LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies

Genotype-Phenotype Correlations in Charcot-Marie-Tooth Disease Due to MTMR2 Mutations and Implications in Membrane Trafficking

Charcot-Marie-Tooth type 4 (CMT4) is an autosomal recessive severe form of neuropathy with genetic heterogeneity. CMT4B1 is caused by mutations in the myotubularin-related 2 (MTMR2) gene and as a member of the myotubularin family, the MTMR2 protein is crucial for the modulation of membrane trafficking. To enable future clinical trials, we performed a detailed review of the published cases with MTMR2 mutations and describe four novel cases identified through whole-exome sequencing (WES). The four unrelated families harbor novel homozygous mutations in MTMR2 (NM_016156, Family 1: c.1490dupC; p.Phe498IlefsTer2; Family 2: c.1479+1G>A; Family 3: c.1090C>T; p.Arg364Ter; Family 4: c.883C>T; p.Arg295Ter) and present with CMT4B1-related severe early-onset motor and sensory neuropathy, generalized muscle atrophy, facial and bulbar weakness, and pes cavus deformity. The clinical description of the new mutations reported here overlap with previously reported CMT4B1 phenotypes caused by mutations in the phosphatase domain of MTMR2, suggesting that nonsense MTMR2 mutations, which are predicted to result in loss or disruption of the phosphatase domain, are associated with a severe phenotype and loss of independent ambulation by the early twenties. Whereas the few reported missense mutations and also those truncating mutations occurring at the C-terminus after the phosphatase domain cause a rather mild phenotype and patients were still ambulatory above the age 30 years. Charcot-Marie-Tooth neuropathy and Centronuclear Myopathy causing mutations have been shown to occur in proteins involved in membrane remodeling and trafficking pathway mediated by phosphoinositides. Earlier studies have showing the rescue of MTM1 myopathy by MTMR2 overexpression, emphasize the importance of maintaining the phosphoinositides equilibrium and highlight a potential compensatory mechanism amongst members of this pathway. This proved that the regulation of expression of these proteins involved in the membrane remodeling pathway may compensate each other's loss- or gain-of-function mutations by restoring the phosphoinositides equilibrium. This provides a potential therapeutic strategy for neuromuscular diseases resulting from mutations in the membrane remodeling pathway

Influence of Inertia on the Dynamic Compressive Strength of Concrete

The rate sensitivity of concrete material is closely related to the inertia and viscous effects. However, the effect of inertia on the dynamic strength of concrete remains unclear. In this paper, digital image correlation technology was applied to study the strain variation of dry and saturated concrete with different loading rates. The test results indicated that the strain gradually decreased with the distance from the load end, and the strain gradient around the load region increased with the strain rate, especially for saturated concrete. Then, a single degree of freedom model was established to evaluate the dynamic compressive strength of elastic concrete. The calculated results indicated that the influence of inertia on the dynamic increase factor (DIF) was negligible for concrete within a low strain rate. When the strain rate is larger than 100/s, the inertial effect on the strength of concrete should be considered. After that, a quasi-static concrete damaged plasticity (CDP) model was employed to simulate the influence of inertia on the stress distribution and axial reaction force at the loaded end of concrete under different rates of compressive loading and verified with experimental results. The results obtained in this study indicated that the dynamic nominal strength of concrete obtained from the tests could not be directly used for structural analysis which may overestimate the effect of inertia on the dynamic response of the structure

Insights from genotype-phenotype correlations by novel SPEG mutations causing centronuclear myopathy

