Objective: 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.
Materials and methods: 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.
Results: 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.
Conclusions: 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.