Missense mutation in non-recombinase domain of RAG1 results in B-cell developmental defect

Abstract

Abstract Objective To examine how a single amino acid change to a region of RAG1 that is not related to VDJ recombination affects B-cell development. Methods We studied B-cells within a Rag1 P326G missense mutant mouse (Rag1PG) utilizing flow cytometric analysis to phenotype B-cell development and peripheral populations, as well as biochemical studies examining the B-cell Receptor (BCR) molecular complex and signaling function. We also created a mixed bone marrow chimera to determine which alterations were cell-intrinsic. Results We find that overall there is an ~50% decrease in total B-cells in the Rag1PG mouse compared to wildtype (WT) due to a Pro-B to Pre-B-cell developmental block. Peripheral B cell cytopenias are in the Follicular Zone B-cell compartment, with sparing of the Marginal Zone B-cells. There is decreased Syk expression in Rag1PG—however no differences in BCR signaling strength were appreciated in Rag1PG. In the mixed bone marrow chimera, we find a competitive advantage in the WT developing B-cells, but peripheral B cell populations survive equally well. Conclusion Despite the Rag1PG protein being a single amino acid change to a region of RAG1 not involved in recombination, we nevertheless find a developmental defect consistent with leaky SCID. Given that there is no evidence of altered BCR signaling, and the developmental block occurs at the stage in B-cell development in which VDJ recombination is first required, these data suggest a recombination defect. The mutation is in a ubiquitin ligase domain, and it is possible this alters currently unappreciated roles of RAG1, such as histone degradation to allow RAG1 access to DNA. Our findings illustrate how mutations in the non-recombinase domain of RAG1 may result in leaky SCID.