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Test Code NGENZ Red Blood Cell Enzyme Sequencing, Varies

Useful For

Providing a comprehensive genetic evaluation for patients with a personal or family history suggestive of an underlying RBC enzymopathy

 

Identifying mutations within genes associated with phenotypic severity, allowing for predictive testing and further genetic counseling

Genetics Test Information

See Targeted Genes Interrogated by NGHHA Next-Generation Sequencing in Special Instructions for a list of the genes and exons targeted by this test.

Method Name

Hereditary Mutation Detection by Next-Generation Sequencing (NGS)

Reporting Name

RBC Enzyme Sequencing, V

Specimen Type

Varies


Shipping Instructions


Peripheral blood specimens must arrive within 30 days of collection.



Necessary Information


The following information is required on patient information or test request form:

1. Clinical diagnosis

2. Pertinent clinical history (submit CBC results and relevant clinical notes)

3. Differentials based on clinical or morphologic presentation

4. Date of collection

5. Specimen type, whole blood or extracted DNA



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Peripheral blood (preferred)

Container/Tube:

Preferred: Lavender top (EDTA) or Yellow top or (ACD)

Acceptable: Green top (heparin)

Specimen Volume: 3 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send specimen in original tube.

3. Label specimen as blood

Specimen Stability: Refrigerated ≤30 days

 

Specimen Type: Extracted DNA

Container/Tube: 1.5- to 2-mL tube

Specimen Volume: Entire specimen

Collection Instructions:

1. Indicate volume and concentration of the DNA.

2. Label specimen as extracted DNA and source of specimen.

Specimen Stability: Frozen/Refrigerated/Ambient ≤30 days


Specimen Minimum Volume

Blood, Bone Marrow: 1 mL/Extracted DNA: 100 mcL at 20 ng/mcL concentration

Specimen Stability Information

Specimen Type Temperature Time
Varies Varies

Reject Due To

Hemolysis

Mild OK; Gross reject

Lipemia

Mild OK; Gross OK

Icterus

NA

Other

Bone marrow biopsies, slides, paraffin shavings, frozen tissues, paraffin-embedded tissues, paraffin-embedded bone marrow aspirates

Clinical Information

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

 

This panel aids in the diagnosis and genetic counseling of individuals with inherited RBC enzymopathies, possible carrier states, or compound mutations with severity modulating interactions. This panel always should be interpreted in the context of protein functional findings by enzymatic assay and complete blood count and peripheral blood findings. This complete interpretation can be provided by also ordering the EEEVP / RBC Enzyme Evaluation. Please fill out the information sheet and indicate that NGS testing was ordered. Providing CBC data and clinical notes will also allow more precise interpretation of results.

 

Mature erythrocytes are dependent upon glycolysis for energy production and the hexose monophosphate shunt for oxidation-reduction stability. Hereditary deficiencies in RBC enzymes within these pathways cause nonspherocytic hemolytic anemia (NSHA) with variable clinical presentations, therapeutic considerations and inheritance patterns.(1-3 Most of these deficiencies cause chronic hemolysis with little to no pathognomonic morphologic changes in the peripheral blood smear making correlation with enzyme activity critical for diagnosis. Some are associated with acute episodic anemia triggered by medications, food, or viral illness. Variable additional symptoms may be present for some deficiency types, including myopathy, neuropathy, and developmental delay. Because a subset of clinically significant RBC enzyme disorders can have indeterminate to normal enzyme activity (masking in the presence of increased reticulocytes), the protein  (enzymatic activity) studies are more sensitive when performed as a panel of RBC enzymes, which allows comparison of multiple enzyme activities. This genetic panel can aid in the interpretation of equivocal protein findings and genetically confirm an enzyme deficiency. Additionally, there are genes interrogated on this panel for which an enzyme test is not clinically available for correlation.

Reference Values

An interpretive report will be provided.

Interpretation

Evaluation and categorization of variants is performed using the most recent published American College of Medical Genetics recommendations as a guideline.(4,5) Variants are classified based on known, predicted, or possible pathogenicity and reported with interpretive comments detailing their potential or known significance.

 

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 predictions made by these tools may change over time. Results from in silico evaluation tools should be interpreted with caution and professional clinical judgment.

Cautions

Clinical:

Some individuals may have a mutation that is not identified by the methods performed. The absence of a mutation, therefore, does not eliminate the possibility of hereditary hemolytic anemia or a related disorder. This assay does not distinguish between germline and somatic alterations, particularly with variant allele frequencies (VAF) significantly lower than 50%. Test results should be interpreted in context of clinical findings, family history, and other laboratory data. Misinterpretation of results may occur if the information provided is inaccurate or incomplete.

 

If there is a family history of hereditary hemolytic anemia or a related disorder, it is often useful to test first-degree family members to help establish the clinical significance of variants of unknown significance.

 

At this time, it is not standard practice for the laboratory to systematically review likely pathogenic variants or variants of uncertain significance that have been previously detected and reported. The laboratory encourages health care providers to contact the laboratory at any time to learn how the status of a particular variant may have changed over time.

 

Technical:

Some genetic or genomic alterations, such as large insertion/deletion (indel) events, copy number alterations and gene translocation events are not detected by this assay. The depth of coverage may be variable for some target regions, but assay performance below the minimum acceptable criteria or for failed regions will be noted. Additionally, rare polymorphisms may be present that could lead to false negative or positive results. If results do not match clinical findings, consider alternative methods for analyzing these genes, such as Sanger sequencing or large deletion/duplication analysis. If the patient has had an allogenic blood transfusion, these results may be inaccurate due to the presence of donor DNA.

Method Description

This next-generation sequencing assay is performed to test for the presence of a pathogenic mutation in targeted regions of the following 17 genes: AK1, ALDOA, G6PD, GCLC, GPI, GSR, GSS, HBB, HBD, HK1, HMOX1, NT5C3A, PFKM, PGK1, PKLR, TPI1, and UGT1A1. See Targeted Genes Interrogated by NGENZ Next-Generation Sequencing in Special Instructions for details regarding the targeted gene regions identified by this test. This is a laboratory-developed test using Research Use Only reagents.

 

Next-generation sequencing (NGS) is performed using the Illumina MiSeq instrument with paired-end, 151-base pair (bp) reads. The DNA is prepared for NGS using a custom Agilent SureSelect Target Enrichment System. Data is analyzed with the CLC Genomics Server Program. Supplemental or confirmatory Sanger sequencing is performed when necessary.(Unpublished Mayo method)

Day(s) and Time(s) Performed

Monday

Analytic Time

8 weeks

Specimen Retention Time

1 year

Performing Laboratory

Mayo Medical Laboratories in Rochester

Test Classification

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

CPT Code Information

81350-UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) (eg, irinotecan metabolism), gene analysis, common variants (eg, *28, *36, *37)

81364-HBB

81405-PKLR

81479-AK1, ALDOA, GPI, GSR, GSS, HBD, HK1, HMOX1, NT5C3A, PGK1 and TPI1

81249-G6PD

LOINC Code Information

Test ID Test Order Name Order LOINC Value
NGENZ RBC Enzyme Sequencing, V In Process

 

Result ID Test Result Name Result LOINC Value
NGENS Specimen Type 31208-2
NGEND Indication for Test 42349-1
40560 Alterations Detected 82939-0
40561 Interpretation 59465-5
40562 Additional Notes 48767-8
40563 Method Summary 49549-9
40564 Disclaimer 62364-5
40566 Panel Gene List 36908-2
40567 Reviewed By 18771-6

Forms

Hemolytic Anemia Patient Information Form (T705)