Focus on cell therapy to treat corneal endothelial diseases.

The cornea is a multi-layered structure which allows fine refraction and provides both resistance to external insults and adequate transparency. The corneal endothelium ensures stromal hydration, failure of which, such as in Fuchs endothelial corneal dystrophy, after trauma or in aging, may lead to loss of corneal transparency and induce blindness. Currently, no efficient therapeutic alternatives exist except for corneal grafting. Thus corneal tissue engineering represents a valuable alternative approach, which may overcome cornea donor shortage. Several studies describe protocols to isolate, differentiate, and cultivate corneal endothelial cells (CEnCs) in vitro. Two main in vitro strategies can be described: expansion of eye-native cell populations, such as CEnCs, or the production and expansion of CEnCs from non-eye native cell populations, such as induced Pluripotent Stem Cells (iPSCs). The challenge with these cells is to obtain a monolayer of CEnCs on a biocompatible carrier, with a specific morphology (flat hexagonal cells), and with specific functions such as programmed cell cycle arrest. Another issue for this cell culture methodology is to define the adapted protocol (media, trophic factors, timeframe) that can mimic physiological development. Additionally, contamination by other cell types still represents a huge problem. Thus, purification methods, such as Fluorescence Activated Cell Sorting (FACS), Magnetic Ativated Cell Sorting (MACS) or Sedimentation Field Flow Fractionation (SdFFF) are useful. Animal models are also crucial to provide a translational approach for these therapies, integrating macro- and microenvironment influences, systemic hormonal or immune responses, and exogenous interactions. Non-eye native cell graft protocols are constantly improving both in efficacy and safety, with the aim of being the most suitable candidate for corneal therapies in future routine practice. The aim of this work is to review these different aspects with a special focus on issues facing CEnC culture in vitro, and to highlight animal graft models adapted to screen the efficacy of these different protocols.

Oxidative Stress Evaluation in Ischemia Reperfusion Models: Characteristics, Limits and Perspectives.

Ischemia reperfusion injury is a complex process consisting of a seemingly chaotic but actually organized and compartmentalized shutdown of cell function, of which oxidative stress is a key component. Studying oxidative stress, which results in an imbalance between reactive oxygen species (ROS) production and antioxidant defense activity, is a multi-faceted issue, particularly considering the double function of ROS, assuming roles as physiological intracellular signals and as mediators of cellular component damage. Herein, we propose a comprehensive overview of the tools available to explore oxidative stress, particularly in the study of ischemia reperfusion. Applying chemistry as well as biology, we present the different models currently developed to study oxidative stress, spanning the vitro and the silico, discussing the advantages and the drawbacks of each set-up, including the issues relating to the use of in vitro hypoxia as a surrogate for ischemia. Having identified the limitations of historical models, we shall study new paradigms, including the use of stem cell-derived organoids, as a bridge between the in vitro and the in vivo comprising 3D intercellular interactions in vivo and versatile pathway investigations in vitro. We shall conclude this review by distancing ourselves from "wet" biology and reviewing the in silico, computer-based, mathematical modeling, and numerical simulation options: (a) molecular modeling with quantum chemistry and molecular dynamic algorithms, which facilitates the study of molecule-to-molecule interactions, and the integration of a compound in a dynamic environment (the plasma membrane...); (b) integrative systemic models, which can include many facets of complex mechanisms such as oxidative stress or ischemia reperfusion and help to formulate integrated predictions and to enhance understanding of dynamic interaction between pathways.

Hypercholesterolemia-induced increase in plasma oxidized LDL abrogated pro angiogenic response in kidney grafts.

