International Journal of Molecular Sciences Article Pathological Features in Paediatric Patients with TK2 Deficiency Cristina Jou 1,2 , Andres Nascimento 1,2 , Anna Codina 1 , Julio Montoya 2,3 , Ester López-Gallardo 2,3 , Sonia Emperador 2,3 , Eduardo Ruiz-Pesini 2,3 , Raquel Montero 1 , Daniel Natera-de Benito 1 , Carlos I. Ortez 1 , Jesus Marquez 1 , Maria V. Zelaya 4 , Alfonso Gutierrez-Mata 5 , Carmen Badosa 1 , Laura Carrera-García 1 , Jesica Expósito-Escudero 1 , Monica Roldán 6 , Yolanda Camara 2,7 , Ramon Marti 2,7 , Isidre Ferrer 8,9,10 , Cecilia Jimenez-Mallebrera 1,2,10, * and Rafael Artuch 1,2, * 1 Pathology, Biobank, Pediatric Neurology, Neuromuscular Unit and Clinical Biochemistry Departments, Hospital Sant Joan de Déu and Institut de Recerca Sant Joan de Déu, 08950 Barcelona, Spain 2 Biomedical Center for Research in Rare Diseases CIBERER-ISCIII, 28029 Madrid, Spain 3 Instituto de Investigación Sanitaria de Aragón (IISA), Universidad de Zaragoza, 50011 Zaragoza, Spain 4 Department of Pathology, Complejo Hospitalario de Navarra-IdiSNA (Navarra Institute for Health Research), 31008 Pamplona, Spain 5 Pediatric Neurology Department, Hospital Nacional Niños “Dr Carlos Sáenz Herrera”, San José 267-1005, Costa Rica 6 Unitat de Microscòpia Confocal i Imatge Cel·lular, Servei de Medicina Genètica i Molecular, Institut Pediàtric de Malaties Rares (IPER), Hospital Sant Joan de Déu, Esplugues de Llobregat, 08950 Barcelona, Spain 7 Research Group on Neuromuscular and Mitochondrial Disorders, Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain 8 Department of Pathology and Experimental Therapeutics, University of Barcelona, 08007 Barcelona, Spain 9 Biomedical Center for Research in Neurodegenerative Diseases (CIBERNED), Bellvitge Institute of Biomedical Research (IDI-BELL), Hospitalet de Llobregat, 08007 Barcelona, Spain 10 Department of Genetics, Microbiology and Statistics, University of Barcelona, 08007 Barcelona, Spain * Correspondence: cecilia.jimenez@sjd.es (C.J.-M.); rafael.artuch@sjd.es (R.A.) Citation: Jou, C.; Nascimento, A.; Abstract: Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We Codina, A.; Montoya, J.; aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of López-Gallardo, E.; Emperador, S.; Ruiz-Pesini, E.; Montero, R.; a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle Natera-de Benito, D.; Ortez, C.I.; et al. biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing Pathological Features in Paediatric from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence Patients with TK2 Deficiency. Int. J. was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and Mol. Sci. 2022, 23, 11002. https:// cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in doi.org/10.3390/ijms231911002 younger patients and was accompanied by an overexpression of major histocompatibility complex Academic Editor: Gail B. Mahady 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 Received: 12 August 2022 three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, Accepted: 14 September 2022 electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle Published: 20 September 2022 showed substantial differences in the youngest patients when compared with those that had a later Publisher’s Note: MDPI stays neutral onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as with regard to jurisdictional claims in sarcomeric de-structuration in the youngest patients with a more severe phenotype. published maps and institutional affil- iations. Keywords: TK2 deficiency; paediatric patients; ultrastructural studies; muscle biopsy; mitochondrial myopathies; ragged red fibres Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and The mitochondria contains the most intricate metabolic pathways within the cellu- conditions of the Creative Commons lar metabolism. This complexity is partially explained by the double genetic origin that Attribution (CC BY) license (https:// controls oxidative phosphorylation, where proteins are codified by either nuclear or mito- creativecommons.org/licenses/by/ chondrial DNA, and by the fundamental pathways that occur in mitochondria [1,2]. All 4.0/). manners of clinical presentations, ages of disease onset, and inheritance types are possible Int. J. Mol. Sci. 2022, 23, 11002. https://doi.org/10.3390/ijms231911002 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 11002 2 of 11 in mitochondrial diseases, which are a group of rare genetic disorders that impair different mitochondrial biological functions, including ATP biosynthesis [2,3]. Thus, mitochondrial diseases can be caused by mutations in nuclear and mitochondrial genes, and defects in over 300 genes are reported to cause mitochondrial disease [3]. Mitochondrial thymidine kinase (TK2) is a nuclear DNA-encoded mitochondrial en- zyme that catalyses the phosphorylation of thymidine in mitochondria. Its function is essential for thymidine and deoxycytidine triphosphate (dTTP and dCTP) synthesis in noncycling cells to maintain and control mitochondrial DNA (mtDNA) replication. Thus, mutations in the TK2 gene cause mtDNA depletion or multiple mtDNA deletion syn- dromes [1], which has a deep