Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 https://doi.org/10.1186/s13023-019-1071-z RESEARCH Open Access Late-onset thymidine kinase 2 deficiency: a review of 18 cases Cristina Domínguez-González1,2,3, Aurelio Hernández-Laín4, Eloy Rivas5, Ana Hernández-Voth6, Javier Sayas Catalán6, Roberto Fernández-Torrón7, Carmen Fuiza-Luces2,3,8, Jorge García García9, Germán Morís10, Montse Olivé11, Frances Miralles12, Jordi Díaz-Manera3,13, Candela Caballero14, Bosco Méndez-Ferrer15, Ramon Martí3,17, Elena García Arumi3,16, María Carmen Badosa17, Jesús Esteban1,2,3, Cecilia Jimenez-Mallebrera3,17, Alberto Blazquez Encinar2,3,8, Joaquín Arenas2,3,8, Michio Hirano18, Miguel Ángel Martin2,3,8 and Carmen Paradas19,20* Abstract Background: TK2 gene encodes for mitochondrial thymidine kinase, which phosphorylates the pyrimidine nucleosides thymidine and deoxycytidine. Recessive mutations in the TK2 gene are responsible for the ‘myopathic form’ of the mitochondrial depletion/multiple deletions syndrome, with a wide spectrum of severity. Methods: We describe 18 patients with mitochondrial myopathy due to mutations in the TK2 gene with absence of clinical symptoms until the age of 12. Results: The mean age of onset was 31 years. The first symptom was muscle limb weakness in 10/18, eyelid ptosis in 6/18, and respiratory insufficiency in 2/18. All patients developed variable muscle weakness during the evolution of the disease. Half of patients presented difficulty in swallowing. All patients showed evidence of respiratory muscle weakness, with need for non-invasive Mechanical Ventilation in 12/18. Four patients had deceased, all of them due to respiratory insufficiency. We identified common radiological features in muscle magnetic resonance, where the most severely affected muscles were the gluteus maximus, semitendinosus and sartorius. On muscle biopsies typical signs of mitochondrial dysfunction were associated with dystrophic changes. All mutations identified were previously reported, being the most frequent the in-frame deletion p.Lys202del. All cases showed multiple mtDNA deletions but mtDNA depletion was present only in two patients. Conclusions: The late-onset is the less frequent form of presentation of the TK2 deficiency and its natural history is not well known. Patients with late onset TK2 deficiency have a consistent and recognizable clinical phenotype and a poor prognosis, due to the high risk of early and progressive respiratory insufficiency. Keywords: TK2 deficiency, Mitochondrial myopathy, Multiple deletions Background This group includes defects in enzymes involved in the Defects in the maintenance and repair of mitochondrial maintenance of the balanced pool of deoxynucleotides of DNA (mtDNA) result in an emerging and heterogeneous the mitochondria, which are crucial in the biosynthesis of group of mitochondrial disorders, caused by alterations of the mitochondrial genome and have therapeutic implica- the nuclear genes involved in mtDNA replication [1–3]. tions [4, 5]. The disrupted synthesis of mtDNA results in qualitative (multiple deletions) and/or quantitative (a drastic decrease in the number of copies or depletion) defects of * Correspondence: cparadas@us.es 19 Neurology Department, Neuromuscular Disorders Unit, Instituto de the mtDNA. In particular, one of the ‘myopathic forms’ of Biomedicina de Sevilla, Hospital U. Virgen del Rocío, CSIC, Universidad de the mitochondrial depletion/multiple deletions syndromes is Sevilla, Avd. Manuel Siurot s/n, 41013 Sevilla, Spain 20 caused by mutations in the TK2 gene which encodes for Biomedical Network Research Centre on Neurodegenerative Diseases (CIBERNED), Madrid, Spain mitochondrial thymidine kinase, which phosphorylates the Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 2 of 10 pyrimidine nucleosides thymidine (dT) and deoxycytidine extra-muscular symptoms. We gathered information (dC) [1, 6]. from the latest neurological examination registered in- Recessive mutations in the TK2 gene (MIM# 609560) are cluding, when available, the Muscle Research Council responsible for diverse clinical presentations mainly charac- (MRC) scale to assess the muscle strength and the 6 min terized by progressive muscle weakness, dysphagia and re- walk test (6MWT) for functional evaluation. spiratory involvement with a wide spectrum severity and of age of onset. TK2 deficiency was initially described by Saada, Respiratory assessment et al. in 2001 [6] in four children with a severe myopathy as- The latest value of the forced vital capacity (FVC) in sociated with depletion of the mtDNA. Since then, a number seated and supine position, maximum inspiratory pres- of cases have been