Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1

Traducció RAN; Transcripció antisentit; Moduladors fenotípicsTraducción RAN; Transcripción antisentido; Moduladores fenotípicosRAN translation; Antisense transcription; Phenotypic modulatorsMyotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.The research of G. Nogales-Gadea and A. Ramos-Fransi is funded by Instituto de Salud Carlos III (grant numbers PI15/01756 and PI18/00713) and co-financed by Fondos FEDER. G. Nogales-Gadea is supported by a Miguel Servet research contract (ISCIII CD14/00032, ISCIII CPII19/00021, and FEDER) and by a Trampoline Grant #21108 from AFM Telethon. E. Koehorst is funded by the La Caixa Foundation (ID 100010434), fellowship code LCF/BQ/IN18/11660019, cofunded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 713673. The research of M. Suelves is funded by Ministerio de Ciencia e Innovación (grant number PID2020-118730RB-I00) and co-financed by Fondos FEDER. J. Núñez-Manchón is funded by Instituto de Salud Carlos III I-PFIS fellowship (grant number IFI20/00022). G. Lucente is supported by a Rio Hortega contract (ISCIII CM16/00016 and FEDER). J. Chojnacki is supported by European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 793830. The work of A.P. Gómez-Escribano and R.P. Vázquez-Manrique is funded by the ISCIII (CPII16/00004, PI17/00011 and PI20/00114) and the Fundación Ramón Areces (CIVP19S8119). This work was supported by the CERCA program/ Government of Catalonia. The funding bodies had no role in the design of the study and the collection, analysis, and interpretation of data

Myotonic Dystrophy Type 1: the heterogeneity of a complex disease in a global research approach

[eng] Myotonic Dystrophy Type 1 (DM1) is a complex disease with a dominant autosomic inheritance caused by a CTG expansion at the end of the DMPK gene. This expansion is very unstable and it is known that is correlated with the severity of the disease. However, DM1 is a multisystemic disease, with a high heterogeneity in the clinical manifestation and with no cure yet. The main pathomolecular mechanism, although the is incomplete information, it is caused by the accumulation of toxic RNA aggregates called RNA foci, product from the CTG expansion. In the present thesis we analyzed the genetic complexity of DM1. First, studying different methods that are currently used to size the CTG expansion in DM1 patients and compare the results between them. We have seen that different methodologies yield different results, and thus, manifesting the need of establishing a universal method in the DM1 community for sizing the CTG expansion in patients with DM1. We also studied the genetic instability present in three tissues of DM1 patients: blood, muscle and skin. We have seen that the three tissues have CTG instability, with longer expansions present in muscle and skin compared to blood. Moreover, we have seen that only the estimated progenitor Resumen de la Tesis: Myotonic Dystrophy Type 1 (DM1) is a complex disease with a dominant autosomic inheritance caused by a CTG expansion at the end of the DMPK gene. This expansion is very unstable and it is known that is correlated with the severity of the disease. However, DM1 is a multisystemic disease, with a high heterogeneity in the clinical manifestation and with no cure yet. The main pathomolecular mechanism, although the is incomplete information, it is caused by the accumulation of toxic RNA aggregates called RNA foci, product from the CTG expansion. In the present thesis we analyzed the genetic complexity of DM1. First, studying different methods that are currently used to size the CTG expansion in DM1 patients and compare the results between them. We have seen that different methodologies yield different results, and thus, manifesting the need of establishing a universal method in the DM1 community for sizing the CTG expansion in patients with DM1. We also studied the genetic instability present in three tissues of DM1 patients: blood, muscle and skin. We have seen that the three tissues have CTG instability, with longer expansions present in muscle and skin compared to blood. Moreover, we have seen that only the estimated progenitor CTG expansion found in muscle correlates with the age of disease onset of these patients. We studied as well the presence of interruptions in the CTG expansion and how these variant repeats can affect the genetic transmission in between generations and the phenotype of the patients. After analyzing a cohort of 49 DM1 patients we have seen that 10% of the patients carried CCG interruptions. Moreover, the presence of interruptions resulted, in one case, in a contraction of the CTG expansion in between generations, but also resulted in an expansion of the CTG track in the other case, linked to anticipation. As it has been previously described, we have seen that the presence of interruptions can be linked to non-typical phenotypic traits of DM1, such as proximal weakness. However, contrary of what is believed, we have seen that patients carrying interruptions can develop a clinical phenotype with a high severity. We also studied the pathomolecular mechanism of RNA gain-of function, trough 3D imaging. We studied myoblasts cells derived from DM1 patients, analyzing the presence of RNA toxic aggregates. 3D imaging allowed us to have a full characterization of the cell, quantifying the number of the RNA toxic aggregates and studying the relationship between the main molecular players. We have seen that the CTG expansion leads the number of RNA foci that is accumulated in the cell and the number of the RNA foci correlates with the DMPK expression. We have seen as well that the RNA foci is not only present in the nucleus, but also in the cytoplasm, and the area of these toxic aggregates is related with its presence in the cytoplasm. Finally, we studied the efficacy (reduction of RNA foci number) and toxicity (cell mortality) of a promising therapeutic approach for DM1, using antisense oligonucleotides with BNANC modifications, in cells derived from DM1 patients. We have studied the treatment response in three different subtypes, since due to the multisystemic nature of DM1, it is necessary to know how the different cells would react to a specific treatment. We have seen that the RNA foci reduction and the cell mortality is different in fibroblasts, lymphoblasts and myoblasts of Dm1 patients. Myoblasts, derived from one of the most affected tissues in DM1 patients, are the cells responding less to the treatment, which highlights the importance to perform experiments in order to improve the delivery in these cells. Moreover, we have seen that the treatment response seems to not be related with the CTG expansion size