impact on mitochondrial energy metabolism. Regarding the natural history of the disease, a large series of patients have been previously reported [4–9], and the clinical spectrum has been extensively described, with three main phenotypes: (i) infantile-onset myopathy with severe mtDNA depletion, frequent neurological involve- ment, and a rapid progression to early mortality; (ii) childhood-onset myopathy, with mtDNA depletion and a moderate-to-severe progression of generalized weakness; and (iii) late-onset myopathy, with mild limb weakness at onset and a progression to respi- ratory insufficiency [6]. Although skeletal muscle seems to be the target organ in TK2 deficiency, it has been suggested that tissues other than muscles may be involved, such as the brain, eyes, heart, and liver [10]. Muscle biopsies have traditionally been considered the gold standard for the analysis of multiple mtDNA deletions and general depletion, including those caused by the TK2 deficiency [11]. In all cases, the main histopathological features in TK2 deficiency are ragged-red fibres (RRFs), described in variable numbers with cytochrome C oxidase (COX) defects [6]. RRFs are hyperreactive muscular fibres with succinate dehydrogenase (SDH) reactions indicating mitochondrial proliferation, and they are usually COX-deficient [11]. To the best of our knowledge, the presence of a dystrophic pattern, myonecrosis, increased endomysial connective tissue, or inflammatory features have scarcely been reported in TK2 deficiency cases [6,12]. Ultrastructural features in the cohorts of TK2-deficient patients have not been published. With this background, we aimed to report the histopathological, immunohistochemical, and ultrastructural features of a cohort of paediatric patients, including the infantile- and childhood-onset forms of the TK2 deficiency. 2. Results 2.1. Clinical, Biochemical, and Genetic Data of the Patients The main clinical, biochemical, and genetic data are presented in Table S1. We report on eight affected non-consanguineous children (five males and three females) from eight families. Two cases corresponded to the infantile-onset form (below 1 year of age) and the other six corresponded to the childhood-onset form (between 1 and 12 years old). The first symptoms occurred before the age of 36 months in all cases. Based on the clinical characteristics, we divided the patients into two subgroups: those with an onset before 2 years of age who showed a very rapid progression of symptoms (n = 5), and a second group that debuted after the age of 2, of which the patients showed a moderate progression, with a loss of ambulation before adolescence (n = 3). In infants younger than 24 months of age, the main symptom was failure to thrive, with feeding problems and severe muscular hypotonia. In children over 24 months of age, the clinical picture was more subtle with progressive weakness and facial involvement. The patients did not have dysmorphic features or associated malformations. No patient showed intellectual disability. Two of the youngest two-year-old patients (one was not treated) died. The rest of the patients were treated with oral pyrimidine deoxyribonucleosides (thymidine and deoxycytidine), and their clinical outcome has been previously reported, with all of them being alive [6,13]. Regarding the biochemical and genetic features, while the blood lactate was normal in most cases, the level of creatine kinase was elevated in all cases but one, as was the level of GDF-15 [14]. Other biochemical analyses disclosed normal results. The measurement of mtDNA content in the muscle was abnormal in most cases (from 55 to 90% of mtDNA Int. J. Mol. Sci. 2022, 23, 11002 3 of 11 depletion). Six out of eight patients presented an mtDNA percentage of depletion equal to or higher than 70%. Multiple deletions were also found in the two patients presenting lower percentages of depletion (case 6 and 8). All patients presented pathogenic mutations in the TK2 gene: five compound heterozygotes and three homozygotes. The nomenclature of the TK2 variants follows the numeration of the GenBank accession number NM_004614.4 sequence, where one corresponds to the A nucleotide of the initial ATG codon. All patients had previously reported pathogenic mutations [6,13]. The TK2 activity was frankly reduced in almost all patients for which cultured fibroblasts were available for this determination. Only in one case (case 6) was the reduction of TK2 activity mild. The substrate-saturating conditions for the TK2 activity determination that we have used are more sensitive for detecting reductions in the maximum velocity (Vmax) of the enzyme than for detecting alterations in the affinity for the substrates [15,16]. In fact, the enzyme kinetics of the homozygous mutation for this particular patient (p.His121Asn) markedly increases the Km for ATP [13], which is in agreement with this explanation. 