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We have developed a technique to detect accurately heterozygous carriers of a deletion. Specific target sequences were amplified by the polymerase chain reaction (PCR), and the products subsequently were analyzed by high-performance liquid chromatography. Examples from four loci demonstrated that 24-27 cycles of amplification for a single-copy DNA, based on 50 ng of genomic DNA, results in excellent quantitation that readily permits the detection of heterozygous carriers of a deletion. We have demonstrated that triplex PCR (three targets in a single PCR) entails no loss of precision. We also have demonstrated that this method can accurately differentiate the heterozygous carriers of a deletion from normal individuals in four family studies, three for Duchenne muscular dystrophy patients and one for a hemophilia B patient.

A phosphoglycerate kinase (PGKase: ATP:3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3; X chromosome-linked) variant, PGKase-Tokyo, is associated with enzyme deficiency, nonspherocytic hemolytic anemia, and neurological disturbances. Because a sufficient amount of the patient's erythrocytes was not available, the variant enzyme was purified to homogeneity from the cultured lymphoblastoid cells of the patient. The enzyme activity of the variant lymphoblastoid cells was about 16% of that of the normal lymphoblastoid cells. PGKase-Tokyo, compared to the normal enzyme, had a lower specific activity (31% of normal in the backward reaction and 15% of normal in the forward reaction), higher than normal Michaelis constants for ATP and 3-phosphoglycerate, a more acidic pH optimum, and increased thermal instability. Microscale peptide mapping analysis revealed that the structural abnormality of PGKase-Tokyo is a single amino acid substitution from valine to methionine at position 266. Thus, the use of the cultured lymphoblastoid cells is proven to be useful for the study of structural and functional abnormalities of mutant enzymes.