Combined malonic and methylmalonic aciduria


Combined malonic and methylmalonic aciduria , also called combined malonic and methylmalonic acidemia is an inherited metabolic disease biochemically characterized by elevated levels of malonic acid and methylmalonic acid. However, the methylmalonic acid levels exceed those of malonic acid. CMAMMA is not only an organic aciduria but is also defined by defects of mitochondrial fatty acid synthesis and mitochondrial lysine malonylation. Researchers have suggested that CMAMMA might be one of the most common forms of methylmalonic acidemias, and possibly one of the most common inborn errors of metabolism. As CMAMMA does not show up in routine newborn screening, and symptoms are variable and often appear only in adulthood, diagnoses are frequently delayed or entirely missed, making genetic methods the key to its detection.

Symptoms and signs

The clinical phenotypes of CMAMMA are highly heterogeneous and range from asymptomatic, mild to severe symptoms. In the literature, the following symptoms have been reported:
When the first symptoms appear in childhood, they are more likely to be intermediary metabolic disorders, whereas in adults they are usually neurological symptoms.

Cause

CMAMMA is an inborn, autosomal-recessive metabolic disorder caused by homozygous or compound heterozygous variants in the ACSF3 gene. This results in a defect of the mitochondrial enzyme acyl-CoA synthetase family member 3, leading to reduced or complete loss of its activity. The ACSF3 gene, located on chromosome 16 at locus q24.3, consists of 14 exons and produces four alternatively spliced mRNAs that encode two isoforms of the ACSF3 protein:
  • a 576-amino-acid protein and
  • a shorter 311-amino-acid protein, which begins translation at a downstream start codon relative to isoform 1.
Based on minor allele frequency, a prevalence of ~ 1: 30 000 can be predicted for CMAMMA.

Pathophysiology

ACSF3 encodes an acyl-CoA synthetase, which is localized in the mitochondrial matrix and has a high specificity for malonic acid and methylmalonic acid. These substrates are activated by ACSF3 through an ATP-dependent reaction, linking them to coenzyme A and generating the thioesters malonyl-CoA and methylmalonyl-CoA. The corresponding biochemical reactions are in vivo as follows:
  • as malonyl-CoA synthetase:
  • as methylmalonyl-CoA synthetase:
ACSF3, whose activity also depends on magnesium, most efficiently converts malonate and activates methylmalonate at about 70% of that rate. Lignoceric acid has been reported as an additional substrate, although in vitro studies provide conflicting results.

Primary effects

Based on the ACSF3-catalyzed reactions described above, the following subsections outline the metabolic consequences of CMAMMA, beginning with the accumulation of the upstream substrates malonate and methylmalonate, and followed by deficiencies of the downstream products malonyl-CoA and methylmalonyl-CoA:

Substrate accumulations

The defect in ACSF3 results in impaired detoxification, as malonic acid and methylmalonic acid are not converted into their CoA derivatives and therefore these substrates accumulate.
Malonic acid
The exact mitochondrial origin of malonic acid is unknown, but its transport properties are partly understood: it crosses plasma membranes only to a limited extent, with uptake increasing under acidic conditions, and within cells it enters mitochondria via the dicarboxylate carrier SLC25A10 that also transports succinate, malate, and oxaloacetate. A major proposed source is the non-enzymatic hydrolysis of cytosolic malonyl-CoA generated during de novo fatty acid synthesis, whose levels correlate with lipogenic activity. Enzymatic hydrolysis by acyl-CoA thioesterases may also contribute, alongside other potential routes such as slow carboxylation of acetyl-CoA by propionyl-CoA carboxylase, decarboxylation of oxaloacetate, oxidation of malondialdehyde, and conversion of β-alanine via malonate semialdehyde. Beyond endogenous formation, exogenous sources may also contribute from the diet, with free malonic acid occurring in plants such as legumes.
Malonic acid is an antimetabolite that acts as a classic competitive inhibitor of succinate dehydrogenase in the mitochondrial electron transport chain, thereby blocking succinate oxidation and impairing the citric acid cycle. This inhibition reduces mitochondrial respiration and can be cytotoxic, particularly in cells with high oxidative metabolism such as striatal neurons.
Methylmalonic acid
accumulates to even higher levels than malonic acid, making it the biochemical hallmark of CMAMMA and places it among the methylmalonic acidemias.
The origin of methylmalonic acid is the propionate metabolism pathway in mitochondria, in which the essential amino acids valine, threonine, methionine, and isoleucine, odd-chained fatty acids, propionic acid and the cholesterol side chain are converted into propionyl-CoA. Propionic acid arises from bacterial fermentation in the gut and from dietary intake, being naturally present in certain cheeses or added as a preservative, especially in baked goods. Propionyl-CoA carboxylase forms D-methylmalonyl-CoA, which is epimerized to L-methylmalonyl-CoA and converted by methylmalonyl-CoA mutase to succinyl-CoA for entry into the citric acid cycle, a reaction that requires the coenzyme adenosylcobalamin. However, D-methylmalonyl-CoA may also be hydrolyzed by D-methylmalonyl-CoA hydrolase, releasing coenzyme A and generating methylmalonic acid, which represents a by-product of this pathway. The more unspecific mitochondrial acyl-CoA thioesterase 9 can likewise hydrolyze methylmalonyl-CoA, irrespective of isomer, to methylmalonic acid, with the enzyme's activity being strongly regulated by NADH and free CoA.
But in CMAMMA, methylmalonic acid mainly derives from threonine metabolism, as shown in Acsf3 knockout mice. Moreover, methylmalonic acid was found to impair osteogenesis by inhibiting osteoblast differentiation and reducing mineralization, providing a mechanistic link to the reduced body length observed in these mice.
In vitro, a connection between free methylmalonic acid and neurotoxicity has been established.

