RESUMEN
BACKGROUND: Deep-intronic variants that alter RNA splicing were ineffectively evaluated in the search for the cause of genetic diseases. Determination of such pathogenic variants from a vast number of deep-intronic variants (approximately 1,500,000 variants per individual) represents a technical challenge to researchers. Thus, we developed a Pathogenicity predictor for Deep-Intronic Variants causing Aberrant Splicing (PDIVAS) to easily detect pathogenic deep-intronic variants. RESULTS: PDIVAS was trained on an ensemble machine-learning algorithm to classify pathogenic and benign variants in a curated dataset. The dataset consists of manually curated pathogenic splice-altering variants (SAVs) and commonly observed benign variants within deep introns. Splicing features and a splicing constraint metric were used to maximize the predictive sensitivity and specificity, respectively. PDIVAS showed an average precision of 0.92 and a maximum MCC of 0.88 in classifying these variants, which were the best of the previous predictors. When PDIVAS was applied to genome sequencing analysis on a threshold with 95% sensitivity for reported pathogenic SAVs, an average of 27 pathogenic candidates were extracted per individual. Furthermore, the causative variants in simulated patient genomes were more efficiently prioritized than the previous predictors. CONCLUSION: Incorporating PDIVAS into variant interpretation pipelines will enable efficient detection of disease-causing deep-intronic SAVs and contribute to improving the diagnostic yield. PDIVAS is publicly available at https://github.com/shiro-kur/PDIVAS .
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Empalme del ARN , Humanos , Intrones , Virulencia , MutaciónRESUMEN
Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.
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Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofina/genética , Empalme del ARN/genética , Intrones/genética , Nucleótidos , Análisis de Secuencia de ARNRESUMEN
Down syndrome (DS) caused by trisomy of chromosome 21 is the most common genetic cause of intellectual disability. Although the prenatal diagnosis of DS has become feasible, there are no therapies available for the rescue of DS-related neurocognitive impairment. A growth inducer newly identified in our screen of neural stem cells (NSCs) has potent inhibitory activity against dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) and was found to rescue proliferative deficits in Ts65Dn-derived neurospheres and human NSCs derived from individuals with DS. The oral administration of this compound, named ALGERNON (altered generation of neurons), restored NSC proliferation in murine models of DS and increased the number of newborn neurons. Moreover, administration of ALGERNON to pregnant dams rescued aberrant cortical formation in DS mouse embryos and prevented the development of abnormal behaviors in DS offspring. These data suggest that the neurogenic phenotype of DS can be prevented by ALGERNON prenatal therapy.
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Síndrome de Down/tratamiento farmacológico , Terapias Fetales , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/embriología , Corteza Cerebral/patología , Cognición/efectos de los fármacos , Ciclina D1/metabolismo , Giro Dentado/efectos de los fármacos , Giro Dentado/patología , Síndrome de Down/patología , Síndrome de Down/psicología , Femenino , Células HEK293 , Humanos , Aprendizaje/efectos de los fármacos , Masculino , Ratones , Células-Madre Neurales/patología , Embarazo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Quinasas DyrKRESUMEN
Seckel syndrome (SS) is a rare spectrum of congenital severe microcephaly and dwarfism. One SS-causative gene is Ataxia Telangiectasia and Rad3-Related Protein (ATR), and ATR (c.2101 A>G) mutation causes skipping of exon 9, resulting in a hypomorphic ATR defect. This mutation is considered the cause of an impaired response to DNA replication stress, the main function of ATR, contributing to the pathogenesis of microcephaly. However, the precise behavior and impact of this splicing defect in human neural progenitor cells (NPCs) is unclear. To address this, we established induced pluripotent stem cells (iPSCs) from fibroblasts carrying the ATR mutation and an isogenic ATR-corrected counterpart iPSC clone. SS-patient-derived iPSCs (SS-iPSCs) exhibited cell type-specific splicing; exon 9 was dominantly skipped in fibroblasts and iPSC-derived NPCs, but it was included in undifferentiated iPSCs and definitive endodermal cells. SS-iPSC-derived NPCs (SS-NPCs) showed distinct expression profiles from ATR non-mutated NPCs with negative enrichment of neuronal genesis-related gene sets. In SS-NPCs, abnormal mitotic spindles occurred more frequently than in gene-corrected counterparts, and the alignment of NPCs in the surface of the neurospheres was perturbed. Finally, we tested several splicing-modifying compounds and found that TG003, a CLK1 inhibitor, could pharmacologically rescue the exon 9 skipping in SS-NPCs. Treatment with TG003 restored the ATR kinase activity in SS-NPCs and decreased the frequency of abnormal mitotic events. In conclusion, our iPSC model revealed a novel effect of the ATR mutation in mitotic processes of NPCs and NPC-specific missplicing, accompanied by the recovery of neuronal defects using a splicing rectifier.
