The Biomarker Potential of miRNAs in Myotonic Dystrophy Type I

MicroRNAs (miRNAs) are mostly known for their gene regulation properties, but they also play an important role in intercellular signaling. This means that they can be found in bodily fluids, giving them excellent biomarker potential. Myotonic Dystrophy type I (DM1) is the most frequent autosomal dominant muscle dystrophy in adults, with an estimated prevalence of 1:8000. DM1 symptoms include muscle weakness, myotonia, respiratory failure, cardiac conduction defects, cataracts, and endocrine disturbances. Patients display heterogeneity in both age of onset and disease manifestation. No treatment or cure currently exists for DM1, which shows the necessity for a biomarker that can predict disease progression, providing the opportunity to implement preventative measures before symptoms arise. In the past two decades, extensive research has been conducted in the miRNA expression profiles of DM1 patients and their biomarker potential. Here we review the current state of the field with a tissue-specific focus, given the multi-systemic nature of DM1 and the intracellular signaling role of miRNAs

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

Model-based data analysis of individual human postprandial plasma bile acid responses indicates a major role for the gallbladder and intestine

BACKGROUND: Bile acids are multifaceted metabolic compounds that signal to cholesterol, glucose, and lipid homeostasis via receptors like the Farnesoid X Receptor (FXR) and transmembrane Takeda G protein-coupled receptor 5 (TGR5). The postprandial increase in plasma bile acid concentrations is therefore a potential metabolic signal. However, this postprandial response has a high interindividual variability. Such variability may affect bile acid receptor activation. METHODS: In this study, we analyzed the inter- and intraindividual variability of fasting and postprandial bile acid concentrations during three identical meals on separate days in eight healthy lean male subjects using a statistical and mathematical approach. MAIN FINDINGS: The postprandial bile acid responses exhibited large interindividual and intraindividual variability. The individual mathematical models, which represent the enterohepatic circulation of bile acids in each subject, suggest that interindividual variability results from quantitative and qualitative differences of distal active uptake, colon transit, and microbial bile acid transformation. Conversely, intraindividual variations in gallbladder kinetics can explain intraindividual differences in the postprandial responses. CONCLUSIONS: We conclude that there is considerable inter- and intraindividual variation in postprandial plasma bile acid levels. The presented personalized approach is a promising tool to identify unique characteristics of underlying physiological processes and can be applied to investigate bile acid metabolism in pathophysiological conditions

Three-dimensional imaging in myotonic dystrophy type 1

Altres ajuts: The research of G. Nogales-Gadea, A. Ramos-Fransi, and A. Lucia is funded by Instituto de Salud Carlos III and cofinanced by Fondos FEDER. G. Nogales-Gadea is supported by a Miguel Servet research contract and by a Trampoline Grant #21108 from AFM Telethon. A. Ballester-Lopez is funded by an FI Agaur fellowship and Generalitat de Catalunya. 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. I. Linares-Pardo is funded by CP14/00032 and SGR 1520 (GRC) Generalitat de Catalunya. J. Núñez-Manchón was funded by AFM Telethon Trampoline Grant #21108. G. Lucente was supported by a Rio Hortega contract. J. Chojnacki is supported by European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant . The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data.We aimed to determine whether 3D imaging reconstruction allows identifying molecular:clinical associations in myotonic dystrophy type 1 (DM1). 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. 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]). 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

The need for establishing a universal CTG sizing method in myotonic dystrophy type 1

The number of cytosine-thymine-guanine (CTG) repeats (‘CTG expansion size’) in the 3′untranslated region (UTR) region of the dystrophia myotonica-protein kinase (DMPK) gene is a hallmark of myotonic dystrophy type 1 (DM1), which has been related to age of disease onset and clinical severity. However, accurate determination of CTG expansion size is challenging due to its characteristic instability. We compared five different approaches (heat pulse extension polymerase chain reaction [PCR], long PCR-Southern blot [with three different primers sets—1, 2 and 3] and small pool [SP]-PCR) to estimate CTG expansion size in the progenitor allele as well as the most abundant CTG expansion size, in 15 patients with DM1. Our results indicated variability between the methods (although we found no overall differences between long PCR 1 and 2 and SP-PCR, respectively). While keeping in mind the limited sample size of our patient cohort, SP-PCR appeared as the most suitable technique, with an inverse significant correlation found between CTG expansion size of the progenitor allele, as determined by this method, and age of disease onset (r = −0.734, p = 0.016). Yet, in light of the variability of the results obtained with the different methods, we propose that an international agreement is needed to determine which is the most suitable method for assessing CTG expansion size in DM1

