Molecular diagnosis of hypophosphatasia with severe periodontitis

Hypophosphatasia (HOPS) is an inherited disorder characterized by the defect of skeletal mineralization due to tissue-nonspecific alkaline phosphatase (TNSALP) deficiency. In this study we analyzed the TNSALP gene from a Japanese patient with HOPS, his parents, his brother, and unrelated normal controls. The proband is a 25-year-old Japanese male diagnosed with childhood hypophosphatasia. The patient reported premature exfoliation of the deciduous teeth and severe periodontal destruction of the permanent dentition. Genomic DNA was extracted from peripheral leukocytes of subjects. Eleven pairs of the polymerase chain reaction (PCR) primers were used to amplify the coding exons according to the published sequence data of the TNSALP gene. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis. In PCR-SSCP analysis of the patient's genomic DNA, the fragments containing exons 9 and 10 revealed abnormal mobilities. These abnormal mobilities (exons 9 and 10) were also found from his mother and father's genomic DNA, respectively. The sequencing analysis of the abnormal bands extracted from the SSCP gel showed a T to C transition at nucleotide position 1155 (T1155C) in exon 9 and G1320A in exon 10. PCR-ASO analysis confirmed these missense point mutations. PCR-ASO analysis also confirmed that mutation-specific oligonucleotides corresponded to the new mutations and did not hybridize with PCR products from normal control genomic DNAs. These results indicated that the proband was a compound heterozygote who inherited T1155C mutation in exon 9 from the mother and G1320A mutation in exon 10 from the father. Both of them are new missense point mutations and appear to cause significant changes in the structure and function of TNSALP.