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PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.
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Empalme Alternativo , Enfermedad de Charcot-Marie-Tooth , Enfermedades Mitocondriales , Humanos , Empalme Alternativo/genética , Masculino , Femenino , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Niño , NADH Deshidrogenasa/genética , Linaje , Mutación/genética , Fenotipo , Secuenciación del Exoma , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Mitocondrias/genética , Mitocondrias/patología , Complejo I de Transporte de Electrón/genética , Adulto , Preescolar , AdolescenteRESUMEN
Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.
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Enfermedad de Charcot-Marie-Tooth/genética , Proteínas del Citoesqueleto/genética , Predisposición Genética a la Enfermedad , Proteínas de la Membrana/genética , Adolescente , Alelos , Biopsia , Mapeo Cromosómico , Consanguinidad , Salud de la Familia , Femenino , Fibroblastos/metabolismo , Genes Recesivos , Ligamiento Genético , Marcadores Genéticos , Haplotipos , Humanos , Escala de Lod , Pérdida de Heterocigocidad , Malasia , Masculino , Mutación Missense , Neuronas/metabolismo , Linaje , Secuenciación del ExomaRESUMEN
Defects in mRNA export from the nucleus have been linked to various neurodegenerative disorders. We report mutations in the gene MCM3AP, encoding the germinal center associated nuclear protein (GANP), in nine affected individuals from five unrelated families. The variants were associated with severe childhood onset primarily axonal (four families) or demyelinating (one family) Charcot-Marie-Tooth neuropathy. Mild to moderate intellectual disability was present in seven of nine affected individuals. The affected individuals were either compound heterozygous or homozygous for different MCM3AP variants, which were predicted to cause depletion of GANP or affect conserved amino acids with likely importance for its function. Accordingly, fibroblasts of affected individuals from one family demonstrated severe depletion of GANP. GANP has been described to function as an mRNA export factor, and to suppress TDP-43-mediated motor neuron degeneration in flies. Thus our results suggest defective mRNA export from nucleus as a potential pathogenic mechanism of axonal degeneration in these patients. The identification of MCM3AP variants in affected individuals from multiple centres establishes it as a disease gene for childhood-onset recessively inherited Charcot-Marie-Tooth neuropathy with intellectual disability.
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Acetiltransferasas/genética , Enfermedad de Charcot-Marie-Tooth/genética , Predisposición Genética a la Enfermedad/genética , Discapacidad Intelectual/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Acetiltransferasas/metabolismo , Adolescente , Adulto , Células Cultivadas , Enfermedad de Charcot-Marie-Tooth/complicaciones , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Humanos , Discapacidad Intelectual/complicaciones , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Mutación , Linaje , Adulto JovenRESUMEN
PURPOSE: Homozygosity mapping is an effective approach for detecting molecular defects in consanguineous families by delineating stretches of genomic DNA that are identical by descent. Constant developments in next-generation sequencing created possibilities to combine whole-exome sequencing (WES) and homozygosity mapping in a single step. METHODS: Basic optimization of homozygosity mapping parameters was performed in a group of families with autosomal-recessive (AR) mutations for which both single-nucleotide polymorphism (SNP) array and WES data were available. We varied the criteria for SNP extraction and PLINK thresholds to estimate their effect on the accuracy of homozygosity mapping based on WES. RESULTS: Our protocol showed high specificity and sensitivity for homozygosity detection and facilitated the identification of novel mutations in GAN, GBA2, and ZFYVE26 in four families affected by hereditary spastic paraplegia or Charcot-Marie-Tooth disease. Filtering and mapping with optimized parameters was integrated into the HOMWES (homozygosity mapping based on WES analysis) tool in the GenomeComb package for genomic data analysis. CONCLUSION: We present recommendations for detection of homozygous regions based on WES data and a bioinformatics tool for their identification, which can be widely applied for studying AR disorders.Genet Med 18 6, 600-607.
