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Small-Fiber Neuropathy (SFN) is a disorder of the peripheral nervous system, characterised by neuropathic pain; approximately 11% of cases are linked to variants in Voltage-Gated Sodium Channels (VGSCs). This study aims to broaden the genetic knowledge on painful SFN by applying Whole-Exome Sequencing (WES) in Early-Onset (EO) cases. A total of 88 patients from Italy (n = 52) and the Netherlands (n = 36), with a disease onset at age ≤ 45 years old and a Pain Numerical Rating Score ≥ 4, were recruited. After variant filtering and classification, WES analysis identified 142 potentially causative variants in 93 genes; 8 are Pathogenic, 15 are Likely Pathogenic, and 119 are Variants of Uncertain Significance. Notably, an enrichment of variants in transient receptor potential genes was observed, suggesting their role in pain modulation alongside VGSCs. A pathway analysis performed by comparing EO cases with 40 Italian healthy controls found enriched mutated genes in the "Nicotinic acetylcholine receptor signaling pathway". Targeting this pathway with non-opioid drugs could offer novel therapeutic avenues for painful SFN. Additionally, with this study we demonstrated that employing a gene panel of reported mutated genes could serve as an initial screening tool for SFN in genetic studies, enhancing clinical diagnostics.
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Idade de Início , Sequenciamento do Exoma , Neuropatia de Pequenas Fibras , Humanos , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Neuropatia de Pequenas Fibras/genética , Neuralgia/genética , Mutação , Predisposição Genética para Doença , Itália , Adulto Jovem , Adolescente , Países BaixosRESUMO
Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.
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Imageamento Tridimensional , Microscopia Confocal , Mitocôndrias , Humanos , Mitocôndrias/metabolismo , Microscopia Confocal/métodos , Imageamento Tridimensional/métodos , Corantes Fluorescentes/química , Potencial da Membrana Mitocondrial , Carbocianinas/química , Rodaminas/químicaRESUMO
Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.
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Recém-Nascido Prematuro , Nascimento Prematuro , Gravidez , Recém-Nascido , Feminino , Humanos , Resultado da Gravidez , Gravidez Múltipla , Nascimento Prematuro/epidemiologia , Peso ao Nascer , Mitocôndrias/genética , DNA Mitocondrial/genéticaRESUMO
The European Joint Programme on Rare Diseases (EJPRD) funded the workshop "LAMA2-Muscular Dystrophy: Paving the road to therapy", bringing together 40 health-care professionals, researchers, patient-advocacy groups, Early-Career Scientists and other stakeholders from 14 countries. Progress in natural history, pathophysiology, trial readiness, and treatment strategies was discussed together with efforts to increase patient-awareness and strengthen collaborations. Key outcomes were (a) ongoing natural history studies in 7 countries already covered more than 350 patients. The next steps are to include additional countries, harmonise data collection and define a minimal dataset; (b) therapy development was largely complementary. Approaches included LAMA2-replacement and correction, LAMA1-reactivation, mRNA modulation, linker-protein expression, targeting downstream processes and identifying modifiers, using viral vectors, muscle stem cells, iPSC and mouse models and patient lines; (c) LAMA2-Europe will inform patients (-representatives) worldwide on standards of care and scientific progress, and enable sharing experiences. Follow-up monthly online meetings and research repositories have been established to create sustainable collaborations.
