RESUMEN
Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c+/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10-16, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.
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Trastorno del Espectro Autista , Trastorno Autístico , Modelos Animales de Enfermedad , Haploinsuficiencia , Histona Demetilasas , N-Metiltransferasa de Histona-Lisina , Transcriptoma , Animales , Haploinsuficiencia/genética , Ratones , Trastorno del Espectro Autista/genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Transcriptoma/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Trastorno Autístico/genética , Masculino , Discapacidad Intelectual/genética , Deleción Cromosómica , Anomalías Craneofaciales/genética , Femenino , Ratones Endogámicos C57BL , Conducta Animal , Encéfalo/metabolismo , Cromosomas Humanos Par 9 , Cardiopatías CongénitasRESUMEN
Despite enormous efforts employing various approaches, the molecular pathology in the schizophrenia brain remains elusive. On the other hand, the knowledge of the association between the disease risk and changes in the DNA sequences, in other words, our understanding of the genetic pathology of schizophrenia, has dramatically improved over the past two decades. As the consequence, now we can explain more than 20% of the liability to schizophrenia by considering all analyzable common genetic variants including those with weak or no statistically significant association. Also, a large-scale exome sequencing study identified single genes whose rare mutations substantially increase the risk for schizophrenia, of which six genes (SETD1A, CUL1, XPO7, GRIA3, GRIN2A, and RB1CC1) showed odds ratios larger than ten. Based on these findings together with the preceding discovery of copy number variants (CNVs) with similarly large effect sizes, multiple disease models with high etiological validity have been generated and analyzed. Studies of the brains of these models, as well as transcriptomic and epigenomic analyses of patient postmortem tissues, have provided new insights into the molecular pathology of schizophrenia. In this review, we overview the current knowledge acquired from these studies, their limitations, and directions for future research that may redefine schizophrenia based on biological alterations in the responsible organ rather than operationalized criteria.
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Esquizofrenia , Humanos , Esquizofrenia/genética , Patología Molecular , Estudio de Asociación del Genoma Completo , Predisposición Genética a la Enfermedad/genética , Mutación , Variaciones en el Número de Copia de ADN/genéticaRESUMEN
Large-scale genome-wide association studies (GWASs) on bipolar disorder (BD) have implicated the involvement of the fatty acid desaturase (FADS) locus. These enzymes (FADS1 and FADS2) are involved in the metabolism of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are thought to potentially benefit patients with mood disorders. To model reductions in the activity of FADS1/2 affected by the susceptibility alleles, we generated mutant mice heterozygously lacking both Fads1/2 genes. We measured wheel-running activity over six months and observed bipolar swings in activity, including hyperactivity and hypoactivity. The hyperactivity episodes, in which activity was far above the norm, usually lasted half a day; mice manifested significantly shorter immobility times on the behavioral despair test performed during these episodes. The hypoactivity episodes, which lasted for several weeks, were accompanied by abnormal circadian rhythms and a marked decrease in wheel running, a spontaneous behavior associated with motivation and reward systems. We comprehensively examined lipid composition in the brain and found that levels of certain lipids were significantly altered between wild-type and the heterozygous mutant mice, but no changes were consistent with both sexes and either DHA or EPA was not altered. However, supplementation with DHA or a mixture of DHA and EPA prevented these episodic behavioral changes. Here we propose that heterozygous Fads1/2 knockout mice are a model of BD with robust constitutive, face, and predictive validity, as administration of the mood stabilizer lithium was also effective. This GWAS-based model helps to clarify how lipids and their metabolisms are involved in the pathogenesis and treatment of BD.