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Empalme Alternativo , Proteínas de la Ataxia Telangiectasia Mutada , Enanismo , Facies , Células Madre Pluripotentes Inducidas , Microcefalia , Modelos Biológicos , Mutación , Proteínas de la Ataxia Telangiectasia Mutada/biosíntesis , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular , Enanismo/enzimología , Enanismo/genética , Enanismo/patología , Femenino , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/patología , Masculino , Microcefalia/enzimología , Microcefalia/genética , Microcefalia/patologíaRESUMEN
Coffin-Siris syndrome (CSS, MIM#135900) is a congenital disorder characterized by coarse facial features, intellectual disability, and hypoplasia of the fifth digit and nails. Pathogenic variants for CSS have been found in genes encoding proteins in the BAF (BRG1-associated factor) chromatin-remodeling complex. To date, more than 150 CSS patients with pathogenic variants in nine BAF-related genes have been reported. We previously reported 71 patients of whom 39 had pathogenic variants. Since then, we have recruited an additional 182 CSS-suspected patients. We performed comprehensive genetic analysis on these 182 patients and on the previously unresolved 32 patients, targeting pathogenic single nucleotide variants, short insertions/deletions and copy number variations (CNVs). We confirmed 78 pathogenic variations in 78 patients. Pathogenic variations in ARID1B, SMARCB1, SMARCA4, ARID1A, SOX11, SMARCE1, and PHF6 were identified in 48, 8, 7, 6, 4, 1, and 1 patients, respectively. In addition, we found three CNVs including SMARCA2. Of particular note, we found a partial deletion of SMARCB1 in one CSS patient and we thoroughly investigated the resulting abnormal transcripts.
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Anomalías Múltiples/genética , Cara/anomalías , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Deformidades Congénitas de la Mano/genética , Discapacidad Intelectual/genética , Micrognatismo/genética , Cuello/anomalías , Estudios de Cohortes , Estudios de Asociación Genética/métodos , HumanosRESUMEN
BACKGROUND: Williams syndrome (WS) is a neurodevelopmental disorder that has been attributed to heterozygous deletions in chromosome 7q11.23 and exhibits a variety of physical, cognitive, and behavioral features. However, the genetic basis of this phenotypic variability is unclear. In this study, we identified genetic clues underlying these complex phenotypes. METHODS: Neurobehavioral function was assessed in WS patients and healthy controls. Total RNA was extracted from peripheral blood and subjected to microarray analysis, RNA-sequencing, and qRT-PCR. Weighted gene co-expression network analysis was performed to identify specific alterations related to intermediate disease phenotypes. To functionally interpret each WS-related module, gene ontology and disease-related gene enrichment were examined. We also investigated the micro (mi)RNA expression profiles and miRNA co-expression networks to better explain the regulation of the transcriptome in WS. RESULTS: Our analysis identified four significant co-expression modules related to intermediate WS phenotypes. Notably, the three upregulated WS-related modules were composed exclusively of genes located outside the 7q11.23 region. They were significantly enriched in genes related to B-cell activation, RNA processing, and RNA transport. BCL11A, which is known for its association with speech disorders and intellectual disabilities, was identified as one of the hub genes in the top WS-related module. Finally, these key upregulated mRNA co-expression modules appear to be inversely correlated with a specific downregulated WS-related miRNA co-expression module. CONCLUSIONS: Dysregulation of the mRNA/miRNA network involving genes outside of the 7q11.23 region is likely related to the complex phenotypes observed in WS patients.
