Combined RNA interference and gene replacement therapy targeting MFN2 as proof of principle for the treatment of Charcot-Marie-Tooth type 2A.

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2 molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2 mutations.

Genetic Profiling of Sodium Channels in Diabetic Painful and Painless and Idiopathic Painful and Painless Neuropathies.

Neuropathic pain is a frequent feature of diabetic peripheral neuropathy (DPN) and small fiber neuropathy (SFN). Resolving the genetic architecture of these painful neuropathies will lead to better disease management strategies, counselling and intervention. Our aims were to profile ten sodium channel genes (SCG) expressed in a nociceptive pathway in painful and painless DPN and painful and painless SFN patients, and to provide a perspective for clinicians who assess patients with painful peripheral neuropathy. Between June 2014 and September 2016, 1125 patients with painful-DPN (n = 237), painless-DPN (n = 309), painful-SFN (n = 547) and painless-SFN (n = 32), recruited in four different centers, were analyzed for SCN3A, SCN7A-SCN11A and SCN1B-SCN4B variants by single molecule Molecular inversion probes-Next Generation Sequence. Patients were grouped based on phenotype and the presence of SCG variants. Screening of SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B revealed 125 different (potential) pathogenic variants in 194 patients (17.2%, n = 194/1125). A potential pathogenic variant was present in 18.1% (n = 142/784) of painful neuropathy patients vs. 15.2% (n = 52/341) of painless neuropathy patients (17.3% (n = 41/237) for painful-DPN patients, 14.9% (n = 46/309) for painless-DPN patients, 18.5% (n = 101/547) for painful-SFN patients, and 18.8% (n = 6/32) for painless-SFN patients). Of the variants detected, 70% were in SCN7A, SCN9A, SCN10A and SCN11A. The frequency of SCN9A and SCN11A variants was the highest in painful-SFN patients, SCN7A variants in painful-DPN patients, and SCN10A variants in painless-DPN patients. Our findings suggest that rare SCG genetic variants may contribute to the development of painful neuropathy. Genetic profiling and SCG variant identification should aid in a better understanding of the genetic variability in patients with painful and painless neuropathy, and may lead to better risk stratification and the development of more targeted and personalized pain treatments.

Integrative miRNA-mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy.

Personalized management of neuropathic pain is an unmet clinical need due to heterogeneity of the underlying aetiologies, incompletely understood pathophysiological mechanisms and limited efficacy of existing treatments. Recent studies on microRNA in pain preclinical models have begun to yield insights into pain-related mechanisms, identifying nociception-related species differences and pinpointing potential drug candidates. With the aim of bridging the translational gap towards the clinic, we generated a human pain-related integrative miRNA and mRNA molecular profile of the epidermis, the tissue hosting small nerve fibres, in a deeply phenotyped cohort of patients with sodium channel-related painful neuropathy not responding to currently available therapies. We identified four miRNAs strongly discriminating patients from healthy individuals, confirming their effect on differentially expressed gene targets driving peripheral sensory transduction, transmission, modulation and post-transcriptional modifications, with strong effects on gene targets including NEDD4. We identified a complex epidermal miRNA-mRNA network based on tissue-specific experimental data suggesting a cross-talk between epidermal cells and axons in neuropathy pain. Using immunofluorescence assay and confocal microscopy, we observed that Nav1.7 signal intensity in keratinocytes strongly inversely correlated with NEDD4 expression that was downregulated by miR-30 family, suggesting post-transcriptional fine tuning of pain-related protein expression. Our targeted molecular profiling advances the understanding of specific neuropathic pain fine signatures and may accelerate process towards personalized medicine in patients with neuropathic pain.

Significance and clinical suggestions for the somatosensory evoked potentials increased in amplitude revealed by a large sample of neurological patients.