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

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 a very late onset of symptoms (&gt;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

Unraveling potential disease modifiers of myotonic dystrophy type 1

[spa] La distrofia miotónica tipo 1 (DM1) es un trastorno multisistémico, progresivo y no tratable con una expansión CTG en el gen DMPK que es la base de la patología. La compleja naturaleza multisistémica de la DM1 no puede explicarse únicamente por dicha expansión y la identificación de otros modificadores de la enfermedad es clave para comprender la patología completa de la DM1. Por lo tanto, el objetivo principal de este proyecto de doctorado ha sido investigar los posibles modificadores de la enfermedad y estudiar su relación con el fenotipo clínico de la DM1. Concretamente, la Tesis se ha centrado en el estudio de cinco modificadores potenciales de la enfermedad a nivel genético (repeticiones con variantes), transcripcional (transcripción antisentido), proteico (RAN translation), epigenético (metilación del ADN) y de ARNs no codificantes (miRNAs). Durante mucho tiempo se pensó que la expansión de CTGs era una secuencia ininterrumpida, pero evidencias crecientes mostraron la existencia de variaciones dentro de las repeticiones CTG, aunque su influencia en la patología DM1 aún no está clara. Para dilucidar la relación de estas variantes con el fenotipo clínico de DM1, se examinó una cohorte española de 49 pacientes con DM1, donde se identificó una familia portadora de patrones complejos de repeticiones variantes de CCG. Los estudios clínicos mostraron que estos individuos portadores de variantes repetidas presentaron características clínicas atípicas, lo que dificultaba el diagnóstico de DM1, manifestaron la enfermedad a una edad tardía, pero con síntomas clínicos más graves, relacionada con el envejecimiento. Otro modificador potencial de la enfermedad es el mecanismo de transcripción bidireccional, en el que la transcripción se produce tanto en dirección sentido como antisentido (AS) en el locus del gen. Además, se ha descubierto que la traducción no ATG (RAN) asociada a la repetición, que se origina en la cadena antisentido, produce proteínas de poliglutamina potencialmente tóxicas. Para estudiar la influencia de la transcripción de DM1-AS y la traducción de RAN en la patología de DM1, analizamos tres cultivos celulares primarios de diez pacientes con DM1. En conjunto, los pacientes con DM1 tenían niveles más bajos de tránscritos de DM1-AS en comparación con los controles, que se presentaban como un grupo heterogéneo con y sin la inclusión de la repetición expandida. La traducción no ATG (RAN translation) no estaba presente en las células DM1 derivadas de pacientes, o en cantidades tan bajas que las técnicas actuales no pudieron detectar su presencia. Para investigar la contribución de la epigenética a la complejidad de DM1, comparamos los perfiles de metilación del ADN en las cuatro islas CpG (CpGi 374, dividida en CTCF1 y CTCF2; CpGi 36; CpGi 43; CpGi 74) que residen en el locus DMPK en distintos tejidos DM1 y en células derivadas de pacientes con distintos fenotipos clínicos. Nuestros resultados mostraron un gradiente de hipermetilación del ADN en la región CTCF1 en muestras de sangre con una edad de inicio decreciente y la hipermetilación de CTCF1 se correlacionó con la gravedad de la enfermedad y el tamaño de expansión de CTG. Los casos hipermetilados mostraron una mayor probabilidad de transmisión materna y la hipermetilación de CTCF1 en el progenitor se asoció con una contracción de la expansión de CTG tras la transmisión generacional. Además, las células derivadas de pacientes DM1 conservaron los patrones de metilación observados en los tejidos. Finalmente, nuestros resultados mostraron un patrón epigenético específico del músculo DM1, con una pérdida de metilación en CpGi 43, una región que contiene un promotor DMPK alternativo, acompañada de una hipermetilación de la región CTCF1 en muestras musculares

