Abstract
Incorporation of distant intronic sequences in mature mRNA is an underappreciated cause of genetic disease. Several disease-causing pseudoexons have been found to contain repetitive elements such as Alu elements. This study describes an original pathological mechanism by which a small intronic deletion leads to Alu exonization. We identified an intronic deletion, c.2113+461_2113+473del, in the F8 intron 13, in two individuals with mild hemophilia A. In vivo and in vitro transcript analysis found an aberrant transcript, with an insertion of a 122-bp intronic fragment (c.2113_2114ins2113+477_2113+598) at the exon 13-14 junction. This out-of-frame insertion is predicted to lead to truncated protein (p.Gly705Aspfs(∗)37). DNA sequencing analysis found that the pseudoexon corresponds to antisense AluY element and the deletion removed a part of the poly(T)-tail from the right arm of these AluY. The heterogenous nuclear riboprotein C1/C2 (hnRNP C) is an important antisense Alu-derived cryptic exon silencer and binds to poly(T)-tracts. Disruption of the hnRNP C binding site in AluY T-tract by mutagenesis or hnRNP C knockdown using siRNA in HeLa cells reproduced the effect of c.2113+461_2113+473del. The screening of 114 unrelated families with mild hemophilia A in whom no genetic event was previously identified found a deletion in the poly(T)-tail of AluY in intron 13 in 54% of case subjects (n = 61/114). In conclusion, this study describes a deletion leading to Alu exonization found in 6.1% of families with mild hemophila A in France.