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Trastorno Bipolar , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Femenino , Animales , Ratones , Trastorno Bipolar/genética , Alelos , Actividad Motora , Ácidos Docosahexaenoicos , Ácido Eicosapentaenoico , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Bipolar disorder (BD) is a global medical issue, afflicting around 1% of the population with manic and depressive episodes. Despite various genetic studies, the genetic architecture and pathogenesis of BD have not been fully resolved. Besides germline variants, postzygotic mosaic variants are proposed as new candidate mechanisms contributing to BD. Here, we performed extensive deep exome sequencing (DES, ~300×) and validation experiments to investigate the roles of mosaic variants in BD with 235 BD cases (194 probands of trios and 41 single cases) and 39 controls. We found an enrichment of developmental disorder (DD) genes in the genes hit by deleterious mosaic variants in BD (P = 0.000552), including a ClinVar-registered pathogenic variant in ARID2. An enrichment of deleterious mosaic variants was also observed for autism spectrum disorder (ASD) genes (P = 0.000428). The proteins coded by the DD/ASD genes with non-synonymous mosaic variants in BD form more protein-protein interaction than expected, suggesting molecular mechanisms shared with DD/ASD but restricted to a subset of cells in BD. We also found significant enrichment of mitochondrial heteroplasmic variants, another class of mosaic variants, in mitochondrial tRNA genes in BD (P = 0.0102). Among them, recurrent m.3243 A > G variants known as causal for mitochondrial diseases were found in two unrelated BD probands with allele fractions of 5-12%, lower than in mitochondrial diseases. Despite the limitation of using peripheral tissues, our DES investigation supports the possible contribution of deleterious mosaic variants in the nuclear genome responsible for severer phenotypes, such as DD/ASD, to the risk of BD and further demonstrates that the same paradigm can be applied to the mitochondrial genome. These results, as well as the enrichment of heteroplasmic mitochondrial tRNA variants in BD, add a new piece to the understanding of the genetic architecture of BD and provide general insights into the pathological roles of mosaic variants in human diseases.
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Trastorno del Espectro Autista , Trastorno Bipolar , Enfermedades Mitocondriales , Humanos , Trastorno Bipolar/genética , Trastorno del Espectro Autista/genética , Predisposición Genética a la Enfermedad/genética , Secuenciación del ExomaRESUMEN
De novo variants (DNVs) cause many genetic diseases. When DNVs are examined in the whole coding regions of genes in next-generation sequencing analyses, pathogenic DNVs often cluster in a specific region. One such region is the last exon and the last 50 bp of the penultimate exon, where truncating DNVs cause escape from nonsense-mediated mRNA decay [NMD(-) region]. Such variants can have dominant-negative or gain-of-function effects. Here, we first developed a resource of rates of truncating DNVs in NMD(-) regions under the null model of DNVs. Utilizing this resource, we performed enrichment analysis of truncating DNVs in NMD(-) regions in 346 developmental and epileptic encephalopathy (DEE) trios. We observed statistically significant enrichment of truncating DNVs in semaphorin 6B (SEMA6B) (p value: 2.8 × 10-8; exome-wide threshold: 2.5 × 10-6). The initial analysis of the 346 individuals and additional screening of 1,406 and 4,293 independent individuals affected by DEE and developmental disorders collectively identified four truncating DNVs in the SEMA6B NMD(-) region in five individuals who came from unrelated families (p value: 1.9 × 10-13) and consistently showed progressive myoclonic epilepsy. RNA analysis of lymphoblastoid cells established from an affected individual showed that the mutant allele escaped NMD, indicating stable production of the truncated protein. Importantly, heterozygous truncating variants in the NMD(+) region of SEMA6B are observed in general populations, and SEMA6B is most likely loss-of-function tolerant. Zebrafish expressing truncating variants in the NMD(-) region of SEMA6B orthologs displayed defective development of brain neurons and enhanced pentylenetetrazole-induced seizure behavior. In summary, we show that truncating DNVs in the final exon of SEMA6B cause progressive myoclonic epilepsy.
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Exoma/genética , Exones/genética , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Epilepsias Mioclónicas Progresivas/genética , Semaforinas/genética , Adolescente , Adulto , Alelos , Animales , Femenino , Heterocigoto , Humanos , Masculino , Degradación de ARNm Mediada por Codón sin Sentido/genética , Convulsiones/genética , Adulto Joven , Pez Cebra/genéticaRESUMEN
MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.