reported depicting a heterogeneous clin- sure (MIP), blood gas analysis, nocturnal ventilation ical presentation with a continuum spectrum of the disease, (assessed with nocturnal pulse oximetry and/or capno- which includes early-onset extremely severe and rapidly pro- graphy [11] and the need for mechanical ventilation gressive forms with survival of less than two years, to less se- (MV) type and hours of use were recorded. vere forms with late or very late onset, and a variably slower rate of progression [7, 8]. In 2012, Tyynismaa, et al. reported Laboratory tests the first two cases with mutations in the TK2 gene with onset CK (creatine kinase) and lactate levels were quantified in in the fifth decade of life, manifesting chronic progressive ex- serum in basal conditions, at diagnosis. GDF-15 ternal ophthalmoplegia (CPEO) associated with limb muscle (growth/differentiation factor-15) levels were quantified weakness and dysphagia [9]. A recent publication that in- in plasma samples using human GDF-15 quantitative cluded 92 patients describing the natural history of this dis- ELISA kit (R&D Biosystems) according to the manufac- order proposed the classification of three clinical forms turer’s instructions. according to ages-at-onset: infantile (< 1 year-old), childhood (1–12) and late (> 12 years) onset. Nearly 40% of the reported Muscle MRI TK2 cases presented with the symptoms prior to the age of Muscle MRI was performed in 8 of the 18 patients. All 1, in another 41% the onset occurred between the ages of of them were scanned in a 1.5 T MR scanner (Siemens). one and 12, and only in 19% of patients did the symptoms Lower limb axial T1-weighted sequences were used for appeared after the age of 12 [7]. A subsequent retrospective morphological analysis and short-tau inversion recovery review, with similar frequencies for those three subgroups, (STIR) sequences were examined to detect muscle included eleven new cases of which only three were classified edema. The muscle MRI studies were evaluated by the as late-onset [8]. So far, the natural history of patients with same neurologist (R F-T) with wide experience in neuro- late onset TK2 deficiency has not been defined in detail. muscular disorders. The evaluator was blind regarding Here, we report on the clinical features and assess- the clinical manifestations. He scored pelvic, thigh and ments in a large series of 18 patients with late-onset lower leg muscles in axial T1-sequences with the semi- TK2 deficiency, the less known and poorest defined quantitative Mercuri visual scale (MVS) modified by form of this disease, to further characterize this patient Fisher [12]: 0: Normal appearance; 1: Mild involvement, subgroup. Expanding the natural history and prognosis less than 30% of individual muscle volume; 2: Moderate of late-onset TK2 deficiency will facilitate earlier diagno- involvement, 30–60% of individual muscle volumes; 3: sis and identification for treatment with therapies under Severe involvement, > 60% of individual muscle; 4: End clinical development. stage, all the muscle is severely affected, replaced by in- creased density of connective tissue and fat, with only a Methods rim of fascia and neurovascular structures distinguish- Patients able. We compared median value of muscle fatty re- We describe the phenotypic features of 16 Spanish and 2 placement using the Wilcoxon-Mann-Whitney test. US patients with mitochondrial myopathy due to muta- Statistical analyses were performed using IBM SPSS Sta- tions in the TK2 gene with the absence of clinical symp- tistics, V.22 (IBM, Armonk, New York, USA). toms until the age of 12. The series include three pairs of siblings (P3-P4, P6-P10 and P14-P15). Partial data Aerobic exercise testing from five patients have been previously published else- Exercise testing was performed in 5 patients on a cycle where (P1, P5, P9 [7], P3 and P12 [10]). ergometer, following a ramp-like protocol (workload in- creases of 1 W every 6 s [averaging 10 W·min− 1] starting Clinical evaluation from an initial load of 0 W, with a pedal cadence of 60– The electronic records were reviewed to collect informa- 70 rpm throughout the test). Gas-exchange variables tion about the age of onset, initial symptoms, severity, were collected breath-by-breath with an automated distribution and progression of the muscle weakness and metabolic cart (Quark CPET, COSMED, Rome, Italy). Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 3 of 10 The peak oxygen uptake (VO2 peak) was computed as ability to walk without support. Facial musculature was the highest value obtained for any 10-s period during symmetrically affected in 17 patients (94.4%), with pre- the tests [13]. dominance of the orbicular oculis muscle. 