Forms

Pyruvate Carboxylase Deficiency via the PC Gene

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

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
4827 PC$990.00 81406 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.

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 clinical sensitivity of this test is near 100%. In a collective total of 20 patients from different families with a clinical diagnosis of pyruvate carboxylase (PC) deficiency, pathogenic variants were detected on 39 of 40 alleles (Wang et al. 2008; Monnot et al. 2009; Ostergaard et al. 2013). Analytical sensitivity should also be close to 100% because all reported pathogenic variants are detectable by sequencing.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PC$690.00 81479 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 deletions or duplications have been described in the PC gene.

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Clinical Features

Pyruvate Carboxylase (PC) Deficiency is an inborn error of glucose metabolism. Three different categories of PC Deficiency have been described, and they are differentiated by clinical and biochemical features (Robinson 2014; Wang and De Vivo 2015).

Type A PC Deficiency (also called the infantile or North American form) presents primarily with psychomotor retardation, intellectual disability, failure to thrive, hypotonia, pyramidal tract signs, seizures and mild metabolic acidosis. The majority of patients with this form die in infancy or early childhood, although some patients have lived beyond childhood. Such patients exhibit intellectual disability and continued seizures and require special care. A large proportion of Type A PC Deficient individuals belong to Algonquian-speaking Native American tribes.

Type B PC Deficiency (also called the severe neonatal or French form) presents with failure to thrive, hypotonia, developmental delay, recurrent seizures, hepatomegaly, pyramidal tract signs and abnormal movement. Biochemically, they are found to exhibit severe lactic acidemia, hyperammonemia, citrullinemia and hyperlysinemia. All patients in this category have presented during the neonatal period and the majority have died before three months of age. Type B PC Deficiency was first described in patients from France and the United Kingdom, although patients have been since described from a variety of ethnic backgrounds.

Patients with Type C PC Deficiency (also called the intermittent/benign form) may have frequent episodes of metabolic acidosis in infancy, but are generally well between episodes. Clinically, patients typically develop normally or exhibit only mild delays in neurological development. 

Diagnosis of PC Deficient patients is generally made based on clinical symptoms, analysis of plasma and urine amino acids and organic acids, analysis of lactate, pyruvate, glutamine, glutamic acid and proline concentrations in cerebrospinal fluid (CSF), and based on PC enzyme activity assays (Wang and De Vivo 2015). Treatment focuses primarily on management of symptoms and on prevention of acute symptoms via dietary management. PC Deficient patients are put in a high-carbohydrate, high-protein diet. Fasting and the use of a ketogenic diet are contraindicated in these patients (Wang and De Vivo 2015).

Genetics

Pyruvate Carboxylase Deficiency is typically inherited in an autosomal recessive fashion, although mosaicism of de novo somatic pathogenic variants has also been reported (Wang and De Vivo 2015). The PC gene is the only gene known to be involved in PC Deficiency. To date, over 30 pathogenic variants have been reported in the PC gene (Human Gene Mutation Database). The majority of reported variants are missense, although nonsense, splicing, and small insertions and deletions have also been reported. Variants are spread throughout the coding regions of the gene, with no reported mutational hotspots. In the Ojibwa and Cree tribes, the Ala610Thr missense variant has been reported to be a founder mutation. Carrier frequency of this variant in these populations is thought to be as high as 1 in 10 (Carbone et al. 1998; Robinson 2014).

A genotype-phenotype correlation has been proposed to explain the different clinical outcome of different patients. Patients with Type A or C PC deficiency have all been reported to have variants thought to leave some level of residual enzyme activity, whereas the more severely affected Type B patients are found to have variants that lead to a complete loss of pyruvate carboxylase protein or enzyme activity (Robinson 2014). 

The pyruvate carboxylase enzyme is a homotetramer with a molecule of biotin covalently bound to each subunit. PC is located within the mitochondrial matrix, and is the most active in the liver and kidneys, although it is also active to a lesser extent in other tissues (Robinson 2014). The primary function of the PC enzyme is the conversion of pyruvate to oxaloacetate, which is the first step in gluconeogenesis. Additionally, PC plays an important role in lipogenesis as well as in replenishing the Krebs cycle, which is important for cellular energy production and the generation of neurotransmitters (Wang and De Vivo 2015).

Testing Strategy

For this NextGen test, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for the gene listed below. 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.

Indications for Test

Patients with clinical and biochemical features suggestive of PC Deficiency are good candidates for this test, as are family members of patients with known PC variants. We will also sequence the PC gene to determine carrier status.

Gene

Official Gene Symbol OMIM ID
PC 608786
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Pyruvate Carboxylase Deficiency 266150

Related Test

Name
Glycogen Storage Disease and Disorders of Glucose Metabolism Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Carbone M.A. et al. 1998. American Journal of Human Genetics. 62: 1312-9.  PubMed ID: 9585612
  • Human Gene Mutation Database (Bio-base).
  • Monnot S. et al. 2009. Human Mutation. 30: 734-40.  PubMed ID: 19306334
  • Ostergaard E. et al. 2013. Jimd Reports. 9: 1-5.  PubMed ID: 23430542
  • Robinson B.H. 2014. Lactic Acidemia: Disorders of Pyruvate Carboxylase and Pyruvate Dehydrogenase. Online Metabolic & Molecular Bases of Inherited Disease, New York, NY: McGraw-Hill.
  • Wang D, De Vivo D. 2015. Pyruvate Carboxylase Deficiency. 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: 20301764
  • Wang D. et al. 2008. Molecular Genetics and Metabolism. 95: 31-8.  PubMed ID: 18676167
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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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