BACKGROUND: Renal transplantation is increasingly associated with the presence of comorbidity factors such as dyslipidemia which could influence the graft outcome. We hypothesized that hypercholesterolemia could affect vascular repair processes and promote post-transplant renal vascular remodeling through the over-expression of the anti-angiogenic thrombospondin-1 interacting with vascular endothelial growth factor-A levels. METHODS: We tested this hypothesis in vitro, in vivo and in a human cohort using (1) endothelial cells; (2) kidney auto-transplanted pig subjected (n = 5) or not (n = 6) to a diet enriched in cholesterol and (3) a renal transplanted patient cohort (16 patients). RESULTS: Cells exposed to oxidized LDL showed reduced proliferation and an increased expression of thrombospondin-1. In pigs, 3 months after transplantation of kidney grafts, we observed a deregulation of the hypoxia inducible factor 1a-vascular endothelial growth factor-A axis induced in cholesterol-enriched diet animals concomitant with an overexpression of thrombospondin-1 and a decrease in cortical microvessel density promoting vascular remodeling. In patients, hypercholesterolemia was associated with decreased vascular endothelial growth factor-A plasma levels during early follow up after renal transplantation and increased chronic graft dysfunction. CONCLUSIONS: These results support a potential mechanism through which a high fat-diet impedes vascular repair in kidney graft and suggest the value of controlling cholesterolemia in recipient even at the early stage of renal transplantation.

Vectisol Formulation Enhances Solubility of Resveratrol and Brings Its Benefits to Kidney Transplantation in a Preclinical Porcine Model.

Current organ shortages have led centers to extend the acceptance criteria for organs, increasing the risk for adverse outcomes. Current preservation protocols have not been adapted so as to efficiently protect these organs. Herein, we target oxidative stress, the key mechanism of ischemia reperfusion injury. Vectisol® is a novel antioxidant strategy based on the encapsulation of resveratrol into a cyclodextrin, increasing its bioavailability. We tested this compound as an additive to the most popular static preservation solutions and machine perfusion (LifePort) in a preclinical pig model of kidney autotransplantation. In regard to static preservation, supplementation improved glomerular filtration and proximal tubular function early recovery. Extended follow-up confirmed the higher level of protection, slowing chronic loss of function (creatininemia and proteinuria) and the onset of histological lesions. Regarding machine perfusion, the use of Vectisol® decreased oxidative stress and apoptosis at the onset of reperfusion (30 min post declamping). Improved quality was confirmed with decreased early levels of circulating SOD (Superoxide Dismutase) and ASAT (asparagine amino transferase). Supplementation slowed the onset of chronic loss of function, as well as interstitial fibrosis and tubular atrophy. The simple addition of Vectisol® to the preservation solution significantly improved the performance of organ preservation, with long-term effects on the outcome. This strategy is thus a key player for future multi-drug therapy aimed at ischemia reperfusion in transplantation.

Subcutaneous cardioverter defibrillator has longer time to therapy but is less cardiotoxic than transvenous cardioverter defibrillator. Study carried out in a preclinical porcine model.

Aims: Totally subcutaneous implantable cardioverter defibrillator (S-ICD) delivers higher shock energy and can have longer time to therapy compared to transvenous implantable cardioverter defibrillator (T-ICD). Aim of the study was to compare time to therapy and to investigate cardiac, cerebral and systemic injuries of S-ICD and T-ICD shocks delivered after ventricular fibrillation (VF) induction. Methods and results: Fourteen pigs were randomly implanted with a S-ICD (n = 7) or a T-ICD (n = 7). Five VF episodes were induced in each pig. For each VF episode, up to two shocks could be delivered by the T-ICD or the S-ICD to terminate the arrhythmia. Cardiac, systemic, and cerebral toxicity were monitored. Mean time to therapy was longer in the S-ICD group compared to the T-ICD group (19[18; 23] s vs. 9 [7; 10] s; P = 0.001, respectively). High-sensitivity troponin T levels were significantly higher in the T-ICD group from 1 to 24 h after the procedure (P ≤ 0.02). Creatine phosphokinase activity levels were significantly higher in the S-ICD group, at 3, 6, and 24 h after the procedure (P ≤ 0.05). Lactate levels were not significantly different between groups. S100 protein level was similar in both groups at 1 h after the procedure and then decreased in the T-ICD group compared to the S-ICD group (P = 0.04). Conclusions: Time to therapy in S-ICD was twice as long as for T-ICD, but didn't induce relevant brain injury. Conversely, S-ICD shocks were less cardiotoxic than T-ICD shocks.

