Twenty-five years after our identification of the genetic defect in GNE Myopathy (GNEM), we review the current state-of-affairs in the research of this unique myopathy. In this narrative review, we describe the clinical aspects of this myopathy, the genetics of this muscle disorder, the biochemistry of the GNE enzyme, and the animal models that have been developed. We critically discuss the accumulating scientific and clinical data that show that hyposialylation cannot be the sole explanation for the disease pathomechanism. The negative or minimal effects of sialic acid supplementation in clinical therapy trials of GNEM call for a re-evaluation of future planned trials. We review the known facts and the current enigmas as well as research controversies in this field. We also discuss the prospects for further basic research, as a reliable animal model of GNEM is lacking, and future genetic therapy of this myopathy.

Background:Glutamate is the major excitatory neurotransmitter in the nervous system, common in neuromuscular junctions, and with abnormally reduced levels in several muscle diseases. Glutamate receptor AMPA GluR3, encoded by the GRIA3 gene, has important neurophysiological roles in regulation of neural networks, sleep, and breathing. GluR3 deletion or abnormal function increases the susceptibility to seizures and disrupts oscillatory networks of sleep, breathing, exploratory activity, and motor coordination. Questions:Do human skeletal muscle cells express GluR3? Are they activated by glutamate? Do autoimmune GluR3B antibodies of Nodding Syndrome (NS) patients, and/or other intractable epilepsy patients, that bind and damage neural cells, also bind and affect skeletal muscle cells? Results:We discovered several original findings: 1) Human primary skeletal muscle cells (myoblasts) express GluR3 RNA and protein, evident by PCR and immunostaining, 2) glutamate (10-8-10-5M) increases intracellular sodium in human skeletal muscle cells and increases muscle cell number (probably by inducing muscle cell proliferation), 3) AMPA and NMDA increase intracellular sodium in skeletal muscle cells, 4) GluR3B monoclonal antibody binds skeletal muscle cells and increases their number, 5) autoimmune affinity-purified GluR3B antibodies of epileptic NS patients, suffering from nodding due to loss of muscle tone and muscle wasting, bind skeletal muscle cells, 6) purified IgGs rich in autoimmune GluR3B antibodies of intractable epilepsy patients bind and kill skeletal muscle cells. Possible implications:Together, the novel findings in this study may have various important implications on muscle physiology and pathology and call for continuation studies on diverse physiological, pathological and therapeutic topics. Meanwhile, we raise few hypotheses: 1) GluR3 has an important physiological role in muscle cells and motor function, 2) impaired GluR3 function (due to genetic/epigenetic/autoimmune/infectious/inflammatory factors?) can cause muscle impairments and motor problems, 3) glutamate, by direct activation of GluR3 and/or other GluRs expressed in skeletal muscle cells, can beneficially affect muscle cell survival, growth, and function, 3) Glutamate, iGluR agonists, and/or GluR3B mAb may have therapeutic effects for muscle diseases, injuries, and age-related sarcopenia, 4) autoimmune GluR3B antibodies of NS patients and/or other epilepsy patients may bind GluR3 in muscle cells, damage these cells, and induce muscle dysfunction and motor problems.

Background: GNE Myopathy is a unique recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, caused by mutations in the GNE gene which is a key enzyme in the biosynthesis of sialic acid. To date, the precise pathophysiology of the disease is not well understood and no reliable animal model is available. Gne KO is embryonically lethal in mice. Objective: To gain insights into GNE function in muscle, we have generated an inducible muscle Gne KO mouse. To minimize the contribution of the liver to the availability of sialic acid to muscle via the serum, we have also induced combined Gne KO in liver and muscle. Methods: A mouse carrying loxp sequences flanking Gne exon3 was generated by Crispr/Cas9 and bred with a human skeletal actin (HSA) promoter driven CreERT mouse. Gne muscle knock out was induced by tamoxifen injection of the resulting homozygote GneloxpEx3loxp/HSA Cre mouse. Liver Gne KO was induced by systemic injection of AAV8 vectors carrying the Cre gene driven by the hepatic specific promoter of the thyroxine binding globulin gene. Results: Characterization of these mice for a 12 months period showed no significant changes in their general behaviour, motor performance, muscle mass and structure in spite of a dramatic reduction in sialic acid content in both muscle and liver. Conclusions: We conclude that post weaning lack of Gne and sialic acid in muscle and liver have no pathologic effect in adult mice. These findings could reflect a strong interspecies versatility, but also raise questions about the loss of function hypothesis in Gne Myopathy. If these findings apply to humans they have a major impact on therapeutic strategies.

