Functional analysis of a novel pathogenic glycine amidinotransferase mutant in hereditary Fanconi syndrome

Glycine amidinotransferase (GATM), a key enzyme in creatine biosynthesis, is localized in the mitochondria of proximal tubular epithelial cells (PTECs). Mutant GATM proteins polymerize into crystalline structures, causing mitochondrial dysfunction and leading to Fanconi syndrome (FS) with progressive kidney failure. To date, 28 patients from five unrelated kindreds have been reported worldwide. We identified a novel hereditary FS in a Japanese family carrying a distinct mutation from known cases. We effectively demonstrated the predicted pathological polymerization of our mutant GATM protein with hypersound-perturbed molecular dynamics simulations (HS-MD). This is the first report in an Asian population.

Two FS patients (father and daughter) were referred to our hospital. Kidney biopsies were examined by light and electron microscopy. Immunohistochemical staining for GATM and ATPB (a mitochondrial marker) was performed. Targeted panel sequencing identified variants in the GATM gene. Because root-mean-square fluctuation (RMSF) analysis using conventional methods failed to detect significant changes, pathogenicity of the GATM mutant was further investigated with HS-MD simulations on the Fugaku supercomputer.

Kidney biopsies from both patients exhibited numerous crystal-like linear aggregations of various sizes in nearly all PTECs. Electron microscopy revealed numerous electron-dense, crystal-like linear aggregations located inside and adjacent to mitochondria in PTECs. GATM staining was clearly detected in crystalline forms, merging with most ATPB staining. Furthermore, pathological staining of GATM revealed that ATPB did not show complete colocalization, suggesting how polymerized mutant GATM damages mitochondria. Targeted panel sequencing identified a novel mutation in the GATM gene (NM_001482.3: c.802C>G, Q268E), distinct from the previously reported cluster sites. Structural analysis confirmed that the GATM molecule consists of five β-sheets (B1–B5). The conventional mutations (P320S, T336A, T336I, P341L) were located on the B4 surface and considered to have adhesive properties, whereas Q268E was situated away from the B4 surface and buried within the protein’s interior. Our HS-MD simulations effectively demonstrated that the Q268E mutation, together with the previously reported mutations, could transition from the native dimer to an abnormal linear polymer via a pathological sequential B2–B2 and B4–B4 interaction. Furthermore, we found that the Q268E mutation significantly enhanced the conformational flexibility of a region spanning residues 290–312, which is spatially positioned between the mutated residue and the B4 surface as an adjacent force. These results also suggested that the Q268E mutation may increase the solvent-accessible surface area (SASA) of several amino acids on the B4 surface, simultaneously confirming the previous hypothesis.

We identified hereditary FS caused by a novel GATM gene mutation. We verified the pathogenicity and molecular dynamics of the Q268E mutation with HS-MD, and effectively demonstrated the possibility of abnormal polymerization through sequential b-sheet interactions (B2–B2 and B4–B4) of the mutant GATM. This study provides valuable insights not only into the pathological mechanisms of abnormal protein structure and misfolding but also into potential therapeutic strategies in the future.