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Congenital myopathies are a broad group of inborn muscle disorders caused by a multitude of genetic factors, often characterized by muscle atrophy and hypotonia.Clinical studies, imaging, histology, whole-exome sequencing (WES) and muscle tissue RNA studies.We describe a severe congenital myopathy manifesting at birth with bilateral clubfeet, delayed motor development and hypotonia, becoming evident by 4 months of age. At 3 years of age, the patient had tongue fasciculations, was bedridden, and was chronically ventilated via tracheostomy. Imaging studies demonstrated severe muscle atrophy and, surprisingly, cerebral atrophy; electromyography demonstrated a myasthenic pattern and histological evaluation did not facilitate a definitive diagnosis. Trio WES did not identify a causative variant, except for a non-canonical intronic TPM3 c.118-12GA variant of uncertain significance. Transcript analysis of muscle tissue from the patient proved the pathogenicity of this homozygous variant, with a 97% reduction in the muscle-specific TPM3.12 transcript.This study broadens the phenotypic spectrum of recessive TPM3 disease, highlighting tongue fasciculations and bilateral clubfoot, as well as possibly-related cerebral atrophy. It also shows the importance of a broad approach to genetic analysis and the utility of RNA-based studies, demonstrating efficacy of early genome and transcriptome queries in facilitating rapid and cost-effective diagnosis of congenital myopathies.

BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59, leading to loss of function, have been described, and overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the expression and possible role of membrane-bound complement regulators including CD59, in the peripheral nervous system (PNS) of mice and humans. MAIN: Methods. We examined the expression of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. Results. We demonstrate that CD59-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express P0, ankyrin G (AnkG), caspr, dystroglycan, and neurofascin. CD59 is colocalized with myelin basic protein (MBP) and P0 and is present in compact myelin and endothelium but absent from noncompact myelin. Furthermore, immunolabeling of WT nerves using antibodies to CD59 and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and Crry, rendering this area highly exposed to complement attack. CONCLUSION: Myelin in the human PNS is protected only by CD59 and CD55, and not by CD46 or CD35, rendering myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barre syndrome, as seen in CD59 deficiency.

Our goal was to measure the absolute differential abundance of key drug transporters in human epileptogenic brain tissue and to compare them between patients and at various distances from the epileptogenic zone within the same patient. Transporter protein abundance was quantified in brain tissue homogenates from patients who underwent epilepsy surgery, using targeted proteomics, and correlations with clinical and tissue characteristics were assessed. Fourteen brain samples (including four epileptogenic hippocampal samples) were collected from nine patients. Among the quantifiable drug transporters, the abundance (median, range) ranked: breast cancer resistance protein (ABCG2/BCRP; 0.55, 0.01-3.26 pmol/g tissue) > P-glycoprotein (ABCB1/MDR1; 0.30, 0.02-1.15 pmol/g tissue) > equilibrative nucleoside transporter 1 (SLC29A1/ENT1; 0.06, 0.001-0.35 pmol/g tissue). The ABCB1/ABCG2 ratio (mean 0.27, range 0.08-0.47) was comparable with literature values from nonepileptogenic brain tissue (mean 0.5-0.8). Transporter abundance was lower in the hippocampi than in the less epileptogenic neocortex of the same patients. ABCG2/BCRP and ABCB1/MDR1 expression strongly correlated with that of glucose transporter 1 (SLC2A1/GLUT1) (r = 0.97, p < 0.001; r = 0.90, p < 0.01, respectively). Low transporter abundance was found in patients with overt vascular pathology, whereas the highest abundance was seen in a sample with normally appearing blood vessels. In conclusion, drug transporter abundance highly varies across patients and between epileptogenic and less epileptogenic brain tissue of the same patient. The strong correlation in abundance of ABCB1/MDR1, ABCG2/BCRP, and SLC2A1/GLUT1 suggests variation in the content of the functional vasculature within the tissue samples. The epileptogenic tissue can be depleted of key drug transport mechanisms, warranting consideration when selecting treatments for patients with drug-resistant epilepsy.

MSTO1 is a cytoplasmic protein that modulates mitochondrial dynamics by promoting mitochondrial fusion. Mutations in the MSTO1 gene are responsible for an extremely rare condition characterized by early-onset myopathy and cerebellar ataxia. We report here two siblings from a large Ashkenazi Jewish family, presenting with a progressive neuromuscular disease characterized by ataxia and myopathy. By whole exome sequencing, we found a novel homozygous missense mutation (c.1403T>A, p.Leu468Gln) in MSTO1. Studies performed on fibroblasts from the index patient demonstrated the pathogenic role of the identified variant; we found that MSTO1 protein level was reduced and that mitochondrial network was fragmented or formed enlarged structures. Moreover, patient's cells showed reduced mitochondrial DNA amount. Our report confirms that MSTO1 mutations are typically recessive, and associated with clinical phenotypes characterized by early-onset muscle impairment and ataxia, often with upper motor neuron signs and varied cognitive impairment.

