Severe haemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing

Background
No F8 genetic abnormality is detected in about 2% of severe haemophilia A patients using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal.

Objective
To characterize, in a genetically unresolved severe haemophilia A patient, a new Xq28 rearrangement disrupting F8 using comprehensive molecular techniques including nanopore sequencing.

Results
Long‐range PCR performed throughout F8 identified a non‐amplifiable region in intron 25 indicating the presence of a genomic rearrangement. F8 mRNA analysis including 3'Rapid Amplification of cDNA Ends and nanopore sequencing found the presence of a F8 fusion transcript in which F8 exon 26 was replaced by a 742 bp pseudo‐exon corresponding to a non‐coding region located at the beginning of the long arm of chromosome X (Xq12; chrX: 66,310,352‐66,311,093, GRCh37/hg19). Cytogenetic Microarray Analysis found the presence of a Xq11.1q12 gain of 3.8 Mb. PCR amplification of junction fragments and FISH analysis found that the Xq11q12 duplicated region was inserted in F8 intron 25 genomic region.

Conclusion
We characterized a novel genomic rearrangement in which a 3.8Mb Xq11.1q12 gain inserted in the F8 intron 25 led to an aberrant fusion transcript in a patient with severe HA, using comprehensive molecular techniques. This study highlights the value of single molecule long‐read sequencing technologies for molecular diagnosis of HA especially when conventional genetic approaches have failed.