Malignant Hyperthermia Susceptibility Sequencing Panel
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test||CPT Code Copy CPT Codes|
|Full Panel Price*||$1590.00|
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.
Sensitivity for ideal test candidates (family history of MH plus either positive in vitro contracture test or an MH event) is approximately 60% (Monnier et al. 2005; Levano et al. 2009).
Deletion/Duplication Testing via aCGH
|Test Code||Test||Individual Gene Price||CPT Code Copy CPT Codes|
|Full Panel Price*||$770.00|
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The great majority of tests are completed within 28 days.
No large deletions or insertions in any of the three genes have been reported in MH patients. aCGH testing is therefore probably not warranted.
Malignant Hyperthermia (MH) is a severe adverse reaction to commonly used anesthetics (halothane, sevoflurane, desflurane, enflurane, isoflurane) or to depolarizing muscle relaxants (succinylcholine) (Nelson and Flewellen 1983; Larach et al. 2010; Rosenberg et al. 2013). In susceptible patients these agents may trigger uncontrolled muscle hypermetabolism. In almost all cases, the first manifestations of MH occur in the operating room. Death can result unless the patient is promptly treated. Alternative anesthetics are available for known MH Susceptible individuals.
Malignant Hyperthermia (MH) Susceptibility may be caused by mutations in at least three genes: RYR1, CACNA1S and STAC3. Causative mutations in RYR1 are much more common than causative mutations in the other two genes.
RYR1 mutations causative for MH are inherited in an autosomal dominant manner, although patients with two causative mutations in trans have been reported (Monnier et al. 2002). All RYR1 MH causative mutations reported in the literature have been either missense or (rarely) in-frame deletion or insertion of one or a few amino acids (Robinson et al. 2006; Ibarra et al. 2006; Levano et al. 2009; www.emhg.org). Premature protein termination, splicing, or large deletion mutations have not been reported in RYR1 for MH (although they are well known in cases of recessive myopathy). Over 300 RYR1 missense variants have been found in MH patients, but only 35-50 have been conclusively demonstrated to be involved in the disease. Most causative RYR1 MH mutations are clustered in three hot spots along the gene. Penetrance of many (and perhaps all) RYR1 MH causative variants is incomplete; that is, MH susceptible patients will not always trigger upon exposure to offending agents (Robinson et al. 2009; Grievink and Stowell 2010). The large RYR1 gene with 106 exons encodes the primary skeletal muscle calcium channel within the sarcoplasmic reticulum membrane. Mutations in RYR1 can also cause a variety of myopathies (see Test #1771).
Autosomal dominant MH may also be caused by mutations in the CACNA1S gene (Monnier et al. 1997; Carpenter et al. 2009; Toppin et al. 2010). Several CACNA1S missense variants have been connected to MH, but to date the only variant conclusively demonstrated to be involved is c.3257G>A (p.Arg1086His). CACNA1S encodes the α1 subunit of the L-type calcium channel in skeletal muscle, also known as the dihydropyridine receptor. The CACNA1S and RYR1 gene products interact to transduce action potentials into contraction of skeletal muscle fibers (Lanner et al. 2010). Missense mutations in the CACNA1S gene can also cause hypokalemic periodic paralysis.
Recently, a founder missense mutation in the STAC3 gene has been reported to cause autosomal recessive Native American myopathy (Horstick et al. 2013). This myopathy is characterized by congenital weakness and arthrogryposis, cleft palate, ptosis, short stature, kyphoscoliosis, talipes deformities and susceptibility to MH (Stamm et al. 2008). STAC3 encodes a T-tubule protein which mediates Ca++ release through the RYR1 channel (Nelson et al. 2013). The involvement of STAC3 in MH is not as well established as the other two genes, but we included this gene in our NGS panel to be inclusive.
For this NGS panel, the full coding regions plus ~20bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below. 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.
Indications for Test
Ideal MH test candidates have a family history of MH along with either a positive in vitro contracture test or a clear MH event. Testing should begin in such a family member. If a causative mutation is identified, other family members can be screened at much reduced cost. Other, less ideal candidates for testing are those with just a family history of MH or those with a “MH-like” event and no family history.
