Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss

Abstract

Background: Pathogenic variants in MFN2 cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some MFN2 variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between MFN2 and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic MFN2 variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions. Method: Several MFN2-mediated functions were characterized in fibroblast cells from a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy, who harbours a novel homozygous MFN2 variant, D414V, which is found in a region of the HR1 domain of MFN2 where few pathogenic variants occur. Results: We found evidence for impairment of several MFN2-mediated functions. Consistent with reduced mitochondrial fusion, patient fibroblasts exhibited more fragmented mitochondrial networks and had reduced mtDNA copy number. Additionally, patient fibroblasts had reduced oxygen consumption, fewer mitochondrial-ER contacts, and altered lipid droplets that displayed an unusual perinuclear distribution. Conclusion: Overall, this work characterizes D414V as a novel variant in MFN2 and expands the phenotypic presentation of MFN2 variants to include cerebellar ataxia.

The Role of Impaired Mitochondrial Dynamics in MFN2-Mediated Pathology

“…For example, a study into motor neurons expressing MFN2 variants T105M, R274W, H361Y and H364W showed mitochondrial fragmentation ( Franco et al, 2020 ). Meanwhile, mitochondrial fragmentation was also noted in fibroblasts from a patient with the MFN2 D414V variant, which is unusual due to its location a region of the HR1 domain that is largely devoid of MFN2 variants ( Sharma et al, 2021b ).…”

“…Further investigation into lipid metabolism and calcium homeostasis showed a variety of differences among the MFN2 variants, consistent with alterations to MERC functions. In a separate study of fibroblasts harbouring a homoplasmic D414V MFN2 variant from a patient showing atypical characteristics of CMT2A, a significant decrease in both the size and the number of MERCs was reported ( Sharma et al, 2021b ). Even though MFN2 is just one of many proteins involved in forming MERCs, the available data looking at pathogenic MFN2 variants all show altered MERCs ( Larrea et al, 2019 ).…”

“…In addition to peripheral neuropathy, patients with MFN2 variants can sometimes also present with additional phenotypes, including optical atrophy ( Leonardi et al, 2015 ), sensorineural hearing loss ( Chung et al, 2006 ), cerebellar ataxia ( Sharma et al, 2021b ) and multiple systemic lipomatosis ( Capel et al, 2018 ), to name a few. The reasons for this phenotypic variability due to different variants in the same gene remain unknown.…”

Functional characterization of two variants in the mitochondrial topoisomerase gene TOP1MT that impact regulation of the mitochondrial genome

“…Briefly, background signal was subtracted, images of mitochondrial networks were skeletonized, and the mitochondrial length was obtained using the analyze skeleton function in ImageJ FIJI. The sum of the lengths of all branches of a mitochondrion was evaluated as the total length of that mitochondrion in every image [19]. For each of WT and KO cells, at least 20 cells were evaluated.…”

Mitochondria Endoplasmic Reticulum Contact Sites (MERCs): Proximity Ligation Assay as a Tool to Study Organelle Interaction

“…A second approach to validate PLA spots is to stain the organelles of interest along with PLA foci ( Figures 1B,C ). This allows to colocalize PLA spots with sites of overlap between the two organelles ( Alpy et al, 2013 ; Gomez-Suaga et al, 2017 ; Sharma et al, 2021 ), thus identifying real PLA foci and allowing further assessment of organelle colocalization using Mander’s coefficients. Co-staining of PLA and organelles can be achieved by expressing fluorescently tagged organelles markers, using fixable stains such as mitotracker or labelling the primary antibodies used for the PLA.…”