Direct nonisotopic assay of 3-methylglutaconyl-CoA hydratase in cultured human skin fibroblasts to specifically identify patients with 3-methylglutaconic aciduria type I.

Abstract

3-Methylglutaconic aciduria (3MGA) type I (McKusick 250950) is biochemically characterized by increased excretion of 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid in urine. Affected individuals display a range of clinical manifestations varying from mildly delayed speech development to severe neurologic involvement (1)(2). 3MGA type I is an autosomal recessive disorder caused by a deficiency of 3-methylglutaconyl-CoA hydratase (3MGH; EC 4.2.1.18). Three additional forms of 3MGA have been recognized—type II (Barth syndrome, McKusick 302060); type III (Costeff syndrome, McKusick 258501); and type IV (“unspecified”, McKusick 250951)—all characterized by normal hydratase activities (3). Recently, the gene encoding 3MGH was identified by two independent groups (4)(5). As shown in Fig. 1A⇓ , this mitochondrial enzyme catalyzes the penultimate step in leucine catabolism, which is the reversible conversion of 3-methylglutaconyl-CoA to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Eleven patients have been described with isolated 3MGH deficiency (1)(2)(5)(6)(7)(8)(9)(10)(11). The hydratase deficiency in these patients was identified by use of a radioactive enzyme assay measuring three consecutive steps of leucine degradation, from 3-methylcrotonyl-CoA to acetoacetic acid (12). However, this procedure lacks specificity and is labor-intensive because of the need to purify the coupling enzyme, 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4), from bovine liver. Furthermore, the assay is not very practical for use in clinical laboratories because it involves the use of radiochemicals. The need to differentiate patients with 3MGA type I from patients with other forms of 3MGA requires the availability of a sensitive and specific enzyme assay. From our knowledge that, in general, hydratase reactions are readily reversible and that 3-methylglutaconyl-CoA is not commercially available, we studied the 3MGH activity in the reverse direction, using HMG-CoA as a substrate. We quantified the formation of 3-methylglutaconyl-CoA by reversed-phase HPLC with ultraviolet …