|Official Gene Symbol||OMIM ID|
|Central Core Disease||117000|
|Malignant Hyperthermia Susceptibility Type 5||601887|
|Minicore Myopathy With External Ophthalmoplegia||255320|
|Native American myopathy||255995|
- Genetic Counselor Team - firstname.lastname@example.org
- James L. Weber, PhD - email@example.com
- Carpenter D, Ringrose C, Leo V, Morris A, Robinson RL, Halsall PJ, Hopkins PM, Shaw M-A. 2009. The role of CACNA1S in predisposition to malignant hyperthermia. BMC Med Genet 10: 104. PubMed ID: 19825159
- Grievink H, Stowell KM. 2010. Research Allele-specific differences in ryanodine receptor 1 mRNA expression levels may contribute to phenotypic variability in malignant hyperthermia. Orphanet J Rare Dis 5: 10. PubMed ID: 20482855
- Horstick EJ, Linsley JW, Dowling JJ, Hauser MA, McDonald KK, Ashley-Koch A, Saint-Amant L, Satish A, Cui WW, Zhou W, Sprague SM, Stamm DS, Powell CM, Speer MC, Franzini-Armstrong C, Hirata H, Kuwada JY. 2013. Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy. Nat Commun 4: 1952. PubMed ID: 23736855
- Ibarra M CA, Wu S, Murayama K, Minami N, Ichihara Y, Kikuchi H, Noguchi S, Hayashi YK, Ochiai R, Nishino I. 2006. Malignant hyperthermia in Japan: mutation screening of the entire ryanodine receptor type 1 gene coding region by direct sequencing. Anesthesiology 104: 1146–1154. PubMed ID: 16732084
- Lanner JT, Georgiou DK, Joshi AD, Hamilton SL. 2010. Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release. Cold Spring Harbor Perspectives in Biology 2: a003996–a003996. PubMed ID: 20961976
- Larach MG, Gronert GA, Allen GC, Brandom BW, Lehman EB. 2010. Clinical presentation, treatment, and complications of malignant hyperthermia in North America from 1987 to 2006. Anesth. Analg. 110: 498-507. PubMed ID: 20081135
- Levano S, Vukcevic M, Singer M, Matter A, Treves S, Urwyler A, Girard T. 2009. Increasing the number of diagnostic mutations in malignant hyperthermia. Human Mutation 30: 590–598. PubMed ID: 19191329
- Monnier N, Kozak-Ribbens G, Krivosic-Horber R, Nivoche Y, Qi D, Kraev N, Loke J, Sharma P, Tegazzin V, Figarella-Branger D, Roméro N, Mezin P, et al. 2005. Correlations between genotype and pharmacological, histological, functional, and clinical phenotypes in malignant hyperthermia susceptibility. Human Mutation 26: 413–425. PubMed ID: 16163667
- Monnier N, Krivosic-Horber R, Payen J-F, Kozak-Ribbens G, Nivoche Y, Adnet P, Reyford H, Lunardi J. 2002. Presence of two different genetic traits in malignant hyperthermia families: implication for genetic analysis, diagnosis, and incidence of malignant hyperthermia susceptibility. Anesthesiology 97: 1067–1074. PubMed ID: 12411788
- Monnier N, Procaccio V, Stieglitz P, Lunardi J. 1997. Malignant-hyperthermia susceptibility is associated with a mutation of the alpha 1-subunit of the human dihydropyridine-sensitive L-type voltage-dependent calcium-channel receptor in skeletal muscle. Am. J. Hum. Genet. 60: 1316–1325. PubMed ID: 9199552
- Nelson BR, Wu F, Liu Y, Anderson DM, McAnally J, Lin W, Cannon SC, Bassel-Duby R, Olson EN. 2013. Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca2+ release and contractility. Proc. Natl. Acad. Sci. U.S.A. 110: 11881–11886. PubMed ID: 23818578
- Nelson TE, Flewellen EH. 1983. Current concepts. The malignant hyperthermia syndrome. N. Engl. J. Med. 309:416-418. PubMed ID: 6348539
- Robinson R, Carpenter D, Shaw M-A, Halsall J, Hopkins P. 2006. Mutations in RYR1 in malignant hyperthermia and central core disease. Hum. Mutat. 27: 977–989. PubMed ID: 16917943
- Robinson RL, Carpenter D, Halsall PJ, Iles DE, Booms P, Steele D, Hopkins PM, Shaw M-A. 2009. Epigenetic allele silencing and variable penetrance of malignant hyperthermia susceptibility. British Journal of Anaesthesia 103: 220–225. PubMed ID: 19454545
- Rosenberg H, Sambuughin N, Riazi S, Dirksen R. 2013. Malignant Hyperthermia Susceptibility. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301325
- Stamm DS, Aylsworth AS, Stajich JM, Kahler SG, Thorne LB, Speer MC, Powell CM. 2008. Native American myopathy: congenital myopathy with cleft palate, skeletal anomalies, and susceptibility to malignant hyperthermia. Am. J. Med. Genet. A 146A: 1832–1841. PubMed ID: 18553514
- Toppin PJ, Chandy TT, Ghanekar A, Kraeva N, Beattie WS, Riazi S. 2010. A report of fulminant malignant hyperthermia in a patient with a novel mutation of the CACNA1S gene. Canadian Journal of Anesthesia/Journal canadien d’anesthésie 57: 689–693. PubMed ID: 20431982
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.
- The first four pages of the requisition form must accompany all specimens.
- Billing information is on the third and fourth pages.
- Specimen and shipping instructions are listed on the fifth and sixth pages.
- All testing must be ordered by a qualified healthcare provider.
(Delivery accepted Monday - Saturday)
- Collect 3-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
- If refrigerated, blood specimen is good for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- NextGen Sequencing Tests: Send in screw cap tube at least 10 µg of purified DNA at a concentration of at least 50 µg/ml
- Sanger Sequencing Tests: Send in a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml. For tests involving the sequencing of more than three genes, send an additional 5 µg DNA per gene. DNA may be shipped at room temperature.
- Deletion/Duplication via aCGH: Send in screw cap tube at least 1 µg of purified DNA at a concentration of at least 100 µg/ml.
- Whole-Genome Chromosomal Microarray: Collect at least 5 µg of DNA in TE (10 mM Tris-cl pH 8.0, 1mM EDTA), dissolved in 200 µl at a concentration of at least 100 ng/ul (indicate concentration on tube label). DNA extracted using a column-based method (Qiagen) or bead-based technology is preferred.
(Delivery accepted Monday - Thursday)
- PreventionGenetics should be notified in advance of arrival of a cell culture.
- Ship at least two T25 flasks of confluent cells.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We do not culture cells.