[Analysis of Emerging Gene Variant and Molecular Mechanism Study of a Patient with Inherited FXI Deficiency]

To investigate the pathogenic mechanisms in a patient with coagulation factor Ⅺ (FⅪ) deficiency caused by a de novo F11 gene mutation.

Next generation sequencing （NGS） was used to identify the mutation sites combined with Sanger sequencing. Patients were tested for coagulation factor Ⅺ activity (FⅪ:C) and coagulation factor Ⅺ antigen (FⅪ:Ag). Predict the mutation pathogenicity conservatism by bioinformatics tools, and analyze the structural changes of proteins using SWISS-MODEL and Pymol software to explore the possible molecular pathogenic mechanisms. Constructed the targeted mutation plasmid, transfected 293T cells (HEK293T), and assessed the mRNA level of F11 gene and FⅪ protein expression by qRT-PCR and Western blot. Stable cell lines were constructed using CRISPR Cas9 technology to further study the molecular mechanism of mutation.

This patient had an FⅪ:C of only 1.54%, the presence of a pure missense mutation c.1249G>A (p.Gly417Arg) in exon 11 of the F11 gene, and the predicted pathogenicity score is 0.999. The 3D protein structure model analysis indicated that a shift from glycine to arginine at position 417, a change in amino acid structure, and an altered hydrogen bonding network. qRT-PCR and Western Blot showed that the difference in the expression levels of F11 mRNA and FⅪ protein between wild-type and mutant cells was not statistically significant, and the expression of secreted proteins in the supernatants of the cells was essentially normal. The trend of qRT-PCR and Western blot results of the stable cell lines was consistent with transient transfection.

This patient's coagulation factor Ⅺ deficiency was caused by the c.1249G>A (p. Gly417Arg) pure missense mutation in exon 11 of the F11 gene, which is the first report of its kind.