From PGT-M discovery to mechanism: functional validation of novel compound heterozygous RAG1 mutations in severe combined immunodeficiency.

PURPOSE: Severe combined immunodeficiency (SCID) is a life-threatening primary immunodeficiency disorder. This study aimed to identify novel recombination activating gene 1 (RAG1) variants in a Chinese pedigree and characterize their impact on protein structure and function, providing a genetic basis for preimplantation genetic testing for monogenic (PGT-M) cycle.

METHODS

Potential RAG1 mutations of the probands were screened by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Configuration predictions of the variants were achieved using SWISS-MODEL. PROVEAN, PolyPhen-2, and MutationTaster were used to predict their pathogenicity. Isogenic pre-B cell lines carrying the mutations were established via CRISPR-Cas9 RNP editing. Functional impacts were assessed through western blotting, proliferation ability, and apoptosis analysis.

RESULTS

We identified novel compound heterozygous RAG1 variants c.946T > G (p.C316G) and c.1197_1199del (p.L400del) in two affected siblings with typical SCID. Familial genotyping confirmed autosomal recessive inheritance, with each parent as an asymptomatic carrier of one variant. Both mutations were highly conserved and predicted to be pathogenic. Structural modeling revealed disruption of RAG1 secondary and tertiary structure, affecting zinc-binding (p.C316G) and hydrogen-bonding (p.L400del) interactions. Functional studies demonstrated markedly reduced RAG1 protein expression, synergistic impairment of RAG2 expression, and significantly elevated apoptosis in double-mutant pre‑B cells. Further investigation indicated dysregulation of the PI3K/AKT1/FOXO1 pathway, evidenced by increased phosphorylation of AKT1 and FOXO1.

CONCLUSIONS

Our study provides genetic and functional evidence that biallelic RAG1 p.C316G and p.L400del mutations act synergistically to cause SCID through protein destabilization, disruption of RAG1/RAG2 complex integrity, and induction of pre‑B cell apoptosis likely mediated by PI3K/AKT1/FOXO1 signaling dysregulation. These findings expand the mutational spectrum of RAG1 and support the clinical application of PGT-M for affected families.