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

Autoři: Simon Krooss aff001;  Sonja Werwitzke aff003;  Johannes Kopp aff001;  Alice Rovai aff002;  Dirk Varnholt aff003;  Amelie S. Wachs aff001;  Aurelie Goyenvalle aff004;  Annemieke Aarstma-Rus aff005;  Michael Ott aff002;  Andreas Tiede aff003;  Jörg Langemeier aff001;  Jens Bohne aff001
Působiště autorů: Institute of Virology, Hannover Medical School, Hannover, Germany aff001;  Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School and Twincore Centre for Experimental and Clinical Infection Research, Hannover, Germany aff002;  Clinic of Hematology, Oncology and Hemostaseology, Hannover Medical School, Hannover, Germany aff003;  Université de Versailles St-Quentin en Yvelines, INSERM U1179, France aff004;  Leiden University Medical Center, Leiden, Netherlands aff005;  Pediatric Intensive Care Unit, Children’s Hospital Bielefeld, Germany aff006
Vyšlo v časopise: Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B. PLoS Genet 16(4): e32767. doi:10.1371/journal.pgen.1008690
Kategorie: Research Article


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.

Klíčová slova:

HeLa cells – CHO cells – Introns – Messenger RNA – Northern blot – Sequence motif analysis – Transfection – Antisense RNA


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