Bruton's Tyrosine Kinase Protein Expression Research Articles

Clinical features and mutational analysis of X-linked agammaglobulinemia patients in Malaysia.

Bruton's tyrosine kinase (BTK) is a cytoplasmic protein involved in the B cell development. X-linked agammaglobulinemia (XLA) is caused by mutation in the BTK gene, which results in very low or absent B cells. Affected males have markedly reduced immunoglobulin levels, which render them susceptible to recurrent and severe bacterial infections. Methods: Patients suspected with X-linked agammaglobulinemia were enrolled during the period of 2010-2018. Clinical summary, and immunological profiles of these patients were recorded. Peripheral blood samples were collected for monocyte BTK protein expression detection and BTK genetic analysis. The medical records between January 2020 and June 2023 were reviewed to investigate COVID-19 in XLA. Twenty-two patients (from 16 unrelated families) were molecularly diagnosed as XLA. Genetic testing revealed fifteen distinct mutations, including four splicing mutations, four missense mutations, three nonsense mutations, three short deletions, and one large indel mutation. These mutations scattered throughout the BTK gene and mostly affected the kinase domain. All mutations including five novel mutations were predicted to be pathogenic or deleterious by in silico prediction tools. Genetic testing confirmed that eleven mothers and seven sisters were carriers for the disease, while three mutations were de novo. Flow cytometric analysis showed that thirteen patients had minimal BTK expression (0-15%) while eight patients had reduced BTK expression (16-64%). One patient was not tested for monocyte BTK expression due to insufficient sample. Pneumonia (n=13) was the most common manifestation, while Pseudomonas aeruginosa was the most frequently isolated pathogen from the patients (n=4). Mild or asymptomatic COVID-19 was reported in four patients. This report provides the first overview of demographic, clinical, immunological and genetic data of XLA in Malaysia. The combination of flow cytometric assessment and BTK genetic analysis provides a definitive diagnosis for XLA patients, especially with atypical clinical presentation. In addition, it may also allow carrier detection and assist in genetic counselling and prenatal diagnosis.

Base editor-mediated correction of a BTK Mutation in XLA Patient Hematopoietic Stem Cells restores Human B cell development

IntroductionX-linked agammaglobulinemia (XLA) due to Bruton's tyrosine kinase (BTK) mutations causes lack of circulating mature B lymphocytes (<1%), lack of serum immunoglobulins (IgG/A/M) and increased susceptibility to infections. Correction of BTK mutation in hematopoietic stem/progenitor cells (HSPC) should restore B cell development and humoral immunity. Here, we develop a novel approach that leverages adenine base editors (ABEs) to correct HSPCs from a XLA patient. Materials and methodsPeripheral blood HSPCs were collected from an XLA BTK:c.46C>T (p.Gln16Ter) patient and healthy donors (HD) after G-CSF/plerixafor mobilization. HSPCs were cultured ex vivo (StemSpan™ SFEM II with SCF, FLT3L, TPO) for 2 days before base editing (BE) with mRNA-encoded ABEs (CELLSCRIPT LLC) and gRNAs (Synthego) including ABE8e-SpRY with a gRNA that positions the target adenine in nucleotide 6 of the target site (A6), and ABE8e-SpG or ABE8e-SpCas9-NG with gRNA A7. BE-HSPCs were transplanted two days later into NSG mice conditioned with busulfan (20 mg/kg). Analyses were performed at 12, 16 weeks and animals sacrificed at 20 weeks to assess engraftment and B cell immune reconstitution. ResultsHSPCs BE with ABE8e-SpRY/gRNA A6(SpRY/A6) achieved highest correction at 86.6% BTKc.46C >T and restored BTK protein expression in ex vivo differentiated (14 days, DMEM+G-CSF) CD33+ myeloid cells (Fig.1A) which restored peripheral blood (PB) CD19+CD20+ B cells to HD level (Fig.1C). Phenotype analysis of PB confirmed predominantly mature naïve B cells in BE comparable with HD instead and mostly transitional B cells in mice with naïve-XLA cells (Fig.1F) with CD179a+ that corresponded to Pre-B cells (Fig.1G). Analysis of bone marrow confirmed restored immature (BE: 16.3 vs naïve:1.0, p = 0.001) and mature B cells (BE: 1.2 vs naïve:0.11 p = 0.002) in mice with BE-HSPCs to HD levels while naïve mice comprised of mostly Pre-B-II cells (mean BE: 64.7% vs naïve:81.3%, p = 0.0006). ConclusionsOur studies demonstrate a novel base editing strategy for a XLA missense mutation that overcame the bone marrow arrest of B cells to restore mature B cells in humanized NSG peripheral blood. These findings demonstrate preclinical efficacy of an ABE-based treatment for BTK. The highly efficient-specific correction with base editing holds great promise for gene therapy for XLA disease. [Display omitted]

