B cells in patients with X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) has been described as a disorder in which pre-B cells fail to differentiate into B cells. However, a small number of B cells have been seen occasionally in patients with this disorder. Because the phenotype of these cells might be helpful in defining the site of the defect in XLA, immunofluorescent staining techniques were used to characterize the B cells that can be found in patients with XLA. Surface IgM-positive B cells could be detected in the peripheral circulation of all seven patients studied. These B cells constituted a very small percentage of the total lymphocytes (0.01 to 0.3% compared with 3.2 to 13.7% in controls) and differed in phenotype from control B cells. They were much more brightly stained for surface IgM (p less than 0.001) and less brightly stained for Ia (p less than 0.01). This phenotype is similar to that described for immature B cells in the mouse. Over 80% of the patients' B cells expressed surface IgD, and all expressed the B cell marker B1, but only 35% expressed the B cell marker B2. This B cell marker, which is the C3d receptor and the Epstein-Barr virus receptor, is expressed later in ontogeny than B1 and can be detected on over 80% of control B cells. All B cells expressed either kappa or lambda light chain. These findings indicate that the defect in differentiation of pre-B cells into B cells is not absolute in patients with XLA. The immature phenotype of the B cells additionally suggests that there may be a block in the maturation of B cells at more than one stage of differentiation in this disorder.     

Mutations of the Btk gene in 12 unrelated families with X-linked agammaglobulinemia in Japan

Alterations of the Bruton's tyrosine kinase(Btk) gene are responsible for X-linked agammaglobulinemia (XLA). Although mutations in various regions were reported mainly in the Caucasian population, correlation between the locations of mutation and the clinical phenotypes remains unclear. We report 12 abnormalities of theBtk gene found in 12 unrelated families out of 14 XLA families in Japan and their clinical features. We utilized Southern blotting and single-strand conformation polymorphism (SSCP) analysis. Gene rearrangement in the kinase domain was identified in two patients by Southern blotting. Seven point mutations, two small deletions, and one small insertion were detected by SSCP and sequencing. The SSCP analysis also provided information about the carriers in these families. We found some clinical heterogeneity in the affected family members with the same gene mutation. Moreover, there is considerable inconsistency between the locations of gene aberrations and the immunological phenotypes. Some patients with a nonsense mutation, which may result in the lack of kinase domain, have detectable B cells and immunoglobulins. These identified alterations will provide valuable clues to theBtk protein function and the pathogenesis of XLA.     

X-linked hypophosphatemia in Polish patients. 1. Mutations in the PHEX gene

We present twenty-nine PHEX gene mutations extending our previous work, giving it to a total of 37 different mutations identified in Polish patients with familial or sporadic X-linked hypophosphatemia. Deletions, insertions and nucleotide substitutions leading to frameshift (27%), stop codon (29%), splice site (24%), and missense mutations (20%) were found. The mutations are distributed along the gene; exons 3, 4, 11, 12, 14, 15, 17, 20 and 22 are regions with the most frequent mutation events. Four mutations, P534L, G579R, R549X and IVS15+1nt, recurred in three, four, two and three unrelated patients, respectively. They have also been detected in affected persons from other countries. Twenty-eight mutations are specific for Polish population and almost all of them are unique. Most of the identified mutations are expected to result in major changes in protein structure and/or function.     

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults

Background  

X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood.     

Ecto-5'-nucleotidase activity in T and B lymphocytes from normal subjects and patients with congenital X-linked agammaglobulinemia.

Ecto-5'-nucleotidase activity was measured in lymphocyte subpopulations isolated from normal subjects and patients with congenital X-linked agammaglobulinemia. B lymphocytes from normal subjects have at least three times more ecto-5'-nucleotidase activity than T lymphocytes. Patients with X-linked agammaglobulinemia have 56% of normal activity in their T cells, and lack a lymphocyte subpopulation high in nucleotidase activity. High activity of ecto-5'-nucleotidase may be a biochemical marker for mature surface immunoglobulin-bearing B cells.     

