Exome sequencing identifies ACSF3 as a cause of combined malonic and methylmalonic aciduria

Abstract

We used exome sequencing to identify the genetic basis of combined malonic and methylmalonic aciduria (CMAMMA). We sequenced the exome of an individual with CMAMMA and followed up with sequencing of eight additional affected individuals (cases). This included one individual who was identified and diagnosed by searching an exome database. We identify mutations in ACSF3, encoding a putative methylmalonyl-CoA and malonyl-CoA synthetase as a cause of CMAMMA. We also examined a canine model of CMAMMA, which showed pathogenic mutations in a predicted ACSF3 ortholog. ACSF3 mutant alleles occur with a minor allele frequency of 0.0058 in ∼1,000 control individuals, predicting a CMAMMA population incidence of ∼1:30,000. ACSF3 deficiency is the first human disorder identified as caused by mutations in a gene encoding a member of the acyl-CoA synthetase family, a diverse group of evolutionarily conserved proteins, and may emerge as one of the more common human metabolic disorders.

Figure 1

Alignment of the motif regions in ACSF3 orthologues and the malonyl-CoA synthase enzymes in bacteria. The sequences (see methods) were aligned using MegAlign via the Clustal W method. An additional three amino acids amino-terminal to Motif I are shown. Motif II was aligned independent of the full-length protein to improve the alignment of the ACSF3 and MCS proteins. The ACSF3 alterations identified in the eight subjects and affected dog are indicated. The asterisk (*) indicates the dog variant p.Gly430Ser, which is orthologous to position p.Gly480 in human ACSF3.

Figure 2

MMA production by CMAMMA fibroblasts and lentiviral complementation with ACSF3. A. Control fibroblasts and fibroblasts from Subjects 1–4 were incubated in medium containing 5 mM sodium propionate at 37°C for 72 hours and the media was removed for GC/MS analysis of MMA. The CMAMMA patient cells showed increased accumulation of MMA in the media, which were 6, 2.4, 5.3 and 2.4 fold elevated compared to the control cell lines. Error bars are +/− 1 standard deviation (n=3 measurements per cell line). B. Fibroblasts from Subjects 1, 3 and 4 were transduced with lentivirus designed to express ACSF3 or GFP and then incubated in medium containing 5 mM sodium propionate as described. Fibroblasts transduced with ACSF3, but not GFP, exhibit MMA production similar to control fibroblasts treated in a similar fashion. Error bars are +/− 1 standard deviation (n=3 replicates per cell line).

Figure 3

ACSF3 mitochondrial localization. A) Control fibroblasts transfected with a plasmid expressing C-terminal GFP-tagged ACSF3 were co-stained with anti-ACSF3 (red) and a mitochondrial antibody (white). Scale bar=10 μm B) Fibroblasts from Subject 4 that express ACSF3 after lentiviral transduction were co-stained with anti-ACSF3 (red) and a mitochondrial antibody (green). The bottom right depicts an enlargement of the area surrounding the arrow. Scale bar=20 μm. All images were collected using a confocal microscope using a 63X objective with 0.7 zoom.