The MRI49372 phenotype is caused by a mutation in Ampd3. (A) Representation of the position of the mutation within the protein. (B) Sequence alignment showing conserved residues in AMPD3. Black letters represent residues conserved in 4/5 listed species. Bold “T” is the position of the Ampd3^T689A mutation. (C) Simplified diagram of purine metabolism showing reaction catabolized by AMPD3. Dotted line indicates reaction that does not proceed in human RBCs. It is not known if this reaction proceeds in mouse RBCs. cAMP, cyclic AMP; cGTP, cyclic GTP; GDP, guanosine 5′-diphosphate; GMP, guanosine 5′-monophosphate; GTP, guanosine 5′-triphosphate; Hypox, hypoxanthine; Ino, inosine; XMP, xanthosine 5′-monophosphate. (D) Representative western blot of (magnetic-activated cell sorting) separated reticulocytes (CD71⁺) and mature RBCs (CD71⁻) showing AMPD3 for WT, Ampd3^T689A/+, and Ampd3^T689A, as well as actin loading control for the same blot. Graph shows AMPD3 band density relative to actin band density (E). Mean ± standard of the mean (SEM) fold change in purine concentration in mature RBCs (CD71⁻) for Ampd3^T689A/+, and Ampd3^T689A compared with WT. Data show average of technical replicates (n = 4) from pooled biological replicates (n = 6). (F) Accumulation of ¹³C/¹⁵N-labeled IMP in mature WT, Ampd3^T689A/+, and Ampd3^T689A RBCs (CD71⁻) incubated at 37°C for 2 hours with labeled AMP. Data points represent technical replicates from pooled biological replicates (n = 3); black bars indicate median.