Abstract P4-09-06: High levels of Bruton's tyrosine kinase (BTK) protein is associated with favorable outcome in breast cancer

Abstract Background: Bruton's tyrosine kinase (BTK) belongs to the TEC family of cytoplasmic tyrosine kinases. BTK promotes survival and proliferation of malignant B-cells. Although BTK is predominantly cytoplasmic in hematopoietic malignancies, BTK may also be localized to cell nuclei. In breast cancer cell lines, BTK was identified as a candidate survival factor based on a large-scale loss-of-function analysis of human tyrosine kinases using RNA interference. We therefore sought to determine whether BTK levels in breast cancer cells were associated with clinical outcome. Methods: For these studies we applied quantitative immunofluorescence-based immunohistochemistry of breast cancer tissue specimens in tissue microarray (TMA) format. All patients underwent upfront breast surgery; hence there are no confounding effects of neoadjuvant treatment on biomarker levels. Within the cancer cell compartment, BTK protein was expressed primarily in nuclei of scattered breast cancer cells. Because of the scattered BTK expression distribution, we developed an analytical approach that considered all percentiles of cell-by-cell BTK protein expression in nuclei of all cancer cells sampled within a tumor tissue. Tumors from patient Cohort A (training set, N=758) was used to define the optimal dichotomized (high vs low) BTK marker by selecting the percentile that dichotomized to have the highest predictive power for progression free survival (PFS). The prognostic value of the BTK marker was subsequently evaluated in tumors of the independent validation Cohort B (N=316) using Kaplan-Meier curves and the Cox proportional hazards model for PFS. Results: The analyses of the training set identified the 94th percentile of BTK cell-by-cell distribution as the optimal predictor of recurrence when dichotomized as High if Qn(0.94)&amp;gt;0.369 and as Low if Qn(0.94)≤0.369. For tissue cores in the training set, 467 of 758 (61.6%) were classified as High BTK and 291 of 758 (38.4%) were classified as Low BTK. Application of the optimal cut point from Cohort A to tumors in the validation Cohort B classified 145/316 cores (45.9%) as High BTK and 171/316 cores (54.1%) as Low BTK. High BTK was associated with longer PFS in both training (p=0.001) and validation sets (p=0.048). The result remained similar and significant in the final multivariate Cox model (HR=0.50; 95% CI 0.26-0.97; p=0.039) after adjusting for pathology and treatment variables. Conclusions: High BTK nuclear protein expression was associated with a significantly more favorable clinical outcome in breast cancer patients, and held up as an independent marker after adjusting for other variables, including hormone receptor status and treatment. Although the association of High BTK is associated with favorable outcome, further studies are warranted to determine whether BTK represents a therapeutic target in breast cancer. Citation Format: Chaudhary LN, Peck AR, Yunguang S, Hu H, Kovatich AJ, Hooke JA, Shriver CD, Mitchell EP, Chervoneva I, Rui H. High levels of Bruton's tyrosine kinase (BTK) protein is associated with favorable outcome in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-09-06.

The Role of Bruton's Tyrosine Kinase in Immune Cell Signaling and Systemic Autoimmunity.

