Dysregulated methionine metabolism represents a novel pathobiological factor for biliary atresia: a gene discovery and patient iPSC(induced pluripotent stem cell)-derived hepatocyte study [Oral presentation]

Abstract

<p><strong>Purpose:</strong></p><p>The pathobiological mechanisms underlying biliary atresia (BA) remain unclear. <em>MAT1A</em> encodes Methionine Adenosyltransferase 1A, which catalyzes methionine (Met) metabolism to form SAMe, which is converted to glutathione (GSH; a main antioxidant) in hepatocytes. In this study, we have functionally characterized a <em>MAT1A</em> mutation in hepatocytes development and in BA.</p><p><strong>Methods:</strong></p><p>A trio-based whole genome sequencing on BA patients (n=120) was performed to elucidate the genetic predisposition to BA. MAT1A level in BA and non-BA livers was quantitated by Western blot. Induced pluripotent stem cells (iPSCs) were generated from patients’ blood and differentiated into hepatocytes to characterize the effects of <em>MAT1A</em> mutation on hepatocytes. (i) Quantitation of cell markers expression, oxidative stress (OS) and mitochondrial membrane potential (MMP); (ii) single cell RNA sequencing (scRNA-seq) were conducted in iPSC differentiation cultures to reveal the underlying mechanisms.</p><p><strong>Results:</strong></p><p>We have identified a <em>de novo</em> G>A missense mutation of <em>MAT1A</em> in a BA patient (BA638C), which resulted in the production of a truncated MAT1A. MAT1A level in BA638C liver was only 60% of that in normal liver. Upon induction of hepatocyte differentiation, in contrast to the formation of hepatocytes expressing HNF1A, A1AT, ALB and AFP (hepatocyte markers) in the <em>MAT1A</em> mutation corrected iPSC (BA638C-Corr) and normal control iPSC (HKUPS-001) cultures, patient’s iPSC (BA638C)-derived hepatocytes lost hepatocyte morphology and became fibroblast-like cells, expressing SMA and FSP1 (fibroblasts markers). ScRNA-seq analysis revealed that BA638C-derived hepatocytes underwent epithelial-mesenchymal transition (EMT). Elevation of OS and loss of MMP was detected in BA638C-derived hepatocytes. Met treatment induced EMT on HKUPS-001-derived hepatocytes, which was reversed by the addition of SAMe and GSH.</p><p>​​​​​​​<strong>Conclusion:</strong></p><p><em>MAT1A</em> mutation leads to a dysregulation of Met metabolism, a high Met environment and low SAMe, which is hepatoxic and aggravates liver damage upon injury contributing to BA initiation/progression in patient BA638C. Therefore, liver deterioration despite Kasai surgery could represent a primary event affecting hepatocytes, but not only due to reactive changes secondary to bile duct injuries in some BA patients.</p>