The efficacy and safety of Bruton tyrosine kinase inhibitors as monotherapy in the treatment of newly diagnosed patients with Waldenström macroglobulinemia / 中华血液学杂志

Objective: To investigate the efficacy and safety of Bruton tyrosine kinase inhibitors (BTKi) ibrutinib or zanubrutinib monotherapy in newly diagnosed patients with Waldenström macroglobulinemia (WM) . Methods: The efficacy and adverse effects of 58 patients with newly diagnosed WM receiving BTKi monotherapy in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine were analyzed retrospectively from January 2018 to August 2022. Results: The response of 55 patients may be examined. Forty patients received ibrutinib monotherapy for a median of 15 months, with an overall response rate (ORR) of 85%, a main remission rate (MRR) of 70%, and a very good partial remission (VGPR) rate of 10%. Fifteen patients received zanubrutinib monotherapy for a median of 13 months, with an ORR of 93%, an MRR of 73%, and a VGPR rate of 0%. For various reasons, 10 patients were converted from ibrutinib to zanubrutinib. Ibrutinib treatment lasted an average of 7.5 months before conversion. The median duration of zanubrutinib therapy after conversion was 3.5 months. The ORRs before and after conversion were 90% and 100%, MRRs were 80% and 80%, and VGPR rates were 10% and 50%, respectively. After a median of 16 months, the 24-month progression-free survival (PFS) rate of patients who received both BTKi was 86%. PFS did not differ statistically across individuals with low, medium, and high-risk ISS scores (P=0.998). All of the patients survived. The most common side effects of BTKi were neutropenia and thrombocytopenia, which occurred in 12% and 10% of all patients, respectively. Ibrutinib accounts for 5% of atrial fibrillation, and zanubrutinib has a 7% risk of bleeding. Conclusions: In treating WM, ibrutinib or zanubrutinib provides good efficacy and tolerable adverse effects.

BRD4 inhibitors kill ABC-DLBCL cells by targeting BTK / 中国病理生理杂志

AIM:To investigate the effect of bromodomain-containing protein 4(BRD4)inhibitors on the via-bility and apoptosis of activated B cell-like diffuse large B-cell lymphoma(ABC-DLBCL)cells and the molecular mecha-nism. METHODS:The ABC-DLBCL cells were treated with BRD4 inhibitors JQ1 and I-BET-762,and Bruton tyrosine kinase(BTK)inhibitor ibrutinib. The viability and death of the cells were determined by CCK-8 assay and PI staining,re-spectively. The mRNA levels of BTK,phospholipase Cγ(PLCγ),LYN,SYK,interleukin-6(IL-6),MYC,protein ki-nase Cβ(PKCβ),mucosa-associated lymphoid tissue lymphoma translocation protein 1(MALT1),MYC and RELA were detected by real-time PCR. The protein levels of BTK,PLCγ,MYC and RELA were determined by Western blot. Super-enhancer around BTK gene was revealed by bioinformatics analysis. RESULTS:The ABC-DLBCL cells were sensitive to BRD4/super-enhancer inhibitors such as JQ1 and I-BET-762. Both JQ1 and I-BET-762 inhibited the chronic active B-cell receptor(BCR)/nuclear factorκB(NFκB)signaling through reducing the transcription of BTK,but they had minimal ef-fect on other components in BCR/NFκB signaling. Interestingly,there was no super-enhancer around BTK gene,and the inhibitory effect of JQ1 was likely due to disruption of BRD4 binding within BTK gene. Inhibition of BRD4 had synergic ef-fect with BTK inhibitor ibrutinib. Moreover,inhibition of BRD4 induced significant cell death in ibrutinib-resistant ABC-DLBCL cells. CONCLUSION:Inhibitors of BRD4 induce ABC-DLBCL cell death via blocking BCR/NFκB signaling and has synergic effect with BTK inhibitor. Inhibition of BRD4 might be a promising strategy for treatment of ABC-DLBCL,es-pecially ibrutinib-resistant ABC-DLBCL.