Muscular A-type lamin-interacting protein (MLIP) is most abundantly expressed in cardiac and skeletal muscle. In vitro and animal studies have shown its regulatory role in myoblast differentiation and in organization of myonuclear positioning in skeletal muscle, as well as in cardiomyocyte adaptation and cardiomyopathy. We report the association of biallelic truncating variation in the MLIP gene with human disease in five individuals from two unrelated pedigrees.Clinical evaluation and exome sequencing were performed in two unrelated families with elevated creatine kinase level.Family 1. A 6-year-old girl born to consanguineous parents of Arab-Muslim origin presented with myalgia, early fatigue after mild-to-moderate physical exertion, and elevated creatine kinase levels up to 16,000 U/L. Exome sequencing revealed a novel homozygous nonsense variant, c.2530CT; p.Arg844Ter, in the MLIP gene. Family 2. Three individuals from two distantly related families of Old Order Amish ancestry presented with elevated creatine kinase levels, one of whom also presented with abnormal electrocardiography results. On exome sequencing, all showed homozygosity for a novel nonsense MLIP variant c.1825AT; p.Lys609Ter. Another individual from this pedigree, who had sinus arrhythmia and for whom creatine kinase level was not available, was also homozygous for this variant.Our findings suggest that biallelic truncating variants in MLIP result in myopathy characterized by hyperCKemia. Moreover, these cases of MLIP-related disease may indicate that at least in some instances this condition is associated with muscle decompensation and fatigability during low-to-moderate intensity muscle exertion as well as possible cardiac involvement.

Background: GNE myopathy is a unique adult onset rare neuromuscular disease caused by recessive mutations in the GNE gene. The pathophysiological mechanism of this disorder is not well understood and to date, there is no available therapy for this debilitating disease. We have previously established proof of concept that AAV based gene therapy can effectively deliver the wild type human GNE into cultured muscle cells from human patients and in mice, using a CMV promoter driven human wild type GNE plasmid delivered through an adeno associated virus (AAV8) based platform. Objective: In the present study we have generated a muscle specific GNE construct, driven by the MCK promoter and packaged with the AAVrh74 serotype for efficacy evaluation in an animal model of GNE Myopathy. Methods: The viral vector was systemically delivered at 2 doses to two age groups of a Gne–/– hGNED207V Tg mouse described as a preclinical model of GNE Myopathy, and treatment was monitored for long term efficacy. Results: In spite of the fact that the full described characteristics of the preclinical model could not be reproduced, the systemic injection of the rAAVrh74.MCK.GNE viral vector resulted in a long term presence and expression of human wt GNE in the murine muscles and in some improvements of their mild phenotype. The Gne–/– hGNED207V Tg mice are smaller from birth, but cannot be differentiated from littermates by muscle function (grip strength and Rotarod) and their muscle histology is normal, even at advanced age. Conclusions: The rAAVrh74.MCK.GNE vector is a robust tool for the development of GNE Myopathy therapies that supply the intact GNE. However, there is still no reliable animal model to fully assess its efficacy since the previously developed Gne–/– hGNED207V Tg mice do not present disease characteristics.

Objective Glatiramer acetate (GA), an agent modulating the immune system, has been shown to cause significantly improved mobility and hind limb muscle strength in the dy2J/dy2J mouse model for LAMA2-congenital muscular dystrophy (LAMA2-CMD). In view of these findings and the prominent peripheral nervous system involvement in this laminin-α2 disorder we evaluated GA’s effect on dy2J/dy2J motor nerve conduction electrophysiologically. Methods Left sciatic-tibial motor nerve conduction studies were performed on wild type (WT) mice (n = 10), control dy2J/dy2J mice (n = 11), and GA treated dy2J/dy2J mice (n = 10) at 18 weeks of age. Results Control dy2J/dy2J mice average velocities (34.49 ± 2.15 m/s) were significantly slower than WT (62.57 ± 2.23 m/s; p Conclusion In this study we show for the first time improvement in motor nerve conduction velocity of LAMA2-CMD dy2J/dy2J mouse model’s hereditary peripheral neuropathy following GA treatment. Significance This study suggests a possible therapeutic effect of glatiramer acetate on hereditary peripheral neuropathy in this laminin-α2 disorder.

Our goal was to measure the absolute differential abundance of key drug transporters in human epileptogenic brain tissue and to compare them between patients and at various distances from the epileptogenic zone within the same patient. Transporter protein abundance was quantified in brain tissue homogenates from patients who underwent epilepsy surgery, using targeted proteomics, and correlations with clinical and tissue characteristics were assessed. Fourteen brain samples (including four epileptogenic hippocampal samples) were collected from nine patients. Among the quantifiable drug transporters, the abundance (median, range) ranked: breast cancer resistance protein (ABCG2/BCRP; 0.55, 0.01–3.26 pmol/g tissue) > P-glycoprotein (ABCB1/MDR1; 0.30, 0.02–1.15 pmol/g tissue) > equilibrative nucleoside transporter 1 (SLC29A1/ENT1; 0.06, 0.001–0.35 pmol/g tissue). The ABCB1/ABCG2 ratio (mean 0.27, range 0.08–0.47) was comparable with literature values from nonepileptogenic brain tissue (mean 0.5–0.8). Transporter abundance was lower in the hippocampi than in the less epileptogenic neocortex of the same patients. ABCG2/BCRP and ABCB1/MDR1 expression strongly correlated with that of glucose transporter 1 (SLC2A1/GLUT1) (r = 0.97, p < 0.001; r = 0.90, p < 0.01, respectively). Low transporter abundance was found in patients with overt vascular pathology, whereas the highest abundance was seen in a sample with normally appearing blood vessels. In conclusion, drug transporter abundance highly varies across patients and between epileptogenic and less epileptogenic brain tissue of the same patient. The strong correlation in abundance of ABCB1/MDR1, ABCG2/BCRP, and SLC2A1/GLUT1 suggests variation in the content of the functional vasculature within the tissue samples. The epileptogenic tissue can be depleted of key drug transport mechanisms, warranting consideration when selecting treatments for patients with drug-resistant epilepsy.