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Encefalopatías/etiología , Anomalías Craneofaciales/etiología , Mutación con Ganancia de Función , Regulación de la Expresión Génica , Eliminación de Secuencia , Transactivadores/genética , Proteínas Supresoras de Tumor/genética , Adolescente , Encefalopatías/patología , Proliferación Celular , Niño , Preescolar , Anomalías Craneofaciales/patología , Femenino , Células HeLa , Humanos , Masculino , Proteolisis , Síndrome , Transactivadores/metabolismo , Transcriptoma , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Bipolar disorder (BD) is a common mental disorder characterized by recurrent mood episodes, which causes major socioeconomic burdens globally. Though its disease pathogenesis is largely unknown, the high heritability of BD indicates strong contributions from genetic factors. In this review, we summarize the recent achievements in the genetics of BD, particularly those from genome-wide association study (GWAS) of common variants and next-generation sequencing analysis of rare variants. These include the identification of dozens of robust disease-associated loci, deepening of our understanding of the biology of BD, objective description of correlations with other psychiatric disorders and behavioral traits, formulation of methods for predicting disease risk and drug response, and the discovery of a single gene associated with bipolar disorder and schizophrenia spectrum with a large effect size. On the other hand, the findings to date have not yet made a clear contribution to the improvement of clinical psychiatry of BD. We overview the remaining challenges as well as possible paths to resolve them, referring to studies of other major neuropsychiatric disorders.
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Trastorno Bipolar , Esquizofrenia , Humanos , Trastorno Bipolar/genética , Estudio de Asociación del Genoma Completo , Predisposición Genética a la Enfermedad , Esquizofrenia/genética , Polimorfismo de Nucleótido Simple , BiologíaRESUMEN
A pentanucleotide TTTCA repeat insertion into a polymorphic TTTTA repeat element in SAMD12 causes benign adult familial myoclonic epilepsy. Although the precise determination of the entire SAMD12 repeat sequence is important for molecular diagnosis and research, obtaining this sequence remains challenging when using conventional genomic/genetic methods, and even short-read and long-read next-generation sequencing technologies have been insufficient. Incomplete information regarding expanded repeat sequences may hamper our understanding of the pathogenic roles played by varying numbers of repeat units, genotype-phenotype correlations, and mutational mechanisms. Here, we report a new approach for the precise determination of the entire expanded repeat sequence and present a workflow designed to improve the diagnostic rates in various repeat expansion diseases. We examined 34 clinically diagnosed benign adult familial myoclonic epilepsy patients, from 29 families using repeat-primed PCR, Southern blot, and long-read sequencing with Cas9-mediated enrichment. Two cases with questionable results from repeat-primed PCR and/or Southern blot were confirmed as pathogenic using long-read sequencing with Cas9-mediated enrichment, resulting in the identification of pathogenic SAMD12 repeat expansions in 76% of examined families (22/29). Importantly, long-read sequencing with Cas9-mediated enrichment was able to provide detailed information regarding the sizes, configurations, and compositions of the expanded repeats. The inserted TTTCA repeat size and the proportion of TTTCA sequences among the overall repeat sequences were highly variable, and a novel repeat configuration was identified. A genotype-phenotype correlation study suggested that the insertion of even short (TTTCA)14 repeats contributed to the development of benign adult familial myoclonic epilepsy. However, the sizes of the overall TTTTA and TTTCA repeat units are also likely to be involved in the pathology of benign adult familial myoclonic epilepsy. Seven unsolved SAMD12-negative cases were investigated using whole-genome long-read sequencing, and infrequent, disease-associated, repeat expansions were identified in two cases. The strategic workflow resolved two questionable SAMD12-positive cases and two previously SAMD12-negative cases, increasing the diagnostic yield from 69% (20/29 families) to 83% (24/29 families). This study indicates the significant utility of long-read sequencing technologies to explore the pathogenic contributions made by various repeat units in complex repeat expansions and to improve the overall diagnostic rate.
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Expansión de las Repeticiones de ADN/genética , Epilepsias Mioclónicas/genética , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia de ADN/métodos , Adulto , Anciano , Anciano de 80 o más Años , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana EdadRESUMEN
We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.
