Detection of Deletion 1154–1156 Hypophosphatasia Mutation Using TNSALP Exon Amplification

Alkaline phosphatases [ALPs; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] are ubiquitously distributed membrane-bound enzymes that hydrolyze monophosphate esters at a high pH optimum, but their physiological roles are not well understood. In humans, four ALP genes, tissue-nonspecific (TNSALP), intestine (IALP), placenta (PALP), and placenta-like, have been cloned. The TNSALP gene is located on chromosome 1p36.1-p34 and is only about 52-57% homologous with the three other ALPs at the amino acid level. The size of the TNSALP gene is more than 50 kb and is bigger than the other ALPs. An autosomal recessive inherited disease, hypophosphatasia, is caused by deficiency of TNSALP and is characterized by skeletal hypomineralization. To date, 10 missense mutations in the TNSALP gene are reported from North America. The method by which the mutations were detected was cloning of the mutant cDNA from cultured fibroblasts of the patients, but this technique is laborious for the screening of mutation sites in the 1.5-kb coding sequence. We applied the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method to screen mutations in the TNSALP gene and found seven new mutations in Japanese patients. This method is quick enough for the screening, but we were not able to screen whole exons and splicing sites because the nucleotide sequences of introns have not been elucidated. One needs primer pairs in the intronic sequence to amplify whole exons in PCR, but to design such primers, the flanking sequence of each exon must be known. In this study, we sequenced the flanking region of each exon by using the long PCR method and found a new mutation using the newly synthesized primers (DBO).