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Proteínas Portadoras/genética , Enfermedad de Charcot-Marie-Tooth/genética , Proteínas del Citoesqueleto/genética , Paraplejía Espástica Hereditaria/genética , beta-Glucosidasa/genética , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/patología , Mapeo Cromosómico , Consanguinidad , Femenino , Glucosilceramidasa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Masculino , Mutación , Linaje , Polimorfismo de Nucleótido Simple/genética , Paraplejía Espástica Hereditaria/diagnóstico , Paraplejía Espástica Hereditaria/patología , Secuenciación del ExomaRESUMEN
Autosomal recessive forms of Charcot-Marie-Tooth disease (ARCMT) are rare but severe disorders of the peripheral nervous system. Their molecular basis is poorly understood due to the extensive genetic and clinical heterogeneity, posing considerable challenges for patients, physicians, and researchers. We report on the genetic findings from a systematic study of a large collection of 174 independent ARCMT families. Initial sequencing of the three most common ARCMT genes (ganglioside-induced differentiation protein 1GDAP1, SH3 domain and tetratricopeptide repeats-containing protein 2SH3TC2, histidine-triad nucleotide binding protein 1HINT1) identified pathogenic mutations in 41 patients. Subsequently, 87 selected nuclear families underwent single nucleotide polymorphism (SNP) genotyping and homozygosity mapping, followed by targeted screening of known ARCMT genes. This strategy provided molecular diagnosis to 22% of the families. Altogether, our unbiased genetic approach identified pathogenic mutations in ten ARCMT genes in a total of 41.3% patients. Apart from a newly described founder mutation in GDAP1, the majority of variants constitute private molecular defects. Since the gene testing was independent of the clinical phenotype of the patients, we identified mutations in patients with unusual or additional clinical features, extending the phenotypic spectrum of the SH3TC2 gene. Our study provides an overview of the ARCMT genetic landscape and proposes guidelines for tackling the genetic heterogeneity of this group of hereditary neuropathies.
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Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Mutación , Mapeo Cromosómico , Análisis Mutacional de ADN , Femenino , Genes Recesivos , Homocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Proteínas del Tejido Nervioso/genética , Fenotipo , Polimorfismo de Nucleótido Simple , Proteínas/genéticaRESUMEN
Charcot-Marie-Tooth disease type 2B (CMT2B) is an inherited axonal peripheral neuropathy. It is characterised by prominent sensory loss, often complicated by severe ulcero-mutilations of toes or feet, and variable motor involvement. Missense mutations in RAB7A, the gene encoding the small GTPase Rab7, cause CMT2B and increase Rab7 activity. Rab7 is ubiquitously expressed and is involved in degradation through the lysosomal pathway. In the neurons, Rab7 plays a role in the long-range retrograde transport of signalling endosomes in the axons. Here we developed the first animal model of CMT2B, modelling one of the mutations (L129F) in Drosophila melanogaster. Behavioural assays show that this model recapitulates several hallmarks of the human disease. Upon expression of mutant Rab7 in the sensory neurons, larvae present with a reduction of temperature and pain perception. Furthermore, the larvae exhibit a crawling defect when the mutant protein is expressed in the motor neurons. Analysis of axonal transport of Rab7 positive vesicles in sensory neurons of Drosophila larvae and in neurites of mammalian neuroblastoma cells demonstrates that mutant vesicles pause less than their wild-type counterparts. This latter finding indicates that alterations in vesicle transport might contribute to the pathomechanism of CMT2B.
