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.

Decoding Non-coding Variants: Recent Approaches to Studying Their Role in Gene Regulation and Human Diseases

“…New methods have been developed to perform high-throughput functional evaluations of variants to determine causal mechanisms linked to human diseases ( Table 1 , Ref. [ 43 – 47 , 82 , 86 , 88 , 89 , 101 , 103 , 104 , 106 , 113 – 118 ]). Changes in chromatin interaction maps, TF–DNA binding affinity, gene expression, and translation efficiency provide evidence to support the role of many disease-associated variants.…”

“…Krooss et al . [ 113 ] described the pathomechanism of a non-GWAS SNP found in four families with moderate to severe hemophilia B. The variant created a U1snRNP binding site in the 3′ UTR region of the coagulation factor 9 ( F9 ) mRNA (c.2545A>G).…”

“…The variant created a U1snRNP binding site in the 3′ UTR region of the coagulation factor 9 ( F9 ) mRNA (c.2545A>G). The binding of U1snRNP inhibited polyadenylation and proper 3′-end processing, which resulted in mRNA degradation and reduced expression of F9 [ 113 ]. Bauwens et al .…”

“…Our recommendation and most strategies reviewed here rely on mining public databases or previous knowledge. When these options are unavailable, pedigree WGS combined with patient-derived iPSCs and transcriptomics of differentiated cells provides an alternative to identify de novo variants in specific cases [ 113 – 115 , 123 – 126 ].…”

Two sides of the same medal: Noncoding mutations reveal new pathological mechanisms and insights into the regulation of gene expression

“…A similar mutation was detected in the F9 gene of some hemophilia B patients (Vielhaber et al, 1993). In both cases, a new U1 snRNP binding site is generated and results in the suppression of the authentic PAS (Krooss et al, 2020; Langemeier et al, 2012). Studying these pathomechanisms helps to decipher the mechanism of U1 snRNP mediated inhibition of 3′ end processing that is still not completely unraveled.…”