Evidence for male X chromosomal mosaicism in X-linked agammaglobulinemia

Summary X-Linked agammaglobulinemia (XLA) is a severe antibody deficiency disease in man, resulting from an arrest in differentiation of pre-B cells. XLA is recessive: female carriers do not exhibit antibody deficiency, but manifest an exclusive inactivation of the XLA-carrying X chromosome in all peripheral blood B lymphocytes. An exclusive inactivation of the paternal X chromosome in the B lymphocytes of all daugthers thers of a male who had no agammalobulineamia demonstrated that the XLA defect can originate from healthy males. These males are X chromosomal mosaics. X-Chromosomal RFLP segregation analyses in other XLA pedigrees suggest a frequent introduction of XLA by healthy males. This implies that XLA often originates from mitotic errors, either at postmeiotic or early postzygotic stages.     

Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.

Bruton X-linked agammaglobulinemia (XLA) is a phenotypically recessive genetic disorder of B lymphocyte development. Female carriers of XLA, although asymptomatic, have a characteristic B cell lineage-specific skewing of the pattern of X inactivation. Skewing apparently results from defective growth and maturation of B cell precursors bearing a mutant active X chromosome. In this study, carrier status was tested in 58 women from 22 families referred with a history of agammaglobulinemia. Primary carrier analysis to examine patterns of X inactivation in CD19+ peripheral blood cells (B lymphocytes) was conducted using quantitative PCR at the androgen-receptor locus. Obligate carriers of XLA demonstrated > 95% skewing of X inactivation in peripheral blood CD19+ cells but not in CD19- cells. Carrier status for mothers of isolated affected males could be assessed in 10 of 11 families: 7 women showed skewing, and 3 did not. Five carriers were found in six families in which there were no living affected males. Among all those tested, one individual's carrier status was considered to be indeterminate and five women were noninformative for the carrier test. Results obtained by the carrier test were congruent with linkage analysis (where applicable) using the RFLPs DXS178 and DXS94 and two newly developed polymorphic microsatellite markers, DXS178CA and DXS101AAT. Refinements in techniques for primary carrier testing and genetic mapping of XLA now make possible an ordered approach to diagnosis, prenatal diagnosis, and genetic counseling.     

Molecular and structural characterization of five novel mutations in the Bruton's tyrosine kinase gene from patients with X-linked agammaglobulinemia.

BACKGROUND: The Btk (Bruton's tyrosine kinase) gene has been shown to be mutated in the human immunodeficiency disease, XLA (X-linked agammaglobulinemia). Btk is a member of the Tec family of cytosolic protein tyrosine kinases with distinct functional domains PH, TH, SH3, SH2, and kinase. Mutations have been observed in each of the Btk subdomains in XLA. We have analyzed the Btk gene in six XLA patients from five unrelated families. MATERIALS AND METHODS: DNA was prepared from the patients peripheral blood. The Btk exons including the junctional sequences were analyzed by single-strand conformation polymorphism (SSCP) followed by direct nucleotide sequencing after PCR-amplification. For structural analysis, the missense mutations were introduced into three-dimensional models of the PH and kinase domains of Btk and the outcome was predicted based on the knowledge of the protein function. RESULTS: Five novel mutations and two novel polymorphisms, all of which resulted from single-base alterations, were identified. Three of the five mutations were in the PH domain and two were in the kinase domain of Btk. Three of these mutations were of the missense type, two of which altered the same codon in the PH domain; the third one was located in the kinase domain. The fourth mutation was a point deletion in the PH domain causing a frameshift followed by premature termination. The fifth mutation was a splice donor-site mutation within the kinase domain which could result in an exon skipping. In four of the five instances, mothers of the patients were shown to be obligate carriers. In one instance, a sibling sister was identified as a heterozygote establishing her as a carrier. CONCLUSIONS: Functional consequences of the mutations causing frameshifts and altered splicing can be inferred directly. Functional consequences of the missense mutations were interpreted by 3-dimensional structural modeling of Btk domains. It is proposed that the two PH domain mutations will interfere with membrane localization while the kinase domain mutation will interfere with the enzymatic function of Btk. This study provides further insight into the role of Btk in XLA.     

