Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity.

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

Phosphorylated neurofilament heavy chain in cerebrospinal fluid and plasma as a Nusinersen treatment response marker in childhood-onset SMA individuals from Serbia.

Introduction: Biomarkers capable of reflecting disease onset and short- and long-term therapeutic effects in individuals with spinal muscular atrophy (SMA) are still an unmet need and phosphorylated neurofilament heavy chain (pNF-H) holds significant promise. Methods: We conducted a longitudinal prospective study to evaluate pNF-H levels in the cerebrospinal fluid (CSF) and plasma of 29 individuals with childhood-onset SMA treated with Nuinersen (SMA type 1: n = 6, 2: n = 17, 3: n = 6). pNF-H levels before and during treatment were compared with the levels of controls (n = 22), patients with Duchenne muscular dystrophy (n = 17), myotonic dystrophy type 1 (n = 11), untreated SMA individuals with chronic type 3 disease (n = 8), and children with presymptomatic SMA (n = 3). Results: SMA type 1 showed the highest mean CSF pNF-H levels before treatment initiation. All Nusinersen-treated individuals (types 1, 2, and 3) showed significantly elevated mean baseline CSF pNF-H compared to controls, which inversely correlated with age at disease onset, age at first dose, disease duration and the initial CHOP INTEND result (SMA type 1 and 2). During 22 months of treatment, CSF pNF-H levels declined during loading doses, stabilizing at reduced levels from the initial maintenance dose in all individuals. Baseline plasma pNF-H levels in type 1 and 2 SMA were significantly increased compared to other cohorts and decreased notably in type 1 after 2 months of treatment and type 2 after 14 months. Conversely, SMA type 3, characterized by lower baseline pNF-H levels, did not show significant fluctuations in plasma pNF-H levels after 14 months of treatment. Conclusion: Our findings suggest that CSF pNF-H levels in untreated SMA individuals are significantly higher than in controls and that monitoring of CSF pNF-H levels may serve as an indicator of rapid short-term treatment response in childhood-onset SMA individuals, irrespective of the subtype of the disease, while also suggesting its potential for assessing long-term suppression of neurodegeneration. Plasma pNF-H may serve as an appropriate outcome measure for disease progression and/or response to treatment in types 1 and 2 but not in type 3. Presymptomatic infants with SMA may show elevated pNF-H levels, confirming early neuronal degeneration.

Main features and disease outcome of congenital myotonic dystrophy - experience from a single tertiary center.

Congenital myotonic dystrophy type 1 (CDM1) is a rare neuromuscular disease. The aim of our study was to evaluate clinical variability of CDM1 and factors that may influence survival in CDM1. Research included 24 pediatric patients with CDM1. Most of our patients had some form of hypoxic ischemic encephalopathy (HIE) (74 %), from mild to severe. Prolonged and complicated deliveries (75 %), high percentage of children resuscitated at birth (57 %) and respiratory insufficiency (46 %) with consequent hypoxia were the main reasons that could explain high percentage of HIE. Therapeutic hypothermia was applied in three children with poor outcome. Median survival of all CDM1 was 14.2 ± 1.5 years. Six patients had a fatal outcome (25 %). Their mean age of death was 3.0 ± 2.8 years. Poor prognostic factors for the survival of our CDM1 patients were: preterm delivery, resuscitation at birth, severe HIE, hypothermia treatment and permanent mechanical ventilation. Respiratory insufficiency was the main life-threatening factor. Our data clearly indicates the need to develop natural history studies in CDM1 in order to enhance the standards of care and to develop clinical trials investigating causative therapies in pediatric patients with CDM1.

Association between Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA-4) Locus and Early-Onset Anti-acetylcholine Receptor-Positive Myasthenia Gravis in Serbian Patients.

Genome-wide association studies (GWAS) have provided strong evidence that early- and late-onset MG have different genetic backgrounds. Recent in silico analysis based on GWAS results revealed rs231735 and rs231770 variants within CTLA-4 locus as possible MG causative genetic factors. We aimed to explore the association of rs231735 and rs231770 with MG in a representative cohort of Serbian patients. We conducted an age-, sex-, and ethnicity-matched case-control study. Using TaqMan allele discrimination assays, the frequency of rs231735 and rs231770 genetic variants was examined in 447 AChR-MG patients and 447 matched controls. There was no significant association of rs231735 and rs231770 with the entire MG cohort (P > 0.05). Nevertheless, when stratifying patients into early-onset (n = 183) and late-onset MG (n = 264), we found early-onset patients had a significantly lower frequency of the rs231735 allele T compared to controls (OR = 0.734, 95% CI = 0.575-0.938, p10e6 permutation < 0.05), and rs231735 genotype TT and rs231770 genotype TT had a protective effect on early-onset MG (OR = 0.548, 95% CI = 0.339-0.888, and OR = 0.563, 95% CI = 0.314-1.011, p10e6 permutation < 0.05). Consequently, we found that individuals with the rs231735-rs231770 haplotype GC had a higher risk for developing early-onset MG (OR = 1.360, P = 0.027, p10e6 permutation < 0.05). Our results suggest that CTLA-4 rs231735 and rs231770 may be risk factors only for patients with early-onset MG in Serbian population.

Single-molecule RNA sizing enables quantitative analysis of alternative transcription termination.

Transcription, a critical process in molecular biology, has found many applications in RNA synthesis, including mRNA vaccines and RNA therapeutics. However, current RNA characterization technologies suffer from amplification and enzymatic biases that lead to loss of native information. Here, we introduce a strategy to quantitatively study both transcription and RNA polymerase behaviour by sizing RNA with RNA nanotechnology and nanopores. To begin, we utilize T7 RNA polymerase to transcribe linear DNA lacking termination sequences. Surprisingly, we discover alternative transcription termination in the origin of replication sequence. Next, we employ circular DNA without transcription terminators to perform rolling circle transcription. This allows us to gain valuable insights into the processivity and transcription behaviour of RNA polymerase at the single-molecule level. Our work demonstrates how RNA nanotechnology and nanopores may be used in tandem for the direct and quantitative analysis of RNA transcripts. This methodology provides a promising pathway for accurate RNA structural mapping by enabling the study of full-length RNA transcripts at the single-molecule level.