Investigations of cardiac fibrosis rheology by in vitro cardiac tissue modeling with 3D cellular spheroids.

Cardiac fibrosis refers to the abnormal accumulation of extracellular matrix within the cardiac muscle, leading to increased stiffness and impaired heart function. From a rheological standpoint, knowledge about myocardial behavior is still lacking, partially due to a lack of appropriate techniques to investigate the rheology of in vitro cardiac tissue models. 3D multicellular cardiac spheroids are powerful and versatile platforms for modeling healthy and fibrotic cardiac tissue in vitro and studying how their mechanical properties are modulated. In this study, cardiac spheroids were created by co-culturing neonatal rat ventricular cardiomyocytes and fibroblasts in definite ratios using the hanging-drop method. The rheological characterization of such models was performed by Atomic Force Microscopy-based stress-relaxation measurements on the whole spheroid. After strain application, a viscoelastic bi-exponential relaxation was observed, characterized by a fast relaxation time (τ1) followed by a slower one (τ2). In particular, spheroids with higher fibroblasts density showed reduction for both relaxation times comparing to control, with a more pronounced decrement of τ1 with respect to τ2. Such response was found compatible with the increased production of extracellular matrix within these spheroids, which recapitulates the main feature of the fibrosis pathophysiology. These results demonstrate how the rheological characteristics of cardiac tissue vary as a function of cellular composition and extracellular matrix, confirming the suitability of such system as an in vitro preclinical model of cardiac fibrosis.

Filamin C Deficiency Impairs Sarcomere Stability and Activates Focal Adhesion Kinase through PDGFRA Signaling in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Truncating mutations in filamin C (FLNC) are associated with dilated cardiomyopathy and arrhythmogenic cardiomyopathy. FLNC is an actin-binding protein and is known to interact with transmembrane and structural proteins; hence, the ablation of FLNC in cardiomyocytes is expected to dysregulate cell adhesion, cytoskeletal organization, sarcomere structural integrity, and likely nuclear function. Our previous study showed that the transcriptional profiles of FLNC homozygous deletions in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are highly comparable to the transcriptome profiles of hiPSC-CMs from patients with FLNC truncating mutations. Therefore, in this study, we used CRISPR-Cas-engineered hiPSC-derived FLNC knockout cardiac myocytes as a model of FLNC cardiomyopathy to determine pathogenic mechanisms and to examine structural changes caused by FLNC deficiency. RNA sequencing data indicated the significant upregulation of focal adhesion signaling and the dysregulation of thin filament genes in FLNC-knockout (FLNCKO) hiPSC-CMs compared to isogenic hiPSC-CMs. Furthermore, our findings suggest that the complete loss of FLNC in cardiomyocytes led to cytoskeletal defects and the activation of focal adhesion kinase. Pharmacological inhibition of PDGFRA signaling using crenolanib (an FDA-approved drug) reduced focal adhesion kinase activation and partially normalized the focal adhesion signaling pathway. The findings from this study suggest the opportunity in repurposing FDA-approved drug as a therapeutic strategy to treat FLNC cardiomyopathy.

Prediction of incident atrial fibrillation in hypertrophic cardiomyopathy.

BACKGROUND AND AIM: Atrial fibrillation (AF) is the most common sustained arrhythmia in hypertrophic cardiomyopathy (HCM) with significant effects on outcome. We aim to compare the left atrial (LA) diameter measurement with HCM-AF Score in predicting atrial fibrillation (AF) development in HCM. METHODS: From the regional cohort of the Campania Region, Italy, 519 HCM patients (38% women, age45 ± 17 years) without history of AF, were enrolled in the study. The primary clinical endpoint was the development of AF, defined as at least 1 episode documented by ECG. RESULTS: During the follow-up (mean 8 ± 6, IQ range 2.5-11.2 years), 99 patients (19%) developed AF. Patients who developed AF were more symptomatic, had higher prevalence of ICD implantation, had larger LA diameter, greater left ventricular (LV) maximal wall thickness and LV outflow tract obstruction (p < 0.01). Both LA diameter and HCM-AF score were higher in patients who developed AF versus those who did not (LA diameter 49 ± 7 versus 43 ± 6 mm; HCM-AF score 22 ± 4 versus 19 ± 4; p < 0.0001); however, ROC curve analysis demonstrated that LA diameter had a significant greater area under the curve than HCM-AF Score (p < 0.0001). At 5 years follow-up, a LA diameter > 46 mm, showed a similar accuracy in predicting AF development of HCM-AF score ≥ 22, which identifies patients at high risk to develop AF. CONCLUSION: Our analysis shows that LA diameter, a worldwide and simple echocardiographic measure, is capable alone to predict AF development in HCM patients.

