Variants in EXOSC9 Disrupt the RNA Exosome and Result in Cerebellar Atrophy with Spinal Motor Neuronopathy.

The exosome is a conserved multi-protein complex that is essential for correct RNA processing. Recessive variants in exosome components EXOSC3, EXOSC8, and RBM7 cause various constellations of pontocerebellar hypoplasia (PCH), spinal muscular atrophy (SMA), and central nervous system demyelination. Here, we report on four unrelated affected individuals with recessive variants in EXOSC9 and the effect of the variants on the function of the RNA exosome in vitro in affected individuals' fibroblasts and skeletal muscle and in vivo in zebrafish. The clinical presentation was severe, early-onset, progressive SMA-like motor neuronopathy, cerebellar atrophy, and in one affected individual, congenital fractures of the long bones. Three affected individuals of different ethnicity carried the homozygous c.41T>C (p.Leu14Pro) variant, whereas one affected individual was compound heterozygous for c.41T>C (p.Leu14Pro) and c.481C>T (p.Arg161∗). We detected reduced EXOSC9 in fibroblasts and skeletal muscle and observed a reduction of the whole multi-subunit exosome complex on blue-native polyacrylamide gel electrophoresis. RNA sequencing of fibroblasts and skeletal muscle detected significant >2-fold changes in genes involved in neuronal development and cerebellar and motor neuron degeneration, demonstrating the widespread effect of the variants. Morpholino oligonucleotide knockdown and CRISPR/Cas9-mediated mutagenesis of exosc9 in zebrafish recapitulated aspects of the human phenotype, as they have in other zebrafish models of exosomal disease. Specifically, portions of the cerebellum and hindbrain were absent, and motor neurons failed to develop and migrate properly. In summary, we show that variants in EXOSC9 result in a neurological syndrome combining cerebellar atrophy and spinal motoneuronopathy, thus expanding the list of human exosomopathies.

Second-generation compound for the modulation of utrophin in the therapy of DMD.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked muscle-wasting disease caused by lack of the cytoskeletal protein dystrophin. There is currently no cure for DMD although various promising approaches are progressing through human clinical trials. By pharmacologically modulating the expression of the dystrophin-related protein utrophin, we have previously demonstrated in dystrophin-deficient mdx studies, daily SMT C1100 treatment significantly reduced muscle degeneration leading to improved muscle function. This manuscript describes the significant disease modifying benefits associated with daily dosing of SMT022357, a second-generation compound in this drug series with improved physicochemical properties and a more robust metabolism profile. These studies in the mdx mouse demonstrate that oral administration of SMT022357 leads to increased utrophin expression in skeletal, respiratory and cardiac muscles. Significantly, utrophin expression is localized along the length of the muscle fibre, not just at the synapse, and is fibre-type independent, suggesting that drug treatment is modulating utrophin transcription in extra-synaptic myonuclei. This results in improved sarcolemmal stability and prevents dystrophic pathology through a significant reduction of regeneration, necrosis and fibrosis. All these improvements combine to protect the mdx muscle from contraction induced damage and enhance physiological function. This detailed evaluation of the SMT C1100 drug series strongly endorses the therapeutic potential of utrophin modulation as a disease modifying therapeutic strategy for all DMD patients irrespective of their dystrophin mutation.

Advances in genetic therapeutic strategies for Duchenne muscular dystrophy.

NEW FINDINGS: What is the topic of this review? This review highlights recent progress in genetically based therapies targeting the primary defect of Duchenne muscular dystrophy. What advances does it highlight? Over the last two decades, considerable progress has been made in understanding the mechanisms underlying Duchenne muscular dystrophy, leading to the development of genetic therapies. These include manipulation of the expression of the gene or related genes, the splicing of the gene and its translation, and replacement of the gene using viral approaches. Duchenne muscular dystrophy is a lethal X-linked disorder caused by mutations in the dystrophin gene. In the absence of the dystrophin protein, the link between the cytoskeleton and extracellular matrix is destroyed, and this severely compromises the strength, flexibility and stability of muscle fibres. The devastating consequence is progressive muscle wasting and premature death in Duchenne muscular dystrophy patients. There is currently no cure, and despite exhaustive palliative care, patients are restricted to a wheelchair by the age of 12 years and usually succumb to cardiac or respiratory complications in their late 20s. This review provides an update on the current genetically based therapies and clinical trials that target or compensate for the primary defect of this disease. These include dystrophin gene-replacement strategies, genetic modification techniques to restore dystrophin expression, and modulation of the dystrophin homologue, utrophin, as a surrogate to re-establish muscle function.

RNA exosome mutations in pontocerebellar hypoplasia alter ribosome biogenesis and p53 levels.

The RNA exosome is a ubiquitously expressed complex of nine core proteins (EXOSC1-9) and associated nucleases responsible for RNA processing and degradation. Mutations in EXOSC3, EXOSC8, EXOSC9, and the exosome cofactor RBM7 cause pontocerebellar hypoplasia and motor neuronopathy. We investigated the consequences of exosome mutations on RNA metabolism and cellular survival in zebrafish and human cell models. We observed that levels of mRNAs encoding p53 and ribosome biogenesis factors are increased in zebrafish lines with homozygous mutations of exosc8 or exosc9, respectively. Consistent with higher p53 levels, mutant zebrafish have a reduced head size, smaller brain, and cerebellum caused by an increased number of apoptotic cells during development. Down-regulation of EXOSC8 and EXOSC9 in human cells leads to p53 protein stabilisation and G2/M cell cycle arrest. Increased p53 transcript levels were also observed in muscle samples from patients with EXOSC9 mutations. Our work provides explanation for the pathogenesis of exosome-related disorders and highlights the link between exosome function, ribosome biogenesis, and p53-dependent signalling. We suggest that exosome-related disorders could be classified as ribosomopathies.

