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Episodic Pain Syndrome 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
3447 SCN10A 81479 Add to Order
SCN11A 81479
SCN9A 81479
Full Panel Price* $1440.00
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

In a cohort of 104 patients who presented with painful, predominantly small-fiber neuropathy, Faber et al. reported 9 individuals (~8.5%) with pathogenic variants in SCN10A (Faber et al. 2012). In a separate study of 345 patients who presented with painful peripheral neuropathy and were negative for causative variants in SCN9A or SCN10A, 12 patients (~3.5%) harbored pathogenic variants within the SCN11A gene (Huang et al. 2014). Clinical sensitivity for the SCN9A-related episodic pain syndromes is difficult to predict due to the absence of large cohort studies and the phenotypic heterogeneity of these disorders. Analytic sensitivity is likely high, however, as all reported pathogenic variants to date are missense changes that are expected to be identified by direct sequencing of genomic DNA.

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 SCN9A$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 Features

The Familial Episodic Pain Syndromes (FEPSs) are small fiber neuropathies characterized by intense, episodic inflammatory pain in specific regions of the body (Faber et al. 2012; Bennett and Woods, 2014). Currently, there are three recognized subtypes of this disorder (FEPS I-III). 

FEPS type I is generally confined to the upper body, including the thorax and arms (Kremeyer et al. 2010). Attacks initially present in infancy and are often triggered by fasting in combination with other physical stressors, such as illness, cold temperature, and fatigue. Other symptoms may include breathing difficulties, tachycardia, sweating, pallor, and peribuccal cyanosis.

FEPS type II typically involves the distal lower extremities, although at least two patients have also reported hand pain (Faber et al. 2012; Han et al. 2014). Individuals may also present with allodynia, hyperalgesia, and/or red discoloration of the feet. While only a few patients with FEPS II have been described to date, onset of this disorder appears to occur in adulthood, and attacks may be triggered by stimuli such as cold temperature or intense physical exercise.

Finally, the pain associated with FEPS type III is primarily concentrated in the distal lower extremities, occasionally arising in the joints of fingers and arms (Zhang et al. 2013). Other symptoms may include numbness and tingling, skin discoloration, dry eyes and mouth, hyperhidrosis, orthostatic dizziness, palpitations, and/or hot flushes (Huang et al. 2014). In contrast to FEPS types I and II, age of onset of FEPS type III appears to be highly variable, although severity of the disorder may diminish with age (Zhang et al. 2013; Huang et al. 2014).

The symptoms of FEPS also overlap with those of other episodic pain syndromes, such as SCN9A-associated paroxysmal extreme pain disorder, primary erythermalgia, and small fiber neuropathy (Emery et al. 2015).

Paroxysmal extreme pain disorder (PEPD) is characterized by episodes of extreme pain that is generally confined to proximal regions of the body, including the rectum, eye, and mandible. Additionally, individuals may present with inflammation, excess secretions from the eyes or nose, and/or tonic attacks with apnea and bradycardia (Bennett and Woods 2014). Episodes may occur at birth or shortly after, and can be triggered by certain types of provoking stimuli, such as yawning, eating, or defecation (Fertleman et al. 2006).
  
The predominant symptom of primary erythermalgia is an episodic burning sensation in the hands and feet, often accompanied by redness and swelling (Bennett and Woods 2014). An increase in temperature further aggravates these symptoms, while a decrease in temperature provides relief. In contrast, while SCN9A-associated small fiber neuropathy is also characterized by a painful burning sensation in the lower extremities, patients are unaffected by alterations in temperature (Faber et al. 2012).

Genetics

The episodic pain syndromes are autosomal dominant diseases caused by gain-of-function variants that result in either enhanced activation of cation channels or impairment of cation channel deactivation (Bennet and Woods 2014; Emery et al. 2015). Our Episodic Pain Syndrome NextGen Panel currently includes the genes SCN9A, SCN10A, and SCN11A. Sequencing of TRPA1, which is associated with Familial Episodic Pain Syndrome (FEPS) Type I, is not available at this time.

SCN9A: The SCN9A gene encodes the alpha subunit of a type IX voltage-gated sodium channel, also referred to as Nav1.7 (Faber et al. 2012). Over 60 pathogenic variants have been reported in SCN9A to date (http://www.hgmd.cf.ac.uk/). Heterozygous gain-of-function missense variants in this gene may cause one of several pain disorders, including erythermalgia, small fiber neuropathy, and paroxysmal extreme pain disorder (Faber et al. 2012; Michiels et al. 2005; Fertleman et al. 2006). In contrast, loss-of-function variants in SCN9A may result in congenital indifference to pain, an autosomal recessive disease (Cox et al. 2006).