GNE myopathy is caused by bi allelic recessive mutations in the GNE gene. The largest identified cohort of GNE myopathy patients carries a homozygous mutation- M743T (the "Middle Eastern" mutation). More than 160 such patients in 67 families have been identified by us. Mean onset in this cohort is 30 years (range 17-48) with variable disease severity. However, we have identified two asymptomatic females, homozygous for M743T in two different families, both with affected siblings. The first showed no myopathy when examined at age 76 years. The second has no sign of disease at age 60 years. Since both agreed only for testing of blood, we performed exome and RNA sequencing of their blood and that of their affected siblings. Various filtering layers resulted in 2723 variant loci between symptomatic and asymptomatic individuals, representing 1364 genes. Among those, 39 genes are known to be involved in neuromuscular diseases, and only in two of them the variant is located in the proper exon coding region, resulting in a missense change. Surprisingly, only 27 genes were significantly differentially expressed between the asymptomatic and the GNE myopathy affected individuals, with three overexpressed genes overlapping between exome and RNA sequencing. Although unable to unravel robust candidate genes, mostly because of the very low number of asymptomatic individuals analyzed, and because of the tissue analyzed (blood and not muscle), this study resulted in relatively restricted potential candidate protective genes, emphasizing the power of using polarized phenotypes (completely asymptomatic vs clearly affected individuals) with the same genotype to unmask those genes which could be used as targets for disease course modifiers.

GNE Myopathy is a unique neuromuscular adult onset disease characterized by slowly progressive distal and proximal muscle weakness, caused by recessive mutations in GNE. Although the function of this protein is known to be a key enzyme in the biosynthesis of sialic acid, no clear definite explanation has been provided to account for the muscle atrophic pathology. The lack of animal models severely impair the comprehensive understanding of GNE function, and, more importantly, prevents the development of an efficient system to assess any kind of treatment. Since a complete knockout of the Gne gene is embryonically lethal, we have recently generated an inducible muscle specific Gne knockout (KO) conditional mouse, where Gne can be knocked out postnatally, after Cre activation. Since the liver is the main source of sialylated glycoconjugates in the serum, that could be a source of sialic acid supply in the organism, in particular muscle, we generated double tissue post weaning Gne KO in both muscle and liver, to minimize the potential supply of sialic acid to muscle through the serum. Indeed 4 weeks post Cre induction, Gne abrogation at both DNA and protein levels is noticeable in the targeted tissues, resulting in a dramatic reduction of sialic acid levels in these tissues. However, these mice show no changes in survival and very mild changes in muscle function, motility, saturated oxygen levels and histology. No correlation between sialic acid content and muscle defects was observed. Interestingly, Gne KO in liver, which does lower dramatically sialic acid levels, does not affect the viability of the mice or their behavior and performance. This could suggest that sialic acid is not essential in post weaning mice. However, it could be that some minimal residual sialic acid potentially supplied from other tissues, is enough to support these mice life. Therefore, a post weaning whole body Gne KO should be generated to answer this question and to potentially produce a mouse with muscle pathology. Such a model could provide better insights for Gne functions in muscle and could eventually be used as a platform for therapy evaluation for this crippling disease. GNE Myopathy is a unique neuromuscular adult onset disease characterized by slowly progressive distal and proximal muscle weakness, caused by recessive mutations in GNE. Although the function of this protein is known to be a key enzyme in the biosynthesis of sialic acid, no clear definite explanation has been provided to account for the muscle atrophic pathology. The lack of animal models severely impair the comprehensive understanding of GNE function, and, more importantly, prevents the development of an efficient system to assess any kind of treatment. Since a complete knockout of the Gne gene is embryonically lethal, we have recently generated an inducible muscle specific Gne knockout (KO) conditional mouse, where Gne can be knocked out postnatally, after Cre activation. Since the liver is the main source of sialylated glycoconjugates in the serum, that could be a source of sialic acid supply in the organism, in particular muscle, we generated double tissue post weaning Gne KO in both muscle and liver, to minimize the potential supply of sialic acid to muscle through the serum. Indeed 4 weeks post Cre induction, Gne abrogation at both DNA and protein levels is noticeable in the targeted tissues, resulting in a dramatic reduction of sialic acid levels in these tissues. However, these mice show no changes in survival and very mild changes in muscle function, motility, saturated oxygen levels and histology. No correlation between sialic acid content and muscle defects was observed. Interestingly, Gne KO in liver, which does lower dramatically sialic acid levels, does not affect the viability of the mice or their behavior and performance. This could suggest that sialic acid is not essential in post weaning mice. However, it could be that some minimal residual sialic acid potentially supplied from other tissues, is enough to support these mice life. Therefore, a post weaning whole body Gne KO should be generated to answer this question and to potentially produce a mouse with muscle pathology. Such a model could provide better insights for Gne functions in muscle and could eventually be used as a platform for therapy evaluation for this crippling disease.