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Distrofias Musculares , Doenças Raras , Animais , Humanos , Camundongos , Europa (Continente) , Laminina/genética , Laminina/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/terapia , Doenças Raras/genética , Doenças Raras/terapia , EspanhaRESUMO
The blood-brain barrier consists of tightly connected endothelial cells protecting the brain's microenvironment from the periphery. These endothelial cells are characterized by specific tight junction proteins such as Claudin-5 and Occludin, forming the endothelial barrier. Disrupting these cells might lead to blood-brain barrier dysfunction. The Wnt/ß-catenin signaling pathway can regulate the expression of these tight junction proteins and subsequent barrier permeability. The aim of this study was to investigate the in vitro effects of Wnt7a mediated ß-catenin signaling on endothelial barrier integrity. Mouse brain endothelial cells, bEnd.3, were treated with recombinant Wnt7a protein or XAV939, a selective inhibitor of Wnt/ß-catenin mediated transcription to modulate the Wnt signaling pathway. The involvement of Wnt/HIF1α signaling was investigated by inhibiting Hif1α signaling with Hif1α siRNA. Wnt7a stimulation led to activation and nuclear translocation of ß-catenin, which was inhibited by XAV939. Wnt7a stimulation decreased Claudin-5 expression mediated by ß-catenin and decreased endothelial barrier formation. Wnt7a increased Hif1α and Vegfa expression mediated by ß-catenin. However, Hif1α signaling pathway did not regulate tight junction proteins Claudin-5 and Occludin. Our data suggest that Wnt7a stimulation leads to a decrease in tight junction proteins mediated by the nuclear translocation of ß-catenin, which hampers proper endothelial barrier formation. This process might be crucial in initiating endothelial cell proliferation and angiogenesis. Although HIF1α did not modulate the expression of tight junction proteins, it might play a role in brain angiogenesis and underlie pathogenic mechanisms in Wnt/HIF1α signaling in diseases such as cerebral small vessel disease.
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Barreira Hematoencefálica , Células Endoteliais , Subunidade alfa do Fator 1 Induzível por Hipóxia , Proteínas Wnt , beta Catenina , Animais , beta Catenina/metabolismo , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Proteínas Wnt/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Encéfalo/metabolismo , Encéfalo/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Claudina-5/metabolismo , Ocludina/metabolismo , Linhagem CelularRESUMO
Mutations in nuclear and mitochondrial genes are responsible for severe chronic disorders such as mitochondrial myopathies. Gene therapy using antisense oligonucleotides is a promising strategy to treat mitochondrial DNA (mtDNA) diseases by blocking the replication of the mutated mtDNA. However, transport vehicles are needed for intracellular, mitochondria-specific transport of oligonucleotides. Nanoparticle (NP) based vectors such as large pore mesoporous silica nanoparticles (LP) often rely on surface complexation of oligonucleotides exposing them to nucleases and limiting mitochondria targeting and controlled release ability. In this work, stable, fluorescent, hollow silica nanoparticles (HSN) that encapsulate and protect oligonucleotides in the hollow core were synthesized by a facile one-pot procedure. Both rhodamine B isothiocyanate and bis[3-(triethoxysilyl)propyl]tetrasulfide were incorporated in the HSN matrix by co-condensation to enable cell tracing, intracellular-specific degradation and controlled oligonucleotide release. We also synthesized LP as a benchmark to compare the oligonucleotide loading and release efficacy of our HSN. Mitochondria targeting was enabled by NP functionalization with cationic, lipophilic Triphenylphosphine (TPP) and, for the first time a fusogenic liposome based carrier, previously reported under the name MITO-Porter. HSN exhibited high oligonucleotide incorporation ratios and release dependent on intracellular degradation. Further, MITO-Porter capping of our NP enabled delayed, glutathione (GSH) responsive oligonucleotide release and mitochondria targeting at the same efficiency as TPP functionalized NP. Overall, our NP are promising vectors for anti-gene therapy of mtDNA disease as well as many other monogenic disorders worldwide.
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Cerebral small vessel disease is characterised by decreased cerebral blood flow and blood-brain barrier impairments which play a key role in the development of white matter lesions. We hypothesised that cerebral hypoperfusion causes local hypoxia, affecting oligodendrocyte precursor cell-endothelial cell signalling leading to blood-brain barrier dysfunction as an early mechanism for the development of white matter lesions. Bilateral carotid artery stenosis was used as a mouse model for cerebral hypoperfusion. Pimonidazole, a hypoxic cell marker, was injected prior to humane sacrifice at day 7. Myelin content, vascular density, blood-brain barrier leakages, and hypoxic cell density were quantified. Primary mouse oligodendrocyte precursor cells were exposed to hypoxia and RNA sequencing was performed. Vegfa gene expression and protein secretion was examined in an oligodendrocyte precursor cell line exposed to hypoxia. Additionally, human blood plasma VEGFA levels were measured and correlated to blood-brain barrier permeability in normal-appearing white matter and white matter lesions of cerebral small vessel disease patients and controls. Cerebral blood flow was reduced in the stenosis mice, with an increase in hypoxic cell number and blood-brain barrier leakages in the cortical areas but no changes in myelin content or vascular density. Vegfa upregulation was identified in hypoxic oligodendrocyte precursor cells, which was mediated via Hif1α and Epas1. In humans, VEGFA plasma levels were increased in patients versus controls. VEGFA plasma levels were associated with increased blood-brain barrier permeability in normal appearing white matter of patients. Cerebral hypoperfusion mediates hypoxia induced VEGFA expression in oligodendrocyte precursor cells through Hif1α/Epas1 signalling. VEGFA could in turn increase BBB permeability. In humans, increased VEGFA plasma levels in cerebral small vessel disease patients were associated with increased blood-brain barrier permeability in the normal appearing white matter. Our results support a role of VEGFA expression in cerebral hypoperfusion as seen in cerebral small vessel disease.
