We used whole exome sequencing of a single patient with combined malonic and methylmalonic aciduria (CMAMMA) to identify mutations in ACSF3, a putative malonyl-CoA and methylmalonyl-CoA synthetase (MCS). Follow-up sequencing of eight additional patients, including an individual who was diagnosed after mining an exome database as well as an affected canine, showed pathogenic mutations. ACSF3 mutant alleles occur with a minor allele frequency (MAF) of 0.0058 in ~1,000 control individuals predicting a CMAMMA population incidence ~ 1:30,000. CMAMMA is the first human disorder caused by mutations in 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.
Methylmalonic acidemias (MMAemias) are heterogeneous disorders that exhibit elevated methylmalonic acid (MMA) in body fluids. Deficiency of methylmalonyl-CoA mutase (MUT) or the enzymes (MMAA, MMAB, MMADHC) that synthesize 5′-adenosylcobalamin comprise most disease subtypes. Some patients have atypical forms of MMAemia, e.g., combined malonic and methylmalonic aciduria (CMAMMA) that lack enzymatic and molecular definition. CMAMMA was first reported in a child with immunodeficiency, failure to thrive, seizures, increased urinary MMA compared to malonic acid (MA) and normal malonyl-CoA decarboxylase activity1. A Labrador retriever with similar biochemical features and neurodegeneration has also been described2.
To determine the cause of CMAMMA, we took a multifaceted approach that included exome and candidate gene sequencing in nine patients, identification of the canine orthologue and mutation analysis in an affected dog, and a novel strategy of hypothesis-generating clinical research in an exome cohort3. We establish ACSF3 mutations as the cause of CMAMMA and describe the first disease association with a member of the acyl-CoA synthetase (ACS) family, enzymes that activate fatty acids for intermediary metabolism4.
Nine subjects with CMAMMA participated and six were evaluated at the NIH. The age of diagnosis and symptoms were variable (Table 1). After uneventful early decades, four patients were diagnosed in adulthood with neurological manifestations (seizures, memory problems, psychiatric disease, and/or cognitive decline) without vitamin B12 deficiency. Five subjects presented during childhood with symptoms suggestive of an intermediary metabolic disorder (coma, ketoacidosis, hypoglycemia, failure to thrive, elevated transaminases, microcephaly, dystonia, axial hypotonia, and/or developmental delay).
Methylmalonic and malonic aciduria with urinary MMA/MA >5 was present in seven of nine affecteds (Table 1). Serum MMA was elevated but serum B12 levels, acylcarnitines, and total homocysteine were normal, as were malonyl-CoA decarboxylase activity, 1-C14-propionate incorporation, malonyl-CoA decarboxylase (MLYCD) genetic testing, and sequencing of known MMAemia genes (Table 1). Plasma MA was measured by GC/MS in six patients and was also markedly elevated (Table 1). We conclude that these subjects all have CMAMMA, which is distinct from other forms of MMAemia.
For Subject 1, we sequenced target-selected libraries in the paired-end 101 bp configuration, yielding 114,467 variant genotypes. We used genetic filters for homozygosity or compound heterozygosity. We included nonsynonymous, splice, frameshifting, and nonsense variants as potential mutations but excluded dbSNP variants. We used control exome data3 to exclude homozygous variants or variants with >10% frequency (Supplementary Table 1 and Online Methods).
The filtering strategy yielded 12 genes, from which we selected for further evaluation ACSF3, an orphan member of the acyl-CoA synthetase family, based on its putative function and predicted mitochondrial localization. We found three ACSF3 exome variants in Subject 1 (c.1385A>C p.Lys462Thr, c.del1394_1411, p.Gln465_Gly470del, and c.1627C>T, p.Arg558Trp) and confirmed them by Sanger sequencing (Table 1, Supplementary Figure 1, Supplementary Table 2). The variants p.Lys462Thr and p.Gln465_Gly470del were in trans with p.Arg558Trp based on parental genotypes and segregated in two unaffected siblings who, like their parents, had normal serum MMA levels. We sequenced ACSF3 exons in seven additional patients with CMAMMA; six had ACSF3 variations (Table 1, Supplementary Figure 1). One patient had no damaging mutations detected.
Next, we identified a putative canine ACSF3 orthologue and sequenced DNA from the CMAMMA Labrador retriever, this showed a homozygous alteration (c.1288G>A, p.Gly430Ser; orthologous to human p.Gly480) in a conserved residue (Figure 1, Table 1, Supplementary Figure 1). This variant was absent in 40 control Labrador DNAs selected for maximum diversity based on American Kennel Club numbers. Finally, we took a novel approach to patient discovery by analyzing exome data of 401 individuals ascertained for cardiovascular phenotypes3. We identified a 66 year-old female, apparently homozygous for a c.1411C>T, p.Arg471Trp ACSF3 variant. She had no known metabolic disease symptoms but reported incontinence and mild memory problems. Her laboratory evaluation showed 48 μM MMA and 11.3 μM MA in plasma and 206 mmol/mol Cr MMA and 26.3 mmol/mol Cr MA in urine, and normal serum B12 levels and acylcarnitines. We did not find mutations in other known MMAemia genes in her exome (Table 1).