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.

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression. Second, no reliable animal model is available for the disease and cellular models from GNE myopathy patients’ muscle cells (expressing the mutated protein) are less informative than expected. In order to broaden our knowledge on GNE functions in muscle, we have taken advantage of the CRISPR/Cas9 method for genome editing to first, add a tag to the endogenous Gne gene in mouse, allowing the determination of the spatiotemporal expression of the protein in the organism, using well established and reliable antibodies against the specific tag. In addition we have generated a Gne knock out murine muscle cell lineage to identify the events resulting from the total lack of the protein. A thorough multi-omics analysis of both cellular systems including transcriptomics, proteomics, phosphoproteomics and ubiquitination, unraveled novel pathways for Gne, in particular its involvement in cell cycle control and in the DNA damage/repair pathways. The elucidation of fundamental mechanisms of Gne in normal muscle may contribute to the identification of the disrupted functions in GNE myopathy, thus, to the definition of novel biomarkers and possible therapeutic targets for this disease.

GNE Myopathy is a rare, recessively inherited neuromuscular worldwide disorder, caused by a spectrum of bi-allelic mutations in the human GNE gene. GNE encodes a bi-functional enzyme responsible for the rate-limiting step of sialic acid biosynthesis pathway. However, the process in which GNE mutations lead to the development of a muscle pathology is not clear yet. Cellular and mouse models for GNE Myopathy established to date have not been informative. Further, additional GNE functions in muscle have been hypothesized. In these studies, we aimed to investigate gne functions using zebrafish genetic and transgenic models, and characterized them using macroscopic, microscopic, and molecular approaches. We first established transgenic zebrafish lineages expressing the human GNE cDNA carrying the M743T mutation, driven by the zebrafish gne promoter. These fish developed entirely normally. Then, we generated a gne knocked-out (KO) fish using the CRISPR/Cas9 methodology. These fish died 8–10 days post-fertilization (dpf), but a phenotype appeared less than 24 h before death and included progressive body axis curving, deflation of the swim bladder and decreasing movement and heart rate. However, muscle histology uncovered severe defects, already at 5 dpf, with compromised fiber organization. Sialic acid supplementation did not rescue the larvae from this phenotype nor prolonged their lifespan. To have deeper insights into the potential functions of gne in zebrafish, RNA sequencing was performed at 3 time points (3, 5, and 7 dpf). Genotype clustering was progressive, with only 5 genes differentially expressed in gne KO compared to gne WT siblings at 3 dpf. Enrichment analyses of the primary processes affected by the lack of gne also at 5 and 7 dpf point to the involvement of cell cycle and DNA damage/repair processes in the gne KO zebrafish. Thus, we have established a gne KO zebrafish lineage and obtained new insights into gne functions. This is the only model where GNE can be related to clear muscle defects, thus the only animal model relevant to GNE Myopathy to date. Further elucidation of gne precise mechanism-of-action in these processes could be relevant to GNE Myopathy and allow the identification of novel therapeutic targets.

We report the long-term response to bariatric surgery in a singular family of four adolescents with severe obesity (41–82 kg/m2), homozygous for the C271R loss-of-function mutation in the melanocortin 4 receptor (MC4R), and three adults heterozygous for the same mutation. All patients had similar sociodemographic backgrounds and were followed for an average of 7 years. Three of the four homozygous patients regained their full weight (42–77 kg/m2), while the fourth lost weight but remained obese with a body mass index of 60 kg/m2. Weight regain was associated with relapse of most comorbidities, yet hyperglycemia did not relapse or was delayed. A1c levels were reduced in homozygous and heterozygous patients. The long-term follow-up data on this very unique genetic setting show that weight loss and amelioration of obesity following bariatric surgery require active MC4R signaling, while the improvement in glycemia is in part independent of weight loss. The study validates animal models and demonstrates the importance of biological signaling in the regulation of weight, even after bariatric surgery.