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Trastorno del Espectro Autista/genética , Perfilación de la Expresión Génica , Expresión Génica/genética , Síndrome de Williams/genética , Niño , Cromosomas Humanos Par 7/genética , Humanos , MicroARNs/genética , ARN Mensajero/genéticaRESUMEN
Aicardi-Goutières syndrome (AGS) is a rare, genetically determined early-onset progressive encephalopathy. To date, mutations in six genes have been identified as etiologic for AGS. Our Japanese nationwide AGS survey identified six AGS-affected individuals without a molecular diagnosis; we performed whole-exome sequencing on three of these individuals. After removal of the common polymorphisms found in SNP databases, we were able to identify IFIH1 heterozygous missense mutations in all three. In vitro functional analysis revealed that IFIH1 mutations increased type I interferon production, and the transcription of interferon-stimulated genes were elevated. IFIH1 encodes MDA5, and mutant MDA5 lacked ligand-specific responsiveness, similarly to the dominant Ifih1 mutation responsible for the SLE mouse model that results in type I interferon overproduction. This study suggests that the IFIH1 mutations are responsible for the AGS phenotype due to an excessive production of type I interferon.
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Enfermedades Autoinmunes del Sistema Nervioso/genética , ARN Helicasas DEAD-box/genética , Mutación Missense , Malformaciones del Sistema Nervioso/genética , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box/química , Femenino , Humanos , Helicasa Inducida por Interferón IFIH1 , Japón , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder (FAOD). However, newborn screening (NBS) for this potentially fatal disease has not been established partly because reliable indices are not available. METHODS: We diagnosed CPT II deficiency in a 7-month-old boy presenting with hypoglycemic encephalopathy, which apparently had been missed in the NBS using C16 and C18:1 concentrations as indices. By referring to his acylcarnitine profile from the NBS, we adopted the (C16+C18:1)/C2 ratio (cutoff 0.62) and C16 concentration (cutoff 3.0nmol/mL) as alternative indices for CPT II deficiency such that an analysis of a dried blood specimen collected at postnatal day five retroactively yielded the correct diagnosis. Thereafter, positive cases were assessed by measuring (1) the fatty acid oxidation ability of intact lymphocytes and/or (2) CPT II activity in the lysates of lymphocytes. The diagnoses were then further confirmed by genetic analysis. RESULTS: The disease was diagnosed in seven of 21 newborns suspected of having CPT II deficiency based on NBS. We also analyzed the false-negative patient and five symptomatic patients for comparison. Values for the NBS indices of the false-negative, symptomatic patient were lower than those of the seven affected newborns. Although it was difficult to differentiate the false-negative patient from heterozygous carriers and false-positive subjects, the fatty acid oxidation ability of the lymphocytes and CPT II activity clearly confirmed the diagnosis. Among several other indices proposed previously, C14/C3 completely differentiated the seven NBS-positive patients and the false-negative patient from the heterozygous carriers and the false-positive subjects. Genetic analysis revealed 16 kinds of variant alleles. The most prevalent, detected in ten alleles in nine patients from eight families, was c.1148T>A (p.F383Y), a finding in line with those of several previous reports on Japanese patients. CONCLUSIONS: These findings suggested that CPT II deficiency can be screened by using (C16+C18:1)/C2 and C16 as indices. An appropriate cutoff level is required to achieve adequate sensitivity albeit at the cost of a considerable increase in the false-positive rate, which might be reduced by using additional indices such as C14/C3.