We identified nine missense, one in-frame deletion and one nonsense mutation (Figure 1). Four subjects were apparently homozygous for ACSF3 variants. Although it is possible that unidentified deletions might play a role in this disorder, this is unlikely for these individuals (Table 1). Most of the variants resided in the C-terminal half of ACSF3. Eight out of nine missense mutations and the in-frame deletion were located in conserved ACS motifs predicted to be involved in AMP binding (Motif I), conformational change and catalytic function (Motif II), substrate binding (Motifs III, IV), or catalysis (Motif V)5 (Figure 1). Western analyses using fibroblasts from Subjects 1–4 and 7 showed the presence of cross-reactive ACSF3 (Supplementary Figure 2). Fibroblasts from Subjects 1–4 produced 2.4- to 6-fold more MMA than control cells (Figure 2A) after chemical stimulation. Viral expression of ACSF3, but not GFP (Figure 2B) restored metabolism, and provided validation of ACSF3 function in a cell culture biochemical assay.
These data establish a candidate gene for CMAMMA using exome sequencing in a single affected with validation using four approaches. First, seven additional probands harbored two mutations in ACSF3. Second, an affected dog had a single, unique sequence variant in the canine ACSF3 orthologue in a conserved residue that was absent in 40 diverse controls. Third, one patient with two ACSF3 mutations was identified in a cohort of subjects not ascertained for metabolic disease and had biochemical features of CMAMMA. Finally, viral complementation of ACSF3 in patient fibroblasts corrected the cellular metabolic defect. Based on these observations, we conclude that mutations in ACSF3 cause CMAMMA.
The ACSF3 gene is an orphan member of the acyl-coenzyme A synthetase gene family, enzymes that thioesterify substrates into CoA derivatives, and weakly activated C24:0 fatty acid4. The biochemical abnormalities in the patients led us to reassess the possible function of ACSF3. When we compared human ACSF3 to Bradyrhizobium japonicum malonyl-CoA synthetase (MCS), a well-characterized enzyme, the proteins were more identical (32%) and similar (50%) to each other than ACSF3 was to the next closest human ACS family member (ACSM3v1, 28% identity). Phylogenetic analyses rooted human ACSF3 with the MCS enzymes versus other ACSs (Supplementary Figure 3). To provide preliminary experimental evidence for predicted function, we examined purified, GST-tagged ACSF3 under MCS assay conditions and found that the enzyme activated malonate and methylmalonate, but not acetate, into the respective coenzyme thioesters (Supplementary Table 3). The specific activity of GST-tagged ACSF3 was higher with malonate as a substrate compared to methylmalonate, similar to its prokaryotic homologues. Because the first 58 amino acids of ACSF3 are predicted to encode a mitochondrial leader sequence (Supplementary Figure 4), we performed immunostaining with fibroblasts overexpressing ACSF3 and a C-terminal GFP-ACSF3 fusion protein. ACSF3 staining showed a distinct mitochondrial distribution and co-localized with a mitochondrial antibody (Figure 3). The comparative sequence analysis, enzymatic data, and subcellular localization lead us to propose that ACSF3 is a mitochondrial malonyl-CoA and methylmalonyl-CoA synthetase (MCS), an enzyme postulated to catalyze the first step of intramitochondrial fatty acid synthesis5.
The assignment of ACSF3 as an MCS provides a framework to understand the consequences of the ACSF3 mutations and the metabolic perturbations of CMAMMA. MCS from R. trifolii and B. japonicum activate malonate and methylmalonate as substrates in vitro6,7 as does ACSF3 (Supplementary Table 3), suggesting that malfunction of this enzyme causes accretion of the proximal substrates that manifests as methylmalonic and malonic aciduria. Site-directed mutagenesis experiments with B. japonicum MCS showed that p.Glu308Gln abolishes malonate binding7. The corresponding human ACSF3 position is the residue mutated in Subject 3, p.Glu359Lys in Motif III, and predicts that this mutation is likely to effect the Km for malonate. Arg471 in motif II is nearly invariant in the ACS family4 and essential for acyl-CoA synthetase activity8–10. Therefore, an Arg471 alteration in ACSF3, as in Subjects 5 and 6, likely affects enzymatic function. Other missense alterations (p.Pro243Leu, p.Thr358Ile, p.Gly430Ser (dog), p.Arg558Trp) map to conserved residues in B. japonicum and R. leguminosarum MCS (Figure 1) or to conserved residues in other ACSF3 family members (p.Met198Arg, p.Lys462Thr), and are likely to be deleterious.
In the ClinSeq™ cohort, there were an additional four participants heterozygous for ACSF3 variants (p.Glu359Lys n=1, p.Arg558Trp n=3) also found in patients with CMAMMA. The 1000 genomes dataset (estimated coverage of 629 genomes) there were six individuals with ACSF3 mutations (p.Glu359Lys n=1, p.Arg558Trp n=5). Combining these data yields an overall MAF of 0.0058 (95% CI, .0033–.0106) for an estimated disease incidence of ~1/30,000 (95% CI, 1/9,000 – 1/92,000). We predict that CMAMMA is one of the most common forms of MMAemia11, and perhaps, one of the most common inborn errors of metabolism. Clearly, the spectrum of symptoms and natural history of this disorder are highly variable and require further delineation. The identification of an affected using exome sequencing highlights an interesting and alterative diagnostic approach because CMAMMA is not identified through routine newborn screening (via elevated propionylcarnitine [C3]). We speculate that CMAMMA and other metabolic disorders that have escaped early diagnosis could be identified using genomic techniques.