We conducted an observational long-term study with a median follow-up of 102 months on the outcomes of bariatric surgery in a family that carries a deleterious mutation in the melanocortin 4 receptor (MC4R) gene. In homozygous patients, bariatric surgery led to transient weight-loss and improvements in associated comorbidities, but in the long-term did not resolve morbid obesity: median presurgical/final body mass index 71.2/64.3 kg/m2. Glycemia however was improved despite regaining: median presurgical/final fasting blood glucose 218/101 mg/dL. Heterozygous patients responded normally to bariatric surgery. This unique case series, the largest and longest of its kind, demonstrates that bariatric surgery is dependent on MC4R signaling for sustained weight-loss, but its anti-diabetic effect is also independent of weight-loss. Bariatric surgery provides temporary relief in comorbidities for super-obese patients, may improve long-term glycemic control, and should be considered as a metabolic treatment for such genetic cases.

GNE Myopathy is a recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, and a typical muscle pathology. Although GNE, which is the mutated gene in the disease, is well known as the key enzyme in the biosynthesis pathway of sialic acid, the pathophysiological pathway leading from GNE mutations to the muscle phenotype in GNE Myopathy is still unclear. The obvious hypothesis of impaired sialylation in patients' skeletal muscle as the cause of the disease is still controversial. In the present study we have investigated whether a distinctive altered pattern of sialylation in GNE Myopathy cultured muscle cells could be attributed to a specific glycoconjugate. Mass spectrometry based glycomic methodologies have been utilized to assess the sialylation level of protein N- and O-linked glycans and glycolipid derived glycans from patient and matched control samples. No consistent change in sialylation was detected in glycoconjugates. These results suggest potential additional roles for GNE that could account for the disease pathology.

Background: Mutations in GNE cause a recessive, adult onset myopathy characterized by slowly progressive distal and proximal muscle weakness. Knock-in mice carrying the most frequent mutation in GNE myopathy patients, Gne M743T/M743T , usually die few days after birth from severe renal failure, with no muscle phenotype. However, a spontaneous sub-colony remains healthy throughout a normal lifespan without any kidney or muscle pathology. Objective: We attempted to decipher the molecular mechanisms behind these phenotypic differences and to determine the mechanisms preventing the kidney and muscles from disease. Methods: We analyzed the transcriptome and proteome of kidneys and muscles of sick and healthy Gne M743T/M743T mice. Results: The sick Gne M743T/M743T kidney was characterized by up-regulation of extra-cellular matrix degradation related processes and by down-regulation of oxidative phosphorylation and respiratory electron chain pathway, that was also observed in the asymptomatic muscles. Surprisingly, the healthy kidneys of the Gne M743T/M743T mice were characterized by up-regulation of hallmark muscle genes. In addition the asymptomatic muscles of the sick Gne M743T/M743T mice showed upregulation of transcription and translation processes. Conclusions: Overexpression of muscle physiology genes in healthy Gne M743T/M743T mice seems to define the protecting mechanism in these mice. Furthermore, the strong involvement of muscle related genes in kidney may bridge the apparent phenotypic gap between GNE myopathy and the knock-in Gne M743T/M743T mouse model and provide new directions in the study of GNE function in health and disease. Keywords GNE myopathy , GNE mouse model , HIBM , RNA-Seq , proteomics

