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Genotype-phenotype correlations
Original article
Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B
  1. Vijaya L Simhadri1,
  2. Nobuko Hamasaki-Katagiri1,
  3. Brian C Lin1,
  4. Ryan Hunt1,
  5. Sujata Jha2,
  6. Sandra C Tseng1,
  7. Andrew Wu1,
  8. Amber A Bentley2,
  9. Ran Zichel1,
  10. Qi Lu3,
  11. Lily Zhu3,
  12. Darón I Freedberg4,
  13. Dougald M Monroe5,
  14. Zuben E Sauna1,
  15. Robert Peters3,
  16. Anton A Komar2,
  17. Chava Kimchi-Sarfaty1
  1. 1Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, USA
  2. 2Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA
  3. 3Hematology Research, Cambridge, Massachusetts, USA
  4. 4Laboratory 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
  5. 5Department of Hematology/Oncology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
  1. Correspondence to Dr Chava Kimchi-Sarfaty, Division of Plasma Protein Therapeutics, FDA|CBER|OTAT, Federal Research Center at White Oak, Building 52/72, Room 4118, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA; Chava.Kimchi-Sarfaty{at}fda.hhs.gov
  2. Dr Anton A Komar, Center for Gene Regulation in Health and Disease, Department of Biological, Geological & Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA; A.Komar{at}csuohio.edu
  3. Dr Zuben E Sauna, Division of Plasma Protein Therapeutics, FDA | CBER | OTAT, Federal Research Center at White Oak, Building 52/72, Room 4120, 10903 New Hampshire Avenue, Silver Spring, MD 20993, USA; Zuben.Sauna{at}fda.hhs.gov

Abstract

Background Haemophilia B is caused by genetic aberrations in the F9 gene. 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). The F9 mRNA 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.

Methods 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.

Results The Val107Val synonymous mutation in F9 was 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.

Conclusions The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.

  • hemophilia
  • synonymous mutation
  • factor IX
  • protein folding
  • disease

https://doi.org/10.1136/jmedgenet-2016-104072

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    Footnotes

    • VLS and NHK contributed equally.

    • Contributors VLS, NHK, ZES, RP, AAK and CK-S conceived and designed the study. QL, LZ, RP and DIF provided materials and contributed analytical tools. VLS, NHK, SJ and BCL performed most of the experiments. VLS, NHK, SJ, BCL, SCT, AW, AAB, RZ, QL, LZ, DIF, ZES, AAK and CK-S conducted analyses and interpreted results. ZES, VLS, NHK, BCL, AAK and CK-S drafted and prepared the manuscript. ZES, AAK, CK-S, RP, BCL, VLS, NHK and DIF edited and finalised the manuscript.

    • Funding American Heart Association (13GRNTI7070025), National Institutes of Health (1R15HL121779-01A1).

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.