Limits...
A mutation in Ampd2 is associated with nephrotic syndrome and hypercholesterolemia in mice.

Helmering J, Juan T, Li CM, Chhoa M, Baron W, Gyuris T, Richards WG, Turk JR, Lawrence J, Cosgrove PA, Busby J, Kim KW, Kaufman SA, Cummings C, Carlson G, Véniant MM, Lloyd DJ - Lipids Health Dis (2014)

Bottom Line: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways.The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2.Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and β-sitosterol.

View Article: PubMed Central - PubMed

Affiliation: Department of Metabolic Disorders, Amgen Inc, One Amgen Center Dr, Thousand Oaks, CA 91320, USA. dlloyd@amgen.com.

ABSTRACT

Background: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype.

Methods: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues.

Results: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and β-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy.

Conclusion: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.

Show MeSH

Related in: MedlinePlus

Gene expression analysis ofAmpd2m/mandAmpd2+/+mice. A) Liver mRNA levels were assessed by quantitative RT-PCR for an array of lipid metabolism genes on fed 9-week old Ampd2+/+ and Ampd2m/m mice (n = 5). Statistical analyses were carried out by unpaired two-tailed t-tests; * p <0.05 vs. Ampd2+/+. B) Liver immunoblot analysis of Ldlr protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4232700&req=5

Fig3: Gene expression analysis ofAmpd2m/mandAmpd2+/+mice. A) Liver mRNA levels were assessed by quantitative RT-PCR for an array of lipid metabolism genes on fed 9-week old Ampd2+/+ and Ampd2m/m mice (n = 5). Statistical analyses were carried out by unpaired two-tailed t-tests; * p <0.05 vs. Ampd2+/+. B) Liver immunoblot analysis of Ldlr protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice.

Mentions: In an attempt to uncover a pathogenic mechanism for the observed hypercholesterolemia we focused our attention on liver lipid metabolism. An array of lipid metabolism genes were analyzed by quantitative RT-PCR in the livers of Ampd2+/+ and Ampd2m/m mice at 9 weeks of age (Figure 3A). Ldlr mRNA was decreased by 36%, which translated to reduced protein levels of Ldlr (Figure 3B), consistent with elevated LDL cholesterol levels (Figure 2F). Transcript levels of 3-hydroxy-3-methylglutaryl-CoA synthase (Hmgcs) were also 47% lower. The cholesterol and bile acid synthesis enzyme cholesterol 7 alpha-hydroxylase (Cyp7a1) mRNA was increased by 55% in Ampd2m/m mice. Insulin-induced gene 1 (Insig1) was decreased by 56% in Ampd2m/m mice. We confirmed normal expression of Ampd2, indicating the mutation had no effect on mRNA stability, whereas it likely resulted in protein instability (Figure 1B). We also confirmed normal expression of the broadly expressed paralog Ampd3, suggesting no overlapping compensation. In general it appeared that the changes in gene expression were a consequence of high cholesterol levels, and not causative, prompting us to employ additional technologies.Figure 3


A mutation in Ampd2 is associated with nephrotic syndrome and hypercholesterolemia in mice.

Helmering J, Juan T, Li CM, Chhoa M, Baron W, Gyuris T, Richards WG, Turk JR, Lawrence J, Cosgrove PA, Busby J, Kim KW, Kaufman SA, Cummings C, Carlson G, Véniant MM, Lloyd DJ - Lipids Health Dis (2014)

Gene expression analysis ofAmpd2m/mandAmpd2+/+mice. A) Liver mRNA levels were assessed by quantitative RT-PCR for an array of lipid metabolism genes on fed 9-week old Ampd2+/+ and Ampd2m/m mice (n = 5). Statistical analyses were carried out by unpaired two-tailed t-tests; * p <0.05 vs. Ampd2+/+. B) Liver immunoblot analysis of Ldlr protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232700&req=5

Fig3: Gene expression analysis ofAmpd2m/mandAmpd2+/+mice. A) Liver mRNA levels were assessed by quantitative RT-PCR for an array of lipid metabolism genes on fed 9-week old Ampd2+/+ and Ampd2m/m mice (n = 5). Statistical analyses were carried out by unpaired two-tailed t-tests; * p <0.05 vs. Ampd2+/+. B) Liver immunoblot analysis of Ldlr protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice.
Mentions: In an attempt to uncover a pathogenic mechanism for the observed hypercholesterolemia we focused our attention on liver lipid metabolism. An array of lipid metabolism genes were analyzed by quantitative RT-PCR in the livers of Ampd2+/+ and Ampd2m/m mice at 9 weeks of age (Figure 3A). Ldlr mRNA was decreased by 36%, which translated to reduced protein levels of Ldlr (Figure 3B), consistent with elevated LDL cholesterol levels (Figure 2F). Transcript levels of 3-hydroxy-3-methylglutaryl-CoA synthase (Hmgcs) were also 47% lower. The cholesterol and bile acid synthesis enzyme cholesterol 7 alpha-hydroxylase (Cyp7a1) mRNA was increased by 55% in Ampd2m/m mice. Insulin-induced gene 1 (Insig1) was decreased by 56% in Ampd2m/m mice. We confirmed normal expression of Ampd2, indicating the mutation had no effect on mRNA stability, whereas it likely resulted in protein instability (Figure 1B). We also confirmed normal expression of the broadly expressed paralog Ampd3, suggesting no overlapping compensation. In general it appeared that the changes in gene expression were a consequence of high cholesterol levels, and not causative, prompting us to employ additional technologies.Figure 3

Bottom Line: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways.The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2.Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and β-sitosterol.

View Article: PubMed Central - PubMed

Affiliation: Department of Metabolic Disorders, Amgen Inc, One Amgen Center Dr, Thousand Oaks, CA 91320, USA. dlloyd@amgen.com.

ABSTRACT

Background: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype.

Methods: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues.

Results: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and β-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy.

Conclusion: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.

Show MeSH
Related in: MedlinePlus