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

Identification of a loss-of-function mutation inAmpd2in hypercholesterolemic mice. A) Sequencing chromatographs of Ampd2 in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. B) Liver immunoblot analysis of Ampd2 protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. C) Amino acid conservation of mouse Ampd2 residues 321–361 to Ampd2+, mouse Ampd1, mouse Ampd3, rat Ampd2, human Ampd2, dog Ampd2, Caenorhabditis elegans Ampd, Arabidopsis thaliana AMPD and Saccharomyces cerevisiae Ampd. Arrow indicates residue 341.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Identification of a loss-of-function mutation inAmpd2in hypercholesterolemic mice. A) Sequencing chromatographs of Ampd2 in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. B) Liver immunoblot analysis of Ampd2 protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. C) Amino acid conservation of mouse Ampd2 residues 321–361 to Ampd2+, mouse Ampd1, mouse Ampd3, rat Ampd2, human Ampd2, dog Ampd2, Caenorhabditis elegans Ampd, Arabidopsis thaliana AMPD and Saccharomyces cerevisiae Ampd. Arrow indicates residue 341.

Mentions: We previously reported [11] the mapping of the mutation for the hypercholesterolemic mice to a 7 Mb region on chromosome 3, with 131 possible candidate genes identified. Forty five were selected for further mutation analysis based on functional relevance. After examining the exons and intronic junctions of these 45 candidate genes by direct sequencing, we identified a guanine-to-thymine transversion resulting in an alanine to serine conversion at amino acid 341 in Ampd2 (Figure 1A). Using exon capture and high-throughput sequencing we analyzed the region from D3Mit102 to rs13477320 in 2 affected animals, three ENU mutations were identified: the A341S missense mutation in Ampd2, and intronic mutations in Magi3 and Slc6a17 (data not shown). Western blotting illustrated the functional consequence of this mutation on Ampd2 protein expression. Complete loss of Ampd2 protein was observed in the livers of homozygous mutant mice (Ampd2m/m), with partial loss evident in the heterozygous mice (Ampd2+/m) relative to wild-type (+/+) (Figure 1B). Alanine 341 was conserved in all animal species including Caenorhabditis elegans, but not in plants and fungi (Figure 1C), yet was restricted to a highly conserved region of the protein.Figure 1


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)

Identification of a loss-of-function mutation inAmpd2in hypercholesterolemic mice. A) Sequencing chromatographs of Ampd2 in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. B) Liver immunoblot analysis of Ampd2 protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. C) Amino acid conservation of mouse Ampd2 residues 321–361 to Ampd2+, mouse Ampd1, mouse Ampd3, rat Ampd2, human Ampd2, dog Ampd2, Caenorhabditis elegans Ampd, Arabidopsis thaliana AMPD and Saccharomyces cerevisiae Ampd. Arrow indicates residue 341.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Identification of a loss-of-function mutation inAmpd2in hypercholesterolemic mice. A) Sequencing chromatographs of Ampd2 in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. B) Liver immunoblot analysis of Ampd2 protein in Ampd2+/+, Ampd2+/m and Ampd2m/m mice. C) Amino acid conservation of mouse Ampd2 residues 321–361 to Ampd2+, mouse Ampd1, mouse Ampd3, rat Ampd2, human Ampd2, dog Ampd2, Caenorhabditis elegans Ampd, Arabidopsis thaliana AMPD and Saccharomyces cerevisiae Ampd. Arrow indicates residue 341.
Mentions: We previously reported [11] the mapping of the mutation for the hypercholesterolemic mice to a 7 Mb region on chromosome 3, with 131 possible candidate genes identified. Forty five were selected for further mutation analysis based on functional relevance. After examining the exons and intronic junctions of these 45 candidate genes by direct sequencing, we identified a guanine-to-thymine transversion resulting in an alanine to serine conversion at amino acid 341 in Ampd2 (Figure 1A). Using exon capture and high-throughput sequencing we analyzed the region from D3Mit102 to rs13477320 in 2 affected animals, three ENU mutations were identified: the A341S missense mutation in Ampd2, and intronic mutations in Magi3 and Slc6a17 (data not shown). Western blotting illustrated the functional consequence of this mutation on Ampd2 protein expression. Complete loss of Ampd2 protein was observed in the livers of homozygous mutant mice (Ampd2m/m), with partial loss evident in the heterozygous mice (Ampd2+/m) relative to wild-type (+/+) (Figure 1B). Alanine 341 was conserved in all animal species including Caenorhabditis elegans, but not in plants and fungi (Figure 1C), yet was restricted to a highly conserved region of the protein.Figure 1

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