Programació perinatal de la obesitat: efectes de la restricció calòrica durant la gestació i la lactància sobre la propensió a patir obesitat

L'obesitat suposa, cada cop més, un problema de salut greu en gran part de la població occidental. Cal tenir en compte que l'obesitat no només és resultat de la genètica de l'individu combinada amb l'estil de vida que aquest pugui dur a terme; nombrosos estudis científics posen de manifest l'existència d'una programació metabòlica durant el desenvolupament fetal la qual juga un paper clau en la predisposició a patir obesitat en l'etapa adulta. Així, existeixen tota una sèrie de mecanismes rellevants que contribuiran en l'establiment d'aquest patró metabòlic en períodes crítics com ho són la gestació i la lactància. Aquests mecanismes de programació són, a la vegada, sensibles a tota una sèrie de condicions a les quals l'individu es veu exposat. Entre aquestes influències claus destaca la nutrició materna. Així doncs, alguns estudis han evidenciat la possible relació existent entre una restricció calòrica materna durant la gestació i la lactància i la programació perinatal de l'obesitat; una restricció calòrica durant la gestació sembla tenir conseqüències negatives en el sentit que la programació afavoreix l'eficiència metabòlica i l'acumulació de greix. En canvi, una restricció calòrica moderada durant la lactància sembla tenir efectes ben diferents. Tanmateix però, encara calen futures investigacions que aportin més evidències. A partir d'ara doncs, l'aplicació d'aquests coneixements podria resultar una estratègia per prevenir des de les etapes més inicials del desenvolupament, l'efecte creixent de l'obesitat

Análisis de aplicaciones móviles para reducir peso y mejorar la relación con los alimentos en personas con sobrepeso/obesidad

Sin financiaciónNo data JCR 20190.180 SJR (2019) Q4, 2033/2754 Medicine (miscellaneous)No data IDR 201

Three-dimensional imaging in myotonic dystrophy type 1: Linking molecular alterations with disease phenotype

Objective: We aimed to determine whether 3D imaging reconstruction allows identifying molecular:clinical associations in myotonic dystrophy type 1 (DM1). Methods: We obtained myoblasts from 6 patients with DM1 and 6 controls. We measured cytosine-thymine-guanine (CTG) expansion and detected RNA foci and muscleblind like 1 (MBNL1) through 3D reconstruction. We studied dystrophia myotonica protein kinase (DMPK) expression and splicing alterations of MBNL1, insulin receptor, and sarcoplasmic reticulum Ca(2+)-ATPase 1. Results: Three-dimensional analysis showed that RNA foci (nuclear and/or cytoplasmic) were present in 45%-100% of DM1-derived myoblasts we studied (range: 0-6 foci per cell). RNA foci represented <0.6% of the total myoblast nuclear volume. CTG expansion size was associated with the number of RNA foci per myoblast (r = 0.876 [95% confidence interval 0.222-0.986]) as well as with the number of cytoplasmic RNA foci (r = 0.943 [0.559-0.994]). Although MBNL1 colocalized with RNA foci in all DM1 myoblast cell lines, colocalization only accounted for 1% of total MBNL1 expression, with the absence of DM1 alternative splicing patterns. The number of RNA foci was associated with DMPK expression (r = 0.967 [0.079-0.999]). On the other hand, the number of cytoplasmic RNA foci was correlated with the age at disease onset (r = -0.818 [-0.979 to 0.019]). Conclusions: CTG expansion size modulates RNA foci number in myoblasts derived from patients with DM1. MBNL1 sequestration plays only a minor role in the pathobiology of the disease in these cells. Higher number of cytoplasmic RNA foci is related to an early onset of the disease, a finding that should be corroborated in future studies.Sin financiación3.485 JCR (2020) Q2, 96/208 Clinical Neurology1.262 SJR (2020) Q1, 92/373 Neurology (clinical)No data IDR 2019UE

Manifesting heterozygotes in McArdle disease: A myth or a reality—role of statins

