http://repub.eur.nl/res/aut/2850/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/15493/ 2009-01-01T00:00:00Z One of the severe combined immunodeficiencies (SCIDs), which is caused by a genetic defect in the signal transduction pathways involved in T-cell activation, is the ZAP70 deficiency. Mutations in ZAP70 lead to both abnormal thymic development and defective T-cell receptor (TCR) signaling of peripheral T-cells. In contrast to the lymphopenia in most SCID patients, ZAP70-deficient patients have lymphocytosis, despite the selective absence of CD8+ T-cells. The clinical presentation is usually before 2 years of age with typical findings of SCID. Here, we present three new ZAP70-deficient patients who vary in their clinical presentation. One of the ZAP70-deficient patients presented as a classical SCID, the second patient presented as a healthy looking wheezy infant, whereas the third patient came to clinical attention for the eczematous skin lesions simulating atopic dermatitis with eosinophilia and elevated immunoglobulin E (IgE), similar to the Omenn syndrome. This study illustrates that awareness of the clinical heterogeneity of ZAP70 deficiency is of utmost importance for making a fast and accurate diagnosis, which will contribute to the improvement of the adequate treatment of this severe immunodeficiency. http://repub.eur.nl/res/pub/35908/ 2007-09-01T00:00:00Z Unusual susceptibility to mycobacterial infections can be caused by deleterious mutations in genes that encode the interferon-γ receptor 1 chain. Such mutations hamper the activation of macrophages by a type 1 immune response and result in enhanced survival of intracellular pathogens. We here report two patients with unusual mycobacterial infections, both diagnosed with homozygous deleterious interferon-γ receptor 1 gene mutations. Patient 1 became ill after Bacillus Calmette-Guérin vaccination at the age of 9 months and died at the age of 18 months. She carried a homozygous C71Y mutation in the extracellular part of the mature interferon-γ receptor 1 protein, resulting in the lack of detectable protein expression and absence of interferon-γ dependent signaling. Patient 2 became ill at the age of 3 years, is still alive at 19 years of age, and has suffered from five successive infection episodes with atypical mycobacteria. A homozygous splice-site mutation in intron 3 was identified, resulting in the deletion of exon 3 at the mRNA level and consequently a truncated interferon-γ receptor 1 protein with absence of the transmembrane domain. Protein expression and interferon-γ dependent signaling were not detectable. http://repub.eur.nl/res/pub/8235/ 2003-01-01T00:00:00Z Severe combined immunodeficiency disease (SCID) can be immunologically classified by the absence or presence of T, B, and natural killer (NK) cells. About 30% of T(-)B(-)NK(+) SCID patients carry mutations in the recombination activating genes (RAG). Some T(-)B(-)NK(+) SCID patients without RAG gene mutations are sensitive to ionizing radiation, and several of these radiosensitive (RS) SCID patients were recently shown to have large deletions or truncation mutations in the Artemis gene, implying a role for Artemis in DNA double-strand break (dsb) repair. We identified 5 RS-SCID patients without RAG gene mutations, 4 of them with Artemis gene mutations. One patient had a large genomic deletion, but the other 3 patients carried simple missense mutations in conserved amino acid residues in the SNM1 homology domain of the Artemis protein. Extrachromosomal V(D)J recombination assays showed normal and precise signal joint formation, but inefficient coding joint formation in fibroblasts of these patients, which could be complemented by the wild-type Artemis gene. The cells containing the missense mutations in the SNM1 homology domain had the same recombination phenotype as the cells with the large deletion, indicating that these amino acid residues are indispensable for Artemis function. Immunogenotyping and immunophenotyping of bone marrow samples of 2 RS-SCID patients showed the absence of complete V(H)-J(H) gene rearrangements and consequently a complete B-cell differentiation arrest at the pre-B-cell receptor checkpoint-that is, at the transition from CyIgmu(-) pre-B-I cells to CyIgmu(+) pre-B-II cells. The completeness of this arrest illustrates the importance of Artemis at this stage of lymphoid differentiation. http://repub.eur.nl/res/pub/8222/ 2002-01-01T00:00:00Z The protein products of the recombination activating genes (RAG1 and RAG2) initiate the formation of immunoglobulin (Ig) and T-cell receptors, which are essential for B- and T-cell development, respectively. Mutations in the RAG genes result in severe combined immunodeficiency disease (SCID), generally characterized by the absence of mature B and T lymphocytes, but presence of natural killer (NK) cells. Biochemically, mutations in the RAG genes result either in nonfunctional proteins or in proteins with partial recombination activity. The mutated RAG genes of 9 patients from 7 families were analyzed for their recombination activity using extrachromosomal recombination substrates, rearrangement of endogenous Ig loci in RAG gene-transfected nonlymphoid cells, or the presence of Ig gene rearrangements in bone marrow (BM). Recombination activity was virtually absent in all 6 patients with mutations in the RAG core domains, but partial activity was present in the other 3 RAG-deficient patients, 2 of them having Omenn syndrome with oligoclonal T lymphocytes. Using 4-color flow cytometry, we could define the exact stage at which B-cell differentiation was arrested in the BM of 5 RAG-deficient SCID patients. In 4 of 5 patients, the absence of recombination activity was associated with a complete B-cell differentiation arrest at the transition from cytoplasmic (Cy) Igmu(-) pre-B-I cells to CyIgmu(+) pre-B-II cells. However, the fifth patient showed low frequencies of precursor B cells with CyIgmu and surface membrane IgM, in line with the partial recombination activity of the patient's mutated RAG gene and the detection of in-frame Ig gene rearrangements in BM. http://repub.eur.nl/res/pub/9829/ 2002-01-01T00:00:00Z X-linked agammaglobulinemia (XLA) is characterized by a severe B-cell deficiency, resulting from a differentiation arrest in the bone marrow (BM). Because XLA is clinically and immunologically