The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency

Abstract

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.

Figure 1

The MCC-catalyzed reaction and its position in the leucine catabolic pathway. The dashed arrow indicates the metabolites that accumulate due to deficiency of MCC.

Figure 2

Expression of the biotin-containing MCCα subunit in fibroblasts. Proteins in mitochondrial enriched fractions from cultured fibroblasts were separated by SDS-PAGE, and the biotin-containing subunits of MCC, PCC, and PC were detected with an avidin alkaline phosphatase conjugate. Most, but not all, CG2-probands lack the MCCα subunit, while it is detectable in all CG1 probands.

Figure 3

Sequence alignment of human MCCα and MCCβ with orthologs from mouse and A. thaliana. Amino acids identical to the human sequence are highlighted. Missense mutations identified in MCC-deficient patients are indicated above with the substituted amino acid. Potential cleavage sites for the NH₂-terminal mitochondrial leader sequences are indicated by vertical arrowheads. (a) Sequence alignment of human, mouse, and A. thaliana MCCα. The predicted ATP-binding site in the NH₂-terminal biotin carboxylation domain and the predicted COOH-terminal biotin carboxyl carrier domain are indicated by solid and dashed over-lines, respectively. The arrow indicates the lysine residue that links covalently to biotin (biocytin). The residues marked with an asterisk within the biotin carboxylation domain are thought to play an important role in catalysis (36, 37). (b) Sequence alignment of human and A. thaliana MCCβ. The putative 3-methylcrotonyl-CoA binding domain is indicated by a solid over-line.

Figure 4

Coomassie-stained SDS-PAGE gel of biotin-containing proteins purified from a mouse kidney extract with streptavidin Dynabeads. We excised the proteins from the gel, digested with trypsin, and analyzed the tryptic peptides using MALDI-TOF and liquid chromatography coupled to ESI-MS/MS. We analyzed all the indicated bands including those that we identified as MCCα and β.

Figure 5

MCCA and B missense mutations that alter splicing. (a) MCCA D532H. The 1594G→C transversion of the last bp of exon 13 results in the missense mutation D532H. The 3′ base of an exon also contributes to donor splice site recognition and, as shown in the lower panel, RT-PCR of MCCA cDNA in this patient with primers corresponding to the 5′ and 3′ UTR resulted in a product smaller than in wild-type. Sequence analysis of this product showed that exon 13 (217 bp) is skipped, which shifts the reading frame. Thus, the deleterious consequences of this missense mutation appear to be entirely due to the splicing defect. (b) MCCB I437V. As shown in the upper panel, the 1309A→G transition in exon 14 results in the replacement of isoleucine by valine, a conservative change. However, the mutation also activates a cryptic splice donor. Use of this new donor splice site deletes the last 64 bp of exon 14 from the mature transcript. As shown in the lower panel, direct sequencing of the RT-PCR product shows that virtually all the transcript present uses the new, more 5′ splice donor. The second allele of this compound heterozygous patient does not produce detectable RNA. wt, wild-type; mut, mutant.