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Doenças de Pequenos Vasos Cerebrais , Células Precursoras de Oligodendrócitos , Substância Branca , Humanos , Camundongos , Animais , Barreira Hematoencefálica/metabolismo , Células Precursoras de Oligodendrócitos/metabolismo , Substância Branca/patologia , Hipóxia/metabolismo , Doenças de Pequenos Vasos Cerebrais/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Primary open-angle glaucoma (POAG) is characterized by optic nerve degeneration and irreversible loss of retinal ganglion cells (RGCs). The pathophysiology is not fully understood. Since RGCs have a high energy demand, suboptimal mitochondrial function may put the survival of these neurons at risk. In the present study, we explored whether mtDNA copy number or mtDNA deletions could reveal a mitochondrial component in POAG pathophysiology. Buffy coat DNA was isolated from EDTA blood of age- and sex-matched study groups, namely POAG patients with high intraocular pressure (IOP) at diagnosis (high tension glaucoma: HTG; n = 97), normal tension glaucoma patients (NTG, n = 37), ocular hypertensive controls (n = 9), and cataract controls (without glaucoma; n = 32), all without remarkable comorbidities. The number of mtDNA copies was assessed through qPCR quantification of the mitochondrial D-loop and nuclear B2M gene. Presence of the common 4977 base pair mtDNA deletion was assessed by a highly sensitive breakpoint PCR. Analysis showed that HTG patients had a lower number of mtDNA copies per nuclear DNA than NTG patients (p-value <0.01, Dunn test) and controls (p-value <0.001, Dunn test). The common 4977 base pair mtDNA deletion was not detected in any of the participants. A lower mtDNA copy number in blood of HTG patients suggests a role for a genetically defined, deficient mtDNA replication in the pathology of HTG. This may cause a low number of mtDNA copies in RGCs, which together with aging and high IOP, may lead to mitochondrial dysfunction, and contribute to glaucoma pathology.
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Glaucoma de Ângulo Aberto , Glaucoma , Glaucoma de Baixa Tensão , Humanos , Glaucoma de Ângulo Aberto/diagnóstico , DNA Mitocondrial/genética , Variações do Número de Cópias de DNA , Pressão Intraocular , Glaucoma de Baixa Tensão/genética , Mitocôndrias/genéticaRESUMO
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.
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Diabetes Mellitus , Neuropatias Diabéticas , Neuralgia , Neuropatia de Pequenas Fibras , Humanos , Neuralgia/patologia , Neuropatias Diabéticas/patologia , Canais de Sódio , Canal de Sódio Disparado por Voltagem NAV1.7/genéticaRESUMO
In 25% of patients with mitochondrial myopathies, pathogenic mitochondrial DNA (mtDNA) mutation are the cause. For heteroplasmic mtDNA mutations, symptoms manifest when the mutation load exceeds a tissue-specific threshold. Therefore, lowering the mutation load is expected to ameliorate disease manifestations. This can be achieved by fusing wild-type mesoangioblasts with mtDNA mutant myotubes. We have tested this in vitro for female carriers of the m.3271T>C or m.3291T>C mutation (mutation load >90%) using wild-type male mesoangioblasts. Individual fused myotubes were collected by a newly-developed laser capture microdissection (LCM) protocol, visualized by immunostaining using an anti-myosin antibody. Fusion rates were determined based on male-female nuclei ratios by fluorescently labelling the Y-chromosome. Using combined 'wet' and 'air dried' LCM imaging improved fluorescence imaging quality and cell yield. Wild-type mesoangioblasts fused in different ratios with myotubes containing either the m.3271T>C or the m.3291T>C mutation. This resulted in the reduction of the mtDNA mutation load proportional to the number of fused wild-type mesoangioblasts for both mtDNA mutations. The proportional reduction in mtDNA mutation load in vitro after fusion is promising in the context of muscle stem cell therapy for mtDNA mutation carriers in vivo, in which we propose the same strategy using autologous wild-type mesoangioblasts.
