Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination

Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increas e the diversity of antigen receptors. Two inborn errors of purine metabolis m, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphor ylase (PNP), result in defective lymphoid development and aberrant pools of 2'-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in alt ered N regions in an extrachromosomal substrate transfected into T-cell or pre-B-cell lines. Exposure of the transfected cells to 2'-deoxyadenosine an d an ADA inhibitor increased the dATP pool and resulted in a marked increas e in A-T insertions at recombination junctions, with an overall decreased f requency of V(D)J recombination. Sequence analysis of V-H-D-H-J(H) junction s from the IgM locus in B-cell lines from ADA-deficient patients demonstrat ed an increase in A-T insertions equivalent to that found in the transfecte d cells. In contrast, elevation of dGTP pools, as would occur in PNP defici ency, did not alter the already rich G-C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertio ns are influenced by increases in dATP levels, potentially leading to alter ations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency.