RESUMEN
Intellectual disability (ID) and autism spectrum disorder (ASD) are genetically heterogeneous with hundreds of identified risk genes, most affecting only a few patients. Novel missense variants in these genes are being discovered as clinical exome sequencing is now routinely integrated into diagnosis, yet most of them are annotated as variants of uncertain significance (VUS). VUSs are a major roadblock in using patient genetics to inform clinical action. We developed a framework to characterize VUSs in Coiled-coil and C2 domain containing 1A (CC2D1A), a gene causing autosomal recessive ID with comorbid ASD in 40% of cases. We analyzed seven VUSs (p.Pro319Leu, p.Ser327Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, p.Arg886His and p.Glu910Lys) from four cases of individuals with ID and ASD. Variants were cloned and overexpressed in HEK293 individually and in their respective heterozygous combination. CC2D1A is a signaling scaffold that positively regulates PKA-CREB signaling by repressing phosphodiesterase 4D (PDE4D) to prevent cAMP degradation. After testing multiple parameters including direct interaction between PDE4D and CC2D1A, cAMP levels and CREB activation, we found that the most sensitive readout was CREB transcriptional activity using a luciferase assay. Compared to WT CC2D1A, five VUSs (p.Pro319Leu, p.Gly441Val, p.Val449Met, p.Thr580Ile, and p.Arg886His) led to significantly blunted response to forskolin induced CREB activation. This luciferase assay approach can be scaled up to annotate ~150 CC2D1A VUSs that are currently listed in ClinVar. Since CREB activation is a common denominator for multiple ASD/ID genes, our paradigm can also be adapted for their VUSs.
Asunto(s)
Trastorno del Espectro Autista , Predisposición Genética a la Enfermedad , Discapacidad Intelectual , Humanos , Trastorno del Espectro Autista/genética , Células HEK293 , Discapacidad Intelectual/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Secuenciación del Exoma/métodos , Transducción de Señal/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Masculino , Femenino , Mutación Missense/genética , AMP Cíclico/metabolismo , Anotación de Secuencia MolecularRESUMEN
Biallelic mutations in Protein O-mannosyltransferase 1 (POMT1) are among the most common causes of a severe group of congenital muscular dystrophies (CMDs) known as dystroglycanopathies. POMT1 is a glycosyltransferase responsible for the attachment of a functional glycan mediating interactions between the transmembrane glycoprotein dystroglycan and its binding partners in the extracellular matrix (ECM). Disruptions in these cell-ECM interactions lead to multiple developmental defects causing brain and eye malformations in addition to CMD. Removing Pomt1 in the mouse leads to early embryonic death due to the essential role of dystroglycan during placental formation in rodents. Here, we characterized and validated a model of pomt1 loss of function in the zebrafish showing that developmental defects found in individuals affected by dystroglycanopathies can be recapitulated in the fish. We also discovered that pomt1 mRNA provided by the mother in the oocyte supports dystroglycan glycosylation during the first few weeks of development. Muscle disease, retinal synapse formation deficits, and axon guidance defects can only be uncovered during the first week post fertilization by generating knock-out embryos from knock-out mothers. Conversely, maternal pomt1 from heterozygous mothers was sufficient to sustain muscle, eye, and brain development only leading to loss of photoreceptor synapses at 30 days post fertilization. Our findings show that it is important to define the contribution of maternal mRNA while developing zebrafish models of dystroglycanopathies and that offspring generated from heterozygous and knock-out mothers can be used to differentiate the role of dystroglycan glycosylation in tissue formation and maintenance.
Asunto(s)
Distroglicanos , Pez Cebra , Animales , Distroglicanos/genética , Distroglicanos/metabolismo , Glicosilación , Fenotipo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Congenital muscular dystrophies (CMDs) are a group of rare muscle disorders characterized by early onset hypotonia and motor developmental delay associated with brain malformations with or without eye anomalies in the most severe cases. In this study, we aimed to uncover the genetic basis of severe CMD in Egypt and to determine the efficacy of whole exome sequencing (WES)-based genetic diagnosis in this population. We recruited twelve individuals from eleven families with a clinical diagnosis of CMD with brain malformations that fell into two groups: seven patients with suspected dystroglycanopathy and five patients with suspected merosin-deficient CMD. WES was analyzed by variant filtering using multiple approaches including splicing and copy number variant (CNV) analysis. We identified likely pathogenic variants in FKRP in two cases and variants in POMT1, POMK, and B3GALNT2 in three individuals. All individuals with merosin-deficient CMD had truncating variants in LAMA2. Further analysis in one of the two unsolved cases showed a homozygous protein-truncating variant in Feline Leukemia Virus subgroup C Receptor 1 (FLVCR1). FLVCR1 loss of function has never been previously reported. Yet, loss of function of its paralog, FLVCR2, causes lethal hydranencephaly-hydrocephaly syndrome (Fowler Syndrome) which should be considered in the differential diagnosis for dystroglycanopathy. Overall, we reached a diagnostic rate of 86% (6/7) for dystroglycanopathies and 100% (5/5) for merosinopathy. In conclusion, our results provide further evidence that WES is an important diagnostic method in CMD in developing countries to improve the diagnostic rate, management plan, and genetic counseling for these disorders.
