RESUMO
PURPOSE: DYRK1A syndrome is among the most frequent monogenic forms of intellectual disability (ID). We refined the molecular and clinical description of this disorder and developed tools to improve interpretation of missense variants, which remains a major challenge in human genetics. METHODS: We reported clinical and molecular data for 50 individuals with ID harboring DYRK1A variants and developed (1) a specific DYRK1A clinical score; (2) amino acid conservation data generated from 100 DYRK1A sequences across different taxa; (3) in vitro overexpression assays to study level, cellular localization, and kinase activity of DYRK1A mutant proteins; and (4) a specific blood DNA methylation signature. RESULTS: This integrative approach was successful to reclassify several variants as pathogenic. However, we questioned the involvement of some others, such as p.Thr588Asn, still reported as likely pathogenic, and showed it does not cause an obvious phenotype in mice. CONCLUSION: Our study demonstrated the need for caution when interpreting variants in DYRK1A, even those occurring de novo. The tools developed will be useful to interpret accurately the variants identified in the future in this gene.
Assuntos
Deficiência Intelectual , Microcefalia , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Animais , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Camundongos , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Quinases DyrkRESUMO
Via whole-exome sequencing, we identified rare autosomal-recessive variants in UBA5 in five children from four unrelated families affected with a similar pattern of severe intellectual deficiency, microcephaly, movement disorders, and/or early-onset intractable epilepsy. UBA5 encodes the E1-activating enzyme of ubiquitin-fold modifier 1 (UFM1), a recently identified ubiquitin-like protein. Biochemical studies of mutant UBA5 proteins and studies in fibroblasts from affected individuals revealed that UBA5 mutations impair the process of ufmylation, resulting in an abnormal endoplasmic reticulum structure. In Caenorhabditis elegans, knockout of uba-5 and of human orthologous genes in the UFM1 cascade alter cholinergic, but not glutamatergic, neurotransmission. In addition, uba5 silencing in zebrafish decreased motility while inducing abnormal movements suggestive of seizures. These clinical, biochemical, and experimental findings support our finding of UBA5 mutations as a pathophysiological cause for early-onset encephalopathies due to abnormal protein ufmylation.
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Alelos , Encefalopatias/genética , Mutação/genética , Proteínas/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Idade de Início , Animais , Mapeamento Encefálico , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Criança , Pré-Escolar , Neurônios Colinérgicos/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Epilepsia/genética , Exoma/genética , Feminino , Fibroblastos , Genes Recessivos/genética , Humanos , Deficiência Intelectual/genética , Imageamento por Ressonância Magnética , Masculino , Microcefalia/genética , Transtornos dos Movimentos , Proteínas/genética , Transmissão Sináptica/genética , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/deficiência , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
A rare syndromic form of intellectual disability with impaired speech was recently found associated with mutations in CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), the protein product of which is directly involved in microtubule-kinetochore attachment. Through whole-exome sequencing in six unrelated nonconsanguineous families having a sporadic case of intellectual disability, we identified six novel de novo truncating mutations in CHAMP1: c.1880C>G p.(Ser627*), c.1489C>T; p.(Arg497*), c.1876_1877delAG; p.(Ser626Leufs*4), c.1043G>A; p.(Trp348*), c.1002G>A; p.(Trp334*), and c.958_959delCC; p.(Pro320*). Our clinical observations confirm the phenotypic homogeneity of the syndrome, which represents therefore a distinct clinical entity. Besides, our functional studies show that CHAMP1 protein variants are delocalized from chromatin and are unable to bind to two of its direct partners, POGZ and HP1. These data suggest a pathogenic mechanism of the CHAMP1-associated intellectual disability syndrome mediated by direct interacting partners of CHAMP1, several of which are involved in chromo/kinetochore-related disorders.