Effects of Bruton's tyrosine kinase on the proliferation and differentiation of osteoclasts / 华西口腔医学杂志

OBJECTIVE@#To observe the effect of Bruton's tyrosine kinase (BTK) on the proliferation and differentiation of osteoclasts and to explore the mechanism of BTK on bone destruction in periapical periodontitis.@*METHODS@#After RAW264.7 cells induced with 100 ng·L⁻¹ receptor activator for nuclear factor-κB ligand (RANKL) for 5 days, osteoclast induction was confirmed by light microscopy, tartrate-resistant acid phosphatase (TRAP) staining, and quantitative real-time PCR (RT-qPCR). Then, BTK-small interfering RNA (BTK-siRNA) was transfected into cells induced for 5 days. After 24 h, the expression of TRAP mRNA was measured using RT-qPCR, and the proliferation and differentiation of osteoclasts were detected using CCK-8 and TRAP activity assay. Statistical analysis was performed.@*RESULTS@#After RAW264.7 was induced with RANKL for 5 days, a large number of round, ellipse, irregularly protuberant, and TRAP-positive macrophages were observed under light microscopy. The expression of TRAP mRNA significantly reduced after 24 h of BTK-siRNA transfection (P<0.05). The detection of CCK-8 and TRAP activities showed that the proliferation and differentiation of osteoclasts significantly decreased (P<0.05).@*CONCLUSIONS@#Silencing of BTK can inhibit the proliferation and differentiation of osteoclasts. BTK can be used as a new target for the inhibition of osteoclasts.

Treatment progress of chronic lymphocytic leukemia / 白血病·淋巴瘤

Chronic lymphocytic leukemia (CLL) is one of the common lymphoid malignancies. Single agent ibrutinib has 74％ progression-free survival (PFS) rate in RESONATE-2 trial, but ibrutinib-based therapy combined with obinutuzumab or rituximab in recent iLLUMINTE trial and ALLIANCE trail have 79％ and 88％ PFS rate respectively, which brings encouraging results. New inhibitors and chimeric antigen receptor T-cell (CAR-T) immunotherapy provide new treatment regimens for CLL patients who relapsed after receiving ibrutinib. The clinical trials have been done in phase Ⅰ/Ⅱ.

X-linked agammaglobulinemia in children / 中华实用儿科临床杂志

X-linked agammaglobulinemia (XLA) is one of the most common types of primary immunodeficiency disease in children,and is an antibody deficiency disease which is seen in men.Most XLA patients carry mutations in Bruton tyrosine kinase (BTK) gene,they typically present with very low numbers of peripheral B cells and a profound deficiency of all immunoglobulin isotypes.XLA is characterized by recurrent bacterial infections within 2 years,sometimes life-threatening.The prognosis of XLA has been improved by the treatment of gammaglobulin that allow normal concentrations of serum IgG.

Effect of Bruton's tyrosine kinase in hyperoxia-induced acute lung injury / 中华急诊医学杂志

Objective To investigate both in mechanism of hyperoxia-induced acute lung injury (HALI) by vivo experiment,to observe the Bruton' s tyrosine kinase (Btk) and nuclear factor kappa B (NF-κB) signals expression level.Methods Total of 72 healthy male Kunming mice were randomly (random number) divided into four groups:air control group,hyperoxia exposure 3 days group (H3d group),hyperoxia exposure 3 days + inhibitor group (H3d + Ⅰ group) and inhibitor groups.Then the pathological changes of lung tissues were observed under light microscope;The total protein content (TP) of bronchoalveolar lavage fluid (BALF) and wet/dry weight ratio (W/D) of lung were detected;The protein expression of Btk,p-Btk,pNF-κB p65 were mersured by Western blot;tlhe mRNA level of IL-6 was determined by real-time polymerase chain reaction (qRT-PCR);the level of monocyte chemoattractant protein-1 (MCP-1) in serum was detected by enzyme-linked immunosorbent assay (ELISA).Statistcal significance was determined by 1-way ANOVA.Results There were no significant difference in the data between the control group and the inhibitor group (P ＞ 0.05).The pathological injury in light microscope,content of total protein in BALF,W/D ratio of lung tissues in H3d group were significantly higher than H3d + Ⅰ group (Respectively P =O.002,P =0.000).Western blot analysis showed that expression of Btk,p-Btk,pNF-κB p65 in H3d group were significantly higher than those in H3d + Ⅰ group (Respectively P =0.002,P =0.013,P =0.000).RT-qPCR results showed that the expression of IL-6 mRNA in H3d group were significantly higher than control group (P =0.004),inhibitor group (P =0.000) and H3d + Ⅰ group (P =0.021).In addition,The serum MCP-1 levels in H3d group were higher markely than the control group (P =0.002),inhibitor group (P =0.000) and H3d + Ⅰ group (P =0.009).The correlation analysis showed that pNF-κB p65 were positively correlated wiht Btk and p-Btk (r =0.902 and 0.954,P ＜ 0.01).Conclusions Btk may trigger the release of IL-6 and MCP-1 by mediating the signaling pathway of NF-κB in vivo study,which was most important in the occurrence of HALI.Therefore,inhibiting the Btk activity would alleviate the severity of lung injury effectively.

