Publications (2)10.53 Total impact
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Article: Array-based sequence capture and next-generation sequencing for the identification of primary immunodeficiencies.
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ABSTRACT: Primary immunodeficiencies are genetic disorders in which components of immunological pathways are either missing or dysregulated. With the advent of next-generation sequencing, testing for genes in conditions with a heterogeneous genetic background seems more promising. We designed a custom microarray with 385K probe capacity to capture exons of 395 human genes, known or predicted to be associated with primary immunodeficiency and immune regulation. Enriched target DNA was sequenced using a GS FLX Titanium 454 platform. The patients selected were likely to have an underlying immunodeficiency. In one patient with hepatosplenomegaly, recurrent infections and an elevated IgM level, sequence analysis of the patient and his two unaffected parents identified ATM (ataxia telangiectasia mutated) as the underlying defect. In a second child with a clinical SCID phenotype, we detected a mutation in the ARTEMIS gene after focusing on SCID-associated genes. 454 sequencing yielded 152,000-397,000 high-quality reads per patient. 78-99% of the targeted nucleotides were covered at least one time, 76-82% at least five times. Array-based sequence capture expands our capacities to sequence large targeted DNA regions in a less laborious and time-consuming approach. Our array was capable to find the underlying genetic defect in two patients with suspected primary immunodeficiency. Upcoming whole-exome sequencing definitely will add more valuable data, but bioinformatical analysis and validation of variants already pose major challenges.Scandinavian Journal of Immunology 03/2012; 75(3):350-4. · 2.23 Impact Factor -
Article: Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases.
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ABSTRACT: The purpose of this study was the appraisal of the clinical and functional consequences of germline mutations within the gene for the IL-2 inducible T-cell kinase, ITK. Among patients with Epstein-Barr virus-driven lymphoproliferative disorders (EBV-LPD), negative for mutations in SH2D1A and XIAP (n=46), we identified two patients with R29H or D500T,F501L,M503X mutations, respectively. Human wild-type (wt) ITK, but none of the mutants, was able to rescue defective calcium flux in murine Itk(-/-) T cells. Pulse-chase experiments showed that ITK mutations lead to varying reductions of protein half-life from 25 to 69% as compared with wt ITK (107 min). The pleckstrin homology domain of wt ITK binds most prominently to phosphatidylinositol monophosphates (PI(3)P, PI(4)P, PI(5)P) and to lesser extend to its double or triple phosphorylated derivates (PIP2, PIP3), interactions which were dramatically reduced in the patient with the ITK(R29H) mutant. ITK mutations are distributed over the entire protein and include missense, nonsense and indel mutations, reminiscent of the situation in its sister kinase in B cells, Bruton's tyrosine kinase.Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 01/2012; 26(5):963-71. · 8.30 Impact Factor
