Heterogeneity in maple syrup urine disease: Aspects of cofactor requirement and complementation in cultured fibroblasts

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Abstract
Fibroblast strains derived from 6 patients with maple syrup urine disease [MSUD] were investigated for their requirements of the cofactors NAD, CoASH [reduced coenzyme A] Mg2+ and TPP [thymine pyrophosphate] in comparison with 10 normal control strains. The reconstitution of the decarboxylase function of branched chain .alpha.-keto acid (BCKA) dehydrogenase complex in lysed cells was studied with respect to the substrates .alpha.-keto-isocaproic acid, .alpha.-keto-isovaleric acid and .alpha.-keto-.beta.-methylvaleric acid (KIC, KIVA, MEVA). The enzyme activity of all normal control strains for the substrates KIC and KIVA was not reconstituted by TPP + Mg2+ alone, but CoASH + NAD could reconstitute the enzyme activity with KIC and KIVA in different degrees. Only 2 control strains were tested with MEVA as substrate, and these showed in contrast that TPP + Mg2+ could partly reconstitute the enzyme activity. In contrast to the relative homogeneity in the reconstitution profiles of normal strains, the 5 classical and 1 intermittent MSUD strains showed heterogeneity in cofactor requirements. Complementation analysis using heterokaryons prepared from fibroblasts of 4 patients with classical MSUD and 1 patient with intermittent MSUD showed, in contrast to experiments with normal controls, a partial amelioration of the defect in 2 combinations; the defect in these strains may be located at different functional subunits of the multienzyme complex.