Strategies to optimize kidney recovery and preservation in transplantation: specific aspects in pediatric transplantation.

In renal transplantation, live donor kidney grafts are associated with optimum success rates due to the shorter period of ischemia during the surgical procedure. The current shortage of donor organs for adult patients has caused a shift towards deceased donors, often with co-morbidity factors, whose organs are more sensitive to ischemia-reperfusion injury, which is unavoidable during transplantation. Donor management is pivotal to kidney graft survival through the control of the ischemia-reperfusion sequence, which is known to stimulate numerous deleterious or regenerative pathways. Although the key role of endothelial cells has been established, the complexity of the injury, associated with stimulation of different cell signaling pathways, such as unfolded protein response and cell death, prevents the definition of a unique therapeutic target. Preclinical transplant models in large animals are necessary to establish relationships and kinetics and have already contributed to the improvement of organ preservation. Therapeutic strategies using mesenchymal stem cells to induce allograft tolerance are promising advances in the treatment of the pediatric recipient in terms of reducing/withdrawing immunosuppressive therapy. In this review we focus on the different donor management strategies in kidney graft conditioning and on graft preservation consequences by highlighting the role of endothelial cells. We also propose strategies for preventing ischemia-reperfusion, such as cell therapy.

Diet-induced increase in plasma oxidized LDL promotes early fibrosis in a renal porcine auto-transplantation model.

BACKGROUND: In kidney transplantation, the prevalence of hypercholesterolemia as a co-morbidity factor known to affect graft function, is rising due to the increased number of older donors in response to organ shortage as well as to the hyperlipidemic effects of immunosuppressors in recipient. This study aimed to characterize the effects of hypercholesterolemia on renal graft outcome, investigating the role of oxidized low-density lipoprotein (OxLDL). METHODS: In vivo, we used a porcine preclinical model of renal auto-transplantation modulated by two experimental diets: a normal (n = 6) or a hyperlipidemic diet (n = 5) maintained during the 3 month follow-up after the surgical procedure. Kidney function and OxLDL levels were monitored as well as fibrosis, LOX-1 and TGF beta signaling pathways. In vitro, we used human artery endothelial cells subjected to OxLDL to investigate the TGF beta profibrotic pathway and the role of the scavenger receptor LOX-1. RESULTS: Hyperlipidemic diet-induced increase in plasma OxLDL levels at the time of surgery correlated with an increase in proteinuria 3 months after transplantation, associated with an early graft fibrosis combined with an activation of renal TGF beta signaling. These data suggest a direct involvement of OxLDL in the hyperlipidemic diet-induced activation of the pro-fibrotic TGF beta pathway which seems to be activated by LOX-1 signaling. These results were supported by studies with endothelial cells incubated in culture medium containing OxLDL promoting TGF beta expression inhibited by LOX-1 antibody. CONCLUSIONS: These results implicate OxLDL in the hyperlipidemic diet-promoted fibrosis in transplanted kidneys, suggesting LOX-1 as a potential therapeutic target and reinforce the need to control cholesterol levels in kidney transplant recipients.

Readthrough Activators and Nonsense-Mediated mRNA Decay Inhibitor Molecules: Real Potential in Many Genetic Diseases Harboring Premature Termination Codons.

Nonsense mutations that generate a premature termination codon (PTC) can induce both the accelerated degradation of mutated mRNA compared with the wild type version of the mRNA or the production of a truncated protein. One of the considered therapeutic strategies to bypass PTCs is their "readthrough" based on small-molecule drugs. These molecules promote the incorporation of a near-cognate tRNA at the PTC position through the native polypeptide chain. In this review, we detailed the various existing strategies organized according to pharmacological molecule types through their different mechanisms. The positive results that followed readthrough molecule testing in multiple neuromuscular disorder models indicate the potential of this approach in peripheral neuropathies.

