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

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

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

Effectiveness and Safety of Imipenem-Clavulanate Added to an Optimized Background Regimen (OBR) Versus OBR Control Regimens in the Treatment of Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis

SCOPUS: le.jinfo:eu-repo/semantics/publishe

Comparison of effectiveness and safety of imipenem/clavulanate-versus meropenem/clavulanate-containing regimens in the treatment of MDR- and XDR-TB

No large study to date has ever evaluated the effectiveness, safety and tolerability of imipenem/clavulanate versus meropenem/clavulanate to treat multidrug- and extensively drug-resistant tuberculosis (MDR- and XDR-TB). The aim of this observational study was to compare the therapeutic contribution of imipenem/clavulanate versus meropenem/clavulanate added to background regimens to treat MDR- and XDR-TB cases. 84 patients treated with imipenem/clavulanate-containing regimens showed a similar median number of antibiotic resistances (8 versus 8) but more fluoroquinolone resistance (79.0% versus 48.9%, p<0.0001) and higher XDR-TB prevalence (67.9% versus 49.0%, p=0.01) in comparison with 96 patients exposed to meropenem/clavulanate-containing regimens. Patients were treated with imipenem/clavulanate- and meropenem/clavulanate-containing regimens for a median (interquartile range) of 187 (60-428) versus 85 (49-156) days, respectively. Statistically significant differences were observed on sputum smear and culture conversion rates (79.7% versus 94.8%, p=0.02 and 71.9% versus 94.8%, p<0.0001, respectively) and on success rates (59.7% versus 77.5%, p=0.03). Adverse events to imipenem/clavulanate and meropenem/clavulanate were reported in 5.4% and 6.5% of cases only. Our study suggests that meropenem/clavulanate is more effective than imipenem/clavulanate in treating MDR/XDR-TB patients