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DNA Mitocondrial , Fibras Musculares Esqueléticas , Humanos , Masculino , Feminino , DNA Mitocondrial/genética , Mutação , Mitocôndrias/genética , Cromossomo YRESUMO
Mitochondrial diseases require customized approaches for reproductive counseling, addressing differences in recurrence risks and reproductive options. The majority of mitochondrial diseases is caused by mutations in nuclear genes and segregate in a Mendelian way. Prenatal diagnosis (PND) or preimplantation genetic testing (PGT) are available to prevent the birth of another severely affected child. In at least 15%-25% of cases, mitochondrial diseases are caused by mitochondrial DNA (mtDNA) mutations, which can occur de novo (25%) or be maternally inherited. For de novo mtDNA mutations, the recurrence risk is low and PND can be offered for reassurance. For maternally inherited, heteroplasmic mtDNA mutations, the recurrence risk is often unpredictable, due to the mitochondrial bottleneck. PND for mtDNA mutations is technically possible, but often not applicable given limitations in predicting the phenotype. Another option for preventing the transmission of mtDNA diseases is PGT. Embryos with mutant load below the expression threshold are being transferred. Oocyte donation is another safe option to prevent the transmission of mtDNA disease to a future child for couples who reject PGT. Recently, mitochondrial replacement therapy (MRT) became available for clinical application as an alternative to prevent the transmission of heteroplasmic and homoplasmic mtDNA mutations.
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Doenças Mitocondriais , Gravidez , Feminino , Humanos , Doenças Mitocondriais/genética , DNA Mitocondrial/genética , Diagnóstico Pré-Natal , Mitocôndrias/genética , MutaçãoRESUMO
Introduction: Primary open-angle glaucoma (POAG) is a characteristic optic neuropathy, caused by degeneration of the optic nerve-forming neurons, the retinal ganglion cells (RGCs). High intraocular pressure (IOP) and aging have been identified as major risk factors; yet the POAG pathophysiology is not fully understood. Since RGCs have high energy requirements, mitochondrial dysfunction may put the survivability of RGCs at risk. We explored in buffy coat DNA whether mtDNA variants and their distribution throughout the mtDNA could be risk factors for POAG. Methods: The mtDNA was sequenced from age- and sex-matched study groups, being high tension glaucoma (HTG, n=71), normal tension glaucoma patients (NTG, n=33), ocular hypertensive subjects (OH, n=7), and cataract controls (without glaucoma; n=30), all without remarkable comorbidities. Results: No association was found between the number of mtDNA variants in genes encoding proteins, tRNAs, rRNAs, and in non-coding regions in the different study groups. Next, variants that controls shared with the other groups were discarded. A significantly higher number of exclusive variants was observed in the D-loop region for the HTG group (~1.23 variants/subject), in contrast to controls (~0.35 variants/subject). In the D-loop, specifically in the 7S DNA sub-region within the Hypervariable region 1 (HV1), we found that 42% of the HTG and 27% of the NTG subjects presented variants, while this was only 14% for the controls and OH subjects. As we have previously reported a reduction in mtDNA copy number in HTG, we analysed if specific D-loop variants could explain this. While the majority of glaucoma patients with the exclusive D-loop variants m.72T>C, m.16163 A>G, m.16186C>T, m.16298T>C, and m.16390G>A presented a mtDNA copy number below controls median, no significant association between these variants and low copy number was found and their possible negative role in mtDNA replication remains uncertain. Approximately 38% of the HTG patients with reduced copy number did not carry any exclusive D-loop or other mtDNA variants, which indicates that variants in nuclear-encoded mitochondrial genes, environmental factors, or aging might be involved in those cases. Conclusion: In conclusion, we found that variants in the D-loop region may be a risk factor in a subgroup of POAG, possibly by affecting mtDNA replication.