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Encéfalo , Secuenciación del Exoma , Distrofias Musculares , N-Acetilglucosaminiltransferasas , Humanos , Masculino , Egipto , Femenino , Distrofias Musculares/genética , Distrofias Musculares/diagnóstico , Preescolar , Encéfalo/anomalías , Encéfalo/patología , Niño , Lactante , Laminina/genética , Receptores Virales/genética , Manosiltransferasas/genética , Linaje , Pentosiltransferasa/genética , Variaciones en el Número de Copia de ADN , Mutación , Adolescente , Malformaciones del Sistema Nervioso/genéticaRESUMEN
ATPase, class 1, type 8 A, member 2 (ATP8A2) is a P4-ATPase with a critical role in phospholipid translocation across the plasma membrane. Pathogenic variants in ATP8A2 are known to cause cerebellar ataxia, impaired intellectual development, and disequilibrium syndrome 4 (CAMRQ4) which is often associated with encephalopathy, global developmental delay, and severe motor deficits. Here, we present a family with two siblings born from a consanguineous, first-cousin union from Sudan presenting with global developmental delay, intellectual disability, spasticity, ataxia, nystagmus, and thin corpus callosum. Whole exome sequencing revealed a homozygous missense variant in the nucleotide binding domain of ATP8A2 (p.Leu538Pro) that results in near complete loss of protein expression. This is in line with other missense variants in the same domain leading to protein misfolding and loss of ATPase function. In addition, by performing diffusion-weighted imaging, we identified bilateral hyperintensities in the posterior limbs of the internal capsule suggesting possible microstructural changes in axon tracts that had not been appreciated before and could contribute to the sensorimotor deficits in these individuals.
RESUMEN
One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.
Asunto(s)
Regulación de la Expresión Génica , Pez Cebra , Animales , Humanos , Inositol , Mutación , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Lissencephaly is a severe brain malformation in which failure of neuronal migration results in agyria or pachygyria and in which the brain surface appears unusually smooth. It is often associated with microcephaly, profound intellectual disability, epilepsy, and impaired motor abilities. Twenty-two genes are associated with lissencephaly, accounting for approximately 80% of disease. Here we report on 12 individuals with a unique form of lissencephaly; these individuals come from eight unrelated families and have bi-allelic mutations in APC2, encoding adenomatous polyposis coli protein 2. Brain imaging studies demonstrate extensive posterior predominant lissencephaly, similar to PAFAH1B1-associated lissencephaly, as well as co-occurrence of subcortical heterotopia posterior to the caudate nuclei, "ribbon-like" heterotopia in the posterior frontal region, and dysplastic in-folding of the mesial occipital cortex. The established role of APC2 in integrating the actin and microtubule cytoskeletons to mediate cellular morphological changes suggests shared function with other lissencephaly-encoded cytoskeletal proteins such as α-N-catenin (CTNNA2) and platelet-activating factor acetylhydrolase 1b regulatory subunit 1 (PAFAH1B1, also known as LIS1). Our findings identify APC2 as a radiographically distinguishable recessive form of lissencephaly.
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Alelos , Lisencefalias Clásicas y Heterotopias Subcorticales en Banda/genética , Proteínas del Citoesqueleto/genética , Discapacidades del Desarrollo/genética , Lisencefalia/genética , Femenino , Humanos , Masculino , LinajeRESUMEN
Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in sensitivity in high-resolution mass spectrometry (HRMS). Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. Here, we systematically evaluate performance limitations in this technology to develop a data acquisition strategy with deeper coverage of the neuroproteome from trace amounts of starting materials than traditional dynamic exclusion. During standard data-dependent acquisition (DDA), compact migration challenged the duty cycle of second-stage transitions and redundant targeting of abundant peptide signals lowered their identification success rate. DDA was programmed to progressively exclude a static set of high-intensity peptide signals throughout replicate measurements, essentially forming rungs of a "DDA ladder." The method was tested for â¼500 pg portions of a protein digest from cultured hippocampal (primary) neurons (mouse), which estimated the total amount of protein from a single neuron. The analysis of â¼5 ng of protein digest over all replicates, approximating â¼10 neurons, identified 428 nonredundant proteins (415 quantified), an â¼35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder enhances CE-HRMS sensitivity to single-neuron equivalent amounts of proteins, thus expanding the analytical toolbox of neuroscience.