Asunto(s)
Proteínas CELF , Epilepsia , Discapacidad Intelectual , Proteínas del Tejido Nervioso , Proteínas CELF/genética , Epilepsia/genética , Femenino , Heterocigoto , Humanos , Discapacidad Intelectual/genética , Proteínas del Tejido Nervioso/genética , Señales de Localización Nuclear/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Disorders of sex development (DSDs) are defined as congenital conditions in which chromosomal, gonadal or anatomical sex is atypical. In many DSD cases, genetic causes remain to be elucidated. Here, we performed a case-control exome sequencing study comparing gene-based burdens of rare damaging variants between 26 DSD cases and 2625 controls. We found exome-wide significant enrichment of rare heterozygous truncating variants in the MYRF gene encoding myelin regulatory factor, a transcription factor essential for oligodendrocyte development. All three variants occurred de novo. We identified an additional 46,XY DSD case of a de novo damaging missense variant in an independent cohort. The clinical symptoms included hypoplasia of Müllerian derivatives and ovaries in 46,XX DSD patients, defective development of Sertoli and Leydig cells in 46,XY DSD patients and congenital diaphragmatic hernia in one 46,XY DSD patient. As all of these cells and tissues are or partly consist of coelomic epithelium (CE)-derived cells (CEDC) and CEDC developed from CE via proliferaiton and migration, MYRF might be related to these processes. Consistent with this hypothesis, single-cell RNA sequencing of foetal gonads revealed high expression of MYRF in CE and CEDC. Reanalysis of public chromatin immunoprecipitation sequencing data for rat Myrf showed that genes regulating proliferation and migration were enriched among putative target genes of Myrf. These results suggested that MYRF is a novel causative gene of 46,XY and 46,XX DSD and MYRF is a transcription factor regulating CD and/or CEDC proliferation and migration, which is essential for development of multiple organs.
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Trastornos del Desarrollo Sexual 46, XX/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Proteínas de la Membrana/genética , Factores de Transcripción/genética , Trastornos del Desarrollo Sexual 46, XX/patología , Adolescente , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Preescolar , Estudios de Cohortes , Biología Computacional , Trastorno del Desarrollo Sexual 46,XY/patología , Femenino , Ontología de Genes , Gónadas/crecimiento & desarrollo , Haploinsuficiencia , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Mutación , Mutación Missense , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Secuenciación del Exoma , Adulto JovenRESUMEN
Intellectual disability (ID) accounts for 1% of the general population, and it is caused by the interplay between the genetic and/or environmental factors. The genetic components responsible for the development of ID are highly heterogeneous, and the phenotype and severity of the disease vary in patients even if they have an identical pathological variant and/or belong to the same family. Herein, we reported two male siblings with ID in an Iranian family. By means of the whole-exome sequencing method, elder brother affected by a moderate form of ID exhibited a de novo missense variant in the KCNQ3 gene, while another sibling afflicted with a severe form of the disease exhibited a de novo in-frame deletion in the UBE3A gene. Both variants have been previously ascribed to similar clinical phenotypes. In addition, a genetic variant in the KCNQ3 gene was transmitted to his son, who had a mild form of ID. To our knowledge, all individuals with KCNQ3-related developmental delay show de novo variants in the KCNQ3 gene. Thus, this familial case exhibit milder phenotype that might extend the clinical spectrum of KCNQ3 pathogenic variants. In addition, the current report highlights the significance of the clinical evaluation and non-biased assessment of the genetic analysis.
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Discapacidades del Desarrollo/patología , Predisposición Genética a la Enfermedad , Discapacidad Intelectual/patología , Canal de Potasio KCNQ3/genética , Mutación , Ubiquitina-Proteína Ligasas/genética , Niño , Discapacidades del Desarrollo/genética , Femenino , Estudios de Asociación Genética , Humanos , Discapacidad Intelectual/genética , Masculino , Linaje , Fenotipo , HermanosRESUMEN
Pontocerebellar hypoplasia (PCH) is currently classified into 13 subgroups and many gene variants associated with PCH have been identified by next generation sequencing. PCH type 1 is a rare heterogeneous neurodegenerative disorder. The clinical presentation includes early-onset severe developmental delay, progressive motor neuronopathy, and cerebellar and pontine atrophy. Recently two variants in the EXOSC9 gene (MIM: 606180), NM_001034194.1: c.41T>C (p.Leu14Pro) and c.481C>T (p.Arg161*) were identified in four unrelated patients with PCH type 1D (PCH1D) (MIM: 618065). EXOSC9 encodes a component of the exosome complex, which is essential for correct processing and degradation of RNA. We report here two PCH1D families with biallelic EXOSC9 variants: c.239T>G (p.Leu80Arg) and c.484dupA (p.Arg162Lysfs*3) in one family and c.151G>C (p.Gly51Arg) in the other family. Although the patients studied here showed similar clinical features as previously described for PCH1D, relatively greater intellectual development (although still highly restricted) and normal pontine structure were recognized. Our findings expand the clinical consequences of biallelic EXOSC9 variants.