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Carnitina O-Palmitoiltransferasa/análisis , Carnitina O-Palmitoiltransferasa/deficiencia , Errores Innatos del Metabolismo/diagnóstico , Tamizaje Neonatal , Palmitoilcarnitina/análisis , Alelos , Carnitina O-Palmitoiltransferasa/genética , Pruebas con Sangre Seca/métodos , Reacciones Falso Negativas , Reacciones Falso Positivas , Femenino , Humanos , Hipoglucemia/complicaciones , Lactante , Recién Nacido , Masculino , Errores Innatos del Metabolismo/genética , Sensibilidad y Especificidad , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Leukoencephalopathy with brain calcifications and cysts (LCC) is neuroradiologically characterized by leukoencephalopathy, intracranial calcification, and cysts. Coats plus syndrome is also characterized by the same neuroradiological findings together with defects in retinal vascular development. Indeed, LCC and Coats plus were originally considered to be the same clinical entity termed cerebroretinal microangiopathy with calcifications and cysts, but evidence suggests that they are genetically distinct. Mutations in CTS telomere maintenance complex component 1 (CTC1) and small nucleolar RNA, C/D box 118 (SNORD118) genes have been found to cause Coats plus and LCC, respectively. MATERIALS AND METHODS: Eight unrelated families with LCC were recruited. These patients typically showed major neuroradiological findings of LCC with no signs of extra-neurological manifestations such as retinal abnormality, gastrointestinal bleeding, or hematological abnormalities. SNORD118 was examined by Sanger sequencing in these families. RESULTS: Seven out of eight probands carry compound heterozygous mutations, suggesting that SNORD118 mutations are the major cause of LCC. We identified a total of eight mutation, including four that were novel. Some of the variants identified in this study present heterozygously in public databases with an extremely rare frequency (<0.1%). CONCLUSION: Biallelic SNORD118 mutations were exclusively found in most unrelated families with LCC.
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Calcinosis/genética , Quistes del Sistema Nervioso Central/genética , Predisposición Genética a la Enfermedad , Leucoencefalopatías/genética , ARN Nucleolar Pequeño/genética , Adulto , Alelos , Encéfalo/fisiopatología , Calcinosis/epidemiología , Calcinosis/fisiopatología , Quistes del Sistema Nervioso Central/epidemiología , Quistes del Sistema Nervioso Central/fisiopatología , Quistes/genética , Bases de Datos Factuales , Femenino , Heterocigoto , Humanos , Leucoencefalopatías/epidemiología , Leucoencefalopatías/fisiopatología , Masculino , Mutación , Proteínas de Unión a Telómeros/genéticaRESUMEN
HPMRS or Mabry syndrome is a heterogeneous glycosylphosphatidylinositol (GPI) anchor deficiency that is caused by an impairment of synthesis or maturation of the GPI-anchor. The expressivity of the clinical features in HPMRS varies from severe syndromic forms with multiple organ malformations to mild nonsyndromic intellectual disability. In about half of the patients with the clinical diagnosis of HPMRS, pathogenic mutations can be identified in the coding region in one of the six genes, one among them is PGAP3. In this work, we describe a screening approach with sequence specific baits for transcripts of genes of the GPI pathway that allows the detection of functionally relevant mutations also including introns and the 5' and 3' UTR. By this means, we also identified pathogenic noncoding mutations, which increases the diagnostic yield for HPMRS on the basis of intellectual disability and elevated serum alkaline phosphatase. In eight affected individuals from different ethnicities, we found seven novel pathogenic mutations in PGAP3. Besides five missense mutations, we identified an intronic mutation, c.558-10G>A, that causes an aberrant splice product and a mutation in the 3'UTR, c.*559C>T, that is associated with substantially lower mRNA levels. We show that our novel screening approach is a useful rapid detection tool for alterations in genes coding for key components of the GPI pathway.
