Phenotype Of A Mouse Model Of Combined Methylmalonic Acidemia Caused By MMACHC Gene C.80A>G(p.G27R) Homozygous Mutation

Objective: The cbl C type of combined methylmalonic acidemia and homocysteinemia caused by MMACHC gene mutation is the most prevalent methylmalonic acidemia subtype in China.The c.80A>G mutation is one of the hotspot mutations of the MMACHC gene in China.We validated the genotype of F1 mice with the MMACHC gene c.80A>G(p.G27R)mutation and established a long-lived animal model for experimental studies.We used mouse model with MMACHC gene c.80A>G(p.G27R)homozygous mutant to mimic the clinical features of cbl C patients.We investigated and summarized the phenotype characteristics of homozygous mice.Methods: The genotype of F1 mice with the MMACHC gene c.80A>G(p.G27R)mutation was determined by PCR product sequencing and Southern blot analysis using mouse tail DNA.F1 mice were intercrossed to obtain the experimental mice for this study: mice with MMACHC gene c.80A>G(p.G27R)homozygous mutant and mice with MMACHC gene c.80A>G(p.G27R)heterozygous mutant and wild-type mice(WT)as controls.The survival time,skin and fur appearance,locomotor and exploratory abilities of homozygous mice were observed.The body weight of model mice was measured and recorded periodically.Liquid chromatography-tandem mass spectrometry was used to quantify the blood levels of methylmalonic acid(MMA)and total homocysteine(t Hcy)in mice at 3,6 and 12 weeks of age.The histopathological changes of tissue organs in 12-week-old mice were examined by hematoxylin-eosin(H&E)staining and compared with wild-type mice.The body weight of mice was plotted as a function of age using the mean weight and standard deviation of each group.The blood levels of MMA and t Hcy in homozygous mice and heterozygous mice were compared by Mann-Whitney U test,with P<0.05 indicating statistical significance.Results: 1)The MMACHC gene mutation c.80A>G in mice is verified by PCR product sequencing and alignment with the wild-type genomic sequence.Southern blot analysis of mouse tail DNA confirmes that 12 F1 mice have the correct recombination and no random insertion of the point mutation.2)F1 mice are intercrossed to successfully generate and maintain MMACHC gene c.80A>G homozygous mutant mice.Homozygous mice show no early mortality and have a long lifespan.No obvious abnormalities in food intake,locomotion,exploration,skin and fur are observed in homozygous mice;there is no significant difference in body weight between homozygous mice(n=18)and wild-type mice(n=9).3)The blood levels of MMA and t Hcy in homozygous mice(n=6)increase with age;both blood metabolites significantly elevate in homozygous mice at 3 weeks and 12 weeks of age compared with heterozygous mice(n=6),with statistical significance(P<0.05).4)Compared with wild-type mice,histopathological examination of H&E-stained sections of homozygous mice reveals vacuolar degeneration of cardiomyocytes,mixed thrombi in left and right ventricles,and hypertrophy of cardiomyocytes in the heart;vacuolar degeneration of hepatocytes and focal necrosis of hepatocytes in the liver;inflammatory cell infiltration and pulmonary artery dilation in the lung;microthrombi formation in glomerular capillary tufts and afferent arteriole narrowing in the kidney.Conclusion: The MMACHC gene c.80A>G mutation in F1 mice demonstrate correct recombination and no random insertion.These mice are interbred to generate viable and fertile MMACHC gene c.80A>G mutant homozygotes.Homozygous mice survive for a long time and accurately mimic the changes in blood metabolites of cbl C type patients,and the biochemical and pathological changes partially resemble the clinical manifestations of cbl C type patients.