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Hereditary Paraganglioma-Pheochromocytoma 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
1329 FH 81405 Add to Order
MAX 81479
MEN1 81405
NF1 81408
RET 81406
SDHA 81479
SDHAF2 81479
SDHB 81405
SDHC 81405
SDHD 81404
TMEM127 81479
VHL 81404
Full Panel Price* $1490.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
1329 Genes x (12) $1490.00 81404(x2), 81405(x4), 81406, 81408, 81479(x4) 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. CPT code 81437 can be used if analysis includes MAX, SDHB, SDHC, SDHD, TMEM127, and VHL. If you would like to order a subset of these genes contact us to discuss pricing.

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

Although the majority of Hereditary Paraganglioma-Pheochromocytoma (PGL/PCC) syndrome tumors are sporadic (i.e. non-familial), approximately 13% of all PGL/PCC tumors are caused by germline pathogenic variants in known PGL/PCC syndrome genes (Welander et al. 2011). Clinical sensitivity is dependent on tumor location. For the SDHB gene, pathogenic variants are detectable in up to 44% of hereditary PGL/PCC cases; pathogenic variants in the SDHC gene are detectable in up to 8% of PGL/PCC hereditary cases; pathogenic variants in the SDHA gene are detectable in up to 3% of hereditary PGL/PCC cases; and pathogenic variants in the SDHD gene are detectable in up to 50% of hereditary PGL/PCC cases (Kirmani and Young 2012). The clinical sensitivity for SDHAF2, and TMEM127 pathogenic variants is currently unknown. Germline pathogenic variants in the MAX gene have been estimated to be responsible for PCC/PGL in 1% of patients (Burnichon et al. 2012). In addition to the five PGL/PCC syndrome genes, germline pathogenic variants in a number of other genes may also predispose to PGL/PCC tumors (Opocher and Schiavi 2010). PGL/PCC tumors can also be found in >10% of other familial syndromes such as multiple endocrine neoplasia type 2 (MEN2), von Hippel–Lindau disease (VHL), and neurofibromatosis type 1 (NF1), and less so in Carney triad, Carney–Stratakis syndrome, and, very rarely, multiple endocrine neoplasia type 1 (MEN1) (Welander et al. 2011). The clinical sensitivity of FH pathogenic variants in predisposition to PGL/PCC is unknown, but pathogenic FH variants been reported previously (Castro-Vega et al. 2014).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FH$690.00 81479 Add to Order
MAX$690.00 81479
MEN1$690.00 81404
NF1$540.00 81479
RET$690.00 81479
SDHA$690.00 81479
SDHAF2$690.00 81479
SDHB$690.00 81479
SDHC$690.00 81479
SDHD$690.00 81479
TMEM127$690.00 81479
VHL$690.00 81403
Full Panel Price* $1290.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (12) $1290.00 81403, 81404, 81479(x10) Add to Order
Pricing Comment

NF1 is analyzed by Multiplex Ligation-dependent Probe Amplification. CPT code 81438 can be used if analysis includes SDHB, SDHC, SDHD, and VHL.

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Sensitivity

Hereditary Paraganglioma-Pheochromocytoma Syndrome deletion and duplication frequencies for the majority of these genes are unknown, however deletions have been reported in the SDHB gene in 12% of patients (Cascón et al. 2006) and have also been reported less frequently in the SDHC, SDHD, and MAX genes (Burnichon et al. 2009).

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Clinical Features

Hereditary Paraganglioma-Pheochromocytoma (PGL/PCC) syndrome is a familial cancer syndrome which results in neuroendocrine tumors. The diagnosis of hereditary PGL/PCC syndrome is based on physical examination, family history, imaging studies, biochemical testing, and molecular genetic testing. Symptoms of PGL/PCC result either from mass effects or catecholamine hypersecretion (e.g. sustained or paroxysmal elevations in blood pressure, headache, episodic profuse sweating, palpitations, pallor, and apprehension or anxiety) (Kirmani and Young 2012). Paraganglia are a group of neuroendocrine cells that originate from the embryonic neural crest and have the ability to secrete catecholamines. In PGL/PCC syndrome, paraganglia arise in either the paravertebral axis (base of the skull to the pelvis) for paragangliomas or the adrenal medulla for pheochromocytomas (Welander et al. 2011). Sympathetic paragangliomas hypersecrete catecholamines, whereas parasympathetic paragangliomas are most often nonsecretory. Extra-adrenal parasympathetic paragangliomas are located predominantly in the head and neck. The sympathetic extra-adrenal paragangliomas are generally located in the thorax, abdomen, and pelvis, and are usually secretory. Pheochromocytomas typically hypersecrete catecholamines (Kirmani and Young 2012). The prevalence of PGL/PCC tumors in the United States has been estimated to be between 1:2500 to 1:6000 (Chen et al. 2010), and for the hereditary PGL/PCC syndrome it has been estimated at 1:25000 to 1:50000 (Welander et al. 2011).

