title: Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.
abstract: Haemophilia B is caused by genetic aberrations in theF9gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). TheF9mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation.
  
  
  
  We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation.
  
  
  
  The Val107Val synonymous mutation inF9was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein.
  
  
  
  The pathogenic basis for one synonymous mutation (Val107Val) in theF9gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Vijaya L
authors.initials: VL
authors.lastname: Simhadri
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Hamasaki-Katagiri
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Lin
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Hunt
authors.affiliation: Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.
authors.forename: Sujata
authors.initials: S
authors.lastname: Jha
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Sandra C
authors.initials: SC
authors.lastname: Tseng
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wu
authors.affiliation: Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.
authors.forename: Amber A
authors.initials: AA
authors.lastname: Bentley
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Zichel
authors.affiliation: Hematology Research, Cambridge, Massachusetts, USA.
authors.forename: Qi
authors.initials: Q
authors.lastname: Lu
authors.affiliation: Hematology Research, Cambridge, Massachusetts, USA.
authors.forename: Lily
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Laboratory of Bacterial Polysaccharides, Division of Bacterial Products and Allergenic Products, Office of Vaccines Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Darón I
authors.initials: DI
authors.lastname: Freedberg
authors.affiliation: Department of Hematology/Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Dougald M
authors.initials: DM
authors.lastname: Monroe
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Zuben E
authors.initials: ZE
authors.lastname: Sauna
authors.affiliation: Hematology Research, Cambridge, Massachusetts, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Peters
authors.affiliation: Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.
authors.forename: Anton A
authors.initials: AA
authors.lastname: Komar
authors.affiliation: Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Chava
authors.initials: C
authors.lastname: Kimchi-Sarfaty
mesh_terms: D045744:Cell Line, Tumor; D003062:Codon / Q000235:genetics; D005164:Factor IX / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015944:Factor VIIIa / Q000737:chemistry; D057809:HEK293 Cells; D002836:Hemophilia B / Q000235:genetics*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D000069456:Silent Mutation / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D003062:Codon; D050505:Mutant Proteins; D012333:RNA, Messenger; D015944:Factor VIIIa; D005164:Factor IX
keywords: disease; factor IX; hemophilia; protein folding; synonymous mutation