The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism

Abstract

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

Figure 1

cDNA nucleotide sequence and intron-exon organization of human MCCA and MCCB. A and B, Nucleotide sequence of MCCA (A) and MCCB (B) cDNAs, with deduced translation sequences. Red arrowheads indicate the position of the introns. C, Intron-exon organization of the MCCA gene (top) and the MCCB (bottom) gene. D, MCCβ protein localizing in mitochondria. A mouse polyclonal antiserum raised against histidine-tagged MCCβ purified from bacteria was used to reveal the mitochondrial localization of MCCβ, in immunohistochemical localization experiments with cultured fibroblasts.

Figure 2

Chromosomal localization of MCCA (A) and MCCB (B). RH-mapping results with the GB4 panel (for MCCA) and with the G3 and GB4 panels (for MCCB) are shown. The nearly telomeric chromosomal localization of MCCA was confirmed by FISH (see insets of individual chromosomes, in panel A).

Figure 3

A, Leucine-degradation pathway. The absence of MCC leads to the accumulation of the upstream compounds isovaleryl-CoA and methylcrotonyl-CoA, which lead to 3-hydroxyisovaleric (3-HIVA) and 3-methylcrotonylglycine (3-MC-Gly), which are biochemical landmarks of the isolated, biotin-insensitive MCC deficiency. B, Multitissue northern blot hybridization, showing coordinated, high-level expression of MCCA and MCCB in liver, kidney, heart, and skeletal muscle. β-Actin was used as a loading control. MCCA and MCCB probes were ESTs “AA605162” and “AI949422,” respectively. B = brain; H = heart; SM = skeletal muscle; C = colon; T = thymus; S = spleen; K = kidney; Li = liver; Si = small intestine; P = placenta; Lu = lung; PBL = peripheral blood lymphocytes. The human multiple-tissue northern filter was purchased from Clontech.

Figure 4

Results of in vivo [³H]-biotin labeling of mitochondrial biotin-containing polypeptides PC, PCCα, and MCCα. Cultured fibroblasts (relevant genotypes are shown at the top of each lane) were incubated in the presence of the labeled vitamin and were lysed, and the protein extracts were loaded onto a 7.5% SDS-polyacrylamide gel. Labeled proteins were revealed by fluorography.

Figure 5

Mutations in MCCA and MCCB. A, Mutations in MCCB: results of direct sequencing of PCR-amplified genomic DNA from controls and from CGB patients who have MCG are shown. Arrows indicate the mutated positions. B, Results of direct sequencing of PCR-amplified genomic DNA from controls and from CGA patients who have MCG. Arrows indicate the mutated positions. C, IVS3+5G→T MCCB mutation, which results in skipping of exon 3. The gel shows the results of agarose-gel electrophoresis and direct sequence analysis of MCCB cDNA from the region surrounding the exon2/exon3 junction, amplified by RT-PCR with fibroblast mRNA as template, from a control (lane C) and from the patient (lane P) carrying this mutation. Lane M, molecular-wieght marker.

Figure 6

Knockout of the gene for MCCα in Aspergillus nidulans. An Aspergillus nidulans ΔmccA strain carrying a complete deletion of the gene encoding fungal MCCα is unable to utilize leucine as the sole carbon source but grows on other branched-chain amino acids. The parental strain and three independent transformants (denoted “#1”–“#3”) deleted for the mccA gene were grown on synthetic minimal medium with ammonium chloride as the nitrogen source and with 30 mM of the indicated amino acids as the sole carbon source. Plates were incubated for 5 d at 37°C. Fungi can synthesize carbohydrates from acetyl-CoA, via the glyoxylate cycle—thereby bypassing the Krebs cycle, which leads to complete oxidation of acetyl-CoA to CO₂ and water.