16/18 of the pa- tients (88.9%) also had symmetrical eyelid ptosis of Muscle biopsy variable severity, with this being the first symptom in 6 pa- Muscle samples were obtained by open biopsy and proc- tients (33.3%). Six of them required surgical blepharo- essed following the standard procedures: Hematoxylin plasty due to vision impairment. Nine patients had CPEO. and eosin (H&E), modified Gomori trichrome, ATPase The majority (11/18) had difficulty in swallowing, (adenosine triphosphatase), NADH (nicotinamide adenine which resulted in severe weight loss and/or detriment to dehydrogenase), SDH (succinate dehydrogenase), COX the safety of oral feeding in 6 cases, requiring percutan- (cytochrome C oxidase), and COX-SDH stains were per- eous gastrostomy tube in 5 cases (27.8%) on average formed in all available samples. Respiratory chain enzyme 19.6 years after the onset of the disease (ranging from 12 activity levels were recorded when available. to 28 years). Other clinical manifestations included sensory axonal Genetic studies polyneuropathy (7/18;38.9%), neurosensory hearing loss Molecular diagnosis was performed either by direct (3/18;16.6%) and dysphonia due to vocal cord palsy (2/ Sanger sequencing of exons and intron/exon boundaries 18;11.1%). No patient had cardiomyopathy. of the TK2 gene, or by customized next generation sequencing (NGS) panels. Patient’s skeletal muscle Respiratory function mtDNA deletions were investigated by long-range PCR FVC at diagnosis from the total cohort was 55.4% (ranging (polymerase chain reaction) and/or Southern blot, and from 17 to 103) with a mean decrease of FVC in supine mtDNA copy number was assessed by quantitative PCR position of 8% (ranging from 0 to 14), and a mean MIP of as previously described [10, 14]. 36.8% (ranging from 20 to 101%), independent of the asso- The study was approved by the institutional review ciated muscle symptoms. From a respiratory perspective, board of every centre and all patients signed an informed the high frequency of complications should be noted, with consent for the anonymous publication of this data. need for non-invasive MV in 12/18 patients (66.6%). The mean use of the MV was 11.6 h per day (ranging from 8 to Results 24 h). Eight out of the 12 patients with MV (66.6%) pre- Clinical manifestations (Table 1) sented with acute respiratory insufficiency following a rou- We included 18 patients (6 male, 12 female). The mean tine upper respiratory infection as the first manifestation of age-at-onset was 31 years (range 12 to 60 years) with a the disease. None of these cases had any prior respiratory mean age at diagnosis of 48.5 years (range 23 to 73 years) symptoms; however, once detected, they required MV due resulting in an average of 17.4 years between the onset to hypercapnia secondary to alveolar hypoventilation. Al- of the disease until reaching a genetic diagnosis (range 1 though limb muscle weakness and/or eyelid ptosis were to 44 years). The mean duration of the disease was 19.8 already present at the onset of respiratory insufficiency, years (range 6 to 44 years). Four patients from the series those neuromuscular symptoms had not prompted a neur- were deceased, all of them due to respiratory insuffi- ology consultation in any of the eight patients. Thus, the re- ciency a mean of two decades after the onset. spiratory involvement resulted in the diagnosis of an The first symptom was muscle limb weakness in 10/18 underlying myopathy in these patients; the mean FVC was (55.6%), eyelid ptosis in 6/18 (33%) (two patients also of 40.8% (range from 28 to 58) at the time of diagnosis. Of presented ophthalmoparesis), and respiratory insuffi- the six patients who did not needed MV, all showed evi- ciency in 2/18 (11.1%). All patients developed muscle dence of respiratory muscle weakness on functional tests, weakness during the evolution of the disease, 17/18 although only one of them (P8) reported respiratory symp- showing proximal and distal limb muscle weakness, 1/18 toms (orthopnoea), suggesting diaphragmatic weakness. with only distal limb weakness, and 16/18 axial involve- This patient displayed ptosis and CPEO at the age of 50 as- ment. It is noteworthy that neck flexor weakness was sociated with moderate axial and proximal limb muscle clearly more severe than limb weakness (mean, 2.14 on weakness (4 on the MRC scale). Strikingly, although the the MRC scale). functional respiratory tests and nocturnal pulse oximetry The following muscle groups were the most frequently were normal (FVC seated 103%, FVC decubitus 100% and affected, in a symmetrical manner: shoulder abductor MIP 101%) nocturnal