ENU mutagenesis identifies the first mouse mutants reproducing human β-thalassemia at the genomic level.

Abstract Forward genetic screens have been performed in many species to identify phenotypes in specific organ systems. We have undertaken a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify dominant mutations that perturb erythropoiesis in mice. Mutant mice that displayed an erythrocyte mean cell volume (MCV) greater than three standard deviations from the population mean were identified. Two of these lines, RBC13 and RBC14 , displayed a hypochromic, microcytic anemia, accompanied by a marked reticulocytosis, splenomegaly and diminished red cell survival. Timed pregnancies from heterozygous intercrosses revealed that a quarter of the embryos displayed severe anemia and did not survive beyond embryonic day (E) 18.5, consistent with homozygous β-thalassemia. Genetic complementation studies with a β-thalassemia mouse line reproduced the embryonic lethality in compound heterozygotes and a genomic custom capture array and massively parallel sequencing of the β-globin locus identified the causative mutations. The RBC13 line displayed a nonsense mutation at codon 40 in exon 2 of the β-major gene, invoking parallels with the common β 0 39 thalassemia mutation seen in humans. The RBC14 line exhibited a mutation at the polyadenylation signal of the β-major gene, exactly replicating a human β-thalassemia mutation. The RBC13 and RBC14 lines are the first β-thalassemia mouse models that reproduce human β-thalassemia at the genomic level, and as such highlight the power of ENU mutagenesis screens in generating mouse models of human disease.