Bruton's tyrosine kinase (BTK) is an intracellular signaling molecule first identified as the molecule affected in X-linked agammaglobulinemia (XLA) patients, who almost completely lack peripheral B cells and serum immunoglobulins. BTK is crucial for B cell development and various B cell functions, including cytokine and natural antibody production. Importantly, it is also expressed in numerous other cells, including monocytes, macrophages, granulocytes, dendritic cells, and osteoclasts. A few rare cases of autoimmune disease in XLA patients have been described. Interestingly, increased BTK protein expression in patients with systemic autoimmune disease appears to be correlated with autoantibody production. In addition, BTK may promote autoimmunity as an important driver of an imbalance in B-T cell interaction. Because of this overwhelming evidence of a pathogenic role of BTK in autoimmunity, several clinical trials in rheumatoid arthritis and systemic lupus erythematosus patients with BTK inhibitors are currently running. Here, we review BTK function in different signaling pathways and in different cell lineages, focusing on the growing body of literature indicating a critical role for BTK in autoimmunity. We also discuss BTK and the promising results of BTK inhibition in animal models of autoimmune disease.

Enhanced Bruton's Tyrosine Kinase Activity in Peripheral Blood B Lymphocytes From Patients With Autoimmune Disease.

Bruton's tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease. Using intracellular flow cytometry, we quantified BTK expression and phosphorylation in subsets of peripheral blood B cells from 30 patients with rheumatoid arthritis (RA), 26 patients with primary Sjögren's syndrome (SS), and matched healthy controls. In circulating B cells, BTK protein expression levels correlated with BTK phosphorylation. BTK expression was up-regulated upon BCR stimulation in vitro and was significantly higher in CD27+ memory B cells than in CD27-IgD+ naive B cells. Importantly, BTK protein and phospho-BTK were significantly increased in B cells from anti-citrullinated protein antibody (ACPA)-positive RA patients but not in B cells from ACPA-negative RA patients. BTK was increased both in naive B cells and in memory B cells and correlated with frequencies of circulating CCR6+ Th17 cells. Likewise, BTK protein was increased in B cells from a major fraction of patients with primary SS and correlated with serum rheumatoid factor levels and parotid gland T cell infiltration. Interestingly, targeting T cell activation in patients with primary SS using the CTLA-4Ig fusion protein abatacept restored BTK protein expression in B cells to normal levels. These data indicate that autoimmune disease in humans is characterized by enhanced BTK activity, which is linked not only to autoantibody formation but also to T cell activity.

The Development and Expansion of Resistant Subclones Precedes Relapse during Ibrutinib Therapy in Patients with CLL

▪The Bruton's Tyrosine Kinase (BTK) inhibitor ibrutinib is very effective in chronic lymphocytic leukemia (CLL). Resistance in CLL is at least in part mediated by acquired mutations in BTK or down-stream PLCG2. Here we describe the largest institutional cohort of CLL patients treated with ibrutinib, focusing on risk factors for relapse, prevalence of known resistance mutations, and development of a monitoring strategy to identify patients at risk for relapse.All 308 patients from The Ohio State University Comprehensive Cancer Center participating in 4 clinical trials of ibrutinib were included. Deep sequencing for BTK and PLCG2 was performed using Ion Torrent Personal Genome Machine and covered coding regions of both genes. Preclinical experiments used XLA cell lines (Coriell Institute) infected with lentiviral constructs containing empty vector, wild type BTK, or C481S BTK.With a median follow-up of 40.5 months (range 4-71 months), 136 patients remain on study, 14 have received transplant or therapy elsewhere, and 158 have discontinued. 83 patients experienced disease progression, classified as Richter’s or PLL transformation (n=28) and progressive CLL (n=55). Using multivariable models, baseline risk factors for ibrutinib discontinuation due to transformation include complex karyotype (p<0.01) and MYC abnormalities on FISH (p=0.051). Age<65, del(17p) by FISH, and complex karyotype all associated with discontinuation due to CLL progression (p<0.05). Relapse generally has a poor prognosis, with a median survival from ibrutinib discontinuation for patients with transformation and progressive CLL of 3.9 (95% CI 2.0 to 10.1) and 22.7 (95% CI 13.5 to not reached) months, respectively.46 patients with progressive CLL had samples at relapse available for deep sequencing. Of these, 39 had mutations in BTK and/or PLCG2 (84.8%; 95% CI 71.1-93.7). Distribution of mutation included patients with BTK C481 mutation only (n=30), mutation in PLCG2 only (n=3) and both BTK/PLCG2 genes (n=6).Given the poor prognosis at relapse and presence of acquired mutations in the majority of progressive CLL patients, we were interested to evaluate whether the emergence of small clones of mutated cells could be used as a biomarker to predict relapse. For 15 patients with BTK or PLCG2 mutations, serial samples were available prior to relapse, and a clone of resistant cells could first be detected a median of 9.3 months prior to clinical relapse (95% CI: 7.6-11.7). Based on the finding that patients who relapsed on ibrutinib often had rapid progression and poor outcomes, we initiated a clinical-grade monitoring strategy in our institutional CLIA-certified molecular lab starting in November 2014. Mutational analysis of the entire coding regions of BTK and PLCG2 was performed on a cohort of 112 patients every 3 months. To date, 8 patients have clinically relapsed and all 8 had BTK C481S mutations with expansion of the clone prior to relapse. BTK C481S mutations of over 1% allelic frequency were detected in an additional 8 patients. All patients have had expansion of the resistant clones and all but one, who discontinued therapy without clinical relapse, have had increasing circulating CLL cells in the peripheral blood. Four of these 7 patients have also had increasing lymph node size on CT scan.In 11 patients who relapsed with BTK C481S, samples were available following discontinuation. In 5 patients treated subsequently with venetoclax, allelic fractions of C481S decreased dramatically or were eliminated, but in 6 patients treated with other agents, the mutant clone persisted. To investigate whether this was due to differences in biology between wild type and C481S BTK, we created XLA cell lines without BTK protein expression stably expressing BTK, or C481S BTK. C481S BTK showed enhanced BCR signaling as evidenced by pERK and cMYC expression, and enhanced migration compared to wild type BTK (p=0.04). This demonstrates for the first time that this acquired resistance mutation fundamentally alters the tumor cell biology.These data confirm our initial findings that relapse on ibrutinib is primarily mediated through mutations in BTK and PLCG2 and suggest that the presence of these mutations may have utility as an early biomarker to predict clinical relapse. Early identification of relapsing patients could improve outcomes by facilitating initiation of adjunct therapies such as venetoclax to eliminate the small resistant clone. DisclosuresWoyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