transcutaneous capnography revealed (mean, 4 on the MRC scale), hip flexor (mean, 3.75 on high mean levels of carbon dioxide (CO2, mean of 48 the MRC scale) and hip extensor (mean, 3.87 for both mmHg, with a maximum peak of 54 mmHg). on the MRC scale) and finger extensor muscles (mean, Four patients died of respiratory insufficiency at mean 4.14 on the MRC scale). Four patients (22%) lost the age of 56 years (ranging from 40 to 68), and a mean of Table 1 Clinical manifestations summary ID Age At Gender Current Clinical Assessments Respiratory Assessments Other Onset Age manifestations Ptosis CPEO Facial Dysphagia Neck flexor Limb Wheelchair- 6MWT PEG Orthopnea BMI Sitting FVC Hours on Weakness weakness Weakness bound (Meters) (AT AGE) FVC (%) Sitting/ MV (AT Supine (%) AGE) 1 12 F 32 + – + Yes +++ + No 530 No No 13.86 46 −8 8 (32) – 2 20 F 33 – – + Yes +++ + No 390 Yes (32) Yes 17.9 26 −22 8–9 (28) Hepatopathy 3 30 F 61 + – + Yes ++ + No 386 No Yes 27.12 71 −14 8 (61) PNP-S 4 60 F 73 + + ++ Yes ++ + No 345 No Yes 26 69 NA 8 (73) Hearing loss PNP-S 5 50 M 50 ++ – + No + + No 475 No Yes 27.6 47 NA 12 (50) PNP-S 6 14 F 58 ++ ++ ++ No +++ + No 450 No Yes 24.5 51 −28 8 (56) – 7 40 M Death ++ ++ + Yes +++ ++ Yes NA Yes (68) Yes 23.7 48 NA 10–12 (49) Hearing loss at 68 PNP-S Domínguez-González et al. Orphanet Journal of Rare Diseases Vocal cord palsy 8 50 F 63 + + ++ No + + No 417 No Yes 28.7 103 −3 – PNP-S 9 23 F Death – – + Yes +++ ++ Yes NA Yes (39) Yes 15 28 NA 22 (30) – at 40 (2019) 14:100 10 14 M Death + – – Yes ++ +++ Yes NA Yes (42) Yes 17 32 NA 24 (42) – at 49 11 40 M 51 + – + No – + No 600 No No 26.3 70 −10 – – 12 30 M 60 ++ – + Yes NA + No NA No No 23 75 NA – Vocal cord palsy 13 48 F Death + + + No +++ +++ Yes NA No Yes NA 43 NA 12 (58) – at 67 14 15 M 26 + + + Yes + + No 413 No No NA 72 −10 – Hearing loss PNP-S 15 12 F 31 + + + No + + No 428 No No NA 75 −12 – PNP-S 16 30 F 43 ++ + + No + + No 425 No Yes NA 69 2 8 (43) – 17 45 F 51 ++ + ++ Yes + + No NA No No NA NA NA – NA 18 25 F 58 ++ ++ ++ Yes ++ ++ No 228 Yes (39) Yes NA 17 NA 11 (59) NA NA Not available. Ptosis: ++ History of eyelid surgery, + mild, − no ptosis. CPEO chronic progressive external ophthalmoplegia: ++ complete, + partial, − no ocular weakness. Facial weakness: ++ severe, + mild, − no facial weakness. Neck flexor weakness; +++ unable to lift head in supine position, ++ 3 on Medical Research Council (MRC) scale, + 4 on MRC scale, − no neck flexor weakness. Limb weakness: +++ 1–2 on MRC scale, ++ 3 on MRC scale, + 4 on MRC scale, − no weakness. 6MWT six-minute walking test, FVC forced vital capacity, MV Mechanical ventilation, PNP-S sensory polyneuropathy, BMI body mass index Page 4 of 10 Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 5 of 10 24 years after the onset of their initial symptoms (ran- were increased in 5 out of 5 cases analysed (100%), ranging ging from 17 to 35). from 1529 to 2438 pg/mL (2113 pg/mL ± 462, mean ± standard deviation, upper limit of normal =550 pg/mL) [16]. CK and lactate levels (Table 2) 94.4% of patients had increased variable serum CK levels Muscle MRI findings ranging from 190 to 2435 UI/l (normal levels < 170 UI/ It was performed in 8 patients. Mean age at muscle MRI l)), and 16.7% showed levels 10-fold above the upper was 46.4 years old (range: 23–73). Mean disease duration normal limit. Serum lactate levels were measured in at the time of the scan was 18 years (range 10–31). The basal conditions in 12 of the 18 cases. Of these, only most severely affected muscles in axial T1-weighted se- three (25%) displayed slightly increased levels (1.4-2x quences were the gluteus maximus, semitendinosus, sar- above the upper normal limit). torius and gastrocnemius medialis (median MVS: 3). Of these, only the gluteus maximus and sartorius were af- GDF-15 levels fected in all patients. Apart from the later, gluteus med- GDF-15, a biomarker identified in the analysis of transcrip- ius, adductor magnus and semitendinosus were also tomic profiling of TK2 deficient human skeletal muscle [15], moderately affected in the thighs and gastrocnemius has been proven useful in the diagnosis of mitochondrial lateralis in the legs (median MVS: 2). No muscle fat in- myopathies [16], being especially increased in patients with filtration was observed in obturator, quadratus femoris, mitochondrial TK2 deficiency [17]. Serum levels of GDF-15 extensoris digitorum and tibialis posterior (Fig. 1). The Table 2 Biochemical and molecular characteristics ID Mutation Muscle Multiple Residual Respiratory Chain CK GDF-15 Lactate Biopsy Deletions mtDNA (%) Enzyme Activity (UI/l) (pg/mL) (mmol/l) Allele 1 Allele 2 1 c.323C > T (p.Thr108Met) c.323C > T (p.Thr108Met) Yes Yes 17 CI, CIII and CIV 2435 2423 1.95 deficit 2 c.323C > T (p.Thr108Met) c.323C > T (p.Thr108Met) Yes Yes 39 Normal 303 2439 2.3 3 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel Yes Yes 60 CIII deficit 294 1695 2.6 (p.Lys202del) 4 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel ND ND NA ND 647 2483 2.2 (p.Lys202del) 5 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel Yes Yes 66 Normal 357 1529 1.5 (p.Lys202del) 6 c.323C > T (p.Thr108Met) c.323C > T (p.Thr108Met) Yes Yes 19 Normal 425 NA 1.6 7 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel Yes Yes 33 NA 568 NA 2.6 (p.Lys202del) 8 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel Yes Yes NA NA 405 NA 2.4 (p.Lys202del) 9 c.323C > T (p.Thr108Met) c.323C > T (p.Thr108Met) Yes Yes 35 CI, CIII and CIV 190 NA NA deficit 10 c.323C > T (p.Thr108Met) c.323C > T (p.Thr108Met) Yes NA NA Normal 405 NA 3 11 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel Yes Yes NA ND 266 NA NA (p.Lys202del) 12 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel Yes Yes NA ND 350 NA NA (p.Lys202del) 13 c.388C > T (p.Arg130Trp) c.415G > A (p.Ala139Thr) Yes Yes NA CI, CIII and CIV 170 NA 4.14 deficit 14 c.623A > G (p.Tyr208Cys) c.623A > G (p.Tyr208Cys) Yes Yes NA CI, CIII and CIV 1739 NA NA deficit 15 c.623A > G (p.Tyr208Cys) c.623A > G (p.Tyr208Cys) ND NA NA ND 381 NA NA 16 c.323C > T (p.Thr108Met) c.323C > T (p.Thr108Met) Yes Yes 53 Normal 233 NA 1.77 17 c.469_470insTGGG c.156 + 6 T > G Yes Yes 50 NA 537 NA NA (p.Asp157Valfs*11) 18 c.604–606 AAGdel (p.Lys202del) c.604–606 AAGdel NA NA NA NA 1348 NA NA (p.Lys202del) NA not available, ND not done, CK creatine kinase, GDF-15 Growth differentiation factor-15 Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 6 of 10 Fig. 1 Axial T1 muscle MRI and bar charts with Mercuri Visual Scale (MVS) distribution for 7 patients and per anatomical region. a, Axial T1 muscle MRI in pelvis: These two consecutive slices from different patients are showing that the gluteus maximus (marked with asterisk) is the most affected muscle. Tensor fascia latae is affected while obturator and quatratus femoris are less affected. b, Bar chart MVS fat replacement in pelvis: MVS (0: no fat replacement, 4: the muscle is completely replaced) for all patients. Gluteus maximus is the most affected muscle, followed by tensor fascia latae. c, Axial T1 muscle MRI in thighs: These two slices from two different patients are showing the fat replacement of sartorius (wide white arrow) and vastus lateralis (thin white arrow). Other muscles like semitendinosus, semimembranosus and gracilis are also moderately affected. d, Bar chart MVS fat replacement in thighs: MVS for all patients. Sartorius, semimembranosus, semitendinosus, gracilis and vastus lateralis are the most affected muscles. Sartorius and gracilis are affected in all patients. e, Axial T1 muscle MRI in legs: These two slices from two different patients are showing the fat replacement of gastrocnemius medialis (white arrow head). Gastrocnemius lateralis and soleus are also moderately affected. Tibialis anterior and tibialis posterior are the least affected. f, Bar chart MVS fat replacement in legs: MVS for all patients. Gastrocnemius medialis and lateralis are the most affected muscles in legs. Tibialis anterior, extensoris digitorum and tibialis posterior are the least affected muscles Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 7 of 10 fat replacement followed a diffuse pattern and no focal copy-number was studied in 9 patients and severe areas of fat infiltration were detected. We did not ob- mtDNA depletion was detected in only two (17% of serve statistical differences regarding asymmetric in- residual mtDNA in P1 and 19% of residual mtDNA volvement. STIR sequence was normal in all patients. in P6). Fourteen out of 14 patients (100%) showed the presence of multiple mtDNA deletions in muscle. Aerobic exercise testing In addition to weakness, one of the most frequent clin- Discussion ical manifestations in the mitochondrial myopathies is The late-onset presentation of TK2 deficiency is the least poor exercise capacity [18].The latter is reflected by low frequent clinical mode of presentation known. These pa- levels of VO2peak or by poor muscle-oxygen extraction tients are considered to have a milder presentation than (as assessed with near-infrared spectroscopy) during those with infancy and childhood onset disease, however, graded cycle-ergometer/treadmill testing [19]. Aerobic few cases have been described to date and those re- exercise testing was performed on a cycle ergometer in ported were not extensively explored. So far, 17 patients five patients. The