SCN10A: The SCN10A gene encodes the alpha subunit of the type X voltage-gated sodium channel. also referred to as Nav1.8 (Faber et al. 2012). SCN10A gain-of-function missense variants are responsible for FEPS type II. To date, four variants have been reported as causative for SCN10A-associated pain syndromes (Faber et al. 2012; Huang et al. 2013; Han et al. 2014).

SCN11A: The SCN11A gene encodes the alpha subunit of the type XI voltage-gated sodium channel, also referred to as Nav1.9 or NaN (Zhang et al. 2013). SCN11A gain-of-function missense variants are responsible for FEPS type III, and at least six pathogenic variants have been described in this gene (http://www.hgmd.cf.ac.uk/). Additionally, one gain-of-function missense change in SCN11A has been associated with hereditary sensory and autonomic neuropathy type VII (HSAN7), a congenital disorder characterized by the inability to perceive pain (Leipold et al. 2013).

Testing Strategy

For this NGS panel, the full coding regions, plus ~20 bp 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

The ideal test candidates for this Episodic Pain Syndrome panel are individuals who present with recurring episodes of pain and have a family history of FEPS type II, FEPS type III, or any of the SCN9A-related pain disorders.

Genes

Official Gene Symbol OMIM ID
SCN10A 604427
SCN11A 604385
SCN9A 603415
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Comprehensive Neuropathy Sequencing Panel
Early Infantile Epileptic Encephalopathy:
Dominant and X-linked Sequencing Panel
Epilepsy: Dravet Syndrome Sequencing Panel
Epilepsy: Generalized Epilepsy with Febrile Seizures Plus (GEFS+) Sequencing Panel
Familial Episodic Pain Type 2 Syndrome via the SCN10A Gene
Familial Episodic Pain Type 3 (FEPS3) Syndrome, Hereditary Sensory and Autonomic Neuropathy Type VII (HSAN7), and other Pain-Related Disorders via the SCN11A Gene
Sodium Channel, Voltage-Gated, Type IX, Alpha Subunit Disorders via the SCN9A Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bennett D.L and Woods C.G. 2014. The Lancet Neurology. 13:587-99. PubMed ID: 24813307
  • Cox J.J. et al. 2006. Nature. 444: 894-8. PubMed ID: 17167479
  • Emery E.C. et al. 2015. The Journal of Neuroscience : the Official Journal of the Society For Neuroscience. 35:7674-81. PubMed ID: 25995458
  • Faber C.G. et al. 2012. Proceedings of the National Academy of Sciences U S A. 109:19444–9. PubMed ID: 23115331
  • Faber, C. G. et al. (2012). PubMed ID: 21698661
  • Fertleman C.R. et al. 2006. Neuron. 52: 767-74. PubMed ID: 17145499
  • Han C. et al. 2014. Journal of Neurology, Neurosurgery, and Psychiatry. 85:499-505. PubMed ID: 24006052
  • Huang J, Han C, Estacion M, Vasylyev D, Hoeijmakers JG, Gerrits MM, Tyrrell L, Lauria G, Faber CG, Dib-Hajj SD, Merkies IS, Waxman SG, PROPANE Study Group. 2014. Gain-of-function mutations in sodium channel Na(v)1.9 in painful neuropathy. Brain 137: 1627-1642. PubMed ID: 24776970
  • Huang J. et al. 2013. The Journal of Neuroscience : the Official Journal of the Society For Neuroscience. 33:14087-97.  PubMed ID: 23986244
  • Kremeyer B. et al. 2010. Neuron. 66: 671-80. PubMed ID: 20547126
  • Leipold E, Liebmann L, Korenke GC, Heinrich T, Giesselmann S, Baets J, Ebbinghaus M, Goral RO, Stödberg T, Hennings JC, Bergmann M, Altmüller J, Thiele H, Wetzel A, Nürnberg P, Timmerman V, De Jonghe P, Blum R, Schaible HG, Weis J, Heinemann SH, Hübner CA, Kurth I. 2013. A de novo gain-of-function mutation in SCN11A causes loss of pain perception. Nature Genetics 45: 1399-1404. PubMed ID: 24036948
  • Michiels, J. J. et al. (2005).   PubMed ID: 16216943
  • Zhang X.Y. et al. 2013. American Journal of Human Genetics. 93:957-66. PubMed ID: 24207120
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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