A multicomponent physical activity home-based intervention for fibromyalgia patients: effects on clinical and skin biopsy features.

OBJECTIVES: Adapted physical activity (APA) has been recommended for fibromyalgia (FM) treatment as an essential component of a biopsychosocial therapeutic approach for patients. Previous studies report that aerobic and resistance training are the most effective programs in improving the quality of life and psycho-physical well-being. Patients with FM are frequently affected by an impairment of small fibers innervation, which is evident in the proximal somatic districts. Therefore, this pilot randomised controlled not pharmacological trial aimed to investigate if a 12-week home-based multicomponent (aerobic and resistance training and mobility) physical activity (PA) intervention was effective in improving pain perception, FM-related disability, and IntraEpidermal Nerve Fibers Density (IENFD) in adult FM patients. METHODS: Thirty-four female subjects with a fibromyalgia diagnosis (51.5±11.88 years) were randomly assigned to an experimental group (n=17) that received a supervised home-based multicomponent PA intervention twice a week and a control group (n=17) that received a generic program of aerobic exercise. Skin biopsy was performed before the physical program and after 18 months with constant execution of the supervised PA intervention or generic aerobic exercise. Both groups assumed pharmacological treatment with duloxetine and/or pregabalin. RESULTS: We found that the group performing physical activity in a supervised and regular way showed a significant improvement in the Fibromyalgia-linked invalidity questionnaire (FIQ) as well as epidermal fibers density at proximal and distal sites. CONCLUSIONS: Physical activity could improve FM outcomes, with a possible beneficial impact on peripheral factors contributing to pain-related disability.

Mechano-energetic efficiency in patients with hypertrophic cardiomyopathy with and without sarcomeric mutations.

Hypertrophic cardiomyopathy (HCM) is mainly caused by sarcomeric mutations which may affect myocardial mechano-energetic efficiency (MEE). We investigated the effects of sarcomeric mutations on MEE. A non-invasive pressure/volume (P/V) analysis was performed. We included 49 genetically screened HCM patients. MEEi was calculated as the ratio between stroke volume and heart rate normalized by LV mass. Fifty-seven percent (57%) HCM patients carried a sarcomeric mutation. Patients with and without sarcomeric mutations had similar LV ejection fraction, heart rate, LV mass, and LV outflow gradient. Younger age at diagnosis, family history of HCM, and lower MEEi were associated with presence of sarcomeric mutation (p = 0.017; p = 0.001 and p = 0.0001, respectively). Lower MEEi in HCM with sarcomeric mutation is not related to significant differences on filling pressure as shown on P/V analysis. Sarcomeric mutations determine a reduction of the LV pump performance as estimated by MEEi in HCM. Lower MEEi may predict a positive genetic analysis.

Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations.

Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

Diagnosis and Clinical Implication of Left Ventricular Aneurysm in Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disease with heterogeneous clinical presentation and prognosis. Within the broad phenotypic expression of HCM, there is a subgroup of patients with a left ventricular (LV) apical aneurysm, which has an estimated prevalence between 2% and 5%. LV apical aneurysm is characterized by an area of apical dyskinesis or akinesis, often associated with regional scarring. To date, the most accepted pathomechanism of this complication is, in absence of coronary artery disease, the high systolic intra-aneurysmal pressure, which, combined with impaired diastolic perfusion from lower stroke volume, results in supply-demand ischemia and myocardial injury. Apical aneurysm is increasingly recognized as a poor prognostic marker; however, the efficacy of prophylactic anticoagulation and/or intracardiac cardioverted defibrillator (ICD) in improving morbidity and mortality is not yet clearly demonstrated. This review aims to elucidate the mechanism, diagnosis and clinical implication of LV aneurysm in patients with HCM.