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Atrofia/patología , Enfermedades Cerebelosas/patología , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Enfermedad de la Neurona Motora/patología , Atrofia Muscular Espinal/patología , Mutación , Atrofias Olivopontocerebelosas/patología , Proteínas de Unión al ARN/genética , Atrofia/complicaciones , Atrofia/genética , Enfermedades Cerebelosas/complicaciones , Enfermedades Cerebelosas/genética , Femenino , Estudios de Asociación Genética , Humanos , Lactante , Masculino , Enfermedad de la Neurona Motora/complicaciones , Enfermedad de la Neurona Motora/genética , Atrofia Muscular Espinal/complicaciones , Atrofia Muscular Espinal/genética , Atrofias Olivopontocerebelosas/complicaciones , Atrofias Olivopontocerebelosas/genética , LinajeRESUMEN
The objective of this study was to evaluate the efficacy of whole exome sequencing (WES) for the genetic diagnosis of cases presenting with fetal structural anomalies detected by ultrasonography. WES was performed on 19 cases with prenatal structural anomalies. Genomic DNA was extracted from umbilical cords or umbilical blood obtained shortly after birth. WES data were analyzed on prenatal phenotypes alone, and the data were re-analyzed after information regarding the postnatal phenotype was obtained. Based solely on the fetal phenotype, pathogenic, or likely pathogenic, single nucleotide variants were identified in 5 of 19 (26.3%) cases. Moreover, we detected trisomy 21 in two cases by WES-based copy number variation analysis. The overall diagnostic rate was 36.8% (7/19). They were all compatible with respective fetal structural anomalies. By referring to postnatal phenotype information, another candidate variant was identified by a postnatal clinical feature that was not detected in prenatal screening. As detailed phenotyping is desirable for better diagnostic rates in WES analysis, we should be aware that fetal phenotype is a useful, but sometimes limited source of information for comprehensive genetic analysis. It is important to amass more data of genotype-phenotype correlations, especially to appropriately assess the validity of WES in prenatal settings.
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Anomalías Congénitas/genética , Secuenciación del Exoma , Feto/anomalías , Ultrasonografía Prenatal , Aborto Eugénico , Adulto , Cesárea , Anomalías Congénitas/diagnóstico por imagen , Anomalías Congénitas/embriología , ADN/sangre , ADN/genética , Variaciones en el Número de Copia de ADN , Síndrome de Down/diagnóstico por imagen , Síndrome de Down/embriología , Síndrome de Down/genética , Femenino , Sangre Fetal/química , Muerte Fetal/etiología , Edad Gestacional , Humanos , Leucocitos/química , Leucocitos/ultraestructura , Polimorfismo de Nucleótido Simple , Embarazo , Resultado del EmbarazoRESUMEN
Calcineurin is a calcium (Ca2+)/calmodulin-regulated protein phosphatase that mediates Ca2+-dependent signal transduction. Here, we report six heterozygous mutations in a gene encoding the alpha isoform of the calcineurin catalytic subunit (PPP3CA). Notably, mutations were observed in different functional domains: in addition to three catalytic domain mutations, two missense mutations were found in the auto-inhibitory (AI) domain. One additional frameshift insertion that caused premature termination was also identified. Detailed clinical evaluation of the six individuals revealed clinically unexpected consequences of the PPP3CA mutations. First, the catalytic domain mutations and frameshift mutation were consistently found in patients with nonsyndromic early onset epileptic encephalopathy. In contrast, the AI domain mutations were associated with multiple congenital abnormalities including craniofacial dysmorphism, arthrogryposis and short stature. In addition, one individual showed severe skeletal developmental defects, namely, severe craniosynostosis and gracile bones (severe bone slenderness and perinatal fractures). Using a yeast model system, we showed that the catalytic and AI domain mutations visibly result in decreased and increased calcineurin signaling, respectively. These findings indicate that different functional effects of PPP3CA mutations are associated with two distinct disorders and suggest that functional approaches using a simple cellular system provide a tool for resolving complex genotype-phenotype correlations.
