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BACKGROUND: Tatton-Brown-Rahman syndrome (TBRS; OMIM 615879), also known as DNA methyltransferase 3 alpha (DNMT3A)-overgrowth syndrome (DOS), was first described by Tatton-Brown in 2014. This syndrome is characterised by overgrowth, intellectual disability and distinctive facial features and is the consequence of germline loss-of-function variants in DNMT3A, which encodes a DNA methyltransferase involved in epigenetic regulation. Somatic variants of DNMT3A are frequently observed in haematological malignancies, including acute myeloid leukaemia (AML). To date, 100 individuals with TBRS with de novo germline variants have been described. We aimed to further characterise this disorder clinically and at the molecular level in a nationwide series of 24 French patients and to investigate the correlation between the severity of intellectual disability and the type of variant. METHODS: We collected genetic and medical information from 24 individuals with TBRS using a questionnaire released through the French National AnDDI-Rares Network. RESULTS: Here, we describe the first nationwide French cohort of 24 individuals with germline likely pathogenic/pathogenic variants in DNMT3A, including 17 novel variants. We confirmed that the main phenotypic features were intellectual disability (100% of individuals), distinctive facial features (96%) and overgrowth (87%). We highlighted novel clinical features, such as hypertrichosis, and further described the neurological features and EEG results. CONCLUSION: This study of a nationwide cohort of individuals with TBRS confirms previously published data and provides additional information and clarifies clinical features to facilitate diagnosis and improve care. This study adds value to the growing body of knowledge on TBRS and broadens its clinical and molecular spectrum.
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OBJECTIVES: The main objective of this study was to evaluate how an apparently minor anomaly of the sphenoid bone, observed in a haploinsufficient mouse model for Sonic Hedgehog (Shh), affects the growth of the adult craniofacial region. This study aims to provide valuable information to orthodontists when making decisions regarding individuals carrying SHH mutation. MATERIALS AND METHODS: The skulls of embryonic, juvenile and adult mice of two genotypes (Shh heterozygous and wild type) were examined and measured using landmark-based linear dimensions. Additionally, we analysed the clinical characteristics of a group of patients and their relatives with SHH gene mutations. RESULTS: In the viable Shh+/ - mouse model, bred on a C57BL/6J background, we noted the presence of a persistent foramen at the midline of the basisphenoid bone. This particular anomaly was attributed to the existence of an ectopic pituitary gland. We discovered that this anomaly led to premature closure of the intrasphenoidal synchondrosis and contributed to craniofacial deformities in adult mice, including a longitudinally shortened skull base. This developmental anomaly is reminiscent of that commonly observed in human holoprosencephaly, a disorder resulting from a deficiency in SHH activity. However, sphenoid morphogenesis is not currently monitored in individuals carrying SHH mutations. CONCLUSION: Haploinsufficiency of Shh leads to isolated craniofacial skeletal hypoplasia in adult mouse. This finding highlights the importance of radiographic monitoring of the skull base in all individuals with SHH gene mutations.
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Proteínas Hedgehog , Holoprosencefalia , Adulto , Animales , Humanos , Ratones , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Ratones Endogámicos C57BL , Mutación , Hueso EsfenoidesRESUMEN
Regarding several cytotoxic agents, it was evidenced that genetic polymorphisms in genes encoding enzymes involved in their metabolism are associated with higher risk of toxicity. Genotyping these genes before treatment is a valuable strategy to prevent side effects and to predict individual response to drug therapy. This pharmacogenetic approach is recommended for chemotherapies such as thiopurines (azathioprine, 6-mercaptopurine, thioguanine), irinotecan, and fluoropyrimidines (capecitabine and 5-fluorouracil). In this study, we aimed at developing and validating a fast, cost-effective, and easily implementable multiplex genotyping method suitable for analyzing a panel of nine variants involved in the pharmacogenetics of widely prescribed anticancer drugs. We designed a multiplex-specific PCR assay where fragments were labeled by two different fluorescent dye markers (HEX/FAM) identifiable by fragment analysis. These two labels were used to discriminate bi-allelic variants, while the size of the fragment allowed the identification of a particular polymorphism location. Variants of interest were TPMT (rs1800462, rs1142345, rs1800460), NUDT15 (rs116855232), DPYD (rs55886062, rs3918290, rs67376798, rs75017182), and UGT1A1 (rs8175347). The assay was repeatable, and genotypes could be determined when DNA sample amounts ranged from 25 to 100 ng. Primers and dye remained stable in a ready-to-use mixture solution after five freeze-thaw cycles. Accuracy was evidenced by the consistency of 187 genotyping results obtained with our multiplex assay and a reference method. The developed method is fast and cost-effective in simultaneously identifying nine variants involved in the pharmacological response of anticancer drugs. This assay can be easily implemented in laboratories for widespread access to pharmacogenetics in clinical practice.