Bruton's tyrosine kinase is a potential therapeutic target in prostate cancer

Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase that has mainly been studied in haematopoietic cells. We have investigated whether BTK is a potential therapeutic target in prostate cancer. We find that BTK is expressed in prostate cells, with the alternate BTK-C isoform predominantly expressed in prostate cancer cells and tumors. This isoform is transcribed from an alternative promoter and results in a protein with an amino-terminal extension. Prostate cancer cell lines and prostate tumors express more BTK-C transcript than the malignant NAMALWA B-cell line or human lymphomas. BTK protein expression is also observed in tumor tissue from prostate cancer patients. Down regulation of this protein with RNAi or inhibition with BTK-specific inhibitors, Ibrutinib, AVL-292 or CGI-1746 decrease cell survival and induce apoptosis in prostate cancer cells. Microarray results show that inhibiting BTK under these conditions increases expression of apoptosis related genes, while overexpression of BTK-C is associated with elevated expression of genes with functions related to cell adhesion, cytoskeletal structure and the extracellular matrix. These results are consistent with studies that show that BTK signaling is important for adhesion and migration of B cells and suggest that BTK-C may confer similar properties to prostate cancer cells. Since BTK-C is a survival factor for these cells, it represents both a potential biomarker and novel therapeutic target for prostate cancer.

