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1.
Nat Commun ; 15(1): 3698, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38693102

RESUMO

Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of polycystin-1. The signaling pathways underlying this process, termed cilia-dependent cyst activation (CDCA), remain unknown. Using translating ribosome affinity purification RNASeq on mouse kidneys with polycystin-1 and cilia inactivation before cyst formation, we identify the differential 'CDCA pattern' translatome specifically dysregulated in kidney tubule cells destined to form cysts. From this, Glis2 emerges as a candidate functional effector of polycystin signaling and CDCA. In vitro changes in Glis2 expression mirror the polycystin- and cilia-dependent changes observed in kidney tissue, validating Glis2 as a cell culture-based indicator of polycystin function related to cyst formation. Inactivation of Glis2 suppresses polycystic kidney disease in mouse models of ADPKD, and pharmacological targeting of Glis2 with antisense oligonucleotides slows disease progression. Glis2 transcript and protein is a functional target of CDCA and a potential therapeutic target for treating ADPKD.


Assuntos
Cílios , Modelos Animais de Doenças , Rim Policístico Autossômico Dominante , Transdução de Sinais , Canais de Cátion TRPP , Animais , Humanos , Masculino , Camundongos , Cílios/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligonucleotídeos Antissenso/farmacologia , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/tratamento farmacológico , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/genética
2.
Front Genet ; 13: 1067168, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36386820
3.
Circ Genom Precis Med ; 13(6): e003000, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33196317

RESUMO

BACKGROUND: Human heterotaxy is a group of congenital disorders characterized by misplacement of one or more organs according to the left-right axis. The genetic causes of human heterotaxy are highly heterogeneous. METHODS: We performed exome sequencing in a cohort of 26 probands with heterotaxy followed by gene burden analysis for the enrichment of novel rare damaging mutations. Transcription activator-like effector nuclease was used to generate somatic loss-of-function mutants in a zebrafish model. Ciliary defects were examined by whole-mount immunostaining of acetylated α-tubulin. RESULTS: We identified a significant enrichment of novel rare damaging mutations in the CC2D1A gene. Seven occurrences of CC2D1A mutations were found to affect 4 highly conserved amino acid residues of the protein. Functional analyses in the transcription activator-like effector nuclease-mediated zebrafish knockout models were performed, and heterotaxy phenotypes of the cardiovascular and gastrointestinal systems in both somatic and germline mutants were observed. Defective cilia were demonstrated by whole-mount immunostaining of acetylated α-tubulin. These abnormalities were rescued by wild-type cc2d1a mRNA but not cc2d1a mutant mRNA, strongly suggesting a loss-of-function mechanism. On the other hand, overexpression of cc2d1a orthologous mutations cc2d1a P559L and cc2d1a G808V (orthologous to human CC2D1A P532L and CC2D1A G781V) did not affect embryonic development. CONCLUSIONS: Using a zebrafish model, we were able to establish a novel association of CC2D1A with heterotaxy and ciliary dysfunction in the F2 generation via a loss-of-function mechanism. Future mechanistic studies are needed for a better understanding of the role of CC2D1A in left-right patterning and ciliary dysfunction.


Assuntos
Alelos , Cílios/patologia , Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Síndrome de Heterotaxia/genética , Mutação/genética , Animais , Variações do Número de Cópias de DNA/genética , Modelos Animais de Doenças , Mutação em Linhagem Germinativa/genética , Heterozigoto , Humanos , Penetrância , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Sequenciamento do Exoma , Peixe-Zebra/genética
4.
Epilepsia Open ; 4(1): 63-72, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30868116