Introduction: Immune-checkpoint inhibitors have demonstrated a significant survival benefit in metastatic and non-resectable head and neck squamous cell carcinoma (HNSCC). Patients with a combined positivity score (CPS) of 20 and higher benefit the most from therapy. Inaccurate definition of the CPS category might lead to the incorrect stratification of patients to immunotherapy. This study’s main aim was to investigate programmed death-ligand 1 (PD-L1) antigen expression in HNSCC in diverse clinical situations and histological settings. Materials and Methods: This is a prospective cohort study conducted in a tertiary referral medical center. Tissues were investigated for PD-L1 expression using the FDA-approved 22C3 immunohistochemistry assay (Dako). We analyzed potential associations between the CPS category and meaningful demographic, clinical, and outcome metrics. Furthermore, we investigated morphologically separate sites for CPS scores in whole surgical tissue specimens and matched preoperative biopsies. Results: We analyzed 36 patients, of whom 26 had oral cavity SCC and 10 had laryngeal SCC. The overall, disease-specific, and progression-free survival of the HNSCC group of patients were not associated with the CPS category (p = 0.45, p = 0.31, and p = 0.88, respectively). There was a significant (18%, 95% CI 0.65–0.9) inconsistency between the CPS category determined in biopsies versus whole carcinoma analyses. We also found an uneven distribution of whole-tumor CPS attributed to spatial carcinoma invasiveness, tumor differentiation, and inflammatory cell infiltration heterogeneity. Discussion and Conclusions: Our data suggest that careful selection of tumor area for CPS analysis is important. PD-L1 antigen expression, clinically represented by CPS, may be up- or down-categorized in different clinical and pathological circumstances. The high whole-tissue CPS category scatter may clinically result in potential treatment modifications. We argue that CPS analysis requires not only adequacy (at least 100 viable tumor cells), but also correct representation of the tumor microenvironment.

We previously reported the development of a novel formulation of an ultra-long-acting local anesthetic based on bupivacaine encapsulated in large multivesicular liposomes (Bupisomes) embedded in hydrogel. This formulation (Bupigel) prolonged bupivacaine release from the formulation in dissolution-like studies in vitro and analgesia in vivo in mouse, rat, and pig models. In this study we assessed Bupigel neurotoxicity on rabbit sciatic nerve using histopathology and electrophysiologic testing. Sciatic nerves of both hind limbs were injected dropwise with different formulations. Nerve conduction studies and needle electromyography two weeks after perineural administration showed signs of neural damage after injection of free lidocaine and bupivacaine, while there was no sign of neural damage after injection with saline, demonstrating the validity of the method. This test also did not show evidence of motor or sensory nerve damage after injection with liposomal bupivacaine at a dose 10-times higher than free bupivacaine. Histologically, signs of neural damage could be observed with lidocaine. Nerves injected with Bupigel showed mild signs of inflammation and small residues of hydrogel in granulomas, indicating a long residence time of the hydrogel at the site of injection, but no histopathological signs of nerve damage. This demonstrated that early signs of neural damage were detected electrophysiologically, showing the usefulness and sensitivity of electrodiagnostic testing in detection of neural damage from new formulations.

We report a multiplex family with extended multisystem neurological phenotype associated with a CRYAB variant. Two affected siblings were evaluated with whole exome sequencing, muscle biopsy, laser microdissection, and mass spectrometry-based proteomic analysis. Both patients and their mother manifested a combination of early-onset cataracts, cardiomyopathy, cerebellar ataxia, optic atrophy, cognitive impairment, and myopathy. Whole exome sequencing identified a heterozygous c.458C>T variant mapped to the C-terminal extension domain of the Alpha-crystallin B chain, disrupting its function as a molecular chaperone and its ability to suppress protein aggregation. In accordance with the molecular findings, muscle biopsies revealed subsarcolemmal deposits that appeared dark with H&E and trichrome staining were negative for the other routine histochemical staining and for amyloid with the Congo-red stain. Electron microscopy demonstrated that the deposits were composed of numerous parallel fibrils. Laser microdissection and mass spectrometry-based proteomic analysis revealed that the inclusions are almost exclusively composed of crystallized chaperones/heat shock proteins. Moreover, a structural model suggests that Ser153 could be involved in monomer stabilization, dimer association, and possible binding of partner proteins. We propose that our report potentially expands the complex phenotypic spectrum of alpha B-crystallinopathies with possible effect of a CRYAB variant on the central nervous system.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10&ndash, 15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14&ndash, 17 duplication. Two strategies were employed, the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.

To date, classical deterministic Newtonian physics has been used by biologists to describe living processes. However, it is increasingly appreciated that the probabilistic view offered by quantum mechanics more accurately describes the behavior of atoms and materials in all systems. Here, we discuss how the concepts of quantum mechanics can be applied to biological processes involved in cancer. We present a concise summary inspired by Heisenberg's Uncertainty Principle to describe our «Genetic Environmental Field Hypothesis». Combining the uncertainties of genetic changes as expressed by epigenetic changes and/or somatic mutations with the uncertainties of environmental changes, cells may become cancerous as a way to increase entropy. Throughout the paper we will utilize the H19 gene system as an example. Using the concepts of quantum mechanics to describe oncological processes may provide novel directions in our understanding of cancer.

Sarcoidosis is uncommon multiple organ granulomatous disease of unknown etiology. Neurosarcoidosis occurs in about 5% of cases and most frequently follows systemic disease. We present a case of 52 -years -old woman with a progressive hemifacial paresthesia and multiple enhancing dural based lesions. Resection of the right frontal mass allowed for the diagnosis to be made. The patient had no other features of sarcoidosis. Therefore, the diagnosis of neurosarcoidosis, especially when unaccompanied by systemic features can be challenging but should be considered in the differential diagnosis of multiple enhancing dural based tumours.