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Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias/patología , Farmacogenética , Polimorfismo de Nucleótido Simple , Pruebas Genéticas , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genéticaRESUMEN
Synonymous single nucleotide variants (sSNVs) have been implicated in various genetic disorders through alterations of pre-mRNA splicing, mRNA structure and miRNA regulation. However, their impact on synonymous codon usage and protein translation remains to be elucidated in clinical context. Here, we explore the functional impact of sSNVs in the Sonic Hedgehog (SHH) gene, identified in patients affected by holoprosencephaly, a congenital brain defect resulting from incomplete forebrain cleavage. We identified eight sSNVs in SHH, selectively enriched in holoprosencephaly patients as compared to healthy individuals, and systematically assessed their effect at both transcriptional and translational levels using a series of in silico and in vitro approaches. Although no evidence of impact of these sSNVs on splicing, mRNA structure or miRNA regulation was found, five sSNVs introduced significant changes in codon usage and were predicted to impact protein translation. Cell assays demonstrated that these five sSNVs are associated with a significantly reduced amount of the resulting protein, ranging from 5% to 23%. Inhibition of the proteasome rescued the protein levels for four out of five sSNVs, confirming their impact on protein stability and folding. Remarkably, we found a significant correlation between experimental values of protein reduction and computational measures of codon usage, indicating the relevance of in silico models in predicting the impact of sSNVs on translation. Considering the critical role of SHH in brain development, our findings highlight the clinical relevance of sSNVs in holoprosencephaly and underline the importance of investigating their impact on translation in human pathologies.
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Uso de Codones/genética , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Biosíntesis de Proteínas/genética , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
CONTEXT: In human, Sonic hedgehog (SHH) haploinsufficiency is the predominant cause of holoprosencephaly, a structural malformation of the forebrain midline characterized by phenotypic heterogeneity and incomplete penetrance. The NOTCH signaling pathway has recently been associated with holoprosencephaly in humans, but the precise mechanism involving NOTCH signaling during early brain development remains unknown. OBJECTIVE: The aim of this study was to evaluate the relationship between SHH and NOTCH signaling to determine the mechanism by which NOTCH dysfunction could cause midline malformations of the forebrain. DESIGN: In this study, we have used a chemical inhibition approach in the chick model and a genetic approach in the mouse model. We also reported results obtained from the clinical diagnosis of a cohort composed of 141 holoprosencephaly patients. RESULTS: We demonstrated that inhibition of NOTCH signaling in chick embryos as well as in mouse embryos induced a specific downregulation of SHH in the anterior hypothalamus. Our data in the mouse also revealed that the pituitary gland was the most sensitive tissue to Shh insufficiency and that haploinsufficiency of the SHH and NOTCH signaling pathways synergized to produce a malformed pituitary gland. Analysis of a large holoprosencephaly cohort revealed that some patients possessed multiple heterozygous mutations in several regulators of both pathways. CONCLUSIONS: These results provided new insights into molecular mechanisms underlying the extreme phenotypic variability observed in human holoprosencephaly. They showed how haploinsufficiency of the SHH and NOTCH activity could contribute to specific congenital hypopituitarism that was associated with a sella turcica defect.