Improvement of Charcot-Marie-Tooth Phenotype with a Nanocomplex Treatment in Two Transgenic Models of CMT1A.

Curcumin has been shown to exert beneficial effects in peripheral neuropathies. Despite its known biological activities, curcumin has unfavorable pharmacokinetics. Its instability has been linked to its failure in clinical trials of curcumin for the treatment of human pathologies. For this reason, we developed curcumin-loaded cyclodextrin/cellulose nanocrystals (NanoCur) to improve its pharmacokinetics. The present study aims to assess the potency of a low dose of NanoCur in 2 Charcot-Marie-Tooth disease type 1A (CMT1A) rodent models at different stages of the disease. The efficiency of NanoCur is also compared to that of Theracurmin (Thera), a commercially available curcumin formulation. The toxicity of a short-term and chronic exposure to the treatment is investigated both in vitro and in vivo, respectively. Furthermore, the entry route, the mechanism of action and the effect on the nerve phenotype are dissected in this study. Overall, the data support an improvement in sensorimotor functions, associated with amelioration in peripheral myelination in NanoCur-treated animals; an effect that was not evident in the Thera-treated group. That was combined with a high margin of safety both in vivo and in vitro. Furthermore, NanoCur appears to inhibit inflammatory pathways that normally include macrophage recruitment to the diseased nerve. This study shows that NanoCur shows therapeutic benefits with minimal systemic toxicity, suggesting that it is a potential therapeutic candidate for CMT1A and, possibly, for other neuropathies.

Role of warm ischemia on innate and adaptive responses in a preclinical renal auto-transplanted porcine model.

BACKGROUND: Deceased after cardiac arrest donor are an additional source of kidney graft to overcome graft shortage. Deciphering the respective role of renal warm and cold ischemia is of pivotal interest in the transplantation process. METHODS: Using a preclinical pig model of renal auto-transplantation, we investigated the consequences of warm and cold ischemia on early innate and adaptive responses as well as graft outcome. Kidneys were subjected to either 60 min-warm ischemia (WI) or auto-transplanted after cold storage for 24 h at 4°C (CS), or both conditions combined (WI+CS). Renal function, immune response and cytokine expression, oxidative stress and cell death were investigated at 3 h, 3 and 7 days (H3, D3 and D7) after reperfusion. At 3 months, we focused on cell infiltration and tissue remodelling. RESULTS: WI + CS induced a delayed graft function linked to higher tubular damage. Innate response occurred at D3 associated to a pro-oxidative milieu with a level dependent on the severity of ischemic injury whereas adaptive immune response occurred only at D7 mainly due to CS injuries and aggravated by WI. Graft cellular death was an early event detected at H3 and seems to be one of the first ischemia reperfusion injuries. These early injuries affect graft outcome on renal function, cells infiltration and fibrosis development. CONCLUSIONS: The results indicate that the severe ischemic insult found in kidneys from deceased after cardiac arrest donor affects kidney outcome and promotes an uncontrolled deleterious innate and adaptive response not inhibited 3 months after reperfusion.

[Ischemia reperfusion control: the key of kidney graft outcome]. / L'ischémie reperfusion - Acteur essentiel du devenir du greffon rénal.

During the transplantation procedure, ischemia reperfusion is an inevitable situation characterized by specific pathophysiological processes, which ultimately act synergistically to create injuries in the graft. These injuries are involved in early graft dysfunctions which promote chronic dysfunction and compromise graft outcome. Progresses in immunosuppressive drug regimens now place ischemia reperfusion injury control at the forefont for innovative therapeutic strategy to improve the quality of the graft. This review details these different processes and its consequences on renal graft function underlying the interest of novel therapeutic strategy.

Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot-Marie-Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene.

Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot-Marie-Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy.

The First Large Deletion of ATL3 Identified in a Patient Presenting with a Sensory Polyneuropathy.

Hereditary sensory neuropathies (HSN) are a heterogenous group of sensory neuropathies. Mutations in ATL3 have been described in patients presenting with hereditary sensory neuropathy IF (HSN1F), a subtype of HSN. Herein, by analyzing targeted-NGS data of a patient presenting with sensory neuropathy symptoms using the CovCopCan bioinformatic tool, we discovered the presence of a deletion of around 3kb in ATL3 from Chr11:63,401,422 to Chr11:63,398,182. This deletion affects ATL3 exons 11 and 12 and could lead to the mutation c.(1036-861_1539+329del), p.(Ala346_Gln513del). In addition, an analysis of the breakpoints' sequences revealed the presence of Alu transposable elements at the position of the breakpoints, which pointed to a possible erroneous recombination event following a non-allelic-homologous-recombination mechanism in this area. Moreover, electronic microscopy analysis of the patient's nerve biopsy revealed a severe rarefaction of the myelinated fibers, a demyelinating-remyelinating process, and an abnormal aspect of the endoplasmic reticulum. These findings suggest that this structural variation could potentially be responsible for the HSN symptoms of the patient. Research of structural variations in ATL3 in numerous other patients presenting similar symptoms should be broadly investigated in order to improve patients' diagnoses.

An analytical procedure for the determination of aluminum used in antiperspirants on human skin in Franz™ diffusion cell.

A local case report of hyperaluminemia (aluminum concentration: 3.88 µmol/L) in a woman using an aluminum-containing antiperspirant for 4 years raises the question of possible transdermal uptake of aluminum salt as a future public health problem. Prior to studying the transdermal uptake of three commercialized cosmetic formulas, an analytical assay of aluminum (Al) in chlorohydrate form (ACH) by Zeeman Electrothermal Atomic Absorption Spectrophotometer (ZEAAS) in a clean room was optimized and validated. This analysis was performed with different media on human skin using a Franz(™) diffusion cell. The detection and quantification limits were set at ≤ 3 µg/L. Precision analysis as within-run (n = 12) and between-run (n = 15-68 days) yield CV ≤ 6%. The high analytic sensitivity (2-3 µg/L) and low variability should allow an in vitro study of the transdermal uptake of ACH.

From Negative to Positive Diagnosis: Structural Variation Could Be the Second Mutation You Are Looking for in a Recessive Autosomal Gene.

Next-generation sequencing (NGS) allows the detection of plentiful mutations increasing the rate of patients getting a positive diagnosis. However, while single-nucleotide variants (SNVs) or small indels can be easily detected, structural variations (SVs) such as copy number variants (CNVs) are often not researched. In Charcot-Marie-Tooth disease (CMT), the most common hereditary peripheral neuropathy, the PMP22-duplication was the first variation detected. Since then, more than 90 other genes have been associated with CMT, with point mutations or small indels mostly described. Herein, we present a personalized approach we performed to obtain a positive diagnosis of a patient suffering from demyelinating CMT. His NGS data were aligned to the human reference sequence but also studied using the CovCopCan software, designed to detect large CNVs. This approach allowed the detection of only one mutation in SH3TC2, the frequent p.Arg954*, while SH3TC2 is known to be responsible for autosomal recessive demyelinating CMT forms. Interestingly, by modifying the standard CovCopCan use, we detected the second mutation of this patient corresponding to a 922 bp deletion in SH3TC2 (Chr5:148,390,609-Chr5:148,389,687), including only one exon (exon 14). This highlights that SVs, different from PMP22 duplication, can be responsible for peripheral neuropathy and should be searched systematically. This approach could also be employed to improve the diagnosis of all inherited diseases.

Early Diagnosis in Cerebellar Ataxia, Neuropathy, Vestibular Areflexia Syndrome (CANVAS) by Focusing on Major Clinical Clues: Beyond Ataxia and Vestibular Impairment.

