Finding new immune regulatory genes by ENU mutagenesis

IntroductionThe Thousand Genomes Project has revealed the extraor-dinary scale of human genetic variation such as each per-son inherits ~12,000 protein-changing single nucleotidevariations (SNVs) including up to 100 premature STOPcodons creating an immense challenge to investigate thephysiological consequence of this type of genetic variationin experimental animals.AimTo develop a new strategy to meet this need by usingwhole exome capture, massively parallel DNA sequenc-ing and computational analysis to sensitively and specifi-cally identify 40-50 de novo mis-sense SNV mutationsspread across the genome of individual offspring fromENU-mutagenized C57BL/6 mice [1].ResultsIn this presentation we demonstrate how direct se-quencing of animals with indepe ndent defects in B celldevelopment resulted in the identification of the causa-tive mutation both in genes of previously known andunknown function without meiotic mapping. Thisapproach has resulted in the identification of a newphospholipid transport pathway that is crucial for nor-mal B cell development in the bone marrow [2] and therealization that an endopeptidase of previously unknownin vivo function is essential for normal processing ofMHC invariant chain and terminal B cell and DCmaturation.By sequencing animals from defined strains and alsofounder animals of ENU mutant pedigrees two generationsbefore the phenotypic screens we have started to build adatabase of missense mutations containing currently closeto 7000 mutations in mouse pedigrees actively breedingand immediately available for experimental analysis.All data is made available from the missense variant data-base (pb.apf.edu.au/phenbank).ConclusionThis approach transforms mammalian experimentalgenetics and opens up an unparalleled source for experi-mental models of human disease and validation of humandisease associated SNVs.