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Neuropathic pain is a characteristic feature of small fiber neuropathy (SFN), which in 18% of the cases is caused by genetic variants in voltage-gated sodium ion channels. In this study, we assessed the role of fifteen other ion channels in neuropathic pain. Patients with SFN (n = 414) were analyzed for ANO1, ANO3, HCN1, KCNA2, KCNA4, KCNK18, KCNN1, KCNQ3, KCNQ5, KCNS1, TRPA1, TRPM8, TRPV1, TRPV3 and TRPV4 variants by single-molecule molecular inversion probes-next-generation sequencing. These patients did not have genetic variants in SCN3A, SCN7A-SCN11A and SCN1B-SCN4B. In twenty patients (20/414, 4.8%), a potentially pathogenic heterozygous variant was identified in an ion-channel gene (ICG). Variants were present in seven genes, for two patients (0.5%) in ANO3, one (0.2%) in KCNK18, two (0.5%) in KCNQ3, seven (1.7%) in TRPA1, three (0.7%) in TRPM8, three (0.7%) in TRPV1 and two (0.5%) in TRPV3. Variants in the TRP genes were the most frequent (n = 15, 3.6%), partly in patients with high mean maximal pain scores VAS = 9.65 ± 0.7 (n = 4). Patients with ICG variants reported more severe pain compared to patients without such variants (VAS = 9.36 ± 0.72 vs. VAS = 7.47 ± 2.37). This cohort study identified ICG variants in neuropathic pain in SFN, complementing previous findings of ICG variants in diabetic neuropathy. These data show that ICG variants are central in neuropathic pain of different etiologies and provides promising gene candidates for future research.
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Canais Iônicos , Neuralgia , Neuropatia de Pequenas Fibras , Humanos , Anoctaminas , Estudos de Coortes , Neuropatias Diabéticas/genética , Neuralgia/genética , Canais de Potássio/genética , Neuropatia de Pequenas Fibras/genética , Canais Iônicos/genéticaRESUMO
Alzheimer's disease (AD) is a neurodegenerative disease that eventually affects memory and behavior. The identification of biomarkers based on risk factors for AD provides insight into the disease since the exact cause of AD remains unknown. Several studies have proposed microRNAs (miRNAs) in blood as potential biomarkers for AD. Exposure to heavy metals is a potential risk factor for onset and development of AD. Blood cells of subjects that are exposed to lead detected in the circulatory system, potentially reflect molecular responses to this exposure that are similar to the response of neurons. In this study we analyzed blood cell-derived miRNAs derived from a general population as proxies of potentially AD-related mechanisms triggered by lead exposure. Subsequently, we analyzed these mechanisms in the brain tissue of AD subjects and controls. A total of four miRNAs were identified as lead exposure-associated with hsa-miR-3651, hsa-miR-150-5p and hsa-miR-664b-3p being negatively and hsa-miR-627 positively associated. In human brain derived from AD and AD control subjects all four miRNAs were detected. Moreover, two miRNAs (miR-3651, miR-664b-3p) showed significant differential expression in AD brains versus controls, in accordance with the change direction of lead exposure. The miRNAs' gene targets were validated for expression in the human brain and were found enriched in AD-relevant pathways such as axon guidance. Moreover, we identified several AD relevant transcription factors such as CREB1 associated with the identified miRNAs. These findings suggest that the identified miRNAs are involved in the development of AD and might be useful in the development of new, less invasive biomarkers for monitoring of novel therapies or of processes involved in AD development.