X-chromosome inactivation and mutation pattern in the Bruton's tyrosine kinase gene in patients with X-linked agammaglobulinemia. Italian XLA Collaborative Group

BACKGROUND: The diagnosis of X-linked agammaglobulinemia (XLA) is not always clearcut. Not all XLA conform to the classic phenotype and less than 50% of affected boys have a family history of immunodeficiency. Mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for the majority of agammaglobulinemia cases. However, a certain proportion of patients may have mutations involving other genes, although they show with an XLA phenotype. We performed BTK gene mutation analysis in 37 males with presumed XLA and analyzed the pattern of X-chromosome inactivation (XCI) in 31 mothers to evaluate the relevance of these approaches to diagnosis and genetic counseling. MATERIALS AND METHODS: Twenty affected males with a sporadic occurrence and 17 familial cases belonging to nine families were enrolled within the framework of the Italian Multicenter Clinical Study on XLA. We used non-isotopic RNase cleavage assay (NIRCA), followed by cDNA sequence determination to screen for BTK mutations and X-chromosome inactivation analysis for carrier detection. RESULTS: Using the cDNA-based approach, the identification of BTK gene abnormalities confirmed the clinical diagnosis of XLA in 31 of 37 affected infants. Missense was the most frequent mutational event and the kinase domain was mostly involved. In addition, nine novel mutations were identified. In sporadic cases, BTK gene abnormalities were identified in 9 out of 10 patients whose mothers had a nonrandom pattern of XCI and in 5 out of 6 patients whose mother had a random pattern of XCI. With the exception of one family, all patients with a familial occurrence and born to mothers with a nonrandom pattern of XCI had mutations of the BTK gene. CONCLUSIONS: Our findings indicate that in sporadic cases BTK gene sequencing is the only reliable tool for a definitive diagnosis of XLA and support XCI as the first diagnostic tool in the mothers of affected males in multiple generations. Furthermore, our molecular analysis confirms that 10-20% of BTK-unaltered patients have disorders caused by defects in other genes.     

Mutations, clinical findings and survival estimates in South American patients with X-linked adrenoleukodystrophy

In this study, we analyzed the ABCD1 gene in X-linked adrenoleukodystrophy (X-ALD) patients and relatives from 38 unrelated families from South America, as well as phenotypic proportions, survival estimates, and the potential effect of geographical origin in clinical characteristics.

Methods

X- ALD patients from Brazil, Argentina and Uruguay were invited to participate in molecular studies to determine their genetic status, characterize the mutations and improve the genetic counseling of their families. All samples were screened by SSCP analysis of PCR fragments, followed by automated DNA sequencing to establish the specific mutation in each family. Age at onset and at death, male phenotypes, genetic status of women, and the effect of family and of latitude of origin were also studied.

Results

We identified thirty-six different mutations (twelve novel). This population had an important allelic heterogeneity, as only p.Arg518Gln was repeatedly found (three families). Four cases carried de novo mutations. Intra-familiar phenotype variability was observed in all families. Out of 87 affected males identified, 65% had the cerebral phenotype (CALD). The mean (95% CI) ages at onset and at death of the CALD were 10.9 (9.1–12.7) and 24.7 (19.8–29.6) years. No association was found between phenotypic manifestations and latitude of origin. One index-case was a girl with CALD who carried an ABCD1 mutation, and had completely skewed X inactivation.

Conclusions

This study extends the spectrum of mutations in X-ALD, confirms the high rates of de novo mutations and the absence of common mutations, and suggests a possible high frequency of cerebral forms in our population.     

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus. Received: 20 August 1996     

BTKbase, mutation database for X-linked agammaglobulinemia (XLA).

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton's agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations has been compiled and the recent update lists 463 mutation entries from 406 unrelated families showing 303 unique molecular events. In addition to mutations, the database also lists variants or polymorphisms. Each patient is given a unique patient identity number (PIN). Information is included regarding the phenotype including symptoms. Mutations in all the five domains of BTK have been noticed to cause the disease, the most common event being missense mutations. The mutations appear almost uniformly throughout the molecule and frequently affect CpG sites that code for arginine residues. The putative structural implications of all the missense mutations are given in the database. The improved version of the registry having a number of new features is available at http://www. helsinki.fi/science/signal/btkbase.html