2.2. Pathological Characteristics of Patients The main pathological and ultrastructural findings are stated in Table 1 and Figures 1–4. Table 1. Histopathological (n = 8) features in the muscle biopsies from paediatric patients with TK2 deficiency. Case 1 2 3 4 5 6 7 8 % RRF 70% 30% 40% 40% 25% 10% 8% 5% % SDH 20% 12% 15% 5% 10% 10% 3% 8% % COX negative 90% 65% 60% 90% 70% 25% 14% 5% Increased lipid content Yes Yes Yes N.A. Yes Yes Yes No Cytoplasmic bodies No No Yes Yes Yes No No No Expression of neonatal myosin 20% 25% 20% 25% N.A. N.A. 4% 2% Sarcolemmal vacuoles Yes Yes No Yes Yes Yes Yes No Inflammation Severe Severe Mild Severe Moderate Mild Mild Mild Macrophages infiltration Yes Yes Yes Yes N.A. N.A. Yes Yes T-lymphocyte infiltration Isolated Isolated Isolated No N.A. N.A. No Isolated B-lymphocyte infiltration No Isolated No Yes N.A. N.A. Yes No MHC I overexpression Yes Yes Yes Yes N.A. N.A. Yes Yes Myofagocytosis Yes Yes Yes Yes No No Yes Yes Variation size fibres Yes Yes Yes Yes Yes Yes Yes Yes Predominate type I fibres Yes No Yes Yes N.A. N.A. Yes Yes The muscle histological and histochemical analysis showed two main patterns related to the age of onset regarding symptoms: one pattern displayed more severe features corresponding to patients in whom first symptoms were observed before the age of 2 years, and the other with a mild pathological involvement corresponding to three cases older than 24 months. (Figure 1). All muscle biopsies revealed a myopathic pattern with variable muscle fibre size and occasionally internal small myonuclei, as well as the presence of a variable number of RRFs. The number of RRFs and COX-negative fibres was greater at younger ages. In general, an increase in SDH activity was observed in all muscle biopsies, indicating mitochondrial proliferation (Figure 1(1c,2c)). The fibres displayed small sarcolemmal vacuoles, which were observed as areas devoid of staining in the haematoxylin-eosin slides (Figure 1(1a)). These vacuoles showed a high lipid content. No endomysial or perimysal connective tissue were seen. The fibres displayed regenerative changes with an overexpression of neonatal myosin (Figure 1(1e)) and alteration in fibre typing with a predominance of type I muscle fibres; these findings were more intense in the infantile- onset patients. In general, an increase in SDH activity was observed in all muscle biopsies, indicating mitochondrial proliferation (Figure 1(1c,2c)). The fibres displayed small sarcolemmal vac- uoles, which were observed as areas devoid of staining in the haematoxylin-eosin slides (Figure 1(1a)). These vacuoles showed a high lipid content. No endomysial or perimysal connective tissue were seen. The fibres displayed regenerative changes with an overex- Int. J. Mol. Sci. 2022, 23, 11002 pression of neonatal myosin (Figure 1(1e)) and alteration in fibre typing with a predomi- 4 of 11 nance of type I muscle fibres; these findings were more intense in the infantile-onset pa- tients. Figure Figure 1. 1.Muscular Muscular biopsy biopsyfrom patients from with infantile-onset patients (row 1, corresponding with infantile-onset to case 1), child- (row 1, corresponding to case 1), hood-onset (row 2, corresponding to case 8), and a healthy control (row 3). Columns: (a) haematox- childhood-onset (row 2, corresponding to case 8), and a healthy control ylin-eosin stain; (b) Gomori’s modified trichrome stain; (c) SDH stain; (d) COX stain; (e) immuno- (row 3). Columns: (a) haematoxylin-eosin histochemical stain for stain; (b)myosin; neonatal Gomori’s and modified trichrome stain; (f) immunohistochemical stain(c) forSDH MCHstain; I. The (d) COX stain; infan- tile-onset patient showed stain (e) immunohistochemical a marked, unstructured for neonatal muscular myosin; andpattern with variability in the stain (f) immunohistochemical size offor MCH the fibres, internal myonuclei, and regenerative fibres. (1a) Many ragged red fibres and numerous I. The infantile-onset patient showed a marked, unstructured muscular pattern with variability in fibres with large vacuoles; (1b) proliferation of mitochondria is visible as blue-coloured fibres; (1c) the size of thefibres numerous fibres, with internal myonuclei, COX depletion; and regenerative (1d) small fibres. fibres with positive (1a) Many ragged immunostaining red fibres and for neonatal numerous myosin fibres indicatedwiththelarge vacuoles; presence (1b) proliferation of regenerative of mitochondria fibres; and (1e) is visibleexpressed MHC I is physiologically as blue-coloured in the fibres; (1c)capillaries. numerous The patient fibres withpresents a staining of(1d) COX depletion; the sarcoplasm small fibresand the positive with sarcolemma in all the immunostaining for muscle fibres. Scale bar: 100 µm. The childhood-onset patient shows a more conserved pattern with neonatal myosin indicated the presence of regenerative fibres; and (1e) MHC I is physiologically a moderate variability in the measurement of fibres, with the presence of a population of hypo- expressed in the(2a) trophic fibres. capillaries. The patient Isolated ragged presents red fibres without asubsarcolemmal staining of the sarcoplasm and reinforcements. the sarcolemma No increased endomysial or perimysial connective tissue; (2b) occasional fibres with mitochondrial in all the muscle fibres. Scale bar: 100 µm. The childhood-onset patient shows a more conserved