OBJECTIVES: To investigate the relationship between N20-P25 peak-to-peak amplitude (N20p-P25p) of somatosensory evoked potentials (SEPs) and the occurrence of abnormalities of the peripheral and/or central sensory pathways and of myoclonus/epilepsy, in 308 patients with increased SEPs amplitude from upper limb stimulation. METHODS: We compared cortical response (N20p-P25p) in different groups of patients identified by demographic, clinical, and neurophysiological factors and performed a cluster analysis for classifying the natural occurrence of subgroups of patients. RESULTS: No significant differences of N20p-P25p were found among different age-dependent groups, and in patients with or without PNS/CNS abnormalities of sensory pathways, while myoclonic/epileptic patients showed higher N20p-P25p than other groups. Cluster analysis identified four clusters of patients including myoclonus/epilepsy, central sensory abnormalities, peripheral sensory abnormalities, and absence of myoclonus and sensory abnormalities. CONCLUSIONS: Increased N20p-P25p prompts different possible pathophysiological substrates: larger N20p-P25p in patients with cortical myoclonus and/or epilepsy is likely sustained by strong cortical hyperexcitability, while milder increase of N20p-P25p could be underpinned by plastic cortical changes following abnormalities of sensory pathways, or degenerative process involving the cortex. SEPs increased in amplitude cannot be considered an exclusive hallmark of myoclonus/epilepsy. Indeed, in several neurological disorders, it may represent a sign of adaptive, plastic, and/or degenerative cortical changes.

SARS-COV-2 comorbidity network and outcome in hospitalized patients in Crema, Italy.

We report onset, course, correlations with comorbidities, and diagnostic accuracy of nasopharyngeal swab in 539 individuals suspected to carry SARS-COV-2 admitted to the hospital of Crema, Italy. All individuals underwent clinical and laboratory exams, SARS-COV-2 reverse transcriptase-polymerase chain reaction on nasopharyngeal swab, and chest X-ray and/or computed tomography (CT). Data on onset, course, comorbidities, number of drugs including angiotensin converting enzyme (ACE) inhibitors and angiotensin-II-receptor antagonists (sartans), follow-up swab, pharmacological treatments, non-invasive respiratory support, ICU admission, and deaths were recorded. Among 411 SARS-COV-2 patients (67.7% males) median age was 70.8 years (range 5-99). Chest CT was performed in 317 (77.2%) and showed interstitial pneumonia in 304 (96%). Fatality rate was 17.5% (74% males), with 6.6% in 60-69 years old, 21.1% in 70-79 years old, 38.8% in 80-89 years old, and 83.3% above 90 years. No death occurred below 60 years. Non-invasive respiratory support rate was 27.2% and ICU admission 6.8%. Charlson comorbidity index and high C-reactive protein at admission were significantly associated with death. Use of ACE inhibitors or sartans was not associated with outcomes. Among 128 swab negative patients at admission (63.3% males) median age was 67.7 years (range 1-98). Chest CT was performed in 87 (68%) and showed interstitial pneumonia in 76 (87.3%). Follow-up swab turned positive in 13 of 32 patients. Using chest CT at admission as gold standard on the entire study population of 539 patients, nasopharyngeal swab had 80% accuracy. Comorbidity network analysis revealed a more homogenous distribution 60-40 aged SARS-COV-2 patients across diseases and a crucial different interplay of diseases in the networks of deceased and survived patients. SARS-CoV-2 caused high mortality among patients older than 60 years and correlated with pre-existing multiorgan impairment.

Life with chronic pain during COVID-19 lockdown: the case of patients with small fibre neuropathy and chronic migraine.

OBJECTIVE: We aimed at investigating the impact of COVID-19-related distress on patients with chronic pain, highlighting the effects of changes in individual habits and public health care reconfiguration on physical and psychological health. METHODS: During the pandemic, 80 participants (25 patients with small fibre neuropathy (SFN), 42 patients with chronic migraine (CM) and 13 patients' healthy family members (HFM)) were asked to evaluate their COVID-19 complains, changes in habits and clinical management, behaviour, mood, loneliness, quality of life (QoL), physical and mental health and coping strategies. Data were analysed by Spearman rho correlations and Mann-Whitney U tests. RESULTS: Patients had lower QoL, lower physical health and higher catastrophizing attitude towards pain than HFM. During the pandemic, SFN patients referred greater decline in clinical symptoms, worries about contagion and discomfort for disease management changes than CM patients. In the SFN group, the higher levels of disability were associated with suffering from changes in neurologist-patient relationship. CM patients complained of agitation/anxiety that was related to feelings of loneliness, depressive mood and catastrophism. DISCUSSION: Despite similar complains of change in habits and worries about COVID-19 pandemic, SFN and CM patients had distinct reactions. In SFN patients, pandemic distress impacted on physical health with worsening of clinical conditions, especially suffering from changes in their care. In CM patients, pandemic distress affected behaviour, mainly with psychological frailty. This suggests the need to customize public health care for patients with distinct chronic pain conditions.

