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Case Reports
. 2020 Oct;88(4):830-842.
doi: 10.1002/ana.25854. Epub 2020 Aug 29.

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

Alexander Fay #  1 , Yngo Garcia #  2   3 , Marta Margeta  4 , Sunita Maharjan  5 , Claudia Jürgensen  6 , Jose Briceño  7 , Mariaelena Garcia  6 , Sitao Yin  5 , Laia Bassaganyas  8 , Thomas McMahon  1 , Ya-Ming Hou  5 , Ying-Hui Fu  1 , Louis J Ptáček  1
Affiliations
Case Reports

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

Alexander Fay et al. Ann Neurol. 2020 Oct.

Abstract

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.

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Conflict of interest statement

Potential Conflicts of Interest: None

Figures

Fig. 1.
Fig. 1.
First and Second Generations of the Pedigree (A) and a Representative Branch “D” of the Pedigree (B).
Figure 2.
Figure 2.
A-B Dual immunohistochemistry for slow myosin (yellow; type 1 fibers) and fast myosin (pink; type 2 fibers) shows a much greater degree of fiber-type grouping in a muscle biopsy from a moderately-severely affected family member (D 6.178; panel B) compared to a biopsy from an asymptomatic family member (D 5.67; panel A). Both biopsies show a significant population of hybrid fibers that co-express both myosin proteins (orange), evidence of recent reinnervation. C-D Coarsened internal architecture is noted on NADH-TR stain in the more affected individual (D) compared to the asymptomatic Individual (C). E-G Representative electron micrographs from nerve biopsies obtained from two other affected family members (B 5.88 and D 5.111) show mitochondrial hyperplasia and hypertrophy in the cytoplasm of a Schwann cell (E) and a small myelinated axon (F). Crystalline arrays are seen in an abnormal Schwann cell mitochondrion (arrow in G). For orientation, axonal cytoplasm is marked with an asterisk in each image. Scale bars: A-B, 10 μm; C-D, 50 μm; E, 0.4 μm; F, 1.0 μm; G, 0.3 μm.
Figure 3.
Figure 3.
Relative abundance of mt-tRNAVal in control and patient muscle. Two control samples (#2 and #3) and four patient samples (D 6.146, female 24, 18/01/1994; B 6.28, female 27, 25/01/1991; D 6.178, female 45, 14/08/1972; and D 5.67, female 41, 14/10/1978) were collected and analyzed. (A-C) A representative Northern blot analysis of (A) mt-tRNAVal, (B) mt-tRNAPhe, and (C) mt-tRNALeu(UUR) in muscle. The abundance of each tRNA relative to 5S rRNA in each sample is calculated and normalized to that in control #2 as the percentage at the bottom of each lane. (D) Bar graphs showing the 5S rRNA-normalized levels of mt-tRNAVal vs. mt-tRNAPhe. Data are the average of three Northern blot experiments as shown in (A-C). (E) Bar graphs showing the mt-tRNALeu(UUR)-normalized levels of mt-tRNAVal vs. mt-tRNAPhe. Data are the average of three Northern blot experiments as shown in (A-C).
Figure 4.
Figure 4.
Human mt-tRNAVal. (A) Sequence and cloverleaf structure of human mt-tRNAVal in the standard nucleotide numbering framework of tRNA, showing the acceptor stem, the D (dihydrouridine)-loop, the anticodon, the variable region, and the T (thymidine)-loop. The site of m.1661A>G mutation is marked by an arrow, corresponding to an A64G mutation in mt-tRNAVal. (B) Location of human mt-tRNAVal within the cryo-EM (electron microscopy) structure of the human mitoribosome near the central protuberance of the mitoribosome large subunit (mt-LSU, shown in green). The human mitoribosome small subunit (mt-SSU, shown in yellow) is next to the mt-LSU, marked by the head domain and the body domain. (C) The tertiary structure of human mt-tRNAVal as derived from the cryo-EM structure of the human mitoribosome in (B). The backbone of m.1661A in the structure of the tRNA (shown in cyan) is broken.
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