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Disorders Related to Metabolism of Cobalamin, Folate and Homocysteine Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
4505 ABCD4 81479 Add to Order
ACSF3 81479
AHCY 81479
ALDH6A1 81479
AMN 81479
CBS 81406
CD320 81479
CUBN 81479
FOLR1 81479
GIF 81479
GNMT 81479
HCFC1 81479
LMBRD1 81479
MAT1A 81479
MCEE 81479
MLYCD 81479
MMAA 81405
MMAB 81405
MMACHC 81404
MMADHC 81479
MTHFR 81479
MTR 81479
MTRR 81479
MUT 81406
PCCA 81406
PCCB 81406
SLC46A1 81479
SUCLA2 81479
SUCLG1 81479
TCN1 81479
TCN2 81479
Full Panel Price* $1590.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
4505 Genes x (31) $1590.00 81404, 81405(x2), 81406(x4), 81479(x24) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

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 clinical sensitivity of this specific grouping of genes is difficult to estimate as we are unaware of any reports in the literature in which all of these genes have been sequenced together in a patient cohort. One study of 131 patients with elevated methylmalonic acid in the blood or urine but no molecular diagnosis based on initial testing has been published. A 24 gene sequencing panel, of which 22 genes overlap with our test, was run for each of these patients to attempt to obtain a diagnosis. Eight patients were reported with pathogenic variants consistent with their diagnosis (Pupavac et al. 2016).

The clinical sensitivity of sequencing the individual genes in this test is high in patient groups with biochemical and/or enzymatic diagnoses of the relevant disorders; details are available on the individual test description pages. Analytical sensitivity is expected to be high as nearly all variants reported in these genes are detectable via 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
CBS$690.00 81479
FOLR1$690.00 81479
LMBRD1$690.00 81479
MMAA$690.00 81479
MMAB$690.00 81479
MMACHC$690.00 81479
MMADHC$690.00 81479
MTHFR$690.00 81479
MTR$690.00 81479
MTRR$690.00 81479
MUT$690.00 81479
PCCA$690.00 81405
PCCB$690.00 81479
SUCLA2$690.00 81479
SUCLG1$690.00 81479
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (16) $1290.00 81405, 81479(x15) 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 large (exonic level, usually multi-exon) deletions or duplications have been documented in the following genes: FOLR1, MMAB, MMADHC, MTHFR, MTR and SUCLG1 (Human Gene Mutation Database).

Large deletions or duplications have been documented, but appear to be relatively uncommon, in the following genes: ACSF3, CBS, LMBRD1, MMAA, MMACHC, MTRR, MUT, PCCB and SUCLA2 (HGMD).

Deletions and duplications are reported to account for ~20% of causative alleles in the PCCA gene (Yang et al. 2004; Kaya et al. 2008; Desviat et al. 2009).

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Clinical Features

The disorders caused by defects in the genes in this sequencing panel are all inborn errors of metabolism related to the transport and metabolism of cobalamin, propionic acid, methylmalonyl-CoA, folate and methionine. Clinical features vary with the different disorders, but can include symptoms such as feeding difficulties, vomiting, failure to thrive, lethargy, hypotonia and myopathy along with increased creatine kinase (CK) levels. Liver abnormalities, such as hepatomegaly and elevation of serum transaminases, are also common. Neurologic abnormalities often occur, and may include intellectual impairment, cerebral atrophy and developmental delay, seizures, microcephaly, increased tendon reflexes, ataxia, dystonia, and nystagmus. Hematological abnormalities are also common, and may include megaloblastic anemia, neutropenia and/or coagulopathy. Visual defects such as ectopia lentis, strabismus, severe myopia or blindness may occur. Less common features may include skeletal abnormalities, vascular disease, behavioral issues, and dysmorphic features. Biochemically, patients may have hypoglycemia, ketosis, metabolic acidosis, hyperammonemia, and abnormally low or high levels of serum and/or urine homocysteine, methylmalonic acid, methionine, and/or propionic acid; the specific biochemical profile will vary with each of the different disorders. Onset for the majority of patients is early in life, though later-onset cases do occur.

For details on specific disorders, please visit the individual gene test description page.

Genetics

This sequencing panel currently includes genes that have been associated with inborn errors of cobalamin, propionic acid, methylmalonyl-CoA, folate and methionine metabolism. The majority of the disorders associated with these genes are inherited in an autosomal recessive manner, with the exception of cblX type methylmalonic aciduria and homocystinuria which is an X-linked disorder caused by pathogenic variants in the HCFC1 gene, and methionine adenosyltransferase I/III deficiency, which primarily autosomal recessive but is sometimes associated with autosomal dominant inheritance.

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

Testing Strategy

For this Next Generation Sequencing (NGS) test, 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 reported pathogenic, likely pathogenic, and 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.

In addition to the regions described above, this testing includes coverage of the following variants that reside in untranslated or deep intronic regions: AMN c.514-34 G>A, CUBN c.3330-439 C>G, HCFC1 c.-970T>C, TCN2 c.580+624 A>T, and MCEE c.379-644A>G.

This test does not include sequencing of exons 41 through 50 or 61 through 67 of the CUBN gene because of high sequence similarity to other chromosomal regions. To date, only one pathogenic variant has been reported within these exons (Human Gene Mutation Database).

Other than exons 41 through 50 and 61 through 67 of the CUBN, this panel provides full coverage of all coding exons of the genes listed, plus ~20 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads for coding regions 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 features consistent with inborn errors of cobalamin, propionic acid, methylmalonyl-CoA, folate and methionine metabolism are good candidates for this test.

