C. I. E. Smith

Mutations of the human BTK gene coding for Bruton tyrosine kinase in X-linked agammaglobulinemia

X‐linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the gene coding for Bruton agammaglobulinemia tyrosine kinase (BTK). A database (BTKbase) of BTK mutations lists 544 mutation entries from 471 unrelated families showing 341 unique molecular events. In addition to mutations, a number of variants or polymorphisms have been found. Mutations in all the five domains of BTK cause the disease, the single most common event being missense mutations. Most mutations lead to truncation of the enzyme. The mutations appear almost uniformly throughout the molecule. About one‐third of point mutations affect CpG sites, which usually code for arginine residues. The putative structural implications of all the missense mutations are provided in the database. BTKbase is available at http://www.uta.fi/imt/bioinfo. Hum Mutat 13:280–285, 1999.

The life situations of patients with primary antibody deficiency untreated or treated with subcutaneous gammaglobulin infusions.

The life situations of 25 patients with hypogammaglobulinaemia were studied before and after the initiation of subcutaneous replacement therapy by using medical records, data registers and questionnaires (a study and a disease‐specific questionnaire, the Sickness Impaet Profile and the General Health Rating Index). Before treatment, the patients perceived more dysfunctions with regard to ambulation. mobility, emotional behaviour, social interaction, sleep and rest, household management, work and recreation or pastime activities compared with a Swedish reference group (P= 0.0001). A significant increase in the perceived frequency of infections was also seen in untreated patients compared with a group of healthy individuals (P=0.0001) After 18 months of weekly subcutaneous infusions of an intramuscular gammaglobulin preparation (100 mg/kg), the patients reported a significantly increased, health‐related function and improved self‐rated health. A significantly higher pre‐infusion IgG level was also found.

Primary immunodeficiency mutation databases

Primary immunodeficiencies are intrinsic defects of immune systems. Mutations in a large number of cellular functions can lead to impaired immune responses. More than 80 primary immunodeficiencies are known to date. During the last years genes for several of these disorders have been identified. Here, mutation information for 23 genes affected in 14 immunodefects is presented. The proteins produced are employed in widely diverse functions, such as signal transduction, cell surface receptors, nucleotide metabolism, gene diversification, transcription factors, and phagocytosis. Altogether, the genetic defect of 2,140 families has been determined. Diseases with X-chromosomal origin constitute about 70% of all the cases, presumably due to full penetrance and because the single affected allele causes the phenotype. All types of mutations have been identified; missense mutations are the most common mutation type, and truncation is the most common effect on the protein level. Mutational hotspots in many disorders appear in CPG dinucleotides. The mutation data for the majority of diseases are distributed on the Internet with a special database management system, MUTbase. Despite large numbers of mutations, it has not been possible to make genotype-phenotype correlations for many of the diseases.

Prevalence, genetics and clinical presentation of chronic granulomatous disease in Sweden

To estimate the prevalence of chronic granulomatous disease (CGD) in Sweden, an inquiry asking for known and possible CGD cases was mailed to paediatric, internal medicine and infectious disease departments all over Sweden. The detected patients were characterized as to genetics and the clinical presentation. Twenty–one patients (belonging to 16 different families) were found, corresponding to a prevalence of ∼ 1/450 000 individuals. The patients with X–linked disease, lacking a functional gp91phox protein (n= 12), comprised 57% and 43% of the patients had an autosomal recessive (AR) disease lacking p47phox (n= 7) or p67phox (n=1), respectively. All unrelated patients with X–linked disease displayed different gene abnormalities such as point mutations predicting nonsense (n= 3), missense (n= 1) or splice site mutations (n = 2), but also a total deletion and a unique 40 base pair duplicature insertion. The patients with p47phox–deficiency showed a GT deletion at a GTGT tandem repeat, and the p67phox–deficient patient displayed a heterozygous in–frame deletion of AAG combined with a large deletion in the other allele. Three patients died during the study period, two from Pseudomonas cepacia infections. Patients with X–linked disease had more frequent infections (mean of 1.7 per year), than the patients with AR inheritance (0.5 infections per year). The most common infections were dermal abscesses (n=111), followed by lymphadenitis (n=82) and pneumonias (n=73). Inflammatory bowel disease–like symptoms, mimicking Crohns disease of the colon, was seen in three CGD patients.

The Role of Adherent Cells in B and T Lymphocyte Activation

Macrophages have been found to be of importance in several difierent immunological reactions. Thus, macrophages are required for induction of a primary immune response to heterologous red blood cells in vitro (Mosier & Coppleson 1968), and for a proliferative response induced in vitro to alloantigens (Alter & Bach 1970, Rode & Gordon 1970, Twormey et al. 1970), as well as for the developement of cytotoxic cells (Wagner et al. 1972, McDonald et al. 1973). Fxirthermore, macrophage and factors released by macrophages can induce proliferation in spleen cells and cause production of polyclonal immunoglobuiins (Lemke & Opitz 1976, Moller et al. 1976, Opitz et al. 1976). In all these systems, however, fetal calf serum together with 2-mercaptoethanol (2-ME) can substitute for macrophages, indicating a nonspecific function of adherent cells (Chen & Hirsh 1972, Bevan et al. 1974, Lemke & Opitz 1976). In this paper, we report on some effects of adherent cells on B and T cell activation induced by polyclonal cell activators, and we discuss the possible mechanisms behind the synergistic effect observed between B and T cell mitogens and adherent cells. In the experiments shown, Fi hybrids of the inbred mouse strains A/Sn and C57B1 or B10.5M were used, if not otherwise indicated. Cells were cultured senimfree in microcultures (usually triplicate cultures with 5 X 10* cells/0.2 ml of medium) and incorporation of ^H-thymidine was measured on day 2.

