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Test Code NHEP Hereditary Erythrocytosis Gene Panel, Next-Generation Sequencing, Varies


Ordering Guidance


Polycythemia vera and acquired causes of erythrocytosis should be excluded before ordering this evaluation. See Erythrocytosis Genotyping Comparison Chart for a comparison of erythrocytosis testing options. If this test is ordered in the setting of erythrocytosis and suspicion of polycythemia vera, interpretation requires correlation with a concurrent or recent prior bone marrow evaluation.

 

For an evaluation including hemoglobin electrophoresis testing and hereditary erythrocytosis variant analysis of the most common gene regions associated with hereditary erythrocytosis in an algorithmic fashion, order REVE2 / Erythrocytosis Evaluation, Blood.

 

The hemoglobin genes, HBA1/HBA2 and HBB are not interrogated in this assay.

 

Multiple gene panels are available. For more information see NHEP and Subpanel Comparison Gene List.

 

Customization of this panel and single gene analysis for any gene present on this panel are available. For more information see CGPH / Custom Gene Panel, Hereditary, Next-Generation Sequencing, Varies.

 

Targeted testing for familial variants (also called site-specific or known variants testing) is available for the genes on this panel. See FMTT / Familial Variant, Targeted Testing, Varies. To obtain more information about this testing option, call 800-533-1710.



Shipping Instructions


Specimen preferred to arrive within 96 hours of collection.


Necessary Information


1. Erythrocytosis Patient Information is required. Testing may proceed without the patient information, however, the information aids in providing a more thorough interpretation. Ordering providers are strongly encouraged to fill out the form and send with the specimen.

2. If form not provided, include the following information with the test request: clinical diagnosis, pertinent clinical history (ie, complete blood cell count results and relevant clinical notes), and differentials based on clinical presentation and/or laboratory findings.



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Whole blood

Patient Preparation: A previous bone marrow transplant from an allogenic donor will interfere with whole blood testing. Call 800-533-1710 for instructions for testing patients who have received a bone marrow transplant.

Container/Tube:

Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD)

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.

Specimen Stability Information: Ambient (preferred) 4 days/Refrigerated

 

Specimen Type: Skin biopsy

Supplies: Fibroblast Biopsy Transport Media (T115)

Container/Tube: Sterile container with any standard cell culture media (eg, minimal essential media, RPMI 1640). The solution should be supplemented with 1% penicillin and streptomycin.

Specimen Volume: 4-mm punch

Specimen Stability Information: Refrigerated (preferred)/Ambient

Additional Information: A separate culture charge will be assessed under CULFB / Fibroblast Culture for Biochemical or Molecular Testing, Chorionic Villi/Products of Conception/Tissue. An additional 3 to 4 weeks is required to culture fibroblasts before genetic testing can occur.

 

Specimen Type: Cultured fibroblast

Container/Tube: T-25 flask

Specimen Volume: 2 Flasks

Collection Instructions: Submit confluent cultured fibroblast cells from a skin biopsy from another laboratory. Cultured cells from a prenatal specimen will not be accepted.

Specimen Stability Information: Ambient (preferred)/Refrigerated (<24 hours)

Additional Information: A separate culture charge will be assessed under CULFB / Fibroblast Culture for Biochemical or Molecular Testing, Chorionic Villi/Products of Conception/Tissue. An additional 3 to 4 weeks is required to culture fibroblasts before genetic testing can occur.


Forms

1. Erythrocytosis Patient Information (T694) is required.

2. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing (Spanish) (T826)

3. If not ordering electronically, complete, print, and send a Benign Hematology Test Request (T755) with the specimen.

Secondary ID

619019

Useful For

Evaluating an individual with JAK2-V617F negative erythrocytosis associated with lifelong sustained increased red blood cell (RBC) mass, elevated RBC count, hemoglobin, or hematocrit

 

Providing an extensive genetic evaluation for patients with a personal or family history suggestive of hereditary erythrocytosis

 

Comprehensive testing for patients in whom previous targeted gene variant analyses were negative for a specific hereditary erythrocytosis

 

Establishing a diagnosis of a hereditary erythrocytosis or related disorder, allowing for appropriate management and surveillance of disease features based on the gene involved

Testing Algorithm

This evaluation is recommended for patients presenting with lifelong elevation in hemoglobin or hematocrit, usually with a positive family history of similar symptoms. Polycythemia vera should be excluded prior to testing as it is much more common than hereditary erythrocytosis and can be present even in young patients. A JAK2 V617F or JAK2 exon 12 variant should not be present. More sensitive, variant-specific testing for JAK2 V617F is highly recommended prior to ordering this test. Additionally, alpha and beta chain high-oxygen affinity hemoglobin variants should be excluded prior to ordering this test panel.

