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Methylmalonic Acidemia Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
Order Kits
TEST METHODS

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
3401 ACSF3 81479 Add to Order
ALDH6A1 81479
CD320 81479
MCEE 81479
MLYCD 81479
MMAA 81405
MMAB 81405
MMADHC 81479
MUT 81406
SUCLA2 81479
SUCLG1 81479
Full Panel Price* $1440.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
3401 Genes x (11) $1440.00 81405(x2), 81406, 81479(x8) Add to Order
Pricing Comment

Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information. If you would like to order a subset of these genes contact us to discuss pricing.

For Sanger Sequencing click here.
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 28 days.

Clinical Sensitivity

The overall clinical sensitivity of this sequencing panel is expected to be high. Based on enzymatic studies of patients with isolated methylmalonic acidemia, Hörster et al. (2007) reported that ~63% of patients can be classified as mut0 or mut -, ~24% as cblA type, and ~13% as cblB type. Two molecular studies of patients diagnosed biochemically, enzymatically and via complementation analysis with isolated methylmalonic acidemia have been fairly consistent with these results. First, Martinez et al. (2005) evaluated 25 MMA patients and found MUT pathogenic variants in 13 (52%), MMAA pathogenic variants in 7 (28%), and MMAB pathogenic variants in 2 (8%). Merinero et al. (2008) studied 32 patients with isolated MMA and found MUT causative variants in 19 (59%), MMAA causative variants in 9 (28%), and MMAB causative variants in 4 (13%).

Pathogenic variants in the MMADHC, MCEE, ACSF3, ALDH6A1, CD320, SUCLA2, SUCLG1 and MLYCD genes are expected to make up some fraction of the remaining patients. In one recent study of 131 methylmalonic acidemia/aciduria patients without a diagnosis based on testing of the more commonly involved genes, 8 patients were found to have pathogenic variants in less commonly mutated genes (2 patients with SUCLG1 variants and 5 patients with ACSF3 variants) (Pupavac et al. 2016). Of note, the ALDH6A1 and MLYCD genes were not included in this study.

Nearly all reported pathogenic variants in the eleven genes in this panel (MUT, MMAA, MMAB, MMADHC, MCEE, ACSF3, ALDH6A1, CD320, MLYCD, SUCLA2 and SUCLG1) are detectable via direct sequencing, so the analytical sensitivity of this sequencing panel is expected to be high.

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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
MMAA$690.00 81479
MMAB$690.00 81479
MMADHC$690.00 81479
MUT$690.00 81479
SUCLA2$690.00 81479
SUCLG1$690.00 81479
Full Panel Price* $840.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (7) $840.00 81479(x7) Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote
Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

To date, no gross deletions or insertions have been reported in the MMAB, MMADHC or SUCLG1 genes, and only 1-2 gross deletions have been reported in the MMAA and ACSF3 genes (Nizon et al. 2013; Pupavac et al. 2016). Deletions and duplications appear to be somewhat more common in the MUT and SUCLA2 genes (Acquaviva et al. 2005; Chu et al. 2016; Forny et al. 2016), though it is difficult to precisely estimate the fraction of patients carrying such large copy number variants. In summary, the sensitivity of duplication/deletion testing for these rare disorders, although not precisely known, is low.

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Clinical Features

Methylmalonic acidemia (MMA) is typically a severe disease with onset in infancy. Patients may present with lethargy, vomiting, hepatomegaly, acidosis, hypoglycemia and neutropenia. Many patients die in childhood; those that survive often experience neurological and renal complications. Milder forms of the disease are also known. Today, many cases are detected through routine neonatal screening with tandem mass spectrometry (Fenton et al. 2014; Watkins and Rosenblatt 2014; Manoli et al. 2016).

The MMADHC, MMAA, MMAB, MCEE, MUT and CD320 genes encode proteins directly related to cobalamin transport or metabolism, or methylmalonyl-CoA metabolism. Defects in these genes have been shown to lead to the development of isolated MMA. Defects in each gene are associated with a specific type of MMA disorder (cblD, cblA, cblB, MCEE, MUT or CD320 deficiency, respectively). The majority of MMA patients have defects in the MUT gene, followed by the MMAA and then MMAB genes (Manoli et al. 2016). Defects in the MMADHC, MCEE and CD320 genes are rare, having only been reported in a small number of families (Bikker et al. 2006; Dobson et al 2006; Carrillo et al. 2013; Manoli et al. 2016). It should be noted that pathogenic variants in the CD320 gene lead to transient methylmalonic aciduria and/or homocystinuria, which has not been associated with the neurological or hematological symptoms of the cblD, cblA, cblB, mcee or mut deficiency disorders. However, CD320 deficient individuals may be detected via newborn screening (Quadros et al. 2010; Karth et al. 2012; Watkins and Rosenblatt 2014).