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Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Mutación/genética , Proteínas de Unión al GTP rab/genética , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Diferenciación Celular , Línea Celular Transformada , Dendritas/patología , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Laminopatías , Larva , Masculino , Actividad Motora/genética , Neuroblastoma/patología , Células Receptoras Sensoriales/patología , Transducción Genética , Proteínas de Unión a GTP rab7RESUMEN
Defects in mitochondrial dynamics are a common cause of Charcot-Marie-Tooth disease (CMT), while primary deficiencies in the mitochondrial respiratory chain (MRC) are rare and atypical for this etiology. This study aims to report COX18 as a novel CMT-causing gene. This gene encodes an assembly factor of mitochondrial Complex IV (CIV) that translocates the C-terminal tail of MTCO2 across the mitochondrial inner membrane. Exome sequencing was performed in four affected individuals. The patients and available family members underwent thorough neurological and electrophysiological assessment. The impact of one of the identified variants on splicing, protein levels, and mitochondrial bioenergetics was investigated in patient-derived lymphoblasts. The functionality of the mutant protein was assessed using a Proteinase K protection assay and immunoblotting. Neuronal relevance of COX18 was assessed in a Drosophila melanogaster knockdown model. Exome sequencing coupled with homozygosity mapping revealed a homozygous splice variant c.435-6A>G in COX18 in two siblings with early-onset progressive axonal sensory-motor peripheral neuropathy. By querying external databases, we identified two additional families with rare deleterious biallelic variants in COX18 . All affected individuals presented with axonal CMT and some patients also exhibited central nervous system symptoms, such as dystonia and spasticity. Functional characterization of the c.435-6A>G variant demonstrated that it leads to the expression of an alternative transcript that lacks exon 2, resulting in a stable but defective COX18 isoform. The mutant protein impairs CIV assembly and activity, leading to a reduction in mitochondrial membrane potential. Downregulation of the COX18 homolog in Drosophila melanogaster displayed signs of neurodegeneration, including locomotor deficit and progressive axonal degeneration of sensory neurons. Our study presents genetic and functional evidence that supports COX18 as a newly identified gene candidate for autosomal recessive axonal CMT with or without central nervous system involvement. These findings emphasize the significance of peripheral neuropathy within the spectrum of primary mitochondrial disorders and the role of mitochondrial CIV in the development of CMT. Our research has important implications for the diagnostic workup of CMT patients.
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Dominant mutations in tyrosyl-tRNA synthetase (YARS1) and six other tRNA ligases cause Charcot-Marie-Tooth peripheral neuropathy (CMT). Loss of aminoacylation is not required for their pathogenicity, suggesting a gain-of-function disease mechanism. By an unbiased genetic screen in Drosophila, we link YARS1 dysfunction to actin cytoskeleton organization. Biochemical studies uncover yet unknown actin-bundling property of YARS1 to be enhanced by a CMT mutation, leading to actin disorganization in the Drosophila nervous system, human SH-SY5Y neuroblastoma cells, and patient-derived fibroblasts. Genetic modulation of F-actin organization improves hallmark electrophysiological and morphological features in neurons of flies expressing CMT-causing YARS1 mutations. Similar beneficial effects are observed in flies expressing a neuropathy-causing glycyl-tRNA synthetase. Hence, in this work, we show that YARS1 is an evolutionary-conserved F-actin organizer which links the actin cytoskeleton to tRNA-synthetase-induced neurodegeneration.
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Actinas , Tirosina-ARNt Ligasa , Animales , Humanos , Actinas/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Drosophila/genética , Glicina-ARNt Ligasa/genética , Mutación , ARN de Transferencia , Tirosina-ARNt Ligasa/genética , Tirosina-ARNt Ligasa/metabolismo , Línea Celular TumoralRESUMEN
Polyelectrolyte microcapsules (MCs) are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells (DCs). We investigated the immune stimulatory capacity of monocyte-derived DC (Mo-DC) loaded with these MCs, containing p24 antigen from human immunodeficiency virus type 1 (HIV-1) alone [p24-containing MC (MCp24)] or with the Toll-like receptor ligand 3 (TLR3) ligand poly I:C (MCp24pIC) as a maturation factor. MO-DC, loaded with MCp24pIC, upregulated CCR7, CD80, CD83, and CD86 and produced high amounts of interleukin-12 (IL-12) cytokine, to a similar extent as MCp24 in the presence of an optimized cytokine cocktail. MO-DC from HIV-infected patients under highly active antiretroviral therapy (HAART) exposed to MCp24 together with cytokine cocktail or to MCp24pIC expanded autologous p24-specific CD4(+) and CD8(+) T-cell responses as measured by interferon-gamma (IFN-gamma) and IL-2 cytokine production and secretion. In vivo relevance was shown by immunizing C57BL/6 mice with MCp24pIC, which induced both humoral and cellular p24-specific immune responses. Together these data provide a proof of principle that both antigen and DC maturation signal can be delivered as a complex with polyelectrolyte capsules to stimulate virus-specific T cells both in vitro and in vivo. Polyelectrolyte MCs could be useful for in vivo immunization in HIV-1 and other infections.
