Simhadri Vijaya L, Hamasaki-Katagiri Nobuko, Lin Brian C, Hunt Ryan, Jha Sujata, Tseng Sandra C, Wu Andrew, Bentley Amber A, Zichel Ran, Lu Qi, Zhu Lily, Freedberg Darón I, Monroe Dougald M, Sauna Zuben E, Peters Robert, Komar Anton A, Kimchi-Sarfaty Chava
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.
Department of Biological, Geological & Environmental Sciences, Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio, USA.
J Med Genet. 2017 May;54(5):338-345. doi: 10.1136/jmedgenet-2016-104072. Epub 2016 Dec 22.
Haemophilia B is caused by genetic aberrations in the 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 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.
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 in 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.
The pathogenic basis for one synonymous mutation (Val107Val) in the gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.
B型血友病由该基因的遗传畸变引起。其中大多数是改变凝血因子IX（FIX）一级结构的非同义突变。然而，临床报道一种同义突变c.459G>A（Val107Val）导致轻度B型血友病（FIX凝血活性为正常的15%-20%）。这些患者的mRNA未显示内含子跳跃或保留和/或mRNA水平变化，这表明mRNA完整性对受影响个体疾病的起源没有影响。本研究的目的是阐明能够解释这种同义突变患者疾病表现的分子机制。
我们通过计算机分析并在稳定细胞系中重现c.459G>A（Val107Val）突变，分析FIX缺乏的分子机制。使用构象和非构象敏感抗体、有限胰蛋白酶消化、FIX活性测定、与其他蛋白质的相互作用以及翻译后修饰来评估同义突变的生物物理和生化后果。
发现该基因中的Val107Val同义突变显著降低FIX表达。我们的结果表明，这种突变减缓FIX翻译并影响其构象，导致细胞外蛋白水平降低。构象改变并未改变突变蛋白的比活性。
确定了与B型血友病相关的该基因中一种同义突变（Val107Val）的致病基础。对这种同义变异的机制理解为指导和开发未来的治疗方法提供了潜力。
