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1.
Life Sci Alliance ; 6(9)2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37414529

RESUMEN

Impaired development and maintenance of Schlemm's canal (SC) are associated with perturbed aqueous humor outflow and intraocular pressure. The angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and maintenance, whereas the molecular mechanisms of crosstalk between SC and the neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular pressure. Visible-light optical coherence tomography analysis further demonstrated functional impairment of the SC in response to changes in intraocular pressure in NC-Foxc2 -/- mice, suggesting altered TM biomechanics. Single-cell RNA-sequencing analysis identified that this phenotype is predominately characterized by transcriptional changes associated with extracellular matrix organization and stiffness in TM cell clusters, including increased matrix metalloproteinase expression, which can cleave the TIE2 ectodomain to produce soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC morphogenesis because of reduced TIE2 expression, which was rescued by deleting the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and morphogenesis via TM-SC crosstalk.


Asunto(s)
Glaucoma , Malla Trabecular , Animales , Ratones , Humor Acuoso/fisiología , Glaucoma/genética , Glaucoma/patología , Presión Intraocular , Canal de Schlemm , Malla Trabecular/patología , Malla Trabecular/fisiología
2.
EMBO Rep ; 24(7): e56030, 2023 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-37154714

RESUMEN

Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia-induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia-reperfusion (I/R) injury. Vascular endothelial cell (EC)- and lymphatic EC (LEC)-specific deletions of Foxc1, Foxc2, or both in mice worsen I/R-induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R-spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R-induced intestinal damage in EC- and LEC-Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.


Asunto(s)
Intestinos , Daño por Reperfusión , Ratones , Animales , Células Endoteliales/metabolismo , Vía de Señalización Wnt , Mucosa Intestinal , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo
3.
J Neurosci Methods ; 383: 109730, 2023 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-36280087

RESUMEN

BACKGROUND: Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain remains unclear. NEW METHOD: We generated C-terminal 2xHA-tag knock-in Cnpy3 mice by i-GONAD in vivo genome editing system to investigate the expression and function of Cnpy3 in the mouse brain. RESULTS: 2xHA-tagged Cnpy3 was confirmed by immunoblot analysis using anti-HA and CNPY3 antibodies, although HA tagging caused the decreased Cnpy3 protein level. Immunohistochemical analysis of Cnpy32xHA knock-in mice showed that Cnpy3-2xHA was predominantly expressed in the neuron. In addition, Cnpy3 and Cnpy3-2xHA were both localized in the endoplasmic reticulum and synaptosome and showed age-dependent expression changes in the brain. COMPARISON WITH EXISTING METHODS: Conventional Cnpy3 antibodies could not allow us to investigate the distribution of Cnpy3 expression in the brain, while HA-tagging revealed the expression of CNPY3 in neuronal cells. CONCLUSIONS: Taken together, we demonstrated that Cnpy32xHA knock-in mice would be useful to further elucidate the property of Cnpy3 in brain function and neurological disorders.


Asunto(s)
Epilepsia , Neuronas , Animales , Ratones , Neuronas/fisiología , Encéfalo/metabolismo , Electroencefalografía , Ratones Noqueados , Anticuerpos
4.
Int J Mol Sci ; 23(19)2022 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-36233218

RESUMEN

Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIß mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and ß proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable.


Asunto(s)
Electroporación , Edición Génica , Animales , Anticuerpos/metabolismo , Western Blotting , Sistemas CRISPR-Cas/genética , Epítopos/genética , Epítopos/metabolismo , Gónadas/metabolismo , Ratones
5.
Int J Mol Sci ; 23(18)2022 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-36142589

RESUMEN

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.