heterogeneous, we investigated whether the B-cell differentiation arrest in BM of XLA patients is heterogeneous as well. First, we analyzed BM samples from 19 healthy children by flow cytometry. This resulted in a normal B-cell differentiation model with eight consecutive stages. Subsequently, we analyzed BM samples from nine XLA patients. Eight patients had amino acid substitutions in the Bruton's tyrosine kinase (BTK) domain or premature stop codons, resulting in the absence of functional BTK proteins. In seven of these eight patients a major differentiation arrest was observed at the transition between cytoplasmic Ig(mu-) pre-B-I cells and cytoplasmic Ig(mu+) pre-B-II cells, consistent with a role for BTK in pre-B-cell receptor signaling. However, one patient exhibited a very early arrest at the transition between pro-B cells and pre-B-I cells, which could not be explained by a different nature of the BTK mutation. We conclude that the absence of functional BTK proteins generally leads to an almost complete arrest of B-cell development at the pre-B-I to pre-B-II transition. The ninth XLA patient had a splice site mutation associated with the presence of low levels of wild-type BTK mRNA. His BM showed an almost normal composition of the precursor B-cell compartment, suggesting that low levels of BTK can rescue the pre-B-cell receptor signaling defect, but do not lead to sufficient numbers of mature B lymphocytes in the peripheral blood. http://repub.eur.nl/res/pub/9583/ 2001-01-01T00:00:00Z Regulation of allelic and isotypic exclusion of human immunoglobulin (Ig) light-chain genes was studied in 113 chronic B-cell leukemias as a "single-cell" model that allowed complete analysis of each light chain allele. Our data show that monospecific Ig light chain expression is in about 90% of cases determined by ordered recombination: Igkappa gene (IGK) rearrangements, followed by IGK deletions and Iglambda gene (IGL) rearrangements, resulting in the presence of only one functional Ig light chain rearrangement. In about 10% (10 cases), 2 functional Ig light chain rearrangements (IGK/IGL or IGL/IGL, but not IGK/IGK) were identified. This might be explained by the fact that regulation of the ordered recombination process is not fully strict, particularly when the IGL locus is involved. Unfavorable somatic mutations followed by receptor editing might have contributed to this finding. Eight of these 10 cases indeed contained somatic mutations. In cases with 2 functional Ig light chain rearrangements, both alleles were transcribed, but monospecific Ig expression was still maintained. This suggests that in these cases allelelic exclusion is not regulated at the messenger RNA level but either at the level of translation or protein stability or via preferential pairing of Ig light and Ig heavy chains. Nevertheless, ordered rearrangement processes are the main determinant for monospecific Ig light chain expression. http://repub.eur.nl/res/pub/9310/ 2000-01-01T00:00:00Z Age-matched reference values for lymphocyte subpopulations are generally obtained via cross-sectional studies, whereas patients are followed longitudinally. We performed a detailed longitudinal analysis of the changes in lymphocyte subpopulations in a group of 11 healthy infants followed from birth up to 1 y of age, with special attention for early developmental markers, markers of maturation, and markers of activation. We found that T and B lymphocytes increased at 1 and 6 wk of age, respectively. In contrast, NK cells showed a sharp decline directly after birth, suggesting that they are more important during pregnancy than thereafter. CD45RA+--mainly CD4+--naive T lymphocytes were high at birth, and increased further during the first year of life; they form a large expanding pool of cells, ready for participation in primary immune responses. The absolute counts of CD45RO+ memory T lymphocytes were similar in infants and adults, albeit with a lower level of expression of CD45RO on infant T lymphocytes. Almost all infant T lymphocytes expressed CD38 throughout the first year of life. The abundant expression of CD38 on an infant's T lymphocytes might be related to a greater metabolic need of the large population of naive untriggered cells that are continually involved in primary immune responses during the first year of life. The high B lymphocyte counts in infants mainly concerned CD38+ B lymphocytes throughout the first year of life. Also, the relative frequencies of CD1c+ and CD5+ B lymphocytes were higher throughout the first year of life than in adults. Therefore, CD1c, CD5, and CD38 could be markers of untriggered B lymphocytes. In conclusion, our longitudinal survey of T and B lymphocytes, NK cells, and their subpopulations during the first year of life helps to complete the picture of lymphocyte development in infants. This information contributes to the correct interpretation of data from infants with possible immune disorders. http://repub.eur.nl/res/pub/9083/ 1999-01-01T00:00:00Z Immunophenotyping of blood lymphocyte subpopulations in neonates and young infants is hampered by the limited amount of blood that can be collected. Contamination of the flow cytometric "lympho-gate" by normoblasts and analysed erythrocytes, and therefore the underestimation of the relative frequencies of lymphocyte subpopulations, interferes with the precise calculation of absolute counts. A microassay was developed by adapting the lysed whole blood technique. Triple immunostaining in a single antibody staining step was used to reduce washing steps and cell loss. Introduction of a triple staining for CD71 (expressed by erythroid precursors), glycophorin A (GpA, expressed by all erythroid cells), and CD45 (expressed by all leucocytes) permitted the relative frequencies of normoblasts (CD71(+)/GpA+/CD45(-) population) and unlysed erythrocytes (CD71(-)/GpA+/CD45(-) population)to be identified and measured within the "lympho-gate" of neonatal cord blood samples. Particularly high frequencies were found (median: 31%) in cord blood samples from preterm neonates. These erythroid cells disappear rapidly by 1 week of age The relative frequencies of erythroid cells can be used to calculate correct lymphocyte subpopulation values. Using only 0.5-0.8 ml of blood, this micro- assay would also be suitable for rapid prenatal immunodiagnosis of congenital immunodeficiencies.