T-B-NK+severe combined immunodeficiency (SCID) is caused by a defect in V(D)J recombination. A subset of these patients has a mutation in one of the non-homologous end joining (NHEJ) genes, most frequently the Artemis gene. Artemis is involved in opening of hairpin-sealed coding ends. The low levels of residual DH-JHjunctions that could be amplified from patients' bone marrow precursor B cells showed high numbers of palindromic (P)-nucleotides. In 25% of junctions, microhomology was observed in the P-nucleotide regions, whereas this phenomenon was never observed in junctions amplified from bone marrow precursor B cells from healthy controls. We utilized this difference between Artemis-deficient cells and normal controls to develop a V(D)J recombination assay to detern-dne hairpin-opening activity. Mutational analysis of the Artemis gene confirmed and extended the mapping of an N-terminal nuclease active site, which contains several indispensable aspartate residues. C-terminal deletion mutants did not show such severe defects in the V(D)J recombination assay using transient overexpression of (mutated) Artemis protein. However, a C-terminal deletion mutation causes T-B-NK+SCID, indicating that the Artemis C terminus is essential for V(D)J recombination at the normal Artemis expression level. The V(D)J recombination assays used in this study contribute to the diagnostic strategy for T-B-NK+SCID patients.

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Keywords Gene rearrangement, Human, Immunodeficiency diseases, Molecular biology
Persistent URL dx.doi.org/10.1002/eji.200737624, hdl.handle.net/1765/35081
Journal European Journal of Immunology
van der Burg, M, Verkaik, N.S, den Dekker, A.T, Barendregt, B.H, Pico-Knijnenburg, I, Tezcan, I, … van Gent, D.C. (2007). Defective Artemis nuclease in characterized by coding joints with microhomology in long palindromic-nucleotide stretches. European Journal of Immunology, 37(12), 3522–3528. doi:10.1002/eji.200737624