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Anomalías Múltiples/genética , Anomalías Múltiples/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Mutación Missense , Trastornos del Metabolismo del Fósforo/genética , Trastornos del Metabolismo del Fósforo/patología , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Regiones no Traducidas 3' , Adolescente , Adulto , Hidrolasas de Éster Carboxílico , Células Cultivadas , Niño , Preescolar , Femenino , Predisposición Genética a la Enfermedad , Humanos , Intrones , Masculino , Linaje , Análisis de Secuencia de ADN/métodos , Adulto JovenRESUMEN
Irlen syndrome is a proposed perceptual processing disorder characterized by visual distortions while reading. Patients with this syndrome may experience light sensitivity, visual stress, and other related problems such as dyslexia. Tinted lenses and colored overlays have been designed to help individuals with the symptoms of Irlen syndrome. However, there is still debate over the effectiveness of these interventions and whether this syndrome actually exists. In this report, we describe a case involving an 8-year-old girl with dyslexia who experienced severe visual hypersensitivity and whose symptoms completely resolved after wearing tinted lenses. While it is possible that she experienced a psychogenic visual disturbance that was relieved because of the placebo effect, the clinical course of her symptoms matched the findings previously described by Irlen. The patient was unable to read without tinted lenses. With tinted lenses, she could read at the appropriate age level, suggesting that her difficulty was due to a problem in optical information processing. The concepts underlying Irlen syndrome are vaguely defined, and several groups insist that the visual stress associated with this syndrome might be responsible for dyslexia as well as other disorders. These ambiguous criteria may be responsible for the criticism over the validity of this condition. Although this was only an anecdotal case, our patient exhibited the core functional deficit described in Irlen syndrome and showed a dramatic improvement with tinted lenses; therefore, this case may facilitate investigations into the mechanism underlying Irlen syndrome, if it actually exists. Although further studies are required to confirm the validity of this syndrome and the treatment approach, Irlen syndrome should be recognized as a disorder since its symptoms can be easily relieved by wearing tinted lenses or color filters.
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Dislexia/terapia , Trastornos de la Visión/terapia , Niño , Color , Dislexia/etiología , Anteojos , Femenino , Humanos , Trastornos de la Visión/complicacionesRESUMEN
OBJECTIVE: Topiramate (TPM), lamotrigine (LTG), and levetiracetam (LEV) are three new-generation antiepileptic drugs (AEDs) which have recently come into use in add-on therapy for refractory childhood epilepsy in Japan. The aim of this study was to evaluate their efficacy and tolerability, and to clarify the role of these three AEDs in childhood epilepsy therapy. METHODS: Three separate audits were conducted between July 2007 and July 2012. All patients studied had epilepsy refractory to other AEDs. Efficacy was confirmed if a patient became seizure-free or achieved > 50% reduction (50% responder rate: 50% RR) in seizure frequency for 12 months after starting add-on therapy. RESULTS: A total of 55 children received TPM, 44 LTG, and 38 LEV. The 50% RR of partial epilepsy was 31.8% for LTG, 41.8% for TPM, and 52.6% for LEV. The 50% RR of generalized epilepsy was 28.6% for LTG, 26.7% for TPM, and 44.4% for LEV. The incidence of adverse events was 9.1% for LTG, 43.6% for TPM, and 15.8% for LEV. CONCLUSION: LEV was the most effective of the three add-on therapies in refractory childhood epilepsy with partial and generalized onset. Regarding seizure-free, TPM was more effective than the other therapies, but it had many side effects. LTG tended to be more effective for generalized epilepsy, particularly idiopathic epilepsy, than partial epilepsy. We conclude that it is necessary to develop a treatment plan for pediatric epilepsies after considering the advantages and disadvantage of these new AEDs.
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Anticonvulsivantes/uso terapéutico , Epilepsia Refractaria/tratamiento farmacológico , Fructosa/análogos & derivados , Piracetam/análogos & derivados , Triazinas/uso terapéutico , Adolescente , Anticonvulsivantes/efectos adversos , Niño , Femenino , Fructosa/efectos adversos , Fructosa/uso terapéutico , Humanos , Lamotrigina , Levetiracetam , Masculino , Piracetam/efectos adversos , Piracetam/uso terapéutico , Estudios Retrospectivos , Topiramato , Triazinas/efectos adversosRESUMEN
OBJECTIVES: Aicardi-Goutières syndrome (AGS) is a rare, genetically determined, early onset progressive encephalopathy associated with autoimmune manifestations. AGS is usually inherited in an autosomal recessive manner. The disease is rare, therefore the clinical manifestations and genotype-phenotype correlations, particularly with regard to autoimmune diseases, are still unclear. Here we performed a nationwide survey of AGS patients in Japan and analysed the genetic and clinical data. METHODS: Patients were recruited via questionnaires sent to paediatric or adult neurologists in Japanese hospitals and institutions. Genetic analysis was performed and clinical data were collected. RESULTS: Fourteen AGS patients were identified from 13 families; 10 harboured genetic mutations. Three patients harboured dominant-type TREX1 mutations. These included two de novo cases: one caused by a novel heterozygous p.His195Tyr mutation and the other by a novel somatic mosaicism resulting in a p.Asp200Asn mutation. Chilblain lesions were observed in all patients harbouring dominant-type TREX1 mutations. All three patients harbouring SAMHD1 mutations were diagnosed with autoimmune diseases, two with SLE and one with SS. The latter is the first reported case. CONCLUSION: This study is the first to report a nationwide AGS survey, which identified more patients with sporadic AGS carrying de novo dominant-type TREX1 mutations than expected. There was a strong association between the dominant-type TREX1 mutations and chilblain lesions, and between SAMHD1 mutations and autoimmunity. These findings suggest that rheumatologists should pay attention to possible sporadic AGS cases presenting with neurological disorders and autoimmune manifestations.
