RESUMEN
Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs.
Asunto(s)
Anomalías Congénitas/genética , Metilación de ADN , Enfermedades Genéticas Congénitas/diagnóstico , Estudio de Asociación del Genoma Completo , Estudios de Cohortes , Simulación por Computador , Anomalías Congénitas/diagnóstico , Variaciones en el Número de Copia de ADN , Epigenómica , Dosificación de Gen , Enfermedades Genéticas Congénitas/genética , Variación Genética , Impresión Genómica , Humanos , Fenotipo , Análisis de Secuencia de ADN , Síndrome , Expansión de Repetición de TrinucleótidoRESUMEN
MN1 encodes a transcriptional co-regulator without homology to other proteins, previously implicated in acute myeloid leukaemia and development of the palate. Large deletions encompassing MN1 have been reported in individuals with variable neurodevelopmental anomalies and non-specific facial features. We identified a cluster of de novo truncating mutations in MN1 in a cohort of 23 individuals with strikingly similar dysmorphic facial features, especially midface hypoplasia, and intellectual disability with severe expressive language delay. Imaging revealed an atypical form of rhombencephalosynapsis, a distinctive brain malformation characterized by partial or complete loss of the cerebellar vermis with fusion of the cerebellar hemispheres, in 8/10 individuals. Rhombencephalosynapsis has no previously known definitive genetic or environmental causes. Other frequent features included perisylvian polymicrogyria, abnormal posterior clinoid processes and persistent trigeminal artery. MN1 is encoded by only two exons. All mutations, including the recurrent variant p.Arg1295* observed in 8/21 probands, fall in the terminal exon or the extreme 3' region of exon 1, and are therefore predicted to result in escape from nonsense-mediated mRNA decay. This was confirmed in fibroblasts from three individuals. We propose that the condition described here, MN1 C-terminal truncation (MCTT) syndrome, is not due to MN1 haploinsufficiency but rather is the result of dominantly acting C-terminally truncated MN1 protein. Our data show that MN1 plays a critical role in human craniofacial and brain development, and opens the door to understanding the biological mechanisms underlying rhombencephalosynapsis.
Asunto(s)
Anomalías Múltiples/genética , Anomalías Craneofaciales/genética , Discapacidad Intelectual/genética , Trastornos del Desarrollo del Lenguaje/genética , Malformaciones del Sistema Nervioso/genética , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Anomalías Múltiples/diagnóstico por imagen , Adolescente , Arteria Basilar/anomalías , Arteria Basilar/diagnóstico por imagen , Arterias Carótidas/anomalías , Arterias Carótidas/diagnóstico por imagen , Vermis Cerebeloso/anomalías , Vermis Cerebeloso/diagnóstico por imagen , Cerebelo/anomalías , Cerebelo/diagnóstico por imagen , Niño , Preescolar , Estudios de Cohortes , Hibridación Genómica Comparativa , Anomalías Craneofaciales/diagnóstico por imagen , Femenino , Fibroblastos/metabolismo , Humanos , Imagenología Tridimensional , Lactante , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mutación , Malformaciones del Sistema Nervioso/diagnóstico por imagen , Degradación de ARNm Mediada por Codón sin Sentido , Polimicrogiria/diagnóstico por imagen , Polimicrogiria/genética , RNA-Seq , Reacción en Cadena en Tiempo Real de la Polimerasa , Síndrome , Tomografía Computarizada por Rayos X , Secuenciación del Exoma , Secuenciación Completa del GenomaRESUMEN
Intracellular membrane fusion is mediated by the concerted action of N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. During fusion, SM proteins bind the N-terminal peptide (N-peptide) motif of the SNARE subunit syntaxin, but the function of this interaction is unknown. Here, using FRET-based biochemical reconstitution and Caenorhabditis elegans genetics, we show that the N-peptide of syntaxin-1 recruits the SM protein Munc18-1/nSec1 to the SNARE bundle, facilitating their assembly into a fusion-competent complex. The recruitment is achieved through physical tethering rather than allosteric activation of Munc18-1. Consistent with the recruitment role, the N-peptide is not spatially constrained along syntaxin-1, and it is functional when translocated to another SNARE subunit SNAP-25 or even when simply anchored in the target membrane. The N-peptide function is restricted to an early initiation stage of the fusion reaction. After association, Munc18-1 and the SNARE bundle together drive membrane merging without further involving the N-peptide. Thus, the syntaxin N-peptide is an initiation factor for the assembly of the SNARE-SM membrane fusion complex.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Fusión de Membrana , Fosfoproteínas/metabolismo , Proteínas SNARE/metabolismo , Sintaxina 1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Microscopía Confocal , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Proteínas Munc18/química , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Mutación , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas SNARE/química , Proteínas SNARE/genética , Sintaxina 1/química , Sintaxina 1/genética , Factores de Tiempo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
Split hand/foot malformation (SHFM) is a rare limb abnormality with clefting of the fingers and/or toes. For many individuals, the genetic etiology is unknown. Through whole-exome and targeted sequencing, we detected three novel variants in a gene encoding a transcription factor, PRDM1, that arose de novo in families with SHFM or segregated with the phenotype. PRDM1 is required for limb development; however, its role is not well understood and it is unclear how the PRDM1 variants affect protein function. Using transient and stable overexpression rescue experiments in zebrafish, we show that the variants disrupt the proline/serine-rich and DNA-binding zinc finger domains, resulting in a dominant-negative effect. Through gene expression assays, RNA sequencing, and CUT&RUN in isolated pectoral fin cells, we demonstrate that Prdm1a directly binds to and regulates genes required for fin induction, outgrowth and anterior/posterior patterning, such as fgfr1a, dlx5a, dlx6a and smo. Taken together, these results improve our understanding of the role of PRDM1 in the limb gene regulatory network and identified novel PRDM1 variants that link to SHFM in humans.