BTK expression and its clinical significance in 32 cases of mantle cell lymphoma / 中国肿瘤临床

Objective:To detect Bruton tyrosin kinase (BTK) expression in patients with mantle cell lymphoma (MCL) and analyze its correlation with clinical features and prognosis. Methods:A total of 32 cases of MCL tissues and 10 cases of benign lymph nodes were sampled and stained with immunohistochemical (IHC) staining. Clinical data of these patients were analyzed using SPSS 17.0. Results:BTK was positively expressed in MCL and normal lymphoid tissues and was more strongly expressed in MCL tissue than in normal lym-phoid tissue. Moreover, BTK expression level was correlated with Ki-67 and MIPI scores. Prognosis analysis showed that patients with high BTK expression exhibited shorter progression-free survival (PFS) than patients with low expression levels (P=0.030);however, no significant difference in overall survival (OS) was observed (P=0.073). Single-factor analysis of PFS showed that age≥65 years, ECOG score≥2, bone marrow involvement, strongly positive BTK expression, Ki-67>30%, and MIPI score≥6 are poor prognostic factors for patients with MCL. Only MIPI score≥6 is considered an independent poor prognostic factor in the multivariate analysis. Conclusion:BTK is strongly and positively expressed in patients with MCL, and its expression level is correlated with Ki-67 and MIPI scores. Patients with high-level BTK expression usually exhibit shorter PFS than those with low-level BTK expression;however, owing to short follow-up time and limited sample size, high-level BTK expression cannot be considered an independent poor prognostic factor for PFS.

Agammaglobulinemia ligada al cromosoma X en adultos: Evolucion clínica / X-linked agammaglobulinemia in adults. Clinical evolution

La agammaglobulinemia ligada al cromosoma X (XLA) se caracteriza por la ausencia o reducción significativa de linfocitos B, niveles bajos o indetectables de inmunoglobulinas y, clínicamente, por infecciones principalmente respiratorias por bacterias capsuladas extracelulares y diarrea recurrente. El tratamiento de reemplazo con gammaglobulina ha permitido a la mayor parte de los enfermos llegar a adultos con una buena calidad de vida. Analizamos las características clínicas de 14 pacientes mayores de 18 años con diagnóstico de XLA asistidos en nuestra Unidad desde 2003, fecha en que fue derivado el primer paciente, hasta 2015. La edad promedio en el momento de la derivación fue de 20.4 años, en el momento de la última consulta de 25.5. El tiempo promedio de seguimiento fue de 59.8 meses. Previo al diagnóstico todos habían presentado infecciones, las más frecuentes fueron las respiratorias. Posteriormente al diagnóstico todos iniciaron tratamiento de reemplazo con gammaglobulina endovenosa, y a pesar de que las infecciones disminuyeron en frecuencia y gravedad, en este período se presentaron enfermedades con secuelas graves. Al comenzar el seguimiento en nuestra Unidad, 35.7% presentaban deterioro de la función respiratoria, solo grave en un paciente. Durante el seguimiento ninguno presentó deterioro de la función respiratoria ni complicaciones clínicas importantes. Tres pasaron a gammaglobulina subcutánea con buena tolerancia. El número de adultos con XLA es cada vez mayor, la mayoría llegan a la segunda década de la vida sin complicaciones graves y bajo tratamiento se mantienen libres de enfermedades infecciosas graves y de progresión de sus secuelas pulmonares.