Product deficiencies

Defective ACSF3 leads not only to accumulation of its substrates but also to reduced levels of its mitochondrial products, malonyl-CoA and methylmalonyl-CoA.
Malonyl-CoA
is an intermediate that cannot cross membranes and therefore requires local synthesis within mitochondria. Although the exact origin of mitochondrial malonyl-CoA remains debated, the pool is thought to be provided by ACSF3 from malonic acid and by mitochondrial acetyl-CoA carboxylase 1 from acetyl-CoA. Partial compensation of defective ACSF3 by mtACC1 could explain the broad clinical heterogeneity of CMAMMA. Mitochondrial malonyl-CoA is required for lysine malonylation, mitochondrial fatty acid synthesis, acetyl-CoA synthesis and incorporation into cellular lipids.
Lysine malonylation is a dynamically regulated post-translational modification in which malonyl groups are added to lysine residues of proteins, reversing their positive charge into a negative one and increasing their steric bulk. This can influence protein conformation, enzyme activity, and protein–protein interactions and has been linked to the regulation of energy metabolism, in particular glycolysis and β-oxidation. ACSF3 expression, tightly coupled to feeding cycles, controls the extent of mitochondrial lysine malonylation by regulating the availability of malonyl-CoA, which serves as the donor of malonyl groups.
In ACSF3 and Acsf3 knockout models, mitochondrial lysine malonylation was shown to be markedly reduced, confirming that ACSF3-derived malonyl-CoA is required for this modification. It has been proposed that reduction in lysine malonylation contributes more than malonic acid accumulation to the widespread mitochondrial dysfunction observed in CMAMMA.
Mitochondrial fatty acid synthesis has been described as a nutrient-responsive signaling pathway linked to acetyl-CoA utilization, respiratory chain function, iron–sulfur cluster biogenesis, mitochondrial translation, and llipid-mediated signaling processes. In this pathway, malonyl-CoA serves as the precursor of the chain extender unit malonyl-ACP, which, in a condensation reaction with CO2 release, elongates the ACP-bound fatty acid chain by two carbons per round. It generates acyl-ACP species of different chain lengths, which fulfill distinct functions: Octanoyl-ACP is one such mtFAS product and a direct precursor of lipoic acid biosynthesis, which serves as a cofactor for several mitochondrial multienzyme complexes involved in energy metabolism, including the pyruvate dehydrogenase complex, the α-ketoglutarate dehydrogenase complex, the branched-chain α-ketoacid dehydrogenase complex, the 2-oxoadipate dehydrogenase complex, and the glycine cleavage system. In contrast, longer-chain acyl-ACP species allosterically activate the network of LYRM proteins. In humans, this network comprises at least 12 proteins and regulates mitochondrial translation, iron–sulfur cluster biogenesis, and the assembly of electron transport chain complexes.
It is unclear whether lipoic acid biosynthesis is impaired in CMAMMA, as in-vitro studies have produced conflicting results on this point. However, in fibroblasts from CMAMMA patients showed lower concentrations of octanoyl-carnitine, indicating impaired mtFAS with reduced octanoyl-ACP availability. This is also consistent with decreased lipoylation of α-KGDH in all cases and PDH in some, but not all cases. Experimental knockdown models suggest that lipoylation becomes impaired only when the remaining ACSF3 activity falls below a critical threshold. This could explain the variability observed both in in-vitro studies and in the clinical presentation of CMAMMA. The overall extent of impaired lipoylation in CMAMMA is likely underestimated, since only α-KGDH and PDH have been investigated so far.
Malonyl-CoA can also be converted to acetyl-CoA by malonyl-CoA decarboxylase, providing a minor additional pathway of acetyl-CoA synthesis for oxidation in the citric acid cycle. When ACSF3 is defective, mitochondrial malonyl-CoA is reduced, which may impair the supplementary pathway from malonic acid to acetyl-CoA via malonyl-CoA. The clinical similarities between CMAMMA and malonic aciduria support the view that ACSF3 and MCD act within the same pathway.
ACSF3-derived malonyl-CoA is required for lipid synthesis, as shown by the reduced incorporation of malonate into cellular lipids in ACSF3-knockout HEK293 cells.