CANVAS, a rare disorder responsible for late-onset ataxia of autosomal recessive inheritance, can be misdiagnosed. We investigated a series of eight patients with sensory neuropathy and/or an unexplained cough, who appeared to suffer from CANVAS, and we emphasized the clinical clues for early diagnosis. Investigations included clinical and routine laboratory analyses, skin biopsy, nerve biopsy and molecular genetics. The eight patients had clinical and/or laboratory evidence of sensory neuronopathy. All but one had neuropathic pain that had started in an asymmetric fashion in two patients. A chronic cough was a prominent feature in our eight patients and had started years before neuropathic symptoms in all but one. The course of the disease was slow, and ataxia remained mild in all. Five patients were initially thought to have immune-mediated sensory neuronopathy and received immunotherapy. Skin biopsies showed a near complete and non-length-dependent loss of intraepidermal nerve fibers. Moreover, nerve biopsy findings suggested a prominent involvement of small myelinated and unmyelinated fibers. The burden of CANVAS extends far beyond cerebellar ataxia and vestibular manifestations. Indeed, our study shows that a chronic cough and neuropathic pain may represent a major source of impairment in these patients and should not be overlooked to allow an early diagnosis and prevent unnecessary immunotherapy.

Early experimental hypertension preserves the myocardial microvasculature but aggravates cardiac injury distal to chronic coronary artery obstruction.

Coronary artery disease is a leading cause of death. Hypertension (HT) increases the incidence of cardiac events, but its effect on cardiac adaptation to coexisting coronary artery stenosis (CAS) is unclear. We hypothesized that concurrent HT modulates microvascular function in chronic CAS and aggravates microvascular remodeling and myocardial injury. Four groups of pigs (n=6 each) were studied: normal, CAS, HT, and CAS+HT. CAS and HT were induced by placing local irritant coils in the left circumflex coronary artery and renal artery, respectively. Six weeks later multidetector computerized tomography (CT) was used to assess systolic and diastolic function, microvascular permeability, myocardial perfusion, and responses to adenosine in the "area at risk." Microvascular architecture, inflammation, and fibrosis were then explored in cardiac tissue. Basal myocardial perfusion was similarly decreased in CAS and CAS+HT, but its response to adenosine was significantly more attenuated in CAS. Microvascular permeability in CAS+HT was greater than in CAS and was accompanied by amplified myocardial inflammation, fibrosis, and microvascular remodeling, as well as cardiac systolic and diastolic dysfunction. On the other hand, compared with normal, micro-CT-derived microvascular (20-200 µm) transmural density decreased in CAS but not in HT or CAS+HT. We conclude that the coexistence of early renovascular HT exacerbated myocardial fibrosis and vascular remodeling distal to CAS. These changes were not mediated by loss of myocardial microvessels, which were relatively preserved, but possibly by exacerbated myocardial inflammation and fibrosis. HT modulates cardiac adaptive responses to CAS and bears cardiac functional consequences.

Analysis of machine perfusion benefits in kidney grafts: a preclinical study.

BACKGROUND: Machine perfusion (MP) has potential benefits for marginal organs such as from deceased from cardiac death donors (DCD). However, there is still no consensus on MP benefits. We aimed to determine machine perfusion benefits on kidney grafts. METHODS: We evaluated kidney grafts preserved in ViaspanUW or KPS solutions either by CS or MP, in a DCD pig model (60 min warm ischemia+24 h hypothermic preservation). Endpoints were: function recovery, quality of function during follow up (3 month), inflammation, fibrosis, animal survival. RESULTS: ViaspanUW-CS animals did not recover function, while in other groups early follow up showed similar values for kidney function. Alanine peptidase and ß-NAG activities in the urine were higher in CS than in MP groups. Oxydative stress was lower in KPS-MP animals. Histology was improved by MP over CS. Survival was 0% in ViaspanUW-CS and 60% in other groups. Chronic inflammation, epithelial-to-mesenchymal transition and fibrosis were lowest in KPS-MP, followed by KPS-CS and ViaspanUW-MP. CONCLUSIONS: With ViaspanUW, effects of MP are obvious as only MP kidney recovered function and allowed survival. With KPS, the benefits of MP over CS are not directly obvious in the early follow up period and only histological analysis, urinary tubular enzymes and red/ox status was discriminating. Chronic follow-up was more conclusive, with a clear superiority of MP over CS, independently of the solution used. KPS was proven superior to ViaspanUW in each preservation method in terms of function and outcome. In our pre-clinical animal model of DCD transplantation, MP offers critical benefits.