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Artrogriposis/genética , Calcineurina/genética , Anomalías Craneofaciales/genética , Enanismo/genética , Mutación con Ganancia de Función , Mutación con Pérdida de Función , Espasmos Infantiles/genética , Secuencia de Aminoácidos , Artrogriposis/metabolismo , Artrogriposis/patología , Secuencia de Bases , Calcineurina/química , Calcineurina/metabolismo , Niño , Preescolar , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/patología , Enanismo/metabolismo , Enanismo/patología , Femenino , Regulación de la Expresión Génica , Estudios de Asociación Genética , Humanos , Masculino , Modelos Moleculares , Linaje , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Espasmos Infantiles/metabolismo , Espasmos Infantiles/patología , Adulto JovenRESUMEN
BACKGROUND: Rett syndrome (RTT) is a characteristic neurological disease presenting with regressive loss of neurodevelopmental milestones. Typical RTT is generally caused by abnormality of methyl-CpG binding protein 2 (MECP2). Our objective to investigate the genetic landscape of MECP2-negative typical/atypical RTT and RTT-like phenotypes using whole exome sequencing (WES). METHODS: We performed WES on 77 MECP2-negative patients either with typical RTT (n=11), atypical RTT (n=22) or RTT-like phenotypes (n=44) incompatible with the RTT criteria. RESULTS: Pathogenic or likely pathogenic single-nucleotide variants in 28 known genes were found in 39 of 77 (50.6%) patients. WES-based CNV analysis revealed pathogenic deletions involving six known genes (including MECP2) in 8 of 77 (10.4%) patients. Overall, diagnostic yield was 47 of 77 (61.0 %). Furthermore, strong candidate variants were found in four novel genes: a de novo variant in each of ATPase H+ transporting V0 subunit A1 (ATP6V0A1), ubiquitin-specific peptidase 8 (USP8) and microtubule-associated serine/threonine kinase 3 (MAST3), as well as biallelic variants in nuclear receptor corepressor 2 (NCOR2). CONCLUSIONS: Our study provides a new landscape including additional genetic variants contributing to RTT-like phenotypes, highlighting the importance of comprehensive genetic analysis.
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Secuenciación del Exoma , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Fenotipo , Síndrome de Rett/diagnóstico , Síndrome de Rett/genética , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Ontología de Genes , Redes Reguladoras de Genes , Estudios de Asociación Genética/métodos , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: The diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20-40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants. METHODS: Aberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES. RESULTS: We identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases. CONCLUSION: These results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.
Asunto(s)
Miopatías Nemalínicas/genética , Análisis de Secuencia de ARN , Humanos , Japón , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Mutación , Empalme del ARNRESUMEN
We identified a de novo frameshift variant (NM_015048.1:c.5644_5647del:p.(Ile1882Serfs*118)) in the last exon of SETD1B in a Japanese patient with autistic behavior, developmental delay, intellectual disability, and myoclonic seizures. This variant is predicted to disrupt a well-conserved carboxyl-terminus SET domain, which is known to modulate gene activities and/or chromatin structure. Previously, two de novo missense mutations in SETD1B were reported in two patients with epilepsy. All three patients including the current patient share similar clinical features. Herein, we report a first epilepsy patient with a frameshift variant in SETD1B, emphasizing a possible pathomechanistic association of SETD1B abnormality with neurodevelopmental delay with epilepsy.
