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1.
J Clin Invest ; 131(12)2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-33945503

RESUMEN

BACKGROUNDDeciphering the function of the many genes previously classified as uncharacterized open reading frame (ORF) would complete our understanding of a cell's function and its pathophysiology.METHODSWhole-exome sequencing, yeast 2-hybrid and transcriptome analyses, and molecular characterization were performed in this study to uncover the function of the C2orf69 gene.RESULTSWe identified loss-of-function mutations in the uncharacterized C2orf69 gene in 8 individuals with brain abnormalities involving hypomyelination and microcephaly, liver dysfunction, and recurrent autoinflammation. C2orf69 contains an N-terminal signal peptide that is required and sufficient for mitochondrial localization. Consistent with mitochondrial dysfunction, the patients showed signs of respiratory chain defects, and a CRISPR/Cas9-KO cell model of C2orf69 had similar respiratory chain defects. Patient-derived cells revealed alterations in immunological signaling pathways. Deposits of periodic acid-Schiff-positive (PAS-positive) material in tissues from affected individuals, together with decreased glycogen branching enzyme 1 (GBE1) activity, indicated an additional impact of C2orf69 on glycogen metabolism.CONCLUSIONSOur study identifies C2orf69 as an important regulator of human mitochondrial function and suggests that this gene has additional influence on other metabolic pathways.


Asunto(s)
Glucógeno/metabolismo , Mutación con Pérdida de Función , Microcefalia/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Sistemas de Lectura Abierta , Animales , Línea Celular , Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Humanos , Ratones , Ratones Noqueados , Microcefalia/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética
2.
Sci Rep ; 11(1): 6934, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767215

RESUMEN

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene. The neuropathology of HD is characterized by the decline of a specific neuronal population within the brain, the striatal medium spiny neurons (MSNs). The origins of this extreme vulnerability remain unknown. Human induced pluripotent stem cell (hiPS cell)-derived MSNs represent a powerful tool to study this genetic disease. However, the differentiation protocols published so far show a high heterogeneity of neuronal populations in vitro. Here, we compared two previously published protocols to obtain hiPS cell-derived striatal neurons from both healthy donors and HD patients. Patch-clamp experiments, immunostaining and RT-qPCR were performed to characterize the neurons in culture. While the neurons were mature enough to fire action potentials, a majority failed to express markers typical for MSNs. Voltage-clamp experiments on voltage-gated sodium (Nav) channels revealed a large variability between the two differentiation protocols. Action potential analysis did not reveal changes induced by the HD mutation. This study attempts to demonstrate the current challenges in reproducing data of previously published differentiation protocols and in generating hiPS cell-derived striatal MSNs to model a genetic neurodegenerative disorder in vitro.


Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Enfermedad de Huntington , Neuronas/fisiología , Potenciales de Acción , Animales , Calcio/metabolismo , Estudios de Casos y Controles , Línea Celular , Humanos , Células Madre Pluripotentes Inducidas , Ratones Endogámicos C57BL , Subunidad beta-4 de Canal de Sodio Activado por Voltaje/metabolismo , Ácido gamma-Aminobutírico/metabolismo
3.
Nat Commun ; 10(1): 4919, 2019 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-31664039

RESUMEN

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements.


Asunto(s)
Expansión de las Repeticiones de ADN , Epilepsias Mioclónicas/genética , Proteínas de la Membrana/genética , Ubiquitina-Proteína Ligasas/genética , Adolescente , Adulto , Anciano , Mapeo Cromosómico , Femenino , Humanos , Intrones , Masculino , Persona de Mediana Edad , Linaje , Adulto Joven
4.
PLoS Genet ; 12(12): e1006461, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27923065

RESUMEN

Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.


Asunto(s)
Proteínas de Transporte de Membrana/genética , Degeneración Nerviosa/genética , Estrés Oxidativo/genética , Dolor/genética , Receptores Virales/genética , Apoptosis/genética , Línea Celular , Exoma/genética , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Mutación del Sistema de Lectura/genética , Hemo/genética , Humanos , Inmunoprecipitación , Masculino , Degeneración Nerviosa/patología , Nociceptores/metabolismo , Nociceptores/patología , Dolor/patología , Cultivo Primario de Células , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/patología
5.
Brain ; 137(Pt 3): 683-92, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24459106

RESUMEN

Many neurodegenerative disorders present with sensory loss. In the group of hereditary sensory and autonomic neuropathies loss of nociception is one of the disease hallmarks. To determine underlying factors of sensory neurodegeneration we performed whole-exome sequencing in affected individuals with the disorder. In a family with sensory neuropathy with loss of pain perception and destruction of the pedal skeleton we report a missense mutation in a highly conserved amino acid residue of atlastin GTPase 3 (ATL3), an endoplasmic reticulum-shaping GTPase. The same mutation (p.Tyr192Cys) was identified in a second family with similar clinical outcome by screening a large cohort of 115 patients with hereditary sensory and autonomic neuropathies. Both families show an autosomal dominant pattern of inheritance and the mutation segregates with complete penetrance. ATL3 is a paralogue of ATL1, a membrane curvature-generating molecule that is involved in spastic paraplegia and hereditary sensory neuropathy. ATL3 proteins are enriched in three-way junctions, branch points of the endoplasmic reticulum that connect membranous tubules to a continuous network. Mutant ATL3 p.Tyr192Cys fails to localize to branch points, but instead disrupts the structure of the tubular endoplasmic reticulum, suggesting that the mutation exerts a dominant-negative effect. Identification of ATL3 as novel disease-associated gene exemplifies that long-term sensory neuronal maintenance critically depends on the structural organisation of the endoplasmic reticulum. It emphasizes that alterations in membrane shaping-proteins are one of the major emerging pathways in axonal degeneration and suggests that this group of molecules should be considered in neuroprotective strategies.


