Maternal germinal mosaicism of X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by abnormalities in 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 (BTK) in peripheral blood mononuclear cells from two siblings with XLA and additional family members. Cytoplasmic expression of BTK protein in monocytes was not detected in either patient with XLA. A single base deletion (C563) in BTK-exon 6, which encodes the TH domain, was identified in both XLA patients. However, normal cytoplasmic expression of BTK protein in monocytes was detected in their mother without any BTK mutation. These results strongly suggest germinal mosaicism in the mother.     

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 7bp 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.     

Maternal germinal mosaicism of X‐linked agammaglobulinemia

X‐linked agammaglobulinemia (XLA) is an immunodeficiency caused by abnormalities in 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 (BTK) in peripheral blood mononuclear cells from two siblings with XLA and additional family members. Cytoplasmic expression of BTK protein in monocytes was not detected in either patient with XLA. A single base deletion (C563) in BTK‐exon 6, which encodes the TH domain, was identified in both XLA patients. However, normal cytoplasmic expression of BTK protein in monocytes was detected in their mother without any BTK mutation. These results strongly suggest germinal mosaicism in the mother. © 2001 Wiley‐Liss. Inc.     

Interaction of human papillomavirus type 11 E7 protein with TAP-1 results in the reduction of ATP-dependent peptide transport

X-linked agammaglobulinemia (XLA), caused by mutations in Bruton's tyrosine kinase (BTK), typically presents in early childhood. We report here the case of a male diagnosed at age 23 years with hypogammaglobulinemia, originally classified as common variable immunodeficiency (CVID). On further analysis at age 40, flow cytometric analysis of lymphocytes showed only 0.1% B cells and Western blot analysis showed a deficiency of BTK protein in peripheral blood mononuclear cells, indicating the patient has XLA. BTK cDNA and genomic DNA analysis revealed a splice site mutation at the 3' end of intron 13. Multiple abnormally spliced mRNA species were identified, one of which was predicted to produce a protein with a 24-amino-acid insertion between the SH2 and kinase domains. In vitro kinase assay of this product showed weak kinase activity, perhaps resulting in milder than usual disease. XLA can present in adult males, and sporadic cases may be misdiagnosed as CVID.     

BTK: 22 novel and 25 recurrent mutations in European patients with X-linked agammaglobulinemia

X linked agammaglobulinemia (XLA) is an immunodeficiency disease caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK), that is involved in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B lineage lymphoid cells. XLA is a primary immunodeficiency disorder characterized by lack of mature, circulating B lymphocytes, and recurrent infections. Using Single Strand Conformation Polymorphism (SSCP) followed by direct sequencing we investigated 57 patients with XLA phenotype, with or without a positive family history, from 52 unrelated families enrolled in the Italian XLA Multicenter Clinical Study. We have identified 25 recurrent mutations, 22 novel mutations including one large deletion comprising the coding sequence from exon 11 to 18. Among the mutations identified, three were detected in different unrelated families, whereas all the others were private mutations.     

中国X连锁无丙种球蛋白血症40例基因型表型相关性分析

目的通过中国X连锁无丙种球蛋白血症（XLA）患儿临床表现、免疫功能评价、Bruton＇s酪氨酸激酶（BTK）的表达及BTK基因突变分析,分析基因型和表型间可能存在的关系。方法选取拟诊为XLA患儿,使用抗BTK单克隆抗体通过流式细胞技术分析单核细胞BTK蛋白表达。采用RT-PCR获得患儿cDNA,使用8对不同引物分2步扩增BTKcDNA,PCR产物测序。突变结果通过对DNA外显子相应部位扩增、测序证实。并对确诊XLA患儿的母亲及家族中部分亲属进行BTK蛋白表达和BTK基因分析。结果①40/50例原发性低丙种球蛋白血症患儿经BTK基因突变分析确诊为XLA,以错义突变（16例,40.0%）和无义突变（13例,32.5%）为主。②突变类型为错义突变的患儿平均起病年龄为（1.4±1.1）岁,其他突变类型患儿为（1.4±0.7）岁,差异无统计学意义（P=0.45）。错义突变的发生率随年龄的增长呈上升趋势,无义突变的发生率呈下降趋势。③34/40例（85.0%）B细胞〈0.1%;4例（10.0%）B细胞在1%～2%,其中错义突变2例,无义突变1例,剪接突变1例;2例（5.0%）B细胞为2%,均为错义突变。④血清IgG〈3g·L-1患儿BTK基因突变类型以错义突变和无义突变为主。⑤错义突变患儿BTK蛋白表达水平与其他突变类型无显著差异。⑥6/21例（28.6%）2031C/T多态性患儿伴有严重的关节炎,3/19例（15.8%）无多态性患儿有关节炎表现。⑦28/32例（87.5%）XLA患儿母亲为BTK基因杂合型。结论错义突变可能与确诊年龄较大有关,且某些位点的错义突变可能与较高的外周血B细胞数量和血清IgG水平及正常的BTK蛋白表达水平有关。BTK基因多态性（2031C/T）可能增加关节炎的风险。     