Consistency in reference radiotherapy dosimetry: resolution of an apparent conundrum when (60)Co is the reference quality for charged-particle and photon beams.

Substantial changes in ion chamber perturbation correction factors in (60)Co γ-rays, suggested by recent Monte Carlo (MC) calculations, would cause a decrease of about 1.5% in the reference dosimetry of all types of charged particles (electrons, protons and heavier ions) based on calculated kQ values. It has gone largely unnoticed that the ratio of calibration coefficients ND, w, Co60 and NK, air, Co60 yields an experimental value of Fch, Co60 = (sw-air pch)Co60 through ND, air, Co60. Coefficients provided by the IAEA and traceable to the BIPM for 91 NE-2571 chambers result in an average Fch, Co60 which is compared with published (and new) MC simulations and with the value in IAEA TRS-398. It is shown that TRS-398 agrees within 0.12% with the experimental Fch, Co60. The 1.5% difference resulting from MC calculations (1.1% for the new simulations) cannot be justified using current fundamental data and BIPM standards if consistency in the entire dosimetry chain is sought. For photons, MC kQ factors are compared with TRS-398. Using the same uncertainty for Wair, the two sets of data overlap considerably. Experimental kQ values from standards laboratories lie between the two sets of calculated values, showing no preference for one set over the other. Observed chamber-to-chamber differences, that include the effect of waterproof sleeves (also seen for (60)Co), justify the recommendation in TRS-398 for kQ values specifically measured for the user chamber. Current developments on I-values for the stopping powers of water and graphite are presented. A weighted average Iwater = 78 ± 2 eV is obtained from published experimental and DRF-based values; this would decrease sw-air for all types of radiotherapy beams between 0.3% and 0.6%, and would consequently decrease the MC derived Fch, Co60. The implications of a recent proposal for Igraphite = 81 eV are analysed, resulting in a potential decrease of 0.7% in NK, air, Co60 which would raise the experimental Fch, Co60; this would result in an increase of about 0.8% in the current TRS-398 value when referred to the BIPM standards. MC derived Fch, Co60 using new stopping powers would then agree at a level of 0.1% with the experimental value, confirming the need for consistency in the dosimetry chain data. Should world average standards be used as reference, the figures would become +0.4% for TRS-398 and -0.3% for the MC calculation. Fch, Q calculated for megavoltage photons using new stopping powers would decrease by between 0.2% and 0.5%. When they enter as a ratios in kQ, differences with MC values based on current key data would be within 0.2% but their discrepancy with kQ experimental photon values remains unresolved. For protons the new data would require an increase in Wair, Q of about 0.6%, as this is inferred from a combination of calorimetry and ionometry. This consistent scenario would leave unchanged the current TRS-398 kQ (NE-2571) data for protons, as well as for ions heavier than protons unless new independent Wair, Q values become available. Also in these advanced radiotherapy modalities, the need for maintaining data consistency in an analysis that unavoidably must include the complete dosimetry chain is demonstrated.

On the uncertainties of photon mass energy-absorption coefficients and their ratios for radiation dosimetry.

A systematic analysis of the available data has been carried out for mass energy-absorption coefficients and their ratios for air, graphite and water for photon energies between 1 keV and 2 MeV, using representative kilovoltage x-ray spectra for mammography and diagnostic radiology below 100 kV, and for ¹9²Ir and 6°Co gamma-ray spectra. The aim of this work was to establish 'an envelope of uncertainty' based on the spread of the available data. Type A uncertainties were determined from the results of Monte Carlo (MC) calculations with the PENELOPE and EGSnrc systems, yielding mean values for µ(en)/ρ with a given statistical standard uncertainty. Type B estimates were based on two groupings. The first grouping consisted of MC calculations based on a similar implementation but using different data and/or approximations. The second grouping was formed by various datasets, obtained by different authors or methods using the same or different basic data, and with different implementations (analytical, MC-based, or a combination of the two); these datasets were the compilations of NIST, Hubbell, Johns-Cunningham, Attix and Higgins, plus MC calculations with PENELOPE and EGSnrc. The combined standard uncertainty, u(c), for the µ(en)/ρ values for the mammography x-ray spectra is 2.5%, decreasing gradually to 1.6% for kilovoltage x-ray spectra up to 100 kV. For 6°Co and ¹9²Ir, u(c) is approximately 0.1%. The Type B uncertainty analysis for the ratios of µ(en)/ρ values includes four methods of analysis and concludes that for the present data the assumption that the data interval represents 95% confidence limits is a good compromise. For the mammography x-ray spectra, the combined standard uncertainties of (µ(en)/ρ)(graphite,air) and (µ(en)/ρ)(graphite,water) are 1.5%, and 0.5% for (µ(en)/ρ)(water,air), decreasing gradually down to u(c) = 0.1% for the three µ(en)/ρ ratios for the gamma-ray spectra. The present estimates are shown to coincide well with those of Hubbell (1977 Rad. Res. 70 58-81), except for the lowest energy range (radiodiagnostic) where it is concluded that current databases and their systematic analysis represent an improvement over the older Hubbell estimations. The results for (µ(en)/ρ)(graphite,air) for the gamma-ray dosimetry range are moderately higher than those of Seltzer and Bergstrom (2005 private communication).