Asunto(s)
Enfermedades Óseas/genética , Retículo Endoplásmico/genética , GTP Fosfohidrolasas/genética , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Adulto , Edad de Inicio , Enfermedades Óseas/etiología , Enfermedades Óseas/fisiopatología , Estudios de Cohortes , Tos/genética , Tos/patología , Tos/fisiopatología , Retículo Endoplásmico/patología , Exoma/genética , Femenino , Fracturas Óseas/genética , Fracturas Óseas/patología , Reflujo Gastroesofágico/genética , Reflujo Gastroesofágico/patología , Reflujo Gastroesofágico/fisiopatología , Genes Dominantes/genética , Haplotipos/genética , Neuropatías Hereditarias Sensoriales y Autónomas/complicaciones , Neuropatías Hereditarias Sensoriales y Autónomas/patología , Neuropatías Hereditarias Sensoriales y Autónomas/fisiopatología , Humanos , Espacio Intracelular/genética , Masculino , Mutación , Mutación Missense/genética , Linaje , Fenotipo , Adulto Joven
6.
Nat Genet ; 45(11): 1399-404, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24036948

RESUMEN

The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.


Asunto(s)
Percepción del Dolor/fisiología , Dolor/genética , Potenciales de Acción/genética , Animales , Canalopatías/genética , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Canal de Sodio Activado por Voltaje NAV1.9/genética , Nociceptores/fisiología
7.
Am J Hum Genet ; 93(4): 727-34, 2013 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-24035193

RESUMEN

In guanosine diphosphate (GDP)-mannose pyrophosphorylase A (GMPPA), we identified a homozygous nonsense mutation that segregated with achalasia and alacrima, delayed developmental milestones, and gait abnormalities in a consanguineous Pakistani pedigree. Mutations in GMPPA were subsequently found in ten additional individuals from eight independent families affected by the combination of achalasia, alacrima, and neurological deficits. This autosomal-recessive disorder shows many similarities with triple A syndrome, which is characterized by achalasia, alacrima, and variable neurological deficits in combination with adrenal insufficiency. GMPPA is a largely uncharacterized homolog of GMPPB. GMPPB catalyzes the formation of GDP-mannose, which is an essential precursor of glycan moieties of glycoproteins and glycolipids and is associated with congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-dystroglycan. Surprisingly, GDP-mannose pyrophosphorylase activity was unchanged and GDP-mannose levels were strongly increased in lymphoblasts of individuals with GMPPA mutations. This suggests that GMPPA might serve as a GMPPB regulatory subunit mediating feedback inhibition of GMPPB instead of displaying catalytic enzyme activity itself. Thus, a triple-A-like syndrome can be added to the growing list of congenital disorders of glycosylation, in which dysregulation rather than mere enzyme deficiency is the basal pathophysiological mechanism.


Asunto(s)
Codón sin Sentido , Genes Recesivos/genética , Guanosina Difosfato Manosa/genética , Discapacidad Intelectual/genética , Nucleotidiltransferasas/genética , Adolescente , Insuficiencia Suprarrenal/genética , Adulto , Niño , Consanguinidad , Acalasia del Esófago/genética , Enfermedades Hereditarias del Ojo/genética , Glicosilación , Guanosina Difosfato Manosa/metabolismo , Homocigoto , Humanos , Discapacidad Intelectual/enzimología , Enfermedades del Aparato Lagrimal/genética , Enfermedades del Sistema Nervioso/genética , Nucleotidiltransferasas/metabolismo , Linaje , Adulto Joven
8.
Am J Hum Genet ; 88(5): 621-7, 2011 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-21549336

RESUMEN

The fact that hereditary hearing loss is the most common sensory disorder in humans is reflected by, among other things, an extraordinary allelic and nonallelic genetic heterogeneity. X-chromosomal hearing impairment represents only a minor fraction of all cases. In a study of a Spanish family the locus for one of the X-chromosomal forms was assigned to Xp22 (DFNX4). We mapped the disease locus in the same chromosomal region in a large German pedigree with X-chromosomal nonsyndromic hearing impairment by using genome-wide linkage analysis. Males presented with postlingual hearing loss and onset at ages 3-7, whereas onset in female carriers was in the second to third decades. Targeted DNA capture with high-throughput sequencing detected a nonsense mutation in the small muscle protein, X-linked (SMPX) of affected individuals. We identified another nonsense mutation in SMPX in patients from the Spanish family who were previously analyzed to map DFNX4. SMPX encodes an 88 amino acid, cytoskeleton-associated protein that is responsive to mechanical stress. The presence of Smpx in hair cells and supporting cells of the murine cochlea indicates its role in the inner ear. The nonsense mutations detected in the two families suggest a loss-of-function mechanism underlying this form of hearing impairment. Results obtained after heterologous overexpression of SMPX proteins were compatible with this assumption. Because responsivity to physical force is a characteristic feature of the protein, we propose that long-term maintenance of mechanically stressed inner-ear cells critically depends on SMPX function.


Asunto(s)
Cromosomas Humanos X/genética , Codón sin Sentido , Pérdida Auditiva/genética , Proteínas Musculares/genética , Adolescente , Edad de Inicio , Alelos , Animales , Niño , Preescolar , Cóclea , Oído Interno/embriología , Oído Interno/metabolismo , Femenino , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Células Ciliadas Auditivas/metabolismo , Haplotipos , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Linaje
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