mean ± SD VO2 peak obtained was with late-onset were reported to harbour TK2 biallelic 14.8 ± 3.2 mL/kg− 1/min− 1, with normal consumption mutations [7–10, 22]. However clinical details were values of 40.0 ± 9.5 mL/kg− 1/min− 1 [20]. scarce, heterogeneous, and reports did not clearly define the phenotype or rate of progression of the disease. In Muscle biopsies some cases, clinical presentation is similar to that de- Muscle biopsies were performed in 16 patients, 11 were scribed in the childhood onset patients, with progressive available to re-analysis. The morphological study re- limb, facial, extraocular, oropharyngeal and respiratory vealed numerous ragged-red fibers in 100% of the biop- muscle weakness, but with a slower progression, whereas sies, which were hyper-reactive with SDH reaction and in other cases, CPEO is the main manifestation [9]. Re- usually COX-deficient. COX-deficient fibers accounted spiratory insufficiency has been mentioned as a potential for approximately 5–15% of all fibers. Frequently these cause of death although comprehensive data about the muscles also showed dystrophic features with frequent respiratory involvement is not available for all the previ- necrotic fibers, some with phagocytosis, and increased ously published patients: severe respiratory insufficiency endomysial connective tissue (present in 7 out of 11 bi- is described in 41% of the reported cases but in the opsies revised). Marked type I fiber predominance was remaining 59% this data is unavailable or superficially also observed in 2 patients (Fig. 2). These findings differ described [7, 8, 22]. from the usual pattern displayed in other mitochondrial We identified 16 Spanish and two North American pa- myopathies, where the typical signs of mitochondrial tients, from 13 different families, with TK2 mutations and proliferation and dysfunction are not associated with a late-onset presentation. Exhaustive clinical description is other relevant changes in muscle histology structure here provided to facilitate earlier and accurate diagnosis [21]. We have results of the analysis of the enzymatic ac- and to improve the knowledge of the natural history of tivity of respiratory chain complexes of 10 patients. Only this rare, and probably underdiagnosed disorder. in half of them a reduction in the activity of one or more The clinical features and results of the diagnostic tests enzymatic complexes were identified (Table 2). described in our series show a homogeneous phenotypic pattern in late-onset TK2 deficiency consisting of pro- Genetic studies gressive proximal limb, axial neck flexor and facial All patients harbored biallelic mutations in the TK2 muscle weakness frequently associated with ptosis, gene (Ref.Seq. NM_004614.4) (Table 2). Most patients ophthalmoparesis and bulbar weakness, along with an (16/18;88.9%) were homozygous. All mutations were early and severe, although unrecognized, respiratory in- previously reported [7, 8], with the in-frame deletion volvement. Diaphragmatic weakness is very characteris- p.Lys202del (c.604_606AAGdel) being the most fre- tic, occurring in all of our cases, showing an early onset quent (16/36 alleles; 44.4%), followed by the missense but slow progression; 12/18 (66.6%) required MV during mutation p.Thr108Met (c.323C > T) (12/36;27.8%). the evolution of the disease and in 8/18 (44.4%) was the Additionally, three missense mutations were identified cause for the first medical consultation. This pattern of in 3 patients: p.Arg130Trp (c.388C > T), p.Ala139Thr respiratory involvement was found even in patients who (c.415G > A), and p.Tyr208Cys (c.623A > G). Finally, only had an apparently isolated CPEO phenotype. one patient harbored a frameshift mutation p.Asp157 Therefore, it is critical to identify signs of nocturnal Valfs*11(c.469-470insTGGG) in compound heterozy- hypoventilation during clinical evaluation of these pa- gosis with a splice site mutation c.156 + 6 T > G. tients, regardless of the severity of the skeletal myopathy. Genetic data from patients P1, P2, P5, P9 and P12, This discrepancy between diaphragmatic and limb weak- were previously reported [7, 10]. Muscle mtDNA ness was also reflected in some patients with virtually Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 8 of 10 Fig. 2 Morphological alterations in muscle biopsies from patients P1 (a, f, k, p), P5 (b, g, l, q), P9 (c, h, m, r), P14 (D, i, n, s) and P16 (e, j, o, t). a-e H&E shows dystrophic features in all cases with mild endomysal fibrosis, adipose tissue replacement, atrophy and necrotic fibers. Ragged-red fibers are frequently identified in all muscle samples (arrows). f-j Gomori trichrome showed the characteristic