 

If skin biopsy (fresh) is received, fibroblast culture will be added at an additional charge. If viable cells are not obtained, the client will be notified.

Method Name

Sequence Capture and Targeted Next-Generation Sequencing (NGS) followed by Polymerase Chain Reaction (PCR) and Sanger Sequencing

Reporting Name

Erythrocytosis Full Panel, NGS

Specimen Type

Varies

Specimen Minimum Volume

1 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Varies Varies

Reject Due To

All specimens will be evaluated at Mayo Clinic Laboratories for test suitability.

Clinical Information

Next-generation sequencing is a methodology that can interrogate large regions of genomic DNA in a single assay. The presence and pattern of gene variants can provide critical diagnostic, prognostic, and therapeutic information for managing physicians.

 

Erythrocytosis (ie, increased red blood cell [RBC] mass or polycythemia) may be primary, due to an intrinsic defect of bone marrow stem cells (ie, polycythemia vera: PV), or secondary, in response to increased serum erythropoietin (EPO) levels. Secondary erythrocytosis is associated with a number of disorders, including chronic lung disease, chronic increase in carbon monoxide (due to smoking), cyanotic heart disease, high-altitude living, kidney cysts and tumors, hepatoma, and other EPO-secreting tumors. When these common causes of secondary erythrocytosis are excluded, a heritable cause involving hemoglobin or erythrocyte regulatory mechanisms may be suspected.

 

Unlike polycythemia vera, hereditary erythrocytosis is not associated with the risk of clonal evolution and should present with isolated erythrocytosis that has been present since birth. A small subset of cases are associated with neoplasia (eg, pheochromocytoma or paraganglioma formation). It is caused by variations in several genes and may be inherited in either an autosomal dominant or autosomal recessive manner. A family history of erythrocytosis would be expected in these cases, although it is possible for new variants to arise in an individual.

 

The genes coding for hemoglobin, beta globin and alpha globin (high-oxygen-affinity hemoglobin variants), hemoglobin-stabilization proteins (2,3 bisphosphoglycerate mutase: BPGM), and the erythropoietin receptor (EPOR) and oxygen-sensing pathway enzymes (hypoxia-inducible factor [HIF2A/EPAS1], prolyl hydroxylase domain 2 [PHD2/EGLN1], and von Hippel Lindau [VHL]) can result in hereditary erythrocytosis. High-oxygen-affinity hemoglobin variants and BPGM abnormalities result in a decreased p50 result, whereas those affecting EPOR, HIF2A, PHD2, and VHL have normal p50 results. The true prevalence of hereditary erythrocytosis-causing variants is unknown. Due to high homology, the hemoglobin genes, HBA1/HBA2 and HBB, are not interrogated in this panel.

 

The oxygen-sensing pathway functions through an enzyme, HIF, which regulates RBC mass. A heterodimer protein comprised of alpha and beta subunits, HIF functions as a marker of depleted oxygen concentration. When present, oxygen becomes a substrate mediating HIF-alpha subunit degradation. In the absence of oxygen, degradation does not take place, and the alpha protein component is available to dimerize with a HIF-beta subunit. The heterodimer then induces transcription of many hypoxia response genes, including EPO. HIF-alpha is regulated by VHL protein-mediated ubiquitination and proteasomal degradation, which requires prolyl hydroxylation of HIF proline residues. The HIF-alpha subunit is encoded by the HIF2A (EPAS1) gene. Enzymes important in the hydroxylation of HIF-alpha are the prolyl hydroxylase domain proteins, of which the most significant isoform is PHD2, which is encoded by the PHD2 (EGLN1) gene. Variations resulting in altered HIF-alpha, PHD2, and VHL proteins can lead to clinical erythrocytosis. A small subset of variants, in PHD2/EGLN1 and HIF2A/EPAS1, has also been detected in erythrocytic patients presenting with paragangliomas or pheochromocytomas. Truncating variants in the EPOR gene coding for the erythropoietin receptor can result in erythrocytosis through loss of the negative regulatory cytoplasmic SHP-1 binding domain leading to EPO hypersensitivity. All currently known variants have been localized to exon 8 and are heterozygous truncating variants. EPOR variants are associated with decreased EPO levels and normal p50 values. Gain-of-function variants in EPO have also been associated with hereditary erythrocytosis.