High Levels of Interferon Gamma in the Plasma of Children With Complete Interferon Gamma Receptor Deficiency

We have found that children with complete interferon gamma (IFNγ) receptor deficiency, unlike patients with other genetic defects predisposing them to mycobacterial diseases, have very high levels of IFNγ in their plasma. This unexpected observation provides a simple and accurate diagnostic method for complete IFNγ receptor deficiency in children with clinical disease caused by bacille Calmette-Guérin vaccines or environmental nontuberculous mycobacteria.

Thermal unfolding of small proteins with SH3 domain folding pattern

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two‐state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec). At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk‐, Itk‐, and Tec‐SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12–16 kJ/mol but show quite high melting temperatures.

Six X-linked agammaglobulinemia-causing missense mutations in the src homology 2 domain of Bruton's tyrosine kinase: Phosphotyrosine-binding and circular dichroism analysis

Src homology 2 (SH2) domains recognize phosphotyrosine (pY)-containing sequences and thereby mediate their association to ligands. Bruton’s tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase, in which mutations cause a hereditary immunodeficiency disease, X-linked agammaglobulinemia (XLA). Mutations have been found in all Btk domains, including SH2. We have analyzed the structural and functional effects of six disease-related amino acid substitutions in the SH2 domain: G302E, R307G, Y334S, L358F, Y361C, and H362Q. Also, we present a novel Btk SH2 missense mutation, H362R, leading to classical XLA. Based on circular dichroism analysis, the conformation of five of the XLA mutants studied differs from the native Btk SH2 domain, while mutant R307G is structurally identical. The binding of XLA mutation-containing SH2 domains to pY-Sepharose was reduced, varying between 1 and 13% of that for the native SH2 domain. The solubility of all the mutated proteins was remarkably reduced. SH2 domain mutations were divided into three categories: 1) Functional mutations, which affect residues presumably participating directly in pY binding (R307G); 2) structural mutations that, via conformational change, not only impair pY binding, but severely derange the structure of the SH2 domain and possibly interfere with the overall conformation of the Btk molecule (G302E, Y334S, L358F, and H362Q); and 3) structural-functional mutations, which contain features from both categories above (Y361C).

Intranasal inoculation of Mycoplasma pulmonis in mice with severe combined immunodeficiency (SCID) causes a wasting disease with grave arthritis

Mycoplasma pulmonis or Myc. pneumoniae were inoculated intranasally to C.B‐17 scid/scid mice (severe combined immunodeficient (SCID) mice). Immunocompetent C.B‐17 mice were inoculated as controls. During the observation period of 5 weeks the mice were killed and necropsied. Mycoplasma pulmonis was recovered from all of the inoculated mice, and dissemination to various tissues increased with time. SCID mice, unlike immunocompetent mice, did not show lung lesions but exhibited severe inflammatory changes of the joints. Mycoplasma pulmonis, however, was isolated both from the lungs and the articular lesions. In addition, SCID mice infected for more than 3 weeks suffered from a pronounced loss of weight and emaciation. In the experiment with Myc. pneumoniae the agent could be reisolated, but lesions were not found in any of the infected mice. Mycoplasma pulmonis infection in SCID mice may be useful as a model of arthritis in immunodeficient humans.

Signalling of Bruton's tyrosine kinase, Btk.

Brutons tyrosine kinase, which is encoded by the BTK gene, is a cytoplasmic protein tyrosine kinase (PTK) crucial for B‐cell development and differentiation. It belongs to the Tec family of PTKs containing several domains that are characteristic of signalling molecules. In humans, mutations that disrupt the function of this gene lead to the classical XLA syndrome (X‐linked agammaglobulinaemia), a primary immunodeficiency mainly characterized by lack of mature B cells as well as low levels of immunoglobulins. In contrast, animal models of this disease such as the xid mice display profoundly milder XLA phenotype. BTK phosphorylation and activation in response to engagement of the B‐cell receptor (BCR) by antigen is a dynamic process whereby a variety of proteins interact with each other and recruit signalling molecules resulting in a physiological response such as B‐cell proliferation and antibody production. The main players, however, that participate in the intracellular downstream cascade have not yet been identified and are therefore under intense scrutiny in several laboratories. This review discusses certain aspects of BTK activation following receptor stimulation by agonists and how this event is translated into the biochemical signals within the cell that eventually lead to nuclear responses.