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Proteínas Hedgehog/genética , Holoprosencefalia/genética , Sistema Hipotálamo-Hipofisario/metabolismo , Receptores Notch/genética , Animales , Células Cultivadas , Embrión de Pollo , Estudios de Cohortes , Modelos Animales de Enfermedad , Embrión de Mamíferos , Femenino , Haploinsuficiencia/genética , Proteínas Hedgehog/metabolismo , Holoprosencefalia/metabolismo , Holoprosencefalia/patología , Holoprosencefalia/fisiopatología , Humanos , Sistema Hipotálamo-Hipofisario/patología , Masculino , Ratones , Ratones Transgénicos , Embarazo , Receptores Notch/deficiencia , Estudios Retrospectivos , Transducción de Señal/genéticaRESUMEN
Congenital uterine anomalies (CUA) may have major impacts on the health and social well-being of affected individuals. Their expressivity is variable, with the most severe end of the spectrum being the absence of any fully or unilaterally developed uterus (aplastic uterus), which is a major feature in Mayer-Rokitansky-Kuster-Hauser syndrome (MRKH). So far, etiologies of CUA remain largely unknown. As reports of familial occurrences argue for strong genetic contributors in some cases, we performed whole exome sequencing in nine multiplex families with recurrence of uterine and kidney malformations, a condition called hereditary urogenital adysplasia. Heterozygous likely causative variants in the gene GREB1L were identified in four of these families, confirming GREB1L as an important gene for proper uterine and kidney development. The apparent mode of inheritance was autosomal dominant with incomplete penetrance. The four families included fetuses with uterovaginal aplasia and bilateral renal agenesis, highlighting the importance to investigate GREB1L in such phenotypes. Subsequent sequencing of the gene in a cohort of 68 individuals with MRKH syndrome or uterine malformation (mostly sporadic cases) identified six additional variants of unknown significance. We therefore conclude that heterozygous GREB1L variants contribute to MRKH syndrome and this probably requires additional genetic or environmental factors for full penetrance.
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Trastornos del Desarrollo Sexual 46, XX/genética , Anomalías Múltiples/genética , Anomalías Congénitas/genética , Riñón/anomalías , Conductos Paramesonéfricos/anomalías , Proteínas de Neoplasias/genética , Útero/anomalías , Trastornos del Desarrollo Sexual 46, XX/patología , Anomalías Múltiples/patología , Adulto , Estudios de Cohortes , Anomalías Congénitas/patología , Femenino , Feto/anomalías , Feto/patología , Predisposición Genética a la Enfermedad , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Conductos Paramesonéfricos/patología , Penetrancia , Útero/patología , Secuenciación del ExomaRESUMEN
Tacrolimus (TAC) is the cornerstone of immunosuppressive therapy in liver transplantation. This study aimed at elucidating the interplay between pharmacogenetic determinants of TAC whole blood and intracellular exposures as well as the pharmacokinetic-pharmacodynamic relationship of TAC in both compartments. Complete pharmacokinetic profiles (Predose, and 20 min, 40 min, 1h, 2h, 3h, 4h, 6h, 8h, 12h post drug intake) of twice daily TAC in whole blood and peripheral blood mononuclear cells (PBMC) were collected in 32 liver transplanted patients in the first ten days post transplantation. A non-parametric population pharmacokinetic model was applied to explore TAC pharmacokinetics in blood and PBMC. Concurrently, calcineurin activity was measured in PBMC. Influence of donor and recipient genetic polymorphisms of ABCB1, CYP3A4 and CYP3A5 on TAC exposure was assessed. Recipient ABCB1 polymorphisms 1199G>A could influence TAC whole blood and intracellular exposure (p<0.05). No association was found between CYP3A4 or CYP3A5 genotypes and TAC whole blood or intracellular concentrations. Finally, intra-PBMC calcineurin activity appeared incompletely inhibited by TAC and less than 50% of patients were expected to achieve intracellular IC50 concentration (100 pg/millions of cells) at therapeutic whole blood concentration (i.e.: 4-10 ng/mL). Together, these data suggest that personalized medicine regarding TAC therapy might be optimized by ABCB1 pharmacogenetic biomarkers and by monitoring intracellular concentration whereas the relationship between intracellular TAC exposure and pharmacodynamics biomarkers more specific than calcineurin activity should be further investigated.