Assuntos
Proteínas Cromossômicas não Histona/genética , Deficiência Intelectual/genética , Fosfoproteínas/genética , Deleção de Sequência , Alelos , Criança , Pré-Escolar , Exoma , Fácies , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Deficiência Intelectual/diagnóstico , Masculino , Fenótipo , SíndromeRESUMO
OBJECT: Experimental circuits for biomaterial surface testing are frequently limited by the tested blood volume, composition of the circuit, flow conditions and the use of animal blood. This report describes an ex vivo set-up for simulated cardiopulmonary bypass with human blood perfusion. We investigated the clinical generalizability of the observed effects on hematological and metabolic parameters and the hemocompatibility of the system. METHODS: The simulated cardiopulmonary bypass circuit consisted of a heparin-coated tubing system connected to an oxygenator and a venous reservoir. Normothermic flow of blood obtained from healthy donors was maintained at 2.4 L/min/m(2) by a roller pump. Heparin was dosed to obtain a target activated clotting time (ACT) ⩾500 s. Blood was drawn at baseline and 0, 10, 60 and 120 minutes following the initiation of blood flow to determine hematological and metabolic parameters and the hemocompatibility of the extracorporeal system. Data were analyzed using repeated measures ANOVA. RESULTS: Two hours of blood perfusion resulted in a small, but clinically unimportant reduction in hematocrit, whereas hemoglobin levels and red blood cell, platelet and leukocyte counts remained stable. There was a significant increase in ACT throughout the experiment. While pO2 levels and the pH remained unaltered during the experiment, pCO2 values decreased from 51 ± 6 mmHg at T0 to 41 ± 3 mmHg at T120 (p<0.001). Simulated cardiopulmonary bypass induced a two-fold increase in C3a (p=0.001) while tissue factor was decreased from 44 ± 14 pg/mL at T0 to 38 ± 13 pg/mL at T120 (p=0.009). Levels of CD40L, prothrombin fragment 1+2, ß-thromboglobulin and factor VIIa remained stable over time. CONCLUSION: The ex vivo set-up for simulated cardiopulmonary bypass mimicked the clinical cardiosurgical setting. Exposure of fresh donor blood to the extracorporeal circuit showed a good hemocompatibility, indicated by maintained hematological parameters and a mild immune response.
Assuntos
Ponte Cardiopulmonar , Teste de Materiais/métodos , Heparina/administração & dosagem , Humanos , Tempo de Coagulação do Sangue TotalAssuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Fator Estimulador de Colônias de Granulócitos/imunologia , Interleucina-3/imunologia , Poliarterite Nodosa/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Poliarterite Nodosa/diagnóstico , Adulto JovemRESUMO
OBJECTIVES: Bioactive Carmeda® heparin-coated extracorporeal circuits (ECCs) have been shown to reduce contact phase and coagulation activation during cardiopulmonary bypass (CPB). Heparin coating is therefore effective in safely reducing coagulation during routine CPB. Balance® Biosurface is a new, recently developed biopassive coating containing negatively charged sulphonated polymers. This study sought to compare the clotting activation and thromboresistance of the Balance® (B) circuit with that of the Carmeda® (C) with full-dose systemic heparin (FDH) and reduced-dose systemic heparin (RDH). METHODS: This ex vivo study set-up comprising 40 experiments consisted of simplified ECC and circulation of freshly donated human blood. RDH and FDH regimens were obtained with 0.5 IU/ml and 1 IU/ml heparin administered to reach target activated clotting times (ACTs) of 250 and 500 s, respectively. The study design comprised four groups: FDH-C, FDH-B, RDH-C and RDH-B (all n = 10). Blood was sampled prior to and during the 2-h CPB. Coagulation activation was assessed (FXIIa, F1.2) and electron microscope scan imaging of oxygenators enabled determination of adhesion scores. RESULTS: With a biopassive compared with bioactive surface, mean ACT was lower, regardless of the heparin regimen applied (P < 0.001), whereas the total heparin dose required to maintain ACT was above target level (P < 0.001). However, FXIIa and F1.2 values were similar in all groups throughout, as were pressure gradients among oxygenators. All groups demonstrated similar adhesion scores following ultrastructural oxygenator assessment. CONCLUSIONS: In the absence of surgical-related haemostatic disturbances and based on target ACT levels under reduced- or full-dose heparin, the clotting process was similar to heparin-coated and new sulphonated polymer-coated ECC, both demonstrating similar thromboresistance.