Molecular analysis of myotonic dystrophy type 1 patients with an unusual molecular diagnosis

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterised by multiple tissue involvement and caused by an expansion of a (CTG)n repeat within the 3’-UTR of the DMPK gene (19q13.3). Normal individuals contain between 5 and 35 CTG repeats, whereas the repeats in DM1 patients expand in the range of 50 to several thousands. Longer alleles are very unstable and generally always increase in size when transmitted from parent to child, explaining the phenomenon of anticipation defined by earlier age of onset and an increase in the severity of the symptoms. Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous, hereditary motor and sensory neuropathy of the peripheral nervous system. To date, 30 different loci have been mapped and mutations have been identified in more than 20 different genes. The DM1+CMT++ family is a very unusual three generation family in which all patients co-segregate both DM1 and CMT (LOD score = 7.03). It was postulated that either a single or two closely linked mutations near the APOC2 marker must be the cause of DM1 and CMT. Southern blot analysis of restriction digested genomic DNA revealed a fragment equivalent to a small CTG expansion (~200-400) at the DM1 locus, but an expanded allele could not be amplified by PCR. We postulated that the expanded repeats may have predisposed the repeat tract and the flanking regions to further DNA instability, leading to a secondary deletion, insertion and/or rearrangement. These novel mutations might modify the expression of DMPK and/or nearby genes explaining the unusual clinical presentation. To identify the lesion in the DM1+CMT++ family, a variety of molecular approaches was performed. The molecular lesion identified was an insertionof a GC rich region within the CTG repeats. The allele was comprised of a variable number of CTGs at the 5'-end followed by (GGC)3 G (CCG)20 (CCGCTG)14 (CTG)35. Analysis of single molecule separated alleles revealed 3 that the interrupted 3'-end of the array was stable, while the CTG repeats at the 5'-end were unstable. Postulated mechanisms to explain the DM1 and CMT symptoms in the family were: a novel RNA gain-of-function, and/or a novel effect on the downstream genes. Finding an imperfect CTG repeat allele in the DM1+CMT++ family led us to suggest that imperfect CTG repeat alleles may not be unique events and other DM1 patients may also contain similar alleles. To investigate this DNA samples from 14 DM1 patients with an unusual molecular diagnosis were analysed. The majority of these patients presented with an imperfect CTG repeat allele containing CCGCTG hexamers and/or CCG repeats. Five patients contained two or three higher order repeats containing between 18 and 30 bp such as ((CTG)5 (CCG)5), ((CTG)2 (CCGCTG)4) and ((CTG)5 (CCG)2 (CCGCTG)). These findings further suggest that imperfect CTG repeat alleles might not be as rare as was previously believed. The results of this project point out the importance of performing a more detailed molecular characterisation of the DM1 patients, which could lead to the provision of more accurate prognoses and the development of effective therapies

Epileptiform Activity and Cognitive Deficits in SNAP-25+/− Mice are Normalized by Antiepileptic Drugs

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25+/−) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25+/− mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drug

Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity

Deciphering the contribution of genetic instability in somatic cells is critical to our understanding of many human disorders. Myotonic dystrophy type 1 (DM1) is one such disorder that is caused by the expansion of a CTG repeat that shows extremely high levels of somatic instability. This somatic instability has compromised attempts to measure intergenerational repeat dynamics and infer genotype–phenotype relationships. Using single-molecule PCR, we have characterized more than 17 000 de novo somatic mutations from a large cohort of DM1 patients. These data reveal that the estimated progenitor allele length is the major modifier of age of onset. We find no evidence for a threshold above which repeat length does not contribute toward age at onset, suggesting pathogenesis is not constrained to a simple molecular switch such as nuclear retention of the DMPK transcript or haploinsufficiency for DMPK and/or SIX5. Importantly, we also show that age at onset is further modified by the level of somatic instability; patients in whom the repeat expands more rapidly, develop the symptoms earlier. These data establish a primary role for somatic instability in DM1 severity, further highlighting it as a therapeutic target. In addition, we show that the level of instability is highly heritable, implying a role for individual-specific trans-acting genetic modifiers. Identifying these trans-acting genetic modifiers will facilitate the formulation of novel therapies that curtail the accumulation of somatic expansions and may provide clues to the role these factors play in the development of cancer, aging and inherited disease in the general population

