Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array

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mRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies

To gain insight on dystrophin (DMD) gene transcription dynamics and spatial localization, we assayed the DMD mRNA amount and defined its compartmentalization in myoblasts, myotubes, and skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients. Using droplet digital PCR, Real-time PCR, and RNAscope in situ hybridization, we showed that the DMD transcript amount is extremely reduced in both DMD patients' cells and muscle biopsies and that mutation-related differences occur. We also found that, compared to controls, DMD transcript is dramatically reduced in the cytoplasm, as up to 90% of it is localized in nuclei, preferentially at the perinuclear region. Using RNA/protein colocalization experiments, we showed that about 40% of nuclear DMD mRNA is localized in the nucleoli in both control and DMD myogenic cells. Our results clearly show that mutant DMD mRNA quantity is strongly reduced in the patients' myogenic cells and muscle biopsies. Furthermore, mutant DMD mRNA compartmentalization is spatially unbalanced due to a shift in its localization towards the nuclei. This abnormal transcript repartition contributes to the poor abundance and availability of the dystrophin messenger in cytoplasm. This novel finding also has important repercussions for RNA-targeted therapies

Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2 -O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules

Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2  -O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules

Tumor Necrosis Factor Receptor SF10A (TNFRSF10A) SNPs Correlate With Corticosteroid Response in Duchenne Muscular Dystrophy

Background Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment. Methods and Findings We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in theTNFRSF10Agene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N= 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results. Conclusion We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker

A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures

Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR

Dystrophin regulates peripheral circadian SRF signalling

Dystrophin is a sarcolemmal protein essential for muscle contraction and maintenance, absence of which leads to the devastating muscle wasting disease Duchenne muscular dystrophy (DMD)[1, 2]. Dystrophin has an actin-binding domain [3–5], which specifically binds and stabilises filamentous (F)-actin[6], an integral component of the RhoA-actin-serum response factor (SRF)-pathway[7]. The RhoA-actin-SRF-pathway plays an essential role in circadian signalling whereby the hypothalamic suprachiasmatic nucleus, transmits systemic cues to peripheral tissues, activating SRF and transcription of clock target genes[8, 9]. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised that dystrophin loss causes circadian deficits. Here we show for the first time alterations in the RhoA-actin-SRF-signalling-pathway, in both dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios and nuclear MRTF, dysregulation of core clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from DMD patients harbouring an array of mutations. Further, disrupted circadian locomotor behaviour was observed in dystrophic mice indicative of disrupted SCN signalling, and indeed dystrophin protein was absent in the SCN of dystrophic animals. Dystrophin is thus a critically important component of the RhoA-actin-SRF-pathway and a novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation

Spatial RNA-mapping reveals the unique patterns of expression and localisation of dystrophin isoforms in the human adult normal brain to improve understanding of brain involvement in muscular dystrophy.