 

However, the cause of erythrocytosis remains unknown in greater than 70% of cases after testing genes most associated with hereditary erythrocytosis. Therefore, additional genes associated with erythropoiesis are also interrogated in this assay. These include additional oxygen sensing erythrocytosis pathway genes (EGLN2, EGLN3, HIF1A, HIF1AN, HIF3A), some genes associated with myeloid proliferation (JAK2, SH2B3, SOCS3), and those associated with other conditions that also variably present with erythrocytosis (ANKRD26, PFKM, PIEZO1, PKLR), or methemoglobinemia (CYB5A, CYB5R3). In addition, genes identified by whole genome studies (ACO1, GFI1B, KDM6A, and BHLHE41) are included.(1)

Reference Values

An interpretive report will be provided.

Interpretation

All detected variants are evaluated according to American College of Medical Genetics and Genomics recommendations.(2) Variants are classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance.

Cautions

Clinical Correlations:

Test results should be interpreted in the context of clinical findings, family history, and other laboratory data. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

 

If testing was performed because of a clinically significant family history, it is often useful to first test an affected family member. Detection of a reportable variant in an affected family member would allow for more informative testing of at-risk individuals.

 

To discuss the availability of additional testing options or for assistance in the interpretation of these results, contact the Mayo Clinic Laboratories genetic counselors at 800-533-1710.

 

Technical Limitations:

Next-generation sequencing may not detect all types of genomic variants. In rare cases, false-negative or false-positive results may occur. The depth of coverage may be variable for some target regions; assay performance below the minimum acceptable criteria or for failed regions will be noted. Given these limitations, negative results do not rule out the diagnosis of a genetic disorder. If a specific clinical disorder is suspected, evaluation by alternative methods can be considered.

 

There may be regions of genes that cannot be effectively evaluated by sequencing or deletion and duplication analysis as a result of technical limitations of the assay, including regions of homology, high guanine-cytosine (GC) content, and repetitive sequences. Confirmation of select reportable variants will be performed by alternate methodologies based on internal laboratory criteria.

 

This test is validated to detect 95% of deletions up to 75 base pairs (bp) and insertions up to 47 bp. Deletions-insertions (delins) of 40 or more bp, including mobile element insertions, may be less reliably detected than smaller delins.

 

Deletion/Duplication Analysis:

This analysis targets single and multi-exon deletions/duplications; however, in some instances single exon resolution cannot be achieved due to isolated reduction in sequence coverage or inherent genomic complexity. Balanced structural rearrangements (such as translocations and inversions) may not be detected.

 

This test is not designed to detect low levels of mosaicism or to differentiate between somatic and germline variants. If there is a possibility that any detected variant is somatic, additional testing may be necessary to clarify the significance of results.

 

Genes may be added or removed based on updated clinical relevance. For detailed information regarding gene-specific performance and technical limitations, see Method Description or contact a laboratory genetic counselor.

 

If the patient has had an allogeneic hematopoietic stem cell transplant or a recent heterologous blood transfusion, results may be inaccurate due to the presence of donor DNA. Call Mayo Clinic Laboratories for instructions for testing patients who have received a bone marrow transplant.

 

Reclassification of Variants:

Currently, it is not standard practice for the laboratory to systematically review previously classified variants on a regular basis. The laboratory encourages health care providers to contact the laboratory at any time to learn how the classification of a particular variant may have changed over time. Due to broadening genetic knowledge, it is possible that the laboratory may discover new information of relevance to the patient. Should that occur, the laboratory may issue an amended report.

 

Variant Evaluation:

Evaluation and categorization of variants are performed using published American College of Medical Genetics and Genomics and the Association for Molecular Pathology recommendations as a guideline.(2) Other gene-specific guidelines may also be considered. Variants are classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance. Variants classified as benign or likely benign are not reported.

 

Multiple in silico evaluation tools may be used to assist in the interpretation of these results. The accuracy of predictions made by in silico evaluation tools is highly dependent upon the data available for a given gene, and periodic updates to these tools may cause predictions to change over time. Results from in silico evaluation tools should be interpreted with caution and professional clinical judgment.

 

Rarely, incidental or secondary findings may implicate another predisposition or presence of active disease. These findings will be carefully reviewed to determine whether they will be reported.