Abstract 3112: Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma

Abstract BACKGROUND: Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children and adolescents (Cairo et al., Blood, 2007; Miles/Cairo et al., BJH, 2012). Children with BL who relapsed or progressed have chemotherapy resistant disease and can rarely be cured after salvage therapy (Cairo et al.,JCO, 2012). Bruton's tyrosine Kinase (BTK) is a major regulator of normal B-cell development and functions through activation of the BCR, MAPK, NF-κB and AKT signaling pathways (Qiu et al., Oncogene, 2000). Chronic active B-cell receptor signaling through activation of the BTK, can be specifically inhibited by a selective BTK inhibitor, ibrutinib (Davis et al., Nature, 2010). However, the mechanism(s) and function of BTK is still poorly understood in B-cells, especially in BL. Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in a variety of organisms and stem cells for new experimental and therapeutic tools (Sander et al, Nat. Biotech., 2011; Carlson et al., Mol Ther., 2012). OBJECTIVES: We hypothesize that 1) TALEN nuclease technology is suitable for the modification of endogenous BTK gene expression in BL cells, and 2) a TALENs-mediated BTK knockout model will provide a useful tool for understanding the mechanism and function of BTK and the development of therapeutic agents that target BTK in BL. METHODS: BTK TALENs were constructed based on REAL (restriction enzyme and ligation) assembly methods (Sander et al., Nat. Biotech, 2011; Lee/Cairo et al., ASH, 2012) for BTK gene disruption. BTK TALENs validities in 293 and Raji cells were confirmed by surveyor assay and sequencing analysis. Western blotting was performed and statistical significance was determined by one-tailed Student t test using GraphPad Prism software. RESULTS: Two pairs of functional BTK TALENs (BTK-T1 and BTK-T2) targeting endogenous BTK gene were constructed and confirmed their validities with gene modification caused by non-homologous end joining (NHEJ) in TALENs mediated 293 cells. We confirmed that entire BTK coding sequences were deleted by PCR genotyping and sequencing analysis in stable BTK-knockout Raji cell clones (BTK+/-). BTK-T12-25 and BTK-T12-29 showed a significant decrease of BTK protein expression (50±1.1% reduction, p&amp;lt;0.0005; 27±0.7% reduction, p&amp;lt;0.0005, respectively) compared to empty vector-transfected Raji cells as mock control. There was a significant decrease in Erk phosphorylation in BTK knockout Raji cells compared to mock control (30±1.6%, p&amp;lt;0.001). CONCLUSIONS: These preliminary data suggest that BTK-TALENs are useful tools for the disruption of endogenous BTK gene expression. This TALEN mediated BTK knockout model will be used for studying BTK function in BL and the efficacy of BTK inhibitors as potential therapeutic agents in BL. Citation Format: Sanghoon Lee, Changhong Yin, Timmy O'Connell, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. Transcription activator-like effector nucleases (TALENs) mediated silencing of Bruton's tyrosine kinase (BTK) inhibits phosphorylation of Erk in Burkitt lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3112. doi:10.1158/1538-7445.AM2014-3112

The bruton tyrosine kinase inhibitor ibrutinib (PCI‐32765) blocks hairy cell leukaemia survival, proliferation and B cell receptor signalling: a new therapeutic approach

B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, as well as B cell migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited also CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL.

A novel Bruton’s tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1G<C. This mutation resulted in exon 9 skipping. This defect rendered the patient susceptible to asthma, failure to thrive, recurrent pyogenic infections, otitis media and bronchopneumonia. His mother and sister were heterozygous for this mutation. The combination of flow cytometry and genetic study is necessary in the diagnosis of X-linked agammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

Role of Bruton's tyrosine kinase in myeloma cell migration and induction of bone disease

Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Bruton's tyrosine kinase (BTK), of the TEC family, is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. We demonstrated BTK expression in clinical myeloma plasma cells, interleukin (IL)-6- or stroma-dependent cell lines and osteoclasts. SDF-1 induced BTK activation in myeloma cells and BTK inhibition by small hairpin RNA or the small molecule inhibitor, LFM-A13, reduced their migration toward stromal cell-derived factor-1 (SDF-1). Pretreatment with LFM-A13 also reduced in vivo homing of myeloma cells to bone using bioluminescence imaging in the SCID-rab model. Enforced expression of BTK in myeloma cell line enhanced cell migration toward SDF-1 but had no effect on short-term growth. BTK expression was correlated with cell-surface CXCR4 expression in myeloma cells (n = 33, r = 0.81, P < 0.0001), and BTK gene and protein expression was more profound in cell-surface CXCR4-expressing myeloma cells. BTK was not upregulated by IL-6 while its inhibition had no effect on IL-6 signaling in myeloma cells. Human osteoclast precursors also expressed BTK and cell-surface CXCR4 and migrated toward SDF-1. LFM-A13 suppressed migration and differentiation of osteoclast precursors as well as bone-resorbing activity of mature osteoclasts. In primary myeloma-bearing SCID-rab mice, LFM-A13 inhibited osteoclast activity, prevented myeloma-induced bone resorption and moderately suppressed myeloma growth. These data demonstrate BTK and cell-surface CXCR4 association in myeloma cells and that BTK plays a role in myeloma cell homing to bone and myeloma-induced bone disease. Am. J. Hematol. 88:463-471, 2013. © 2013 Wiley Periodicals, Inc.

