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

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

Long-term outcomes of the global tuberculosis and COVID-19 co-infection cohort

Background: Longitudinal cohort data of patients with tuberculosis (TB) and coronavirus disease 2019 (COVID-19) are lacking. In our global study, we describe long-term outcomes of patients affected by TB and COVID-19. Methods: We collected data from 174 centres in 31 countries on all patients affected by COVID-19 and TB between 1 March 2020 and 30 September 2022. Patients were followed-up until cure, death or end of cohort time. All patients had TB and COVID-19; for analysis purposes, deaths were attributed to TB, COVID-19 or both. Survival analysis was performed using Cox proportional risk-regression models, and the log-rank test was used to compare survival and mortality attributed to TB, COVID-19 or both. Results: Overall, 788 patients with COVID-19 and TB (active or sequelae) were recruited from 31 countries, and 10.8% (n=85) died during the observation period. Survival was significantly lower among patients whose death was attributed to TB and COVID-19 versus those dying because of either TB or COVID-19 alone (p<0.001). Significant adjusted risk factors for TB mortality were higher age (hazard ratio (HR) 1.05, 95% CI 1.03-1.07), HIV infection (HR 2.29, 95% CI 1.02-5.16) and invasive ventilation (HR 4.28, 95% CI 2.34-7.83). For COVID-19 mortality, the adjusted risks were higher age (HR 1.03, 95% CI 1.02-1.04), male sex (HR 2.21, 95% CI 1.24-3.91), oxygen requirement (HR 7.93, 95% CI 3.44-18.26) and invasive ventilation (HR 2.19, 95% CI 1.36-3.53). Conclusions: In our global cohort, death was the outcome in >10% of patients with TB and COVID-19. A range of demographic and clinical predictors are associated with adverse outcomes

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Os principais objetivos deste trabalho são apresentar e discutir os benefícios que podem ser alcançados com a aplicação da metodologia Design for Manufacturability and Assembly (DFMA) e propor a sua disseminação na indústria automotiva. O trabalho inicia-se com uma revisão teórico dos conceitos e princípios da metodologia Design for Manufacturability and Assembly (DFMA) e Design for Excellence (DFX), bem como de outras metodologias complementares. Em seguida, o trabalho apresenta o ambiente da indústria automotiva, incluindo o processo de desenvolvimento e implementação de projetos neste segmento. Para verificar a aplicabilidade da metodologia DFMA, o trabalho contou com a análise de dados de produção que comprovou a necessidade de aplicação da metodologia DFMA durante o desenvolvimento de produtos. Na seqüência são apresentados os resultados de uma pesquisa realizada com um grupo focal, composto por funcionários da empresa, que serviu para levantar os requisitos necessários para a elaboração de uma ferramenta de análise de DFMA. O mesmo grupo focal contribuiu para identificação dos principais benefícios buscados e principais dificuldades encontradas na aplicação desta metodologia.The main objectives of this study are to outline and discuss the benefits to be achieved through the application of Design for Manufacturability and Assembly (DFMA) methods and to propose the dissemination of the same in the automotive industry. The study begins with a theoretical review of the principles and concepts of the Design for Manufacturability and Assembly (DFMA) and Design for Excellence (DFX) methodologies, as well as that of other related methodologies. Next, the study describes the automotive industry environment, including the process for developing and implementing projects in this sector. In order to verify the applicability of DFMA methods, the study analyzes production data that proves the necessity of applying DFMA methods during product development. Subsequently, the study presents the results of research carried out with a focus group made up of company employees that was used to establish the necessary requirements for the development of DFMA analytical tools. The same focus group helped to identify the principal benefits sought and difficulties likely to be encountered in the application of this methodology. Based on the theoretical review and the research conducted with the focus group, the study concludes with the development and presentation of a tool to aid the dissemination of DFMA methods

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

Abstract 062: N20 Potential And Imaging Biomarkers Of Functional Recovery In Acute Ischemic Stroke Patients And EVT