This sequencing panel also includes coverage for several genes that do not encode proteins directly involved in cobalamin or methylmalonyl-CoA metabolism. Defects in the ALDH6A1 gene lead to methylmalonate semialdehyde dehydrogenase deficiency (MMSDH), which is an inborn error of valine and thymine metabolism (Chambliss et al. 2000; Marcadier et al. 2013). Defects in the ACSF3 and MLYCD genes lead to combined malonic and methylmalonic aciduria (CMAMMA) (FitzPatrick et al. 1999; Alfares et al. 2011; Sloan et al. 2011; Baertling et al. 2014). Defects in the SUCLA2 and SUCLG1 genes lead to mitochondrial DNA depletion syndromes (MDSs) (Elpeleg et al. 2005; Carrozzo et al. 2007; Ostergaard et al. 2007; Carrozzo et al. 2016). These five genes are included in this panel as defects in all of them have been shown to result in methylmalonic acidemia in at least some patients. Phenotypically, these disorders may be similar or quite different from the cblD, cblA, cblB, mcee or mut types of methylmalonic acidemia.

More details regarding each disorder are available on individual gene test pages.

Genetics

Isolated methylmalonic acidemia (MMA) can result from pathogenic variants in at least six different genes directly associated with inborn errors of cobalamin metabolism or transport, or methylmalonyl-CoA metabolism (MMADHC, MMAA, MMAB, MCEE, MUT and CD320). In addition, defects in an additional five genes (ACSF3, ALDH6A1, MLYCD, SUCLA2 and SUCLG1) may also lead to methylmalonic acidemia, although these genes are not directly related to cobalamin metabolism or transport or methylmalonyl-CoA metabolism. All of these disorders are inherited in an autosomal recessive manner.

Massively parallel sequencing plus Sanger confirmation will detect the vast majority of sequence variants in the MMADHC, MMAA, MMAB, MCEE, MUT, CD320, ACSF3, ALDH6A1, MLYCD, SUCLA2 and SUCLG1 genes. It should be noted that large deletions that may not be detectable via sequencing have been reported in the MMAA, MUT, ACSF3 and SUCLA2 genes (Human Gene Mutation Database).

See individual gene test descriptions for information on molecular biology of gene products.

Testing Strategy

For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the genes listed. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

In addition to the regions described above, this testing includes coverage of the following deep intronic variant: MCEE c.379-644A>G.

This panel provides 100% coverage of the aforementioned regions of the indicated genes. We define coverage as > 20X NGS reads for exons and 0-10 bases of flanking DNA, > 10X NGS reads for 11-20 bases of flanking DNA, or Sanger sequencing.

Indications for Test

Patients with biochemical and/or clinical symptoms suggestive of isolated methylmalonic acidemia (MMA) are good candidates for this test.

