RESUMO
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-JH junctions 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 determine 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.