A Mitochondrial tRNA Mutation Causes Axonal CMT in a Large Venezuelan Family.

OBJECTIVE: The objective of this study was to identify the genetic cause for progressive peripheral nerve disease in a Venezuelan family. Despite the growing list of genes associated with Charcot-Marie-Tooth disease, many patients with axonal forms lack a genetic diagnosis. METHODS: A pedigree was constructed, based on family clinical data. Next-generation sequencing of mitochondrial DNA (mtDNA) was performed for 6 affected family members. Muscle biopsies from 4 family members were used for analysis of muscle histology and ultrastructure, mtDNA sequencing, and RNA quantification. Ultrastructural studies were performed on sensory nerve biopsies from 2 affected family members. RESULTS: Electrodiagnostic testing showed a motor and sensory axonal polyneuropathy. Pedigree analysis revealed inheritance only through the maternal line, consistent with mitochondrial transmission. Sequencing of mtDNA identified a mutation in the mitochondrial tRNAVal (mt-tRNAVal ) gene, m.1661A>G, present at nearly 100% heteroplasmy, which disrupts a Watson-Crick base pair in the T-stem-loop. Muscle biopsies showed chronic denervation/reinnervation changes, whereas biochemical analysis of electron transport chain (ETC) enzyme activities showed reduction in multiple ETC complexes. Northern blots from skeletal muscle total RNA showed severe reduction in abundance of mt-tRNAVal , and mildly increased mt-tRNAPhe , in subjects compared with unrelated age- and sex-matched controls. Nerve biopsies from 2 affected family members demonstrated ultrastructural mitochondrial abnormalities (hyperplasia, hypertrophy, and crystalline arrays) consistent with a mitochondrial neuropathy. CONCLUSION: We identify a previously unreported cause of Charcot-Marie-Tooth (CMT) disease, a mutation in the mt-tRNAVal , in a Venezuelan family. This work expands the list of CMT-associated genes from protein-coding genes to a mitochondrial tRNA gene. ANN NEUROL 2020;88:830-842.

Lipid peroxidation measurement by thiobarbituric acid assay in rat cerebellar slices.

Lipid peroxidation by reactive oxygen species (ROS) is known to be involved in the damaging mechanism of several acute and chronic brain disorders. The most prominent and currently used assay as an index for lipid peroxidation products is the thiobarbituric acid assay (TBA test). It is based on the reactivity of an end product of lipid peroxidation, malondialdehyde (MDA) with TBA to produce a red adduct. However, it is known that the MDA levels are frequently overestimated, that the reaction lacks specificity and mainly reflects the susceptibility of brain tissue to the generation and degradation of newly formed lipid hydroperoxides under the TBA test conditions. The present paper shows that artifactual lipid peroxidation by TBA test conditions can be prevented and that the MDA level overestimation can be minimized in cerebellar slices. This can be done by incubating the slices in a continuous tissue perfusion system, by adding butylated hydroxytoluene (BHT) to the homogenization solutions and by carrying out the assay anaerobically on deproteinizated supernatants of cerebellar slice homogenates. The present research also showed that lipid peroxidation products generated during incubation of the slices by hydrogen peroxide (H2O2) could be measured without artifactual interference by the TBA test conditions.