WOS: 000411302400005PubMed ID: 28624463Centronuclear myopathies (CNM) are a clinically and genetically heterogeneous group of congenital myopathies, defined histologically by increased number of fibres with centrally located nuclei, and type I fibre predominance in muscle biopsy. Myotubular myopathy, the X-linked form of CNM caused by mutations in the phosphoinositide phosphatase MTM1, is histologically characteristic since muscle fibres resemble myotubes. Here we present two unrelated patients with CNM and typical myotubular fibres in the muscle biopsy caused by mutations in striated muscle preferentially expressed protein kinase (SPEG). Next generation sequencing revealed novel biallelic homozygous mutations in SPEG in both cases. Patient 1 showed the c.1627_1628insA (p.Thr544Aspfs*48) mutation and patient 2 the c.9586C>T (p.Arg3196*) mutation. The clinical phenotype was distinctive in the two patients since patient 2 developed a dilated cardiomyopathy with milder myopathy features, while patient 1 showed only myopathic features without cardiac involvement. These findings expand the genotype phenotype correlations after the initial report. Additionally, we describe whole body muscle MRI of patient 2 and we argue on the different SPEG isoforms in skeletal muscle and heart as the possible explanation leading to variable phenotypes of SPEG mutations. (C) 2017 Elsevier B.V. All rights reserved.Muscular Dystrophy Association, USAMuscular Dystrophy Association [255889]; Deutsche Forschungsgemeinschaft, GermanyGerman Research Foundation (DFG) [CI 218/1-1]; Clinica Las Condes, Santiago, Chile [PIDA. 2014-002]; Ministry of HealthMinistry of Health - Turkey [Finalizzata 201201X002951]; TelethonFondazione Telethon [GUP13004]; FONDECYTComision Nacional de Investigacion Cientifica y Tecnologica (CONICYT)CONICYT FONDECYT [1151383]Our patients and their parents are gratefully acknowledged. This work was supported by Muscular Dystrophy Association (255889), USA, and Deutsche Forschungsgemeinschaft (CI 218/1-1), Germany grants to Dr. Sebahattin Cirak. Clinica Las Condes, Santiago, Chile: PIDA. 2014-002, grants to Dr. Claudia Castiglioni, Ministry of Health Grant Finalizzata 201201X002951 and Telethon GUP13004 grants to Fabiana Fattori and Enrico Bertini and Grant FONDECYT 1151383 to Jorge Bevilacqua

A Genome-Wide View of the Transcriptome Dynamics of Fresh-Cut Potato Tubers

Fresh fruits and vegetable products are easily perishable during postharvest handling due to enzymatic browning reactions. This phenomenon has contributed to a significant loss of food. To reveal the physiological changes in fresh-cut potato tubers at the molecular level, a transcriptome analysis of potato tubers after cutting was carried out. A total of 10,872, 10,449, and 11,880 differentially expressed genes (DEGs) were identified at 4 h, 12 h and 24 h after cutting, respectively. More than 87.5% of these DEGs were classified into the categories of biological process (BP) and molecular function (MF) based on Gene Ontology (GO) analysis. There was a difference in the response to cutting at different stages after the cutting of potato tubers. The genes related to the phenol and fatty biosynthesis pathways, which are responsible for enzymatic browning and wound healing in potato tubers, were significantly enriched at 0–24 h after cutting. Most genes related to the enzymatic browning of potato tubers were up-regulated in response to cut-wounding. Plant hormone biosynthesis, signal molecular biosynthesis and transduction-related genes, such as gibberelin (GA), cytokinin (CK), ethylene (ET), auxin (IAA), jasmonic acid (JA), salicylic (SA), and Respiratory burst oxidase (Rboh) significantly changed at the early stage after cutting. In addition, the transcription factors involved in the wound response were the most abundant at the early stage after cutting. The transcription factor with the greatest response to injury was MYB, followed by AP2-EREBP, C3H and WRKY. This study revealed the physiological changes at the molecular level of fresh-cut potato tubers after cutting. This information is needed for developing a better approach to enhancing the postharvest shelf life of fresh processed potato and the breeding of potato plants that are resistant to enzymatic browning

Shorter Phosphorodiamidate Morpholino Splice-Switching Oligonucleotides May Increase Exon-Skipping Efficacy in DMD.

