ENU-induced phenovariance in mice: inferences from 587 mutations

Abstract

BackgroundWe present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.FindingsIn the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.ConclusionsCollectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Quantitative Reduction of the TCR Adapter Protein SLP-76 Unbalances Immunity and Immune Regulation

“…Studying the effect of null alleles in mice and humans has defined our understanding of physiological gene function, as it has for TCR signaling, and will continue to do so (2). The use of mouse conditional alleles (52) and hypomorphic variants such as Lcp2 twimp (53, 54) will complement these efforts, and play an important role in understanding the immunological consequences of genetic variation between wild-type and null.…”

Real-time resolution of point mutations that cause phenovariance in mice

“…Because the majority of causative ENU-induced mutations exist in protein-coding exons rather than in cis-acting gene regulatory elements (7,8), this was accomplished by exome sequencing of the G1 male progenitor of each pedigree to identify all coding and splice site mutations that could possibly be present in the G3 mice. The G2 and G3 mice were then genotyped at each mutation site before phenotypic screening.…”

“…The number of genes for which homozygosity for putative null alleles (the gold standard for loss of function) has been analyzed can be taken as the lower limit of saturation, whereas the number of genes with homozygosity for putative null alleles or alleles judged probably damaging by the program PolyPhen-2, which may or may not impair protein function (8), can be taken as the upper limit of saturation. Minimum and maximum estimates of saturation can be offered even with a relatively small dataset.…”

Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection

“…Fifty microliters of blood was subjected to two rounds of red blood cell lysis with ammonium chloride before staining. Lymphocyte suspensions from blood, bone marrow (femurs and tibias from one hind leg), spleen, and thymus were counted (Z2 Coulter Counter; Beckman Coulter) and stained with a combination of the following mouse-reactive antibodies: FITCconjugated IgM (goat polyclonal; 1020-02; Southern Biotech); FITC-conjugated F4/80 (BM8); PE-conjugated IgD (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26); PerCP-Cy5.5-conjugated TCRβ (H57-597); APC-conjugated CD8α (53-6.7), CD11b (M1/70), CD93 (AA4.1), IgM (II/41), Ly6G (RB6-8C5), NK1.1 (PK136), TER119 (TER-119; eBioscience); FITC-conjugated CD4 (GK1.5), CD23 (B3B4), HY-TCR (T3.70), PE-conjugated CD11b (M1/70), CD19 (1D3), CD21/35 (7G6), CD44 (IM7), γδ TCR (GL3), Vα2 TCR (B20.1), PerCP-Cy5.5-conjugated B220 (RA3-6B2), CD8α (53-6.7), CD19 (1D3), NK1.1 (PK136); CD44 (IM7; Horizon V500; BD); APC-conjugated CD3e (145-2C11); APCCy7-conjugated CD45.2 (104), PE-Cy7-conjugated CD19 (6D5), CD45.1 (A20; Pacific blue), and CD16/32 (93; purified; BioLegend). 7-Aminoactinomycin D (7-AAD) was purchased from eBioscience, and EdU labeling and staining were performed according to the manufacturer's instructions (Invitrogen) 4 h after a single i.p.…”