Characterization of liver GSD IX γ2 pathophysiology in a novel Phkg2-/- mouse model

Abstract

Introduction: Liver Glycogen Storage Disease IX is a rare metabolic disorder of glycogen metabolism caused by deficiency of the phosphorylase kinase enzyme (PhK). Variants in the PHKG2 gene, encoding the liver-specific catalytic γ2 subunit of PhK, are associated with a liver GSD IX subtype known as PHKG2 GSD IX or GSD IX γ2. There is emerging evidence that patients with GSD IX γ2 can develop severe and progressive liver disease, yet research regarding the disease has been minimal to date. Here we characterize the first mouse model of liver GSD IX γ2.

Methods: A Phkg2^-/- mouse model was generated via targeted removal of the Phkg2 gene. Knockout (Phkg2^-/-, KO) and wild type (Phkg2^+/+, WT) mice up to 3 months of age were compared for morphology, Phkg2 transcription, PhK enzyme activity, glycogen content, histology, serum liver markers, and urinary glucose tetrasaccharide Glcα1-6Glcα1-4Glcα1-4Glc (Glc₄).

Results: When compared to WT controls, KO mice demonstrated significantly decreased liver PhK enzyme activity, increased liver: body weight ratio, and increased glycogen in the liver, with no glycogen accumulation observed in the brain, quadricep, kidney, and heart. KO mice demonstrated elevated liver blood markers as well as elevated urine Glc₄, a commonly used biomarker for glycogen storage disease. KO mice demonstrated features of liver structural damage. Hematoxylin & Eosin and Masson's Trichrome stained KO mice liver histology slides revealed characteristic GSD hepatocyte architectural changes and early liver fibrosis, as have been reported in liver GSD patients.

Discussion: This study provides the first evidence of a mouse model that recapitulates the liver-specific pathology of patients with GSD IX γ2. The model will provide the first platform for further study of disease progression in GSD IX γ2 as well as for the evaluation of novel therapeutics.

Keywords: GSD IX γ2; Liver glycogen storage disease type IX; Mouse model; Phkg2; Phosphorylase kinase deficiency.

Figure 1 –. Generation of Phkg2^−/− mouse model verified at DNA, mRNA, and enzyme activity level. (A) Illustration of the Phkg2^tm1.1 knockout allele.

Targeted deletion of the critical exons containing the entire PhK protein coding regions was achieved via a non-conditional knockout approach followed by Cre-mediated excision of the neomycin selection cassette. (B) Genotyping by PCR. F1, R1 primers added to genomic DNA of knockout (KO) mice generate a PCR fragment at 210 bp in length. F2, R2 primers added to genomic DNA of wild type (WT) mice generate a PCR fragment at 136 bp in length. Primers added to genomic DNA of Heterozygous (HT) mice generate both PCR fragments. M indicates the size marker. (C) Phkg2 mRNA. RT-qPCR was performed to assess transcription of the Phkg2 gene. b-actin served as the loading control. The level of transcription was significantly higher in KO mice than WT controls. KO n=3, WT n=3. ***p<0.001. (D) PhK Enzyme Activity. Enzyme activity was assayed with liver lysates from KO mice and WT controls. Phosphorylase kinase (PhK) activity was significantly less in KO mice compared to WT controls. 6 WT (3 male, 3 female), 6 KO (3 male, 3 female). ****p<0.0001.

Figure 2 –. Morphological measurements demonstrate differences in body weight and hepatomegaly in Phkg2^−/− mice.

(A) Body weight. Total body weight was measured for WT and KO mice at 1 month, 2 months, and 3 months of age. At 1 month, KO mice have significantly lower body weight than WT controls. 1 month 20 WT (13 female, 7 male), 20 KO (13 female, 7 male). 2 months 14 WT (7 male, 7 female), 14 KO (7 male, 7 female). 3 months 14 WT (7 male, 7 female), 14 KO (7 male, 7 female). Mean with SEM. ***p<0.001. (B) Percent liver weight. Liver weight (LW) was measured and normalized to body weight (BW) as a quantitative marker of hepatomegaly. Percent liver weight was significantly higher in KO mice compared to WT controls. 17 WT (7 male, 10 female), 17 KO (7 male, 10 female). Min to max demonstrating all points. ****p < 0.0001.

Figure 3 –. Glycogen level was significantly elevated in liver of Phkg2^−/− mice and similar to WT in other tissues. (A) PAS Stain.

Slides were stained with Periodic acid-Schiff (PAS) to identify the presence of glycogen in tissues. Brain, quadricep, kidney, and heart tissue demonstrated no differences on PAS stain between KO mice and WT controls. Liver tissue demonstrated a remarkable difference on PAS stain between KO mice and WT controls. Images were captured at 20x. Scale bar is 50 μm. (B) Liver glycogen content assay. To verify PAS stain, glycogen assay was performed on liver lysates from WT and KO mice. Liver tissue from KO mice demonstrate significantly higher glycogen content levels than WT controls. Male and Female. 6 WT (3 male, 3 female), 6 KO (3 male, 3 female). Min to max demonstrating all points. ****p < 0.0001.

Figure 4 –. Phkg2^−/− mice demonstrated characteristic GSD hepatocyte architectural changes and early perisinusoidal fibrosis.

(A) H&E Stain. Slides were stained with Hematoxylin & Eosin (H&E) to identify hepatocyte architecture. Per pathologist review, KO mice demonstrated heterogeneously enlarged hepatocytes with pale cytoplasm and distinct cell membranes as well as lateralized, pyknotic nuclei. 6 WT (4 male, 2 female), 5 KO (2 male, 3 female). 2 WT and 2 KO mice displayed. (B) Masson’s Trichrome Stain. Slides were stained with Masson’s Trichrome to identify tissue fibrosis. Per pathologist review, 3 KO mice demonstrated features of early perisinusoidal fibrosis. 6 WT (4 male, 2 female), 8 KO (5 male, 3 female). 2 WT and 2 KO mice displayed. Images were captured at 20x. Scale bar is 50 μm.

Figure 5 –. Blood and urine analyses demonstrated significantly increased ALT, AST, and Hex₄ levels in Phkg2^−/− mice.

Fasted whole blood samples were collected to measure blood glucose and blood ketones (A) Fasting Blood glucose curve Blood glucose was significantly lower in KO mice than WT controls for 0 and 8 hours of fasting ****p<0.0001. (B) Fasting Blood ketones curve Blood ketones in KO mice were not significantly elevated compared to WT controls. 10 WT (5 male, 5 female) and 10 KO (5 male, 5 female). (C) Urine Hex₄. Urine Glc₄, also known as Hex₄, was significantly elevated in KO mice compared to WT controls. 6 WT (3 male, 3 female), 6 KO (3 male, 3 female). **p<0.01. (D) ALT Alanine aminotransferase (ALT) was significantly elevated in KO mice compared to WT controls ****p<0.0001. (E) AST Aspartate aminotransferase (AST) was significantly elevated in KO mice compared to WT controls ****p<0.0001. 5 WT (2 male, 3 female), 5 KO (2 male, 3 female). Min to max demonstrating all points.