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Inmunosupresores/farmacocinética , Inmunosupresores/uso terapéutico , Leucocitos Mononucleares/metabolismo , Tacrolimus/farmacocinética , Tacrolimus/uso terapéutico , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Anciano , Citocromo P-450 CYP3A/genética , Femenino , Genotipo , Humanos , Terapia de Inmunosupresión/métodos , Trasplante de Hígado/métodos , Masculino , Persona de Mediana Edad , Farmacogenética/métodos , Pruebas de Farmacogenómica/métodos , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Neural tube defect disorders are developmental diseases that originate from an incomplete closure of the neural tube during embryogenesis. Despite high prevalence-1 out of 3000 live births-their etiology is not yet established and both environmental and genetic factors have been proposed, with a heritability rate of about 60%. Studies in mouse models as well as in human have further suggested a multifactorial pattern of inheritance for neural tube defect disorders. Here, we report results obtained from clinical diagnosis and NGS analysis of a cohort composed of 52 patients. Using a candidate gene panel approach, we identified variants in known genes of planar cell polarity (PCP) pathway, although with higher prevalence than previously reported. Our study also reveals variants in novel genes such as FREM2 and DISP1. Altogether, these results confirm the implication of the PCP genes and involve the FRAS/FREM2 complex and Sonic Hedgehog signaling as novel components in the appearance of NTDs.
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Polaridad Celular/genética , Estudios de Asociación Genética/métodos , Defectos del Tubo Neural/genética , Análisis de Secuencia de ADN/métodos , Adulto , Animales , Niño , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Defectos del Tubo Neural/patología , Embarazo , Transducción de Señal/genética , TranscriptomaRESUMEN
Holoprosencephaly is a pathology of forebrain development characterized by high phenotypic heterogeneity. The disease presents with various clinical manifestations at the cerebral or facial levels. Several genes have been implicated in holoprosencephaly but its genetic basis remains unclear: different transmission patterns have been described including autosomal dominant, recessive and digenic inheritance. Conventional molecular testing approaches result in a very low diagnostic yield and most cases remain unsolved. In our study, we address the possibility that genetically unsolved cases of holoprosencephaly present an oligogenic origin and result from combined inherited mutations in several genes. Twenty-six unrelated families, for whom no genetic cause of holoprosencephaly could be identified in clinical settings [whole exome sequencing and comparative genomic hybridization (CGH)-array analyses], were reanalysed under the hypothesis of oligogenic inheritance. Standard variant analysis was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis. Statistical validation was performed on 248 ancestrally similar control trios provided by the Genome of the Netherlands project and on 574 ancestrally matched controls provided by the French Exome Project. Variants of clinical interest were identified in 180 genes significantly associated with key pathways of forebrain development including sonic hedgehog (SHH) and primary cilia. Oligogenic events were observed in 10 families and involved both known and novel holoprosencephaly genes including recurrently mutated FAT1, NDST1, COL2A1 and SCUBE2. The incidence of oligogenic combinations was significantly higher in holoprosencephaly patients compared to two control populations (P < 10-9). We also show that depending on the affected genes, patients present with particular clinical features. This study reports novel disease genes and supports oligogenicity as clinically relevant model in holoprosencephaly. It also highlights key roles of SHH signalling and primary cilia in forebrain development. We hypothesize that distinction between different clinical manifestations of holoprosencephaly lies in the degree of overall functional impact on SHH signalling. Finally, we underline that integrating clinical phenotyping in genetic studies is a powerful tool to specify the clinical relevance of certain mutations.
