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Background <p>Hemophilia B (HB), an X-linked recessive disorder, results from variants in the coagulation factor IX gene (<i>F9</i>). The <i>F9</i> c.520 + 13&#xa0;A &gt; G variant is a recurrent intronic variant in HB patients, accounting for 15.05% of all documented <i>F9</i> intronic variants. Despite prior predictions of its impact, the molecular mechanism associated with moderate to mild HB remains undissected.</p> Materials and methods <p>In silico predictions and splicing-competent cDNA constructs were used to assess the impact of <i>F9</i> c.520 + 13&#xa0;A &gt; G on mRNA splicing. Factor IX (FIX) variant (p.V174delinsGHNLM) expression in HEK293T cells was evaluated using activated partial thromboplastin time, enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence analyses. Structural modeling and molecular dynamics simulations were performed to evaluate the structural impact of the variant. Engineered U1 small nuclear RNA (U1snRNA) was challenged with <i>F9</i> full-length splicing-competent constructs to evaluate splicing correction.</p> Results <p>The <i>F9</i> c.520 + 13&#xa0;A &gt; G variant caused nearly complete aberrant splicing, producing the <i>F9</i> c.520_521insGTCATAATCTGA insertion and the in-frame FIX p.V174delinsGHNLM variant. A small amount (approximately 10%) of wild-type FIX was also detected. We characterize the p.V174delinsGHNLM variant, which exhibited impaired secretion and increased intracellular accumulation. Interestingly, an engineered U1snRNA partially rescued aberrant splicing, restoring functional FIX levels to approximately 40%.</p> Conclusion <p>This study elucidates the molecular mechanism of the <i>F9</i> c.520 + 13&#xa0;A &gt; G variant, which activates a cryptic 5′ splice site in intron 5, leading to an in-frame FIX (p.V174delinsGHNLM) with secretion defects and loss of protein function. And Engineered U1snRNA partially rescued the splicing defect.</p> Graphical abstract <p></p>

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Molecular mechanisms and therapeutic strategies for the recurrent F9 (c.520 + 13 A > G) variant in hemophilia B

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月8日

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  • Huayang Zhang,
  • Chong Wang,
  • Meixiu Gu,
  • Zhimin Meng,
  • Yichao Guo,
  • Weitao Zhang,
  • Dario Balestra,
  • Wei Guo,
  • Beili Wang

摘要

Background

Hemophilia B (HB), an X-linked recessive disorder, results from variants in the coagulation factor IX gene (F9). The F9 c.520 + 13 A > G variant is a recurrent intronic variant in HB patients, accounting for 15.05% of all documented F9 intronic variants. Despite prior predictions of its impact, the molecular mechanism associated with moderate to mild HB remains undissected.

Materials and methods

In silico predictions and splicing-competent cDNA constructs were used to assess the impact of F9 c.520 + 13 A > G on mRNA splicing. Factor IX (FIX) variant (p.V174delinsGHNLM) expression in HEK293T cells was evaluated using activated partial thromboplastin time, enzyme-linked immunosorbent assay, Western blot analysis, and immunofluorescence analyses. Structural modeling and molecular dynamics simulations were performed to evaluate the structural impact of the variant. Engineered U1 small nuclear RNA (U1snRNA) was challenged with F9 full-length splicing-competent constructs to evaluate splicing correction.

Results

The F9 c.520 + 13 A > G variant caused nearly complete aberrant splicing, producing the F9 c.520_521insGTCATAATCTGA insertion and the in-frame FIX p.V174delinsGHNLM variant. A small amount (approximately 10%) of wild-type FIX was also detected. We characterize the p.V174delinsGHNLM variant, which exhibited impaired secretion and increased intracellular accumulation. Interestingly, an engineered U1snRNA partially rescued aberrant splicing, restoring functional FIX levels to approximately 40%.

Conclusion

This study elucidates the molecular mechanism of the F9 c.520 + 13 A > G variant, which activates a cryptic 5′ splice site in intron 5, leading to an in-frame FIX (p.V174delinsGHNLM) with secretion defects and loss of protein function. And Engineered U1snRNA partially rescued the splicing defect.

Graphical abstract