Background: Mutations in GNE cause a recessive, adult onset myopathy characterized by slowly progressive distal and proximal muscle weakness.Knock-in mice carrying the most frequent mutation in GNE myopathy patients, Gne M743T/M743T , usually die few days after birth from severe renal failure, with no muscle phenotype.However, a spontaneous sub-colony remains healthy throughout a normal lifespan without any kidney or muscle pathology.Objective: We attempted to decipher the molecular mechanisms behind these phenotypic differences and to determine the mechanisms preventing the kidney and muscles from disease.Methods: We analyzed the transcriptome and proteome of kidneys and muscles of sick and healthy Gne M743T/M743T mice.Results: The sick Gne M743T/M743T kidney was characterized by up-regulation of extra-cellular matrix degradation related processes and by down-regulation of oxidative phosphorylation and respiratory electron chain pathway, that was also observed in the asymptomatic muscles.Surprisingly, the healthy kidneys of the Gne M743T/M743T mice were characterized by up-regulation of hallmark muscle genes.In addition the asymptomatic muscles of the sick Gne M743T/M743T mice showed upregulation of transcription and translation processes.Conclusions: Overexpression of muscle physiology genes in healthy Gne M743T/M743T mice seems to define the protecting mechanism in these mice.Furthermore, the strong involvement of muscle related genes in kidney may bridge the apparent phenotypic gap between GNE myopathy and the knock-in Gne M743T/M743T mouse model and provide new directions in the study of GNE function in health and disease.

GNE myopathy is an autosomal recessive adult onset disorder caused mainly by missense mutations in the GNE gene. Although GNE function in the biosynthesis pathway of sialic acid is well established, the mechanism leading from GNE mutation to this myopathy is unclear. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, it is crucial to monitor GNE expression, tissue distribution and subcellular localization that might shed some light on GNE additional function(s) in muscle. To date, no reliable antibodies can detect the endogenous levels of the GNE protein. Therefore we have targeted the mouse endogenous Gne locus with two different independent tags to monitor Gne's specific expression and subcellular localization. We used the crRNA:tracrRNA Crispr/Cas complex strategy to engineer the carboxi-terminus of the Gne gene and introduced either a 3X FLAG or a 3X HA tag in mouse zygotes using a ssDNA donor for homologous recombination. The resulting mice were analyzed by western blot with specific antibody for each tag. A Gne product of ∼80 kDa, as expected, was detected in the various tissues examined. The endogenous Gne was successfully detected even in tissues with low expression level, like skeletal muscles. BothGne alleles were found to be expressed, resulting in a 2-fold increase in protein detection in homozygous versus heterozygous Gne tagged mice. These Gne-tagged mice will be a useful platform for the follow up of the spatio-temporal expression of Gne, for comparison between wild type and mutant protein expression and for the establishment of a GNE myopathy model. Further, it can be used as the basis of a conditional knock-out mice that might shed some light about the Gne mechanisms leading to the disease in muscle.

GNE Myopathy is a recessive adult-onset myopathy, characterized by slowly progressive distal and proximal muscle weakness. A knock in mouse carrying the most frequent mutation in patients, GneM743T/M743T, usually dies few days after birth from severe renal failure, with no muscle phenotype. In contrast, a spontaneously established subcolony of these GneM743T/M743Tmice lacks any kidney pathology, muscle weakness or histopathology changes, even at very advanced age. In order to elucidate the molecular mechanisms behind the phenotypic differences of sick, healthy-mutated and wild-type kidney, we have established and analyzed the transcriptome and proteome of sick and healthy kidneys in these GneM743T/M743T mice. Notably, the differentially up and down regulated genes and the subsequent enriched pathways were consistent in RNA and protein,and resulted from overlapping genes. The sick GneM743T/M743T kidney was characterized by up-regulation of cytoskeleton organization and extra-cellular matrix related processes and by down-regulation of metabolic processes. These findings are in line with previous data from GNE Myopathy patients muscle cells. In contrast, the main cluster identified in healthy kidneys of healthy GneM743T/M743T mice, involved hallmark muscle genes. Overexpression of these genes may define the protecting mechanism occurring in the healthy GneM743T/M743T kidney. Further, these findings strongly support the hypothesis that GNE has functions specific to muscle. The strong involvement of muscle related genes in kidney may bridge the apparent phenotypic gap between GNE Myopathy and the knock in GneM743T/M743T mouse model. Most importantly it may give a clue for the protective mechanisms occurring in the muscles which remain uncompromised in the usual GneM743T/M743T knock in mice, thus providing new directions in the study of Gne function in health and disease and new possibilities for modulating the disease phenotype in GNE myopathy patients.

