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HEREDITARY PARAGANGLIOMA-PHEOCHROMOCYTOMA SYNDROME NEXTGEN SEQUENCING (NGS) PANEL

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 and most often are nonsecretory. 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).

Citations
  • Chen H, Sippel RS, O’Dorisio MS, Vinik AI, Lloyd RV, Pacak K. 2010. The North American Neuroendocrine Tumor Society Consensus Guideline for the Diagnosis and Management of Neuroendocrine Tumors: Pheochromocytoma, Paraganglioma, and Medullary Thyroid Cancer. Pancreas 39: 775–783. PubMed ID: 20664475
  • 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
  • Welander J, Soderkvist P, Gimm O. 2011. Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas. Endocrine Related Cancer 18: R253–R276. PubMed ID: 22041710

Genetics

Hereditary Paraganglioma-Pheochromocytoma syndrome is an autosomal dominant disorder that presents with variable expressivity and age-related penetrance. This syndrome can be caused by mutations in the SDHD, SDHAF2, SDHC and SDHB genes, which are also known by their syndromic names PGL1, PGL2, PGL3, PGL4, and PGL5 respectively.  SDHB, SDHC, and SDHD are nuclear genes which encode three of the four subunits of the mitochondrial enzyme succinate dehydrogenase (SDH). A fourth nuclear gene, SDHAF2 (also known as SDH5) encodes a protein that appears to be required for flavination of SDHA subunit (Kirmani and Young 2012).  Mutations in the MAX gene, which encodes a transcription factor that regulates cell proliferation, differentiation, and apoptosis, also predispose individuals to PGL and PCC (Comino-Méndez et al. 2011; Burnichon et al. 2012). Mutations in MAX, SDHD and SDHAF2 demonstrate parent-of-origin effects and generally cause disease only when the mutation is inherited from the father. A proband with a hereditary PGL/PCC syndrome may have inherited the mutation 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 mutation from his/her mother has a low risk of developing disease; 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 mutation from his/her father is at high risk of manifesting PGL/PCC. Germline predisposing mutations 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 NF1, VHLRET, 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 that it is involved in many vital processes, including transcriptional regulation, DNA replication, and DNA repair (Larsson et al. 1988; Lemos and Thakker 2008).

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

Citations
  • Burnichon N, Cascon A, Schiavi F, Morales NP, Comino-Mendez I, Abermil N, Inglada-Perez L, Cubas AA de, Amar L, Barontini M, Quiros SB de, Bertherat J, et al. 2012. MAX Mutations Cause Hereditary and Sporadic Pheochromocytoma and Paraganglioma. Clinical Cancer Research 18: 2828–2837. PubMed ID: 22452945
  • Comino-Méndez I, Gracia-Aznárez FJ, Schiavi F, Landa I, Leandro-García LJ, Letón R, Honrado E, Ramos-Medina R, Caronia D, Pita G, Gómez-Graña Á, Cubas AA de, et al. 2011. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. 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, Skogseid B, Oberg K, Nakamura Y, Nordenskjöld M. 1988. Multiple endocrine neoplasia type 1 gene maps to chromosome 11 and is lost in insulinoma. Nature 332: 85–87. PubMed ID: 2894610
  • Lemos MC, Thakker RV. 2008. Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Hum. Mutat. 29: 22–32. PubMed ID: 17879353
  • Maher ER, Neumann HP, Richard S. 2011. von Hippel–Lindau disease: a clinical and scientific review. European Journal of Human Genetics 19: 617–623. PubMed ID: 21386872
  • Santoro M. 2004. Minireview: RET: Normal and Abnormal Functions. Endocrinology 145: 5448–5451. PubMed ID: 15331579
  • Welander J, Soderkvist P, Gimm O. 2011. Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas. Endocrine Related Cancer 18: R253–R276. PubMed ID: 22041710

Testing Strategy

The Hereditary Paraganglioma-Pheochromocytoma Syndrome NextGen Sequencing Panel analyzes 10 genes that have been associated with hereditary paragangliomas and pheochromocytomas. 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 Sanger sequencing or 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

Individuals with a clinical history of Hereditary PGL/PCC syndrome or individuals with a family history of Hereditary PGL/PCC syndrome should be tested early for the latter (i.e. <10 years of age). Hereditary PGL/PCC syndrome should be considered in all individuals with paragangliomas and/or pheochromocytomas, especially those with multiple, multifocal, recurrent or early onset tumors (i.e. <40 years) (Young. 2008). This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Earlier 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.