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Proteómica , Espectrometría de Masa por Ionización de Electrospray , Animales , Electroforesis Capilar , Ratones , Péptidos , ProteínasRESUMEN
Neurodevelopmental disorders (NDDs) such as intellectual disability and autism spectrum disorder consistently show a male bias in prevalence, but it remains unclear why males and females are affected with different frequency. While many behavioral studies of transgenic NDD models have focused only on males, the requirement by the National Institutes of Health to consider sex as a biological variable has promoted the comparison of male and female performance in wild-type and mutant animals. Here, we review examples of rodent models of NDDs in which sex-specific deficits were identified in molecular, physiological, and/or behavioral responses, showing sex differences in susceptibility to disruption of genes mutated in NDDs. Haploinsufficiency in genes involved in mechanisms such as synaptic function (GABRB3 and NRXN1), chromatin remodeling (CHD8, EMHT1, and ADNP), and intracellular signaling (CC2D1A and ERK1) lead to more severe behavioral outcomes in males. However, in the absence of behavioral deficits, females can still present with cellular and electrophysiological changes that could be due to compensatory mechanisms or differential allocation of molecular and cellular functions in the two sexes. By contrasting these findings with mouse models where females are more severely affected (MTHFR and AMBRA1), we propose a framework to approach the study of sex-specific deficits possibly leading to sex bias in NDDs.
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Modelos Animales de Enfermedad , Trastornos del Neurodesarrollo , Caracteres Sexuales , Animales , Femenino , Masculino , Ratones , RatasRESUMEN
Digging behavior is often used to test motor function and repetitive behaviors in mice. Different digging paradigms have been developed for behaviors related to anxiety and compulsion in mouse lines generated to recapitulate genetic mutations leading to psychiatric and neurological disorders. However, the interpretation of these tests has been confounded by the difficulty of determining the motivation behind digging in mice. Digging is a naturalistic mouse behavior that can be focused toward different goals, that is foraging for food, burrowing for shelter, burying objects, or even for recreation as has been shown for dogs, ferrets, and human children. However, the interpretation of results from current testing protocols assumes the motivation behind the behavior often concluding that increased digging is a repetitive or compulsive behavior. We asked whether providing a choice between different types of digging activities would increase sensitivity to assess digging motivation. Here, we present a test to distinguish between burrowing and exploratory digging in mice. We found that mice prefer burrowing when the option is available. When food restriction was used to promote a switch from burrowing to exploration, males readily switched from burrowing to digging outside, while females did not. In addition, when we tested a model of intellectual disability and autism spectrum disorder that had shown inconsistent results in the marble burying test, the Cc2d1a conditional knockout mouse, we found greatly reduced burrowing only in males. Our findings indicate that digging is a nuanced motivated behavior and suggest that male and female rodents may perform it differently.
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Aprendizaje Discriminativo/fisiología , Conducta Exploratoria/fisiología , Privación de Alimentos/fisiología , Caracteres Sexuales , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Congenital muscular dystrophies display a wide phenotypic and genetic heterogeneity. The combination of clinical, biochemical, and molecular genetic findings must be considered to obtain the precise diagnosis and provide appropriate genetic counselling. Here we report five individuals from four families presenting with variable clinical features including muscular dystrophy with a reduction in dystroglycan glycosylation, short stature, intellectual disability, and cataracts, overlapping both the dystroglycanopathies and Marinesco-Sjögren syndrome. Whole-exome sequencing revealed homozygous missense and compound heterozygous mutations in INPP5K in the affected members of each family. INPP5K encodes the inositol polyphosphate-5-phosphatase K, also known as SKIP (skeletal muscle and kidney enriched inositol phosphatase), which is highly expressed in the brain and muscle. INPP5K localizes to both the endoplasmic reticulum and to actin ruffles in the cytoplasm. It has been shown to regulate myoblast differentiation and has also been implicated in protein processing through its interaction with the ER chaperone HSPA5/BiP. We show that morpholino-mediated inpp5k loss of function in the zebrafish results in shortened body axis, microphthalmia with disorganized lens, microcephaly, reduced touch-evoked motility, and highly disorganized myofibers. Altogether these data demonstrate that mutations in INPP5K cause a congenital muscular dystrophy syndrome with short stature, cataracts, and intellectual disability.