X-linked agammaglobulinemia (XLA) is characterized by absent or severely reduced B cells, low or undetectable immunoglobulin levels and clinically by extracellular bacterial infections which mainly compromise the respiratory tract as well as recurrent diarrheas. The mainstay of treatment is gammaglobulin replacement therapy, which allows most patients to reach adulthood with high quality of life. We analyzed the clinical features of 14 patients over 18 years of age with XLA diagnosis that received treatment in our unit from the year 2003, the date the first patient was derived, until 2015. The average age at which patients were referred was 20.4 years old; age at the last consult was 25.5. The average follow-up time was 59.8 months. Previously to being diagnosed all patients had suffered infections, most frequently respiratory. After diagnosis all were started on intravenous gammaglobulin replacement treatment and in spite of infections being reduced in severity and frequency, there were cases of severe disease with long term sequelae. At the beginning of our follow-up 35.7% presented impaired respiratory function with only one case being severe. In no cases during this period did the respiratory function worsen, nor were there severe clinical complications. Three patients were switched to subcutaneous immunoglobulin treatment with good tolerance. The number of XLA cases is increasing, as most reach the second decade of life without serious complications and remain free of severe infectious disease and further impairment of their respiratory functions with the treatment.

Effect of Bruton’s tyrosine kinase inhibitors combined with bortezomib on human multiple myeloma cell lines and its mechanism / 第二军医大学学报

Objective To explore the effect of Bruton’s tyrosine kinase (BTK) inhibitors, ibrutinib and AVL-292 alone or in combination with proteasome inhibitor bortezomib on human multiple myeloma (MM) cell lines H929 and RPMI8226 and the related mechanism. Methods H929 and RPMI8226 cells were treated with ibrutinib or AVL-292 alone or in combination with bortezomib in vitro. The cell viability was detected by CCK-8 assay after treatment, the apoptosis levels were analyzed by flow cytometry, the expression and phosphorylation levels of BTK pathway proteins and apoptosis-related proteins were measured by Western blotting analysis. Results The proliferation of H929 and RPMI8226 cells was inhibited by ibrutinib and AVL-292 in a dose-dependent manner. The 48 h median inhibitory concentration (IC50) values of ibrutinib in the two cell lines were (10. 41±3. 29) μmol/L and (51. 65±13. 58) μmol/L, respectively, and the 48 h IC50 values of AVL-292 were (7. 77±2. 99) μmol/L and (6. 44±1. 06) μmol/L, respectively. The inhibition effects of different concentrations of ibrutinib (5 μmol/L, 10 μmol/L) and AVL-292 (5 μmol/L, 10 μmol/L) combined with different concentrations of bortezomib (5 nmol/L, 10 nmol/L, 20 nmol/L, and 50 nmol/L) were significantly higher than those of the corresponding single agents (P<0. 05, P<0. 01); the coefficients of concordance (R) of different combinations were all above 1. 0. After treatment with 10 μmol/L ibrutinib, 10 μmol/L AVL-292 and 20 nmol/L bortezomib alone for 48 h, the apoptosis levels of H929 cells were (15. 12±1. 59)%, (18. 23±6. 38)% and (10. 71±1. 62)%, respectively, which were all significantly higher than that of the control group ([6. 46±1. 18]%; P<0. 05, P<0. 01); the apoptosis levels of RPMI8226 cells were (9. 29±1. 44)%, (15. 01±4. 99)% and (7. 58±1. 13)%, respectively, and those of treated with 10 μmol/L ibrutinib and 10 μmol/L AVL-292 were significantly higher than that of the control group ([5. 54±1. 61]%, P<0. 05). The apoptosis levels of H929 cells in the 20 nmol/L bortezomib+10 μmol/L ibrutinib and 20 nmol/L bortezomib+10 μmol/L AVL-292 groups were (40. 31±3. 94)% and (51. 55±6. 39)%, respectively, and those of RPMI8226 cells were (31. 86±1. 93)% and (43. 23±4. 03)%, respectively, and they were all significantly higher than those in their corresponding single agent groups (P<0. 01). Compared with control group, the phosphorylation levels of BTK, NF-κB p65, Akt and ERK and expression of Bcl-xL protein were significantly decreased in H929 cells treated with 10 μmol/L ibrutinib or 10 μmol/L AVL-292 for 24 h (P<0. 05), and the expression of cleaved caspase-3 was significantly increased (P<0. 01). The regulation effects on the above indices were significantly more evident when the two agents were combined with 20 nmol/L bortezomib (P<0. 05, P<0. 01). Conclusion Both ibrutinib and AVL-292 can inhibit proliferation and induce apoptosis of human MM cell lines H929 and RPMI8226; there are significant synergistic effects between them and proteasome inhibitor bortezomib, which may be related to inhibition of BTK activity and the downstream pathways (NF-κB, Akt and ERK), down-regulation of Bcl-xL and activation of caspase-3 apoptotic pathway.