RESUMO

OBJECTIVE: Early onset drug-resistant epilepsy is a neurologic disorder in which 2 antiepileptic drugs fail to maintain the seizure-free status of the patient. Heterogeneous clinical presentations make the diagnosis challenging. We aim to identify the underlying genetic causes of a pediatric cohort with drug-resistant epilepsy and evaluate whether the findings can provide information on patient management. METHODS: We include patients with drug-resistant epilepsy onset before 18 years of age. Singleton clinical chromosomal microarray (CMA) followed by whole exome sequencing (WES) was performed using genomic DNA. In the first-tier analysis of the exome data, we aimed to identify disease-causing mutations in 546 genes known to cause, or to be associated with, epilepsy. For negative cases, we proceeded to exome-wide analysis. Rare coding variants were interrogated for pathogenicity based on the American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: We recruited 50 patients. We identified 6 pathogenic or likely pathogenic mutations, giving a diagnostic yield of 12%. Mutations were found in 6 different genes: SCN8A, SCN1A, MECP2, CDKL5, DEPDC5, and CHD2. The CDKL5 variant was found to be mosaic. One variant of unknown significance (VUS) in KCNT1 was found in a patient with compatible clinical features. Of note, a reported pathogenic SCN5A mutation known to contribute to Brugada syndrome, was also found in the patient with an SCN1A mutation. SIGNIFICANCE: Our study suggests that singleton WES is an effective diagnostic tool for drug-resistant epilepsy. Genetic diagnosis can help to consolidate the clinical diagnosis, to facilitate phenotypic expansion, and to influence treatment and management options for seizure control in our patients. In our study, a significant portion of the genetic findings are known to be associated with an increased risk of sudden unexpected death in epilepsy (SUDEP). These findings could assist with more appropriate management in patients with epilepsy.

5.
Mol Autism ; 8: 66, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29296277

RESUMO

Background: Macrocephaly, which is defined as a head circumference greater than or equal to + 2 standard deviations, is a feature commonly observed in children with developmental delay and/or autism spectrum disorder. Although PTEN is a well-known gene identified in patients with this syndromic presentation, other genes in the PI3K-AKT-mTOR signalling pathway have also recently been suggested to have important roles. The aim of this study is to characterise the mutation spectrum of this group of patients. Methods: We performed whole-exome sequencing of 21 patients with macrocephaly and developmental delay/autism spectrum disorder. Sources of genomic DNA included blood, buccal mucosa and saliva. Germline mutations were validated by Sanger sequencing, whereas somatic mutations were validated by droplet digital PCR. Results: We identified ten pathogenic/likely pathogenic mutations in PTEN (n = 4), PIK3CA (n = 3), MTOR (n = 1) and PPP2R5D (n = 2) in ten patients. An additional PTEN mutation, which was classified as variant of unknown significance, was identified in a patient with a pathogenic PTEN mutation, making him harbour bi-allelic germline PTEN mutations. Two patients harboured somatic PIK3CA mutations, and the level of somatic mosaicism in blood DNA was low. Patients who tested positive for mutations in the PI3K-AKT-mTOR pathway had a lower developmental quotient than the rest of the cohort (DQ = 62.8 vs. 76.1, p = 0.021). Their dysmorphic features were non-specific, except for macrocephaly. Among the ten patients with identified mutations, brain magnetic resonance imaging was performed in nine, all of whom showed megalencephaly. Conclusion: We identified mutations in the PI3K-AKT-mTOR signalling pathway in nearly half of our patients with macrocephaly and developmental delay/autism spectrum disorder. These patients have subtle dysmorphic features and mild developmental issues. Clinically, patients with germline mutations are difficult to distinguish from patients with somatic mutations, and therefore, sequencing of buccal or saliva DNA is important to identify somatic mosaicism. Given the high diagnostic yield and the management implications, we suggest implementing comprehensive genetic testing in the PI3K-AKT-mTOR pathway in the clinical evaluation of patients with macrocephaly and developmental delay and/or autism spectrum disorder.


Assuntos
Transtorno do Espectro Autista/genética , Análise Mutacional de DNA/métodos , Deficiências do Desenvolvimento/genética , Redes Reguladoras de Genes , Microcefalia/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Lactente , Masculino , PTEN Fosfo-Hidrolase , Fosfatidilinositol 3-Quinases/genética , Proteína Fosfatase 2/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Sequenciamento do Exoma
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