Genotype-phenotype correlations in specific granule deficiency: loss of DNA-binding ability and impaired nuclear localization cause severe manifestations due to the c.655_665del CEBPE variant.

Introduction: Specific granule deficiency (SGD)-a rare innate immune disorder-is classified into types 1 and 2 (SGD-1 and -2). SGD-1 is caused by variants of the CCAAT/enhancer-binding protein epsilon (C/EBPε) gene.

Methods: We assessed the molecular mechanisms underlying C/EBPε dysfunction in SGD-1, caused by the frameshift variant (c.655_665del; del11) that we previously reported. We compared the functions of del11 with those of the previously reported p.Arg247_Ser248del (ΔRS) variant and wild-type (WT) C/EBPε.

Results: Forced expression in embryonic stem cells revealed that both the del11 and ΔRS variants inhibited C/EBPε-mediated target gene induction, indicating a loss of transcriptional activity. In NIH3T3 cells, WT and ΔRS C/EBPε were localized to the nucleus, whereas del11 C/EBPε showed cytoplasmic retention and induced morphological changes in expressing cells. Protein-protein interaction analyses demonstrated that both mutants failed to interact with the transcription factors GATA-binding protein 1 and purine-rich box-1. DNA-binding assays revealed that del11 C/EBPε completely lost its ability to bind to target DNA sequences, whereas WT and ΔRS C/EBPε retained binding capacity. Thus, del11 disrupts multiple C/EBPε functions, including nuclear localization, DNA-binding, and protein interactions.

Conclusion: Based on these functional differences, we propose a novel classification of SGD-1 into types 1a and 1b (SGD-1a and -1b). Patients with SGD-1b,-associated with nonsense and frameshift variants, such as del11,-exhibit more severe clinical phenotypes than those with SGD-1a,-associated with missense variants and in-frame deletions. This study offers novel insights into the pathogenesis of SGD and genotype-phenotype correlations, potentially informing the development of future therapeutic strategies.