Citations
  • Young. Williams Textbook of Endocrinology, 11 ed. Pp.:505-37, 2008

Gene(s)

Offical Gene Symbol OMIM Id
MAX 154950
MEN1 613733
NF1 613113
RET 164761
SDHAF2 613019
SDHB 185470
SDHC 602413
SDHD 602690
TMEM127 613403
VHL 608537

Disease(s)

Name OMIM Id
Paragangliomas 1 168000
Paragangliomas 2 601650
Paragangliomas 3 605373
Paragangliomas 4 115310
Paraganglioma And Gastric Stromal Sarcoma 606864
Pheochromocytoma 171300
Carcinoid Tumors, Intestinal 114900
Cowden-Like Syndrome 612359

Related Tests

Contacts

Genetic Counselors

Geneticist(s)

Test Methods

NextGen Sequencing

Test Number Test Price CPT Code(s)
1329 NextGen Sequencing (10 genes) $2,290 81405 (x3), 81404 (x2), 81479 (x3), 81406, 81408

In addition, Targeted Familial Mutation testing via Sanger sequencing is available for any gene in the panel:

Test Number Test Price CPT Code
100 Targeted Familial Mutations - Single Exon Sequencing $250 81479
200 Targeted Familial Mutations - Double Exon Sequencing $370 81479
300 Targeted Familial Mutations - Triple Exon Sequencing $440 81479

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the genes listed, plus ~20 bases of non-coding DNA flanking each exon.  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, rare, undocumented and suspect NGS variant calls are confirmed by Sanger sequencing.

Patient DNA sequence is aligned to the genomic reference sequence for each gene. ALL differences from the reference sequences (sequence variants) are reported in four tables:
(1) Variants Documented or Expected to be Pathogenic
(2) Variants Suspected to be Pathogenic or of Unknown Significance
(3) Variants Suspected to be Benign
(4) Common Variants Unlikely to be a Primary Cause of Disease.
Human Genome Variation Society (HGVS) recommendations are used to describe variants (http://www.hgvs.org).

As required, DNA is extracted from the patient specimen is using an ArchivePure DNA Blood Kit (5 PRIME, Inc. Gaithersburg, MD, USA).  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.

Clinical Sensitivity

Although the majority of PGL/PCC tumors are sporadic (i.e. non-familial), approximately 13% of all PGL/PCC tumors are caused by germline mutations known PGL/PCC syndrome genes (Welander et al. 2011). Clinical sensitivity for mutations is dependent on tumor location. For the SDHB gene, mutations are detectable in up to 44% of hereditary PGL/PCC cases; mutations in the SDHC gene are detectable in up to 8% of PGL/PCC hereditary cases; and mutations for 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 gene mutations is currently unknown. Germline mutations 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 mutations 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).

Citations
  • Burnichon N, Cascon A, Schiavi F, Morales NP, Comino-Mendez I, Abermil N, Inglada-Perez L, Cubas AA de, Amar L, Barontini M, Quiros SB de, Bertherat J, et al. 2012. MAX Mutations Cause Hereditary and Sporadic Pheochromocytoma and Paraganglioma. Clinical Cancer Research 18: 2828–2837. PubMed ID: 22452945
  • Burnichon N, Cascon A, Schiavi F, Morales NP, Comino-Mendez I, Abermil N, Inglada-Perez L, Cubas AA de, Amar L, Barontini M, Quiros SB de, Bertherat J, et al. 2012. MAX Mutations Cause Hereditary and Sporadic Pheochromocytoma and Paraganglioma. Clinical Cancer Research 18: 2828–2837. PubMed ID: 22452945
  • 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
  • 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
  • Opocher G, Schiavi F. 2010. Genetics of pheochromocytomas and paragangliomas. Best Practice & Research Clinical Endocrinology & Metabolism 24: 943–956. PubMed ID: 21115163
  • Opocher G, Schiavi F. 2010. Genetics of pheochromocytomas and paragangliomas. Best Practice & Research Clinical Endocrinology & Metabolism 24: 943–956. PubMed ID: 21115163
  • Welander J, Soderkvist P, Gimm O. 2011. Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas. Endocrine Related Cancer 18: R253–R276. PubMed ID: 22041710
  • Welander J, Soderkvist P, Gimm O. 2011. Genetics and clinical characteristics of hereditary pheochromocytomas and paragangliomas. Endocrine Related Cancer 18: R253–R276. PubMed ID: 22041710

Analytical Validity

As of October 2012, 0.84 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 1438 total sequence variants (differences from the reference sequences). Of these, 1261 were nucleotide substitutions and 177 were insertions or deletions. About 56% of the variants were heterozygous and 44% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides. Our NextGen methodology is generally not capable of detecting larger insertions (duplications) or larger heterozygous deletions. 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.