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.

Outcome of small fibre pathology in fibromyalgia: a real life longitudinal observational study.

OBJECTIVES: Small fibre pathology is frequently described in fibromyalgia (FM), but its evolution and its role in clinical outcome of the disease are unclear. This longitudinal observational real-life study aimed to monitor the evolution of skin nerve fibre density in FM, in view of the clinical data. METHODS: Sixty-two FM patients were controlled by means of skin biopsy and clinical assessment after 18 months of follow-up. RESULTS: At T0 intraepidermal nerve fibre density (IENFD) was normal in 10 patients, reduced at thigh-proximal-site in 46 cases and decreased at proximal and foot-distal-site in 6 patients. At follow up-T1-the IENFD was unchanged, while Brief Pain Inventory-BPI-pain sub score, DN4 and fatigue were improved. Reduced IENFD at proximal and distal sites, together with fatigue and BPI-motor and work sub scores were predictors of more severe disability measured with Fibromyalgia Impact Questionnaire (FIQ) at T1. Reduced IENFD influenced a minor effect of drugs-antiepileptics and/or antidepressants, and physical exercise on fatigue. CONCLUSIONS: Small fibre impairment seems stable in medium term in FM. A possible influence of small fibre dysfunction on motor performance could have a role in FM evolution. The beneficial effect of physical exercise could be limited in patients with reduced IENFD.

Next-Generation Sequencing Gene Panels in Inheritable Cardiomyopathies and Channelopathies: Prevalence of Pathogenic Variants and Variants of Unknown Significance in Uncommon Genes.

The diffusion of next-generation sequencing (NGS)-based approaches allows for the identification of pathogenic mutations of cardiomyopathies and channelopathies in more than 200 different genes. Since genes considered uncommon for a clinical phenotype are also now included in molecular testing, the detection rate of disease-causing variants has increased. Here, we report the prevalence of genetic variants detected by using a NGS custom panel in a cohort of 133 patients with inherited cardiomyopathies (n = 77) or channelopathies (n = 56). We identified 82 variants, of which 50 (61%) were identified in genes without a strong or definitive evidence of disease association according to the NIH-funded Clinical Genome Resource (ClinGen; "uncommon genes"). Among these, 35 (70%) were variants of unknown significance (VUSs), 13 (26%) were pathogenic (P) or likely pathogenic (LP) mutations, and 2 (4%) benign (B) or likely benign (LB) variants according to American College of Medical Genetics (ACMG) classifications. These data reinforce the need for the screening of uncommon genes in order to increase the diagnostic sensitivity of the genetic testing of inherited cardiomyopathies and channelopathies by allowing for the identification of mutations in genes that are not usually explored due to a currently poor association with the clinical phenotype.

Medical treatment of patients with hypertrophic cardiomyopathy: An overview of current and emerging therapy.

Several treatments have demonstrated safety and effectiveness in the treatment of patients with hypertrophic cardiomyopathy; however, no drug has been shown to modify the natural history of the disease or to decrease maximal wall thickness. Improvement in our knowledge of the physiopathology of the disease has permitted the development of new therapeutical approaches, including sarcomere modulators and gene therapy. A sarcomere modulator - mavacamten - has been shown to improve exercise capacity, left ventricular outflow tract obstruction, New York Heart Association functional class and health status in a phase 3 trial. Gene therapy - although still far from human experimentation - also has promising characteristics that may radically revolutionize the treatment of hypertrophic cardiomyopathy in the future. This therapy is currently approved for the treatment of select haematological malignancies, inherited retinal dystrophy and spinal muscular atrophy, and could potentially correct the genetic alterations of the most frequent sarcomeric forms of hypertrophic cardiomyopathy. This review provides an overview of current conventional therapies for the management of patients with hypertrophic cardiomyopathy, discusses emerging therapeutic approaches and presents future perspectives.

Deletion of the Lmna gene in fibroblasts causes senescence-associated dilated cardiomyopathy by activating the double-stranded DNA damage response and induction of senescence-associated secretory phenotype.

