Pathological Features in Paediatric Patients with TK2 Deficiency

Abstract

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.

Keywords: TK2 deficiency; mitochondrial myopathies; muscle biopsy; paediatric patients; ragged red fibres; ultrastructural studies.

Figure 1

Muscular biopsy from patients with infantile-onset (row 1, corresponding to case 1), childhood-onset (row 2, corresponding to case 8), and a healthy control (row 3). Columns: (a) haematoxylin-eosin stain; (b) Gomori’s modified trichrome stain; (c) SDH stain; (d) COX stain; (e) immunohistochemical stain for neonatal myosin; and (f) immunohistochemical stain for MCH I. The infantile-onset patient showed a marked, unstructured muscular pattern with variability in the size of the fibres, internal myonuclei, and regenerative fibres. (1a) Many ragged red fibres and numerous fibres with large vacuoles; (1b) proliferation of mitochondria is visible as blue-coloured fibres; (1c) numerous fibres with COX depletion; (1d) small fibres with positive immunostaining for neonatal myosin indicated the presence of regenerative fibres; and (1e) MHC I is physiologically expressed in the capillaries. The patient presents a staining of the sarcoplasm and the sarcolemma in all the muscle fibres. Scale bar: 100 µm. The childhood-onset patient shows a more conserved pattern with a moderate variability in the measurement of fibres, with the presence of a population of hypotrophic fibres. (2a) Isolated ragged red fibres without subsarcolemmal reinforcements. No increased endomysial or perimysial connective tissue; (2b) occasional fibres with mitochondrial proliferation; (2c) isolated COX-negative fibres; (2d) very occasional regenerative fibres visible with immunohistochemical staining for neonatal myosin; (2e) mild MCH type I overexpression; and (2f) scale bar: 100–200 μm. (3a–f) Muscular biopsy from a healthy control. Scale bar: 50–100 μm.

Figure 2

Immunohistochemical studies for CD68 in case 2 (a), 4 (b), 7 (c), and 8 (d). The infantile-onset patients (a,b) presented a diffused inflammatory infiltration in the endomysium and perimysium, while the childhood-onset cases (c,d) have CD68-positive inflammatory cells isolated in the endomysium mostly related to myonecrosis. Scale bar: 100 µm.

Figure 3

Muscular biopsy from patients with infantile-onset (row 1, corresponding to case 1), childhood-onset (row 2, corresponding to case 8), and a healthy control (row 3). Columns: (a) mitochondrial complex I immunofluorescence stain; (b) mitochondrial porin immunofluorescence stain (surrogate for mitochondrial mass); (c) merge (mitochondrial co-localization); (d) mitochondrial complex IV immunofluorescence stain; (e) mitochondrial porin immunofluorescence stain; and (f) mitochondrial co-localization. In the patient with infantile-onset, a severe deficiency of CI and CIV are shown in almost all the muscular fibres. Red fibres are present in the co-localization images, indicating a deficiency in mitochondrial complexes. In the childhood-onset case, a decrease in staining intensity are observed with the presence of some negative fibres, which are labelled in red in the co-localization image. In the healthy control, the mitochondrial complexes and porin have normal expression and the co-localization is coloured yellow, showing a correct co-expression of both proteins. Scale bar: 50 µm.

Figure 4

Transmission electron micrograph of the infantile-onset form (case 1) in the skeletal muscle region, showing a marked distortion of the intermyofibrillary pattern with an increase in the number of mitochondria and content of lipids (1a: scale bar of 10 µm was chosen to better observe a single muscular fibre). The mitochondria are enlarged with abnormal cristae (blue arrow) (1b: scale bar of 2 µm) and concentric, “onion-shaped” cristae with electrodense granule (blue arrow) (1c: scale bar of 1 µm was chosen to detect the electrodense inclusions that are not appreciable with lower magnification). The childhood-onset form (case 7) shows a relative conserved sarcomeric structure with a mild increased lipid content and lipid droplet size (2a: scale bar of 2 µm). Mitochondria are arranged around the nucleus with a slight increase in quantity (blue arrow) (2b: scale bar of 2 µm). The isolated mitochondria show mild abnormalities (2c: scale bar of 0.5 µm was chosen to rule out the presence of electrodense inclusions and intramitochondrial vacuoles). In the other infantile-onset patient (case 4), we observed intramitochondrial electrodense inclusions (blue arrow) (3a: scale bar of 1 µm); there is an intimate relationship between the mitochondria and lipid droplets (star) (3b: scale bar of 2 µm), and the intramitochondrial vacuoles contain fine granular material (star) (3c: scale bar on 2 µm). The healthy control child showed normal-sized mitochondria with unaltered cristae networks (4a: scale bar of 2 µm; 4b: scale bar of 1 µm; 4c: scale bar of 1 µm). Electrodense inclusions are not appreciable at a 1 µm scale bar.