Asunto(s)
Deformidades Congénitas de las Extremidades , Pez Cebra , Animales , ADN , Deformidades Congénitas de las Extremidades/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Factores de Transcripción/genética , Pez Cebra/genética , Dedos de ZincRESUMEN
BACKGROUND: ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome. RESULTS: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The "epi-ADNP-1" episignature included ~ 6000 mostly hypomethylated CpGs, and the "epi-ADNP-2" episignature included ~ 1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N- and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model. CONCLUSIONS: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome.
Asunto(s)
Metilación de ADN , Proteínas de Homeodominio/genética , Mutación , Proteínas del Tejido Nervioso/genética , Trastornos del Neurodesarrollo/diagnóstico , Trastornos del Neurodesarrollo/genética , Trastorno del Espectro Autista/genética , Niño , Preescolar , Biología Computacional/métodos , Islas de CpG , Diagnóstico Precoz , Epigénesis Genética , Femenino , Humanos , Discapacidad Intelectual/genética , Masculino , Modelos GenéticosRESUMEN
Coffin-Siris and Nicolaides-Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS (ARID1B, SMARCB1, and SMARCA4) and NCBRS (SMARCA2). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening.
Asunto(s)
Anomalías Múltiples/genética , Proteínas Cromosómicas no Histona/genética , Metilación de ADN , Factores de Transcripción/genética , Anomalías Múltiples/diagnóstico , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Epigenómica , Cara/anomalías , Facies , Deformidades Congénitas del Pie/diagnóstico , Deformidades Congénitas del Pie/genética , Deformidades Congénitas de la Mano/diagnóstico , Deformidades Congénitas de la Mano/genética , Humanos , Hipotricosis/diagnóstico , Hipotricosis/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Micrognatismo/diagnóstico , Micrognatismo/genética , Mutación , Cuello/anomalías , Proteínas Nucleares/genética , Proteína SMARCB1/genética , SíndromeRESUMEN
The calcium release channel (CRC) of the skeletal sarcoplasmic reticulum is rich in thiol groups and is strongly regulated by covalent modification of these thiols. Oxidizing reagents activate the release channel, whereas reducing reagents inhibit the channel. However, most CRC regulators are not thiol reagents. Here, we propose that reversible redox interactions are involved in regulation of the CRC by nonthiol reagents. This hypothesis was tested with several CRC regulators. The local anesthetics tetracaine, procaine and QX-314, which block the CRC, behaved as electron donors in reactions with dye free radicals. In contrast, ryanodine, caffeine, doxorubicin and daunorubicin, compounds known to activate the release channel, all accepted electrons from dye anion radicals. Moreover, release of Ca2+ from SR initiated by doxorubicin (acceptor) was antagonized by local anesthetics (donors). Based on the redox characterization of CRC modulators, we propose a functional model in which channel inhibitors and activators act as weak electron donors and acceptors, respectively, and shift the thiol-disulfide balance within the release protein. The consequence of this model is that, in spite of the chemical diversity of CRC modulators, a common mechanism of channel regulation involves the transient exchange of electrons between the activator/inhibitor and the CRC.