ragged-red fibers in all the biopsies. k-o Succinate dehydrogenase (SDH) reveals an increase of the oxidative staining in numerous fibers. p-t Frequent cytochrome C oxidase (COX) deficient fibers are present in variable proportion in the different cases (p and r, COX staining; o, s and t, COX-SDH combined staining). Scale bar =100 μm normal 6MWT results, despite using MV (see Table 1). myopathies. Muscle degeneration in MRI was described In our series, the capnography was the most sensitive in five MERRF patients with the m.8344A > G mutation test for detecting the respiratory dysfunction, since it [23], and more recently fatty infiltration has been com- was abnormal even before basal FVC and MIP revealed municated in patients with single, large-scale deletions alterations. of mitochondrial DNA [24]. However, no extensive stud- Muscle biopsies showed the typical findings of mito- ies have been published trying to define muscle MRI chondrial dysfunction described in most mitochondrial patterns in different mitochondrial myopathies. So, there myopathies. However, as in other TK2 deficiency forms, is no specific MRI pattern for any mitochondrial myop- they also revealed dystrophic features which are distinct athy described so far. In our series of TK2 patients al- from the majority of other mitochondrial myopathies. though no clear pattern of fat infiltration was detected, Thus, our data support that the association of both we have identified some radiological common features, mitochondrial and dystrophic pattern strongly suggest as the involvement of the sartorius muscle in all cases. mutations in the TK2 gene as the underlying cause. This muscle is usually spared until late stages in many All previously published late-onset patients showed genetic muscle diseases (is only affected early in some multiple mtDNA deletions, while mtDNA depletion was myofibrillar myopathies, in the Laing distal myopathy found only in one of the five cases in whom the mtDNA and in RYR1-related myopathies (encodes for ryanodine copy-number was quantified. Our findings corroborate receptor 1 protein) [12, 25–27]), so this finding could be the previous results indicating the presence of multiple helpful for differential diagnosis. mtDNA deletions is more frequent than mtDNA deple- Serum GDF-15 levels have recently been revealed as a tion in the late-onset TK2 deficient patients. Previous re- sensitive and specific biomarker for the diagnosis of ports showed that mtDNA depletion is found in the mitochondrial myopathies [16, 17]. In our series, it most of early onset patients [7], but our data support proved to be very high in all analysed cases, so it could that it cannot be considered a valid prognostic marker orientate the molecular diagnosis in a proper clinical since it can also be found in late-onset cases. context, before the muscle biopsy was performed. In muscle MRI the fat muscle replacement was diffuse, As in other mitochondrial myopathies [19], in our resembling many muscular dystrophies and congenital series the cardiopulmonary exercise testing identified a Domínguez-González et al. Orphanet Journal of Rare Diseases (2019) 14:100 Page 9 of 10 very reduced consumption of oxygen, even in patients risk of early and progressive respiratory insufficiency. Yet, with CPEO as a predominant clinical manifestation some patients may present with a severe acute respiratory (P8). This indicates that, although the weakness may failure. Early detection of respiratory involvement requires not be severe in late-onset TK2 deficiency patients, an active search in the clinics, even in asymptomatic pa- the exercise capacity is abnormally low, ultimately tients. A small number of rationally designed treatments impairing physical activity. are being developed for mitochondrial disorders [29], in- Noticeably, the p.Lys202del was the most frequent cluding nucleoside substrate enhancement therapy de- mutation in the TK2 gene in our series of late-onset pa- signed specifically for TK2 deficiency [4]. Therefore, early tients, which is consistent with the finding that this mu- diagnosis of TK2 deficiency is important as patients could tation appears to be restricted to adult-onset cases, since benefit from the existence of a potential therapy. it has not been reported in any infantile-onset patients Abbreviations who have not even harbouring this mutation in a single 6MWT: 6-min walking test; ATPase: Adenosine triphosphatase; BMI: Body allele [8]. Nevertheless, it was reported in one patient mass index; CK: Creatine kinase; CO2: Carbon dioxide; COX: Cytochrome C with childhood-onset, who was compound heterozygous oxidase; CPEO: Chronic progressive external