Disease circuit verification
A) B) C)
Figure 1. Sanger sequencing of the edited PNPLA3 gene using CRISPR/Cas9 in the reference hiPSC line. (A) PNPLA3 gene in the wild-type line. (B) PNPLA3 homozygous knock-in clone with I148M mutation (Isoleucine to methionine at position 148, exon3). (C) PNPLA3 homozygous knock-out with a 6bp deletion and no frameshift mutation.
Key hepatocyte marker analysis
Figure 2. Functional analysis of Def-HEP PNPLA3 cells. QPCR analysis indicates that the PNPLA3 CRISPR-modified cells can be successfully differentiated into hepatocyte cells that express key hepatocyte markers at similar levels to primary human hepatocytes (PHH).
Fatty acid accumulation
Figure 3. Fatty acid accumulation in Def-HEP PNPLA3 cells. BODIPY staining shows a higher accumulation of lipids in Def-HEP I148M when cultured in low-lipid media. When cells are treated with 0.25mM of Oleic acid for 7 days all cell variants demonstrate an increase in lipid accumulation. A moderate increase in lipid accumulation can be observed with treatment of 0.25mM of Palmitic acid for 7 days.
Principal component analysis
Figure 4. Principal component analysis of lipid metabolism-related genes. PCA analysis of Def-HEP cells using a subset of 129 lipid metabolism associated genes demonstrates that the wild-type control and CRISPR modified PNPLA3 disease model hepatocyte cells cluster differently independently of fatty acid treatment.
Figure 5. Gene ontology of differentially expressed genes in PNPLA3 I148M relative to Def-HEP WT. Gene ontology analysis was performed using large-scale genome and gene function analysis using the PANTHER classification system. Pathways related to lipid, cholesterol and triglyceride metabolism are observed to be affected.
Figure 6. Heatmap analysis showing the differential expression of relevant lipid metabolism and accumulation genes. PNPLA3 I148M variant shows a significantly altered gene expression pattern when compared to Def-HEP WT.
A subset of potential causal gene variants have been determined which are currently a significant focus on NAFLD studies
NAFLD gene variants
PNPLA3 rs738409 impaired hepatocellular triglycerides hydrolysis and increased lipogenesis associated to the 148 M allele
GCKR rs1260326 increased glycolysis favours an increase in triglyceride levels
TM6SF2 rs58542926 impaired mobilization of neutral lipids for very low-density lipoprotein (VLDL) assembly and secretion by the liver in E167K carriers
MBOAT7 rs641738 variant causing decreased MBOAT7 expression, predisposes to NAFLD/NASH by affecting the acyl remodelling of phosphatidylinositol in the liver
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