Research progress of Bruton tyrosine kinase inhibitors in mantle cell lymphoma / 白血病·淋巴瘤

Mantle cell lymphoma (MCL), is a disease with high heterogeneity, which is insensitivity to the chemotherapy and presents poor prognosis. Appearance of the Bruton tyrosine kinase (BTK) targeted inhibitors, such as ibrutinib, provides novel treatment strategy for MCL. This review focused on the latest achievements of BTK inhibitors in MCL treatment.

Research progress of Bruton tyrosine kinase in B cell malignances / 白血病·淋巴瘤

Bruton tyrosine kinase (Btk) is a member of the non-receptor protein tyrosine kinases of Tec family.It plays an important role in growth,proliferation,differentiation and signal transduction of B lymphocyte.After activation of B-cell receptor (BCR) by diverse stimuli,Btk is activated through tyrosine phosphorylation.Activated Btk can stimulate several downstream signaling proteins.Btk and BCR signaling pathways play important roles in initiation and maintenance of B cell malignancies.This review focuses on the recent findings in association of Btk and B cell malignancies.

Bruton tyrosine kinase inhibitor in B cell malignancies / 白血病·淋巴瘤

Bruton tyrosine kinase (BTK) is a non-receptor tyrosine kinase which belongs to the Tec kinase family and plays an important role in B cell receptor signaling.Nowadays,BTK has been a nove] target for treating some B cell malignancies.Recently,some studies have confirmed that BTK inhibitor,PCI-32765 (ibrutinib),can effectively treat chronic lymphocytic leukemia,mantle cell lymphoma and so on.This review will discuss the preclinical and clinical development of this BTK inhibitor in B cell malignancies.

Mutations of Bruton's tyrosine kinase gene in Brazilian patients with X-linked agammaglobulinemia

Mutations in Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in peripheral blood. We evaluated 5 male Brazilian patients, ranging from 3 to 10 years of age, from unrelated families, whose diagnosis was based on recurrent infections, markedly reduced levels of IgM, IgG and IgA, and circulating B cell numbers <2 percent. BTK gene analysis was carried out using PCR-SSCP followed by sequencing. We detected three novel (Ala347fsX55, I355T, and Thr324fsX24) and two previously reported mutations (Q196X and E441X). Flow cytometry revealed a reduced expression of BTK protein in patients and a mosaic pattern of BTK expression was obtained from mothers, indicating that they were XLA carriers.

A Case of X-Linked Agammaglobulinemia with Btk Gene Intron 2 Mutation / 결핵및호흡기질환

X-linked agammaglobulinemia is the most common type of primary immunodeficiency disorder. Mutation ofthe cytoplasmic tyrosine kinase gene, Btk (Bruton's tyrosine kinase), is known to be the etiology of X-linked agammaglobulinemia. The patients with this disease manifest a B-cell deficiency and low levels of serum immunoglobulin; due to the deficient antibodies, they suffers from recurrent upper and lower respiratory infections. We report here a 24-year-old male with an initial clinical impression of recurrent pneumonia and bronchiectasis. The patient presented with marked pan-hypogammaglobulinemia and the absence of circulating B-lymphocytes on the immunologic study, and he carried a splicing mutation of intron 2 in the Btk gene (IVS2 -3C>G).