Asunto(s)
Edición Génica , Ácidos Nucleicos , Animales , Sistemas CRISPR-Cas , Electroporación/métodos , Femenino , Edición Génica/métodos , Gónadas , Humanos , Hialuronoglucosaminidasa/genética , Mamíferos/genética , Ratones , Oviductos , Embarazo , Ratas , Ribonucleoproteínas/genética , Cigoto
6.
J Neurosci Res ; 100(3): 880-896, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-35043465

RESUMEN

Recently, we have identified CaMKIIα and CaMKIIß mutations in patients with neurodevelopmental disorders by whole exome sequencing study. Most CaMKII mutants have increased phosphorylation of Thr286/287, which induces autonomous activity of CaMKII, using cell culture experiments. In this study, we explored the pathological mechanism of motor dysfunction observed exclusively in a patient with Pro213Leu mutation in CaMKIIß using a mouse model of the human disease. The homozygous CaMKIIß Pro213Leu knockin mice showed age-dependent motor dysfunction and growth failure from 2 weeks after birth. In the cerebellum, the mutation did not alter the mRNA transcript level, but the CaMKIIß protein level was dramatically decreased. Furthermore, in contrast to previous result from cell culture, Thr287 phosphorylation of CaMKIIß was also reduced. CaMKIIß Pro213Leu knockin mice showed similar motor dysfunction as CaMKIIß knockout mice, newly providing evidence for a loss of function rather than a gain of function. Our disease model mouse showed similar phenotypes of the patient, except for epileptic seizures. We clearly demonstrated that the pathological mechanism is a reduction of mutant CaMKIIß in the brain, and the physiological aspects of mutation were greatly different between in vivo and cell culture.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Cerebelo , Animales , Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cerebelo/metabolismo , Humanos , Ratones , Mutación/genética , Fosforilación
7.
J Hum Genet ; 67(7): 387-392, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35067677

RESUMEN

Exome sequencing and panel testing have improved diagnostic yield in genetic analysis by comprehensively detecting pathogenic variants in exonic regions. However, it is important to identify non-exonic pathogenic variants to further improve diagnostic yield. Here, we present a female proband and her father who is diagnosed with Marfan syndrome, a systemic connective tissue disorder caused by pathogenic variants in FBN1. There are also two affected individuals in the siblings of the father, indicating the genetic basis in this family. However, panel testing performed by two institutions reported no causal variants. To further explore the genetic basis of the family, we performed genome sequencing of the proband and RNA sequencing of urinary cells derived from urine samples of the proband and her father because FBN1 is strongly expressed in urinary cells though it is poorly expressed in peripheral blood mononuclear cells. Genome sequencing identified a rare intronic variant (c.5789-15G>A) in intron 47 of FBN1 (NM_000138.4), which was transmitted from her father. RNA sequencing revealed allelic imbalance (monoallelic expression) of FBN1, retention of intron 47, and fewer aberrant transcripts utilizing new acceptor sites within exon 48, which were confirmed by RT-PCR. These results highlighted urinary cells as clinically accessible tissues for RNA sequencing if disease-causing genes are not sufficiently expressed in the blood, and the usefulness of multi-omics analysis for molecular diagnosis of genetic disorders.


Asunto(s)
Fibrilina-1 , Síndrome de Marfan , Empalme del ARN , Orina , Femenino , Fibrilina-1/genética , Humanos , Leucocitos Mononucleares , Masculino , Síndrome de Marfan/diagnóstico , Síndrome de Marfan/genética , Mutación , Análisis de Secuencia de ARN , Orina/citología
8.
Brain Dev ; 44(2): 178-183, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-34598833

RESUMEN

BACKGROUND: Heterozygous variants in TMEM63A have been recently identified as the cause of infantile-onset transient hypomyelination. To date, four TMEM63A variants have been reported in five patients. These patients exhibited favorable clinical course, developmental progress, and completion of myelination. CASE REPORT: The patient was a 5-year-old girl with severe global developmental delay, absent speech, no turning over, no gazing, hypotonia, and daily episodes of autonomic seizures. Brain MRI showed hypomyelination of deep and subcortical white matter that appeared hyperintense in T2-weighted imaging from 2 months of age and that showed no change at 4 years of age. Exome sequencing of the patient and her parents revealed a novel de novo missense variant, NM_014698.3:c.1658G>T, p.(Gly553Val), in the TMEM63A gene, which was confirmed by Sanger sequencing. The variant has not been registered in public databases, and it substitutes a highly conserved glycine residue located in a pore-lining transmembrane helix. No other candidate variants were identified. CONCLUSIONS: Although TMEM63A variants are generally thought to cause transient hypomyelination with favorable developmental progress, identification of a de novo TMEM63A variant in our patient suggests that the TMEM63A-related clinical spectrum is broad and includes severe developmental delay with seizures.