proliferation; (2c) with pattern isolated COX-negative a moderate fibres; (2d) variability verymeasurement in the occasional regenerative of fibres, fibres visible with the with immuno- presence of a population histochemical staining for neonatal myosin; (2e) mild MCH type I overexpression; and (2f) scale bar: of hypotrophic fibres. (2a) Isolated ragged red fibres without 100–200 µm. (3a–f) Muscular biopsy from a healthy control. Scale bar: 50–100 µm. subsarcolemmal reinforcements. No increased endomysial or perimysial connective tissue; (2b) occasional fibres with mitochondrial Occasional proliferation; endomysial (2c) isolated and perimysial COX-negative fibres;mononucleated (2d) very occasionalinflammatory cells fibres regenerative were ob-visible with served, mainly corresponding to macrophages with myonecrosis, immunohistochemical staining for neonatal myosin; (2e) mild MCH type I overexpression; and (2f) and they were accom- scalepanied by isolated bar: 100–200 µm. lymphocytes. (3a–f) Muscular In the infantile-onset biopsy from a healthycases,control. the macrophagic Scale bar: inflamma- 50–100 µm. Occasional endomysial and perimysial mononucleated inflammatory cells were ob- Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW served, mainly corresponding to macrophages with myonecrosis, and they were 5 of 12 accompa- nied by isolated lymphocytes. In the infantile-onset cases, the macrophagic inflammatory infiltrate was distributed in the perimysium, endomysium, and in relation to the muscle torywhile fibres, infiltrate in was the distributed in the perimysium, childhood-onset cases this endomysium, and in infiltration was relationto limited tonecrotic the mus- muscle cle fibres, while fibres (Figure 2). in the childhood-onset cases this infiltration was limited to necrotic mus- cle fibres (Figure 2). Figure Figure 2. Immunohistochemical studies 2. Immunohistochemical studiesforfor CD68 in case CD68 2 (a),24(a), in case (b),47 (b), (c), and 8 (d). 7 (c), andThe infantile- 8 (d). The infantile- onset onset patients patients (a,b)presented (a,b) presented aadiffused diffused inflammatory inflammatoryinfiltration in the endomysium infiltration and perimys- in the endomysium and perimy- ium, while the childhood-onset cases (c,d) have CD68-positive inflammatory cells isolated in the sium, while the childhood-onset cases (c,d) have CD68-positive endomysium mostly related to myonecrosis. Scale bar: 100 µm. inflammatory cells isolated in the endomysium mostly related to myonecrosis. Scale bar: 100 µm. These findings are accompanied by an overexpression of MHC I in the sarcolemma ofThese findings practically all ofare the accompanied by an overexpression fibres with sarcoplasmic of MHC I infibres expression in regenerative the sarcolemma (Figure of practically all oldest 1(1f)). The of thepatients fibres with sarcoplasmic presented expression fewer regenerative in regenerative fibres (Figure 1(2e)),fibres and MHC(Figure I 1(1f)). Theoverexpression oldest patients was identified fewer presented in the isolated fibres (Figure regenerative fibres 1(2f)). (Figure 1(2e)), and MHC I overex- pression was identified in the isolated fibres (Figure 1(2f)). 2.3. Immunofluorescence Studies for Mitochondrial Complexes Immunofluorescence studies specific to the mitochondrial respiratory chain com- plexes showed a marked reduction in both complexes, CI and CIV (Figure 3(1a,d,2a,d)), supporting the findings that we observed during the COX staining (Figure 1(1d,2d)). The mitochondrial mass was evaluated by means of porin immunostaining, showing a lower 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 perimys- ium, while the childhood-onset cases (c,d) have CD68-positive inflammatory cells isolated in the endomysium mostly related to myonecrosis. Scale bar: 100 µm. Int. J. Mol. Sci. 2022, 23, 11002 These findings are accompanied by an overexpression of MHC I in the sarcolemma 5 of 11 of practically all of the fibres with sarcoplasmic expression in regenerative fibres (Figure 1(1f)). The oldest patients presented fewer regenerative fibres (Figure 1(2e)), and MHC I overexpression was identified in the isolated fibres (Figure 1(2f)). 2.3. Immunofluorescence Studies for Mitochondrial Complexes 2.3.Immunofluorescence studies Immunofluorescence Studies specific to the for Mitochondrial mitochondrial respiratory chain complexes Complexes showed a marked reduction Immunofluorescence in both studies complexes, specific CI and CIVrespiratory to the mitochondrial (Figure 3(1a,d,2a,d)), chain com- sup- porting plexes the findings showed that reduction a marked we observed during in both the COX complexes, CI andstaining (Figure CIV (Figure 1(1d,2d)). The 3(1a,d,2a,d)), mitochondrial supporting themass wasthat findings evaluated by means we observed duringofthe porin COXimmunostaining, showing staining (Figure 1(1d,2d)). Thea lower mitochondrial expression in TK2masspatients was evaluated (Figure by3(1b,e,2b,e)) means of porin immunostaining, when compared with showing a lower healthy controls expression (Figure in TK2 3(3b,e). patients In the cases(Figure 3(1b,e,2b,e)) that presented when number a lower