Introduction: Mutations in the LMNA gene, encoding Lamin A/C (LMNA), are established causes of dilated cardiomyopathy (DCM). The phenotype is typically characterized by progressive cardiac conduction defects, arrhythmias, heart failure, and premature death. DCM is primarily considered a disease of cardiac myocytes. However, LMNA is also expressed in other cardiac cell types, including fibroblasts. Aim: The purpose of the study was to determine the contribution of the fibroblasts to DCM caused by LMNA deficiency. Methods and Results: The Lmna gene was deleted by crossing the platelet-derived growth factor receptor α-Cre recombinase (Pdgfra-Cre) and floxed Lmna (Lmna F/F) mice. The LMNA protein was nearly absent in ~80% of the cardiac fibroblasts and ~25% of cardiac myocytes in the Pdgfra-Cre:Lmna F/F mice. The Pdgfra-Cre:Lmna F/F mice showed an early phenotype characterized by cardiac conduction defects, arrhythmias, cardiac dysfunction, myocardial fibrosis, apoptosis, and premature death within the first six weeks of life. The Pdgfra-Cre:Lmna wild type/F (Lmna W/F) mice also showed a similar but slowly evolving phenotype that was expressed within one year of age. RNA sequencing of LMNA-deficient and wild-type cardiac fibroblasts identified differential expression of ~410 genes, which predicted activation of the TP53 and TNFA/NFκB and suppression of the cell cycle pathways. In agreement with these findings, levels of phospho-H2AFX, ATM, phospho-TP53, and CDKN1A, markers of the DNA damage response (DDR) pathway, were increased in the Pdgfra-Cre:Lmna F/F mouse hearts. Moreover, expression of senescence-associated beta-galactosidase was induced and levels of the senescence-associated secretory phenotype (SASP) proteins TGFß1, CTGF (CCN2), and LGLAS3 were increased as well as the transcript levels of additional genes encoding SASP proteins in the Pdgfra-Cre:Lmna F/F mouse hearts. Finally, expression of pH2AFX, a bonafide marker of the double-stranded DNA breaks, was increased in cardiac fibroblasts isolated from the Pdgfra-Cre:Lmna F/F mouse hearts. Conclusion: Deletion of the Lmna gene in fibroblasts partially recapitulates the phenotype of the LMNA-associated DCM, likely through induction of double-stranded DNA breaks, activation of the DDR pathway, and induction of expression of the SASP proteins. The findings indicate that the phenotype in the LMNA-associated DCM is the aggregate consequence of the LMNA deficiency in multiple cardiac cells, including cardiac fibroblasts.

DNAJB2-related Charcot-Marie-Tooth disease type 2: Pathomechanism insights and phenotypic spectrum widening.

BACKGROUND AND PURPOSE: Mutations in DNAJB2 are associated with autosomal recessive hereditary motor neuropathies/ Charcot-Marie-Tooth disease type 2 (CMT2). We describe an Italian family with CMT2 due to a homozygous DNAJB2 mutation and provide insight into the pathomechanisms. METHODS: Patients with DNAJB2 mutations were characterized clinically, electrophysiologically and by means of skin biopsy. mRNA and protein levels were studied in lymphoblastoid cells (LCLs) from patients and controls. RESULTS: Three affected siblings were found to carry a homozygous DNAJB2 null mutation segregating with the disease. The disease manifested in the second to third decade of life. Clinical examination showed severe weakness of the thigh muscles and complete loss of movement in the foot and leg muscles. Sensation was reduced in the lower limbs. All patients had severe hearing loss and the proband also had Parkinson's disease (PD). Nerve conduction studies showed an axonal motor and sensory length-dependent polyneuropathy. DNAJB2 expression studies revealed reduced mRNA levels and the absence of the protein in the homozygous subject in both LCLs and skin biopsy. Interestingly, we detected phospho-alpha-synuclein deposits in the proband, as already seen in PD patients, and demonstrated TDP-43 accumulation in patients' skin. CONCLUSIONS: Our results broaden the clinical spectrum of DNAJB2-related neuropathies and provide evidence that DNAJB2 mutations should be taken into account as another causative gene of CMT2 with hearing loss and parkinsonism. The mutation likely acts through a loss-of-function mechanism, leading to toxic protein aggregation such as TDP-43. The associated parkinsonism resembles the classic PD form with the addition of abnormal accumulation of phospho-alpha-synuclein.