Identification of mutations of Bruton's tyrosine kinase gene (BTK) in Brazilian patients with X-linked agammaglobulinemia

Mutations in the Bruton 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 the peripheral blood. We evaluated 17 male Brazilian patients from 13 unrelated families who showed markedly reduced numbers of blood B cells and hypogammaglobulinemia. BTK gene analysis detected mutations in 10 of the 13 presumed XLA families. Seven mutations (Q196X, G613D, R28L, 251-273del, Q234X, H364P, and R13X) had been reported previously, whereas the remaining three mutations (M501T, IVS15+1G>C, and IVS14+1G>A) were novel. Mutation IVS15+1G>C occurred in a splice donor site and caused exons 15 and 16 to be skipped, and IVS14+1G>A might cause exon 14 to be skipped. Flow cytometry revealed deficient expression of BTK protein in 10 of the 13 families. This is the first report of the diagnosis of XLA by analysis of mutations of the BTK gene in Brazilian patients.     

De novo mutation in the BTK gene of atypical X-linked agammaglobulinemia in a patient with recurrent pyoderma.

BACKGROUND: X-linked agammaglobulinemia (XLA), characterized by a profound deficiency of all immunoglobulins and the absence of mature B cells, is caused by mutations in the gene encoding Bruton tyrosine kinase (BTK). Most patients have recurrent sinopulmonary infection. Infections usually occur in multiple locations across time, but single infection may be limited to one anatomic location. OBJECTIVES: To report a case of atypical XLA with recurrent pyoderma and to observe the immunologic changes in the patient in 10 years. METHODS: Immunologic investigations, skin wound culture, and molecular study with DNA sequencing were performed. RESULTS: The patient was originally diagnosed as having common variable immunodeficiency disease because of the presence of circulating B cells (CD19+ B cells: 7%) at 11 years old. On further evaluation at the age of 20 years, flow cytometric analysis of lymphocytes showed only 0.4% B cells. The molecular study with DNA sequencing of the patient showed a point mutation in complementary DNA 1630 A>G(p.R544G) in the BTK gene, indicating that the patient has XLA. The mutation analysis of the BTK gene revealed a normal DNA sequence in the other family members. CONCLUSIONS: This case is an important example of a possible presentation of XLA with a predominant skin manifestation, and it demonstrates that maintaining a high level of clinical suspicion is essential for the diagnosis of XLA in a child with recurrent pyoderma.     

Identification of Bruton tyrosine kinase mutations in 12 Chinese patients with X-linked agammaglobulinaemia by long PCR-direct sequencing

X-linked agammaglobulinaemia (XLA) is an immunodeficiency caused by Bruton tyrosine kinase (BTK) gene mutations. The disease is characterized by recurrent bacterial infections and profound hypogammaglobulinemia with marked reduction or lack of mature B-cells in the peripheral blood. Molecular characterization of BTK gene provides an opportunity for definitive diagnosis of XLA patients, especially for those with atypical phenotype resulting in a milder or late-onset form of the disease. The diagnosis allows accurate carrier detection with subsequent genetic counselling and prenatal diagnosis. In this study, long polymerase chain reaction (PCR)-direct sequencing analysis of the BTK gene in 12 unrelated Chinese XLA patients had been performed. Eight recurrent mutations and four novel mutations were identified. This is the first report of Chinese cases from three different East Asia regions together, including Hong Kong, Singapore and mainland China. Future clinical and genetic information from the undiagnosed Chinese XLA patients may provide insight into the genotype-phenotype correlations of BTK gene.     

Clinical, immunological and molecular characteristics of 37 Iranian patients with X-linked agammaglobulinemia

BACKGROUND: X-linked agammaglobulinemia (XLA) is a hereditary immunodeficiency characterized by an early onset of recurrent bacterial infections, a profound deficiency of all immunoglobulin isotypes and a markedly reduced number of peripheral B lymphocytes. Eighty-five percent of the patients with this phenotype have mutations in Bruton's tyrosine kinase (BTK) gene. METHODS: To provide an informative outlook of clinical and immunological manifestations of XLA in Iran, 37 Iranian male patients with an age range of 1-34 years, followed over a period of 25 years, were studied. Twenty-four of the 37 patients were screened for BTK gene mutation using PCR-SSCP followed by direct sequencing. BTK protein expression assay was done by flow cytometry in 9 families. RESULTS: All patients first presented with infectious diseases, the most common of which were respiratory tract infections. Eighteen different mutations were identified, 13 of which were novel: IVS1+5G>C, 1896G>A, 349delA, 1618C>T, 1783T>C, 2084A>G, 1346delT, 1351delGAG, 587A>G, IVS14-1G>A, IVS3+2T>C, 1482G>A, 1975C>A. CONCLUSION: The fact that we found a great number of novel mutations in a relatively limited number of patients underlines the heterogeneity of BTK mutations in the Iranian population. The large number of new mutations indicates that extended studies in this region would be rewarding.     

Identification of nine novel mutations in the Bruton's tyrosine kinase gene in X-linked agammaglobulinaemia patients

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. Non Isotopic RNAse Cleavage Assay (NIRCA), followed by sequencing was used to screen for BTK mutations in 11 Italian XLA patients. Nine novel mutations were identified: 6 missense (Y39S, L512P, L512Q, R544G, S578Y, E589K), one non-sense (Q260X), one frameshift (1599-1602del GCGC) and one in-frame insertion (2037-2038insTTTTAG), that represents the first case of premature stop codon introduction in the BTK coding frame. These data support the high molecular heterogeneity of BTK gene in XLA disease and provide new insight to the diagnosis and to the role of BTK domain in XLA and in B cell signal transduction and development. Hum Mutat 15:117, 2000.     

Novel Igalpha (CD79a) gene mutation in a Turkish patient with B cell-deficient agammaglobulinemia

Mutations that impair early B cell development result in profound antibody deficiency, which is characterized by a paucity of mature B cells and the early onset of recurrent pyogenic infections. Among these inherited early B cell defects, X-linked agammaglobulinemia (XLA) with mutations in Bruton's tyrosine kinase (BTK) gene is mostly identified. Recent studies have shown that mutations in the gene for mu heavy chain (IGHM) and for other components of the pre-B cell receptor complex, including lambda5/14.1 (IGLL1) or Igalpha (CD79a), can cause a disorder that is clinically similar to XLA. In a genetic survey of XLA in Turkey, we examined possible mutations in the IGHM, IGLL1, and Igalpha genes in some male patients with presumed XLA who did not have identifiable BTK mutations. We found an eight-year-old boy with a novel homozygous mutation in the Igalpha gene (IVS2+1G>A) causing B cell defect. This is the second case of agammaglobulinemia due to an Igalpha (CD79a) deficiency in the world.     

Delayed diagnosis in X-linked agammaglobulinemia and its relationship to the occurrence of mutations in BTK non-kinase domains

Background: X-linked agammaglobulinemia (XLA) is characterized by the absence of immunoglobulin and B cells. Patients suffer from recurrent bacterial infections from early childhood, and require lifelong immunoglobulin replacement therapy. Mutations in BTK (Bruton’s Tyrosine Kinase) are associated with this phenotype. Some patients that present XLA do not show typical clinical symptoms, resulting in delayed diagnosis due to the lack of a severe phenotype. This study presents a report of five XLA patients from four different families and attempts to determine a relationship between delayed diagnosis and the occurrence of BTK mutations.

Methods: Samples from patients with antibody deficiency were analyzed to determine BTK expression, immunophenotyping and mutation analysis. Clinical and laboratory data was analyzed and presented for each patient.

Results: Most patients presented here showed atypical clinical and laboratory data for XLA, including normal IgM, IgG, or IgA levels. Most patients expressed detectable BTK protein. Sequencing of BTK showed that these patients harbored missense mutations in the pleckstrin homology and Src-homology-2 domains. When it was compared to public databases, BTK sequencing exhibited a new change, along with three other previously reported changes.

Conclusions: Delayed diagnosis and atypical manifestations in XLA might be related to mutation type and BTK expression.     