Asunto(s)
Distrofia Muscular de Cinturas/genética , Monoéster Fosfórico Hidrolasas/genética , Degeneraciones Espinocerebelosas/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Niño , Modelos Animales de Enfermedad , Distroglicanos/metabolismo , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Femenino , Estudio de Asociación del Genoma Completo , Glicosilación , Trastornos del Crecimiento/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Microcefalia/genética , Músculo Esquelético/metabolismo , Mutación , Linaje , Adulto Joven , Pez Cebra/genéticaRESUMEN
PURPOSE: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. RESULTS: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. CONCLUSIONS: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.
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Aspartato Carbamoiltransferasa , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante) , Línea Celular , Dihidroorotasa , Humanos , UridinaRESUMEN
BACKGROUND: Biallelic mutations in TBC1-domain containing kinase (TBCK) lead to hypotonia, global developmental delay with severe cognitive and motor deficits, and variable presentation of dysmorphic facial features and brain malformations. It remains unclear whether hypotonia in these individuals is purely neurogenic, or also caused by progressive muscle disease. METHODS: Whole exome sequencing was performed on a family diagnosed with nonspecific myopathic changes by means of histological analysis and immunohistochemistry of muscle biopsy samples. RESULTS: A novel homozygous truncation in TBCK was found in two sisters diagnosed with muscle disease and severe psychomotor delay. TBCK was completely absent in these patients. CONCLUSIONS: Our findings identify a novel early truncating variant in TBCK associated with a severe presentation and add muscle disease to the variability of phenotypes associated with TBCK mutations. Inconsistent genotype/phenotype correlation could be ascribed to the multiple roles of TBCK in intracellular signaling and endolysosomal function in different tissues.
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Encefalopatías/genética , Mutación con Pérdida de Función , Hipotonía Muscular/genética , Músculo Esquelético/patología , Enfermedades Musculares/genética , Proteínas Serina-Treonina Quinasas/genética , Trastornos Psicomotores/genética , Convulsiones/genética , Adolescente , Encéfalo/diagnóstico por imagen , Encefalopatías/diagnóstico por imagen , Niño , Discapacidades del Desarrollo/genética , Femenino , Homocigoto , Humanos , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/genética , Imagen por Resonancia Magnética , Debilidad Muscular/genética , Debilidad Muscular/patología , Enfermedades Musculares/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Reflejo Anormal/genética , Índice de Severidad de la Enfermedad , Hermanos , Síndrome , Secuenciación del ExomaRESUMEN
Genetic mutations associated with brain malformations can lead to a spectrum of severity and it is often difficult to determine whether there are additional pathogenic variants contributing to the phenotype. Here, we present a family affected by a severe brain malformation including bilateral polymicrogyria, hydrocephalus, patchy white matter signal changes, and cerebellar and pontine hypoplasia with elongated cerebellar peduncles leading to the molar tooth sign. While the malformation is reminiscent of bilateral frontoparietal polymicrogyria (BFPP), the phenotype is more severe than previously reported and also includes features of Joubert syndrome (JBTS). Via exome sequencing, we identified homozygous truncating mutations in both ADGRG1/GPR56 and KIAA0556, which are known to cause BFPP and mild brain-specific JBTS, respectively. This study shows how two independent mutations can interact leading to complex brain malformations.