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Holoprosencefalia/genética , Herencia Multifactorial/genética , Enfermedades Raras/genética , Estudios de Casos y Controles , Hibridación Genómica Comparativa , Exoma/genética , Femenino , Humanos , Masculino , Mutación , Linaje , FenotipoRESUMEN
Juvenile hemochromatosis is a rare autosomal recessive disease due to variants in the Hemojuvelin (HJV) gene. Although biological features mimic HFE hemochromatosis, clinical presentation is worst with massive iron overload diagnosed during childhood. Our study describes clinical features and results of genetic testing for a group of patients initially referred for a hepcidino-deficiency syndrome and for whom HJV hemochromatosis was finally diagnosed. 662 patients with iron overload and high serum transferrin saturation were tested, and five genes (HFE, HJV, HAMP, TFR2, SLC40A1) were sequenced. Among our cohort, ten unrelated patients were diagnosed with HJV hemochromatosis. Genetic testing revealed five previously published and five undescribed variants: p.Arg41Pro, p.His180Arg, p.Lys299Glu, p.Cys361Arg and p.Ala384Val. Surprisingly, this study revealed a late age of onset in some patients, contrasting with the commonly accepted definition of "juvenile" hemochromatosis. Five of our patients were 30â¯years old or older, including two very late discoveries. Biological features and severity of iron overload were similar in younger and older patients. Our study brings new insight on HJV hemochromatosis showing that mild phenotype and late onset are possible. Genetic testing for HJV variants should thus be performed for all patients displaying a non-p.Cys282Tyr homozygous HFE hemochromatosis with hepcidin deficiency phenotype.
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Proteínas Ligadas a GPI , Hemocromatosis/congénito , Hemocromatosis/diagnóstico , Adulto , Edad de Inicio , Niño , Femenino , Variación Genética , Proteína de la Hemocromatosis , Hepcidinas/deficiencia , Humanos , Sobrecarga de Hierro , Masculino , Transferrina , Adulto JovenRESUMEN
Extreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fÅtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fÅtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fÅtus amniocytes vs amniocytes from age-matched control fÅtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2-/- mice. In Adgrl2+/- mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly.
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Microcefalia/genética , Microcefalia/patología , Mutación/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Rombencéfalo/patología , Adulto , Animales , Ciclo Celular/genética , Células Cultivadas , Embrión de Pollo , Análisis Mutacional de ADN , Embrión de Mamíferos , Femenino , Feto , Regulación del Desarrollo de la Expresión Génica/genética , Edad Gestacional , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microcefalia/complicaciones , Microcefalia/diagnóstico por imagen , Persona de Mediana Edad , Neuroglía/metabolismo , Neuroglía/patología , Rombencéfalo/diagnóstico por imagenRESUMEN
ZIC2 mutation is known to cause holoprosencephaly (HPE). A subset of ZIC2 HPE probands harbour cardiovascular and visceral anomalies suggestive of laterality defects. 3D-imaging of novel mouse Zic2 mutants uncovers, in addition to HPE, laterality defects in lungs, heart, vasculature and viscera. A strong bias towards right isomerism indicates a failure to establish left identity in the lateral plate mesoderm (LPM), a phenotype that cannot be explained simply by the defective ciliogenesis previously noted in Zic2 mutants. Gene expression analysis showed that the left-determining NODAL-dependent signalling cascade fails to be activated in the LPM, and that the expression of Nodal at the node, which normally triggers this event, is itself defective in these embryos. Analysis of ChiP-seq data, in vitro transcriptional assays and mutagenesis reveals a requirement for a low-affinity ZIC2 binding site for the activation of the Nodal enhancer HBE, which is normally active in node precursor cells. These data show that ZIC2 is required for correct Nodal expression at the node and suggest a model in which ZIC2 acts at different levels to establish LR asymmetry, promoting both the production of the signal that induces left side identity and the morphogenesis of the cilia that bias its distribution.
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Mesodermo/embriología , Morfogénesis , Proteína Nodal/metabolismo , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Tipificación del Cuerpo , Cilios , Holoprosencefalia/genética , Ratones , Mutación , Proteínas Nucleares/genética , Fenotipo , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
Holoprosencephaly (HPE) is a complex genetic disorder of the developing forebrain characterized by high phenotypic and genetic heterogeneity. HPE was initially defined as an autosomal dominant disease, but recent research has shown that its mode of transmission is more complex. The past decade has witnessed rapid development of novel genetic technologies and significant progresses in clinical studies of HPE. In this review, we recapitulate genetic epidemiological studies of the largest European HPE cohort and summarize the novel genetic discoveries of HPE based on recently developed diagnostic methods. Our main purpose is to present different inheritance patterns that exist for HPE with a particular emphasis on oligogenic inheritance and its implications in genetic counseling.