Previous studies suggest that 14–30% of glioblastoma multiforme (GBM) patients and 5–24% of patients with brain metastases demonstrate imaging treatment-effects in the first few months after treatment [1], [2], [3]. These treatment-induced imaging changes, often termed pseudoprogression/radiation-necrosis, are depicted as increasing volumes of contrast-enhancing lesions on MRI, mimicking progression. Therefore, treatment decisions, such as whether to operate on a patient with radiographic deterioration, continue current treatment or change treatment is a daily unsolved struggle. In addition, radiation-based treatments may also induce vascular malformations such as radiation-induced Cavernous malformations (RICM) and capillary telangiectasias. Cavernous malformations are low-flow vascular malformations, characterized by the lack of mural elements of mature vascular structures and intervening parenchymal neural tissue [4]. Radiation induced capillary telangiectasias (RIT), thin-walled ectatic capillaries with intervening normal brain parenchyma, usually occur 3–9 months after irradiation. Cavernomas take a longer time to develop (1–35 years) after radiation [5]. RICMs mostly develop in the pediatric population [6], but are also observed in adults. In a literature search from 2006 by Nimjee et al [7], 76 cases of RICMs were found. A retrospective study conducted at Mayo clinic found 32 RICMs [5]. RICMs latency median was 12.0 years with only 3 diagnosed in the first two years post radiation (9.3%). Kleinschmidt-DeMasters and Lillehei [8] found 13 cases between 2000 and 2016 in their surgical neuropathology databases. The latency median was 18 years. Strenger et al calculated a cumulative index of 2.24%, 3.86%, 4.95%, and 6.74% at 5, 10, 15, and 20 years following radiotherapy of children, respectively [9]. Vinchon et al [10] identified cumulative indices for their cohort in children at 10 years of 8.9%. Although RICMs are rare in adults with astrocytoma, mainly due to poor survival, three cases of long term survivals were reported in the literature to develop RICMs 10, 19 and 26 years post radiotherapy [11]. Gaensler and colleagues reported a series of 20 patients with RITs (6 proven pathologically) for whom the latency was only 2.7 years [12]. 70% of the 20 patients were 1 h (60–105 min) after a conventional injection of contrast agent. Blue/tumor regions in the TRAMs represent efficient clearance of contrast from the tissue (delayed signal early signal). The TRAMs were validated histologically in 51 resected lesions from patients with primary and metastatic brain tumors reaching 92% positive predictive value (PPV) and 100% sensitivity to morphologically active tumor. When studying the histological samples, we found that the common vessels morphology in the blue regions was undamaged vessels lumens, while vessels in the red regions presented different stages of vessel necrosis. Therefore, one explanation for the difference between the two populations may be that vessels in blue/tumor regions provide efficient contrast clearance from the tissue, while the damaged lumens in the red/treatment-effects regions are unable to clear the accumulating contrast, resulting in contrast accumulation. Over 400 adult patients have been recruited thus far to our ongoing TRAMs-based studies in Israel since 2010. As the TRAMs cannot differentiate blood vessels from active tumor (both appearing blue), we studied here whether RCIMs/RITs may mimic tumors in the TRAMs.

The median survival time of patients with glioblastoma is still poor (14.6 month), partly due to a lack of effective treatment. We have observed that androgen receptor (AR) is amplified in glioblastomas at the DNA, RNA and protein levels. The AR gene was amplified in 27% of glioblastoma specimens from men (n=22) and of 38.2% from women (n=21). AR-RNA was overexpressed (>2.5 fold) in 93% (n=30), and AR-protein was induced (>two fold) in 56% of the glioblastomas samples (n=16). Thirty percent of the glioblastomas (n=21) also expressed a constitutively active AR-splice-variant (AR-V7/AR3) lacking the Ligand-Binding-Domain. Following these findings, we examined the effect of pharmacological inhibition of androgen receptor in vitro and in vivo, as well as of genetic silencing of the receptor in glioblastoma cell lines. AR antagonists, induced concentration-dependent death in three glioblastoma cell lines, as well as in two glioma initiating cell lines. Silencing of AR expression by siRNA induced cell death in the three tested glioblastoma cell lines. Enzalutamide given orally to nude mice bearing subcutaneous human glioma xenografts resulted in a 72% reduction in tumor volume (p=0.0027). The presence of AR-V7/AR3 in glioblastoma, together with the present data showing that genetic silencing of the full length AR in cell lines and pharmacological inhibition of AR, induce GBM cell death in vivo and in vitro, point to the important role of AR in GBM survival and render a potential therapeutic target for this devastating disease.

Deep Brain Stimulation (DBS) is an established surgical therapy for movement disorders, epilepsy and more recently certain psychiatric disorders. The procedure is safe and complications fortunately few. Bowstringing as a complication has not been well documented and is most probably underreported. Bowstringing describes an entity where the subcutaneous electrode extensions running from head to chest cause a sensation of unpleasant tension, pulling and tightness. Patients may even complain of difficulty with turning their head to the contralateral side. This unpleasant feeling may continue to occur even after the removal of DBS extension leads as the cord like scar still remains. The underlying pathological process and why this occurs in a subset of patients is poorly understood. As such, to date in the literature, there are but a few proposed methods to manage bowstringing in DBS patients and certainly no consensus. We present an illustrative case report with histopathological correlation and suggestions for how this complication might be prevented and managed. Keywords: Deep brain stimulation, Bowstringing, Complication, Pseudocapsule