Analysis of Clinical Presentations of Bruton Disease: A Review of 20 Years of Accumulated Data from Pediatric Patients at Severance Hospital

PURPOSE: X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency disease caused by a mutation in the Bruton tyrosine kinase (BTK) gene resulting in defective B cell differentiation. Because it is a relatively rare disorder, it is difficult for clinicians to have a comprehensive understanding of XLA due to a lack of exposure to the disease. Clinical presentations of patients with XLA were analyzed and discussed to improve care plans. MATERIALS AND METHODS: During a 20 year period, from January 1987 to June 2006, a total of 19 patients were diagnosed as XLA in the Department of Pediatrics at Severance Hospital, Seoul, Korea. A retrospective analysis of the clinical presentations of those patients was performed. RESULTS: The mean age of the XLA patients included in the study was 4.89 years, with a range of 6 months to 13 years. Twelve patients were diagnosed before age 5, while the other 7 patients were diagnosed after age 5. Recurrent infections observed in the patients included pneumonia, acute otitis media, septic arthritis, skin infection, sepsis, sinusitis, acute gastroenteritis, cervical lymphadenitis, epididymitis, meningitis, osteomyelitis, urinary tract infection and encephalitis. Frequency of admissions was variable from 0 to 12 times, depending on the time at which immunoglobulin therapy was started. Six cases had family histories positive for XLA. BTK gene mutations were found in 8 cases. CONCLUSION: The overall prognosis of XLA is good as long as patients are diagnosed and treated early with regular intra venous gamma globulin therapy before the sequelae of recurrent infections appear.

A case of X-linked agammaglobulinemia with deletion of introns 15-18 of Btk gene mediated by Alu-Alu recombination / 대한내과학회지

X-linked agammaglobulinemia (XLA) is characterized by early onset of recurrent bacterial infection, markedly reduced levels of all major classes of immunoglobulins in the serum and few mature B cells in the blood. XLA is known to be associated with mutations in Bruton's tyrosin kinase (Btk). The Btk protein consists of 5 functional domains; the pleckstrin homology (PH) domain, the Tec homology (TH) domain, the Src homology 3 (SH3) domain, the SH2 domain, and the kinase (SH1) domain. Mutations in all domains of the Btk gene have been shown to cause XLA. The large number of Alu elements within the human genome provides abundant opportunities for unequal homologous recombination events between Alu repeats, resulting in human disease. We present a case of XLA with deletion of introns 15-18 of Btk gene which were mediated by an Alu-Alu recombination event.

A case of X-linked agammaglobulinemia with deletion of introns 15-18 of Btk gene mediated by Alu-Alu recombination / 대한내과학회지

X-linked agammaglobulinemia (XLA) is characterized by early onset of recurrent bacterial infection, markedly reduced levels of all major classes of immunoglobulins in the serum and few mature B cells in the blood. XLA is known to be associated with mutations in Bruton's tyrosin kinase (Btk). The Btk protein consists of 5 functional domains; the pleckstrin homology (PH) domain, the Tec homology (TH) domain, the Src homology 3 (SH3) domain, the SH2 domain, and the kinase (SH1) domain. Mutations in all domains of the Btk gene have been shown to cause XLA. The large number of Alu elements within the human genome provides abundant opportunities for unequal homologous recombination events between Alu repeats, resulting in human disease. We present a case of XLA with deletion of introns 15-18 of Btk gene which were mediated by an Alu-Alu recombination event.