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Doença de Alzheimer , MicroRNAs , Doenças Neurodegenerativas , Doença de Alzheimer/genética , Biomarcadores , Humanos , Chumbo/toxicidade , MicroRNAs/metabolismo , Fatores de TranscriçãoRESUMO
Neuropathic pain is common in diabetic peripheral neuropathy (DN), probably caused by pathogenic ion channel gene variants. Therefore, we performed molecular inversion probes-next generation sequencing of 5 transient receptor potential cation channels, 8 potassium channels and 2 calcium-activated chloride channel genes in 222 painful- and 304 painless-DN patients. Twelve painful-DN (5.4%) patients showed potentially pathogenic variants (five nonsense/frameshift, seven missense, one out-of-frame deletion) in ANO3 (n = 3), HCN1 (n = 1), KCNK18 (n = 2), TRPA1 (n = 3), TRPM8 (n = 3) and TRPV4 (n = 1) and fourteen painless-DN patients (4.6%-three nonsense/frameshift, nine missense, one out-of-frame deletion) in ANO1 (n = 1), KCNK18 (n = 3), KCNQ3 (n = 1), TRPA1 (n = 2), TRPM8 (n = 1), TRPV1 (n = 3) and TRPV4 (n = 3). Missense variants were present in both conditions, presumably with loss- or gain-of-functions. KCNK18 nonsense/frameshift variants were found in painless/painful-DN, making a causal role in pain less likely. Surprisingly, premature stop-codons with likely nonsense-mediated RNA-decay were more frequent in painful-DN. Although limited in number, painful-DN patients with ion channel gene variants reported higher maximal pain during the night and day. Moreover, painful-DN patients with TRP variants had abnormal thermal thresholds and more severe pain during the night and day. Our results suggest a role of ion channel gene variants in neuropathic pain, but functional validation is required.
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Diabetes Mellitus , Neuropatias Diabéticas , Neuralgia , Canais de Potencial de Receptor Transitório , Anoctaminas , Humanos , Canais de Potássio , Canais de Cátion TRPV/genética , Canais de Potencial de Receptor Transitório/fisiologiaRESUMO
Mutations of the mitochondrial DNA are an important cause of inherited diseases that can severely affect the tissue's homeostasis and integrity. The m.3243A > G mutation is the most commonly observed across mitochondrial disorders and is linked to multisystemic complications, including cognitive deficits. In line with in vitro experiments demonstrating the m.3243A > G's negative impact on neuronal energy production and integrity, m.3243A > G patients show cerebral grey matter tissue changes. However, its impact on the most neuron dense, and therefore energy-consuming brain structure-the cerebellum-remains elusive. In this work, we used high-resolution structural and functional data acquired using 7â T MRI to characterize the neurodegenerative and functional signatures of the cerebellar cortex in m.3243A > G patients. Our results reveal altered tissue integrity within distinct clusters across the cerebellar cortex, apparent by their significantly reduced volume and longitudinal relaxation rate compared with healthy controls, indicating macroscopic atrophy and microstructural pathology. Spatial characterization reveals that these changes occur especially in regions related to the frontoparietal brain network that is involved in information processing and selective attention. In addition, based on resting-state functional MRI data, these clusters exhibit reduced functional connectivity to frontal and parietal cortical regions, especially in patients characterized by (i) a severe disease phenotype and (ii) reduced information-processing speed and attention control. Combined with our previous work, these results provide insight into the neuropathological changes and a solid base to guide longitudinal studies aimed to track disease progression.
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In a Dutch non-consanguineous patient having mitochondrial encephalomyopathy with complex I and complex IV deficiency, whole exome sequencing revealed two compound heterozygous variants in SLIRP. SLIRP gene encodes a stem-loop RNA-binding protein that regulates mitochondrial RNA expression and oxidative phosphorylation (OXPHOS). A frameshift and a deep-intronic splicing variant reduced the amount of functional wild-type SLIRP RNA to 5%. Consequently, in patient fibroblasts, MT-ND1, MT-ND6, and MT-CO1 expression was reduced. Lentiviral transduction of wild-type SLIRP cDNA in patient fibroblasts increased MT-ND1, MT-ND6, and MT-CO1 expression (2.5-7.2-fold), whereas mutant cDNAs did not. A fourfold decrease of citrate synthase versus total protein ratio in patient fibroblasts indicated that the resulting reduced mitochondrial mass caused the OXPHOS deficiency. Transduction with wild-type SLIRP cDNA led to a 2.4-fold increase of this ratio and partly restored OXPHOS activity. This confirmed causality of the SLIRP variants. In conclusion, we report SLIRP variants as a novel cause of mitochondrial encephalomyopathy with OXPHOS deficiency.
