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Mayo Clinic Laboratories

Test ID GNHMA Hemophilia A, F8 Gene, Next-Generation Sequencing, Varies

Ordering Guidance

For male patients, this test should only be considered if clinical and family history, initial coagulation screens, and/or initial activity tests indicate a diagnosis of hemophilia A. For female patients, this test should only be considered if there is a confirmed diagnosis of hemophilia A in a family member or the patient has abnormally low factor VIII (FVIII) activity.

This test does not measure FVIII activity levels. For assessment of FVIII activity, order F8A / Coagulation Factor VIII Activity Assay, Plasma.

For individuals with bleeding symptoms and no known personal or family history of hemophilia A, consider ALBLD / Bleeding Diathesis Profile, Limited, Plasma or the specific factor assays.

This test does not evaluate for the presence of inversions in the F8 gene that can cause hemophilia A. If testing for possible inversions in the F8 gene is desired, order F8INV / Hemophilia A F8 Gene, Intron 1 and 22 Inversion Mutation Analysis, Whole Blood.

If genetic testing for hereditary bleeding disorders using a larger panel is desired, both a 6-gene focused bleeding panel and a 25-gene comprehensive bleeding panel are available. For more information see GNBLF / Bleeding Disorders, Focused Gene Panel, Next-Generation Sequencing, Varies or GNBLC / Bleeding Disorders, Comprehensive Gene Panel, Next-Generation Sequencing, Varies

Testing for the F8 gene as part of a customized panel is 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 F8 gene. See FMTT / Familial Variant, Targeted Testing, Varies. To obtain more information about this testing option, call 800-533-1710.

Additional Testing Requirements

All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen as this must be a different order number than the prenatal specimen.

Shipping Instructions

Specimen preferred to arrive within 96 hours of collection.

Necessary Information

Hemophilia A 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.

Specimen Required

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

Submit only 1 of the following specimens:

Specimen Type: Whole blood

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

Prenatal Specimens

Due to its complexity, consultation with the laboratory is required for all prenatal testing; call 800-533-1710 to speak to a genetic counselor.

Specimen Type: Amniotic fluid

Container/Tube: Amniotic fluid container

Specimen Volume: 20 mL

Specimen Stability Information: Refrigerated (preferred)/Ambient

Additional information:

1. A separate culture charge will be assessed under CULAF / Culture for Genetic Testing, Amniotic Fluid.

2. All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.

Specimen Type: Chorionic villi

Container/Tube: 15-mL tube containing 15 mL of transport media

Specimen Volume: 20 mg

Specimen Stability Information: Refrigerated

Additional Information:

1. A separate culture charge will be assessed under CULFB / Fibroblast Culture for Biochemical or Molecular Testing.

2. All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.

Acceptable:

Specimen Type: Confluent cultured cells

Container/Tube: T-25 flask

Specimen Volume: 2 Flasks

Collection Instructions: Submit confluent cultured cells from another laboratory.

Specimen Stability Information: Ambient (preferred)/Refrigerated

Additional Information:

All prenatal specimens must be accompanied by a maternal blood specimen; order MATCC / Maternal Cell Contamination, Molecular Analysis, Varies on the maternal specimen.

Forms

1. Hemophilia A Patient Information (T712) 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 an Coagulation Test Request (T753) with the specimen.

Useful For

Confirming a clinical diagnosis of hemophilia A in affected male patients with the identification of a disease-causing variant in the F8 gene

Determining the disease-causing alteration within the F8 gene to delineate the underlying molecular defect in a male patient with a laboratory diagnosis of hemophilia A

Identifying the causative alteration for prognostic and genetic counseling purposes

Assessing hemophilia A carrier status for female patients with a family history of hemophilia A

Prenatal testing for hemophilia A when a familial F8 variant has been previously identified in a family member

Genetics Test Information

This test utilizes next-generation sequencing to detect single nucleotide and copy number variants in the F8 gene associated with hemophilia A (also known as factor VIII deficiency). See Method Description for additional details.

Identification of a disease-causing variant may assist with diagnosis, prognosis, clinical management, recurrence risk assessment, familial screening, and genetic counseling for hemophilia A.

Disease States

Hemophilia A

Reflex Tests

Test ID	Reporting Name	Available Separately	Always Performed
CULFB	Fibroblast Culture for Genetic Test	Yes	No
CULAF	Amniotic Fluid Culture/Genetic Test	Yes	No
MATCC	Maternal Cell Contamination, B	Yes	No

Testing Algorithm

The clinical workup for hemophilia A in symptomatic male patients should begin with special coagulation testing for factor VIII (FVIII) activity. Genetic testing is indicated if FVIII activity is less than 40% of normal (Note: reference range may vary depending on the locally established reference range) and von Willebrand factor antigen testing is normal. For more information see Hemophilia Testing Algorithm.