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Encéfalo/diagnóstico por imagen , Holoprosencefalia/genética , Encéfalo/anomalías , Encéfalo/embriología , Aberraciones Cromosómicas , Femenino , Genes Recesivos , Asesoramiento Genético , Pruebas Genéticas/métodos , Proteínas Hedgehog/genética , Holoprosencefalia/etiología , Humanos , Patrón de Herencia , Masculino , Linaje , Embarazo , Diagnóstico PrenatalRESUMEN
BACKGROUND: Neurons arise in very specific regions of the neural tube, controlled by components of the Notch signalling pathway, proneural genes, and other bHLH transcription factors. How these specific neuronal areas in the brain are generated during development is just beginning to be elucidated. Notably, the critical role of proneural genes during differentiation of the neuronal populations that give rise to the early axon scaffold in the developing brain is not understood. The regulation of their downstream effectors remains poorly defined. RESULTS: This study provides the first overview of the spatiotemporal expression of proneural genes in the neuronal populations of the early axon scaffold in both chick and mouse. Overexpression studies and mutant mice have identified a number of specific neuronal genes that are targets of proneural transcription factors in these neuronal populations. CONCLUSION: Together, these results improve our understanding of the molecular mechanisms involved in differentiation of the first neuronal populations in the brain.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Encéfalo/embriología , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neurogénesis , Animales , Diferenciación Celular , Pollos , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Receptores Notch/genética , Transducción de Señal , Especificidad de la EspecieRESUMEN
Holoprosencephaly (HPE) is the most common congenital cerebral malformation in humans, characterized by impaired forebrain cleavage and midline facial anomalies. It presents a high heterogeneity, both in clinics and genetics. We have developed a novel targeted next-generation sequencing (NGS) assay and screened a cohort of 257 HPE patients. Mutations with high confidence in their deleterious effect were identified in approximately 24% of the cases and were held for diagnosis, whereas variants of uncertain significance were identified in 10% of cases. This study provides a new classification of genes that are involved in HPE. SHH, ZIC2, and SIX3 remain the top genes in term of frequency with GLI2, and are followed by FGF8 and FGFR1. The three minor HPE genes identified by our study are DLL1, DISP1, and SUFU. Here, we demonstrate that fibroblast growth factor signaling must now be considered a major pathway involved in HPE. Interestingly, several cases of double mutations were found and argue for a polygenic inheritance of HPE. Altogether, it supports that the implementation of NGS in HPE diagnosis is required to improve genetic counseling.
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Factores de Crecimiento de Fibroblastos/genética , Holoprosencefalia/genética , Mutación , Femenino , Predisposición Genética a la Enfermedad , Proteínas Hedgehog/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Análisis de Secuencia de ADN/métodos , Transducción de SeñalRESUMEN
Ocular developmental anomalies (ODA) such as anophthalmia/microphthalmia (AM) or anterior segment dysgenesis (ASD) have an estimated combined prevalence of 3.7 in 10,000 births. Mutations in SOX2 are the most frequent contributors to severe ODA, yet account for a minority of the genetic drivers. To identify novel ODA loci, we conducted targeted high-throughput sequencing of 407 candidate genes in an initial cohort of 22 sporadic ODA patients. Patched 1 (PTCH1), an inhibitor of sonic hedgehog (SHH) signaling, harbored an enrichment of rare heterozygous variants in comparison to either controls, or to the other candidate genes (four missense and one frameshift); targeted resequencing of PTCH1 in a second cohort of 48 ODA patients identified two additional rare nonsynonymous changes. Using multiple transient models and a CRISPR/Cas9-generated mutant, we show physiologically relevant phenotypes altering SHH signaling and eye development upon abrogation of ptch1 in zebrafish for which in vivo complementation assays using these models showed that all six patient missense mutations affect SHH signaling. Finally, through transcriptomic and ChIP analyses, we show that SOX2 binds to an intronic domain of the PTCH1 locus to regulate PTCH1 expression, findings that were validated both in vitro and in vivo. Together, these results demonstrate that PTCH1 mutations contribute to as much as 10% of ODA, identify the SHH signaling pathway as a novel effector of SOX2 activity during human ocular development, and indicate that ODA is likely the result of overactive SHH signaling in humans harboring mutations in either PTCH1 or SOX2.
