F9 variant is not secreted

In healthy individuals, the coagulation factor IX (FIX) is synthesized as a 461 amino acid precursor (primarily in the liver) and then secreted into plasma where it converts factor X to its active form. FIX zymogen undergoes extensive co- and post-translational modifications, including but not limited to glycosylation and γ-carboxylation. A deficiency or dysfunction of FIX caused by mutations in the F9 gene is associated with a blood clotting disorder hemophilia B (HB). The FIX protein level may be decreased in the circulation by F9 mutations affecting FIX protein synthesis, stability, or secretion. The carboxyl-terminal region of FIX contains several natural missense mutations such as Y450C, W453R and T458K, which impair secretion of FIX and result in mild to severe forms of HB (Kurachi S et al. 1997; Branchini A et al. 2013). In addition, exon 5 of the F9 gene contains dense splicing regulatory information that overlap with the amino acid code (Tajnik M et al. 2016). Under normal conditions exon 5 is correctly recognized by the spliceosome and mostly included in the final transcript. This leads to the production of a normal FIX protein that folds correctly in the endoplasmic reticulum (ER) and is efficiently secreted into the blood to activate coagulation. Several F9 mutations in exon 5 result in exon skipping (Tajnik M et al. 2016; Odaira K et al. 2019; Katneni UK et al. 2019). The exonic splicing mutations (ESM) can be divided into three major groups which define their molecular basis. In the first group, two synonymous variants, V153V and R162R, affect binding of splicing factors and induce severe exon skipping with the production of a non-functional mRNA (Tajnik M et al. 2016). The second type of ESM, such as A164V and Q167H, showed partial splicing defects producing low amounts of normally spliced transcript that, when translated, resulted in a defective FIX protein with a significantly reduced, but not completely abolished, secretion. However, the lower amounts of the secreted proteins maintained a normal specific coagulant activity. Lastly, mutation L163F (ESM group 3) showed a splicing defect but the resulting amino acid change severely affected FIX secretion (Tajnik M et al. 2016). Another HB-associated F9 variant caused abnormal mRNA splicing, r.83_88del, and produced the mutant FIX protein (p.C28_V30delinsF), which is an in-frame mutant at the signal peptide cleavage site (Odaira K et al. 2019). The FIX C28_V30delinsF variant was found to be retained in the ER without being secreted (Odaira K et al. 2019). Studies also showed defective secretion of HB-associated F9 nonsense mutations such as R294*, R298* and R384* (Branchini A et al. 2017; Pinotti M et al. 2012). The mechanism through which nonsense mutations impair gene expression and cause human genetic disease consists of premature translation termination, and the synthesis of truncated proteins with loss‐of‐function features (Mort M et al. 2008). These mutations can trigger nonsense‐mediated decay of mRNA, that degrades mRNA transcripts that harbor a premature translation-termination codon (PTC), thus reducing the synthesis of truncated proteins (Khajavi et al. 2006; Kurosaki T & Maquat LE 2016). However, the mechanism of ribosome readthrough, which consists of misrecognition of the premature stop codon by an aminoacyl‐tRNA instead of the termination factors can restore translation impaired by nonsense mutations (Rospert S et al. 2005). The occurrence of spontaneous ribosome readthrough over F9 R294* and F9 R298* nonsense mutations led to the synthesis of traces of full‐length FIX in HB patients (Pinotti M et al. 2012). A drug-induced ribosome readthrough targeting nonsense variants is considered as a potential treatment of inherited coagulation factor disorders (Branchini A et al. 2017; Ferrarese M et al. 2018; Balestra D & Branchini A 2019).<p>The Reactome event describes intracellular accumulation and/or decreased secretion of FIX due to different HB-related genetic alterations spread throughout the F9 gene. The F9 variants are described in relation to changes in the protein sequence. Defective splicing events induced by F9 mutations are not shown here.

在健康个体中，凝血因子IX（FIX）作为一种由461个氨基酸组成的先导蛋白（主要在肝脏合成）被合成，随后分泌至血浆中，将其转化为活性形式。FIX原酶经历广泛的共翻译和翻译后修饰，包括但不限于糖基化和γ-羧化。由F9基因突变引起的FIX缺乏或功能障碍与血友病B（HB）这一凝血障碍相关。F9突变可能通过影响FIX蛋白的合成、稳定或分泌，导致循环中FIX蛋白水平降低。FIX的羧基末端区域含有多个自然错义突变，如Y450C、W453R和T458K，这些突变损害FIX的分泌，导致血友病B的轻度至重度形式（Kurachi S等，1997；Branchini A等，2013）。此外，F9基因的第5外显子含有密集的剪接调控信息，与氨基酸编码重叠（Tajnik M等，2016）。在正常条件下，第5外显子被剪接体正确识别，主要包含在最终转录本中。这导致产生正常的FIX蛋白，在粗面内质网（ER）中正确折叠，并高效分泌至血液以激活凝血。F9基因第5外显子的多个突变导致外显子跳跃（Tajnik M等，2016；Odaira K等，2019；Katneni UK等，2019）。外显子剪接突变（ESM）可分为三大类，定义了它们的分子基础。在第一组中，两个同义变异体V153V和R162R影响剪接因子的结合，并诱导严重的跳跃外显子，产生非功能性mRNA（Tajnik M等，2016）。第二种类型的ESM，如A164V和Q167H，表现出部分剪接缺陷，产生少量正常剪接的转录本，翻译后导致具有显著降低但非完全丧失分泌功能的FIX蛋白。然而，分泌蛋白的较低量保持了正常的特异性凝血活性。最后，突变L163F（ESM组3）表现出剪接缺陷，但导致的氨基酸改变严重影响了FIX的分泌（Tajnik M等，2016）。另一种与HB相关的F9变异体引起异常mRNA剪接，r.83_88del，并产生突变的FIX蛋白（p.C28_V30delinsF），这是一种在信号肽切割位点处的内含子突变（Odaira K等，2019）。FIX C28_V30delinsF变异体被发现保留在ER中，未分泌（Odaira K等，2019）。研究还显示，HB相关的F9无义突变（如R294*、R298*和R384*）导致缺陷分泌（Branchini A等，2017；Pinotti M等，2012）。无义突变损害基因表达并导致人类遗传疾病的发生机制包括翻译终止的提前终止和具有功能丧失特征的截断蛋白的合成（Mort M等，2008）。这些突变可以触发mRNA的无义介导的降解，降解携带提前终止密码子（PTC）的mRNA转录本，从而减少截断蛋白的合成（Khajavi等，2006；Kurosaki T & Maquat LE，2016）。然而，核糖体读通机制，即由氨基酸酰-tRNA而非终止因子误识别提前终止密码子，可以恢复由无义突变引起的翻译损伤（Rospert S等，2005）。F9 R294*和F9 R298*无义突变上自发核糖体读通的发生导致HB患者中全长度FIX的微量合成（Pinotti M等，2012）。针对无义变体的药物诱导的核糖体读通被认为是一种治疗遗传性凝血因子障碍的潜在治疗方法（Branchini A等，2017；Ferrarese M等，2018；Balestra D & Branchini A，2019）。Reactome事件描述了由于F9基因中的不同HB相关遗传变异导致FIX在细胞内积累和/或分泌减少。F9变异体与蛋白质序列的变化相关。由F9突变引起的缺陷剪接事件在此未显示。