Pathogenic mitochondrial tRNA point mutations: nine novel mutations affirm their importance as a cause of mitochondrial disease

Abstract

Mutations in the mitochondrial genome, and in particular the mt-tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies-outlined in a validated pathogenicity scoring system--is therefore invaluable in determining whether novel or rare mt-tRNA variants are pathogenic. Here, we describe the identification of nine novel mt--tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT-TI; m.5541C>T, MT-TW; m.5690A>G, MT-TN; m.7451A>T, MT-TS1; m.7554G>A, MT-TD; m.8304G>A, MT-TK; m.12206C>T, MT-TH; m.12317T>C, MT-TL2; m.16023G>A, MT-TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as "definitely pathogenic" mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as 'possibly pathogenic' (m.4289T>C, m.7554G>A, and m.8304G>A).

Keywords: mitochondrial disease; mitochondrial tRNA; segregation; single-fiber studies.

Figure 1

Sequential COX–SDH histochemistry. Sequential cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) histochemistry was performed on skeletal muscle biopsies from all patients with the exception of patients 1 and 6. A mosaic pattern of COX activity is visible in each of the images, with COX-deficient fibers shown in blue and COX-positive fibers shown in brown. For patient 6, the individual COX histochemical reaction demonstrates a large number of COX-deficient fibers.

Figure 2

Location of mt-tRNA variants. The location of each of the identified mt-tRNA variants within the tRNA cloverleaf structure is shown. Each of the nine novel variants occurs within a different mt-tRNA, in a variety of stems, loops, and other regions. The affected position and the substitution that occurs are highlighted in bold.

Figure 3

Single fiber mutation load segregation. This graph shows the mutation load measured in individual COX-positive (closed circles) and COX-deficient fibers (open circles) laser-micro dissected from a skeletal muscle biopsy taken from all the patients with the exception of patients 1 and 6, which were omitted due to a lack of available tissue. In each patient studied, the identified variant segregates with the biochemical deficiency.

Figure 4

Location of “definitely pathogenic” mutations across the mt-tRNAs. The number of “definitely pathogenic” mt-tRNA point mutations found at various locations throughout the cloverleaf are shown by the use of a gradient color scale; lighter colors equate to fewer mutations, whereas darker colors specify more mutations. “Definitely pathogenic” mutations occur mainly in the stems and to a lesser degree in the loops with the notable exception of a position in the DHU-loop, which includes the m.3243A>G MT-TL1 mutation, and the third base of the anticodon.