comparedofwith RRFs healthy (casescontrols 7 and 8), the (Figureof3(3b,e). analysis In the cases that for immunofluorescence presented a lower number CIV complexes showedofaRRFs (cases greater 7 andof8),fibres, number the with analysis of immunofluorescence for CIV complexes showed a greater number a decrease in staining when compared with the COX activity. These cases corresponded to of fibres, with a decrease in staining when compared with the COX activity. These cases corre- the childhood-onset patients in our series (Figure 3(2d)). sponded to the childhood-onset patients in our series (Figure 3(2d)). Figure Figure 3.3. Muscular Muscular biopsy biopsyfrom patients from with infantile-onset patients (row 1, corresponding with infantile-onset to case 1), child- (row 1, corresponding to case 1), hood-onset (row 2, corresponding to case 8), and a healthy control (row 3). Columns: (a) mitochon- childhood-onset (row 2, corresponding to case 8), and a healthy control (row 3). Columns: (a) mito- drial complex I immunofluorescence stain; (b) mitochondrial porin immunofluorescence stain (sur- chondrial 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) mi- tochondrial 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. 2.4. Ultrastructural Examination An ultrastructural study was performed for four of the cases (Table 2 and Figure 4). Table 2. Ultrastructural features (n = 4) in the muscle biopsies from the paediatric patients with TK2 deficiency. Case 1 4 7 8 Electron microscopy Increase in mitochondrial number Yes Yes Yes Yes Changes in size and shape Yes Yes Yes Yes Anomalous cristae Yes Yes Yes Yes Presence of electron-dense inclusions No Yes No No Lipid accumulation Yes Yes Yes No Atrophic fibres Yes No Yes No Cytoplasmatic bodies Yes Yes No No Autophagic vacuoles Yes No Yes No Int. J. Mol. Sci. 2022, 23, 11002 6 of 11 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 12 Figure4.4.Transmission Figure Transmissionelectron electronmicrograph micrographof ofthe theinfantile-onset infantile-onset form form (case 1) in (case 1) in the the skeletal skeletalmuscle muscle region, region,showing showinga amarked markeddistortion distortionofofthe theintermyofibrillary intermyofibrillarypattern patternwith withanan increase increase ininthe thenumber num- ofber mitochondria of mitochondriaand content of lipids and content (1a: scale of lipids bar ofbar (1a: scale 10 µm of 10was µmchosen to better was chosen observe to better a single observe a single muscular muscular fibre). fibre). The The mitochondria mitochondria are enlarged are enlarged with abnormal with abnormal cristae cristae (blue(blue arrow)arrow) (1b:(1b: scalescale bar ofbar of 2and 2 µm) µm)concentric, and concentric, “onion-shaped” “onion-shaped” cristaecristae with electrodense with electrodense granulegranule (blue(blue arrow) arrow) (1c: (1c: scale scale bar of 1 µm was chosen to detect the electrodense inclusions that are bar of 1 µm was chosen to detect the electrodense inclusions that are not appreciable with lower not appreciable with lower magnification). The childhood-onset form (case 7) shows a relative conserved sarcomeric magnification). The childhood-onset form (case 7) shows a relative conserved sarcomeric structure structure with a mild increased lipid content and lipid droplet size (2a: scale bar of 2 µm). Mito- with a mild increased lipid content and lipid droplet size (2a: scale bar of 2 µm). Mitochondria are chondria are arranged around the nucleus with a slight increase in quantity (blue arrow) (2b: scale arranged around bar of 2 µm). Thethe nucleus isolated with a slight mitochondria showincrease in quantity (blue mild abnormalities arrow) (2c: scale bar(2b: scale of 0.5 µmbarwasofchosen 2 µm). The isolated mitochondria show mild abnormalities (2c: scale bar of 0.5 µm was to rule out the presence of electrodense inclusions and intramitochondrial vacuoles). In the other chosen to rule out the presence of electrodense infantile-onset patient (caseinclusions and intramitochondrial 4), we observed intramitochondrialvacuoles). electrodenseIn the other infantile-onset inclusions (blue arrow) (3a: scale patient bar4), (case ofwe 1 µm); there isintramitochondrial observed an intimate relationship betweeninclusions electrodense the mitochondria (blue arrow)and lipid (3a: droplets scale bar of(star) (3b: 1 µm); scaleisbar there of 2 µm),relationship an intimate and the intramitochondrial vacuoles contain between the mitochondria fine droplets and lipid granular(star) material (3b: (star) (3c: scale bar on 2 µm). The healthy control child showed normal-sized mitochondria with scale bar of 2 µm), and the intramitochondrial vacuoles contain fine granular material (star) (3c: scale unaltered cristae networks (4a: scale bar of 2 µm; 4b: scale bar of 1 µm; 4c: scale bar of 1 µm). Elec- bar on 2 µm). The healthy control child showed normal-sized mitochondria with unaltered cristae trodense inclusions are not appreciable at a 1 µm scale bar. networks (4a: scale bar of 2 µm; 4b: scale bar of 1 µm; 4c: scale bar of 1 µm). Electrodense inclusions are 3. not appreciable at a 1 µm scale bar. Discussion Themain