Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

Abstract

Loss-of-function mutations in the human coagulation factor 9 (F9) gene lead to hemophilia B. Here, we dissected the consequences and the pathomechanism of a non-coding mutation (c.2545A>G) in the F9 3’ untranslated region. Using wild type and mutant factor IX (FIX) minigenes we revealed that the mutation leads to reduced F9 mRNA and FIX protein levels and to lower coagulation activity of cell culture supernatants. The phenotype could not be compensated by increased transcription. The pathomechanism comprises the de novo creation of a binding site for the spliceosomal component U1snRNP, which is able to suppress the nearby F9 poly(A) site. This second, splicing-independent function of U1snRNP was discovered previously and blockade of U1snRNP restored mutant F9 mRNA expression. In addition, we explored the vice versa approach and masked the mutation by antisense oligonucleotides resulting in significantly increased F9 mRNA expression and coagulation activity. This treatment may transform the moderate/severe hemophilia B into a mild or subclinical form in the patients. This antisense based strategy is applicable to other mutations in untranslated regions creating deleterious binding sites for cellular proteins. The elucidation of the pathomechanisms of non-coding variants yields important insights into diseases as well as cellular processes causing the defect. Although these variants may account for the majority of phenotypic variation, only a minority of them can be explained mechanistically. The human coagulation factor 9 3’ UTR variant described here converts a non-essential sequence motif into a U1snRNP-binding site with deleterious effects on RNA 3’ end processing at the nearby poly(A) site. Poly(A) site suppression by U1snRNP was described before and it normally protects cellular mRNAs from premature termination. However, if misled by creation of a U1 site close the authentic poly(A) site as in the F9 3’ UTR, this nuclear surveillance mechanism results in the opposite. Since recognition by U1snRNP depends on sequence complementarity we were able to use antisense oligonucleotides to mask the mutant site and partially restored F9 mRNA levels. This antisense based strategy may be applicable to other variants in untranslated regions, which create deleterious binding sites for cellular proteins.