A novel ENU-induced Cpox mutation causes microcytic hypochromic anemia in mice

Abstract

Mouse models of red blood cell abnormalities are important for understanding the underlying molecular mechanisms of human erythrocytic diseases. DBA.B6-Mha (Microcytic hypochromic anemia) congenic mice were generated from the cross between N-ethyl-N-nitrosourea (ENU)-mutagenized male C57BL/6J and female DBA/2J mice as part of the RIKEN large-scale ENU mutagenesis project. The mice were established by backcrossing with DBA/2J mice for more than 20 generations. These mice showed autosomal-dominant microcytic hypochromic anemia with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels and increased red blood cell distribution width (RDW) and plasma ferritin levels. Linkage analysis indicated that the Mha locus was located within an interval of approximately 1.95-Mb between D16Nut1 (58.35 Mb) and D16Mit185 (60.30 Mb) on mouse chromosome 16. Mutation analysis revealed that DBA.B6-Mha mice had a point mutation (c.921-2A>G) at the acceptor site of intron 4 in the coproporphyrinogen oxidase (Cpox) gene, a heme-synthesizing gene. RT-PCR revealed that the Cpox mRNA in DBA.B6-Mha mice caused splicing errors. Our results suggest that microcytic hypochromic anemia in DBA.B6-Mha mice is owing to impaired heme synthesis caused by splice mutations in Cpox. Therefore, the DBA.B6-Mha mice may be used to elucidate the molecular mechanisms underlying microcytic hypochromic anemia caused by mutations in Cpox. Although low MCV levels are known to confer malarial resistance to the host, there were no marked changes in the susceptibility of DBA.B6-Mha mice to rodent malarial (Plasmodium yoelii 17XL) infection.

Keywords: N-ethyl-N-nitrosourea (ENU) mutagenesis; coproporphyrinogen oxidase (Cpox) gene; malaria; mice; microcytic hypochromic anemia.

Fig. 1.

Positional cloning in DBA.B6-Mha mice. (A) Linkage analysis of genes from N₃ to N₂₀, obtained by backcrossing M100835 with DBA/2J mice, between the D16Mit171 (56.12 Mb) and D16Mit76 (67.52 Mb) region on mouse chromosome 16. Markers, marker positions, and χ² values are shown on the left. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. Open boxes and black boxes represent the homozygous DBA/2J (D2J/D2J) and heterozygous DBA/2J and C57BL/6J (D2J/B6J) genotypes, respectively. The dotted area indicates the non-recombinant interval region. (B) Mutation analysis of Cpox in DBA.B6-Mha mice. Sequencing analysis of intron 4 to exon 5 of Cpox in DBA.B6-Mha (top), DBA/2J (middle), and C57BL/6J (bottom) mice. A point mutation (A to G) at the acceptor site of intron 4 in Cpox is found in DBA.B6-Mha mice (red arrowhead). (C) Detection of Cpox^Mha mutation using PCR-restriction fragment length polymorphism. C57BL/6J-derived Cpox alleles in DBA.B6-Mha mice lack the XspI restriction endonuclease sites in intron 4 to exon 5. The amplicon from DBA.B6-Mha mice was not partially digested by XspI and yielded 301, 185, and 116 bp bands. In contrast, DBA/2J and C57BL/6J mice carry the XspI restriction site, and the products obtained from both mice did not show the 301 bp band. M, 100 bp DNA ladder.

Fig. 2.

Cpox mRNA transcription in DBA.B6-Mha mice. RT-PCR analysis of Cpox in the bone marrow cells of C57BL/6J, DBA/2J, and DBA.B6-Mha mice. Upper panel shows the RT-PCR products from Cpox-specific primers located in exons 3 and 7. A 560 bp band was detected in C57BL/6J and DBA/2J mice, whereas a 560 bp band, and multiple larger and smaller bands were detected in DBA.B6-Mha mice. Asterisks indicate aberrant transcripts. The integrity of the cDNA was confirmed using an Hprt control band (234 bp, bottom panel). M, 100 bp DNA ladder; RT, reverse transcription.

Fig. 3.

Total Cpox expression levels in DBA.B6-Mha mice. (A) Relative expression levels of Cpox mRNA in the bone marrow cells in C57BL/6J (black box), DBA/2J (gray box), and DBA.B6-Mha (plaid box) mice. Cpox mRNA expression was measured by quantitative RT-PCR analysis using Cpox-specific primers located in exons 1 and 2. The expression levels in C57BL/6J mice were assigned an arbitrary value of 1 for comparative purposes. ^###P<0.001 vs. C57BL/6J mice (one-way analysis of variance with Tukey’s post-hoc multiple comparison test). (B) cDNA sequence waveform of Cpox 3′-untranslated region (UTR) in DBA.B6-Mha mice. An SNP (rs4190612, guanine or adenine) is found in the Cpox 3′-UTR region in C57BL/6J and DBA/2J mice. In double-peaks of this SNP obtained by cDNA sequencing analysis in four DBA.B6-Mha mice, the C57BL/6J-derived guanine peaks (black) were lower than the DBA/2J-derived adenine peaks (green) in each individual.

Fig. 4.

Comparison of the susceptibility to rodent malarial parasite, Plasmodium yoelii 17XL infection of DBA/2J and DBA.B6-Mha mice. (A, B) Parasitemia 3 and 5 days post-infection with P. yoelii 17XL of (A) female DBA/2J (gray circles) and DBA.B6-Mha (half open gray circles), and (B) male DBA/2J (gray squares) and DBA.B6-Mha (half open gray squares) mice. Bold and thin lines represent the mean and SD, respectively. (C, D) Survival rates post-infection with P. yoelii 17XL of (C) female and (D) male DBA/2J (gray line) and DBA.B6-Mha (doted gray line) mice.