Characterization of Mutations in Bruton's Tyrosine Kinase(Btk)Gene from Unrelated 3 X-linked Agammaglobulinemia(XLA) Families in Korea

PURPOSE: X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells(PBMC) from three XLA families in Korea. METHODS: Heparinized venous blood samples were collected from four XLA patients and additional family members in three unrelated XLA families. Mononuclear cells were separated from their blood and the intracellular Btk protein was characterized by a flow cytometry. The mutation analysis was performed using direct sequencing. RESULTS: Cytoplasmic expression of Btk protein in monocytes was not detected in the patients with XLA. We observed a novel deletion and two point mutations within introns(intron 1 and intron 18) resulting in alternative splicings. In XLA family 2, a 980 bp deletion(from intron 9+191 T to intron 10-215 C) including exon 10 was found in patient P2. He was the only sporadic case in this study, because his mother and brother showed a normal Btk expression by flow cytometry. CONCLUSION: These identified genetic alterations support the molecular heterogeneity of Btk gene in XLA disease. Additionally, by means of flow cytometric analysis, we diagnosed three hypogammaglobulinemia patients as XLA. Advancements in diagnostic methods has facilitated a prompt and definite diagnosis of this disease.

Characterization of Bruton's Tyrosine Kinase Genetic Mutations in One Korean X-linked Agammaglobulinemia Family

PURPOSE: X-linked agammaglobulinemia(XLA) is an immunodeficiency caused by abnormalities in Bruton's tyrosine kinase(Btk), and is characterized by a deficiency of peripheral blood B cells. We studied the cytoplasmic expression of Btk protein and analyzed the Btk gene in peripheral blood mononuclear cells from two siblings and one cousin with XLA, as well as additional family members. METHODS: Btk protein expression was analyzed by flow cytometry. Isolation of the coding sequence of the Btk gene was performed by amplification using the reverse transcription-polymerase chain reaction(RT-PCR) technique. Sequence alterations were screened by the single-stranded conformation polymorphism(SSCP) method and characterized by standard sequencing protocols. RESULTS: Cytoplasmic expression of Btk protein in monocytes was not detected in three patients with XLA. In addition, Btk protein analysis clearly showed cellular mosaicism in monocytes from four obligate carriers, findings further supported by SSCP. A single base pair mutation(T to C) in Btk-exon three, which encodes the PH domain, was identified in four XLA patients. A diagnostic sequencing analysis was established to detect heterozygotic pattern in 4 carrier females. Furthermore, we found significant clinical heterogeneity in individuals with the same gene mutation. CONCLUSION: The implicating genetic alteration provided valuable clues to the pathogenesis of XLA in Korea and the flow cytometric analysis was suggested as a useful tool for rapid detection of XLA patients and carriers. The present study has identified a genetic mutation in the Btk coding region and demonstrated heterogeneity in clinical manifestations among patients with the same mutation. A flow cytometric analysis was found to be informative in establishing a deficiency of Btk protein in both patients and carriers and is recommended as a frontline procedure in the molecular diagnosis and work-up of XLA.

Protein and Genetic Analysis of Bruton's Tyrosine Kinase(Btk) in Three Korean X-linked gammaglobulinemia(XLA) Families

PURPOSE: Mutations in the Bruton's tyrosine kinase(Btk) gene are responsible for X-linked agammaglobulinemia(XLA), an immunodeficiency caused by a block in B cell differentiation. In this report we characterize the protein expression and genetic mutations of Btk in four Korean patients with three unrelated XLA families. METHODS: The resulting Btk proteins were characterized by a flow cytometry and the mutations were analyzed using single strand conformation polymorphism(SSCP) and direct sequencing. RESULTS: Two deletions, including one novel genetic alteration, and one splicing error, were found in these three XLA families. Along with the identification of mutations, Btk protein analysis using flow cytometry clearly showed cellular mosaicism in monocytes from five obligate carriers, findings consistent with those by SSCP. We attempted to determine the origin of mutation in an XLA family with a novel 4-bp deletion of exon eight, suggesting a germline mutation in this family. In addition, we found some clinical heterogeneities in the affected brothers with the same gene mutation. CONCLUSION: These identified genetic alterations provided valuable clues to the pathogenesis of XLA in Korea. The flow cytometric analysis is suggested as a useful tool for rapid detection of XLA patients and carriers.