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Pueblo Asiatico/genética , Enfermedades Autoinmunes del Sistema Nervioso/genética , Eritema Pernio/genética , Exodesoxirribonucleasas/genética , Encuestas Epidemiológicas , Mutación/genética , Malformaciones del Sistema Nervioso/genética , Fosfoproteínas/genética , Adolescente , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes del Sistema Nervioso/epidemiología , Eritema Pernio/epidemiología , Niño , Preescolar , Estudios de Cohortes , Femenino , Genotipo , Humanos , Japón/epidemiología , Masculino , Proteínas de Unión al GTP Monoméricas/genética , Malformaciones del Sistema Nervioso/epidemiología , Fenotipo , Proteína 1 que Contiene Dominios SAM y HD , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Ataxia-telangiectasia-like disorder (ATLD) is a rare autosomal recessive disorder, and has symptoms similar to ataxia-telangiectasia (AT). ATLD is caused by mutations in the MRE11 gene, involved in DNA double-strand break repair (DSBR). In contrast to AT, ATLD patients lack key clinical features, such as telangiectasia or immunodeficiency, and are therefore difficult to be diagnosed. We report a female ATLD patient presenting with hypergonadotropic hypogonadism and hypersegmented neutrophils, previously undescribed features in this disorder, and potential diagnostic clues to differentiate ATLD from other conditions. The patient showed slowly progressive cerebellar ataxia from 2 years of age, and MRI revealed atrophy of the cerebellum, oculomotor apraxia, mild cognitive impairment, writing dystonia, hypergonadotropic hypogonadism with primary amenorrhea, and hypersegmented neutrophils. Western blot assay demonstrated total loss of MRE11 and reduction of ATM-dependent phosphorylation; thus, we diagnosed ATLD. Genetically, a novel missense mutation (c.140C>T) was detected in the MRE11 gene, but no other mutation was found in the patient. Our presenting patient suggests that impaired DSBR may be associated with hypergonadotropic hypogonadism and neutrophil hypersegmentation. In conclusion, when assessing patients with ataxia of unknown cause, ATLD should be considered, and the gonadal state and peripheral blood smear samples evaluated.
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Ataxia Telangiectasia/diagnóstico , Hipogonadismo/diagnóstico , Neutrófilos/patología , Fenotipo , Ataxia Telangiectasia/genética , Encéfalo/patología , Preescolar , Proteínas de Unión al ADN/genética , Femenino , Humanos , Proteína Homóloga de MRE11 , Imagen por Resonancia Magnética , Mutación Missense , Análisis de Secuencia de ADNRESUMEN
Objectives: Collagen VI-related myopathy spans a clinical continuum from severe Ullrich congenital muscular dystrophy to milder Bethlem myopathy, caused by genetic variants in COL6A1, COL6A2, and COL6A3 genes. Our objective was to report a newly identified patient with the pathogenic variants restricted to a polyadenylation signal in the 3'-untranslated region, which have not been reported in hereditary muscle disease. Methods: We performed clinicopathologic diagnosis and analysis using whole-genome and RNA sequencing. Results: We report Ullrich congenital muscular dystrophy caused by a homozygous deletion, c.*198_*466del, which includes a polyadenylation signal in the canonical last exon of the COL6A2 gene. The parents were consanguineously married and had the heterozygous variant, but they were completely asymptomatic. In the patient's muscles, collagen VI was deficient in the sarcolemma, but present in the interstitium, showing the pattern of sarcolemma-specific collagen VI deficiency rather than a pattern of complete deficiency despite the lack of a polyadenylation signal. The RNA sequencing of the patient's muscle showed that alternative last exons were raised in COL6A2 transcript. Discussion: Our case provides a valuable example of the mechanism of alternative splicing switches for polyadenylation selection.