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Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Receptor Patched-1/genética , Factores de Transcripción SOXB1/metabolismo , Alelos , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Sitios Genéticos , Heterocigoto , Humanos , Mutación , Receptor Patched-1/metabolismo , Fenotipo , Análisis de Secuencia de ADN , Pez CebraRESUMEN
Holoprosencephaly (HPE) is a frequent congenital malformation of the brain characterized by impaired forebrain cleavage and midline facial anomalies. Heterozygous mutations in 14 genes have been identified in HPE patients that account for only 30% of HPE cases, suggesting the existence of other HPE genes. Data from homozygosity mapping and whole-exome sequencing in a consanguineous Turkish family were combined to identify a homozygous missense mutation (c.2150G>A; p.Gly717Glu) in STIL, common to the two affected children. STIL has a role in centriole formation and has previously been described in rare cases of microcephaly. Rescue experiments in U2OS cells showed that the STIL p.Gly717Glu mutation was not able to fully restore the centriole duplication failure following depletion of endogenous STIL protein indicating the deleterious role of the mutation. In situ hybridization experiments using chick embryos demonstrated that expression of Stil was in accordance with a function during early patterning of the forebrain. It is only the second time that a STIL homozygous mutation causing a recessive form of HPE was reported. This result also supports the genetic heterogeneity of HPE and increases the panel of genes to be tested for HPE diagnosis.
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Holoprosencefalia/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Microcefalia/genética , Animales , Encéfalo/diagnóstico por imagen , Línea Celular , Centriolos , Embrión de Pollo , Pollos/metabolismo , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Holoprosencefalia/patología , Homocigoto , Humanos , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Imagen por Resonancia Magnética , Masculino , Microcefalia/patología , Mutación Missense , Prosencéfalo/metabolismo , Radiografía , HermanosRESUMEN
The establishment of a functional nervous system requires a highly orchestrated process of neural proliferation and differentiation. The evolutionary conserved Notch signaling pathway is a key regulator of this process, regulating basic helix-loop-helix (bHLH) transcriptional repressors and proneural genes. However, little is known about downstream Notch targets and subsequently genes required for neuronal specification. In this report, the expression pattern of Transgelin 3 (Tagln3), Chromogranin A (Chga) and Contactin 2 (Cntn2) was described in detail during early chick embryogenesis. Expression of these genes was largely restricted to the nervous system including the early axon scaffold populations, cranial ganglia and spinal motor neurons. Their temporal and spatial expression were compared with the neuronal markers Nescient Helix-Loop-Helix 1 (Nhlh1), Stathmin 2 (Stmn2) and HuC/D. We show that Tagln3 is an early marker for post-mitotic neurons whereas Chga and Cntn2 are expressed in mature neurons. We demonstrate that inhibition of Notch signaling during spinal cord neurogenesis enhances expression of these markers. This data demonstrates that Tagln3, Chga and Cntn2 represent strong new candidates to contribute to the sequential progression of vertebrate neurogenesis.
RESUMEN
BACKGROUND: The generation of diverse neuronal types and subtypes from multipotent progenitors during development is crucial for assembling functional neural circuits in the adult central nervous system. It is well known that the Notch signalling pathway through the inhibition of proneural genes is a key regulator of neurogenesis in the vertebrate central nervous system. However, the role of Notch during hypothalamus formation along with its downstream effectors remains poorly defined. RESULTS: Here, we have transiently blocked Notch activity in chick embryos and used global gene expression analysis to provide evidence that Notch signalling modulates the generation of neurons in the early developing hypothalamus by lateral inhibition. Most importantly, we have taken advantage of this model to identify novel targets of Notch signalling, such as Tagln3 and Chga, which were expressed in hypothalamic neuronal nuclei. CONCLUSIONS: These data give essential advances into the early generation of neurons in the hypothalamus. We demonstrate that inhibition of Notch signalling during early development of the hypothalamus enhances expression of several new markers. These genes must be considered as important new targets of the Notch/proneural network.