// Nir Pillar 1 , Osnat Bairey 1, 2 , Neta Goldschmidt 3 , Yakov Fellig 4 , Yevgenia Rosenblat 5 , Itchak Shehtman 6 , Danielle Haguel 1 , Pia Raanani 1, 2 , Noam Shomron 1, * and Tali Siegal 7, * 1 Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Institute of Hemathology, Davidoff Institute of Oncology, Rabin Medical Center, Petach Tikva, Israel 3 Department of Hemathology, Hadassah Hebrew University Medical Center, Jerusalem, Israel 4 Department of Pathology, Hadassah Hebrew University Medical Center, Jerusalem, Israel 5 Department of Pathology, Rabin Medical Center, Petach Tikva, Israel 6 Department of Pathology, Meir Medical Center, Kefar Saba, Israel 7 Neuro-Oncology Center, Davidoff Institute of Oncology, Rabin Medical Center, Petach Tikva, Israel * These authors have contributed equally to this work Correspondence to: Tali Siegal, email: talisi1@clalit.org.il Noam Shomron, email: nshomron@post.tau.ac.il Keywords: systemic DLBCL, CNS relapse prediction, microRNA, miR-20a, miR-30d Received: June 06, 2017 Accepted: August 04, 2017 Published: September 15, 2017 ABSTRACT Systemic diffuse large B-cell lymphoma (DLBCL) is a potentially curable disease using current regimen of immunochemotherapy. Central nervous system (CNS) relapse is a complication that occurs in approximately 5% of DLBCL patients and is associated with a high fatality rate. Early identification of molecular markers for CNS involvement may serve for the highly needed accurate stratification of patients into risk groups regarding CNS relapse. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level and are known to be involved in DLBCL pathophysiology. In this study, we utilized miRNA multiplex reading of systemic newly diagnosed DLBCL samples obtained from patients with clinical risk factors for CNS involvement whose disease course was distinguished by the presence or absence of subsequent CNS relapse. The analysis detected two differentially expressed miRNAs, miR-20a and miR-30d, that predict for CNS involvement. Replication of these results in different samples was used for validation. We performed bioinformatics miRNA-target enrichment analysis to reveal a number of putative mechanisms for these miRNAs regulation of CNS relapse, including neuronal plasticity and WNT signaling pathway. Altogether, we show that the expression level of two miRNAs may have valuable information that may refine stratification for patients-at-risk for relapse with CNS involvement in DLBCL. Further larger scale studies are needed to shed light on the pathways involved in this disease.

Mutations in the ryanodine receptor-1 (RYR1) may cause disorders inherited in an autosomal dominant/recessive fashion. Sequencing of RYR1 in an infant of Ashkenazi Jewish descent with severe hypotonia, dislocation of hip, torticollis and scoliosis, and paternal family history of autosomal dominant mild disease. The child was compound heterozygote for a missense variant c.7042G > A inherited from her father associated with autosomal dominant disease, and a missense variant of unknown significance c.5309C > T inherited from an asymptomatic mother. This case raises the possibility of a dominant disease complicated by a second variant in the other allele serving as a modifier.

Congenital muscular dystrophy type-1A (Lama2-CMD) and Duchenne muscular dystrophy (DMD) result from deficiencies of laminin-α2 and dystrophin proteins, respectively. Although both proteins strengthen the sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA-deep sequencing (RNA-Seq) of dy2J/dy2J, Lama2-CMD mouse model, skeletal muscle at 8 weeks of age to elucidate disease pathophysiology. This study is the first report of dy2J/dy2J model whole transcriptome profile. RNA-Seq of the mdx mouse model of DMD and wild-type (WT) mouse was carried as well in order to enable a novel comparison of dy2J/dy2J to mdx. A large group of shared differentially expressed genes (DEGs) was found in dy2J/dy2J and mdx models (1834 common DEGs, false discovery rate [FDR]

Glatiramer acetate (GA), an agent modulating the immune system, has been shown to cause significantly improved mobility and hind limb muscle strength in the dyLeft sciatic-tibial motor nerve conduction studies were performed on wild type (WT) mice (n = 10), control dyControl dyIn this study we show for the first time improvement in motor nerve conduction velocity of LAMA2-CMD dyThis study suggests a possible therapeutic effect of glatiramer acetate on hereditary peripheral neuropathy in this laminin-α2 disorder.

Objectives To identify the minocycline anti-inflammatory and antiapoptotic mechanisms through which it is believed to exert spinal cord protection during aortic occlusion in the rabbit model. Design An animal model of aortic occlusion-related spinal cord ischemia. Randomized study with a control group and pre-ischemia and post-ischemia escalating doses of minocycline to high-dose minocycline in the presence of either hyperglycemia, a pro-apoptotic maneuver, or wortmannin, a specific phosphatidylinositol 3-kinase antagonist. Setting Tertiary medical center and school of medicine laboratory. Participants Laboratory animals–rabbits. Interventions Balloon obstruction of infrarenal aorta introduced via femoral artery incision. Results Severe hindlimb paralysis (mean Tarlov score 0.36±0.81 out of 3) was observed in all the control group animals (9 of 11 with paraplegia and 2 of 11 with paraparesis) compared with 11 of 12 neurologically intact animals (mean Tarlov score 2.58±0.90 [p = 0.001 compared with control]) in the high-dose minocycline group. This protective effect was observed partially during a state of hyperglycemia and was completely abrogated by wortmannin. Minocycline administration resulted in higher neurologic scores (p = 0.003) and a shift to viable neurons and more apoptotic-stained nuclei resulting from reduced necrosis (p = 0.001). Conclusions In a rabbit model of infrarenal aortic occlusion, minocycline effectively reduced paraplegia by increasing the number of viable neurons in a dose-dependent manner. Its action was completely abrogated by inhibiting the phosphatidylinositol 3-kinase pathway and was inhibited partially by the pro-apoptotic hyperglycemia maneuver, indicating that the activation of cell salvage pathways and mitochondrial sites are possible targets of minocycline action in an ischemic spinal cord.