The clinical, biochemical, feature and was in all cases molecular featuresof the presence have been consistently a population reported of abnormal in mito- TK2-deficient patients. Here, we report on a cohort of infantile-onset TK2-deficient chondria. In three out of four patients (two cases with onset before 24 months of age), pa- tients, muscle several focusingfibres on the histopathological demonstrated findings. a distinct Some of the clinical subsarcolemmal and molecular accumulation data of abnormal in this cohort of patients have been previously reported [6,13,14]. In our mitochondria. Frequently, the mitochondria were markedly enlarged or had an abnormalseries, all patients presented the morphology, first and symptoms they exhibitedof densely the disease beforeconcentrically packed, 36 months of age, but showed arranged lamellardifferent cristae. Int. J. Mol. Sci. 2022, 23, 11002 7 of 11 Occasionally, the mitochondrial matrix displaced spaces with electron-dense, osmophilic granules. We found three major types of mitochondrial alterations, mainly consisting of con- centrically arranged lamellar cristae (Figure 4(1c)), electron-dense granules (Figure 4(3a)), and intramitochondrial vacuoles with no clear membranes (Figure 4(3c)) and composed of fine granular material. Furthermore, in patients, fibres showed an accumulation of lipid droplets (Figure 4(1a,2a)) in association with altered mitochondria, and these findings were more pronounced in the infantile-onset. An electron microscopy examination also showed that the youngest case (case 1), when compared with the oldest case (case 8), had a greater involvement of the sarcomeric muscle fibre with respect to regenerative changes, autophagic vacuoles, and the alteration of the intermyofibrillar pattern with the presence of cytoplasmic bodies (Figure 4(1a)). The oldest case (case 8) showed near-normal ultrastruc- tural results, with minor alterations of the myofibrils, including their focal disorganization (Figure 4(2c)). 3. Discussion The clinical, biochemical, and molecular features have been consistently reported in TK2-deficient patients. Here, we report on a cohort of infantile-onset TK2-deficient patients, focusing on the histopathological findings. Some of the clinical and molecular data in this cohort of patients have been previously reported [6,13,14]. In our series, all patients presented the first symptoms of the disease before 36 months of age, but showed different degrees of severity and progression according to the age groups (onset before or after 24 months of age). It is important to note that with the possibility of treating patients, the natural history of this disease has changed, especially in the childhood-onset form, as described in previous works [6,8,9,14]. Thus, the histopathological and ultrastructural data of the muscle fibres can lead us to a better response capacity, and the data highlights the importance of starting this therapy before the muscular alterations are more difficult to reverse. Histopathological features have been described in some TK2 deficiency cohorts [6,8,17], but a deep histological analysis including an ultrastructural study focused on paediatric patients has scarcely been performed. In infantile-onset patients, we observed regenerative fibres with myonecrosis. Regenerative changes with an overexpression of neonatal myosin and myonecrosis are frequently observed in muscular dystrophies [18]. However, this is an unusual feature in mitochondrial diseases [19]. On the other hand, there was no observed increase in endomysial or perimysial connective tissue, nor was there any increase in fat infiltration observed in the infantile-onset form of our TK2 patients, unlike what was reported in the series of adult patients [8]. These observations are relevant as they can help us determine differential diagnoses for muscular dystrophies in the paediatric population. Another finding in these patients, which is not typical of mitochondrial myopathies, is an alteration in fibre typing with a predominance of type I fibres, which are more commonly seen in congenital myopathies [20]. The pathological analysis of the COX fibres are one of the cornerstones in the diagnosis and research of mitochondrial diseases [11]. In our series of patients, the mitochondrial respiratory chain complex immunostaining findings reinforced the observations of the COX histochemistry stains. Furthermore, according to our results, the immunostaining seems to be more sensitive than classical COX histochemistry methods. The immunohistochemical detection of OXPHOS complexes represents a valuable additional diagnostic tool for the evaluation of mitochondrial diseases [21,22]. This is especially relevant when there are few RRF, since OXPHOS complex immunostaining reveals more fibres with CI and CIV depletion than what is observed with the COX histochemistry. Another interesting difference in our TK2-deficient cohort of patients compared with other pediatric mitochondrial myopathies is that the inflammatory infiltrates that were present were mainly constituted by macrophages with few T-lymphocytes that were associ- ated with