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Células Dendríticas/inmunología , Portadores de Fármacos/química , Proteína p24 del Núcleo del VIH/inmunología , Poli I-C/inmunología , Polímeros/química , Animales , Femenino , Proteína p24 del Núcleo del VIH/química , VIH-1/inmunología , Humanos , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Poli I-C/químicaRESUMEN
BACKGROUND AND OBJECTIVES: Inherited peripheral neuropathies (IPNs) are a group of genetic disorders of the peripheral nervous system in which neuropathy is the only or the most predominant clinical feature. The most common type of IPN is Charcot-Marie-Tooth (CMT) disease. Autosomal recessive CMT (ARCMT) is generally more severe than dominant CMT and its genetic basis is poorly understood due to high clinical and genetic diversity. Here, we report clinical and genetic findings from 56 consanguineous Turkish families initially diagnosed with CMT disease. METHODS: We initially screened the GDAP1 gene in our cohort as it is the most commonly mutated ARCMT gene. Next, whole-exome sequencing and homozygosity mapping based on whole-exome sequencing (HOMWES) analysis was performed. To understand the molecular impact of candidate causative genes, functional analyses were performed in patient primary fibroblasts. RESULTS: Biallelic recurrent mutations in the GDAP1 gene have been identified in 6 patients. Whole-exome sequencing and HOMWES analysis revealed 16 recurrent and 13 novel disease-causing alleles in known IPN-related genes and 2 novel candidate genes: 1 for a CMT-like disease and 1 for autosomal recessive cerebellar ataxia with axonal neuropathy. We have achieved a potential genetic diagnosis rate of 62.5% (35/56 families) in our cohort. Considering only the variants that meet the American College for Medical Genetics and Genomics (ACMG) classification as pathogenic or likely pathogenic, the definitive diagnosis rate was 55.35% (31/56 families). DISCUSSION: This study paints a genetic landscape of the Turkish ARCMT population and reports additional candidate genes that might help enlighten the mechanism of pathogenesis of the disease.
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OBJECTIVE: To identify the unknown genetic cause in a nuclear family with an axonal form of peripheral neuropathy and atypical disease course. METHODS: Detailed neurologic, electrophysiologic, and neuropathologic examinations of the patients were performed. Whole exome sequencing of both affected individuals was done. The effect of the identified sequence variations was investigated at cDNA and protein level in patient-derived lymphoblasts. The plasma sphingoid base profile was analyzed. Functional consequences of neuron-specific downregulation of the gene were studied in Drosophila. RESULTS: Both patients present an atypical form of axonal peripheral neuropathy, characterized by acute or subacute onset and episodes of recurrent mononeuropathy. We identified compound heterozygous mutations cosegregating with disease and absent in controls in the SGPL1 gene, encoding sphingosine 1-phosphate lyase (SPL). The p.Ser361* mutation triggers nonsense-mediated mRNA decay. The missense p.Ile184Thr mutation causes partial protein degradation. The plasma levels of sphingosine 1-phosphate and sphingosine/sphinganine ratio were increased in the patients. Neuron-specific downregulation of the Drosophila orthologue impaired the morphology of the neuromuscular junction and caused progressive degeneration of the chemosensory neurons innervating the wing margin bristles. CONCLUSIONS: We suggest SPL deficiency as a cause of a distinct form of Charcot-Marie-Tooth disease in humans, thus extending the currently recognized clinical and genetic spectrum of inherited peripheral neuropathies. Our data emphasize the importance of sphingolipid metabolism for neuronal function.