Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation

Abstract

The RAG1/2 endonuclease initiates programmed DNA rearrangements in progenitor lymphocytes by generating doublestrand breaks at specific recombination signal sequences. This process, known as V(D)J recombination, assembles the vastly diverse antigen receptor genes from numerous V, D, and J coding segments. In vitro biochemical and cellular transfection studies suggest that RAG1/2 may also play postcleavage roles by forming complexes with the recombining ends to facilitate DNA end processing and ligation. In the current study, we examine the in vivo consequences of a mutant form of RAG1, RAG1-S723C, that is proficient for DNA cleavage, yet exhibits defects in postcleavage complex formation and end joining in vitro. We generated a knockin mouse model harboring the RAG1-S723C hypomorphic mutation and examined the immune system in this fully in vivo setting. RAG1-S723C homozygous mice exhibit impaired lymphocyte development and decreased V(D)J rearrangements. Distinct from RAG nullizygosity, the RAG1-S723C hypomorph results in aberrant DNA double-strand breaks within rearranging loci. RAG1-S723C also predisposes to thymic lymphomas associated with chromosomal translocations in a p53 mutant background, and heterozygosity for the mutant allele accelerates age-associated immune system dysfunction. Thus, our study provides in vivo evidence that implicates aberrant RAG1/2 activity in lymphoid tumor development and premature immunosenescence. IntroductionThe immense diversity of genes encoding the variable regions of antigen receptors is generated through rearrangement of component V, D, and J segments via a cut-and-paste mechanism known as V(D)J recombination. 1 These site-specific chromosomal rearrangements are initiated in progenitor lymphocytes by the recombination activating gene 1 (RAG1) and RAG2 proteins that comprise a lymphoid-specific endonuclease, RAG1/2. 2,3 DNA double-strand breaks (DSBs) are generated by RAG1/2 at specific recombination signal sequences (RSSs) adjacent to the numerous rearranging V, D, and J coding exons. 4 The broken ends are then processed and joined by ubiquitously expressed nonhomologous end-joining (NHEJ) DNA repair factors, including Ku70, Ku80, the DNAdependent protein kinase catalytic subunit (DNA-PKcs), Artemis, DNA ligase IV, XRCC4 and Cernunnos/XLF. 1,5,6 The RAG1/2 endonuclease initially binds to RSSs that are composed of a conserved heptamer and nonamer separated by relatively nonconserved spacer sequences of either 12 or 23 nucleotides. 4 Efficient cleavage requires recombination between one 12 RSS and one 23 RSS in the context of a synaptic, paired complex mediated by RAG1/2. RAG1/2 nicks the duplex DNA at the junction between the RSS heptamer and coding sequences then uses the resulting 3Ј OH in a direct transesterification reaction with the phosphodiester backbone of the opposing strand. The products of RAG1/2 cleavage are blunt, 5Ј phosphorylated signal ends and covalently closed, hairpin coding ends. Sequence elements within the nonconserved 12 and 23 spacers 7 and coding ...

GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

“…In detail, after initial purification of DN4 stage T cells by flow sorting, single cells were resorted or isolated by using the CellCelector automated cell picking system (ALS Automated Lab Solutions GmbH, Jena, Germany); digested in a 20-l solution containing 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl 2 , 3.0 g proteinase K (EM Science), and 0.1% Triton X-100 (Sigma); and incubated at 55°C for 60 min followed by 95°C for 15 min. First-round PCR amplified both V(D)Jrearranged alleles together in a 60-l final reaction mixture volume consisting of 20 l of the single-cell isolation solution described above, PCR buffer (10 mM Tris-HCl [pH 9.0], 50 mM KCl, and 2.0 mM MgCl 2 ), 200 M deoxynucleoside triphosphates (dNTPs), 0.5 U Taq DNA polymerase (New England BioLabs), and an oligonucleotide mixture (each at a 100 nM final concentration) (25,26,42,82) (Table 4) containing 22 forward primers corresponding to 23 V␤ exon families (2 primers were designed to amplify 2 V␤5 and 3 V␤8 multisequence families, respectively), the D␤1 and D␤2 diversity segment genes, 2 reverse primers matching joining-segment J␤1 and J␤2 sequences, as well as 1 primer set for ␤-actin. The PCR conditions were 94°C for 1 min and 5 cycles with the annealing temperature ramped from 66°C to 58°C, followed by 25 cycles of 94°C for 30 s, 56°C for 60 s, and 72°C for 60 s, with a final extension step at 72°C for 5 min.…”

Tp53 deletion in B lineage cells predisposes mice to lymphomas with oncogenic translocations

“…Second, germline NHEJ/Tp53-deficient mice succumb to pro-B lymphomas with RAG-dependent Igh/c-myc or Igh/N-myc translocations (Difilippantonio et al, 2002;Zhu et al, 2002;Gladdy et al, 2003;Rooney et al, 2004). Third, Tp53 À/À mice expressing a RAG1 mutant that exhibits defects in DNA end joining develop thymic lymphomas with clonal antigen receptor locus translocations (Giblin et al, 2009). Fourth, Tp53 À/À mice containing a genetic block in thymocyte development succumb to thymic lymphomas with RAG-dependent genomic instability (Haines et al, 2006).…”

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies

“…14,15 Mouse models of OS and of leaky SCID have been generated, such as Rag1 R972Q, 16 the Rag1 S723C, 7 , and Rag2 R229Q 17 mice. In addition to the mouse, SCID and SCID variants have also been modeled in the dog and horse.…”

“…We and others have shown that hypomorphic RAG mutations that affect, but do not abolish, V(D)J recombination, are often associated with distinct immunologic and clinical phenotypes with residual presence of T, and in some cases B, lymphocytes. [6][7][8][9] The presence of autologous, auto-reactive, activated, and oligoclonal T cells that infiltrate and damage peripheral organs is a hallmark of Omenn syndrome (OS). In other cases, hypomorphic RAG mutations may cause delayed disease onset, granuloma formation, autoimmunity, and/or dysgammaglobulinemia.…”