ophthalmoplegia; dC: Deoxycytidine; dT: Thymidine; FVC: Forced vital capacity; GDF-15: Growth for this mutation and a frameshift mutation, and began differentiation factor 15; H&E: Hematoxylin and eosin; MIP: Maximal showing symptoms at 2.5 years but survived until 8.5 inspiratory pressure; MRC: Muscle Research Council; MRI: Magnetic resonance years-old [28]. The eight cases with this mutation in our imaging; mtDNA: Mitochondrial DNA; MV: Mechanical ventilation; MVS: Mercury visual scale; NADH: Nicotinamide adenine dehydrogenase; series were all homozygous supporting the idea that this NGS: Next generation sequencing; PCR: Polymerase chain reaction; mutation is associated with a milder effect (age at onset SDH: Succinate dehydrogenase; STIR: Shor tau inversion recovery; ranging from 25 to 60 years). Interestingly, this mutation VO2peak: Peak oxygen uptake has only been identified in 13 unrelated Spanish patients Acknowledgements ((11, 13, 26, 27, and this study), 2 related patients from Not applicable. Hispanic ethnic background [10], and one patient from Venezuela (this study) suggesting that it could be a pri- Funding vate mutation and that Spanish/Hispanic candidate pa- This work was supported by research grants of Plan Nacional de I + D + I and Instituto de Salud Carlos III (ISCIII), Subdirección General de Evaluación y tients may be amenable for a rapid genetic screening of Fomento de la Investigación Sanitaria”, project PI16–01843 (CP), PI16/00579 this mutation. However, haplotype analysis would be re- and CP09/00011 for CJM and the European Regional Development Fund quired to confirm the possible founder effect of this mu- (FEDER a way to achieve Europe). MAM has received funding from the Spanish ISCIII (grant PI 15/00431). A multicentric grant funded by the ISCIII tation. The p.Thr108Met mutation was the second most (PMP15/00025 to MAM, RM, MO, CP). common mutation in this study, however it has been found in infantile and childhood onset cases [6, 7] of dif- Availability of data and materials The datasets used and/or analysed during the current study are available ferent geographic origin. from the corresponding author on reasonable request. TK2 deficiency is a severe disorder causing premature death. In recent pre-clinical studies, it has been demon- Authors’ contributions strated that treatment with pyrimidine nucleosides (dC CDG, AHV, JSC, JG, GM, MO, FM, JDM, CC, JBM, JE, MH and CP handled patients and recollected clinical data for the manuscript. RT collected and analysed the + dT) in the H126N knock-in mouse model of TK2 defi- MRI data of the patients. EG and ABE performed molecular analysis; CB and CJ ciency, leads to a prolonged life span in the animals and provided GDF-15 analysis. AH and ER collected and review muscle biopsy data. a restored mtDNA copy number, without significant tox- CD and CP coordinated all the study. CD, MAM and CP wrote the initial manu- script. RM, JA, MAM, CP, and MH provided critical discussion of the research. All icity [4]. This opens the door to a potential therapeutic authors contributed to the final version of the manuscript. All authors read and intervention in humans with this metabolic hereditary approved the final manuscript. disorder, making it necessary to define sensitive and ob- jective outcomes to assess an eventual response to treat- Ethics approval and consent to participate Written informed consent was obtained from patients for publication of ment. Our findings suggest that functional respiratory anonymised clinical data. tests, serum GDF-15 level and the stress cyclometer evaluation are potentially good candidates for monitor- Consent for publication Not applicable. ing the progression of disease. Competing interests Conclusion MH and RM are co-inventors on patent applications filed by Columbia Univer- In summary, our study shows that late-onset patients with sity Medical Center (CUMC) for deoxynucleoside therapy for mitochondrial DNA depletion syndromes including TK2 deficiency. The patent applications and mitochondrial TK2 deficiency have a consistent and other intellectual property have been licensed by CUMC to Meves Pharmaceuti- recognizable clinical phenotype, characterized by a pro- cals, Inc. CUMC may be eligible to receive payments related to the develop- gressive myopathy with predominant facial and axial neck ment and commercialization of the technology. Any potential licensing fees earned will be paid to CUMC and are shared with inventors through CUMC dis- flexor weakness, and respiratory involvement, often asso- tribution policy. MH and RM are paid consultants to Meves Pharmaceutical, Inc. ciated to CPEO. 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