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Hereditary Neuroblastoma via the PHOX2B Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1173 PHOX2B$540.00 81404 Add to Order
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 18 days.

Clinical Sensitivity
Taken together, ALK and PHOX2B germline mutations account for 90% of hereditary neuroblastoma, with the majority being in the ALK gene (Fisher and Tweddle. Seminars in Fetal & Neonatal Medicine 17: 207-215, 2012).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PHOX2B$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
Neuroblastoma is the most common type of childhood cancer that occurs before 1 year of age, accounting for 10-15% of cancer deaths in children. Approximately 90% of neuroblastomas are detected by 5 years of age, while 30% are found in the first year of life with the median age of diagnosis of 22 months (Esiashvili et al. Curr Probl Cancer 33:333-60, 2009). This tumor type can occur in adolescence and adulthood, although the prognosis is poorer compared to a childhood incidence (Colon and Chung. Advances in Pediatrics 58:297-311, 2011). The majority of neuroblastomas (65%) arise in the abdomen, with half of these in the medulla of the adrenal gland . They can also occur in the chest (20%), neck (5%), pelvis (5%), and 1% of cases have an unknown primary (Colon and Chung. Advances in Pediatrics 58:297-311, 2011). Symptoms of patients with neuroblastoma include malaise, fevers, weight loss, enlarging mass, pain, and abdominal distention. Other symptoms can include early-onset hypertension and tachycardia due to the secretion of catecholamines. Neuroblastomas commonly occur sporadically in a family, but 1-2% of cases occur with family histories of neuroblastoma. Hereditary neuroblastomas tend to have earlier presentations and lead to multiple primary cancers. They also show significant clinical heterogeneity, whereby a pedigree may show an individual with spontaneous cancer regression, whereas another individual with metastatic spread (Deyell and Attiyeh.  Cancer Genetics 204:113-121, 2011).  Siblings of an affected patient with neuroblastoma have a 10-fold increase in developing neuroblastoma (Friedman et al. Cancer Epidemiol Biomarkers Prev 14:1922-7, 2005). Neuroblastomas can also be found with other conditions such as Hirschsprung disease, congenital hypoventilation disorder, and neurofibromatosis type 1 (Johnson and Park. "ALK-Related Neuroblastoma Susceptibility." GeneReviews, 2012).
Genetics
Hereditary neuroblastoma is an autosomal dominant disorder that shows incomplete penetrance (Fisher and Tweddle. Seminars in Fetal & Neonatal Medicine 17: 207-215, 2012). Neuroblastomas show whole-chromosome gains and segmental chromosomal aberrations. The former results from hyperdiploidy and has a favorable prognosis, whereas the latter is associated with MYCN amplification and associated with worse outcomes (Colon and Chung. Advances in Pediatrics 58:297-311, 2011).  The most frequent genetic aberration is an unbalanced chromosome 17q gain found in 70% of neuroblastomas, which has a poor prognosis (Bown et al. N Engl J Med 340:1954-1961, 1999).  In cases of hereditary neuroblastoma the most common etiology are mutations in the anaplastic lymphoma kinase (ALK) oncogene. Another less frequent cause of hereditary neuroblastomas are from PHOX2B mutations, which are often associated with Hirschsprung's disease and/or congenital hypoventilation (Mosse et al. Am J Hum Genet 75:727-730, 2004). PHOX2B encodes a transcription factor that is involved in the normal sympathetic neuronal development and catecholamine synthesis. PHOX2B mutations are rarely found in sporadic neuroblastomas (van Limpt et al. Oncogene 23:9280-8, 2004). The majority of causative PHOX2B mutations include missense and small insertions and deletions (Human Gene Mutation Database).
Testing Strategy
The paired mesoderm homeobox protein 2B is encoded by 3 exons (1-3) from the PHOX2B gene on chromosome 4p12. Testing is accomplished by amplifying each coding exon and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard dideoxy Sanger sequencing methods and a capillary electrophoresis instrument. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.
Indications for Test
Hereditary neuroblastoma should be suspected in neonates where there is a family history of neuroblastoma in two or more 1st degree relatives. Genetic testing for PHOX2B mutations should especially be carried out for individuals with neuroblastomas associated with Hirschsprung's disease and/or congenital hypoventilation (Mosse et al. Am J Hum Genet 75:727, 2004). Persons with PHOX2B germline mutations may also have dysmorphic features, including downslanting palpebral fissures, small nose, triangular shaped mouth, or low-set, posteriorly rotated ears (Johnson and Park. GeneReviews. 2012). This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Gene

Official Gene Symbol OMIM ID
PHOX2B 603851
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Congenital Central Hypoventilation Syndrome (CCHS) Sequencing Panel
Congenital Central Hypoventilation Syndrome (CCHS) via the PHOX2B Gene
Hereditary Neuroblastoma via the ALK Gene
Hereditary Neuroblastoma via the KIF1B Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bown et al. (1999). "Gain of chromosome arm 17q and adverse outcome in patients with neuroblastoma." N Engl J Med 340:1954-1961. PubMed ID: 10379019
  • Colon and Chung. (2011). "Neuroblastoma." Advances in Pediatrics 58:297-311. PubMed ID: 21736987
  • Deyell and Attiyeh. (2011). "Advances in the understanding of constitutional and somatic genomic alterations in neuroblastoma." Cancer Genetics 204:113-121. PubMed ID: 21504710
  • Esiashvili et al. (2009) "Neuroblastoma." Curr Probl Cancer 33:333-60. PubMed ID: 20172369
  • Fisher and Tweddle. (2012). "Neonatal neuroblastoma." Seminars in Fetal & Neonatal Medicine 17: 207-215. PubMed ID: 22673527
  • Friedman et al. (2005). "Increased risk of cancer among siblings of long-term childhood cancer survivors: a report from the childhood cancer survivor study." Cancer Epidemiol Biomarkers Prev 14:1922-7. PubMed ID: 16103438
  • Human Gene Mutation Database.
  • Johnson and Park. (2012). "ALK-Related Neuroblastoma Susceptibility." GeneReviews. PubMed ID: 20301782
  • Mosse et al. (2004). "Germline PHOX2B mutation in hereditary neuroblastoma." Am J Hum Genet 75:727-730. PubMed ID: 15338462
  • van Limpt et al. (2004). "The Phox2B homeobox gene is mutated in sporadic neuroblastomas." Oncogene 23:9280-8. PubMed ID: 15516980
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TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

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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