Duchenne muscular dystrophy is a fatal muscle disease, caused by mutations in DMD, leading to loss of dystrophin expression. Phosphorodiamidate morpholino splice-switching oligonucleotides (PMO-SSOs) have been used to elicit the restoration of a partially functional truncated dystrophin by excluding disruptive exons from the DMD messenger. The 30-mer PMO eteplirsen (EXONDYS51) developed for exon 51 skipping is the first dystrophin-restoring, conditionally FDA-approved drug in history. Clinical trials had shown a dose-dependent variable and patchy dystrophin restoration. The main obstacle for efficient dystrophin restoration is the inadequate uptake of PMOs into skeletal muscle fibers at low doses. The excessive cost of longer PMOs has limited the utilization of higher dosing. We designed shorter 25-mer PMOs directed to the same eteplirsen-targeted region of exon 51 and compared their efficacies in vitro and in vivo in the mdx52 murine model. Our results showed that skipped-dystrophin induction was comparable between the 30-mer PMO sequence of eteplirsen and one of the shorter PMOs, while the other 25-mer PMOs showed lower exon-skipping efficacies. Shorter PMOs would make higher doses economically feasible, and high dosing would result in better drug uptake into muscle, induce higher levels of dystrophin restoration in DMD muscle, and, ultimately, increase the clinical efficacy. Keywords: DMD, dystrophin, exon skipping, eteplirsen, phosphorodiamidate morpholino, PMO, shorter PMO-SSOs, phosphorothiorate, myopathy, exondys51, md

Bi-allelic mutations in PRUNE lead to neurodegeneration with spinal motor neuron involvement and hyperCKaemia

We aimed to systematically investigate the neuromuscular involvement of individuals with PRUNE mutations who may have a major spinal motor neuron involvement as part of the PRUNE-associated neurodegenerative phenotype. The complex neurological phenotypes associated with Prune mutations include microcephaly with brain abnormalities, spasticity, seizures, severe developmental delay and developmental regression. We used whole exome sequencing to identify the mutation and electrophysiological and muscle biopsy studies to evaluate the signs of spinal motor neuron involvement. The affected individuals carry homozygous PRUNE mutation (NM_021222.1, c.316G>A, p.D106N), showing the signs of spinal motor neuron involvement supported by electrophysiological and muscle biopsy findings and also persistent high creatine kinase levels. We confirm that individuals with PRUNE mutations may have a major spinal motor neuron involvement as part of the PRUNE-associated neurodegenerative phenotype. The PRUNE gene should be considered in all the individuals with non-5q spinal muscular atrophy. High creatine kinase values may be a part of PRUNE disease spectrum

Clinical outcomes of two patients with a novel pathogenic variant in ASNS: response to asparagine supplementation and review of the literature

Asparagine synthetase deficiency (ASNSD, OMIM #615574) is a rare autosomal recessive neurometabolic inborn error that leads to severe cognitive impairment. It manifests with microcephaly, intractable seizures, and progressive cerebral atrophy. Currently, there is no established treatment for this condition. In our pediatric cohort, we discovered, by whole-exome sequencing in two siblings from Turkey, a novel homozygous missense mutation in asparagine synthetase at NM_133436.3 (ASNS_v001): c.1108C>T that results in an amino acid exchange p.(Leu370Phe), in the C-terminal domain. After identification of the metabolic defect, treatment with oral asparagine supplementation was attempted in both patients for 24 months. Asparagine supplementation was well tolerated, and no further disease progression was observed during treatment. One of our patients showed mild developmental progress with increased levels of attention and improved nonverbal communication. These results support our hypothesis that asparagine supplementation should be further investigated as a treatment option for ASNSD. We further reviewed all previously reported ASNSD cases with regard for their clinical phenotypes and brain imaging findings to provide an essential knowledge base for rapid diagnosis and future clinical studies

Lifespans in wild-type and mutants exposed to clenbuterol or ractopamine.

<p>Exposures were performed from L1-larvae to adult (prolonged exposure) at the concentration of 10 µg/L. Thirty nematodes were examined per treatment. clen, clenbuterol; rac, ractopamine. Bars represent mean ± S.E.M. **<i>P</i><0.01.</p