Divergent processing of FVIII light chain variants: secretory potential versus proteasomal retention.

In 20-30% of severe Haemophilia A (HA) patients, FVIII replacement therapy is hindered by inhibitory antibodies. Nonsense mutations in the FVIII light chain (A3-C1-C2) carry a higher risk of inhibitor formation than those in the heavy chain (A1-A2-B). The underlying molecular mechanism remains unclear. Using induced pluripotent stem (iPS) cells from HA patients, we developed two types of endothelial cell models (iLEC and iLSEC) that mimic native F8 mRNA expression and protein synthesis. Immunoassays detected FVIII protein in wild-type, I22I, and two high inhibitor risk light chain variants (R1960X, R2228X). Co-staining with ER markers (PDI, BiP) revealed differential processing: R1960X exhibit enhanced proteasomal degradation with SEL1L, essential for MHC-I peptide loading, possibly contributing to higher immunogenicity. In contrast, R2228X showed a pattern more similar to wild type, suggesting partial secretory potential. Although a mild colocalization with SEL1L was observed, it was not significant. Clinically, this patient did not develop inhibitors. In addition, exploratory in silico peptide binding predictions suggested that R1960X may generate a higher number of FVIII-derived epitopes presented via patient-specific HLA alleles compared to R2228X, further supporting differential immunogenicity. The intron 22 inversion variant also showed detectable FVIII protein, which was deglycosylated and retained in the ER but did not co-localize with SEL1L; no inhibitor was observed in this case either. This cellular model shows reduced variability compared to primary cells, enabling patient specific FVIII variant analyses, including intracellular processing, within the genetic background of the respective patient.

PMID: 41307134