X-linked agammaglobulinemia (XLA) is an X-linked inborn error of immunity caused by variants in the BTK gene. Variants in the 5’-untranslated region (UTR) of the BTK gene account for approximately 2% of all cases, but their detailed molecular pathogenesis remains unclear. In this study, we identified 5’-UTR variants in two XLA families and examined promoter activity. Whole-exome sequencing revealed the 5’-UTR variants (patient 1: c. -358-3G>T; patients 2 and 3: c. -356-6_-3del AAAG) in three patients from two families with XLA. Reduced BTK protein expression by flow cytometry and reduced mRNA expression by quantitative PCR were observed in all three patients. Quantitative PCR on nascent RNA identified a marked decrease in BTK transcriptional activity. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) confirmed that the region near the variants adopts a closed chromatin structure. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) results confirmed that the variants reduce the binding of the transcription factor PU.1. The 5’-UTR variants observed in these two families were identified as critical sites for PU.1 binding, suggesting that these sites are important for promoter activity. Further elucidation of the pathogenesis is anticipated through the accumulation of cases with variants in the 5’-UTR of the BTK gene.
