Homozygous DNA ligase IV R278H mutation in mice leads to leaky SCID and represents a model for human LIG4 syndrome

Abstract

DNA ligase IV (LIG4) is an essential component of the nonhomologous end-joining (NHEJ) repair pathway and plays a key role in V(D)J recombination. Hypomorphic LIG4 mutations in humans are associated with increased cellular radiosensitivity, microcephaly, facial dysmorphisms, growth retardation, developmental delay, and a variable degree of immunodeficiency. We have generated a knock-in mouse model with a homozygous Lig4 R278H mutation that corresponds to the first LIG4 mutation reported in humans. The phenotype of homozygous mutant mice Lig4 R278H/R278H (Lig4 R/R ) includes growth retardation, a decreased life span, a severe cellular sensitivity to ionizing radiation, and a very severe, but incomplete block in T and B cell development. Peripheral T lymphocytes show an activated and anergic phenotype, reduced viability, and a restricted repertoire, reminiscent of human leaky SCID. Genomic instability is associated with a high rate of thymic tumor development. Finally, Lig4 R/R mice spontaneously produce low-affinity antibodies that include autoreactive specificities, but are unable to mount high-affinity antibody responses. These findings highlight the importance of LIG4 in lymphocyte development and function, and in genomic stability maintenance, and provide a model for the complex phenotype of LIG4 syndrome in humans.genomic instability | immunodeficiency | lymphocytes | nonhomologous end joining | immune dysregulation N onhomologous end joining (NHEJ) is one of the two major DNA repair pathways in mammalian cells that protect the genome against DNA double-stranded breaks (DSBs) generated by genomic insults such as ionizing radiation (IR) or reactive oxygen species or that arise during V(D)J recombination (1) and Ig heavy chain (IGH) class switch recombination (CSR) (2). V(D)J recombination is the process by which developing T and B lymphocytes assemble their antigen receptor variable region exons. In this process, the recombinase activating gene (RAG)1 and RAG2 proteins introduce DSBs at recombination signal sequences (RSS) that flank coding variable (V), diversity (D), and joining (J) elements. This process generates hairpin-sealed coding ends and blunt phosphorylated signal ends. These DNA ends are recognized and resolved by proteins of the NHEJ pathway, with formation of coding joins and recombination signal (RS) joins, respectively. In particular, the KU70/KU80 heterodimer binds directly the DNA ends, allowing activation of the DNA-PK catalytic subunit (DNA-PKcs) (3) and phosphorylation of Artemis, which mediates opening of hairpin-sealed DNA coding ends (4,5

A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors

“…19,20,21 Hypomorphic Lig4 mice display a profound immunodeficiency, an age-dependent decrease in HSC numbers, impaired self-renewal function of long-term adult HSCs, reduced bone marrow cellularity and lower numbers of erythrocyte precursors, as well as a severe block in B and T cell development. 13,22,23 Together these data suggest that HSCs accumulate DSBs during ageing, but most importantly rely on the NHEJmediated-DSB repair pathway in maintaining homoeostasis. Indeed, a recent report has shown that NHEJ is the main DSB repair pathway in quiescent HSCs.…”

Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

“…After the Variable (V), Diversity (D), and Joining (J) elements of the T cell receptor (TCR) and immunoglobulin genes have been targeted at recombination signal sequences by the recombination-activating gene 1 (RAG1) and RAG2 endonuclease complex [2], DNA ends are stabilized by the Ku70 and Ku80 proteins. The holoenzyme DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is then recruited to stabilize the complex and to activate the endonuclease ARTEMIS, which opens the hairpins formed at the DNA coding ends [3].…”

Repair of Double-Strand Breaks by End Joining

“…Mouse models and genetic diseases, including DNA-PKcs, XLF, and Artemis mutations in humans, further show that c-NHEJ dysfunction leads to immunodeficiency and B-and T-cell loss due to failed V(D)J recombination as well as varying degrees of dysmorphology, growth retardation, and developmental delay that correlate with the degree of c-NHEJ dysfunction Moshous et al 2001;Buck et al 2006;Li et al 2008a;Kerzendorfer and O'Driscoll 2009;van der Burg et al 2009). One notable case is the ligase IV syndrome caused by hypomorphic mutations in Lig4 (Chistiakov 2010), which, like mouse models Rucci et al 2010), suggests that complete loss of this obligatory and specific c-NHEJ factor is incompatible with mammalian life. Apoptosis of developing neurons and progressive loss of hematopoietic stem cells are two specific features of hypomorphic mouse Lig4 mutation that reveal its importance to viability of different cell systems (Nijnik et al 2007;Gatz et al 2011).…”