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Methylmalonic Aciduria and Homocystinuria Sequencing Panel

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

Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
3287 ABCD4 81479 Add to Order
CD320 81479
HCFC1 81479
LMBRD1 81479
MMACHC 81404
MMADHC 81479
TCN2 81479
Full Panel Price* $640.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
3287 Genes x (7) $640.00 81404, 81479(x6) Add to Order
Pricing Comments

We are happy to accommodate requests for single genes or a subset of these genes. The price will remain the list price. If desired, free reflex testing to remaining genes on panel is available. Alternatively, a single gene or subset of genes can also be ordered on our PGxome Custom Panel.

Targeted Testing

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

Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

Although the overall sensitivity of this test panel is not precisely known, most pathogenic variants reported for the genes in this panel are of the type which can be detected using standard PCR and automated sequencing methods.

In one large study examining 118 patients with confirmed cblC disease, 227 pathogenic variants were identified on 236 alleles, for an overall sensitivity of ~96% (Lerner-Ellis et al. 2009). Regarding MMADHC mutation detection, two studies which examined a total of 10 patients with cblD defect found MMADHC pathogenic variants in either a compound heterozygous or homozygous state in all affected individuals (Coelho et al. 2008; Miousse et al. 2009). Similarly, in the largest published study of patients with cblF deficiency, all 12 affected patients were found to have LMBRD1 pathogenic variants in either a compound heterozygous or homozygous state (Rutsch et al. 2009). Regarding HCFC1 mutation detection, Yu and colleagues (2013) reported pathogenic variants in 13 out of 17 male patients who were suspected of or cellularly diagnosed with cblC type methylmalonic aciduria and homocystinuria but did not harbor pathogenic variants in the MMACHC gene. Finally, based on collective totals of transcobalamin II deficient patients reported in the literature, the clinical sensitivity of TCN2 sequencing is estimated to be ~73% (29 pathogenic TCN2 alleles out of 40 total alleles, with only one patient counted if multiple family members were affected) (Li et al. 1994a; Li et al. 1994b; Namour et al. 2003; Prasad et al. 2008; Häberle et al. 2009; Ratschmann et al. 2009; Nissen et al. 2010; Schiff et al. 2010; Ünal et al. 2015; Pupavac et al. 2016).

Clinical sensitivity of the cblJ disorder and CD320 deficiency cannot be estimated because only a small number of patients with ABCD4 and CD320 pathogenic variants have been reported.

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CNV via aCGH

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
600 LMBRD1$990.00 81479 Add to Order
MMACHC$990.00 81479
MMADHC$990.00 81479
Full Panel Price* $1190.00
Pricing Comments

# of Genes Ordered

Total Price

1

$990

2-5

$1190

6-10

$1290

11-100

$1490

Over 100

Call for quote
Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

To date, no gross deletions or duplications have been reported in the MMADHC gene, and only a single gross deletion has been reported in each of the LMBRD1 and MMACHC genes (Miousse et al. 2011; Weisfeld-Adams and Baker 2015). Therefore, the sensitivity of duplication/deletion testing for these rare disorders although unknown, appears to be low.

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Clinical Features

Cobalamin (Cbl or vitamin B12) is an important cofactor in homocysteine metabolism and in branched-chain amino acid and odd-chain fatty acid catabolism. Cbl is necessary for the production of two vital cofactors: Adenosyl-cobalamin (AdoCbl), which is required in the mitochondria for methylmalonyl CoA mutase to convert L-methylmalonyl-CoA to succinyl CoA, and Methyl-cobalamin (MeCbl), which is required in the cytoplasm for methionine synthase to convert homocysteine to methionine. A series of inherited disorders of cobalamin (Cbl) metabolism and transport, designated cblA through cblJ and cblX as well as transcobalamin II (TC II) deficiency, can lead to elevated levels of methylmalonic acid in the blood and urine and/or hyperhomocysteinemia/homocystinuria. While cblC, cblD (classic), cblF, cblJ, cblX and TC II deficiencies all present with combined methylmalonic aciduria and homocystinuria, the remaining disorders present with either isolated methylmalonic aciduria or homocystinuria (Gailus et al. 2010; Carrillo-Carrasco et al. 2013; Yu et al. 2013; Watkins and Rosenblatt 2014). CblC, cblD, cblF and cblJ show clinical variability, but all may be associated with developmental delay, hematological abnormalities, and severe neurological problems such as seizures (Watkins and Rosenblatt 2014). Patients diagnosed with cblX tend to be more severely affected and may also present with microcephaly, brain abnormalities, facial dysmorphism and other congenital abnormalities, though none have been reported to have hematological abnormalities (Yu et al. 2013). TC II deficient patients typically present in early infancy with gastrointestinal symptoms and potentially severe hematological deficiencies, and may eventually develop neurological complications if treatment is delayed (Häberle et al. 2009; Ünal et al. 2015). Most affected individuals are diagnosed as infants and children, but a few patients with cblC disorder are not diagnosed until the teenage years or as young adults (Watkins and Rosenblatt 2014). Some patients with increased propionylcarnitine concentrations are identified through newborn screening (Rutsch et al. 2009; Coelho et al. 2012). For further clinical information, please see individual gene test descriptions for each of these deficiencies (MMACHC for cblC disease, MMADHC for cblD disease, LMBRD1 for cblF disease, ABCD4 for cblJ disease, HCFC1 for cblX disease and TCN2 for TC II deficiency).

In addition to the disorders detailed above, this sequencing panel also covers the CD320 gene, which has been associated with transient methylmalonic aciduria and/or homocystinuria in a small number of individuals. CD320 deficiency has not been associated with the neurological or hematological symptoms associated with the cblC, cblD, cblF, cblJ or cblX types of methylmalonic aciduria and homocystinuria. However, individuals carrying pathogenic variants in CD320 may be detected via newborn screening (Quadros et al. 2010; Karth et al. 2012; Watkins and Rosenblatt 2014).

Genetics

Combined methylmalonic aciduria and homocystinuria can result from pathogenic variants in at least six different genes associated with inborn errors of cobalamin transport or metabolism. Six of the cobalamin disorders covered by this sequencing panel (cblC, cblD, cblF, cblJ, TC II deficiency, and transient methylmalonic aciduria and/or homocystinuria due to CD320 deficiency) are inherited in an autosomal recessive manner, while the cblX disorder is inherited in an X-linked recessive manner.

Massively parallel sequencing plus Sanger confirmation will detect the vast majority of sequence variants in the MMACHC, MMADHC, LMBRD1, ABCD4, HCFC1, TCN2 and CD320 genes. It should be noted that a large deletion that may not be detectable via sequencing has been reported in the MMACHC gene (Weisfeld-Adams and Baker 2015), and ~27% of causative variants in the TCN2 gene have been reported to be exonic or larger deletions (Schiff et al. 2010; Human Gene Mutation Database).

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

Testing Strategy

For this NGS test, the full coding regions, plus ~10 bp of non-coding DNA flanking each exon, are sequenced for each of the genes listed. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, 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, undocumented and questionable variant calls are confirmed by Sanger sequencing.

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 symptoms suggestive of combined methylmalonic aciduria and homocystinuria are good candidates for this test.

Genes

Official Gene Symbol OMIM ID
ABCD4 603214
CD320 606475
HCFC1 300019
LMBRD1 612625
MMACHC 609831
MMADHC 611935
TCN2 613441
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
HCFC1-Related Disorders via the HCFC1 Gene
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection
Methylmalonic Aciduria and Homocystinuria, cblF type, via the LMBRD1 Gene
Methylmalonic Aciduria and Homocystinuria, cblJ Type, via the ABCD4 Gene
Methylmalonic Aciduria and Homocystinuria, cblD Type, via MMADHC Gene Sequencing with CNV Detection
Methylmalonic Aciduria and/or Homocystinuria via CD320 Gene Sequencing with CNV Detection
Transcobalamin II Deficiency via the TCN2 Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Carrillo-Carrasco N., Adams D., Venditti C.P. 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
  • Coelho D. et al. 2008. The New England Journal of Medicine. 358: 1454-64.  PubMed ID: 18385497
  • Coelho D. et al. 2012. Nature Genetics. 44: 1152-5. PubMed ID: 22922874
  • Gailus S. et al. 2010. Journal of Inherited Metabolic Disease. 33: 17-24. PubMed ID: 20127417
  • Häberle J. et al. 2009. Journal of Human Genetics. 54: 331-4. PubMed ID: 19373259
  • Human Gene Mutation Database (Bio-base).
  • Karth P. et al. 2012. Journal of AAPOS. 16: 398-400.  PubMed ID: 22819238
  • Lerner-Ellis J.P. et al. 2009. Human Mutation. 30: 1072-81. PubMed ID: 19370762
  • Li N. et al. 1994a. Human Molecular Genetics. 3: 1835-40. PubMed ID: 7849710
  • Li N. et al. 1994b. Biochemical and Biophysical Research Communications. 204: 1111-8. PubMed ID: 7980584
  • Miousse I.R. et al. 2009. The Journal of Pediatrics. 154: 551-6.  PubMed ID: 19058814
  • Miousse I.R. et al. 2011. Molecular Genetics and Metabolism. 102: 505-7. PubMed ID: 21303734
  • Namour F. et al. 2003. British Journal of Haematology. 123: 915-20. PubMed ID: 14632784
  • Nissen P.H. et al. 2010. Journal of Inherited Metabolic Disease. 33 Suppl 3: S269-74. PubMed ID: 20607612
  • Prasad C. et al. 2008. Journal of Inherited Metabolic Disease. 31 Suppl 2: S287-92. PubMed ID: 18956254
  • 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
  • Ratschmann R. et al. 2009. Molecular Genetics and Metabolism. 98: 285-8. PubMed ID: 19581117
  • Rutsch F. et al. 2009. Nature Genetics. 41: 234-9. PubMed ID: 19136951
  • Schiff M. et al. 2010. Journal of Inherited Metabolic Disease. 33: 223-9. PubMed ID: 20352340
  • Ünal S. et al. 2015. Turkish Journal of Haematology. 32: 317-22. PubMed ID: 25914105
  • 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.
  • Weisfeld-Adams J.D., Baker P.R. 2015. Journal of Inherited Metabolic Disease. 38: 365-6. PubMed ID: 25388550
  • Yu H.C. et al. 2013. American Journal of Human Genetics. 93: 506-14. PubMed ID: 24011988
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 ~10 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 often covered 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 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 ~10 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 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.

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.

In the case of duplications, aCGH will not determine the chromosomal location of the duplicated DNA. Most duplications will be tandem, but in some cases the duplicated DNA will be inserted at a different locus. This method will also not determine the orientation of the duplicated segment (direct or inverted).

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

The sensitivity of this assay is dependent upon the quality of the input DNA. In particular, highly degraded DNA will yield poor results.

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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