Clinical Reference

1. Camps C, Petousi N, Bento C. Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations. Haematologica. 2016;101(11):1306-1318. doi:10.3324/haematol.2016.144063

2. Richards S, Aziz N, Bale S, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17(5):405-424

3. Patnaik MM, Tefferi A. The complete evaluation of erythrocytosis: congenital and acquired. Leukemia. 2009;23(5):834-844. doi:10.1038/leu.2009.54

4. Prchal JF, Prchal JT. Polycythemia vera. In: Kaushansky K, Lichtman MA, Prchal JT, eds. Williams Hematology. 9th ed. McGraw Hill; 2015

5. Oliveira JL, Coon LM, Frederick LA, et al. Genotype-phenotype correlation of hereditary erythrocytosis mutations, a single center experience. Am J Hematol. 2018 May 23. doi:10.1002/ajh.25150

6. Zhuang Z, Yang C, Lorenzo F, et al. Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. N Engl J Med. 2012;367(10):922-930. doi:10.1056/NEJMoa1205119

7. Tarade D, Robinson CM, Lee JE, Ohh M. HIF-2 alpha-pVHL complex reveals broad genotype-phenotype correlations in HIF-2 alpha-driven disease. Nat Commun. 2018;9(1):3359. doi:10.1038/s41467-018-05554-1

8. Oliveira JL. Algorithmic evaluation of hereditary erythrocytosis: Pathways and caveats. Int J Lab Hematol. 2019;41 Suppl 1:89-94. doi:10.1111/ijlh.13019

9. Gangat N, Oliveira JL, Porter TR, et al. Erythrocytosis associated with EPAS1(HIF2A), EGLN1(PHD2), VHL, EPOR or BPGM mutations: The Mayo Clinic experience. Haematologica. 2022;107(5):1201-1204. doi:10.3324/haematol.2021.280516

10. Iolascon A, Bianchi P, Andolfo I, et al. Recommendations for diagnosis and treatment of methemoglobinemia. Am J Hematol. 2021;96(12):1666-1678. doi:10.1002/ajh.26340

Method Description

Next-generation sequencing (NGS) and/or Sanger sequencing are performed to test for the presence of variants in coding regions and intron/exon boundaries of the genes analyzed, as well as some other regions that have known disease-causing variants. The human genome reference GRCh37/hg19 build was used for sequence read alignment. At least 99% of the bases are covered at a read depth over 30X. Sensitivity is estimated at above 99% for single nucleotide variants, above 94% for deletions-insertions (delins) less than 40 base pairs (bp), above 95% for deletions up to 75 bp, and insertions up to 47 bp. NGS and/or a polymerase chain reaction-based quantitative method is performed to test for the presence of deletions and duplications in the genes analyzed.

 

There may be regions of genes that cannot be effectively evaluated by sequencing or deletion and duplication analysis as a result of technical limitations of the assay, including regions of homology, high guanine-cytosine content, and repetitive sequences.(Unpublished Mayo method)

 

See Targeted Genes and Methodology Details for Hereditary Erythrocytosis Gene Panel for details regarding the targeted genes analyzed for each test and specific gene regions not routinely covered.

 

Reference transcript numbers may be updated due to transcript re-versioning. Always refer to the final patient report for gene transcript information referenced at the time of testing. Confirmation of select reportable variants may be performed by alternate methodologies based on internal laboratory criteria.

 

Genes analyzed: ACO1, ANKRD26, BHLHE41, BPGM, CYB5A, CYB5R3, EGLN1, EGLN2, EGLN3, EPAS1, EPO, EPOR, GFI1B, HIF1A, HIF1AN, HIF3A, JAK2, KDM6A, PFKM, PIEZO1, PKLR, SH2B3, SOCS3, and VHL

Day(s) Performed

Varies

Report Available

28 to 42 days

Specimen Retention Time

Whole blood: 2 weeks (if available); Extracted DNA: 3 months

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

81404

81405

81479

81479 (if appropriate for government payers)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
NHEP Erythrocytosis Full Panel, NGS 103735-7

 

Result ID Test Result Name Result LOINC Value
619020 Test Description 62364-5
619021 Specimen 31208-2
619022 Source 31208-2
619023 Result Summary 50397-9
619024 Result 82939-0
619025 Interpretation 59465-5
619026 Additional Results 82939-0
619027 Resources 99622-3
619028 Additional Information 48767-8
619029 Method 85069-3
619030 Genes Analyzed 82939-0
619031 Disclaimer 62364-5
619032 Released By 18771-6

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
CULFB Fibroblast Culture for Genetic Test Yes No