Genetics

Hereditary Paraganglioma-Pheochromocytoma syndrome is an autosomal dominant disorder and is mainly caused by pathogenic variants in three genes, SDHD, SDHC, and SDHB, which are known by their syndromic names PGL1, PGL3, and PGL4, respectively. The next most commonly mutated gene is SDHA (PGL5), which encodes a catalytic subunit of the succinate-ubiquinone oxidoreductase. Hereditary PGL/PCC syndrome presents variable expressivity and age-related penetrance. SDHA, SDHB, SDHC, and SDHD are nuclear genes which encode the four subunits of the mitochondrial enzyme succinate dehydrogenase (SDH). Another gene, SDHAF2 (also known as SDH5) encodes a protein that appears to be required for flavination of the SDHA subunit. Pathogenic variants in the MAX gene, which encodes a transcription factor that regulates cell proliferation, differentiation, and apoptosis, can also predispose individuals to PGL and PCC (Comino-Méndez et al. 2011; Burnichon et al. 2012). Pathogenic variants in MAX, SDHD and SDHAF2 demonstrate parent-of-origin effects and generally cause disease only when inherited from the father. A proband with a hereditary PGL/PCC syndrome may have inherited it from a parent or have a de novo mutation, although the latter’s frequency is not known. An individual who inherits a MAX, SDHD or SDHAF2 pathogenic variant from his/her mother has a low risk of developing disease; however, each of his/her offspring is at a 50% risk of inheriting the disease-causing allele. An individual who inherits an MAX, SDHD or SDHAF2 pathogenic variant from his/her father is at high risk of manifesting PGL/PCC. Germline predisposing pathogenic variants have also been found in the gene TMEM127, which is a negative regulator of mechanistic target of rapamycin, and has an important role in cellular proliferation and cell death (Kirmani and Young 2012; Welander et al. 2011). Other genes that are causative for Hereditary Paraganglioma-Pheochromocytoma syndrome include FH, NF1, VHL, RET, and MEN1. NF1 encodes for the protein Neurofibromin, which is a tumor suppressor that activates GTPase and controls cellular proliferation (Friedman 2012). The VHL gene is also a tumor suppressor. Inactivation of both alleles at the cellular level leads to abnormal activation of genes involved in hypoxia (Maher et al. 2011). The RET proto-oncogene is one of many receptor tyrosine kinases, a cell-surface molecule that transduce signals for cell growth and differentiation via RET autophosphorylation and intracellular signaling (Santoro 2004). MEN1 is a tumor suppressor gene whose gene product is involved in many vital processes, including transcriptional regulation, DNA replication, and DNA repair (Larsson et al. 1988; Lemos and Thakker 2008). The FH gene is thought to be a tumor suppressor encoding fumurate hydratase, which is involved in the conversion of fumurate to L-malate in the tricarboxylic acid (Krebs) cycle (Maher. 2011; Sudarshan et al. 2007).

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

Testing Strategy

For this Next Generation (NextGen) 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 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.

Due to homology issues, the entire coding region of SDHA (exons 1-15), in addition to ~20 bp of adjacent noncoding sequence of each exon, is bi-directionally sequenced using Sanger sequencing technology.

Please note that for deletion/duplication testing, NF1 is analyzed by Multiplex Ligation-dependent Probe Amplification.

Indications for Test

Individuals with a clinical or family history of Hereditary PGL/PCC syndrome should be tested early. Early diagnosis may improve patient prognosis through regular screening and treatment for early-onset malignancies. Early detection through surveillance and removal of tumors may prevent or minimize complications related to mass effects, catecholamine hypersecretion, and malignant transformation.

Genes

Official Gene Symbol OMIM ID
FH 136850
MAX 154950
MEN1 613733
NF1 613113
RET 164761
SDHA 600857
SDHAF2 613019
SDHB 185470
SDHC 602413
SDHD 602690
TMEM127 613403
VHL 608537
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

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CONTACTS

Genetic Counselors
Geneticist
Citations
  • Burnichon N. et al. 2012. Clinical Cancer Research. 18: 2828-37. PubMed ID: 22452945
  • Cascón Alberto et al. 2006. Genes, Chromosomes and Cancer. 45: 213-219. PubMed ID: 16258955
  • Castro-Vega .LJ. et al. 2014. Human Molecular Genetics. 23: 2440-6. PubMed ID: 24334767
  • Chen H. et al. 2010. Pancreas. 39: 775-83. PubMed ID: 20664475
  • Comino-Méndez Iñaki et al. 2011. Nature Genetics. 43: 663-667. PubMed ID: 21685915
  • Friedman J. 2012. Neurofibromatosis 1. 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: 20301288
  • Kirmani S, Young WF. 2012. Hereditary Paraganglioma-Pheochromocytoma Syndromes. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301715
  • Larsson C. et al. 1988. Nature. 332: 85-7. PubMed ID: 2894610
  • Lemos M.C., Thakker R.V. 2008. Human Mutation. 29: 22-32. PubMed ID: 17879353
  • Maher E.R. 2011. Nephron. Experimental Nephrology. 118: e21-6. PubMed ID: 21071978
  • Maher E.R. et al. 2011. European Journal of Human Genetics : Ejhg. 19: 617-23. PubMed ID: 21386872
  • Opocher G., Schiavi F. 2010. Best Practice & Research. Clinical Endocrinology & Metabolism. 24: 943-56. PubMed ID: 21115163
  • Santoro M. et al. 2004. Endocrinology. 145: 5448-51. PubMed ID: 15331579
  • Sudarshan S. et al. 2007. Nature Clinical Practice. Urology. 4: 104-10. PubMed ID: 17287871
  • Welander J. et al. 2011. Endocrine Related Cancer. 18: R253-R276. PubMed ID: 22041710
Order Kits
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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