Genes

Official Gene Symbol OMIM ID
ACSF3 614245
ALDH6A1 603178
CD320 606475
MCEE 608419
MLYCD 606761
MMAA 607481
MMAB 607568
MMADHC 611935
MUT 609058
SUCLA2 603921
SUCLG1 611224
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
SUCLA2 -Related Encephalomyopathic Form of Mitochondrial DNA Depletion Syndrome via the SUCLA2 Gene
SUCLG1-Related Encephalomyopathic Form of Mitochondrial DNA Depletion Syndrome via the SUCLG1 Gene
SUCLG2-Related Mitochondrial DNA Depletion Syndrome via the SUCLG2 Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Combined Malonic and Methylmalonic Aciduria (CMAMMA) via the ACSF3 Gene
Disorders of Fatty Acid Oxidation Sequencing Panel
Disorders Related to Metabolism of Cobalamin, Folate and Homocysteine Sequencing Panel
Homocystinuria Sequencing Panel
Hyperammonemia Sequencing Panel
Malonyl-CoA Decarboxylase Deficiency via the MLYCD Gene
Metabolic Hypoglycemia Sequencing Panel
Metabolic Myopathies, Rhabdomyolysis and Exercise Intolerance Sequencing Panel
Methlymalonyl-CoA Epimerase Deficiency via the MCEE Gene
Methylmalonate Semialdehyde Dehydrogenase Deficiency via the ALDH6A1 Gene
Methylmalonic Acidemia via the MUT Gene
Methylmalonic Acidemia, cblA type, via the MMAA Gene
Methylmalonic Acidemia, cblB type, via the MMAB Gene
Methylmalonic Aciduria and Homocystinuria Sequencing Panel
Methylmalonic Aciduria and Homocystinuria, cblD type, via the MMADHC Gene
Methylmalonic Aciduria and/or Homocystinuria via the CD320 Gene
Mitochondrial Genome Maintenance/Integrity Nuclear Genes Sequencing Panel
Organic Aciduria Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Acquaviva C. et al. 2005. Human Mutation. 25: 167-76. PubMed ID: 15643616
  • Alfares A. et al. 2011. Journal of Medical Genetics. 48:602-5. PubMed ID: 21785126
  • Baertling F. et al. 2014. European Journal of Pediatrics. 173: 1719-22. PubMed ID: 25233985
  • Bikker H. et al. 2006. Human Mutation. 27: 640-3. PubMed ID: 16752391
  • Carrillo N. et al. 2013. Disorders of Intracellular Cobalamin Metabolism. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301503
  • Carrozzo R. et al. 2007. Brain 130: 862–74. PubMed ID: 17301081
  • Carrozzo R. et al. 2016. Journal of Inherited Metabolic Disease. 39:243-52 PubMed ID: 26475597
  • Chambliss K.L. et al. 2000. Journal of Inherited Metabolic Disease. 23: 497-504. PubMed ID: 10947204
  • Chu J. et al. 2016. Molecular Genetics and Metabolism. 118: 264-71. PubMed ID: 27233228
  • Dobson C.M. et al. 2006. Molecular Genetics and Metabolism. 88: 327-33. PubMed ID: 16697227
  • Elpeleg O. et al. 2005. American Journal of Human Genetics. 76: 1081-6. PubMed ID: 15877282
  • Fenton W.A. et al. 2014. Disorders of Propionate and Methylmalonate Metabolism. In: Valle D, Beaudet A.L., Vogelstein B, et al., editors. New York, NY: McGraw-Hill. OMMBID.
  • FitzPatrick D.R. et al. 1999. American Journal of Human Genetics. 65: 318-26. PubMed ID: 10417274
  • Forny P. et al. 2016. Human Mutation. 37: 745-54. PubMed ID: 27167370
  • Hörster F. et al. 2007. Pediatric Research. 62: 225-30. PubMed ID: 17597648
  • Human Gene Mutation Database (Bio-base).
  • Karth P. et al. 2012. Journal of AAPOS. 16: 398-400.  PubMed ID: 22819238
  • Manoli I. et al. 2016. Isolated Methylmalonic Acidemia. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 20301409
  • Marcadier J.L. et al. 2013. Orphanet Journal of Rare Diseases. 8: 98. PubMed ID: 23835272
  • Martínez M.A. et al. 2005. Molecular Genetics and Metabolism. 84: 317-25. PubMed ID: 15781192
  • Merinero B. et al. 2008. Journal of Inherited Metabolic Disease. 31: 55-66. PubMed ID: 17957493
  • Nizon M. et al. 2013. Orphanet Journal of Rare Diseases. 8: 148. PubMed ID: 24059531
  • Ostergaard E. et al. 2007. Brain. 130: 853-61. PubMed ID: 17287286
  • Pupavac M. et al. 2016. Molecular Genetics and Metabolism. 117: 363-8. PubMed ID: 26827111
  • Quadros E.V. et al. 2010. Human Mutation. 31: 924-9.  PubMed ID: 20524213
  • Sloan J.L. et al. 2011. Nature Genetics. 43:883-6. PubMed ID: 21841779
  • Watkins D. and Rosenblatt D.S. 2014. Inherited Disorders of Folate and Cobalamin Transport and Metabolism. In: Valle D, Beaudet A.L., Vogelstein B, et al., editors. New York, NY: McGraw-Hill. OMMBID.
Order Kits
TEST METHODS

NextGen Sequencing using PG-Designed Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon.  As required, genomic DNA is extracted from the patient specimen.  For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes.  Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA).  Regions with insufficient coverage by NGS are covered by Sanger sequencing.  All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions.  After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).

(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants

Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org).  Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.

Analytical Validity

As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.

In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.   

Analytical Limitations

Interpretation of the test results is limited by the information that is currently available.  Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles.  Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion.   In these cases, the report will contain no information about the second allele.  Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).

We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon.  Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.

In most 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 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.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood).   Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics.  However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.

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.

Order Kits

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.
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.
SPECIMEN TYPES
WHOLE BLOOD

(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.
DNA

(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.
CELL CULTURE

(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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