Diseases

Name Inheritance OMIM ID
Cerebral Folate Deficiency AR 613068
Combined Malonic And Methylmalonic Aciduria AR 614265
Folate Malabsorption, Hereditary AR 229050
Glycine N-Methyltransferase Deficiency AR 606664
Homocystinuria Due To Cbs Deficiency AR 236200
Homocystinuria due to MTHFR Deficiency AR 236250
Homocystinuria-Megaloblastic Anemia Due To Defect In Cobalamin Metabolism, cblE Complementation Type AR 236270
Homocystinuria-Megaloblastic Anemia Due To Defect In Cobalamin Metabolism, cblG Complementation Type AR 250940
Hypermethioninemia With S-Adenosylhomocysteine Hydrolase Deficiency AR 613752
Intrinsic Factor Deficiency AR 261000
Malonyl-CoA Decarboxylase Deficiency AR 248360
Megaloblastic Anemia Due To Inborn Errors Of Metabolism AR 261100
Mental Retardation, X-Linked 3 (Methylmalonic Acidemia and Homocysteinemia, cblX Type) XL 309541
Methionine Adenosyltransferase I/III Deficiency AD,AR 250850
Methylmalonate Semialdehyde Dehydrogenase Deficiency AR 614105
Methylmalonic Aciduria and Homocystinuria, cblC Type AR 277400
Methylmalonic Aciduria and Homocystinuria, cblD Type AR 277410
Methylmalonic Aciduria and Homocystinuria, cblF Type AR 277380
Methylmalonic Aciduria and Homocystinuria, cblJ Type AR 614857
Methylmalonic Aciduria Cbla Type AR 251100
Methylmalonic Aciduria Cblb Type AR 251110
Methylmalonic Aciduria Due To Methylmalonyl-CoA Mutase Deficiency AR 251000
Methylmalonic Aciduria Due To Transcobalamin Receptor Defect AR 613646
Methylmalonyl-CoA Epimerase Deficiency AR 251120
Mitochondrial DNA Depletion Syndrome 5 (Encephalomyopathic with or without Methylmalonic Aciduria) AR 612073
Mitochondrial DNA Depletion Syndrome 9 (Encephalomyopathic With Methylmalonic Aciduria) AR 245400
Propionic Acidemia AR 606054
Transcobalamin II Deficiency AR 275350

Related Tests

Name
HCFC1-Related Disorders via the HCFC1 Gene
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 Sequencing Panel with CNV Detection
Cerebral Folate Deficiency via the FOLR1 Gene
Combined Malonic and Methylmalonic Aciduria (CMAMMA) via the ACSF3 Gene
Comprehensive Cardiology Sequencing Panel with CNV Detection
Disorders of Fatty Acid Oxidation Sequencing Panel
Disorders of Folate Metabolism and Transport Sequencing Panel
Early Infantile Epileptic Encephalopathy Sequencing Panel
Early Infantile Epileptic Encephalopathy, Recessive Sequencing Panel
Glycine N-Methyltransferase Deficiency via the GNMT Gene
Hereditary Folate Malabsorption via the SLC46A1 Gene
Homocystinuria Sequencing Panel
Homocystinuria via the CBS Gene
Homocystinuria, cblE Type, via the MTRR Gene
Homocystinuria, cblG Type, via the MTR Gene
Hyperammonemia Sequencing Panel
Hypermethioninemia Sequencing Panel
Malonyl-CoA Decarboxylase Deficiency via the MLYCD Gene
Marfan Syndrome and Related Aortopathies Sequencing Panel
Metabolic Hypoglycemia Sequencing Panel
Metabolic Myopathies, Rhabdomyolysis and Exercise Intolerance Sequencing Panel
Methionine Adenosyltransferase I/III deficiency via the MAT1A Gene
Methlymalonyl-CoA Epimerase Deficiency via the MCEE Gene
Methylmalonate Semialdehyde Dehydrogenase Deficiency via the ALDH6A1 Gene
Methylmalonic Acidemia Sequencing Panel
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, cblC type, via the MMACHC Gene
Methylmalonic Aciduria and Homocystinuria, cblD type, via the MMADHC Gene
Methylmalonic Aciduria and Homocystinuria, cblF type, via the LMBRD1 Gene
Methylmalonic Aciduria and Homocystinuria, cblJ Type, via the ABCD4 Gene
Methylmalonic Aciduria and/or Homocystinuria via the CD320 Gene
Mitochondrial Genome Maintenance/Integrity Nuclear Genes Sequencing Panel
Nephrotic Syndrome (NS)/Focal Segmental Glomerulosclerosis (FSGS) Sequencing Panel
Organic Aciduria Sequencing Panel
Propionic Acidemia Sequencing Panel
Propionic Acidemia via the PCCA Gene
Propionic Acidemia via the PCCB Gene
S-Adenosylhomocysteine Hydrolase Deficiency via the AHCY Gene
Severe MTHFR Deficiency via the MTHFR Gene
Transcobalamin II Deficiency via the TCN2 Gene
X-Linked Intellectual Disability Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Desviat L.R. et al. 2009. Molecular Genetics and Metabolism. 96: 171-6. PubMed ID: 19157943
  • Human Gene Mutation Database (Bio-base).
  • Kaya N. et al. 2008. European Journal of Medical Genetics. 51: 558-65. PubMed ID: 18790721
  • Pupavac M. et al. 2016. Molecular Genetics and Metabolism. 117: 363-8. PubMed ID: 26827111
  • Yang X. et al. 2004. Molecular Genetics and Metabolism. 81: 335-42. PubMed ID: 15059621
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select 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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