色体的1p34 - 36.1区域，由12个外显子和11个内含子组成。在本研究中，我们分析了一名HOPS患者及其家族成员以及无关正常对照者的TNSALP基因。
先证者是一名52岁成年发病的日本女性HOPS患者。该患者表现出碱性磷酸酶（ALP）活性缺乏、磷酸乙醇胺尿排泄增加以及严重的牙周疾病。从受试者外周血白细胞中提取基因组DNA。根据TNSALP基因已发表的序列数据，使用11对聚合酶链反应（PCR）引物扩增编码外显子。对PCR扩增产物进行PCR - 单链构象多态性（SSCP）分析和PCR - 等位基因特异性寡核苷酸（ASO）分析。
通过对患者基因组DNA进行PCR - SSCP分析，包含外显子5的片段显示出异常迁移。在她母亲的姐妹的基因组DNA中也发现了这种异常迁移（外显子5），但在她的父亲、兄弟、姐妹以及无关正常对照者中未检测到。对从SSCP凝胶中提取的异常条带进行测序分析，发现外显子5中核苷酸位置571处发生了C到T的转换（C571T）。该突变导致成熟TNSALP多肽中第115位的丙氨酸被缬氨酸取代。PCR - ASO分析也证实了这种错义点突变。本研究结果表明，先证者仅从其母亲那里继承了外显子5中的C571T突变，且该家族中的疾病呈常染色体显性遗传。
C571T突变是一种新的错义点突变，似乎导致了TNSALP的结构和功能发生显著变化，因为第115位的丙氨酸在大鼠TNSALP以及人类组织非特异性、肠道和胎盘碱性磷酸酶中高度保守。