Syncope at SARS-CoV-2 onset.

We describe clinical and laboratory findings in 35 patients tested positive for SARS-CoV-2 by reverse transcriptase-polymerase chain reaction on nasopharyngeal swab experiencing one or multiple syncope at disease onset. Clinical neurologic and cardiologic examination, and electrocardiographic findings were normal. Chest computed tomography showed findings consistent with interstitial pneumonia. Arterial blood gas analysis showed low pO2, pCO2, and ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) indicating hypocapnic hypoxemia. Patients who presented with syncope showed significantly lower heart rate as compared to 68 SARS-CoV-2 positive that did not. Such poorer than expected compensatory heart rate increase may have led to syncope based on individual susceptibility. We speculate that SARS-CoV-2 could have caused angiotensin-converting enzyme-2 (ACE2) receptor internalization in the nucleus of the solitary tract and other midbrain nuclei, impairing baroreflex and chemoreceptor response, and inhibiting the compensatory tachycardia during acute hypocapnic hypoxemia.

Clinical diagnosis and management of small fiber neuropathy: an update on best practice.

INTRODUCTION: Small fiber neuropathy (SFN) is a heterogeneous group of disorders affecting thin myelinated Aδ and unmyelinated C fibers. Common symptoms include neuropathic pain and autonomic disturbances, and the typical clinical presentation is that of a length-dependent polyneuropathy, although other distributions could be present. AREA COVERED: This review focuses on several aspects of SFN including etiology, clinical presentation, diagnostic criteria and tests, management, and future perspectives. Diagnostic challenges are discussed, encompassing the role of accurate and standardized assessment of symptoms and signs and providing clues for the clinical practice. The authors discuss the evidence in support of skin biopsy and quantitative sensory testing as diagnostic tests and present an overview of other diagnostic techniques to assess sensory and autonomic fibers dysfunction. The authors also suggest a systematic approach to the etiology including a set of laboratory tests and genetic examinations of sodium channelopathies and other rare conditions that might drive the therapeutic approach based on underlying cause or symptoms treatment. EXPERT OPINION: SFN provides a useful model for neuropathic pain whose known mechanisms and cause could pave the way toward personalized treatments.

Expanding clinical spectrum of Caspr2 antibody-associated disease: warning on brainstem involvement and respiratory failure.

We report the case of a 68-year-old man who presented with ataxia, insomnia, rapidly developing cognitive decline, seizures and small vessel vasculitis. Both serum and cerebro-spinal fluid samples showed positive titre of anti-CASPR2 antibodies. Limbic encephalitis was diagnosed and immunomodulatory therapy was started with benefit. After one-year follow-up, the patient relapsed with a difficult-to-treat respiratory failure, brainstem involvement, neuropathic pain and severe dysautonomia with esophageal dysfunction. We discuss here the occurrence of life-threating complication such as respiratory dysfunction in CASPR2 limbic encephalitis. Furthermore, we showed different phenotype and treatment response during disease onset compared to relapse. This case expands the clinical spectrum of anti-CASPR2 associated disease, underlying the need for respiratory and sleep evaluation.

Diagnostic criteria for small fibre neuropathy in clinical practice and research.

