3-Methylglutaconic aciduria type I is caused by mutations in AUH

Abstract

3-Methylglutaconic aciduria type I is an autosomal recessive disorder clinically characterized by various symptoms ranging from delayed speech development to severe neurological handicap. This disorder is caused by a deficiency of 3-methylglutaconyl-CoA hydratase, one of the key enzymes of leucine degradation. This results in elevated urinary levels of 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid. By heterologous expression in Escherichia coli, we show that 3-methylglutaconyl-CoA hydratase is encoded by the AUH gene, whose product had been reported elsewhere as an AU-specific RNA-binding protein. Mutation analysis of AUH in two patients revealed a nonsense mutation (R197X) and a splice-site mutation (IVS8-1G-->A), demonstrating that mutations in AUH cause 3-methylglutaconic aciduria type I.

Figure 1

The metabolic pathway of l-leucine. The different enzymes involved are numbered as follows: 1, Branched-chain amino acid transaminase; 2, Branched-chain 2-keto acid dehydrogenase complex; 3, Isovaleryl-coa dehydrogenase; 4, Methylcrotonyl-CoA carboxylase; 5, 3-Methylglutaconyl-CoA hydratase; and 6, 3-Hydroxy-3-methylglutaryl-CoA lyase.

Figure 2

Expression and purification of MBP-AUH fusion protein in E. coli. Total E. coli extract (T) and affinity-purified fusion protein (P) were analyzed by 10% SDS-PAGE and stained with Coomassie brilliant blue.

Figure 3

Mutation analysis of AUH in two patients. A, PCR fragment B amplified from cDNA of two control subjects (C1 and C2) and patient 1 (P) showing the 42-bp deletion (exon 9) as a consequence of a IVS8-1G→A mutation. B, PCR-RFLP analysis of the 589C→T mutation. Part of intron 5 and exon 5 containing the c.589C→T mutation was amplified using a mismatch primer (558AUHfNdeI 5′-TGG CTT TAG CCT GTG ACA TA-3′) creating an NdeI restriction site in the 589T allele. For convenient control of the restriction, the reverse primer (In5AUHrM13NdeI 5′-cag gaa aca gct atg acc CAT ATG ACC ATT AGG ACC AAC AAG TG-3′) contained an M13 extension and a second NdeI restriction site. PCR products from genomic DNA of a control subject (C) and patient 2 (P) were loaded directly (−) or digested with NdeI (+) before electrophoresis.