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Encefalomiopatias Mitocondriais/genética , Proteínas de Ligação a RNA/genética , Células Cultivadas , Criança , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Genes Recessivos , Humanos , Masculino , Encefalomiopatias Mitocondriais/patologia , Mutação , Proteínas de Ligação a RNA/metabolismoRESUMO
Whole exome sequencing (WES), analyzed with GENESIS and WeGET, revealed a homozygous deletion in the C1QBP gene in a patient with progressive external ophthalmoplegia (PEO) and multiple mtDNA deletions. The gene encodes the mitochondria-located complementary 1 Q subcomponent-binding protein, involved in mitochondrial homeostasis. Biallelic mutations in C1QBP cause mitochondrial cardiomyopathy and/or PEO with variable age of onset. Our patient showed only late-onset PEO-plus syndrome without overt cardiac involvement. Available data suggest that early-onset cardiomyopathy variants localize in important structural domains and PEO-plus variants in the coiled-coil region. Our patient demonstrates that C1QBP mutations should be considered in individuals with PEO with or without cardiomyopathy.
Assuntos
Proteínas de Transporte/genética , Sequenciamento do Exoma , Proteínas Mitocondriais/genética , Oftalmoplegia Externa Progressiva Crônica/genética , Adulto , DNA Mitocondrial/genética , Feminino , Homozigoto , Humanos , Mitocôndrias/genética , Mutação , Deleção de SequênciaRESUMO
BACKGROUND: SELENON (SEPN1)-related myopathy (SELENON-RM) is a rare congenital myopathy characterized by slowly progressive proximal muscle weakness, early onset spine rigidity and respiratory insufficiency. A muscular dystrophy caused by mutations in the LAMA2 gene (LAMA2-related muscular dystrophy, LAMA2-MD) has a similar clinical phenotype, with either a severe, early-onset due to complete Laminin subunit α2 deficiency (merosin-deficient congenital muscular dystrophy type 1A (MDC1A)), or a mild, childhood- or adult-onset due to partial Laminin subunit α2 deficiency. For both muscle diseases, no curative treatment options exist, yet promising preclinical studies are ongoing. Currently, there is a paucity on natural history data and appropriate clinical and functional outcome measures are needed to reach trial readiness. METHODS: LAST STRONG is a natural history study in Dutch-speaking patients of all ages diagnosed with SELENON-RM or LAMA2-MD, starting August 2020. Patients have four visits at our hospital over a period of 1.5 year. At all visits, they undergo standardized neurological examination, hand-held dynamometry (age ≥ 5 years), functional measurements, questionnaires (patient report and/or parent proxy; age ≥ 2 years), muscle ultrasound including diaphragm, pulmonary function tests (spirometry, maximal inspiratory and expiratory pressure, sniff nasal inspiratory pressure; age ≥ 5 years), and accelerometry for 8 days (age ≥ 2 years); at visit one and three, they undergo cardiac evaluation (electrocardiogram, echocardiography; age ≥ 2 years), spine X-ray (age ≥ 2 years), dual-energy X-ray absorptiometry (DEXA-)scan (age ≥ 2 years) and full body magnetic resonance imaging (MRI) (age ≥ 10 years). All examinations are adapted to the patient's age and functional abilities. Correlation between key parameters within and between subsequent visits will be assessed. DISCUSSION: Our study will describe the natural history of patients diagnosed with SELENON-RM or LAMA2-MD, enabling us to select relevant clinical and functional outcome measures for reaching clinical trial-readiness. Moreover, our detailed description (deep phenotyping) of the clinical features will optimize clinical management and will establish a well-characterized baseline cohort for prospective follow-up. CONCLUSION: Our natural history study is an essential step for reaching trial readiness in SELENON-RM and LAMA2-MD. TRIAL REGISTRATION: This study has been approved by medical ethical reviewing committee Region Arnhem-Nijmegen (NL64269.091.17, 2017-3911) and is registered at ClinicalTrial.gov ( NCT04478981 ).