Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Sep;29(5):1320-1333.
doi: 10.1111/hae.14824. Epub 2023 Jul 6.

Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non-severe haemophilia A

Amy Dericquebourg  1   2 , Mathilde Fretigny  1 , Alexandre Leuci  2 , Christophe Zawadzki  3 , Yoann Huguenin  4 , Sabine-Marie Castet  4 , Yesim Dargaud  2   5 , Christine Vinciguerra  1   2 , Yohann Jourdy  1   2
Affiliations

Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non-severe haemophilia A

Amy Dericquebourg et al. Haemophilia. 2023 Sep.

Abstract

Introduction: Conventional genetic investigation fails to identify the F8 causal variant in 2.5%-10% of haemophilia A (HA) patients with non-severe phenotypes. In these cases, F8 deep intronic variants could be causal.

Aim: To identify pathogenic F8 deep intronic variants in genetically unresolved families with non-severe HA analysed in the haematology laboratory of the Hospices Civils de Lyon.

Methods: The whole F8 was analysed by next generation sequencing. The pathogenic impact of candidate variants identified was assessed using both in silico analysis (MaxEntScan and spliceAI) and functional analysis (RNA or minigene assay).

Results: Sequencing was performed in 49/55 families included for which a DNA sample from a male propositus was available. In total, 33 candidate variants from 43 propositi were identified. These variants corresponded to 31 single nucleotide substitutions, one 173-bp deletion, and an 869-bp tandem triplication. No candidate variant was found in six propositi. The most frequent variants found were the association of [c.2113+1154G>C and c.5374-304C>T], identified in five propositi, and the c.2114-6529C>G identified in nine propositi. Four variants had been previously described as HA-causing. Splicing functional assay found a deleterious impact for 11 substitutions (c.671-94G>A, c.788-312A>G, c.2113+1154G>C, c.2114-6529C>G, c.5999-820A>T, c.5999-786C>A, c.5999-669G>T, c.5999-669G>A, c.5999-669G>C, c.6900+4104A>C, and c.6901-2992A>G). The HA-causing variant was identified in 33/49 (67%) cases. In total, F8 deep intronic variants caused 8.8% of the non-severe HA among the 1643 families analysed in our laboratory.

Conclusion: The results emphasise the value of whole F8 gene sequencing combined with splicing functional analyses to improve the diagnosis yield for non-severe HA.

Keywords: FVIII; RNA; haemophilia A; intron; mutation.

PubMed Disclaimer

References

REFERENCES

    1. White GC 2nd, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost. 2001;85:560. Factor VIII and Factor IX Subcommittee.
    1. Vehar GA, Keyt B, Eaton D, et al. Structure of human factor VIII. Nature. 1984;312:337-342.
    1. Johnsen JM, Fletcher SN, Dove A, et al. Results of genetic analysis of 11 341 participants enrolled in the my life, our future hemophilia genotyping initiative in the United States. J Thromb Haemost. 2022;20:2022-2034.
    1. Jourdy Y, Janin A, Fretigny M, et al. Reccurrent F8 intronic deletion found in mild hemophilia a causes alu exonization. Am J Hum Genet. 2018;102:199-206.
    1. Bogdanova N, Markoff A, Eisert R, et al. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat. 2007;28:54-60.

LinkOut - more resources