Cerebral edema leading to elevated intracranial pressure (ICP) is a fundamental concern after severe traumatic brain injury (TBI), stroke, and severe acute hyponatremia. We describe a swine model of water intoxication and its cerebral histological and physiological sequela. We studied female swine weighing 35–45 kg. Four serum sodium intervals were designated: baseline, mild, moderate, and severe hyponatremia attained by infusing hypotonic saline. Intracranial fluid injections were performed to assess intracranial compliance. At baseline and following water intoxication wedge biopsy was obtained for pathological examination and electron microscopy. We studied 8 swine and found an increase in ICP that was strongly related to the decrease in serum sodium level. Mean ICP rose from a baseline of 6 ± 2 to 28 ± 6 mm Hg during severe hyponatremia, while cerebral perfusion pressure (CPP) decreased from 72 ± 10 to 46 ± 11 mm Hg. Brain tissue oxygen tension (PbtO2) decreased from 18.4 ± 8.9 to 5.3 ± 3.0 mm Hg. Electron microscopy demonstrated intracellular edema and astrocytic foot process swelling following water intoxication. With severe hyponatremia, 2 cc intracranial fluid injection resulted in progressively greater ICP dose, indicating a worsening intracranial compliance. Our model leads to graded and sustained elevation of ICP, lower CPP, and decreased PbtO2, all of which cross clinically relevant thresholds. Intracranial compliance worsens with increased cerebral swelling. This model may serve as a platform to study which therapeutic interventions best improve the cerebral physiological profile in the face of severe brain edema.

Neuromyelitis optica (NMO) and myasthenia gravis (MG) are autoimmune diseases mediated by autoantibodies against either aquaporin 4 (AQP4) or acetylcholine receptor (AChR), respectively. Recently, we and others have reported an increased prevalence of NMO in patients with MG. To verify whether coexisting autoimmune disease may exacerbate experimental autoimmune MG, we tested whether active immunization with AQP4 peptides or passive transfer of NMO-Ig can affect the severity of EAMG. Injection of either AQP4 peptide or NMO-Ig to EAMG or to naive mice caused increased fatigability and aggravation of EAMG symptoms as expressed by augmented muscle weakness (but not paralysis), decremental response to repetitive nerve stimulation, increased neuromuscular jitter, and aberration of immune responses. Thus, our study shows increased disease severity in EAMG mice following immunization with the NMO autoantigen AQP4 or by NMO-Ig, mediated by augmented inflammatory response. This can explain exacerbation or increased susceptibility of patients with one autoimmune disease to develop additional autoimmune syndrome.

Identification of CD59 p.Cys89Tyr mutation in 5 patients from North-African Jewish origin presenting with chronic inflammatory demyelinating polyradiculoneuropathy like disease and chronic hemolysis, led us to reinvestigate an unsolved disease in 2 siblings from the same origin who died 17 years ago. The two patients carried the same CD59 gene mutation previously described by our group. These children had quiet similar disease course but in addition developed devastating recurrent brain infarctions, retinal and optic nerve involvement. Revising the brain autopsy of one of these patients confirmed the finding of multiple brain infarctions of different ages. CD59 protein expression was missing on brain endothelial cells by immunohistochemical staining. This new data expands the clinical spectrum of CD59 mutations and further emphasizes the need for its early detection and treatment.

Introduction: Nemaline myopathy is a rare disorder characterized by skeletal muscle weakness of varying severity and onset, with the presence of nemaline rods on muscle biopsy. Congenital nemaline body myopathy due to mutations in TNNT1 has hitherto only been described as a result of a single founder mutation in patients of Amish origin and in 2 other individuals with different recessive mutations. Methods: Autozygosity mapping and whole exome sequencing were applied after we identified 9 Palestinian patients from 7 unrelated families who have nemaline myopathy. Results: All patients were homozygous for a novel complex rearrangement of the TNNT1 gene (c.574_577delinsTAGTGCTGT | NM_003283) leading to C-terminal truncation of the protein (p.L203* | NP_003274.3). Their clinical course was remarkable for early respiratory failure and striking stiffness of the cervical spine. Conclusions: This report exemplifies the utility of combining autozygosity mapping and whole exome sequencing and expands the phenotype associated with TNNT1 mutations. Muscle Nerve, 2015

Lissencephaly comprises a heterogeneous group of developmental brain disorders of varying severity, involving abnormal cortical gyration. We studied a highly consanguineous Israeli Moslem family with a lethal form of autosomal recessive lissencephaly with cerebellar hypoplasia (LCH). Using microarray-based homozygosity mapping in the reported family, combined with whole exome sequencing in one affected infant, we identified a homozygous splice site mutation g.IVS8+1G>A in cyclin-dependent kinase 5 (CDK5), causing complete skipping of exon 8, and leading to a frame shift and premature stop codon (p.V162SfsX19). The mutation co-segregated with the disease phenotype in all 29 study participants (4 patients and 25 healthy relatives), and was not identified in 200 ethnically matched control chromosomes. The p.V162SfsX19 mutation causes lack of endogenous CDK5 expression in affected dermal fibroblasts and brain tissue at the mRNA and protein levels, consistent with nonsense-mediated mRNA decay. Functional analysis of the p.V162SfsX19 mutation, using a yeast complementation assay, showed loss-of-function of the mutant CDK5 gene product, thereby implicating its role in the pathogenesis of autosomal recessive LCH in the studied family.