Asunto(s)
Canales de Calcio/genética , Discapacidades del Desarrollo/genética , Leucoencefalopatías/genética , Preescolar , Femenino , Humanos
9.
J Hum Genet ; 66(12): 1189-1192, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34168248

RESUMEN

Brain malformations have heterogeneous genetic backgrounds. Tubulinopathies are a wide range of brain malformations caused by variants in tubulin and microtubules-associated genes. Recently biallelic variants in TTC5, also known as stress responsive activator of p300, have been reported in 11 patients from seven families with developmental delay, intellectual disability, and brain malformations. Here, we report compound heterozygous frameshift variants in TTC5 in a Japanese boy who showed severe psychomotor developmental delay and pseudobulbar palsy with growth failure. Brain magnetic resonance imaging showed a simplified gyral pattern and undetectable anterior limb of the internal capsule, suggesting tubulinopathies. Immunoblotting using lymphoblastoid cells derived from the patient showed undetectable TTC5 protein. Ttc5 silencing by RNA interference in Neuro2a cells reduced Tubulin ß3 protein level and caused abnormal cell cycle. Our report suggests a possible link between TTC5-related brain malformation and tubulinopathies.


Asunto(s)
Encéfalo/anomalías , Mutación del Sistema de Lectura , Predisposición Genética a la Enfermedad , Malformaciones del Sistema Nervioso/diagnóstico , Malformaciones del Sistema Nervioso/genética , Fenotipo , Factores de Transcripción/genética , Tubulina (Proteína)/genética , Encéfalo/diagnóstico por imagen , Estudios de Asociación Genética , Humanos , Japón , Imagen por Resonancia Magnética , Masculino , Tubulina (Proteína)/metabolismo
10.
Clin Epigenetics ; 13(1): 73, 2021 04 07.
Artículo en Inglés | MEDLINE | ID: mdl-33827678

RESUMEN

BACKGROUND: Parthenogenetic mosaicism is an extremely rare condition identified only in five subjects to date. The previous studies indicate that this condition is mediated by parthenogenetic activation and is free from a specific phenotype ascribed to unmaking of a maternally inherited recessive variant in the parthenogenetic cell lineage. RESULTS: We examined a 28-year-old Japanese 46,XX female with Silver-Russell syndrome and idiopathic hypersomnia. The results revealed (1) predominance of maternally derived alleles for all the differentially methylated regions examined; (2) no disease-related copy-number variant; (3) two types of regions for all chromosomes, i.e., four BAF (B-allele frequency) band regions with single major microsatellite peaks of maternal origin and single minor microsatellite peaks of non-maternal (paternal) origin, and six BAF band regions with single major microsatellite peaks of maternal origin and two minor microsatellite peaks of maternal and non-maternal (paternal) origin; (4) an unmasked extremely rare PER2 variant (c.1403G>A:p.(Arg468Gln)) with high predicted pathogenicity; (5) mildly affected local structure with altered hydrogen bonds of the p.Arg468Gln-PER2 protein; and (6) nucleus-dominant subcellular distribution of the p.Arg468Gln-PER2 protein. CONCLUSIONS: The above findings imply that the second polar body retention occurred around fertilization, resulting in the generation of the parthenogenetic cell lineage by endoreplication of a female pronucleus and the normal cell lineage by fusion of male and female pronuclei, and that the homozygous PER2 variant in the parthenogenetic cells is the likely causative factor for idiopathic hypersomnia.