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Anomalías Múltiples/genética , Cerebelo/anomalías , Anomalías del Ojo/genética , Hidrocefalia/genética , Enfermedades Renales Quísticas/genética , Proteínas Asociadas a Microtúbulos/genética , Polimicrogiria/genética , Receptores Acoplados a Proteínas G/genética , Retina/anomalías , Niño , Exoma , Salud de la Familia , Femenino , Homocigoto , Humanos , Imagen por Resonancia Magnética , Masculino , Mesencéfalo/patología , Mutación , Linaje , Fenotipo , Prosencéfalo/patología , Análisis de Secuencia de ADN , Sudán , Sustancia Blanca/patología , Secuenciación del Exoma , Adulto JovenRESUMEN
Label-free single-cell proteomics by mass spectrometry (MS) is currently incompatible with complex tissues without requiring cell culturing, single-cell dissection, or tissue dissociation. We here report the first example of label-free single-cell MS-based proteomics directly in single cells in live vertebrate embryos. Our approach integrates optically guided in situ subcellular capillary microsampling, one-pot extraction-digestion of the collected proteins, peptide separation by capillary electrophoresis, ionization by an ultrasensitive electrokinetically pumped nanoelectrospray, and detection by high-resolution MS (Orbitrap). With a 700 zmol (420â¯000 copies) lower limit of detection, this trace-sensitive technology confidently identified and quantified â¼750-800 protein groups (<1% false-discovery rate) by analyzing just â¼5 ng of protein digest, viz. <0.05% of the total protein content from individual cells in a 16-cell Xenopus laevis (frog) embryo. After validating the approach by recovering animal-vegetal-pole proteomic asymmetry in the frog zygote, the technology was applied to uncover proteomic reorganization as the animal-dorsal (D11) cell of the 16-cell embryo gave rise to its neural-tissue-fated clone in the embryo developing to the 32-, 64-, and 128-cell stages. In addition to enabling proteomics on smaller cells in X. laevis, we also demonstrated this technology to be scalable to single cells in live zebrafish embryos. Microsampling single-cell MS-based proteomics raises exciting opportunities to study cell and developmental processes directly in complex tissues and whole organisms at the level of the building block of life: the cell.
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Proteómica , Análisis de la Célula Individual , Proteínas de Xenopus/análisis , Proteínas de Pez Cebra/análisis , Animales , Células Clonales/química , Células Clonales/citología , Electroforesis Capilar , Embrión no Mamífero/química , Embrión no Mamífero/citología , Espectrometría de Masas , Xenopus laevis , Pez CebraRESUMEN
PURPOSE: To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients.
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Proteínas de Unión al ADN/genética , Discapacidades del Desarrollo/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Factores de Transcripción/genética , Adolescente , Adulto , Alelos , Trastorno Autístico/genética , Trastorno Autístico/patología , Niño , Preescolar , Discapacidades del Desarrollo/patología , Exoma/genética , Femenino , Estudios de Asociación Genética , Humanos , Discapacidad Intelectual/patología , Trastornos del Desarrollo del Lenguaje/genética , Trastornos del Desarrollo del Lenguaje/patología , Masculino , Microcefalia/patología , Mutación Missense/genética , Adulto JovenRESUMEN
Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knock-out of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knock-out (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment.SIGNIFICANCE STATEMENT Altered regulation of the 5-HT1A autoreceptor has been implicated in human anxiety, major depression, suicide, and resistance to antidepressants. This study uniquely identifies a single transcription factor, Freud-1, as crucial for 5-HT1A autoreceptor expression in vivo Disruption of Freud-1 in serotonin neurons in mice links upregulation of 5-HT1A autoreceptors to anxiety/depression-like behavior and provides a new model of antidepressant resistance. Treatment strategies to reestablish transcriptional regulation of 5-HT1A autoreceptors could provide a more robust and sustained antidepressant response.
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Ansiedad/metabolismo , Autorreceptores/biosíntesis , Trastorno Depresivo Resistente al Tratamiento/metabolismo , Fluoxetina/uso terapéutico , Receptor de Serotonina 5-HT1A/biosíntesis , Proteínas Represoras/deficiencia , Animales , Antidepresivos de Segunda Generación/farmacología , Antidepresivos de Segunda Generación/uso terapéutico , Ansiedad/tratamiento farmacológico , Autorreceptores/antagonistas & inhibidores , Autorreceptores/genética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Trastorno Depresivo Resistente al Tratamiento/tratamiento farmacológico , Femenino , Fluoxetina/farmacología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptor de Serotonina 5-HT1A/genética , Proteínas Represoras/genética , Neuronas Serotoninérgicas/efectos de los fármacos , Neuronas Serotoninérgicas/metabolismoRESUMEN
Loss-of-function (LOF) mutations in CC2D1A cause a spectrum of neurodevelopmental disorders, including intellectual disability, autism spectrum disorder, and seizures, identifying a critical role for this gene in cognitive and social development. CC2D1A regulates intracellular signaling processes that are critical for neuronal function, but previous attempts to model the human LOF phenotypes have been prevented by perinatal lethality in Cc2d1a-deficient mice. To overcome this challenge, we generated a floxed Cc2d1a allele for conditional removal of Cc2d1a in the brain using Cre recombinase. While removal of Cc2d1a in neuronal progenitors using Cre expressed from the Nestin promoter still causes death at birth, conditional postnatal removal of Cc2d1a in the forebrain via calcium/calmodulin-dependent protein kinase II-alpha (CamKIIa) promoter-driven Cre generates animals that are viable and fertile with grossly normal anatomy. Analysis of neuronal morphology identified abnormal cortical dendrite organization and a reduction in dendritic spine density. These animals display deficits in neuronal plasticity and in spatial learning and memory that are accompanied by reduced sociability, hyperactivity, anxiety, and excessive grooming. Cc2d1a conditional knockout mice therefore recapitulate features of both cognitive and social impairment caused by human CC2D1A mutation, and represent a model that could provide much needed insights into the developmental mechanisms underlying nonsyndromic neurodevelopmental disorders.
