E30Q mutation identified in patient's PZ gene. (A) The normal nucleotide sequence of the PZ gene is shown at the top and a nucleotide substitution in exon II is indicated by a bold letter. A primer for PCR-RFLP analysis, the Q30R primer, was designed with 1 base mismatch (in italics) to generate an MfeI endonuclease restriction site in a PCR product from the mutant allele (bottom). (B) PCR products obtained from the patient (P) and healthy controls (1-8) were applied to agarose gel electrophoresis after MfeI digestion. Only the mutant Q30 allele was cleaved into 262–base pair (bp) and 25-bp fragments, although the latter is too small to detect. (C) Alignment of the amino acid sequences of Gla domains among vitamin K–dependent proteins. The position of E30 in PZ is shown in bold. GAS6 indicates growth arrest specific 6; BGP, bone Gla protein; MGP, matrix Gla protein.