Asunto(s)
Pueblo Asiatico/genética , Trastornos de Somnolencia Excesiva/genética , Predisposición Genética a la Enfermedad , Mosaicismo , Partenogénesis/genética , Proteínas Circadianas Period/genética , Cuerpos Polares , Adulto , Trastornos de Somnolencia Excesiva/fisiopatología , Femenino , Variación Genética , Genotipo , Humanos
11.
Nat Commun ; 12(1): 2107, 2021 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-33833240

RESUMEN

Vacuolar H+-ATPases (V-ATPases) transport protons across cellular membranes to acidify various organelles. ATP6V0A1 encodes the a1-subunit of the V0 domain of V-ATPases, which is strongly expressed in neurons. However, its role in brain development is unknown. Here we report four individuals with developmental and epileptic encephalopathy with ATP6V0A1 variants: two individuals with a de novo missense variant (R741Q) and the other two individuals with biallelic variants comprising one almost complete loss-of-function variant and one missense variant (A512P and N534D). Lysosomal acidification is significantly impaired in cell lines expressing three missense ATP6V0A1 mutants. Homozygous mutant mice harboring human R741Q (Atp6v0a1R741Q) and A512P (Atp6v0a1A512P) variants show embryonic lethality and early postnatal mortality, respectively, suggesting that R741Q affects V-ATPase function more severely. Lysosomal dysfunction resulting in cell death, accumulated autophagosomes and lysosomes, reduced mTORC1 signaling and synaptic connectivity, and lowered neurotransmitter contents of synaptic vesicles are observed in the brains of Atp6v0a1A512P/A512P mice. These findings demonstrate the essential roles of ATP6V0A1/Atp6v0a1 in neuronal development in terms of integrity and connectivity of neurons in both humans and mice.


Asunto(s)
Encefalopatías/genética , Encéfalo/crecimiento & desarrollo , Neuronas/fisiología , Neurotransmisores/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Autofagosomas/patología , Mapeo Encefálico/métodos , Catepsina D/metabolismo , Línea Celular , Células HEK293 , Humanos , Mutación con Pérdida de Función/genética , Lisosomas/patología , Imagen por Resonancia Magnética/métodos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Mutación Missense/genética , Neuronas/citología , Vesículas Sinápticas/patología
12.
Dev Dyn ; 250(8): 1125-1139, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33667029

RESUMEN

BACKGROUND: Foxc2 is a member of the winged helix/forkhead (Fox) box family of transcription factors. Loss of function of Foxc2 causes craniofacial abnormalities such as cleft palate and deformed cranial base, but its role during craniofacial development remains to be elucidated. RESULTS: The contributions of Foxc2-positive and its descendant cells to the craniofacial structure at E18.5 were examined using a tamoxifen-inducible Cre driver mouse (Foxc2-CreERT2) crossed with the R26R-LacZ reporter mouse. Foxc2 expression at E8.5 is restricted to the cranial mesenchyme, contributing to specific components including the cranial base, sensory capsule, tongue, upper incisor, and middle ear. Expression at E10.5 was still positively regulated in most of those regions. In situ hybridization analysis of Foxc2 and its closely related gene, Foxc1, revealed that expression domains of these genes largely overlap in the cephalic mesenchyme. Meanwhile, the tongue expressed Foxc2 but not Foxc1, and its development was affected by the neural crest-specific deletion of Foxc2 in mice (Wnt1-Cre; Foxc2fl/fl ). CONCLUSIONS: Foxc2 is expressed in cranial mesenchyme that contributes to specific craniofacial tissue components from an early stage, and it seems to be involved in their development in cooperation with Foxc1. Foxc2 also has its own role in tongue development.