ABSTRACT (English) Background. Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease due to pathogenic variants in the DMD gene causing the absence of dystrophin protein. Dystrophin function is not only limited to muscle but is also relevant in several brain circuits and during brain development. Pathogenic variations occurring in the 3’ of the gene, and thus disrupting both full-length (Dp427) and short 3’ isoforms as Dp140 and Dp71, are known to impact on the neurocognitive and neurobehavioral phenotypes. Aim. We intended to assess the topography of DMD isoforms in relationship to the known brain comorbidities in human adult normal brain areas. Methods. All dystrophin isoforms were firstly analysed in 24 adult human brain areas using TaqMan Real-time PCR on TissueScan cDNA array. The areas showing the highest levels of DMD isoforms were prioritised for Basescope RNA in situ hybridisation analysis with probes designed to detect each selected DMD isoform. This latter technique was used in samples from 8 human adult normal male donor brains obtained from the Edinburgh Brain-Tissue Bank. We analysed formalin fixed paraffin embedded (FFPE) tissues from the cerebellum, vermis, dentate nucleus, paracentral gyrus, corpus callosum, cingulate gyrus, hippocampus, amygdala, and substantia nigra. Results. The most represented isoforms in studied brain areas were Dp427b, Dp427m, Dp427p2, Dp140, Dp71 and Dp40, based on a preliminary Real-time PCR screening. BaseScope showed that these isoforms were transcribed in all brain areas with a different abundance between donors and also between brain areas. Three main DMD isoform clusters were identified, including i) low represented, as Dp427m and Dp427p2, ii) medium represented, as Dp140, Dp71, and Dp40, which are mainly localised in the cerebellum and amygdala, and iii) highly represented, as Dp427b which is enriched in the hippocampus. The corpus callosum, substantia nigra, nucleus dentate, and vermis were the areas with the lowest isoform representation. We could also recognize isoforms with cell-layer specific localisation in the cerebellum, vermis, paracentral gyrus, and hippocampus. Conclusion. We defined spatial and topographic representation of DMD isoforms in human normal adult brain. The enrichment in Dp427b in some areas belonging to the limbic system (hippocampus, amygdala, and cingulate gyrus), Dp140 in the cerebellum, and Dp71 in the hippocampus, amygdala, and cerebellum are well aligned with the brain phenotypes observed in individuals affected by DMD. The identification of a spatial RNA-mapping of dystrophin isoforms into human normal adult brain may help associate some clinical brain comorbidities with specific molecular mechanisms in DMD/BMD and in other conditions with associated neurocognitive and neurobehavioral phenotypes.Introduzione. La distrofia muscolare di Duchenne (DMD) è una malattia neuromuscolare legata al cromosoma X, dovuta a mutazioni nel gene distrofina (DMD) che causano l'assenza della proteina distrofina. La distrofina svolge le sue funzioni non solo a livello muscolare, ma la sua presenza è rilevante anche per la regolazione di diversi circuiti cerebrali e durante lo sviluppo del sistema nervoso centrale. È stato riportato che mutazioni localizzate nella regione distale del gene DMD, alterando l’espressione sia delle isoforme lunghe (Dp427) sia di quelle corte come la Dp140 e la Dp71, influenzano il fenotipo neurocognitivo e neurocomportamentale dei pazienti DMD. Scopo. L’obiettivo di questo lavoro è stato quello di valutare l’espressione e la localizzazione delle isoforme del gene DMD in diverse aree del cervello umano adulto di controllo. Metodi. Tutte le isoforme DMD sono state inizialmente analizzate in 24 principali aree del cervello umano adulto, rappresentate nel TissueScan cDNA array, utilizzando la TaqMan Real-time PCR. Le aree con i più alti livelli di espressione delle isoforme DMD sono state prioritizzate per la successiva analisi di ibridazione in situ Basescope, usando sonde specifiche per il riconoscimento delle singole isoforme DMD. L’analisi Basescope è stata effettuata su diverse aree di cervello provenienti da 8 donatori maschi adulti, ottenute dalla biobanca di Edimburgo. In particolare, abbiamo analizzato tessuti fissati in formalina e inclusi in paraffina (FFPE) di: cervelletto, vermis, nucleo dentato, giro paracentrale, corpo calloso, giro cingolato, ippocampo, amigdala e substantia nigra. Risultati. Uno screening preliminare tramite Real-time PCR ha dimostrato che le isoforme più rappresentate nelle aree cerebrali studiate sono la Dp427b, Dp427m, Dp427p2, Dp140, Dp71 e Dp40. L’analisi BaseScope ha poi mostrato che queste isoforme sono trascritte in tutte le aree cerebrali testate, in quantità diversa sia tra i donatori sia tra le diverse regioni. Sono stati identificati tre principali clusters di espressione: i) livello basso, rappresentato dalle isoforme Dp427m e Dp427p2; ii) livello medio, comprendente le isoforme Dp140, Dp71 e Dp40, principalmente localizzate nel cervelletto e nell'amigdala; iii) livello alto, riferito alla Dp427b, isoforma maggiormente espressa nell'ippocampo. Il corpo calloso, la substantia nigra, il nucleo dentato e il vermis sono risultate le aree con la più bassa rappresentazione delle isoforme DMD. La valutazione della localizzazione dei trascritti DMD in specifiche sub-aree presenti all’interno di alcune regioni ha evidenziato la presenza di uno specifico pattern di localizzazione nelle sub-aree del cervelletto, vermis, giro paracentrale e ippocampo. Conclusioni. In questo lavoro abbiamo definito l’espressione e la localizzazione delle isoforme DMD nel cervello adulto umano di controllo. L'arricchimento della Dp427b in alcune aree appartenenti al sistema limbico (ippocampo, amigdala e giro cingolato), della Dp140 nel cervelletto e della Dp71 nell'ippocampo, amigdala e cervelletto, sono in linea con le comorbidità neurologiche osservate negli individui affetti da DMD. L'identificazione di una mappa di espressione e localizzazione dei trascritti delle isoforme della distrofina nel cervello umano adulto di controllo potrà contribuire ad associare alcuni fenotipi neurologici con specifici meccanismi molecolari non solo nella DMD ma anche in altre patologie con presenza di problematiche neurocognitive e neurocomportamentali

Urinary Stem Cells as Tools to Study Genetic Disease: Overview of the Literature

Urine specimens represent a novel and non-invasive approach to isolate patient-specific stem cells by easy and low-cost procedures, replacing the traditional sources (muscle/skin biopsy/adipose tissue) obtained with invasive and time-consuming methods. Urine-derived stem cells (USCs) can be used in a broad field of applications, such as regenerative medicine, cell therapy, diagnostic testing, disease modelling and drug screening. USCs are a good source of cells for generating induced pluripotent stem cells (iPSCs) and importantly, they can also be directly converted into specific cell lines. In this review, we show the features of USCs and their use as a promising in vitro model to study genetic diseases

Oligomeric formylpeptide activity on human neutrophils

A series of oligomeric formylpeptides were synthesized by cross-linking the prototype fMLP using a Lys residue. They were then investigated for their ability to stimulate chemotaxis, superoxide anion production, and lytic enzyme release in human neutrophils. Although active in stimulating the different receptor isoforms, leading to the different biological responses, these analogues showed a lesser potency and affinity than the standard peptide. On the basis of the results reported here, we can hypothesise that: (i) the increased bulk of these molecules seems to hinder their correct allocation into the receptor pocket, thereby hindering favourable receptor interaction; and that: (ii) fMLP space positions do not seem to allow the ligand to increase biological responses