Unraveling potential disease modifiers of myotonic dystrophy type 1

Programa de Doctorat en Biomedicina / Tesi realitzada a l'Institut de Recerca Germans Trias i Pujol[spa] La distrofia miotónica tipo 1 (DM1) es un trastorno multisistémico, progresivo y no tratable con una expansión CTG en el gen DMPK que es la base de la patología. La compleja naturaleza multisistémica de la DM1 no puede explicarse únicamente por dicha expansión y la identificación de otros modificadores de la enfermedad es clave para comprender la patología completa de la DM1. Por lo tanto, el objetivo principal de este proyecto de doctorado ha sido investigar los posibles modificadores de la enfermedad y estudiar su relación con el fenotipo clínico de la DM1. Concretamente, la Tesis se ha centrado en el estudio de cinco modificadores potenciales de la enfermedad a nivel genético (repeticiones con variantes), transcripcional (transcripción antisentido), proteico (RAN translation), epigenético (metilación del ADN) y de ARNs no codificantes (miRNAs). Durante mucho tiempo se pensó que la expansión de CTGs era una secuencia ininterrumpida, pero evidencias crecientes mostraron la existencia de variaciones dentro de las repeticiones CTG, aunque su influencia en la patología DM1 aún no está clara. Para dilucidar la relación de estas variantes con el fenotipo clínico de DM1, se examinó una cohorte española de 49 pacientes con DM1, donde se identificó una familia portadora de patrones complejos de repeticiones variantes de CCG. Los estudios clínicos mostraron que estos individuos portadores de variantes repetidas presentaron características clínicas atípicas, lo que dificultaba el diagnóstico de DM1, manifestaron la enfermedad a una edad tardía, pero con síntomas clínicos más graves, relacionada con el envejecimiento. Otro modificador potencial de la enfermedad es el mecanismo de transcripción bidireccional, en el que la transcripción se produce tanto en dirección sentido como antisentido (AS) en el locus del gen. Además, se ha descubierto que la traducción no ATG (RAN) asociada a la repetición, que se origina en la cadena antisentido, produce proteínas de poliglutamina potencialmente tóxicas. Para estudiar la influencia de la transcripción de DM1-AS y la traducción de RAN en la patología de DM1, analizamos tres cultivos celulares primarios de diez pacientes con DM1. En conjunto, los pacientes con DM1 tenían niveles más bajos de tránscritos de DM1-AS en comparación con los controles, que se presentaban como un grupo heterogéneo con y sin la inclusión de la repetición expandida. La traducción no ATG (RAN translation) no estaba presente en las células DM1 derivadas de pacientes, o en cantidades tan bajas que las técnicas actuales no pudieron detectar su presencia. Para investigar la contribución de la epigenética a la complejidad de DM1, comparamos los perfiles de metilación del ADN en las cuatro islas CpG (CpGi 374, dividida en CTCF1 y CTCF2; CpGi 36; CpGi 43; CpGi 74) que residen en el locus DMPK en distintos tejidos DM1 y en células derivadas de pacientes con distintos fenotipos clínicos. Nuestros resultados mostraron un gradiente de hipermetilación del ADN en la región CTCF1 en muestras de sangre con una edad de inicio decreciente y la hipermetilación de CTCF1 se correlacionó con la gravedad de la enfermedad y el tamaño de expansión de CTG. Los casos hipermetilados mostraron una mayor probabilidad de transmisión materna y la hipermetilación de CTCF1 en el progenitor se asoció con una contracción de la expansión de CTG tras la transmisión generacional. Además, las células derivadas de pacientes DM1 conservaron los patrones de metilación observados en los tejidos. Finalmente, nuestros resultados mostraron un patrón epigenético específico del músculo DM1, con una pérdida de metilación en CpGi 43, una región que contiene un promotor DMPK alternativo, acompañada de una hipermetilación de la región CTCF1 en muestras musculares

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