Turnaround Time

Maximum of 60 days.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Number Test Price CPT Code
600 MEN1 Deletion/Duplication Testing via aCGH $990 81479
VHL Deletion/Duplication Testing via aCGH $990 81403
SDHAF2 Deletion/Duplication Testing via aCGH $990 81479
SDHB Deletion/Duplication Testing via aCGH $990 81479
SDHC Deletion/Duplication Testing via aCGH $990 81479
SDHD Deletion/Duplication Testing via aCGH $990 81479
RET Deletion/Duplication Testing via aCGH $990 81479
TMEM127 Deletion/Duplication Testing via aCGH $990 81479
NF1 Deletion/Duplication Testing via aCGH $990 81479
MAX Deletion/Duplication Testing via aCGH $990 81479
Full Panel* $1,990

*Prices:  Allele copy number analysis of one gene                                              $ 990
               Allele copy number analysis of two genes                                            $1190
               Allele copy number analysis of three or more genes                          $1290 + $100 for every additional gene
               Reanalysis of existing aCGH for additional genes (up to 5 genes)     $ 690

Test Procedure

As required, DNA is extracted from the patient specimen using a Gentra PUREGENE kit. 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 hours at 65ºC.  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 (HDGC) aCGH is designed to have comprehensive coverage for both coding and non-coding regions for each targeted gene with very high density probe coverage.  The average probe spacing within each exon is 47 bp or a minimum of three probes per exon covering all targeted exons and UTRs.  The average probe spacing is 289 bp covering all intronic, 2kb upstream and downstream regions of each targeted gene.  In addition, the flanking 300-bp intronic sequence on either side of targeted exons has enriched probe coverage.  Therefore, PreventionGenetics’ aCGH enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene.

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 SDHCSDHD, and MAX genes (Burnichon et al. 2009).

Citations
  • Burnichon N, Cascon A, Schiavi F, Morales NP, Comino-Mendez I, Abermil N, Inglada-Perez L, Cubas AA de, Amar L, Barontini M, Quiros SB de, Bertherat J, et al. 2012. MAX Mutations Cause Hereditary and Sporadic Pheochromocytoma and Paraganglioma. Clinical Cancer Research 18: 2828–2837. PubMed ID: 22452945
  • Cascón A, Montero-Conde C, Ruiz-Llorente S, Mercadillo F, Letón R, Rodríguez-Antona C, Martínez-Delgado B, Delgado M, Díez A, Rovira A, Díaz JÁ, Robledo M. 2006. Gross SDHB deletions in patients with paraganglioma detected by multiplex PCR: A possible hot spot? Genes, Chromosomes and Cancer 45: 213–219. PubMed ID: 16258955

Analytical Validity

PreventionGenetics’ high density gene-centric custom designed aCGH enables the detection of relatively small deletion and amplification mutations (down to ~300 bp) within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

Any copy number changes smaller than 300bps (within the targeted region) may not be detected by our array.

This array may not detect deletion and amplification mutations 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 happened 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.

Turnaround Time

Maximum of 40 days, although many tests are completed in 3-4 weeks.

Requisition Form

  • The first three pages of the requisition form must accompany all specimens.
  • Billing information is on the third page.
  • Specimen and shipping instructions are listed on the fourth page.
  • All testing must be ordered by a qualified healthcare provider.

Download the Requisition Form (pdf)

Specimen Types

Whole Blood

(Delivery accepted Monday - Saturday)

  • Collect 2-5 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.

DNA

(Delivery accepted Monday - Saturday)

  • 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.
  • Label the tube with the DNA concentration as well as the patient name, date of birth, and/or ID number.
  • Specify the composition of the solute.
  • We only accept genomic DNA for testing.  We do not accept products of whole genome amplification reactions or other amplification reactions.

Cell Culture

(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.

Version: 1.1
Last Updated 01/07/2014