Asunto(s)
Linaje de la Célula/genética , Anomalías Craneofaciales/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Animales , Anomalías Craneofaciales/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ratones , Ratones Transgénicos , Cresta Neural/embriología , Cresta Neural/metabolismo
13.
Congenit Anom (Kyoto) ; 60(4): 120-125, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31837184

RESUMEN

CRISPR-Cas9 technology has been used in various studies; however, it has also been found to introduce unexpected structural alternations. In this study, we used nanopore sequencing to characterize an unexpected structural alteration of mirror-image duplicated genes in a mouse line, in which we aimed to delete a part of the duplicated genes using genome editing. We removed low-molecular-weight DNA fragments and increased the input, which led to improved sequence performance. With 14.9 Gb input for whole-genome analysis, we detected a complex structural alteration involving inversion and deletion, which appears to be difficult to characterize with short-read sequencers. Therefore, our study clearly showed the utility of nanopore sequencing for characterizing unexpected complex structural alterations caused by genome editing.


Asunto(s)
Sistemas CRISPR-Cas/genética , Duplicación de Gen/genética , Edición Génica , Secuenciación de Nanoporos , Animales , ADN/genética , Humanos , Ratones
14.
Sci Rep ; 8(1): 12059, 2018 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-30104681

RESUMEN

Zygote-microinjection or in vitro electroporation of isolated zygotes are now widely used methods to produce genome-edited mice. However, these technologies require laborious and time-consuming ex vivo handling of fertilized eggs, including zygote isolation, gene delivery into zygotes and embryo transfer into recipients. We recently developed an alternative method called improved genome-editing via oviductal nucleic acids delivery (i-GONAD), which does not require the above-mentioned ex vivo handing of zygotes, but instead involves intraoviductal instillation of genome-editing components, Cas9 protein and synthetic gRNAs, into the oviducts of pregnant females at the late 1-cell embryo stage under a dissecting microscope and subsequent electroporation. With this method, we succeeded in generating genome-edited mice at relatively high efficiencies (for example, knockout alleles were produced at ~97% efficiency). Here, we extended this improved technology to rats, and found that i-GONAD can create genome-edited rats in various strains, including Sprague Dawley and Lewis, and F1 hybrids (between Sprague Dawley and Brown Norway), with efficiencies of ~62% for indel mutations and ~9% for knock-ins. Thus, i-GONAD will be especially useful for the production of genome-edited rats in small laboratories where expensive micromanipulator systems and highly skilled personnel for embryo manipulation are unavailable.


Asunto(s)
Sistemas CRISPR-Cas/genética , Electroporación/métodos , Trompas Uterinas , Edición Génica/métodos , Animales , Proteína 9 Asociada a CRISPR/administración & dosificación , Proteína 9 Asociada a CRISPR/genética , Embrión de Mamíferos , Femenino , Masculino , Mutación , Factor de Transcripción PAX6/genética , Embarazo , ARN Guía de Kinetoplastida/administración & dosificación , ARN Guía de Kinetoplastida/genética , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Ratas Transgénicas
15.
Hum Mutat ; 39(8): 1070-1075, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29768694

RESUMEN

By whole exome sequencing, we identified three de novo RHOBTB2 variants in three patients with epileptic encephalopathies (EEs). Interestingly, all three patients showed acute encephalopathy (febrile status epilepticus), with magnetic resonance imaging revealing hemisphere swelling or reduced diffusion in various brain regions. RHOBTB2 encodes Rho-related BTB domain-containing protein 2, an atypical Rho GTPase that is a substrate-specific adaptor or itself is a substrate for the Cullin-3 (CUL3)-based ubiquitin ligase complex. Transient expression experiments in Neuro-2a cells revealed that mutant RHOBTB2 was more abundant than wild-type RHOBTB2. Coexpression of CUL3 with RHOBTB2 decreased the level of wild-type RHOBTB2 but not the level of any of the three mutants, indicating impaired CUL3 complex-dependent degradation of the three mutants. These data indicate that RHOBTB2 variants are a rare genetic cause of EEs, in which acute encephalopathy might be a characteristic feature, and that precise regulation of RHOBTB2 levels is essential for normal brain function.