Produção de matéria seca de forragem e acúmulo de nutrientes em pastagem anual de inverno tratada com esterco líquido de suínos Forage dry matter production and nutrient uptake of a hibernal pasture under application of pig slurry

O Esterco líquido de suínos (ELS) pode ser usado como fertilizante orgânico, mas seu uso incorreto pode contaminar o solo e os mananciais de água. O objetivo deste trabalho foi avaliar o efeito da adição de nutrientes ao solo, através do ELS, sobre a produção de matéria seca (MS) e o acúmulo de nutrientes de uma pastagem de aveia branca+azevém (Avena sativa + Lolium multiflorum). Um experimento foi realizado de 2004 a 2006, no campo experimental da UTFPR, em Pato Branco, Paraná, Brasil. O solo era um Latossolo Vermelho distroférrico. Diferentes doses (0, 20, 40, 80 e 120m³ ha-1) de ELS foram aplicadas na pastagem, aos 20 e 61 dias após a emergência (DAE) da pastagem, em 2004, e aos 30 e 67DAE, em 2005. Outras duas aplicações foram realizadas nas culturas de verão, milho em 2004 e soja em 2005, respectivamente. A maior produção de MS foi obtida com a dose de 120m³ ha-1 de ELS, tanto no primeiro, quanto no segundo ano. A absorção de nutrientes pelas plantas respondeu de forma linear à aplicação de esterco líquido de suínos.<br>The pig slurry can be used as an organic fertilizer but its improper use can contaminate water and soil. The objective of this study was to evaluate the effect of nutrient addition to the soil on pasture oat + ryegrass (Avena sativa + Lolium multiflorum) dry matter production and nutrient uptake. One experiment was carried out from 2004 to 2006, installed in the experimental farm of UTFPR in Pato Branco, Paraná State, Brazil. The soil was an Oxisol (Latossolo Vermelho distroférrico, Brazil systems). Different rates (0, 20, 40, 80 and 120m³ ha-1) of pig slurry were applied in the pasture. The pig slurry applications were performed 20 and 61 days after emergence (DAE) of pasture, in 2004, and 30 and 67DAE of pasture in 2005. Another two applications were performed in summer crops, corn in 2004 and soybean in 2005. In both years the rate 120m³ ha-1 of pig slurry resulted in the highest dry matter production. The nutrient uptake by plants responded in a linear manner to pig slurry application

Epileptiform Activity and Cognitive Deficits in SNAP-25+/- Mice are Normalized by Antiepileptic Drugs

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a protein that participates in the regulation of synaptic vesicle exocytosis through the formation of the soluble NSF attachment protein receptor complex and modulates voltage-gated calcium channels activity. The Snap25 gene has been associated with schizophrenia, attention deficit hyperactivity disorder, and bipolar disorder, and lower levels of SNAP-25 have been described in patients with schizophrenia. We used SNAP-25 heterozygous (SNAP-25(+/-)) mice to investigate at which extent the reduction of the protein levels affects neuronal network function and mouse behavior. As interactions of genotype with the specific laboratory conditions may impact behavioral results, the study was performed through a multilaboratory study in which behavioral tests were replicated in at least 2 of 3 distinct European laboratories. Reductions of SNAP-25 levels were associated with a moderate hyperactivity, which disappeared in the adult animals, and with impaired associative learning and memory. Electroencephalographic recordings revealed the occurrence of frequent spikes, suggesting a diffuse network hyperexcitability. Consistently, SNAP-25(+/-) mice displayed higher susceptibility to kainate-induced seizures, paralleled by degeneration of hilar neurons. Notably, both EEG profile and cognitive defects were improved by antiepileptic drugs. These results indicate that reduction of SNAP-25 expression is associated to generation of epileptiform discharges and cognitive dysfunctions, which can be effectively treated by antiepileptic drugs