FVIII clotting activity does not correlate as well with bleeding severity in female patients and therefore, is unreliable in the detection of female carriers of hemophilia A. Carrier status is determined by identification of a heterozygous disease-causing variant in F8 by molecular genetic testing. For female patients with a suspected or confirmed diagnosis of hemophilia A in a family member, carrier testing is recommended as specified by the Hemophilia Carrier Testing Algorithm.(1-3)

Acquired (nongenetic) causes of hemophilia A that should be excluded before genetic testing include heparin use, disorders associated with antibodies to clotting factors such as systemic lupus erythematosus or antiphospholipid syndrome, pregnancy/postpartum period, rheumatic disease, solid or hematologic malignancies, and use of certain drugs such as penicillin, sulfamides, phenytoin, interferon, fludarabine.

For prenatal specimens only:

Prenatal genetic testing for hemophilia A is not routinely performed without the prior identification of a familial F8 variant in an affected male relative or a carrier female relative. Requests for prenatal testing for hemophilia A without a known familial F8 variant are performed at the discretion of a Molecular Hematopathology Laboratory Director.

-If amniotic fluid (nonconfluent cultured cells) is received, amniotic fluid culture/genetic test will be added at an additional charge.

-If chorionic villus specimen (nonconfluent cultured cells) is received, fibroblast culture for genetic test will be added at an additional charge.

For any prenatal specimen that is received, maternal cell contamination testing will be performed at an additional charge.

Method Name

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

Reporting Name

F8 Gene, Full Gene NGS

Specimen Type

Varies

Specimen Minimum Volume

Blood: 1 mL; Amniotic fluid: 10 mL; Other specimen types: see Specimen Required

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

Hemophilia A is a hereditary bleeding disorder associated with germline variants in the F8 gene. It is inherited in an X-linked recessive manner with variable expressivity and is estimated to affect 1 in 4000-5000 live male births.(2)

Hemophilia A is characterized by a deficiency in clotting factor VIII (FVIII), an essential blood coagulation protein. Symptomatic male patients may experience mild to severe bleeding problems, including excessive bruising, prolonged epistaxis, post-operative bleeding, hemarthrosis, deep-muscle hematomas, and/or intracranial or gastrointestinal tract bleeding.(2,3) Female carriers are not typically affected but some may experience increased bleeding tendencies especially after medical procedures and surgery. Note that FVIII activity may not correlate with the severity of symptoms in females.(4-6)

Several causes of acquired (nongenetic) hemophilia A should be excluded prior to genetic testing, including heparin use, disorders associated with antibodies to clotting factors such as systemic lupus erythematosus or antiphospholipid syndrome, pregnancy and the postpartum period, rheumatic disease, solid and hematologic malignancies, and use of certain drugs (eg, penicillin, sulfamides, phenytoin, interferon, fludarabine).(7)

The World Federation of Hemophilia provides guidelines regarding diagnosis, management, and laboratory testing for individuals with hemophilia A.(8)

Reference Values

An interpretive report will be provided.

Interpretation

All detected variants are evaluated according to American College of Medical Genetics and Genomics recommendations.(9) 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.

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 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 healthcare 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.(9) 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. Pruthi RK: Hemophilia: a practical approach to genetic testing. Mayo Clin Proc. 2005 Nov;80(11):1485-1499

2. Berntorp E, Fischer K, Hart DP, et al: Haemophilia. Nat Rev Dis Primers. 2021 Jun 24;7(1):45

3. Konkle BA, Fletcher SN: Hemophilia A. In: Adam MP, Everman DB, Mirzaa GM, et al. GeneReviews [Internet]. University of Washington, Seattle; 2000. Updated October 27, 2022. Accessed November 28, 2022. Available at www.ncbi.nlm.nih.gov/books/NBK1404/

4. Plug I, Mauser-Bunschoten EP, Brocker-Vriends AHJT, et al: Bleeding in carriers of hemophilia. Blood. 2006 Jul 1;108(1):52-56

5. Miesbach W, Alesci S, Geisen C, Oldenburg J: Association between phenotype and genotype in carriers of haemophilia A. Haemophilia. 2011 Mar;17(2):246-251

6. Paroskie A, Gailani D, DeBaun MR, Sidonio RF Jr: A cross-sectional study of bleeding phenotype in haemophilia A carriers. Br J Haematol. 2015 Jul;170(2):223-228

7. Haider MZ: Acquired hemophilia. In: Anwer F, ed. StatPearls [Internet]. StatPearls Publishing; 2021. Updated September 26, 2022. Accessed November 28, 2022. Available at www.statpearls.com/articlelibrary/viewarticle/114240

8. Srivastava A, Santagostino E, Dougall A, et al: WFH Guidelines for the Management of Hemophilia, 3rd edition. Haemophilia. 2020 Aug;26 Suppl 6:1-158

9. Richards S, Aziz N, Bale S, et al; ACMG Laboratory Quality Assurance Committee: 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 May;17(5):405-424

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 F8 gene, 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 F8 gene.

There may be regions of the F8 gene 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.(Unpublished Mayo method)

The reference transcript for F8 gene is NM_000132.3. 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.

Day(s) Performed