Thousands of induced germline mutations affecting immune cells identified by automated meiotic mapping coupled with machine learning.

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.

Essential requirement for nicastrin in marginal zone and B-1 B cell development.

γ-secretase is an intramembrane protease complex that catalyzes the proteolytic cleavage of amyloid precursor protein and Notch. Impaired γ-secretase function is associated with the development of Alzheimer's disease and familial acne inversa in humans. In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for defects in adaptive immunity, we identified animals within a single pedigree exhibiting both hypopigmentation of the fur and diminished T cell-independent (TI) antibody responses. The causative mutation was in Ncstn, an essential gene encoding the protein nicastrin (NCSTN), a member of the γ-secretase complex that functions to recruit substrates for proteolysis. The missense mutation severely limits the glycosylation of NCSTN to its mature form and impairs the integrity of the γ-secretase complex as well as its catalytic activity toward its substrate Notch, a critical regulator of B cell and T cell development. Strikingly, however, this missense mutation affects B cell development but not thymocyte or T cell development. The Ncstn allele uncovered in these studies reveals an essential requirement for NCSTN during the type 2 transitional-marginal zone precursor stage and peritoneal B-1 B cell development, the TI antibody response, fur pigmentation, and intestinal homeostasis in mice.