Variant CCG and GGC repeats within the CTG expansion dramatically modify mutational dynamics and likely contribute toward unusual symptoms in some myotonic dystrophy type 1 patients

Myotonic dystrophy type 1 (DM1) is one of the most variable inherited human disorders. It is characterized by the involvement of multiple tissues and is caused by the expansion of a highly unstable CTG repeat. Variation in disease severity is partially accounted for by the number of CTG repeats inherited. However, the basis of the variable tissue-specific symptoms is unknown. We have determined that an unusual Dutch family co-segregating DM1, Charcot-Marie-Tooth neuropathy, encephalopathic attacks and early hearing loss, carries a complex variant repeat at the DM1 locus. The mutation comprises an expanded CTG tract at the 5'-end and a complex array of CTG repeats interspersed with multiple GGC and CCG repeats at the 3'-end. The complex variant repeat tract at the 3'-end of the array is relatively stable in both blood DNA and the maternal germ line, whilst the 5'-CTG tract remains genetically unstable and prone to expansion. Surprisingly though, even the pure 5'-CTG tract is more stable in blood DNA and the maternal germ line than archetypal DM1 alleles of a similar size. Complex variant repeats were also identified at the 3'-end of the CTG array of [~]3-4% of unrelated DM1 patients. The observed polarity and the stabilizing effect of the variant repeats implicate a cis-acting modifier of mutational dynamics in the 3'-flanking DNA. The presence of such variant repeats very likely contributes toward the unusual symptoms in the Dutch family and additional symptomatic variation in DM1 via affects on both RNA toxicity and somatic instability

El tiempo que vivimos : COVID 19 y su impacto en nuestras sociedades

Desde fines de diciembre de 2019, pero, dramáticamente desde febrero de 2020, el mundo ha comenzado a transcurrir un escenario de incertidumbre total, generado por la posibilidad de contagio y la reproducción exponencial del COVID-19.12 Esta Pandemia ha puesto en el tapete dos fenómenos que parecen dialécticos y contradictorios entre sí. Por un lado, la globalización- proceso del cual esta pandemia es producto- y, por el otro, el cierre de fronteras y la aparición de un estado que debe mostrar autoridad para administrar el riesgo. En estos términos, la pandemia vino a ratificar uno de los desafíos que enfrentan nuestros sociedades a inicios del siglo XXI, las tensiones entre lo global y lo estatal, pero también entre la universalización de la democracia y su realización, así como el imperativo de una necesaria colaboración a nivel subnacional, estatal, regional, internacional y global ante temas que afectan a nuestras sociedades sin reconocimiento de niveles de desarrollo, creencias o condición sociocultural.Fil: Colacrai, Miryam. Universidad Nacional de Rosario; Argentina.Fil: Álvarez, Silvia T. Universidad Nacional del Sur; Argentina

Efficacy and safety of the CVnCoV SARS-CoV-2 mRNA vaccine candidate in ten countries in Europe and Latin America (HERALD): a randomised, observer-blinded, placebo-controlled, phase 2b/3 trial