Asunto(s)
Epilepsia/genética , Epilepsia/patología , Proteínas de Unión al GTP/genética , Proteínas Supresoras de Tumor/genética , Proteínas de Unión al GTP rho/genética , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Adulto Joven
16.
Ann Clin Transl Neurol ; 5(3): 280-296, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29560374

RESUMEN

Objective: α (CAMK2A) and ß (CAMK2B) isoforms of Calcium/calmodulin-dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α- and ß-CaMKII variants in neurodevelopmental disorders. Methods: Whole-exome sequencing was performed for 976 individuals with intellectual disability, developmental delay, and epilepsy. The effect of CAMK2A and CAMK2B variants on CaMKII structure and firing of neurons was evaluated by computational structural analysis, immunoblotting, and electrophysiological analysis. Results: We identified a total of five de novo CAMK2A and CAMK2B variants in three and two individuals, respectively. Seizures were common to three individuals with CAMK2A variants. Using a minigene splicing assay, we demonstrated that a splice site variant caused skipping of exon 11 leading to an in-frame deletion of the regulatory segment of CaMKII α. By structural analysis, four missense variants are predicted to impair the interaction between the kinase domain and the regulatory segment responsible for the autoinhibition of its kinase activity. The Thr286/Thr287 phosphorylation as a result of release from autoinhibition was increased in three mutants when the mutants were stably expressed in Neuro-2a neuroblastoma cells. Expression of a CaMKII α mutant in primary hippocampal neurons significantly increased A-type K+ currents, which facilitated spike repolarization of single action potentials. Interpretation: Our data highlight the importance of CaMKII α and CaMKII ß and their autoinhibitory regulation in human brain function, and suggest the enhancement of A-type K+ currents as a possible pathophysiological basis.

17.
Ann Neurol ; 83(4): 794-806, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29534297

RESUMEN

OBJECTIVE: The cytoplasmic fragile X mental retardation 1 interacting proteins 2 (CYFIP2) is a component of the WASP-family verprolin-homologous protein (WAVE) regulatory complex, which is involved in actin dynamics. An obvious association of CYFIP2 variants with human neurological disorders has never been reported. Here, we identified de novo hotspot CYFIP2 variants in neurodevelopmental disorders and explore the possible involvement of the CYFIP2 mutants in the WAVE signaling pathway. METHODS: We performed trio-based whole-exome sequencing (WES) in 210 families and case-only WES in 489 individuals with epileptic encephalopathies. The functional effect of CYFIP2 variants on WAVE signaling was evaluated by computational structural analysis and in vitro transfection experiments. RESULTS: We identified three de novo CYFIP2 variants at the Arg87 residue in 4 unrelated individuals with early-onset epileptic encephalopathy. Structural analysis indicated that the Arg87 residue is buried at an interface between CYFIP2 and WAVE1, and the Arg87 variant may disrupt hydrogen bonding, leading to structural instability and aberrant activation of the WAVE regulatory complex. All mutant CYFIP2 showed comparatively weaker interactions to the VCA domain than wild-type CYFIP2. Immunofluorescence revealed that ectopic speckled accumulation of actin and CYFIP2 was significantly increased in cells transfected with mutant CYFIP2. INTERPRETATION: Our findings suggest that de novo Arg87 variants in CYFIP2 have gain-of-function effects on the WAVE signaling pathway and are associated with severe neurological disorders. Ann Neurol 2018;83:794-806.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Arginina/genética , Mutación/genética , Espasmos Infantiles/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Línea Celular Transformada , Niño , Preescolar , Electroencefalografía , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Ratones , Modelos Moleculares , Linaje , Espasmos Infantiles/diagnóstico por imagen , Transfección , Secuenciación del Exoma
18.
Am J Hum Genet ; 102(2): 321-329, 2018 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-29394991