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Trastorno del Espectro Autista/genética , Discapacidad Intelectual/genética , Neuronas/citología , Prosencéfalo/patología , Proteínas Represoras/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dendritas/metabolismo , Dendritas/patología , Modelos Animales de Enfermedad , Humanos , Ratones Transgénicos , Plasticidad Neuronal/genética , Proteínas Represoras/deficiencia , Transducción de Señal/fisiologíaRESUMEN
Autism spectrum disorder (ASD) is a constellation of neurodevelopmental presentations with high heritability and both phenotypic and genetic heterogeneity. To date, mutations in hundreds of genes have been associated to varying degrees with increased ASD risk. A better understanding of the functions of these genes and whether they fit together in functional groups or impact similar neuronal circuits is needed to develop rational treatment strategies. We will review current areas of emphasis in ASD research, starting from human genetics and exploring how mouse models of human mutations have helped identify specific molecular pathways (protein synthesis and degradation, chromatin remodeling, intracellular signaling), which are linked to alterations in circuit function and cognitive/social behavior. We will conclude by discussing how we can leverage the findings on molecular and cellular alterations found in ASD to develop therapies for neurodevelopmental disorders.
Asunto(s)
Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/terapia , Encéfalo/metabolismo , Terapia Genética/métodos , Proteínas del Tejido Nervioso/genética , Trastorno del Espectro Autista/diagnóstico , Medicina Basada en la Evidencia , Marcadores Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Pruebas Genéticas/métodos , Humanos , Terapia Molecular Dirigida/métodos , Proteínas del Tejido Nervioso/metabolismo , Resultado del TratamientoRESUMEN
Dystroglycan is a transmembrane glycoprotein whose interactions with the extracellular matrix (ECM) are necessary for normal muscle and brain development, and disruptions of its function lead to dystroglycanopathies, a group of congenital muscular dystrophies showing extreme genetic and clinical heterogeneity. Specific glycans bound to the extracellular portion of dystroglycan, α-dystroglycan, mediate ECM interactions and most known dystroglycanopathy genes encode glycosyltransferases involved in glycan synthesis. POMK, which was found mutated in two dystroglycanopathy cases, is instead involved in a glycan phosphorylation reaction critical for ECM binding, but little is known about the clinical presentation of POMK mutations or of the function of this protein in the muscle. Here, we describe two families carrying different truncating alleles, both removing the kinase domain in POMK, with different clinical manifestations ranging from Walker-Warburg syndrome, the most severe form of dystroglycanopathy, to limb-girdle muscular dystrophy with cognitive defects. We explored POMK expression in fetal and adult human muscle and identified widespread expression primarily during fetal development in myocytes and interstitial cells suggesting a role for this protein during early muscle differentiation. Analysis of loss of function in the zebrafish embryo and larva showed that pomk function is necessary for normal muscle development, leading to locomotor dysfuction in the embryo and signs of muscular dystrophy in the larva. In summary, we defined diverse clinical presentations following POMK mutations and showed that this gene is necessary for early muscle development.
Asunto(s)
Estudios de Asociación Genética , Desarrollo de Músculos/genética , Mutación , Enfermedades Neuromusculares/diagnóstico , Enfermedades Neuromusculares/genética , Fenotipo , Proteínas Quinasas/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Distroglicanos/metabolismo , Exoma , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Estudio de Asociación del Genoma Completo , Glicosilación , Humanos , Imagen por Resonancia Magnética , Masculino , Datos de Secuencia Molecular , Linaje , Proteínas Quinasas/química , Alineación de Secuencia , Adulto Joven , Pez CebraRESUMEN
Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in ß-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a ß-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.