Background: Additional safe and efficacious vaccines are needed to control the COVID-19 pandemic. We aimed to analyse the efficacy and safety of the CVnCoV SARS-CoV-2 mRNA vaccine candidate. Methods: HERALD is a randomised, observer-blinded, placebo-controlled, phase 2b/3 clinical trial conducted in 47 centres in ten countries in Europe and Latin America. By use of an interactive web response system and stratification by country and age group (18–60 years and ≥61 years), adults with no history of virologically confirmed COVID-19 were randomly assigned (1:1) to receive intramuscularly either two 0·6 mL doses of CVnCoV containing 12 μg of mRNA or two 0·6 mL doses of 0·9% NaCl (placebo) on days 1 and 29. The primary efficacy endpoint was the occurrence of a first episode of virologically confirmed symptomatic COVID-19 of any severity and caused by any strain from 15 days after the second dose. For the primary endpoint, the trial was considered successful if the lower limit of the CI was greater than 30%. Key secondary endpoints were the occurrence of a first episode of virologically confirmed moderate-to-severe COVID-19, severe COVID-19, and COVID-19 of any severity by age group. Primary safety outcomes were solicited local and systemic adverse events within 7 days after each dose and unsolicited adverse events within 28 days after each dose in phase 2b participants, and serious adverse events and adverse events of special interest up to 1 year after the second dose in phase 2b and phase 3 participants. Here, we report data up to June 18, 2021. The study is registered at ClinicalTrials.gov, NCT04652102, and EudraCT, 2020–003998–22, and is ongoing. Findings: Between Dec 11, 2020, and April 12, 2021, 39 680 participants were enrolled and randomly assigned to receive either CVnCoV (n=19 846) or placebo (n=19 834), of whom 19 783 received at least one dose of CVnCoV and 19 746 received at least one dose of placebo. After a mean observation period of 48·2 days (SE 0·2), 83 cases of COVID-19 occurred in the CVnCoV group (n=12 851) in 1735·29 person-years and 145 cases occurred in the placebo group (n=12 211) in 1569·87 person-years, resulting in an overall vaccine efficacy against symptomatic COVID-19 of 48·2% (95·826% CI 31·0–61·4; p=0·016). Vaccine efficacy against moderate-to-severe COVID-19 was 70·7% (95% CI 42·5–86·1; CVnCoV 12 cases in 1735·29 person-years, placebo 37 cases in 1569·87 person-years). In participants aged 18–60 years, vaccine efficacy against symptomatic disease was 52·5% (95% CI 36·2–64·8; CVnCoV 71 cases in 1591·47 person-years, placebo, 136 cases in 1449·23 person-years). Too few cases occurred in participants aged 61 years or older (CVnCoV 12, placebo nine) to allow meaningful assessment of vaccine efficacy. Solicited adverse events, which were mostly systemic, were more common in CVnCoV recipients (1933 [96·5%] of 2003) than in placebo recipients (1344 [67·9%] of 1978), with 542 (27·1%) CVnCoV recipients and 61 (3·1%) placebo recipients reporting grade 3 solicited adverse events. The most frequently reported local reaction after any dose in the CVnCoV group was injection-site pain (1678 [83·6%] of 2007), with 22 grade 3 reactions, and the most frequently reported systematic reactions were fatigue (1603 [80·0%] of 2003) and headache (1541 [76·9%] of 2003). 82 (0·4%) of 19 783 CVnCoV recipients reported 100 serious adverse events and 66 (0·3%) of 19 746 placebo recipients reported 76 serious adverse events. Eight serious adverse events in five CVnCoV recipients and two serious adverse events in two placebo recipients were considered vaccination-related. None of the fatal serious adverse events reported (eight in the CVnCoV group and six in the placebo group) were considered to be related to study vaccination. Adverse events of special interest were reported for 38 (0·2%) participants in the CVnCoV group and 31 (0·2%) participants in the placebo group. These events were considered to be related to the trial vaccine for 14 (<0·1%) participants in the CVnCoV group and for five (<0·1%) participants in the placebo group. Interpretation: CVnCoV was efficacious in the prevention of COVID-19 of any severity and had an acceptable safety profile. Taking into account the changing environment, including the emergence of SARS-CoV-2 variants, and timelines for further development, the decision has been made to cease activities on the CVnCoV candidate and to focus efforts on the development of next-generation vaccine candidates. Funding: German Federal Ministry of Education and Research and CureVac