With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.N-ethyl-N-nitrosourea | genetic mapping | forward genetics | mutagenesis | massively parallel sequencing P henotypic variation in mice can be induced with N-ethyl-Nnitrosourea (ENU), which creates single base pair substitutions in germ line DNA. However, the positional cloning of ENU-induced mutations causative for phenotypes of interest has historically been a time-consuming process, beginning with generation of an outcrossed recombinant mapping population of phenotypically mutant and WT mice, genotyping individual mice at genetic markers across the genome to create a linkage map, and finally targeted sequencing to identify the causative mutation within the critical region. The advent of massively parallel sequencing techniques has given rise to more rapid "mapping-bysequencing" methods in which genome-wide marker genotyping and DNA sequencing are combined into a single step applied to either individual or pooled groups of organisms (1). For ENUmutagenized mice, early experiments used massively parallel sequencing for mutation identification within a critical region defined by traditional or bulk segregation mapping using recombinant mapping populations produced by outcrossing the mutant to another inbred laboratory strain and backcrossing or intercrossing a second time (2-4). Later reports demonstrated mapping with the identified sequence variants themselves as markers, which eliminated...

Creatine, a nitrogenous organic acid, replenishes cytoplasmic ATP at the expense of mitochondrial ATP via the phosphocreatine shuttle. Creatine levels are maintained by diet and endogenous synthesis from arginine and glycine. Glycine amidinotransferase (GATM) catalyzes the rate-limiting step of creatine biosynthesis: the transfer of an amidino group from arginine to glycine to form ornithine and guanidinoacetate. We screened 36,530 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea–mutagenized grandsires for intestinal homeostasis abnormalities after oral administration of dextran sodium sulfate (DSS). Among 27 colitis susceptibility phenotypes identified and mapped, one was strongly correlated with a missense mutation in Gatm in a recessive model of inheritance, and causation was confirmed by CRISPR/Cas9 gene targeting. Supplementation of homozygous Gatm mutants with exogenous creatine ameliorated the colitis phenotype. CRISPR/Cas9-targeted (Gatmc/c) mice displayed a normal peripheral immune response and immune cell homeostasis. However, the intestinal epithelium of the Gatmc/c mice displayed increased cell death and decreased proliferation during DSS treatment. In addition, Gatmc/c colonocytes showed increased metabolic stress in response to DSS with higher levels of phospho-AMPK and lower levels of phosphorylation of mammalian target of rapamycin (phospho-mTOR). These findings establish an in vivo requirement for rapid replenishment of cytoplasmic ATP within colonic epithelial cells in the maintenance of the mucosal barrier after injury.

Precise control of Wnt signaling is necessary for immune system development. In this study, we detected severely impaired development of all lymphoid lineages in mice, resulting from an N-ethyl-N-nitrosourea–induced mutation in the limb region 1–like gene (Lmbr1l), which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain–containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing “destruction complex” proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/β-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation, acting as a negative regulator of the Wnt/β-catenin pathway.

The SMCR8-WDR41-C9ORF72 complex is a regulator of autophagy and lysosomal function. Autoimmunity and inflammatory disease have been ascribed to loss-of-function mutations of Smcr8 or C9orf72 in mice. In humans, autoimmunity has been reported to precede amyotrophic lateral sclerosis caused by mutations of C9ORF72. However, the cellular and molecular mechanisms underlying autoimmunity and inflammation caused by C9ORF72 or SMCR8 deficiencies remain unknown. Here, we show that splenomegaly, lymphadenopathy, and activated circulating T cells observed in Smcr8−/− mice were rescued by triple knockout of the endosomal Toll-like receptors (TLRs) TLR3, TLR7, and TLR9. Myeloid cells from Smcr8−/− mice produced excessive inflammatory cytokines in response to endocytosed TLR3, TLR7, or TLR9 ligands administered in the growth medium and in response to TLR2 or TLR4 ligands internalized by phagocytosis. These defects likely stem from prolonged TLR signaling caused by accumulation of LysoTracker-positive vesicles and by delayed phagosome maturation, both of which were observed in Smcr8−/− macrophages. Smcr8−/− mice also showed elevated susceptibility to dextran sodium sulfate-induced colitis, which was not associated with increased TLR3, TLR7, or TLR9 signaling. Deficiency of WDR41 phenocopied loss of SMCR8. Our findings provide evidence that excessive endosomal TLR signaling resulting from prolonged ligand–receptor contact causes inflammatory disease in SMCR8-deficient mice.

