title: The DNA Ligase IV Syndrome R278H Mutation Impairs B Lymphopoiesis via Error-Prone Nonhomologous End-Joining.
abstract: Hypomorphic mutations in the nonhomologous end-joining (NHEJ) DNA repair protein DNA ligase IV (LIG4) lead to immunodeficiency with varying severity. In this study, using a murine knock-in model, we investigated the mechanisms underlying abnormalities in class switch recombination (CSR) associated with the human homozygous Lig4 R278H mutation. Previously, we found that despite the near absence of Lig4 end-ligation activity and severely reduced mature B cell numbers, Lig4(R278H/R278H) (Lig4(R/R)) mice exhibit only a partial CSR block, producing near normal IgG1 and IgE but substantially reduced IgG3, IgG2b, and IgA serum levels. In this study, to address the cause of these abnormalities, we assayed CSR in Lig4(R/R) B cells generated via preassembled IgH and IgK V region exons (HL). This revealed that Lig4(R278H) protein levels while intact exhibited a higher turnover rate during activation of switching to IgG3 and IgG2b, as well as delays in CSR kinetics associated with defective proliferation during activation of switching to IgG1 and IgE. Activated Lig4(R/R)HL B cells consistently accumulated high frequencies of activation-induced cytidine deaminase-dependent IgH locus chromosomal breaks and translocations and were more prone to apoptosis, effects that appeared to be p53-independent, as p53 deficiency did not markedly influence these events. Importantly, NHEJ instead of alternative end-joining (A-EJ) was revealed as the predominant mechanism catalyzing robust CSR. Defective CSR was linked to failed NHEJ and residual A-EJ access to unrepaired double-strand breaks. These data firmly demonstrate that Lig4(R278H) activity renders NHEJ to be more error-prone, and they predict increased error-prone NHEJ activity and A-EJ suppression as the cause of the defective B lymphopoiesis in Lig4 patients.
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; Broad Institute of MIT and Harvard, Cambridge, MA 02142; and.
authors.forename: Jihye
authors.initials: J
authors.lastname: Park
authors.affiliation: Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Welner
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215;
authors.forename: Mei-Yee
authors.initials: MY
authors.lastname: Chan
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215;
authors.forename: Logan
authors.initials: L
authors.lastname: Troppito
authors.affiliation: Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115.
authors.forename: Philipp B
authors.initials: PB
authors.lastname: Staber
authors.affiliation: Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Tenen
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02215; Broad Institute of MIT and Harvard, Cambridge, MA 02142; and Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115 cyan@bidmc.harvard.edu.
authors.forename: Catherine T
authors.initials: CT
authors.lastname: Yan
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D001483:Base Sequence; D049109:Cell Proliferation; D002478:Cells, Cultured; D003564:Cytidine Deaminase / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics*; D004195:Disease Models, Animal; D004485:Eczema / Q000235:genetics*; D019066:Facies; D055879:Gene Knock-In Techniques; D006130:Growth Disorders / Q000235:genetics*; D006801:Humans; D007070:Immunoglobulin A / Q000097:blood / Q000235:genetics / Q000276:immunology; D017578:Immunoglobulin Class Switching / Q000235:genetics / Q000276:immunology; D007073:Immunoglobulin E / Q000097:blood / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007136:Immunoglobulins / Q000235:genetics; D008607:Intellectual Disability / Q000235:genetics*; D038041:Lymphopoiesis / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D008831:Microcephaly / Q000235:genetics*; D008969:Molecular Sequence Data; D016511:Severe Combined Immunodeficiency / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: C014609:IgK; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007136:Immunoglobulins; C000607893:LIG4 protein, human; D016159:Tumor Suppressor Protein p53; D007073:Immunoglobulin E; D003564:Cytidine Deaminase; D011088:DNA Ligases; D000072481:DNA Ligase ATP