Introduction PROMISE trial has shown high predictive accuracy of the N20 somatosensory evoked potential (SEP) response on functional recovery in patients with AIS undergoing endovascular thrombectomy (EVT). This secondary study aims to describe the association between the N20 response and imaging biomarkers of ischemic penumbra, infarct volume and collateral flow. Methods Presence and amplitude of the N20 response was recorded before EVT. At baseline, infarct core was automatically calculated establishing a threshold value of ADC6 sec; and hypoperfusion intensity ratio (HIR) as the proportion of ischemic volume that also has a delay in Tmax>10s. Leptomeningeal collaterals were classified according to the Arterial Collateral Grading Scale in CTA as "poor" (absence and 50%, equal to and greater than the contralateral), or according to the American Society of Interventional and Therapeutic Neuroradiology/Society of Interventional Radiology scale when the acquired image was dynamic MR angiography, dichotomized as incomplete collateral fill (poor collateral) (grades 0 to 2) and complete collateral fill (optimal collateral) (grades 3 and 4). Collaterals were not evaluated in the conventional angiography since we did not perform a full study to shorten the time to thrombus access. The adjusted predictive value of N20 for functional recovery was analyzed by logistic regression and compared with imaging variables by using receiver operating characteristic curves. Results From 223 patients studied, 99 patients had multimodal imaging with perfusion studies and N20 assessable recordings at baseline (mean age, 70y; median NIHSS, 18), 63 patients with present (N20+) and 36 with absent N20 response (N20‐). Median infarct core was 12 (0‐25) and 16 (3‐60) cc (p= 0,193), ischemic volume 81 (39‐132) and 111 (54‐188) cc (p=0,082) and the HIR 0,4 (0,2‐0,6) 0,4 (0,3‐0,6) (p=0,572), respectively. N20+ was associated to a better collateral flow (OR 2,5; [1,2‐5,3]; p=0,01). N20+ showed the highest predictive capacity of functional recovery at day 7 compared to imaging variables (Table 1) and increased 15 (4‐103) fold the likelihood of good outcome after adjusting for collateral flow 2.94 (1.10‐8.40), ASPECT score (1,17 (0,74 ‐ 1,87) and infarct core 0.98 (0.95‐1.01). Conclusion N20 SEP response is a powerful biomarker of functional recovery that might surrogate advanced imaging in patients with AIS evaluated for endovascular thrombectomy

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

Myotonic 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

Prognostic Accuracy of N20 Somatosensory Potential in Patients With Acute Ischemic Stroke and Endovascular Thrombectomy

Background Somatosensory evoked potentials may add substantial prognostic value in patients with acute ischemic stroke and contribute to the selection of patients who may benefit from revascularization therapies beyond the accepted therapeutic time windows. We aimed to study the prognostic accuracy of the N20 somatosensory evoked potential component of the ischemic hemisphere in patients with anterior large‐vessel occlusion undergoing endovascular thrombectomy (EVT). Methods Presence and amplitude of the N20 response were recorded before and after EVT. Its adjusted predictive value for functional independence (modified Rankin scale score, ≤2) at day 7 was analyzed by binary logistic regression adjusting by age, mean arterial blood pressure, National Institute of Health Stroke Scale, Alberta Stroke Program Early CT Score, and serum glucose. N20 predictive power was compared with that of clinical and imaging models by using receiver operating characteristics curve analysis. Results A total of 223 consecutive patients were studied (mean age, 70 years; median National Institute of Health Stroke Scale score, 18). Somatosensory evoked potential recordings identified the presence of N20 in 110 (49.3%), absence in 58 (26%), and not assessable in 55 patients due to radiofrequency interferences in the angiography room. Before EVT, N20 predicted functional independence with a sensitivity of 93% (95% CI, 78%–98%) and negative predictive value of 93% (95% CI, 80%–98%). The adjusted odds ratio for functional independence was 9.9 (95% CI, 3.1–44.6). In receiver operating characteristics curve analysis, N20 amplitude showed a higher area under the curve than prehospital or in‐hospital variables, including advanced imaging. Sensitivity increased to 100% (95% CI, 0.85–1) when N20 was present after EVT. Conclusion Somatosensory evoked potential monitoring is a noninvasive and bedside technique that could help eligibility of patients with acute ischemic stroke for EVT and predict functional recovery

An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation

Myotonic dystrophy type 1 (DM1) is a progressive, non-treatable, multi-systemic disorder. To investigate the contribution of epigenetics to the complexity of DM1, we compared DNA methylation profiles of four annotated CpG islands (CpGis) in the DMPK locus and neighbouring genes, in distinct DM1 tissues and derived cells, representing six DM1 subtypes, by bisulphite sequencing. In blood, we found no differences in CpGi 74, 43 and 36 in DNA methylation profile. In contrast, a CTCF1 DNA methylation gradient was found with 100% methylation in congenital cases, 50% in childhood cases and 13% in juvenile cases. CTCF1 methylation correlated to disease severity and CTG expansion size. Notably, 50% of CTCF1 methylated cases showed methylation in the CTCF2 regions. Additionally, methylation was associated with maternal transmission. Interestingly, the evaluation of seven families showed that unmethylated mothers passed on an expansion of the CTG repeat, whereas the methylated mothers transmitted a contraction. The analysis of patient-derived cells showed that DNA methylation profiles were highly preserved, validating their use as faithful DM1 cellular models. Importantly, the comparison of DNA methylation levels of distinct DM1 tissues revealed a novel muscle-specific epigenetic signature with methylation of the CTCF1 region accompanied by demethylation of CpGi 43, a region containing an alternative DMPK promoter, which may decrease the canonical promoter activity. Altogether, our results showed a distinct DNA methylation profile across DM1 tissues and uncovered a novel and dual epigenetic signature in DM1 muscle samples, providing novel insights into the epigenetic changes associated with DM1