Molecular Diagnosis of Congenital Coagulopathies by NGS: Hemophilia A

Clinical information

Diagnostic Utility:

Identifying the molecular defect in the F8 gene in patients diagnosed with HA.

Haemophilia A (HA)

HA is the most common form of haemophilia with an estimated prevalence of 1:5,000 males. This bleeding disorder is caused by a deficiency of coagulation factor VIII (FVIII). Depending on the levels of FVIII, three forms of HA are distinguished: severe if the biological activity of FVIII is <1%; moderate if FVIII activity is between 1-5%; mild if FVIII activity is between 5-40%. The most frequent clinical manifestations are bleeding into the joints (hemarthrosis) and into the muscles (hematomas). The frequency and severity of bleeding is inversely proportional to the amount of FVIII present in the plasma.

HA follows an X-linked recessive inheritance pattern, meaning that males are affected and females are carriers of the condition. This disorder is caused by mutations in the gene encoding the clotting protein factor VIII (F8) which can affect the activity of FVIII or reduce the amount of this protein. Currently, over 1,300 genetic alterations in F8 have been identified, including single nucleotide changes, deletions, or insertions of multiple nucleotides. However, in cases of severe HA, the most frequent mutation is the inversion of intron 22, present in 50% of patients. This mutation occurs due to a homologous recombination between F8 and the telomeric end of the X chromosome because of the high homology between both sequences.

Application of a multi-gene panel based on simultaneous amplification of exons and flanking intronic regions for sequencing through next-generation sequencing (NGS) techniques allows for the simultaneous molecular study of genes related to congenital coagulopathies and inherited bleeding disorders, including the factor VIII (F8) gene.

Method:

Next-generation sequencing (NGS)

Analysis of inversions of intron 1 through conventional PCR and intron 22 through long-range PCR (LR-PCR) in F8.

Next-generation sequencing of the exons and flanking intronic regions of F8 using a panel of genes related to congenital coagulopathies.

Traditional Sanger sequencing to confirm the detected mutation(s) in patients diagnosed with HA, in order to reach an unequivocal result, analysing the specific region where the variant is located.

If no potential or definitively causative mutation is identified, this will be reported and discussed with the medical team requesting the test about the possibility of carrying out complementary studies.

Reference Values

N/A

Diagnostic Algorithm:

Depending on the patient's phenotype, it includes:

• Severe HA (FVIII:C<1%) and moderate HA (1%
• If negative and in patients diagnosed with mild HA (5%F8 using a panel of genes related to congenital coagulopathies.
• The mutation(s) detected by NGS in Haemophilia A studies, to reach an unequivocal result, will be confirmed by Sanger method analysing the specific region where the mutation is located.
• If no potential or definitively causative mutation is identified, this will be reported and discussed with the medical team requesting the test about the possibility of carrying out complementary studies.

Turnaround Time:

20 working days

Specimen information

Sample: Whole blood
Tube: EDTA K3 tube 5-10 ml if it is a blood sample
Minimum essential volume: 3 ml

Stability:

• At room temperature: 4 days
• In the refrigerator: 10 days

Transport instructions: Preferably at room temperature

Reason for rejection: Clotted sample and/or incorrectly identified.

Other accepted sample types:

• Purified DNA, minimum 300 ng (30 ng/µL)
• Buccal mucosa: contact the laboratory for specimen collection specifications.

Administrative information

BST Code: LRD2833
Test Description: Molecular diagnosis of congenital coagulopathies by NGS: Hemophilia A.
Synonyms: Genetic study of HA, molecular study of Hemophilia A, F8 sequencing.
Section: Congenital Coagulopathies
BST Rates: Check the updated rates here.

The checkbox HEMO. A must be ticked on the molecular study request form and the phenotypic data available must be filled in.

Profiles:

Not applicable.

References

• Peter J Hulick. Next-generation DNA sequencing (NGS): Principles and clinical Applications. Waltham, MA: UpToDate Inc. https://www.uptodate.com
• DNA Sequencing by Capillary Electrophoresis. Applied Biosystems Chemistry Guide. Second Edition.
• Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood. 2002;99(1):168-174.
• Bagnall RD, Giannelli F, Green PM. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost. 2006;4(3):591-598.

Base de dades de mutacions

• Hemobase: http://www.hemobase.com/
• Human Gene Mutation Database: http://www.hgmd.cf.ac.uk
• EAHAD Coagulation Factor Variant Databases: http://f8-db.eahad.org/

Quality

BST holds ISO 9001, ISO 14001, and OSHAS 18001 quality certifications, as well as the European Excellence 500+ seal. BST is accredited by CAT, JACIE-FACT, FACT-NETCORD, and EFI, and complies with Good Manufacturing Practice (GMP) and Good Distribution Practice (GDP) guidelines.