Myotonic Dystrophy type 1 cells display impaired metabolism and mitochondrial dysfunction that are reversed by metformin

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. In this study, we characterized the impact of metabolism and mitochondria on fibroblasts and peripheral blood mononuclear cells (PBMCs) derived from patients with DM1 and healthy individuals. Our results revealed a decrease in oxidative phosphorylation system (OXPHOS) activity, oxygen consumption rate (OCR), ATP production, energy metabolism, and mitochondria! dynamics in DM1 fibroblasts, as well as increased accumulation of reactive oxygen species (ROS). PBMCs of DM1 patients also displayed reduced mitochondria! dynamics and energy metabolism. Moreover, treatment with metformin reversed the metabolic and mitochondria! defects as well as additional accelerated aging phenotypes, such as impaired proliferation, in DM1-derived fibroblasts. Our results identify impaired cell metabolism and mitochondria! dysfunction as important drivers of DM1 pathophysiology and, therefore, reveal the efficacy of metformin treatment in a pre-clinical setting.This work was supported by grants from the Instituto Salud Carlos III and FEDER funds (CP16/00039, PI16/01580, PI17/01841) and Health department from Basque Country (2017 and 2018-2017222021)

Late-onset thymidine kinase 2 deficiency: a review of 18 cases

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.Instituto de Salud Carlos III PI16-01843 PI16/00579 CP09/00011Subdirección General de Evaluación y Fomento de la Investigación Sanitaria PI16-01843 PI16/00579 CP09/00011 PI 15/00431 PMP15/0002

Late-onset thymidine kinase 2 deficiency: a review of 18 cases

Mitochondrial myopathy; Multiple deletions; TK2 deficiencyMiopatia mitocondrial; Delecions múltiples; Deficiència de TK2Miopatía mitocondrial; Deleciones múltiples; Deficiencia de TK2BACKGROUND: 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

A Descriptive Analysis of ATTR Amyloidosis in Spain from the Transthyretin Amyloidosis Outcomes Survey.

Introduction Transthyretin amyloidosis (ATTR amyloidosis) is a clinically heterogeneous disease caused by mutations in the transthyretin (TTR) gene or aggregation of wild-type transthyretin (ATTRwt). In Spain, there are two large endemic foci of ATTR amyloidosis caused by the Val30Met variant, with additional cases across the country; however, these data may be incomplete, as there is no centralized patient registry. The Transthyretin Amyloidosis Outcomes Survey (THAOS) is an ongoing, global, longitudinal, observational survey of patients with ATTR amyloidosis, including both inherited and wild-type disease, and asymptomatic patients with TTR mutations. This analysis aimed to gain a deeper understanding of the clinical profile of patients with ATTR amyloidosis in Spain. Methods This was a descriptive analysis of the demographic and clinical characteristics of symptomatic patients enrolled at six sites geographically dispersed throughout Spain (data cutoff: January 6, 2020). Patient data at enrollment, including genotype, demographics, and clinical presentation for symptomatic patients, were recorded. Patients were grouped by predominant phenotype based on clinical measures at enrollment: predominantly cardiac, predominantly neurologic, or mixed (cardiac and neurologic). Results There were 379 patients (58.0% male; 63.3% symptomatic) enrolled in the six THAOS sites in Spain. Predominant genotypes were the Val30Met mutation (69.1%) or ATTRwt (15.6%). Predominant phenotype distribution was neurologic (50.4%), mixed (35.8%), and cardiac (13.8%) for all symptomatic patients (n = 240); neurologic (67.8%), mixed (21.2%), and cardiac (11.0%) for symptomatic Val30Met (n = 146); and mixed (64.9%), cardiac (22.8%), and neurologic (12.3%) for symptomatic ATTRwt (n = 57). Symptomatic patients reported a range of ATTR amyloidosis signs and symptoms at enrollment, with autonomic neuropathy and sensory neuropathy common in all phenotypes. Conclusions These results from THAOS highlight the phenotypic heterogeneity associated with ATTR amyloidosis in Spain and the importance of comprehensive neurologic and cardiac evaluations in all patients with ATTR amyloidosis.post-print392 K

Phenotypic correlations in a large single center cohort of patients with BSCL2 nerve disorders: a clinical, neurophysiological and muscle MRI study

Background: BSCL2 heterozygote mutations are a common cause of distal hereditary motor neuropathies (dHMN). We present a series of BSCL2 patients and correlate clinical, neurophysiological and muscle-MRI findings. Methods: 26 patients from 5 families carrying the p.N88S mutation were ascertained. Age of onset, clinical phenotype (dHMN, Charcot-Marie-Tooth/CMT, spastic paraplegia), physical examination, disability measured as modified Rankin score (mRS) and neurophysiological findings were collected. A whole body muscle-MRI had been performed in 18 patients. We analyzed the pattern of muscle involvement on T1-weighted and STIR sequences. Hierarchical analysis using heatmaps and a MRI Composite Score (MRI CS) were generated. Statistical analysis was carried out with STATA SE v.15. Results Mean age was 51.54+/-19.94 years and 14 patients were males. dHMN was the most common phenotype (50%) and 5 patients (19.23%) showed no findings on examination. Disease onset was commonly in childhood and disability was low (mRS=1.34+/-1.13) although median time since onset of disease was 32 years (range=10-47). CMT-like patients were more disabled and disability correlated with age. On muscle-MRI, thenar eminence, soleus and tibialis anterior were most frequently involved, irrespective of clinical phenotype. MRI CS was strongly correlated with disability. Conclusion: Patients with the p.N88S BSCL2 gene mutation are phenotypically variable, although dHMN is most frequent and generally slowly progressive. Muscle-MRI pattern is consistent regardless of phenotype and correlates with disease severity, probably serving as a reliable outcome measure for future clinical trials