LPS-responsive beige-like anchor (LRBA) protein deficiency in humans causes immune dysregulation resulting in autoimmunity, inflammatory bowel disease (IBD), hypogammaglobulinemia, regulatory T (Treg) cell defects, and B cell functional defects, but the cellular and molecular mechanisms responsible are incompletely understood. In an ongoing forward genetic screen for N-ethyl-N-nitrosourea (ENU)-induced mutations that increase susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice, we identified two nonsense mutations in Lrba. Although Treg cells have been a main focus in LRBA research to date, we found that dendritic cells (DCs) contribute significantly to DSS-induced intestinal inflammation in LRBA-deficient mice. Lrba−/− DCs exhibited excessive IRF3/7- and PI3K/mTORC1-dependent signaling and type I IFN production in response to the stimulation of the Toll-like receptors (TLRs) 3, TLR7, and TLR9. Substantial reductions in cytokine expression and sensitivity to DSS in LRBA-deficient mice were caused by knockout of Unc93b1, a chaperone necessary for trafficking of TLR3, TLR7, and TLR9 to endosomes. Our data support a function for LRBA in limiting endosomal TLR signaling and consequent intestinal inflammation.

T follicular helper cells (TFH) participate in germinal center (GC) development and are necessary for B cell production of high-affinity, isotype-switched antibodies. In a forward genetic screen, we identified a missense mutation in Prkd2, encoding the serine/threonine kinase protein kinase D2, which caused elevated titers of immunoglobulin E (IgE) in the serum. Subsequent analysis of serum antibodies in mice with a targeted null mutation of Prkd2 demonstrated polyclonal hypergammaglobulinemia of IgE, IgG1, and IgA isotypes, which was exacerbated by the T cell–dependent humoral response to immunization. GC formation and GC B cells were increased in Prkd2−/− spleens. These effects were the result of excessive cell-autonomous TFH development caused by unrestricted Bcl6 nuclear translocation in Prkd2−/− CD4+ T cells. Prkd2 directly binds to Bcl6, and Prkd2-dependent phosphorylation of Bcl6 is necessary to constrain Bcl6 to the cytoplasm, thereby limiting TFH development. In response to immunization, Bcl6 repressed Prkd2 expression in CD4+ T cells, thereby committing them to TFH development. Thus, Prkd2 and Bcl6 form a mutually inhibitory positive feedback loop that controls the stable transition from naïve CD4+ T cells to TFH during the adaptive immune response.

In a forward genetic screen of N-ethyl-N-nitrosourea (ENU)–induced mutant mice for aberrant immune function, we identified mice with a syndromic disorder marked by growth retardation, diabetes, premature death, and severe lymphoid and myeloid hypoplasia together with diminished T cell–independent (TI) antibody responses. The causative mutation was in Pdia6, an essential gene encoding protein disulfide isomerase A6 (PDIA6), an oxidoreductase that functions in nascent protein folding in the endoplasmic reticulum. The immune deficiency caused by the Pdia6 mutation was, with the exception of a residual T cell developmental defect, completely rescued in irradiated wild-type recipients of PDIA6-deficient bone marrow cells, both in the absence or presence of competition. The viable hypomorphic allele uncovered in these studies reveals an essential role for PDIA6 in hematopoiesis, but one extrinsic to cells of the hematopoietic lineage. We show evidence that this role is in the proper folding of Wnt3a, BAFF, IL-7, and perhaps other factors produced by the extra-hematopoietic compartment that contribute to the development and lineage commitment of hematopoietic cells.

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation–phenotype association. At this time, CE has evaluated putative mutation–phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus. The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.