Analyzing intracellular F8 mRNA and Protein of a Methionine Missense Mutation (c.[2T>G];[0]) in a Patient-Specific IPS Cell Model

Introduction Hemophilia A (HA) is a common, X-linked, recessive disorder caused by deficiency or dysfunction of coagulant factor VIII. In about 20% of patients with severe HA, treatment with replacement FVIII is complicated due to the development of inhibitory antibodies against FVIII. The intracellular cross-reactive material (CRM) status of a specific mutation is believed to affect the inhibitory risk. F8 null mutations, like large deletions, nonsense mutations and intron 22 inversions (I22I), have the highest risk for inhibitor formation, and had been grouped so far as CRM-negative. Missense mutations that still result in some protein synthesis have the lowest risk for inhibitor development. Here we present one HA-patient (PMet) with a F8 missense mutation located in the start codon methionine (c.[2T>G];[0]), and one HA-patient with a large deletion (ex7-9). The index patient PMet shows FVIII:CChr of <1% and suffered from high inhibitor titers. His sister is a hemophilia carrier and gave birth to a hemophilic son in June 2018 also showing FVIII activity levels of <1%. Here, we analyzed the CRM status (F8 mRNA and protein) of both patients in IPS differentiated vascular endothelial cells (vEC) and compared the data to wild-type vEC.

Materials and Methods Isolated PBMCs from PMet and PDel were expanded for erythroid progenitors (EPCs) and reprogrammed into stable IPS clones. After proof of pluripotency IPS cells were differentiated into day 9 old vascular endothelial cells and analyzed for intracellular F8 mRNA using overlapping RT-PCR. Protein was localized with an ApoTome2 microscope by immunofluorescent staining with a monoclonal antibody targeting FVIII heavy chain (GMA-012), costained with anti-PDI (ER-marker) or anti-TGN46 (trans-Golgi marker).

Results PMet presents complete F8 mRNA, while PDel presents the expected gap between ex7-9. FVIII protein is not detectable in differentiated vEC from PMet and PDel, while 90% of wild-type vECs present the majority of FVIII located in the ER.

Conclusion Our cellular model enables us to detect intracellular FVIII in wild-type differentiated vEC, while both PDel and PMet are missing any intracellular FVIII. We propose that the missense mutation c.[2T>G];[0] can be classified as null mutation not able to produce intracellular truncated protein (alternative start codon exon1 aa37).

Publication History

Article published online:
13 November 2020