RESUMEN

Early-onset epileptic encephalopathies, including West syndrome (WS), are a group of neurological disorders characterized by developmental impairments and intractable seizures from early infancy. We have now identified biallelic CNPY3 variants in three individuals with WS; these include compound-heterozygous missense and frameshift variants in a family with two affected siblings (individuals 1 and 2) and a homozygous splicing variant in a consanguineous family (individual 3). All three individuals showed hippocampal malrotation. In individuals 1 and 2, electroencephalography (EEG) revealed characteristic fast waves and diffuse sharp- and slow-wave complexes. The fast waves were clinically associated with seizures. CNPY3 encodes a co-chaperone in the endoplasmic reticulum and regulates the subcellular distribution and responses of multiple Toll-like receptors. The amount of CNPY3 in lymphoblastoid cells derived from individuals 1 and 2 was severely lower than that in control cells. Cnpy3-knockout mice exhibited spastic or dystonic features under resting conditions and hyperactivity and anxiolytic behavior during the open field test. Also, their resting EEG showed enhanced activity in the fast beta frequency band (20-35 Hz), which could mimic the fast waves in individuals 1 and 2. These data suggest that CNPY3 and Cnpy3 perform essential roles in brain function in addition to known Toll-like receptor-dependent immune responses.


Asunto(s)
Chaperonas Moleculares/genética , Mutación , Convulsiones/genética , Espasmos Infantiles/genética , Adolescente , Secuencia de Aminoácidos , Animales , Niño , Consanguinidad , Electroencefalografía , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Familia , Femenino , Expresión Génica , Heterocigoto , Hipocampo/diagnóstico por imagen , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Convulsiones/diagnóstico por imagen , Convulsiones/fisiopatología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Hermanos , Espasmos Infantiles/diagnóstico por imagen , Espasmos Infantiles/fisiopatología
19.
Hum Genet ; 137(1): 95-104, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29322246

RESUMEN

SETD1B (SET domain containing 1B) is a component of SET1 histone methyltransferase complex, which mediates the methylation of histone H3 on lysine 4 (H3K4). Here, we describe two unrelated individuals with de novo variants in SETD1B identified by trio-based whole exome sequencing: c.5524C>T, p.(Arg1842Trp) and c.5575C>T, p.(Arg1859Cy). The two missense variants occurred at evolutionarily conserved amino acids and are located within the SET domain, which plays a pivotal role in catalyzing histone methylation. Previous studies have suggested that de novo microdeletions in the 12q24.3 region encompassing SETD1B were associated with developmental delays, intellectual disabilities, autism/autistic behavior, large stature and craniofacial anomalies. Comparative mapping of 12q24.3 deletions refined the candidate locus, indicating KDM2B and SETD1B to be the most plausible candidate genes for the pathogenicity of 12q24.3 deletion syndrome. Our cases showed epilepsy, developmental delay, intellectual disabilities, autistic behavior and craniofacial dysmorphic features, which are consistent with those of individuals with de novo 12q24.31 deletions. Therefore, our study suggests that SETD1B aberration is likely to be the core defect in 12q24.3 deletion syndrome.


Asunto(s)
Trastorno Autístico/genética , Epilepsia/genética , N-Metiltransferasa de Histona-Lisina/genética , Discapacidad Intelectual/genética , Secuencia de Aminoácidos , Pueblo Asiatico/genética , Trastorno Autístico/diagnóstico , Niño , Preescolar , Deleción Cromosómica , Cromosomas Humanos Par 12/genética , Anomalías Craneofaciales/diagnóstico , Anomalías Craneofaciales/genética , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/genética , Epilepsia/diagnóstico , Femenino , Variación Genética , Humanos , Discapacidad Intelectual/diagnóstico , Masculino , Mutación Missense , Linaje , Secuenciación del Exoma
20.
Congenit Anom (Kyoto) ; 57(1): 24-31, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-27783871

RESUMEN

Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2CreERT2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2CreERT2 ; R26R), the fate of Foxc2 positive (Foxc2+ ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2+ cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2+ descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2+ cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2+ cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2CreERT2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2+ expressing cells during mouse embryogenesis.


Asunto(s)
Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética , Animales , Linaje de la Célula/genética , Factores de Transcripción Forkhead/metabolismo , Orden Génico , Marcación de Gen/métodos , Sitios Genéticos , Vectores Genéticos/genética , Recombinación Homóloga , Inmunohistoquímica , Riñón/embriología , Riñón/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones Transgénicos
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