Hydroxyglutaric Aciduria Sequencing Panel
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test||CPT Code Copy CPT Codes|
|Full Panel Price*||$1390.00|
If you would like to order a subset of these genes contact us to discuss pricing.
For ordering targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 28 days.
Clinical sensitivity of DNA sequencing is generally high for the genes in this sequencing panel. For D2HGDH, clinical sensitivity is estimated at ~46% if diagnosis is based solely on elevated D2HG levels. However, this rose to nearly 100% in patients with confirmed D2HGDH enzyme deficiency (Kranendijk et al. 2010a).
In D2HGA patients with no identified pathogenic mutations in D2HGDH, ~88% were found to have either the p.Arg140Gln or p.Arg140Gly variant in IDH2 (Kranendijk et al. 2010b).
In patients with elevated L2HG, the clinical sensitivity is expected to range from approximately 83% to nearly 100% (Topçu et al. 2004; Vilarinho et al. 2005; Sass et al. 2008; Vilarinho et al. 2010).
Only a small number of patients with combined D2HGA/L2HGA have been reported in the literature. However, all reported patients to date have been homozygous or compound heterozygous for SLC25A1 presumably pathogenic variants, suggesting a clinical sensitivity of near 100% (Edvardson et al. 2013; Nota et al. 2013; Smith et al. 2016).
Together, pathogenic variants in the D2HGDH, IDH2, L2HGDH and SLC25A1 genes are expected to explain nearly 96% of the cases of hydroxyglutaric aciduria (Struys et al. 2016).
Deletion/Duplication Testing via aCGH
|Test Code||Test||Individual Gene Price||CPT Code Copy CPT Codes|
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The great majority of tests are completed within 28 days.
In a study of 50 patients with elevated D2HG in body fluids, 24 of the patients were found to be homozygous or compound heterozygous for presumed pathogenic D2HGDH sequence variants. Two of these patients carried unique multi-exonic deletions (Kranendijk et al. 2010a).
The hydroxyglutaric acidurias (HGAs) are a group of organic acidurias that result in the elevation of D-2-hydroxyglutaric acid (D2HG), L-2-hydroxyglutaric acid (L2HG), or both metabolites in the body fluids (urine, plasma and cerebrospinal fluid). It is possible to distinguish D2HG and L2HG biochemically, although additional labwork must be performed to separate these two enantiomers. Onset for the HGAs is mostly in childhood. Common clinical features include developmental delay, hypotonia, and seizures. Additional details regarding each specific HGA are below (Struys et al. 2016):
D-2-hydroxyglutaric aciduria type I (D2HGA-I): this HGA is slowly progressive in many patients, and is mostly non-fatal. Some asymptomatic individuals have been identified. D2HGA-I accounts for ~15% of HGA patients (Kranendijk et al. 2010a; Struys et al. 2016).
D-2-hydroxyglutaric aciduria type II (D2HGA-II): approximately half of these patients also present with cardiomyopathy. Life expectancy is lower than D2HGA-I, with about half of the reported patients having died before adolescence. D2HGA-II also accounts for ~15% of HGA patients (Kranendijk et al. 2010b; Struys et al. 2016).
L-2-hydroxyglutaric aciduria (L2HGA): these patients may also present with intellectual disability, cerebellar ataxia, and a characteristic MRI (see details on individual gene test description). L2HGA patients have been reported to have a tendency to develop central nervous system malignancies. L2HGA accounts for ~60% of HGA patients (Steenweg et al. 2010; Struys et al. 2016).
Combined D2HGA and L2HGA: this is the most severe of the HGAs, having been fatal in two-thirds of reported patients in early childhood. These patients present with severe neonatal epileptic encephalopathy and may require artificial ventilation. They also have highly abnormal MRI findings. Combined D2HGA/L2HGA accounts for <6% of HGA patients (Smith et al. 2016; Struys et al. 2016).
Pathogenic variants in other genes may also lead to increased levels of D2HG or L2HG in body fluids. These genes include IDH1 and possibly ALDH5A1 and ETFA, ETFB and ETFDH (Struys et al. 2006; Struys et al. 2016).
D2HGA-I, L2HGA and combined D2HGA/L2HGA are autosomal recessive disorders caused by pathogenic variants in the D2HGDH, L2HGDH and SLC25A1 genes, respectively. D2HGA-II is an autosomal dominant disorder caused by specific missense variants in the IDH2 gene (p.Arg140Gln and p.Arg140Gly). These variants have been reported to reside in the active site of the enzyme, resulting in a gain of function (Kranendijk et al. 2010b).
Massively parallel sequencing plus Sanger confirmation will detect the vast majority of sequence variants in the D2HGDH, IDH2, L2HGDH and SLC25A1 genes. It should be noted that gross deletions, which may not be detected by direct sequencing, have been reported in the D2HGDH and L2HGDH genes.
See individual gene test descriptions for information on molecular biology of gene products.
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 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: D2HGDH c.293-23A>G and L2HGDH c.906+354G>A.
For the IDH2 gene, this test only includes sequencing of the region of exon 4 containing the c.418 and c.419 nucleotides, plus 20 bp of flanking DNA on each side. At this time, IDH2 sequence variants not located at nucleotides c.418 or c.419 will not be reported (see the IDH2 test description for details).
Other than the IDH2 gene, 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 clinical and biochemical features consistent with hydroxyglutaric aciduria are good candidates for this test.
|Official Gene Symbol||OMIM ID|
|Combined D-2- and L-2-HydroxyGlutaric Aciduria||AR||615182|
|D-2-Alpha Hydroxyglutaric Aciduria||AR||600721|
|D-2-Hydroxyglutaric Aciduria 2||AD||613657|
- Genetic Counselor Team - firstname.lastname@example.org
- McKenna Kyriss, PhD - email@example.com
- Edvardson S. et al. 2013. Journal of Medical Genetics. 50: 240-5. PubMed ID: 23393310
- Kranendijk M. et al. 2010a. Human Mutation. 31: 279-83. PubMed ID: 20020533
- Kranendijk M. et al. 2010b. Science. 330: 336. PubMed ID: 20847235
- Nota B. et al. 2013. American Journal of Human Genetics. 92: 627-31. PubMed ID: 23561848
- Sass J.O. et al. 2008. Journal of Inherited Metabolic Disease. 31 Suppl 2: S275-9. PubMed ID: 18415700
- Smith A. et al. 2016. Jimd Reports. PubMed ID: 27306203
- Steenweg M.E. et al. 2010. Human Mutation. 31: 380-90. PubMed ID: 20052767
- Struys E.A. et al. 2006. Molecular Genetics and Metabolism. 88: 53-7. PubMed ID: 16442322
- Struys E.A., van der Knapp M.S., Salomons G.S. 2016. 2-Hydroxyglutaric Acidurias. In: Hollak C.E.M. and Lachmann R.H., editors. Inherited Metabolic Disease in Adults: A Clinical Guide. New York: Oxford University Press, p 145-147.
- Topçu M. et al. 2004. Human Molecular Genetics. 13: 2803-11. PubMed ID: 15385440
- Vilarinho L. et al. 2005. Human Mutation. 26: 395-6. PubMed ID: 16134148
- Vilarinho L. et al. 2010. Journal of Human Genetics. 55: 55-8. PubMed ID: 19911013
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.
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.
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
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.
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.
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.
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.