The diagnostic criteria for small fibre neuropathy are not established, influencing the approach to patients in clinical practice, their access to disease-modifying and symptomatic treatments, the use of healthcare resources, and the design of clinical trials. To address these issues, we performed a reappraisal study of 150 patients with sensory neuropathy and a prospective and follow-up validation study of 352 new subjects with suspected sensory neuropathy. Small fibre neuropathy diagnostic criteria were based on deep clinical phenotyping, quantitative sensory testing (QST) and intraepidermal nerve fibre density (IENFD). Small fibre neuropathy was ruled out in 5 of 150 patients (3.3%) of the reappraisal study. Small fibre neuropathy was diagnosed at baseline of the validation study in 149 of 352 patients (42.4%) based on the combination between two clinical signs and abnormal QST and IENFD (69.1%), abnormal QST alone (5.4%), or abnormal IENFD alone (20.1%). Eight patients (5.4%) had abnormal QST and IENFD but no clinical signs. Further, 38 patients complained of sensory symptoms but showed no clinical signs. Of those, 34 (89.4%) had normal QST and IENFD, 4 (10.5%) had abnormal QST and normal IENFD, and none had abnormal IENFD alone. At 18-month follow-up, 19 of them (56%) reported the complete recovery of symptoms and showed normal clinical, QST and IENFD findings. None of those with one single abnormal test (QST or IENFD) developed clinical signs or showed abnormal findings on the other test. Conversely, all eight patients with abnormal QST and IENFD at baseline developed clinical signs at follow-up. The combination of clinical signs and abnormal QST and/or IENFD findings can more reliably lead to the diagnosis of small fibre neuropathy than the combination of abnormal QST and IENFD findings in the absence of clinical signs. Sensory symptoms alone should not be considered a reliable screening feature. Our findings demonstrate that the combined clinical, functional and structural approach to the diagnosis of small fibre neuropathy is reliable and relevant both for clinical practice and clinical trial design.

Late-onset and fast progressive neuropathy and cardiomyopathy in Val32Ala transthyretin gene mutation.

More than 100 mutations of the transthyretin gene have been reported in autosomal dominant familial amyloid polyneuropathy. This rare disease causes severe motor and sensory disability, dysautonomia, and in some patients also cardiomyopathy. The diagnosis can be challenging mainly in sporadic adult patients showing clinical, laboratory, and neurophysiological findings overlapping other forms of chronic neuropathy. We describe the clinical features and course of a patient harboring the rare p.V32A (c.155T>C) variant that was previously described in only two patients and whose pathogenicity was unclear.

Laser capture microdissection for transcriptomic profiles in human skin biopsies.

BACKGROUND: The acquisition of reliable tissue-specific RNA sequencing data from human skin biopsy represents a major advance in research. However, the complexity of the process of isolation of specific layers from fresh-frozen human specimen by laser capture microdissection, the abundant presence of skin nucleases and RNA instability remain relevant methodological challenges. We developed and optimized a protocol to extract RNA from layers of human skin biopsies and to provide satisfactory quality and amount of mRNA sequencing data. RESULTS: The protocol includes steps of collection, embedding, freezing, histological coloration and relative optimization to preserve RNA extracted from specific components of fresh-frozen human skin biopsy of 14 subjects. Optimization of the protocol includes a preservation step in RNALater® Solution, the control of specimen temperature, the use of RNase Inhibitors and the time reduction of the staining procedure. The quality of extracted RNA was measured using the percentage of fragments longer than 200 nucleotides (DV200), a more suitable measurement for successful library preparation than the RNA Integrity Number (RIN). RNA was then enriched using the TruSeq® RNA Access Library Prep Kit (Illumina®) and sequenced on HiSeq® 2500 platform (Illumina®). Quality control on RNA sequencing data was adequate to get reliable data for downstream analysis. CONCLUSIONS: The described implemented and optimized protocol can be used for generating transcriptomics data on skin tissues, and it is potentially applicable to other tissues. It can be extended to multicenter studies, due to the introduction of an initial step of preservation of the specimen that allowed the shipment of biological samples.

Small fibre neuropathy.