A genotype-phenotype correlation study in a group of 54 patients with X-linked agammaglobulinemia

BACKGROUND: X-linked (Bruton's) agammaglobulinemia (XLA) is a rare immunodeficiency caused by a block in B-cell development caused by mutations in the Bruton's tyrosine kinase (BTK) gene. Many aspects of XLA and BTK function remain unresolved; atypical presentations have been reported, and no clear genotype-phenotype correlation has been established. OBJECTIVES: We sought to contribute to the understanding of XLA through the phenotypic and biochemical characterization of a large group of Spanish patients with agammaglobulinemia. We also sought to classify the mutations according to their severity to analyze a genotype-phenotype correlation. METHODS: Clinical and analytic data were collected from the clinical records. We studied the BTK gene, protein expression, and function, and the findings were correlated with the phenotypic information. RESULTS: Fifty-four patients were given diagnoses of XLA. We identified 38 different mutations in BTK, 26 not described in other patients, and several uncommon clinical phenotypes or analytic characteristics were found. The statistical analysis shows that less severe mutations or minimal detection of protein by means of flow cytometry are associated with decreased severity in clinical and analytic data, demonstrating a clear relation between the type of mutation and the disease expressivity. However, some exemptions to this rule were noted. CONCLUSIONS: XLA is a variable disease. Globally, a genotype-phenotype correlation is observed, but individual discrepancies between the severity of the mutation and the clinical and analytic phenotype suggest that other loci or ambient factors significantly influence the disease presentation and evolution.     

Detection of a novel mutation in the SRC homology domain 2 (SH2) of Bruton's tyrosine kinase and direct female carrier evaluation in a family with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease with a block in differentiation from pre-B to B cells resulting in a selective defect in the humoral immune response. Affected males have very low concentrations of serum immunoglobulins leading predominantly to recurrent bacterial infections beginning at age 6 to 18 months. The gene responsible for XLA was identified recently to encode a cytoplasmatic tyrosine kinase (Bruton's tyrosine kinase, BTK). We have analyzed the BTK gene in a large family in which two brothers presented with the severe phenotype of XLA. Genomic DNA of affected boys and from healthy relatives was amplified by PCR with primers specific for the putative promoter region and for all 19 exons, including flanking intron boundaries, and subsequently screened for mutations using single strand conformation polymorphism (SSCP) analysis. Altered single strand band patterns were found using primers specific for exon 10, 15, and 18. Direct cycle-sequencing of these BTK segments detected two known polymorphisms in intron 14 and in exon 18. Sequencing of exon 10 from two boys with XLA demonstrated a novel point mutation in the SH2 domain of BTK. Direct identification of healthy female carriers in three generations was performed by amplification mutagenesis using PCR with a modified first primer. This method can easily be applied also to prenatal diagnosis. © 1996 Wiley-Liss, Inc.     

Bruton tyrosine kinase gene mutations in Argentina

The block in differentiation from pro-B to pre-B cells results in a selective defect in the humoral immune response characteristic of human X-linked agammaglobulinemia (XLA). Mutations of Bruton tyrosine kinase (BTK) gene have been identified as the cause of XLA. Mutation detection is the most reliable method for making a definitive diagnosis, except when clinical and laboratory findings are distinctive and coupled with history of X-linked inheritance. To provide a definitive diagnosis to 40 families incorporated in the Argentinian Primary Immunodeficiencies Registry we analysed the BTK gene by SSCP analysis as screening method for XLA, followed by direct sequencing. The molecular defect was localized in 45 patients from 34 unrelated families. From the 34 independent mutations identified, 16 were previously undescribed, 31 were unique mutations, 22 were exonic single nucleotide changes (16 missense and 6 nonsense) and four intronic mutations. Because five families had clinical, immunological and inheritance data sufficient for a definitive diagnosis, our study allowed 37 patients from 29 families previously categorized probable/ possible XLA, have now definitive diagnosis leading to appropriate genetic counseling.     

Bruton's tyrosine kinase mutations in 8 Chinese families with X-linked agammaglobulinemia

Bruton's tyrosine kinase (BTK) is involved in B-cell development. Mutation of BTK results in X-linked agammaglobulinemia (XLA). BTK is expressed in most haemopoietic lineages except mature T cells and plasma cells. We identified six novel and two known mutations of BTK in 11 Chinese XLA patients from 8 families. Family 1 had a novel point mutation at the start codon (135G-->T) in exon 2. Family 2 had known mutation of single A insertion in a stretch of 7 A residues (341-347insA) recognized as mutation hotspot in exon 3. Family 3 had a novel point mutation in exon 11 (1074A-->G) which led to aberrant splicing. Family 4 had known mutation in exon 19 (2053C-->T) in CpG mutation hotspot. The novel mutation of family 5 was an A deleted in a run of three As (1017-1019delA) in exon 10. In family 6, exons 2 and 3 were lost in BTK mRNA, a novel deletion. Family 7 had a novel substitution in exon 2 (227T-->C) which led to change of a conserved leucine to serine. Family 8 had a novel point mutation at beginning of intron 14 (IVS14+ 6 T-->G) resulting in aberrant splicing. Hum Mutat 15:385, 2000.