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] < 0.05). Enrichment pathway analysis using ingenuity pathway analysis showed enrichment of inflammation, fibrosis, cellular movement, migration and proliferation of cells, apoptosis and necrosis in both mouse models (P-values 3E-10-9E-37). Via canonical pathway analysis, actin cytoskeleton, integrin, integrin-linked kinase, NF-kB, renin-angiotensin, epithelial-mesenchymal transition, and calcium signaling were also enriched and upregulated in both models (FDR < 0.05). Interestingly, significant downregulation of Pax7 was detected in dy2J/dy2J compared to upregulation of this key regeneration gene in mdx mice. Pax3 and Mamstr genes were also downregulated in dy2J/dy2J compared to WT mice. These results may explain the distinct disease course and severity in these models. While the mdx model at that stage shows massive regeneration, the dy2J/dy2J shows progressive dystrophic process. Our data deepen our understanding of the molecular pathophysiology and suggest new targets for additional therapies to upregulate regeneration in Lama2-CMD.

Purpose: To improve lymph node (LN) staging in patients with colon cancer (CC).The present study describes the selection of CC-specific miRNAs and assesses their utility as a micro metastases detection assay.Methods: 30 miRNAs have been selected from a microarray assay and 16 miRNAs from database mining for their specific upregulation in colon cancer tissues as compared to normal adjacent tissues.Differential expression was validated by RT-qPCR in a larger cohort of samples (n=20) and compared to normal lymphatic tissues (n=6) and normal peripheral blood lymphocytes (PBLs, n=14).The selected miRNA panel was then used for the screening of 84 lymph nodes (LN) obtained from colon cancer patients (n=20) Results: After validation, a panel of 8 miRNAs was found to be significantly upregulated in CC compared to normal adjacent tissues and to normal lymphatic tissues: miR-96, miR-183, miR-194, miR-200a, miR-200b, miR-200c, miR-203 and miR-429.A total of 84 LNs were analysed: 12 LN metastases were detected by H&E, 18 by CK staining whereas 32 were detected by the CC-specific miRNA analysis.This represents an increase of 40% in the detection rate. Conclusion:This study demonstrated the ability of a CC-specific 8 miRNA panel in detecting micro metastases in CC patients.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE. It is possible that this decrease eventually affects the interaction, thus causing functional imbalance of this complex in skeletal muscle that could contribute to the myopathy phenotype. In vivo, using bi-molecular fluorescent complementation, we show the specific binding of the two proteins inside the intact cell, in a unique interaction pattern between the two partners. This interaction is disrupted in the absence of the C-terminal calmodulin-like domain of α-actinin 2, which is altered in α-actinin 1. Moreover, the binding of GNE to α-actinin 2 prevents additional binding of α-actinin 1 but not vice versa. These results suggest that the interaction between GNE and α-actinin 1 and α-actinin 2 occur at different sites in the α-actinin molecules and that for α-actinin 2 the interaction site is located at the C-terminus of the protein.

Duchenne muscular dystrophy (DMD) caused by mutations in Dystrophin gene and congenital muscular dystrophy type 1A (MDC1A) caused by mutations in LAMA2, are incurable childhood forms of muscular dystrophy. Although both disorders result from mutations in proteins strengthening the muscle sarcolemma, they are implicated in clinically distinct phenotypes. We used RNA deep sequencing (RNA-Seq) of dy2J/dy2J, MDC1A mouse model, skeletal muscle to elucidate disease pathophysiology. This study is the first report of comparing the transcriptome profile of dy2J/dy2J mouse model to WT. RNA-Seq of the mdx mouse model of DMD and its control was carried as well in order to enable a comparison of the two mouse models. 3844 genes were significantly differentially expressed in mdx versus WT mice (2695 upregulated and 1149 downregulated, FDR (false detection rate) < 0.05) and 2675 genes in dy2J/dy2J versus WT (1727 upregulated and 948 downregulated, FDR < 0.05). 1834 common differentially expressed genes between mdx and dy2J/dy2J were found (1285 upregulated and 549 downregulated, FDR < 0.05). Enrichment pathway analysis using Ingenuity Pathway Analysis (IPA) software of the common differentially expressed genes showed enrichment of inflammation, fibrosis, adhesion, apoptosis, muscle cell structure and metabolism (p-values 3E-10 – 9E-37). Canonical pathways, such as ILK, NF-kB, Wnt, calcium signaling and others were found to be enriched in both models (FDR < 0.05). Our data deepen our understanding of the molecular pathophysiology of each disease in order to select new pathways or targets for additional therapies in CMD and DMD.