Senescence plays a role in myotonic dystrophy type 1 br

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of an accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from patients with DM1 and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which related to the accumulation of cellular senescence. The data from transcriptome analyses were corroborated in human myoblasts and blood samples, as well as in mouse and Drosophila models of the disease. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts, whereas the DM1 Drosophila model showed reduced longevity and impaired locomotor activity. Moreover, functional studies highlighted the impact of BMI1 and downstream p16INK4A/ RB and ARF/p53/p21CIP pathways in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds Quercetin, Dasatinib, or Navitoclax reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in Drosophila in vivo. Our results identify the accumulation of senescence as part of DM1 pathophysiology and, therefore, demonstrate the efficacy of senolytic compounds in the preclinical setting.MGP and ASA are recipient of predoctoral fellowships from the University of the Basque Country (PIF 15/245) and Carlos III Institute (FI17/00250), respectively. We thank the methodological support service of Biodonostia Institute for help with statistical analysis. This work is supported by grants from the Instituto Salud Carlos III and FEDER funds (PI16/01580, PI17/01841, DTS18/00181, PI19/01355, CPII19/00021, and DTS20/00179), La Caixa, and Health department from Basque Country (2017222021, 2018222021, and 2020333008)

Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1)

Funding Information: This research was supported by the Isabel Gemio Foundation (P18–13) and was also partially supported by the “Fondo Europeo de Desarrollo Regional” (FEDER) from the European Union. E.A.-C. was supported by a pre-doctoral fellowship of Valhondo Calaff Foundation. S.C.-C. and E.U.-C. were supported by FPU fellowships (FPU19/04435 and FPU16/00684, respectively) from the Ministerio de Ciencia, Innovación y Universidades, Spain. M.P.-B. and A.G.-B. received fellowships from the “Plan Propio de Iniciación a la Investigación, Desarrollo Tecnológico e Innovación (Universidad de Extremadura). M.N.-S. was supported by the “Ramon y Cajal” Program (RYC-2016–20883), and P.G.-S., was funded by “Juan de la Cierva Incorporación” Program (IJC2019–039229-I), Spain. S.M.S.Y.-D. was supported by the Isabel Gemio Foundation and CIBERNED (CB06/05/0041). J.M.F received research support from the Isabel Gemio Foundation and the “Instituto de Salud Carlos” III, CIBERNED (CB06/05/0041). Publisher Copyright: © 2022 by the authors.Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.publishersversionpublishe

Late-onset thymidine kinase 2 deficiency: a review of 18 cases

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

Distribution and genotype-phenotype correlation of GDAP1 mutations in Spain

Mutations in the GDAP1 gene can cause Charcot-Marie-Tooth disease. These mutations are quite rare in most Western countries but not so in certain regions of Spain or other Mediterranean countries. This cross-sectional retrospective multicenter study analyzed the clinical and genetic characteristics of patients with GDAP1 mutations across Spain. 99 patients were identified, which were distributed across most of Spain, but especially in the Northwest and Mediterranean regions. The most common genotypes were p.R120W (in 81% of patients with autosomal dominant inheritance) and p.Q163X (in 73% of autosomal recessive patients). Patients with recessively inherited mutations had a more severe phenotype, and certain clinical features, like dysphonia or respiratory dysfunction, were exclusively detected in this group. Dominantly inherited mutations had prominent clinical variability regarding severity, including 29% of patients who were asymptomatic. There were minor clinical differences between patients harboring specific mutations but not when grouped according to localization or type of mutation. This is the largest clinical series to date of patients with GDAP1 mutations, and it contributes to define the genetic distribution and genotype-phenotype correlation in this rare form of CMT

Detection of variants in dystroglycanopathy-associated genes through the application of targeted whole-exome sequencing analysis to a large cohort of patients with unexplained limb-girdle muscle weakness

Abstract Background Dystroglycanopathies are a clinically and genetically heterogeneous group of disorders that are typically characterised by limb-girdle muscle weakness. Mutations in 18 different genes have been associated with dystroglycanopathies, the encoded proteins of which typically modulate the binding of α-dystroglycan to extracellular matrix ligands by altering its glycosylation. This results in a disruption of the structural integrity of the myocyte, ultimately leading to muscle degeneration. Methods Deep phenotypic information was gathered using the PhenoTips online software for 1001 patients with unexplained limb-girdle muscle weakness from 43 different centres across 21 European and Middle Eastern countries. Whole-exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. Genes known to be associated with dystroglycanopathies were analysed for disease-causing variants. Results Suspected pathogenic variants were detected in DPM3, ISPD, POMT1 and FKTN in one patient each, in POMK in two patients, in GMPPB in three patients, in FKRP in eight patients and in POMT2 in ten patients. This indicated a frequency of 2.7% for the disease group within the cohort of 1001 patients with unexplained limb-girdle muscle weakness. The phenotypes of the 27 patients were highly variable, yet with a fundamental presentation of proximal muscle weakness and elevated serum creatine kinase. Conclusions Overall, we have identified 27 patients with suspected pathogenic variants in dystroglycanopathy-associated genes. We present evidence for the genetic and phenotypic diversity of the dystroglycanopathies as a disease group, while also highlighting the advantage of incorporating next-generation sequencing into the diagnostic pathway of rare diseases