Manuscript handled by: D. Lane
Characterization of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique
Version of Record online: 3 JUL 2013
© 2013 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 11, Issue 6, pages 1103–1110, June 2013
How to Cite
Characterization of large deletions in the F8 gene using multiple competitive amplification and the genome walking technique. J Thromb Haemost 2013; 11: 1103–10., , , , , , .
Final decision: D. Lane, 15 March 2013
- Issue online: 3 JUL 2013
- Version of Record online: 3 JUL 2013
- Accepted manuscript online: 29 MAR 2013 06:15AM EST
- Manuscript Accepted: 15 MAR 2013
- Manuscript Received: 7 NOV 2012
- National Basic Research Program of China. Grant Number: 2013CB966800
- Ministry of Health. Grant Number: 201202017
- chromosome breakpoints;
- chromosome deletion;
- DNA copy number variations;
- DNA end-joining repair;
- hemophilia A
Large deletions in the F8 gene are responsible for approximately 3% of severe hemophilia A (HA) cases. However, only a few breakpoints in large deletions have been characterized.
To identify large deletions in the F8 gene and to characterize the molecular mechanisms leading to these deletions.
Patients and methods
We used AccuCopy technology, a copy number variation (CNV) genotyping method based on multiplex competitive amplification, to confirm deletions in index patients and to screen potential female carriers in 10 HA families. Also, breakpoints of these large deletions were characterized by a primer walking strategy and genome walking technique.
Ten large deletions and four female carriers were identified by AccuCopy. The extents of deleted regions ranged from 1.3 to 68.5 kb. Exact breakpoints of these deletions were successfully characterized. Eight of them presented microhomologies at breakpoint junctions and several recombination-associated elements (repetitive elements, non-B conformation forming motifs and sequence motifs) were also observed in close proximity to the junctions.
AccuCopy technology is a reliable and efficient tool for detecting large deletions in the F8 gene and identifying HA female carriers. The genome walking technique is a highly specific, efficient and versatile method for characterizing the deletion breakpoints. Molecular characterization of deletion breakpoints revealed that non-homologous end joining and microhomology-mediated replication-dependent recombination were the major causative mechanisms of the 10 large deletions in the F8 gene.