an overexpression of MHC I, especially in younger patients, where inflammatory infiltrates are not related only to myonecrosis. Inflammatory features have scarcely been Int. J. Mol. Sci. 2022, 23, 11002 8 of 11 reported in mitochondrial diseases; with these findings, we confirmed the previous inflam- matory features detected by our group in TK2-deficient patients using a transcriptomic analysis [12]. The electron microscopy examination in mitochondrial disease shows mitochondrial abnormalities, including an increase in number and size, and altered individual morphol- ogy, such as irregular cristae consisting of short and discontinuous folding throughout a mitochondrion’s matrix; furthermore, the examination depicts mitochondrial swelling, inclusion bodies, and paracrystalline inclusions [23,24]. Hypotheses arise to explain such abnormalities, such as the alteration in the cristae organization system leading to ultrastruc- tural alterations such as concentric cristae or compartmentalization, among others. How- ever, other pathomechanisms can explain some ultrastructural features in mitochondrial diseases, such as the activation of proinflammatory genes or the presence of compensatory responses to mitochondrial stress [23]. The ultrastructural examination of skeletal muscle biopsies from our TK2-deficient pa- tients revealed an increase in the mitochondrial number and size, as well as their abnormal morphology, with densely packed, concentrically arranged lamellar cristae or concentric “onion-shaped” cristae. Occasionally, the mitochondrial matrix displayed spaces which contained electron-dense inclusions. In contrast to other mitochondrial disease patholog- ical features, no paracrystalline inclusions or cristae linearization were observed in our cohort of patients [23,24]. Intramitochondrial vacuoles containing fine granular material also occurred in our series of samples. The artefact of fixation can be excluded, as the biopsies have been processed at different times. The content of the vacuoles mimics the fine granular material of the cytoplasm, but the internalization of the cytoplasm into the mitochondria is not clear, as it is not limited by a clear mitochondrial membrane. Inter- estingly, these newly reported features were only observed in the infantile-onset cases. Sarcomeric de-structuration could explain the severe clinical involvement in the youngest patients. We did not observe mitochondrial fusion images, despite it being associated as a response to mitochondrial stress caused by various reasons, including mitochondrial DNA depletion [25]. Lipid accumulation is another feature frequently observed in muscle biopsies with impaired mitochondrial energy metabolism [11]. In our cohort of TK2 patients, an increase in the amount of lipids was observed, as well as a connection between lipid droplets and abnormal mitochondria, supporting the role of mitochondria in lipid metabolism. 4. Materials and Methods 4.1. Patients and Control Subjects The clinical, biochemical, genetic, histopathological, and ultrastructural data from the eight infantile-onset and childhood-onset form TK2-deficient patients were recorded. Table S1 summarises the clinical, laboratory, and genetic data. Tables 1 and 2 summarises the pathological and ultrastructural characteristics of the muscle biopsy. Some of our cases have been previously reported [6,9,12,13]. Results were compared with muscular biopsies from age-matched control subjects (age range: 9 months–16 years). 4.2. Pathological Studies The skeletal muscle biopsy was obtained by an open surgical biopsy of the quadriceps in the diagnostic process of the disease. The age of onset of the disease and the age at which the biopsy was performed are detailed in Table 1. The skeletal muscle samples were processed and stained according to standard protocols with haematoxylin and eosin (H&E), modified Gomori trichrome, succinate dehydrogenase (SDH), cytochrome c oxidase (COX), oil red O, neonatal, slow and fast myosin, CD68, and major histocompatibility complex type I (MHC I), as published previously [26]. A double immunofluorescence study was also carried out with the porin antibody (clone VDAC-1, dilution of 1/50. Abcam. Cambridge, EE.UU.) as a surrogate of mitochondrial mass, with a complex I subunit NDUFB8 antibody (CI) (clone 20E9DH10C12, dilution of 1/50. Thermo Fisher Scientific. Waltham, MA, USA, Int. J. Mol. Sci. 2022, 23, 11002 9 of 11 EE.UU) and complex IV subunit IV antibody (CIV) (clone 1D6E1A8, dilution of 1/300. Thermo Fisher Scientific. Waltham, EE.UU) of the mitochondrial respiratory chain. The sections were incubated overnight at 4 ◦ C. After incubation with the primary antibodies, the sections were washed with 1% PBS-Tween and then incubated with the secondary antibodies Alexa 448 and Alexa 594 (dilution of 1/500. Thermo Fisher Scientific, EE.UU) for 2 h at room temperature and were protected from light. After secondary incubation, the 1% PBS-Tween washings were performed, and slides were mounted with the Flouromount mounting medium. The images were captured using a TCS SP8 confocal laser scanning microscope (Leica Microsystems GmbH, Mannheim, Germany). For the electronic microscopic analysis, the tissue was fixed in 2.5% glutaraldehyde, washed in cacodylate buffer, fixed in 1% osmium tetroxide, and dehydrated in graded alcohols. The tissue was embedded in resin and semi-thin and ultra-thin sections were performed. Four cases were available to be examined and were photographed using transmission electronic microscopy (JEOL Model 1100). 