PURPOSE OF REVIEW: To provide a review on the state-of-art of clinical features, diagnostics, genetics and treatments of small fibre neuropathy (SFN). RECENT FINDINGS: The spectrum of clinical features has been widened from the classical presentation of burning feet as length-dependent SFN to that of small fibre dysfunction and/or degeneration associated with focal, diffuse and episodic neuropathic pain syndromes. The involvement of small nerve fibres in neurodegenerative diseases has been further defined, challenging the relationship between neuropathic pain symptoms and small fibre loss. The clinical reliability of skin biopsy has been strengthened by the availability of normative values for both the immunohistochemistry techniques used and their comparison, and by side and short-term follow-up analyses. Corneal confocal microscopy has implemented its diagnostic potentiality because of the availability of age-adjusted and sex-adjusted normative values. Genetic studies expanded the panel on genes involved in SFN because of the discovery of new mutations in SCN10A and SCN11A, besides the first found in SCN9A, and identification of mutations in COL6A5 in patients with itching. SUMMARY: In the last 5 years, the chapter of SFN has been widened by new clinical and genetics descriptions leading to a more comprehensive approach to patients in clinical practice and research.

COL6A5 variants in familial neuropathic chronic itch.

Itch is thought to represent the peculiar response to stimuli conveyed by somatosensory pathways shared with pain through the activation of specific neurons and receptors. It can occur in association with dermatological, systemic and neurological diseases, or be the side effect of certain drugs. However, some patients suffer from chronic idiopathic itch that is frequently ascribed to psychological distress and for which no biomarker is available to date. We investigated three multigenerational families, one of which diagnosed with joint hypermobility syndrome/Ehlers-Danlos syndrome hypermobility type (JHS/EDS-HT), characterized by idiopathic chronic itch with predominantly proximal distribution. Skin biopsy was performed in all eight affected members and revealed in six of them reduced intraepidermal nerve fibre density consistent with small fibre neuropathy. Whole exome sequencing identified two COL6A5 rare variants co-segregating with chronic itch in eight affected members and absent in non-affected members, and in one unrelated sporadic patient with type 1 painless diabetic neuropathy and chronic itch. Two families and the diabetic patient carried the nonsense c.6814G>T (p.Glu2272*) variant and another family carried the missense c.6486G>C (p.Arg2162Ser) variant. Both variants were predicted as likely pathogenic by in silico analyses. The two variants were rare (minor allele frequency < 0.1%) in 6271 healthy controls and absent in 77 small fibre neuropathy and 167 JHS/EDS-HT patients without itch. Null-allele test on cDNA from patients' fibroblasts of both families carrying the nonsense variant demonstrated functional haploinsufficiency due to activation of nonsense mediated RNA decay. Immunofluorescence microscopy and western blotting revealed marked disorganization and reduced COL6A5 synthesis, respectively. Indirect immunofluorescence showed reduced COL6A5 expression in the skin of patients carrying the nonsense variant. Treatment with gabapentinoids provided satisfactory itch relief in the patients carrying the mutations. Our findings first revealed an association between COL6A5 gene and familiar chronic itch, suggesting a new contributor to the pathogenesis of neuropathic itch and identifying a new candidate therapeutic target.

Small fiber neuropathy is a common feature of Ehlers-Danlos syndromes.

OBJECTIVE: To investigate the involvement of small nerve fibers in Ehlers-Danlos syndrome (EDS). METHODS: Patients diagnosed with EDS underwent clinical, neurophysiologic, and skin biopsy assessment. We recorded sensory symptoms and signs and evaluated presence and severity of neuropathic pain according to the Douleur Neuropathique 4 (DN4) and ID Pain questionnaires and the Numeric Rating Scale (NRS). Sensory action potential amplitude and conduction velocity of sural nerve was recorded. Skin biopsy was performed at distal leg and intraepidermal nerve fiber density (IENFD) obtained and referred to published sex- and age-adjusted normative reference values. RESULTS: Our cohort included 20 adults with joint hypermobility syndrome/hypermobility EDS, 3 patients with vascular EDS, and 1 patient with classic EDS. All except one patient had neuropathic pain according to DN4 and ID Pain questionnaires and reported 7 or more symptoms at the Small Fiber Neuropathy Symptoms Inventory Questionnaire. Pain intensity was moderate (NRS ≥4 and <7) in 8 patients and severe (NRS ≥7) in 11 patients. Sural nerve conduction study was normal in all patients. All patients showed a decrease of IENFD consistent with the diagnosis of small fiber neuropathy (SFN), regardless of the EDS type. CONCLUSIONS: SFN is a common feature in adults with EDS. Skin biopsy could be considered an additional diagnostic tool to investigate pain manifestations in EDS.