Metabolic and biophysical study of the MFN2Ile213Thr mutant causing Hereditary Motor and Sensory Neuropathy (HMSN)

Abstract

Charcot-Marie-Tooth (CMT) 2A disease, a genetic axonal nervous lesion, results from MFN2 pathogenic variation, and this gene plays a pivotal role in mitochondrial dynamics and calcium signaling. However, the underlying mechanism linking MFN2 defect to progressive dying-back of peripheral nerves is still unclear. The present work focused on analyzing one CMT2A patient from multiple perspectives. Clinical and pathologic evaluation was initially conducted on the recruited case. Subsequently, Sanger sequencing and whole-exome sequencing (WES) were performed for genetic detection. To reveal the cell metabolic alteration caused by the identified variant, this study also established and transfected plasmid vectors in HEK293 cells and analyzed cell metabolites through liquid chromatography in combination with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS). Additionally, we completed structural modeling and molecular dynamic (MD) simulation to investigate the intramolecular impact of the variant. According to our results, the clinical and neuropathologic manifestations of the proband matched with the diagnosis of CMT. The causative variant MFN2: c.638T>C: (p.Ile213Thr) was identified through genetic analysis. Moreover, metabolic pathway enrichment results demonstrated that this variant significantly affected the metabolism of sphingolipids and glycerophospholipids. MD analysis indicated that this variant crippled the binding ability of MFN2 to GTP. Taken together, our study deduced preliminary clues for the underlying mechanism by which mutant MFN2 affects cell metabolism and provided a novel perspective to understand the cellular and molecular impacts of MFN2 variants.

Keywords: CMT2A; MFN2; hereditary neuropathy; metabonomics; molecular dynamic simulation; whole-exome sequencing.

Figure 1

(A) Pedigree diagram of the affected family. II-2 was the pregnant proband with an affected son (III-1). The (B) hands and (C) feet of the proband. (D-F) Hematoxylin-eosin staining of the gastrocnemius nerve sample of the proband showing severe loss of myelinated nerve fibers. (Scale bar, 5 μ; (magnification: ×1000)). (G-I) Formation of numerous “onion bulbs” shown by scanning electron microscopy. (Scale bar, 1 μ; (magnification: ×5000)).

Figure 2

A. The identified variant MFN2: c. c.638T>C and wild-type (WT) control in Sanger sequencing peak diagrams. B. The evolutionary conservation of MFN2: Ile213 residue. C. The location of the c.638T>C (p.Ile213Thr) variant in the gene and protein schematic diagrams. HR, heptad repeat; PR, proline-rich; TM, transmembrane.

Figure 3

A. The GFP fluorescence signal at 48 h after transfection. NC, with plasmid backbone; WT, with wild-type MFN2 cDNA; MUT, with MFN2: c.638T>C mutant cDNA. (Scale bar, 1000 μ; magnification: ×100). B. The relative MFN2 expression levels in 3 transfected cell groups. C. The volcano plot showing significantly different compounds. Red dots represent the difference >2.0 folds and P<0.05, blue font represents the m/z molecular weight information. D. Part of the compounds with vip (variable importance in projection) value >1. The X- and Y-axes stand for vip score and m/z, respectively. E. KEGG pathway enrichment result. Each point represents a pathway, with X- and Y-axes indicating the importance of a compound related to the pathway and-log₁₀(P) value, respectively.

Figure 4

A. The structure models containing WT MFN2 and I213T mutant. Residues related to the formation of hydrogen bonds with residues I213 or I213T are represented by the sticks, and dotted yellow lines stand for hydrogen bonds. B. Superposition of 10 structural snapshots from 4 ns simulations of WT and I213T models. The backbone of the structures is shown as cartoon, with color from red to white to blue. C. The trajectory of RMSD (Cα) of the two proteins. D. RMSF for both proteins determined through every simulation. E. The WT/MUT models correlated according to RMSF. F. Secondary structural components in relevant area with time. G. Hydrogen bond formation between the residues I213 or I213T and the other residues, respectively. H. Hydrogen bond formation between I213 and GTP or I213T models, separately.