Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: Non-homologous recombination associated with a shortened transcript from whole blood

Ting-Chang Hsu; Shelley M. Nakaya; Arthur R. Thompson

doi:10.1160/TH06-10-0592

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 97(02): 176-180
DOI: 10.1160/TH06-10-0592

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: Non-homologous recombination associated with a shortened transcript from whole blood

Authors

Ting-Chang Hsu

¹Puget Sound Blood Center and the University of Washington, Seattle, Washington, USA
Shelley M. Nakaya

¹Puget Sound Blood Center and the University of Washington, Seattle, Washington, USA
Arthur R. Thompson

¹Puget Sound Blood Center and the University of Washington, Seattle, Washington, USA

Abstract

Summary

In genotyping a severe hemophilia B subject, exons 1–3 and 5–8 were normal. Exon 4 did not amplify, suggesting a partial gene deletion. Previously, a French family with an exon 4 deletion had severe haemophilia B with a circulating, dysfunctional factor IX protein missing its first growth factor-like domain; breakpoints were not analyzed. Using a 5’ primer for exon 3 and a 3’ primer for exon 5 fragments, the subject’s factor IX gene amplified a 5 kb fragment whereas 11 kb was predicted, indicating a 6 kb deletion. Restriction endonucleases localized the 3’ intron 4 deletion breakpoint to 1.2 kb 5’ to exon 5. Sequencing through the breakpoints revealed a 5,969 bp deletion that included exon 4 and was accompanied by a 13 bp duplication inserted near the 3’ breakpoint site. Haemophilia was familial; on testing, his mother was confirmed as a heterozygous carrier, whereas his sister was homozygous for the normal, larger fragments. As exons 4 and 5 of the factor IX gene are in frame, this deletion should produce a shortened transcript, missing 114 bp (38 codons from the first growth factor-like domain). Reverse transcription of mRNA prepared from whole blood and PCR identified the shorter cDNA fragment. Western blotting demonstrated a smaller factor IX protein.

Keywords

Deletion breakpoints - ectopic mRNA - exon 4 - factor IX gene - haemophilia B

Full Text

References

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