4.3. Other Laboratory Studies Biochemical analysis: The biomarkers of mitochondrial dysfunction in plasma (amino acids, lactate, pyruvate, alanine, and growth differentiation factor-15 (GDF-15)) were anal- ysed with ultraperformance liquid chromatography-tandem mass spectrometry, automated spectrometric procedures, and ELISA methods, as previously reported [27,28]. Genetic diagnosis: DNA from muscle biopsies was prepared by standard methods: mtDNA depletions were analysed through real-time quantitative polymerase chain reaction (PCR), as previously reported [29,30], and mtDNA deletions were studied by long-PCR. Genomic DNA was isolated from peripheral blood, and the coding exons and intronic boundaries of the TK2 gene were sequenced by Sanger sequencing. TK2 activity determination: The TK2 activity was determined in primary cultured fibroblasts obtained from skin biopsies. Cells were grown in high-glucose Dulbecco’s mod- ified eagle’s medium (DMEM: 4.5 g/L glucose), supplemented with 2 mM L-glutamine, 100 U/mL penicillin and streptomycin, and 10% dialyzed fetal bovine serum (FBS: Invitro- gen). After reaching confluence, the FBS was reduced to 0.1% to induce quiescence. Two weeks later, we obtained cell pellets for the TK2 activity determination as described in Camara et al. and used [50 -3H]5-(2-bromovinyl)-20 -deoxyuridine (Moravek biochemicals) as a substrate [16]. 5. Conclusions We report the histopathological and ultrastructural features of muscular biopsies in a series of paediatric patients with TK2 deficiency. The pathological features in the muscle showed substantial differences in the youngest patients (onset before 24 months of age) when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsies, such as sarcomeric de-structuration in the youngest patient with a more severe phenotype. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms231911002/s1. Author Contributions: Conceptualization, C.J., C.J.-M. and R.A.; methodology, C.J., Y.C., R.M. (Ramón Marti), E.R.-P., I.F. and R.A.; validation, C.J., J.M. (Julio Montoya) and R.A.; formal analysis, C.J. and R.A.; investigation, A.C., J.M. (Jesus Marquez), A.N., E.L.-G., S.E., E.R.-P., R.M. (Raquel Montero), D.N.-d.B., C.I.O., M.V.Z., A.G.-M., C.B., L.C.-G., J.E.-E., M.R., Y.C., R.M. (Ramón Martí) and I.F.; resources, A.C., J.M. (Jesus Marquez), Y.C., S.E., M.R. and J.M. (Julio Montoya); data curation, C.J., A.N., D.N.-d.B., C.I.O., M.V.Z., A.G.-M. and R.A.; writing—original draft preparation, C.J., C.J.-M. and R.A.; writing—review and editing, all authors; visualization, C.J. and R.A.; supervision, R.A.; project administration, R.A., J.M. (Julio Montoya) and R.M. (Ramón Martí); funding acquisition, R.A. All authors have read and agreed to the published version of the manuscript. Int. J. Mol. Sci. 2022, 23, 11002 10 of 11 Funding: This research was funded by: Instituto de Salud Carlos III (Grant no. FIS-PI17-00021, PI17-00166, and PI20-00340) (co-funded by the European Regional Development Fund, “A way to make Europe”); crowdfunding Program Precipita-FECYT (PR194); Gobierno de Aragón (Grupos Consolidados B33_20R) and FEDER 2014-2020, “Construyendo Europa desde Aragón”; and Aso- ciación de Enfermos de Patología Mitocondrial (AEPMI). CIBERER is an initiative from the Carlos III Health Institute. Institutional Review Board Statement: This study was approved by the Ethical Committee of the Hospital San Joan de Déu (HSJD) in accordance with the Helsinki Declaration of 1964, as revised in 2001. The samples from patients and controls were donated by the HSJD Biobank and they provided written informed consent from the patients or from their parents. The study was approved by the ethics committee with the reference no. PIC-97-16.s. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data are available upon reasonable request. Deidentified patient data. Acknowledgments: We are indebted to the Biobanc de l’Hospital Infantil Sant Joan de Déu per a la Investigació integrated in the Spanish Biobank Network of ISCIII for the sample and data procurement. We thank Maria Jesús Melià for her support with the obtaining of the primary cultures and the determination of the TK2 activity. Conflicts of Interest: The authors declare no conflict of interest. References 1. Zeviani, M.; Viscomi, C. Mitochondrial Neurodegeneration. Cells 2022, 11, 637. [CrossRef] [PubMed] 2. Rahman, J.; Rahman, S. Mitochondrial medicine in the omics era. Lancet 2018, 391, 2560–2574. [CrossRef] 3. Stenton, S.L.; Prokisch, H. Genetics of mitochondrial diseases: Identifying mutations to help diagnosis. EBioMedicine 2020, 56, 102784. [CrossRef] 4. Saada, A.; Shaag, A.; Mandel, H.; Nevo, Y.; Eriksson, S.; Elpeleg, O. 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