McArdle disease is an autosomal recessive condition caused by deficiency of the PYGM gene-encoded muscle isoform of glycogen phosphorylase. Some cases of “manifesting” heterozygotes or carriers (i.e., patients who show some McArdle-like symptoms or signs despite being carriers of only one mutated PYGM allele) have been reported in the literature but there is controversy, with misdiagnosis being a possibility. The purpose of our study was to determine if there are actually “manifesting” heterozygotes of McArdle disease and, if existing, whether statin treatment can trigger such condition. Eighty-one relatives of McArdle patients (among a total of 16 different families) were studied. We determined whether they were carriers of PYGM mutations and also collected information on exercise tests (second wind and modified Wingate anaerobic test) and statin intake. We found 50 carriers and 31 non-carriers of PYGM mutations. Although we found existence of heterozygotes manifesting some exercise-related muscle problems such as exacerbated myalgia or weakness, they only accounted for 14% of the carriers and muscle symptoms were milder than those commonly reported in patients. Further, no carrier (whether reporting symptoms or not) showed the second wind phenomenon or a flat blood lactate response to maximal-intensity exercise, both of which are hallmarks of McArdle disease. On the other hand, statin myotoxicity was not associated with muscle symptom onset. © 2018 SSIEMSin financiación4.287 JCR (2018) Q1, 36/174 Genetics & Heredity, 33/145 Endocrinology & Metabolism; Q2, 38/136 Medicine, Research & Experimental,1.560 SJR (2018) Q1, 83/351 Genetics; Q2, 30/102 Genetics (clinical)No data IDR 2018UE

A novel mutation in the valosin-containing-protein gene found in a Spanish family

Sin financiación2.651 JCR (2018) Q2, 99/199 Clinical Neurology; Q3, 160/267 Neurosciences0.990 SJR (2018) Q2, 70/165 Neurology, 126/378 Neurology (clinical)No data IDR 2018UE

Preliminary Findings on CTG Expansion Determination in Different Tissues from Patients with Myotonic Dystrophy Type 1

Myotonic Dystrophy type 1 (DM1) is characterized by a high genetic and clinical variability. Determination of the genetic variability in DM1 might help to determine whether there is an association between CTG (Cytosine-Thymine-Guanine) expansion and the clinical manifestations of this condition. We studied the variability of the CTG expansion (progenitor, mode, and longest allele, respectively, and genetic instability) in three tissues (blood, muscle, and tissue) from eight patients with DM1. We also studied the association of genetic data with the patients' clinical characteristics. Although genetic instability was confirmed in all the tissues that we studied, our results suggest that CTG expansion is larger in muscle and skin cells compared with peripheral blood leukocytes. While keeping in mind that more research is needed in larger cohorts, we have provided preliminary evidence suggesting that the estimated progenitor CTG size in muscle could be potentially used as an indicator of age of disease onset and muscle function impairment.Instituto de Salud Carlos III (Grant Numbers: PI15/01756; P18/00713)AFM Telethon (Trampoline grant number #21108)AFM Telethon Trampoline Grant #21108FI Agaur fellowship FI_B 01090“La Caixa” Foundation (ID 100010434), fellowship code LCF/BQ/IN18/11660019, co-funded by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement n°713673CP14/00032Miguel Servet research contract (ISCIII CD14/00032, CPII19/00021, and FEDER)Rio Hortega contract (ISCIII CM16/00016 and FEDER)Personal honoraria from Shire-Takeda, Amicus, Kyowa-Kirin, and Sanofi-Genzyme4.096 JCR (2020) Q2, 65/175 Genetics & Heredity1.337 SJR (2020) Q2, 99/340 GeneticsNo data IDR 2019UE

A DM1 family with interruptions associated with atypical symptoms and late onset but not with a milder phenotype

Carriage of interruptions in CTG repeats of the myotonic dystrophy protein kinase gene has been associated with a broad spectrum of myotonic dystrophy type 1 (DM1) phenotypes, mostly mild. However, the data available on interrupted DM1 patients and their phenotype are scarce. We studied 49 Spanish DM1 patients, whose clinical phenotype was evaluated in depth. Blood DNA was obtained and analyzed through triplet‐primed polymerase chain reaction (PCR), long PCR‐Southern blot, small pool PCR, AciI digestion, and sequencing. Five patients of our registry (10%), belonging to the same family, carried CCG interruptions at the 3′‐end of the CTG expansion. Some of them presented atypical traits such as very late onset of symptoms ( > 50 years) and a severe axial and proximal weakness requiring walking assistance. They also showed classic DM1 symptoms including cardiac and respiratory dysfunction, which were severe in some of them. Sizes and interrupted allele patterns were determined, and we found a contraction and an expansion in two intergenerational transmissions. Our study contributes to the observation that DM1 patients carrying interruptions present with atypical clinical features that can make DM1 diagnosis difficult, with a later than expected age of onset and a previously unreported aging‐related severe disease manifestation.Sin financiación4.878 JCR (2020) Q1, 44/175 Genetics & Heredity1.981 SJR (2020) Q1, 51/340 GeneticsNo data IDR 2019UE

Myotilinopathy unmasked by statin treatment: A case report

Sin financiación2.393 JCR (2018) Q3, 115/199 Clinical Neurology, 184/261 Neurosciences0.950 SJR (2018) Q2, 134/378 Neurology (clinical), 80/188 Physiology, 45/108 Physiology (medical); Q3, 59/90 Cellular and Molecular NeuroscienceNo data IDR 2018UE