Family studies in ornithine transcarbamylase deficiency.

Six families with at least one infant each with confirmed ornithine transcarbamylase deficiency were investigated by DNA analysis. All the affected sons had died, and no DNA had been stored. Using the restriction endonucleases MspI and Bam HI three restriction fragment length polymorphisms were detected which led to eight distinct haplotypes. Using these results and those of protein loading tests that diagnosed heterozygote (carrier) status in some family members, some carriers were detected, and prenatal diagnosis was offered to two families. In two further families no polymorphisms were found and no prenatal diagnosis was possible. In the remaining two families prenatal diagnosis was impossible because of the lack of DNA from an affected or unaffected son, or in one case from the father, of an obligate carrier. These studies emphasise the importance of preserving tissue for DNA extraction from infants dying of inborn errors of metabolism, and also show the way in which information from conventional biochemical studies can complement diagnostic tests using DNA.

Immunochemical studies on phosphoglycerate kinase deficiency.

Antisera to normal erythrocyte and skeletal muscle PGK, raised in rabbits, were shown to cross-react with extracts from normal tissues and with extracts from a subject with PGK deficiency. Radial immunodiffusion, using the antisera raised against normal human PGK, was used to determine the amount of cross-reacting PGK protein present in extracts of several tissues from an affected subject. For all tissues tested, activity was only a small percentage of the PGK protein concentration. In particular, evidence for normal levels of protein in erythrocytes and myocardium was obtained. The results indicate that the deficiency is due to a structural mutation of the enzyme.

Phosphoglycerate kinase: studies on normal and a mutant human enzyme.

Phosphoglycerate kinase was purified from a number of tissues obtained at autopsy from a subject with the deficiency. Properties of the mutant enzyme were compared to those of PGK purified from tissue obtained from normal subjects. The purified enzyme from the propositus contained two components, a minor fraction (about 2-5%) which behaved similarly to the normal enzyme during the purification procedure, and a major fraction (greater than 95%) which could not be purified by the same procedure. The major fraction demonstrated a number of other properties which differed from the normal. These included a tendency to form aggregates, increased heat sensitivity and altered nucleotide substrate specificity. The smaller fraction appeared to have effectively identical properties to that of the normal enzyme. In keeping with its X-linked mode of inheritance, phosphoglycerate kinase from all normal tissues appeared to have identical kinetic properties, although evidence for minor variations, presumably due to post-translational modifications, was obtained.

Lack of effect of increased 2,3-diphosphoglycerate on flux through the oxidative pathway in phosphoglycerate kinase deficiency.

Erythrocyte enzyme and substrate levels in two subjects with phosphoglycerate kinase deficiency are reported. The effect of the increased 2,3-diphosphoglycerate, which has been reported as an inhibitor of 6-phosphogluconate dehydrogenase, on the flux through the pentose phosphate pathway, was assessed. There was no significant difference in the flux through erythrocytes from one of the affected subjects and normal subjects in the presence and absence of methylene blue.

Tissue levels of glycolytic enzymes in phosphoglycerate kinase deficiency.

Phosphoglycerate kinase deficiency is a rate, X-linked disorder associated with a severe haemolytic anaemia. In general the deficiency has been demonstrated only in erythrocytes and leucocytes. However, in a subject with this condition, the activity of phosphoglycerate kinase in lymphocytes and platelets was also shown to be less than 5% of the normal value. Following the death of this subject in 1979, the deficiency was also found to occur in tissue samples of brain, skeletal muscle, liver and cardiac muscle, obtained at the autopsy. Values for phosphoglycerate kinase were of the order of 0.5-5% of normal controls. Other glycolytic enzymes which were tested were hexokinase, pyruvate kinase, enolase and 2-phosphoglyceromutase. In general, values for these enzymes were either normal or slightly raised.