RESUMEN
PURPOSE: With the current study, we aimed to reveal the similarities and differences in sensory profiles between Williams syndrome (WS) and autism spectrum disorder. METHODS: Using the sensory profile questionnaire completed by the caregivers, we analyzed the WS (n = 60, 3.4-19.8 years) and autistic (n = 39, 4.2-14.0 years) groups. RESULTS: The Severity Analysis revealed a significant group difference in Sensory Sensitivity but not in Low Registration, Sensation Seeking, and Sensation Avoiding subscales. Age can modulate the subscale scores differently across groups. For Sensation Seeking, the scores of both groups decreased with development. However, the scores of Sensory Sensitivity decreased with age in the autistic group but not in the WS group. Sensation Avoiding scores increased with development in the WS group but not in the autistic group. No significant developmental changes were observed in Low Registration. CONCLUSION: This study highlights the cross-syndrome similarities and differences in sensory profiles and developmental changes in autistic individuals and individuals with WS.
RESUMEN
Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon-intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.
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Oligonucleótidos Antisentido , Empalme del ARN , Femenino , Humanos , Adolescente , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Mutación , Exones/genética , Proteínas Portadoras/genéticaRESUMEN
Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-ß-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.
Asunto(s)
Lesión por Frío , Trasplante de Corazón , Animales , Humanos , Ratas , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/farmacología , Receptores de Esfingosina-1-FosfatoRESUMEN
Interstitial lung disease (ILD) represents a large group of diseases characterized by chronic inflammation and fibrosis of the lungs, for which therapeutic options are limited. Among several causative genes of familial ILD with autosomal dominant inheritance, the mutations in the BRICHOS domain of SFTPC cause protein accumulation and endoplasmic reticulum stress by misfolding its proprotein. Through a screening system using these two phenotypes in HEK293 cells and evaluation using alveolar epithelial type 2 (AT2) cells differentiated from patient-derived induced pluripotent stem cells (iPSCs), we identified Cryptotanshinone (CPT) as a potential therapeutic agent for ILD. CPT decreased cell death induced by mutant SFTPC overexpression in A549 and HEK293 cells and ameliorated the bleomycin-induced contraction of the matrix in fibroblast-dependent alveolar organoids derived from iPSCs with SFTPC mutation. CPT and this screening strategy can apply to abnormal protein-folding-associated ILD and other protein-misfolding diseases.
RESUMEN
Fukutin (FKTN) c.647+2084G>T creates a pseudo-exon with a premature stop codon, which causes Fukuyama congenital muscular dystrophy (FCMD). We aimed to ameliorate aberrant splicing of FKTN caused by this variant. We screened compounds focusing on splicing regulation using the c.647+2084G>T splicing reporter and discovered that the branchpoint, which is essential for splicing reactions, could be a potential therapeutic target. To confirm the effectiveness of branchpoints as targets for exon skipping, we designed branchpoint-targeted antisense oligonucleotides (BP-AONs). This restored normal FKTN mRNA and protein production in FCMD patient myotubes. We identified a functional BP by detecting splicing intermediates and creating BP mutations in the FKTN reporter gene; this BP was non-redundant and sufficiently blocked by BP-AONs. Next, a BP-AON was designed for a different FCMD-causing variant, which induces pathogenic exon trapping by a common SINE-VNTR-Alu-type retrotransposon. Notably, this BP-AON also restored normal FKTN mRNA and protein production in FCMD patient myotubes. Our findings suggest that BPs could be potential targets in exon-skipping therapeutic strategies for genetic disorders.