Combined Malonic and Methylmalonic Aciduria (CMAMMA) via the ACSF3 Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
314 ACSF3$810.00 81479 Add to Order
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

Clinical sensitivity is difficult to estimate because only a small number of patients have been reported. Analytical sensitivity should be high because nearly all variants reported are detectable by direct sequencing.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 ACSF3$690.00 81479 Add to Order
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Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

It is difficult to estimate the clinical sensitivity of this test. To date, a single gross deletion encompassing the ACSF3 gene has been reported in the literature (Pupavac et al. 2016).

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Clinical Features

Combined malonic and methylmalonic aciduria (CMAMMA) is an inborn error of metabolism that is characterized by elevated levels of malonic and methylmalonic acid in the urine. To date, the majority of cases have been found to be caused by variants in the MLYCD gene, which encodes the enzyme malonyl-CoA decarboxylase. However, recent research has shown that CMAMMA is a genetically heterogeneous disorder, and that pathogenic variants in the ACSF3 gene can lead to a non-classical form of CMAMMA (Alfares et al. 2011; Sloan et al. 2011).

Patients with the non-classical form have been found to excrete both malonic and methylmalonic acid in the urine, but the level of methylmalonic acid is significantly higher than that of malonic acid. In contrast, in classical CMAMMA the level of malonic acid is higher. As fewer than 20 patients have been described to date, the clinical spectrum of non-classical CMAMMA is not fully understood. Patients have been reported to be clinically asymptomatic (Alfares et al. 2011), or to have clinical symptoms ranging from childhood to adult onset (Sloan et al. 2011; Pupavac et al. 2016). The patients with childhood onset of disease have exhibited symptoms suggestive of a metabolic disorder, such as ketoacidosis, hypoglycemia, coma, failure to thrive, increased transaminases, microcephaly, dystonia, hypotonia and developmental delay. In contrast, the patients with adult onset disease presented with solely neurologic symptoms, such as seizures, memory problems, psychiatric disease and/or cognitive decline (Sloan et al. 2011). A late-onset patient was recently reported to have progressive generalized weakness without psychiatric disease and/or cognitive decline (Pupavac et al. 2016).

Patients with non-classical CMAMMA may be detected by newborn screening programs due to elevated levels of malonic and methylmalonic acid.


Current knowledge about non-classical CMAMMA suggests that this is an autosomal recessive disorder, and ACSF3 is the only gene known to be involved (Alfares et al. 2011; Sloan et al. 2011). To date, the majority of reported pathogenic variants have been missense variants, although one small deletion, two nonsense variants and one splice variant have been reported. In addition, one gross deletion has been detected, though it should be noted that this deletion included several other genes as well (Pupavac et al. 2016). The R558W missense variant has been reported in multiple unrelated individuals, and recently a patient was identified with an R558Q variant. This suggests that variants at this site may be a common cause of disease (Alfares et al. 2011; Sloan et al. 2011; Pupavac et al. 2016).

The ACSF3 gene encodes an acyl-CoA synthetase protein of uncertain function. Initial functional studies have suggested that it may act as a malonyl-CoA and methylmalonyl-CoA synthetase. The ACSF3 protein has been shown to be localized to the mitochondria (Sloan et al. 2011).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the ACSF3 gene plus ~10 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Individuals with elevated urinary methylmalonic (MMA) and malonic acid (MA), particularly if the level of MMA is higher than the level of MA, are good candidates for this test. Family members of patients who have known ACSF3 pathogenic variants are candidates. We will also sequence the ACSF3 gene to determine carrier status.


Official Gene Symbol OMIM ID
ACSF3 614245
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT


Name Inheritance OMIM ID
Combined Malonic And Methylmalonic Aciduria AR 614265

Related Test

Metabolic Hypoglycemia Sequencing Panel


Genetic Counselors
  • Alfares A. et al. 2011. Journal of Medical Genetics. 48:602-5. PubMed ID: 21785126
  • Pupavac M. et al. 2016. Molecular Genetics and Metabolism. 117: 363-8. PubMed ID: 26827111
  • Sloan J.L. et al. 2011. Nature Genetics. 43:883-6. PubMed ID: 21841779
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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