Transcriptional regulatory defects in the first intron of Bruton’s tyrosine kinase

X-linked agammaglobulinemia (XLA), characterized by the early onset of recurrent bacterial infections, profound hypogammaglobulinemia, and a markedly diminished number of peripheral B lymphocytes, is caused by mutations in the Bruton's tyrosine kinase (BTK) gene. The >600 unique mutations identified to date include single base pair substitutions, small insertions or deletions, and gross deletions. A few cases, however, have been found to have no mutations in the coding region even with reduced BTK mRNA or protein expression. Mutations in intron 1 positions +5 (G-->A) and +6 (T-->G) of the BTK gene have been identified, and these changes were associated with reduced transcriptional activity. In the present study a novel mutation in intron 1 position +5 (G-->T) was identified in a Japanese patient with XLA. The reporter constructs containing these mutations were made, and the reporter activities were measured using a luciferase assay. All the mutant constructs were demonstrated to have reduced transcriptional activity. Positions +5 and +6 in intron 1 of the BTK gene are critical for transcriptional activity, and defects in these regions cause XLA.

Characterization of novel Bruton's tyrosine kinase gene mutations in Central European patients with agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency disorder caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). In this study we investigated 10 male patients with XLA-compatible phenotype (agammaglobulinemia and undetectable B cells in peripheral blood) from 9 unrelated Central European families. We identified seven different mutations, six of which were novel. One previously described point mutation caused a premature stop codon (p.C464X), two point mutations resulted in amino acid exchanges (p.W588R; p.G419E), and two point mutations affected splice sites (c.305−1G>A; c.391+1G>A). We further detected one deletion (c.1921_1927del CGTCCCA) and one large duplication. The duplication resulted from Alu element-induced unequal homologous recombination, which was only detectable by extended analysis of cDNA, while direct sequencing of genomic DNA gave a false negative result. Western blot analysis revealed that the patients with the p.W588R and the p.G419E amino acid substitutions, respectively, produced full length BTK, but in clearly diminished amounts. The patient with the 7 bp deletion expressed low amounts of protein which might represent truncated BTK. All other genomic alterations resulted in complete loss of BTK protein. In two patients from unrelated families BTK protein expression was normal and no Btk gene mutation was detected. The results of this study further substantiate the importance of using elaborate molecular analysis with different detection techniques to obtain an explicit molecular diagnosis in patients with suspected XLA.

Defective Expression of Bruton's Tyrosine Kinase in Acute Lymphoblastic Leukemia

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that serves an essential role in B cell signaling and development. We examined the BTK expression profile of primary leukemic cells from infants with newly diagnosed acute lymphoblastic leukemia (ALL) (N =14) and from pediatric patients with newly diagnosed (N =10) or relapsed (N =5) B-lineage ALL. Analysis of BTK protein and mRNA expression in the infant patient cells (N =14) showed variable levels of BTK expression with the majority of samples having reduced to absent BTK expression. Sequence analysis of reverse transcriptase-polymerase chain reaction (RT-PCR) products of Btk mRNA from infant leukemia cells revealed the presence of aberrant transcripts. These Btk transcripts were characterized by either deletion of exon 16 (Δ 16) alone or deletion of both exons 15 and 16 (Δ 15 and 16). These deletions involve exact exon skipping and encode BTK proteins with either a deleted (Δ 16), or truncated (Δ 15 and 16) kinase domain. Extension of these Btk transcript sequencing studies to 15 pediatric B-lineage ALL patients revealed expression of exon 16 deleted Btk transcripts in several pediatric patients, however, none of these pediatric patients expressed transcripts with the exon 15 and 16 deletion. Both reduced expression of Btk message and expression of aberrant deleted Btk transcripts would contribute to reduced BTK protein expression and function in B-lineage leukemia cells. Since BTK is required for radiation induced apoptosis, reduced to absent expression of functional BTK in infant ALL cells could contribute to their radiation resistance.