Androgen receptor (AR) gene is mapped to Xq11-12. It functions as a steroid-hormone-activated-transcription-factor. AR splice variants lacking the Ligand-Binding-Domain (LBD), such as AR-V7/AR3, which arise primarily through exon skipping and cryptic exon inclusion, are activated in a ligand-independent mechanism. The association between sex steroid receptors and brain tumors was described since 1983, but in contrast to the well-established oncogenic role played by androgen receptor (AR) in prostate cancer and the growing evidences on its role in breast cancer, the expression and significance of AR in GBM is controversial and poorly studied. Our previous presented data (Lavon, I. et al. CSIG-13. Neuro-Oncology 18, vi43-vi43 (2016)) and our current results have demonstrated for the first time amplification of AR at the DNA, RNA and protein levels in the majority of GBM samples in patients of both sexes. Our AR-RNA expression results were validated in 703 glioblastomas by analysis of several datasets including the TCGA. We also showed that 30% of glioblastomas express the constitutively active AR-splice-variant AR-V7/AR3. Based on the previously reported activation of AR-V7/AR3 by RTKs, including EGFR, we identified that combination therapy with the AR-antagonist Enzalutamide and EGFR inhibitor is more effective than AR-antagonists alone in cell line expressing AR-V7/AR3. A cell cycle analysis of glioma cells treated with Enzalutamide demonstrated a dose-dependent number of cells in sub-G1 phase suggesting apoptosis as the mechanism for cell death. Enzalutamide given orally (20 mg/kg three time per week) to nude mice bearing human gliomas (U78MG) resulted in reduction of 72% in tumor volume (p=0.0027) as compared to mice treated with vehicle. We hope that the results of this study together with continued laboratory efforts will lead to a new approach for the treatment of human glioblastoma.

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by mutations or rearrangements in PLP1. It presents in infancy with nystagmus, jerky head movements, hypotonia and developmental delay evolving into spastic tetraplegia with optic atrophy and variable movement disorders. A clinically similar phenotype caused by recessive mutations in GJC2 is known as Pelizaeus-Merzbacher-like disease. Both genes encode proteins associated with myelin. We describe three siblings of a consanguineous family manifesting the typical infantile-onset Pelizaeus-Merzbacher disease-like phenotype slowly evolving into a form of complicated hereditary spastic paraplegia with mental retardation, dysarthria, optic atrophy and peripheral neuropathy in adulthood. Magnetic resonance imaging and spectroscopy were consistent with a demyelinating leukodystrophy. Using genetic linkage and exome sequencing, we identified a homozygous missense c.399C>G; p.S133R mutation in MAG. This gene, previously associated with hereditary spastic paraplegia, encodes myelin-associated glycoprotein, which is involved in myelin maintenance and glia-axon interaction. This mutation is predicted to destabilize the protein and affect its tertiary structure. Examination of the sural nerve biopsy sample obtained in childhood in the oldest sibling revealed complete absence of myelin-associated glycoprotein accompanied by ill-formed onion-bulb structures and a relatively thin myelin sheath of the affected axons. Immunofluorescence, cell surface labelling, biochemical analysis and mass spectrometry-based proteomics studies in a variety of cell types demonstrated a devastating effect of the mutation on post-translational processing, steady state expression and subcellular localization of myelin-associated glycoprotein. In contrast to the wild-type protein, the p.S133R mutant was retained in the endoplasmic reticulum and was subjected to endoplasmic reticulum-associated protein degradation by the proteasome. Our findings identify involvement of myelin-associated glycoprotein in this family with a disorder affecting the central and peripheral nervous system, and suggest that loss of the protein function is responsible for the unique clinical phenotype.

A 7-week-old infant presented with persistent noisy breathing and aspirations during swallowing. Neurological examination and brain MRI were normal. His 12-year-old brother underwent pneumonectomy at the age of 10 years due to recurrent aspirations leading to severe lung damage. The older brother developed subsequently ophthalmoplegia and nystagmus along with mild weakness of the neck flexors and proximal muscles. Exome analysis revealed homozygosity for a novel truncating mutation p.G800fs27* in the Myosin Heavy Chain 2 (MYH2) gene in both brothers, while parents and an unaffected sibling were heterozygous. A muscle biopsy from the older brother showed absence of type-2 muscle fibers and predominance of type-1 fibers. The aspirations causing pneumonia likely result from weakness of the laryngeal muscles, normally rich in type-2 fibers. The findings expand the phenotypic spectrum of MYH2 deficiency. MYH2 mutations should be included in the differential diagnosis of infants presenting with recurrent aspirations.

Tumefactive demyelination (TD) is a solitary cerebral demyelinating lesion clinically and radiologically mimicking brain tumors. It can occur in isolation or may be rarely associated with other demyelinating diseases. The underlying pathogenic mechanisms are unknown. We present the first report of TD following in-vitro fertilization (IVF) in a 36-year-old healthy woman who developed subacute right hemiparesis shortly after a scheduled IVF cycle. Evaluation revealed left hemispheric space-occupying lesion pathologically diagnosed as TD. Treatment with intravenous methylprednisolone promptly resulted in a clinical and radiological improvement maintained thereafter. This report confirms and expands the spectrum of inflammatory demyelinating conditions associated with IVF and suggests possible hormonal influence in the development of TD.

Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild-to-moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe for the first time the clinical and morphological characteristics of recessive myosin IIa myopathy associated with MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild-to-moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated small or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that mild muscle weakness and ophthalmoplegia in combination with muscle biopsy demonstrating small or absent type 2A muscle fibers are the hallmark of recessive myopathy associated with MYH2 mutations.