Asunto(s)
Epilepsia/diagnóstico , Epilepsia/genética , Mutación del Sistema de Lectura , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , N-Metiltransferasa de Histona-Lisina/genética , Alelos , Secuencia de Aminoácidos , Deleción Cromosómica , Análisis Mutacional de ADN , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/genética , Exones , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética/métodos , Genotipo , N-Metiltransferasa de Histona-Lisina/química , Humanos , Masculino , Linaje , Fenotipo , Dominios y Motivos de Interacción de ProteínasRESUMEN
The recent advent of long-read sequencing technologies is expected to provide reasonable answers to genetic challenges unresolvable by short-read sequencing, primarily the inability to accurately study structural variations, copy number variations, and homologous repeats in complex parts of the genome. However, long-read sequencing comes along with higher rates of random short deletions and insertions, and single nucleotide errors. The relatively higher sequencing accuracy of short-read sequencing has kept it as the first choice of screening for single nucleotide variants and short deletions and insertions. Albeit, short-read sequencing still suffers from systematic errors that tend to occur at specific positions where a high depth of reads is not always capable to correct for these errors. In this study, we compared the genotyping of mitochondrial DNA variants in three samples using PacBio's Sequel (Pacific Biosciences Inc., Menlo Park, CA, USA) long-read sequencing and illumina's HiSeqX10 (illumine Inc., San Diego, CA, USA) short-read sequencing data. We concluded that, despite the differences in the type and frequency of errors in the long-reads sequencing, its accuracy is still comparable to that of short-reads for genotyping short nuclear variants; due to the randomness of errors in long reads, a lower coverage, around 37 reads, can be sufficient to correct for these random errors.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , ADN Mitocondrial/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Genoma Humano/genética , Genotipo , Técnicas de Genotipaje , Humanos , Análisis de Secuencia de ADNRESUMEN
Pediatric hypertension can cause hypertensive emergencies, including hemorrhagic stroke, contributing to rare but serious childhood morbidity and mortality. Renovascular hypertension (RVH) is one of the major causes of secondary hypertension in children. Grange syndrome (MIM#602531) is a rare disease characterized by multiple stenosis or occlusion of the renal, abdominal, coronary, and cerebral arteries, which can cause phenotypes of RVH and fibromuscular dysplasia (MIM#135580). We report the case of a 7-year-old girl with Grange syndrome who showed RVH and multiple seizure episodes. At 1 year of age, she experienced seizures and sequential hemiparesis caused by a left thalamic hemorrhage without cerebral vascular anomalies. Chronic hypertension was observed, and abdominal computed tomography angiography showed characteristic bilateral renal artery stenosis. Whole-exome sequencing revealed a novel homozygous pathogenic variant in the YY1AP1 gene (NM_001198903.1: c.1169del: p.Lys390Argfs*12). Biallelic YY1AP1 mutations are known to cause Grange syndrome. Unlike previously reported patients, our patient presented with intracerebral hemorrhagic stroke without anomalous brain artery or bone fragility. The phenotype in our patient may help better understand this ultra-rare syndrome. Grange syndrome should be considered in patients presenting with childhood-onset hypertension and/or hemorrhagic stroke for early clinical intervention.
Asunto(s)
Secuencia de Aminoácidos , Arteriopatías Oclusivas/genética , Huesos/anomalías , Braquidactilia/genética , Proteínas de Ciclo Celular/genética , Cardiopatías Congénitas/genética , Hipertensión Renovascular/genética , Hipertensión/genética , Hemorragias Intracraneales/genética , Eliminación de Secuencia , Accidente Cerebrovascular/genética , Sindactilia/genética , Factores de Transcripción/genética , Arteriopatías Oclusivas/patología , Arteriopatías Oclusivas/fisiopatología , Huesos/patología , Huesos/fisiopatología , Braquidactilia/patología , Braquidactilia/fisiopatología , Niño , Femenino , Cardiopatías Congénitas/patología , Cardiopatías Congénitas/fisiopatología , Homocigoto , Humanos , Hipertensión/patología , Hipertensión/fisiopatología , Hipertensión Renovascular/patología , Hipertensión Renovascular/fisiopatología , Hemorragias Intracraneales/patología , Hemorragias Intracraneales/fisiopatología , Accidente Cerebrovascular/patología , Accidente Cerebrovascular/fisiopatología , Sindactilia/patología , Sindactilia/fisiopatologíaRESUMEN
We report the second case of early infantile epileptic encephalopathy (EIEE) arising from a homozygous truncating variant of NECAP1. The boy developed infantile-onset tonic-clonic and tonic seizures, then spasms in clusters. His electroencephalogram (EEG) showed a burst suppression pattern, leading to the diagnosis of Ohtahara syndrome. Whole-exome sequencing revealed the canonical splice-site variant (c.301 + 1 G > A) in NECAP1. In rodents, Necap1 protein is enriched in neuronal clathrin-coated vesicles and modulates synaptic vesicle recycling. cDNA analysis confirmed abnormal splicing that produced early truncating mRNA. There has been only one previous report of a mutation in NECAP1 in a family with EIEE; this was a nonsense mutation (p.R48*) that was cited as EIEE21. Decreased mRNA levels and the loss of the WXXF motif in both the families suggests that loss of NECAP1 function is a common pathomechanism for EIEE21. This study provided additional support that synaptic vesicle recycling plays a key role in epileptogenesis.