New structural variations responsible for Charcot-Marie-Tooth disease: The first two large KIF5A deletions detected by CovCopCan software.

Next-generation sequencing (NGS) allows the detection of mutations in inherited genetic diseases, like the Charcot-Marie-Tooth disease (CMT) which is the most common hereditary peripheral neuropathy. The majority of mutations detected by NGS are single nucleotide variants (SNVs) or small indels, while structural variants (SVs) are often underdiagnosed. PMP22 was the first gene described as being involved in CMT via a SV of duplication type. To date, more than 90 genes are known to be involved in CMT, with mainly SNVs and short indels described. Herein targeted NGS and the CovCopCan bioinformatic tool were used in two unrelated families, both presenting with typical CMT symptoms with pyramidal involvement. We have discovered two large SVs in KIF5A, a gene known to cause axonal forms of CMT (CMT2) in which no SVs have yet been described. In the first family, the patient presented with a large deletion of 12 kb in KIF5A from Chr12:57,956,278 to Chr12:57,968,335 including exons 2-15, that could lead to mutation c.(130-943_c.1717-533del), p.(Gly44_Leu572del). In the second family, two cases presented with a large deletion of 3 kb in KIF5A from Chr12:57,974,133 to Chr12:57,977,210 including exons 24-28, that could lead to mutation c.(2539-605_*36 + 211del), p.(Leu847_Ser1032delins33). In addition, bioinformatic sequence analysis revealed that a NAHR (Non-Allelic-Homologous-Recombination) mechanism, such as those in the PMP22 duplication, could be responsible for one of the KIF5A SVs and could potentially be present in a number of other patients. This study reveals that large KIF5A deletions can cause CMT2 and highlights the importance of analyzing not only the SNVs but also the SVs during diagnosis of neuropathies.

GDAP1 Involvement in Mitochondrial Function and Oxidative Stress, Investigated in a Charcot-Marie-Tooth Model of hiPSCs-Derived Motor Neurons.

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) gene have been associated with demyelinating and axonal forms of Charcot-Marie-Tooth (CMT) disease, the most frequent hereditary peripheral neuropathy in humans. Previous studies reported the prevalent GDAP1 expression in neural tissues and cells, from animal models. Here, we described the first GDAP1 functional study on human induced-pluripotent stem cells (hiPSCs)-derived motor neurons, obtained from normal subjects and from a CMT2H patient, carrying the GDAP1 homozygous c.581C>G (p.Ser194*) mutation. At mRNA level, we observed that, in normal subjects, GDAP1 is mainly expressed in motor neurons, while it is drastically reduced in the patient's cells containing a premature termination codon (PTC), probably degraded by the nonsense-mediated mRNA decay (NMD) system. Morphological and functional investigations revealed in the CMT patient's motor neurons a decrease of cell viability associated to lipid dysfunction and oxidative stress development. Mitochondrion is a key organelle in oxidative stress generation, but it is also mainly involved in energetic metabolism. Thus, in the CMT patient's motor neurons, mitochondrial cristae defects were observed, even if no deficit in ATP production emerged. This cellular model of hiPSCs-derived motor neurons underlines the role of mitochondrion and oxidative stress in CMT disease and paves the way for new treatment evaluation.