Congenital myopathies are heterogeneous inherited diseases of muscle characterized by a range of distinctive histologic abnormalities. We have studied a consanguineous family with congenital myopathy. Genome-wide linkage analysis and whole-exome sequencing identified a homozygous non-sense mutation in 3-hydroxyacyl-CoA dehydratase 1 (HACD1) in affected individuals. The mutation results in non-sense mediated decay of the HACD1 mRNA to 31% of control levels in patient muscle and completely abrogates the enzymatic activity of dehydration of 3-hydroxyacyl-CoA, the third step in the elongation of very long-chain fatty acids (VLCFAs). We describe clinical findings correlated with a deleterious mutation in a gene not previously known to be associated with congenital myopathy in humans. We suggest that the mutation in the HACD1 gene causes a reduction in the synthesis of VLCFAs, which are components of membrane lipids and participants in physiological processes, leading to congenital myopathy. These data indicate that HACD1 is necessary for muscle function.

Resume Contexte La maladie de Behcet (MB) et la maladie de Kimura (MK) sont deux maladies inflammatoires frequentes au Japon mais rares en Israel. Recemment, nous avons observe une famille au sein de laquelle trois membres de la fratrie etaient atteints d’une MB et un de leur frere avait une MK. Cette association familiale souleve la question d’un facteur genetique predisposant commun a ces deux maladies. Objectif L’objectif de cette etude a ete de decrire cette famille et de rechercher un allele predisposant commun a la MB et a la MK, au sein du gene de l’IL-10 dans la mesure ou plusieurs polymorphismes mononucleotidiques (SNP) de ce gene sont connus pour etre associes a la MB. Methodes Trois membres de la fratrie atteints de MB et un de leur frere atteint de MK ont fait l’objet d’un examen clinique. L’ADN genomique a ete prepare a partir d’echantillons sanguins preleves chez les neuf membres de la famille. L’ADN a ete genotype pour les variations de sequences de six SNP sur le gene de l’IL-10. Conclusions Les SNP du gene de l’IL-10, connus comme facteurs de susceptibilite pour la MB, n’etaient pas associes a la MB dans notre famille. La question d’une susceptibilite genetique commune a la MK et a la MB necessite de nouvelles etudes.

CD59 deficiency is a common finding in RBCs and WBCs in patients with chronic hemolysis suffering from paroxysmal nocturnal hemoglobinuria in which the acquired mutation in the PIGA gene leads to membrane loss of glycosylphosphatidylinositol-anchored membrane proteins, including CD59. The objective of the present study was to elucidate the molecular basis of childhood familial chronic Coombs-negative hemolysis and relapsing polyneuropathy presenting as chronic inflammatory demyelinating polyradiculoneuropathy in infants of North-African Jewish origin from 4 unrelated families. A founder mutation was searched for using homozygosity mapping followed by exome sequencing. The expression of CD59, CD55, and CD14 was examined in blood cells by flow cytometry followed by Western blot of the CD59 protein. A homozygous missense mutation, p.Cys89Tyr in CD59, was identified in all patients. The mutation segregated with the disease in the families and had a carrier rate of 1:66 among Jewish subjects of North-African origin. The mutated protein was present in the patients' cells in reduced amounts and was undetectable on the membrane surface. Based on the results of the present study, we conclude that the Cys89Tyr mutation in CD59 is associated with a failure of proper localization of the CD59 protein in the cell surface. This mutation is manifested clinically in infancy by chronic hemolysis and relapsing peripheral demyelinating disease.

Background Behcet's disease (BD) and Kimura's disease (KD) are two inflammatory diseases commonly found in Japan. In Israel, both diseases are quite rare. Recently, we encountered a family whose three siblings suffer from BD and an additional brother suffers from KD. This observation raised the question as to whether both diseases have a common underlying genetic basis. Aim of the study To describe this unique family and to search for possible common alleles in the IL10 gene between BD and KD, as several SNPs in this gene are known to be associated with BD. Methods Three BD siblings and their brother with KD were interviewed and examined. Genomic DNA was prepared from blood samples taken from all nine members of the family. The DNA was genotyped for sequence variations of six SNPs on the IL10 gene. Results The IL10 SNPs did not segregate with BD and KD suggesting that there was no association between the IL10 gene and these diseases in this family. Conclusions SNPs in the IL10 gene shown to be susceptibility factors in adult BD were not associated with BD in this family. The question regarding a possible common genetic basis for KD and BD requires further investigation.

Juvenile xanthogranuloma (JXG) is primarily a benign cutaneous disorder of non-Langerhans hystiocytic proliferation. Systemic involvement occurs in 4% of patients; isolated central nervous system (CNS) lesions are rare. We report solitary CNS-JXG lesions in two patients. A 3.5-year-old boy with a parietal-occipital lesion underwent total resection with no surgical morbidity and no recurrence at 16-month follow-up. A 3.5-year-old girl underwent subtotal resection of a tumor extending from the left Meckel's cave and invading the cavernous sinus and left orbit with extensive cranial nerve involvement. Tumor regrowth with leptomeningeal spread at 9-month and 12-month follow-up was managed with steroids and chemotherapy (vinblastine and later cladribine). We present our experience and review the literature pertaining to rare reports of solitary CNS-JXG.

There is limited data on the genetic origin and natural history of cerebellar liponeurocytoma. To the best of our knowledge there has been only one report of a familial presentation of this rare entity. We report a 72-year-old female with a posterior fossa tumor presenting with progressive cerebellar signs and symptoms. The patient underwent total tumor resection via an uncomplicated sub-occipital craniotomy. Histopathologic examination was diagnostic for cerebellar liponeurocytoma. Her sister was previously treated for a similar tumor. Our report provides further evidence for the possible existence of a hereditary abnormality predisposing afflicted families to cerebellar liponeurocytoma development.