BTK mutations selectively regulate BTK expression and upregulate monocyte XBP1 mRNA in XLA patients

Abstract

Mutations in the Bruton agammaglobulinemia tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). Unfolded or misfolded proteins can trigger stress pathways in the endoplasmic reticulum (ER), known as unfolded protein response (UPR). The aim was to clarify the involvement of UPR in XLA pathophysiology. By reverse transcription-quantitative PCR, we evaluated the expression of BTK and 12 UPR-related genes in eight patients. Moreover, we assessed the BTK protein expression and pattern in the patients' monocytes by flow cytometry and fluorescence immunocytochemistry. We found a reduced BTK expression in patients with stop codon mutations (P < 0.02). However, missense mutations did not affect BTK expression. Flow cytometry showed a reduction of BTK in patients which was corroborated by an absent or nonfunctional protein synthesis revealed by immunocytochemistry. In contrast with the other UPR-related genes, X-box binding protein 1 (XBP1) was markedly upregulated in the patients (P < 0.01), suggesting Toll-like receptor (TLR) activation since BTK directly interacts with TLRs as a negative regulator and XBP1 can be activated in direct response to TLR ligation. Different BTK mutations can be identified by the BTK expression. Inasmuch as UPR-related genes were downregulated or unaltered in patients, we speculate the involvement of the TLRs-XBP1 axis in the XLA pathophysiology. Such data could be the basis for further studies of this novel pathomechanism concerning XLA.

Keywords: Endoplasmic reticulum stress; HSP90B1; X-linked agammaglobulinemia; interleukin 6; unfolded protein response (UPR).

Figure 1

Expression of BTK in PBMC from XLA patients and healthy controls. Relative expression of the BTK gene in XLA patients with mutations that lead to a premature stop codon (XLA-SC; n = 4), XLA patients with missense mutations (XLA-M; n = 4), and controls (C; n = 8). The y-axis represents the quantitative data of the relative mRNA expression of the BTK gene. Data from RT-qPCRs were generated from three reference genes: GAPDH, HPRT1, and GAPDH+HPRT1 (in combination). Data are shown as median with interquartile range (whiskers: minimum to maximum). Statistical analysis of the variance between the different groups was performed using the test of Kruskal–Wallis and Dunn's Multiple Comparison test was used to compare all pairs of data (*P < 0.05).

Figure 2

Immunolabeling of the BTK protein in PBMC (adherent monocytes) from XLA patients and healthy controls. Immunocytochemistry in PBMC from XLA patients with fluorescent labeling of BTK in green (A, FITC), the cell nucleus in blue (B, Hoechst), and merged in C. Mutations in BTK lead to a lack of BTK protein or nonfunctional protein synthesis, evidenced by abnormal subcellular localization of BTK protein in the cell cytoplasm. Patients XLA01, XLA02, XLA03, and XLA08 have missense mutations. Patients XLA04, XLA05, XLA06, and XLA07 have premature terminations caused by nonsense mutation. Original magnification of 63x.

Figure 3

Expression of ER stress sensor genes in monocytes from XLA patients and healthy controls. Relative expression of 12 genes associated with UPR and the ER stress (see legend and Table2) in XLA patients (XLA; n = 6) and healthy controls (C; n = 6). The y-axis represents the quantitative data of the relative mRNA expression of genes. Data from RT-qPCRs were generated from GAPDH+HPRT1 (in combination) as the reference gene. Data are shown as median with interquartile range (whiskers: minimum to maximum). Statistical analyses of the variance between the two groups (C vs. XLA) were performed using Mann–Whitney test (*P < 0.05; **P < 0.01).

Figure 4

Expression of IL6 in PBMC from XLA patients and healthy controls. Relative expression of the IL6 gene in XLA patients (XLA; n = 7) and controls (C; n = 8). The y-axis represents the quantitative data of the relative mRNA expression of the IL6 gene. Data from RT-qPCRs were generated from three reference genes: GAPDH, HPRT1, and the combination of GAPDH and HPRT1 (shown as “Both” in the legend). Data are shown as mean and SD. Statistical analysis of the difference between the two groups was performed using the Mann–Whitney test (*P < 0.05; **P < 0.01).