Stability of several biochemical markers of bone metabolism.

the polyacrylamide gel were visualized by silver staining (Bio-Rad). PCR products were purified by poly-acrylamide gel electrophoresis and used for sequence analyses. The sequencing reactions were carried out using the Taq Dye-Deoxy Termination Cycle Sequencing Kit and DNA Sequencer (Model 373) from Applied Biosys-tems. The mutation screening showed heteroduplex formation only for exon 2. These heteroduplexes were detectable for every hypocatalasemic (n ϭ 23) but not for the normocatalasemic (n ϭ 26) members of families M, D, and G. No heteroduplex formation was detected in the hy-pocatalasemic and normocatalasemic members of the other three hypocatalasemic families. The heteroduplex pattern showed the first band at 268 bp (wild-wild homoduplex), the second at ϳ270 bp (mutant mutant homoduplex), and two heteroduplexes at 273 and 304 bp. It is rare to be able to distinguish these four bands so clearly. Separation of the patterns of the four bands in exon 2 were found in the same well when a smaller gel (150 ϫ 150 ϫ 1.5 mm) was prepared with molecular biology-grade polyacrylamide (Bio-Rad) and no sample treatment was used (Fig. 1, middle panel). The nucleotide sequence analysis showed a GA insertion (Fig. 1, bottom panel) at position 138 of exon 2. This insertion increased the GA repeat number from four to five and caused a frameshift mutation. This frameshift changed the amino acid sequence from position 68 and generated a terminating codon of TGA at position 134. The mutation yielded a truncated protein and the lack of histidine 74, which is required for the binding of a hydrogen peroxide substrate (12). These findings could explain the decreased blood catalase activities of the hypocatalasemic patients (49.2 Ϯ 13.7 MU/L; n ϭ 23) compared with the normocatalasemic family members (107.6 Ϯ 19.5 MU/L; n ϭ 26). The heteroduplex formed from a 268-bp wild-type and a 270-bp mutant PCR product required neither pretreatment nor a special gel for its detection. This simple method was checked when the screening of the 625 normocatalasemic subjects yielded no heteroduplex formation. In conclusion, a simple heteroduplex analysis of PCR products could be used for screening of GA insertions in exon 2 of the catalase gene. This new syndrome-causing mutation was detected in three of the nine hypocata-lasemic families in Hungary. These data confirm the heterogeneity of the acatalasemia/hypocatalasemia detected in Hungary. Detection of a common mutation of the catalase gene in Japanese acata-lasemic patients. A novel human catalase mutation …