Asunto(s)
Colorantes/química , Radicales Libres , Preparaciones Farmacéuticas/química , Anestésicos Locales/química , Cafeína , Daunorrubicina , Doxorrubicina , Oxidación-Reducción , RianodinaRESUMEN
Changes in neuronal activity create local and transient changes in energy demands at synapses. Here we discover a metabolic compartment that forms in vivo near synapses to meet local energy demands and support synaptic function in Caenorhabditis elegans neurons. Under conditions of energy stress, glycolytic enzymes redistribute from a diffuse localization in the cytoplasm to a punctate localization adjacent to synapses. Glycolytic enzymes colocalize, suggesting the ad hoc formation of a glycolysis compartment, or a "glycolytic metabolon," that can maintain local levels of ATP. Local formation of the glycolytic metabolon is dependent on presynaptic scaffolding proteins, and disruption of the glycolytic metabolon blocks the synaptic vesicle cycle, impairs synaptic recovery, and affects locomotion. Our studies indicate that under energy stress conditions, energy demands in C. elegans synapses are met locally through the assembly of a glycolytic metabolon to sustain synaptic function and behavior. VIDEO ABSTRACT.
Asunto(s)
Caenorhabditis elegans/citología , Caenorhabditis elegans/enzimología , Fosfofructoquinasa-1/metabolismo , Terminales Presinápticos/enzimología , Terminales Presinápticos/fisiología , Estrés Fisiológico , Animales , Caenorhabditis elegans/metabolismo , Endocitosis , Hipoxia , Metabolómica , Mutación , Terminales Presinápticos/metabolismo , Vesículas Sinápticas/enzimología , Vesículas Sinápticas/metabolismoRESUMEN
OBJECTIVE: To perform genotype-phenotype analysis in an infant with congenital arthrogryposis due to a de novo missense mutation in the NALCN ion channel and explore the mechanism of pathogenicity using a Caenorhabditis elegans model. METHODS: We performed whole-exome sequencing in a preterm neonate with congenital arthrogryposis and a severe life-threatening clinical course. We examined the mechanism of pathogenicity of the associated NALCN mutation by engineering the orthologous mutation into the nematode C elegans using CRISPR-Cas9. RESULTS: We identified a de novo missense mutation in NALCN, c.1768C>T, in an infant with a severe neonatal lethal form of the recently characterized CLIFAHDD syndrome (congenital contractures of the limbs and face with hypotonia and developmental delay). We report novel phenotypic features including prolonged episodes of stimulus-sensitive sustained muscular contraction associated with life-threatening episodes of desaturation and autonomic instability, extending the severity of previously described phenotypes associated with mutations in NALCN. When engineered into the C elegans ortholog, this mutation results in a severe gain-of-function phenotype, with hypercontraction and uncoordinated movement. We engineered 6 additional CLIFAHDD syndrome mutations into C elegans and the mechanism of action could be divided into 2 categories: half phenocopied gain-of-function mutants and half phenocopied loss-of-function mutants. CONCLUSIONS: The clinical phenotype of our patient and electrophysiologic studies show sustained muscular contraction in response to transient sensory stimuli. In C elegans, this mutation causes neuronal hyperactivity via a gain-of-function NALCN ion channel. Testing human variants of NALCN in C elegans demonstrates that CLIFAHDD can be caused by dominant loss- or gain-of-function mutations in ion channel function.
Asunto(s)
Anomalías Múltiples/genética , Proteínas de Caenorhabditis elegans/genética , Canalopatías/genética , Mutación Missense , Canales de Sodio/genética , Anomalías Múltiples/fisiopatología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Canalopatías/fisiopatología , Femenino , Humanos , Recién Nacido , Canales Iónicos , Proteínas de la Membrana , Modelos Animales , Fenotipo , Canales de Sodio/metabolismo , SíndromeRESUMEN
Many neurodegenerative disorders are associated with mitochondrial defects [1-3]. Mitochondria can play an active role in degeneration by releasing reactive oxygen species and apoptotic factors [4-7]. Alternatively, mitochondria can protect axons from stress and insults, for example by buffering calcium [8]. Recent studies manipulating mitochondria lend support to both of these models [9-13]. Here, we identify a C. elegans mutant, ric-7, in which mitochondria are unable to exit the neuron cell bodies, similar to the kinesin-1/unc-116 mutant. When axons lacking mitochondria are cut with a laser, they rapidly degenerate. Some neurons even spontaneously degenerate in ric-7 mutants. Degeneration can be suppressed by forcing mitochondria into the axons of the mutants. The protective effect of mitochondria is also observed in the wild-type: a majority of axon fragments containing a mitochondrion survive axotomy, whereas those lacking mitochondria degenerate. Thus, mitochondria are not required for axon degeneration and serve a protective role in C. elegans axons.