Activation of PDGFRA signaling contributes to filamin C-related arrhythmogenic cardiomyopathy.

FLNC truncating mutations (FLNCtv) are prevalent causes of inherited dilated cardiomyopathy (DCM), with a high risk of developing arrhythmogenic cardiomyopathy. We investigated the molecular mechanisms of mutant FLNC in the pathogenesis of arrhythmogenic DCM (a-DCM) using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We demonstrated that iPSC-CMs from two patients with different FLNCtv mutations displayed arrhythmias and impaired contraction. FLNC ablation induced a similar phenotype, suggesting that FLNCtv are loss-of-function mutations. Coimmunoprecipitation and proteomic analysis identified ß-catenin (CTNNB1) as a downstream target. FLNC deficiency induced nuclear translocation of CTNNB1 and subsequently activated the platelet-derived growth factor receptor alpha (PDGFRA) pathway, which were also observed in human hearts with a-DCM and FLNCtv. Treatment with the PDGFRA inhibitor, crenolanib, improved contractile function of patient iPSC-CMs. Collectively, our findings suggest that PDGFRA signaling is implicated in the pathogenesis, and inhibition of this pathway is a potential therapeutic strategy in FLNC-related cardiomyopathies.

Review of techniques useful for the assessment of sensory small fiber neuropathies: Report from an IFCN expert group.

Nerve conduction studies (NCS) are an essential aspect of the assessment of patients with peripheral neuropathies. However, conventional NCS do not reflect activation of small afferent fibers, including Aδ and C fibers. A definitive gold standard for laboratory evaluation of these fibers is still needed and therefore, clinical evaluation remains fundamental in patients with small fiber neuropathies (SFN). Several clinical and research techniques have been developed for the assessment of small fiber function, such as (i) microneurography, (ii) laser evoked potentials, (iii) contact heat evoked potentials, (iv) pain-related electrically evoked potentials, (v) quantitative thermal sensory testing, (vi) skin biopsy-intraepidermal nerve fiber density and (vii) corneal confocal microscopy. The first five are physiological techniques, while the last two are morphological. They all have advantages and limitations, but the combined use of an appropriate selection of each of them would lead to gathering invaluable information for the diagnosis of SFN. In this review, we present an update on techniques available for the study of small afferent fibers and their clinical applicability. A summary of the anatomy and important physiological aspects of these pathways, and the clinical manifestations of their dysfunction is also included, in order to have a minimal common background.

Laser evoked potentials in fibromyalgia with peripheral small fiber involvement.

OBJECTIVE: To evaluate multichannel laser evoked potentials (LEPs) in patients with fibromyalgia (FM) and small fiber impairment. METHODS: We recorded LEPs using 65 electrodes in 22 patients with FM and proximal denervation, 18 with normal skin biopsy, and 7 with proximal and distal intraepidermal nerve fiber density (IENFD) reduction. We considered the amplitude and topographical distribution of N1, N2 and P2 components, and habituation of N2 and P2 waves. The sLORETA dipolar analysis was also applied. We evaluated 15 healthy subjects as controls. RESULTS: We observed reduced amplitude of the P2 component in FM group, without a topographic correspondence with the prevalent site of denervation. Decreased habituation of P2 prevailed in patients with reduced IENFD. The cingulate cortex and prefrontal cortex, were activated in the FM group, without correlation between the degree of denervation and the strength of late wave dipoles. A correlation was noted between anxiety, depression, fibromyalgia invalidity, and pain diffusion. CONCLUSIONS: The amplitude and topography of LEPs were not coherent with epidermal nerve fiber density loss. They supposedly reflected the clinical expression of pain and psychopathological factors. SIGNIFICANCE: Multichannel LEPs are not the expression of small fiber impairment in FM. Rather, they reflect the complexity of the disease.

Congenital insensitivity to pain: a novel mutation affecting a U12-type intron causes multiple aberrant splicing of SCN9A.

ABSTRACT: Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction.