scholarly journals Loss of function mutations in CCDC32 cause a congenital syndrome characterized by craniofacial, cardiac and neurodevelopmental anomalies

2020 ◽  
Vol 29 (9) ◽  
pp. 1489-1497 ◽  
Author(s):  
Tamar Harel ◽  
John N Griffin ◽  
Thomas Arbogast ◽  
Tanner O Monroe ◽  
Flavia Palombo ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Congenital Malformations ◽  
Mammalian Cell Culture ◽  
Zebrafish Embryos ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Functional Studies ◽  
Whole Exome ◽  
Cilia Formation ◽  
Congenital Syndrome

Abstract Despite the wide use of genomics to investigate the molecular basis of rare congenital malformations, a significant fraction of patients remains bereft of diagnosis. As part of our continuous effort to recruit and perform genomic and functional studies on such cohorts, we investigated the genetic and mechanistic cause of disease in two independent consanguineous families affected by overlapping craniofacial, cardiac, laterality and neurodevelopmental anomalies. Using whole exome sequencing, we identified homozygous frameshift CCDC32 variants in three affected individuals. Functional analysis in a zebrafish model revealed that ccdc32 depletion recapitulates the human phenotypes. Because some of the patient phenotypes overlap defects common to ciliopathies, we asked if loss of CCDC32 might contribute to the dysfunction of this organelle. Consistent with this hypothesis, we show that ccdc32 is required for normal cilia formation in zebrafish embryos and mammalian cell culture, arguing that ciliary defects are at least partially involved in the pathomechanism of this disorder.

scholarly journals The Genome-Wide Impact of Nipblb Loss-of-Function on Zebrafish Gene Expression

2020 ◽  
Vol 21 (24) ◽  
pp. 9719
Author(s):  
Marco Spreafico ◽  
Eleonora Mangano ◽  
Mara Mazzola ◽  
Clarissa Consolandi ◽  
Roberta Bordoni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
System Development ◽  
Expression Patterns ◽  
Nervous System Development ◽  
Zebrafish Embryos ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Genome Wide ◽  
Transcriptional Effect

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.

scholarly journals Morphological, behavioral and molecular studies on CRISPR/Cas9-mediated knockout of the NOMO1 gene in zebrafish

2021 ◽  
Author(s):  
Fei Li ◽  
Tingting Li ◽  
Jia Lin ◽  
Jing Jian ◽  
Qi Zhang ◽  
...  
Keyword(s):  
Candidate Gene ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Neuropsychiatric Disorders ◽  
Repetitive Behaviors ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Functional Studies ◽  
Vertebrate Model ◽  
Morphological Differences

Abstract Background: Multiple clinical genome-wide analysis identified that chromosome 16p13.11 is a hotspot associated with neuropsychiatric disorders such as autism, schizophrenia and epilepsy. Nodal modulator 1 (NOMO1), located on human chromosome 16p13.11, was considered as a candidate gene with neuropsychiatric disorders. However, it is unknown whether the nomo1 deficiency causes neurological abnormalities, and the molecular mechanisms and pathogenesis of the NOMO1 gene remain unclear. To study the effects of nomo1 deficiency on brain development and neuropsychiatric system, a nomo1 knockout zebrafish model was established.Methods: We developed a viable vertebrate model of nomo1 loss-of-function using CRISPR/Cas9 technology and characterized nomo1 mutant zebrafish. Phenotypic and functional studies of developing nomo1 mutant zebrafish, including morphological measurements, behavioral assays, and functional mechanistic analyses, were performed.Results: Morphological differences in the phenotype of nomo1-/- zebrafish gradually became less noticeable during development, however, the enlarged interstitial spaces in midbrain and hindbrain were detected in nomo1 mutant zebrafish. Meanwhile, the nomo1 deficiency caused the change of expression levels in neurotransmitters of γ-aminobutyrate, glutamate and serotonin. Interestingly, the nomo1 loss-of-function zebrafish model exhibited social defects and repetitive behaviors in juvenile, which represented autism-like behaviors. The transcriptome analysis showed different gene expression patterns in mutant zebrafish at the genetic level. Further results revealed that the neuroactive drug PTZ recovered the decreased locomotor phenotype in larval mutant zebrafish.Conclusions: In this study, we established a nomo1 vertebrate animal model using CRISPR/Cas9 gene editing approach. The loss-of-function of nomo1 displayed autism-like behaviors and altered levels of the γ-aminobutyrate, glutamate and serotonin in zebrafish, which provide evidence that nomo1 as a candidate gene for autism. The versatility of zebrafish model is contributed to studying NOMO1-related disorders and conducting drug screening in future.Limitations: Further studies are needed to determine whether an intervention with a neuroactive drug in nomo1-/- zebrafish to alter the behavioral phenotype is applicable to the behavior of human patients.

scholarly journals Biallelic DAB1 Variants Are Associated With Mild Lissencephaly and Cerebellar Hypoplasia

2021 ◽  
Vol 7 (2) ◽  
pp. e558
Author(s):  
Daphne J. Smits ◽  
Rachel Schot ◽  
Martina Wilke ◽  
Marjon van Slegtenhorst ◽  
Marie Claire Y. de Wit ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Splice Variants ◽  
Brain Mri ◽  
Cerebellar Vermis ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Whole Exome

ObjectiveWe aimed to identify pathogenic variants in a girl with epilepsy, developmental delay, cerebellar ataxia, oral motor difficulty, and structural brain abnormalities with the use of whole-exome sequencing.MethodsWhole-exome trio analysis and molecular functional studies were performed in addition to the clinical findings and neuroimaging studies.ResultsBrain MRI showed mild pachygyria, hypoplasia of the cerebellar vermis, and abnormal foliation of the cerebellar vermis, suspected for a variant in one of the genes of the Reelin pathway. Trio whole-exome sequencing and additional functional studies were performed to identify the pathogenic variants. Trio whole-exome sequencing revealed compound heterozygous splice variants in DAB1, both affecting the highly conserved functional phosphotyrosine-binding domain. Expression studies in patient-derived cells showed loss of normal transcripts, confirming pathogenicity.ConclusionsWe conclude that these variants are very likely causally related to the cerebral phenotype and propose to consider loss-of-function DAB1 variants in patients with RELN-like cortical malformations.

scholarly journals SOX19b regulates the premature neuronal differentiation of neural stem cells through EZH2-mediated histone methylation in neural tube development of zebrafish

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Xian Li ◽  
Wenjuan Zhou ◽  
Xinyue Li ◽  
Ming Gao ◽  
Shufang Ji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Neural Tube ◽  
Neural Tube Defects ◽  
Histone Methylation ◽  
Zebrafish Embryos ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Proliferation And Differentiation ◽  
Neural Tube Development

Abstract Objective Neural tube defects (NTDs) are the most serious and common birth defects in the clinic. The SRY-related HMG box B1 (SoxB1) gene family has been implicated in different processes of early embryogenesis. Sox19b is a maternally expressed gene in the SoxB1 family that is found in the region of the presumptive central nervous system (CNS), but its role and mechanism in embryonic neural stem cells (NSCs) during neural tube development have not yet been explored. Considering that Sox19b is specific to bony fish, we intended to investigate the role and mechanism of Sox19b in neural tube development in zebrafish embryos. Material and methods Morpholino (MO) antisense oligonucleotides were used to construct a Sox19b loss-of-function zebrafish model. The phenotype and the expression of related genes were analysed by in situ hybridization and immunolabelling. Epigenetic modifications were detected by western blot and chromatin immunoprecipitation. Results In this study, we found that zebrafish embryos exhibited a reduced or even deleted forebrain phenotype after the expression of the Sox19b gene was inhibited. Moreover, we found for the first time that knockdown of Sox19b reduced the proliferation of NSCs; increased the transcription levels of Ngn1, Ascl1, HuC, Islet1, and cyclin-dependent kinase (CDK) inhibitors; and led to premature differentiation of NSCs. Finally, we found that knockdown of Sox19b decreased the levels of EZH2/H3K27me3 and decreased the level of H3K27me3 at the promoters of Ngn1 and ascl1a. Conclusion Together, our data demonstrate that Sox19b plays an essential role in early NSC proliferation and differentiation through EZH2-mediated histone methylation in neural tube development. This study established the role of transcription factor Sox19b and epigenetic factor EZH2 regulatory network on NSC development, which provides new clues and theoretical guidance for the clinical treatment of neural tube defects.

scholarly journals Whole-exome sequencing identifies multiple loss-of-function mutations of NF-κB pathway regulators in nasopharyngeal carcinoma

2016 ◽  
Vol 113 (40) ◽  
pp. 11283-11288 ◽  
Author(s):  
Hong Zheng ◽  
Wei Dai ◽  
Arthur Kwok Leung Cheung ◽  
Josephine Mun Yee Ko ◽  
Rebecca Kan ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Somatic Mutations ◽  
Cytidine Deaminase ◽  
Cycle Phase ◽  
Loss Of Function ◽  
Functional Studies ◽  
Whole Exome ◽  
Multiple Loss

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy with a unique geographical distribution. The genomic abnormalities leading to NPC pathogenesis remain unclear. In total, 135 NPC tumors were examined to characterize the mutational landscape using whole-exome sequencing and targeted resequencing. An APOBEC cytidine deaminase mutagenesis signature was revealed in the somatic mutations. Noticeably, multiple loss-of-function mutations were identified in several NF-κB signaling negative regulators NFKBIA, CYLD, and TNFAIP3. Functional studies confirmed that inhibition of NFKBIA had a significant impact on NF-κB activity and NPC cell growth. The identified loss-of-function mutations in NFKBIA leading to protein truncation contributed to the altered NF-κB activity, which is critical for NPC tumorigenesis. In addition, somatic mutations were found in several cancer-relevant pathways, including cell cycle-phase transition, cell death, EBV infection, and viral carcinogenesis. These data provide an enhanced road map for understanding the molecular basis underlying NPC.

scholarly journals Abnormal expression of GABAA receptor sub-units and hypomotility upon loss of gabra1 in zebrafish

2020 ◽  
Author(s):  
Nayeli Reyes-Nava ◽  
Hung-Chun Yu ◽  
Curtis R. Coughlin ◽  
Tamim H. Shaikh ◽  
Anita M. Quintana
Keyword(s):  
Nervous System ◽  
De Novo ◽  
Functional Approach ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Behavioral Deficits ◽  
Whole Exome ◽  
System Disorder

ABSTRACTWe used whole exome sequencing (WES) to determine the genetic etiology of a patient with a multi-system disorder characterized by a seizure phenotype. WES identified a heterozygous de novo missense mutation in the GABRA1 gene (c.875C>T). GABRA1 encodes the alpha subunit of the Gamma-Aminobutyric Acid receptor A (GABAAR). The GABAAR is a ligand gated ion channel that mediates the fast inhibitory signals of the nervous system and mutations in the sub-units that compose the GABAAR have been previously associated with human disease. To understand the mechanisms by which GABRA1 regulates brain development, we developed a zebrafish model of gabra1 deficiency. gabra1 expression is restricted to the nervous system and behavioral analysis of morpholino injected larvae suggests that the knockdown of gabra1 results in hypoactivity and defects in the expression of other sub-units of the GABAAR. Expression the human GABRA1 protein in morphants partially restored the hypomotility phenotype. In contrast, the expression of the c.875C>T variant did not restore these behavioral deficits. Collectively, these results represent a functional approach to understand the mechanisms by which loss of function alleles cause disease.

scholarly journals Comprehensive Analysis of LIN28A in Chinese Patients With Early Onset Parkinson’s Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaojing Gu ◽  
Yanbing Hou ◽  
Yongping Chen ◽  
Ruwei Ou ◽  
Bei Cao ◽  
...  
Keyword(s):  
Early Onset ◽  
Chinese Patient ◽  
Chinese Patients ◽  
Loss Of Function ◽  
Developmental Defects ◽  
Functional Studies ◽  
Whole Exome ◽  
Increased Risk ◽  
Related Phenotype ◽  
Early Onset Parkinson’S Disease

A loss-of-function variant in Lin-28 Homolog A gene (LIN28A p. R192G, rs558060339) has been identified in two East Asian ancestry patients with early-onset PD (EOPD). Functional studies revealed that such a variant could lead to developmental defects and PD-related phenotype, and the phenotypes could be rescued after correction of the variant. The aim of the study was to screen the variants of LIN28A in Chinese patients with EOPD. A total of 682 EOPD patients were sequenced with whole exome sequencing and the coding and flanking region of LIN28A were analyzed. We identified a rare coding variant, p. P182L, of LIN28A in a Chinese patient with EOPD. Moreover, we also found a 3′-UTR polymorphism (rs4659441) to be associated with an increased risk for PD. However, our rare variant burden analysis did not support a role for LIN28A as a major causal gene for PD.

Abstract WMP26: Missense Mutation in COL22A1 is Associated With Intracranial Aneurysms

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Quynh Ton ◽  
Daniel Leino ◽  
Joseph Broderick ◽  
Tatiana Foroud ◽  
Saulius Sumanas
Keyword(s):  
Missense Mutation ◽  
Intracranial Aneurysms ◽  
Zebrafish Embryos ◽  
Loss Of Function ◽  
Tail Region ◽  
Particle Injection ◽  
Vascular Integrity ◽  
Whole Exome ◽  
Mutant Variant ◽  
Increased Sensitivity

Background: Both environmental and genetic factors have been linked to the formation of intracranial aneurysms (IAs), but animal models to explore mechanisms underlying genetic risk factors of IAs in humans are lacking. Methods: To identify additional genes responsible for IA development, whole exome sequencing of the patients from seven selected families was performed. One of the identified variants found in all five affected members of one family was a missense mutation in the highly conserved triple-helix region of the collagen COL22A1 isoform, the function of which has not been previously known. This mutant variant was examined in zebrafish embryonic and adult models. Results: We demonstrated that COL22A1 is expressed in cranial tissues as well as in the cells between myotomes in the trunk and tail region of the zebrafish embryos. Early global overexpression of the human mutant but not human wild-type COL22A1 in the zebrafish embryos interfered with epiboly movements during gastrulation suggesting that the mutation has a deleterious dominant effect. We then used TALEN-engineered nucleases to generate a loss-of-function mutant in zebrafish col22a1 . The homozygous null mutants were viable as adults but exhibited increased abnormal blood accumulations in the eyes and cranial regions, suggestive of hemorrhages. We further notice abnormal lesions existed in the trunks of these adults caused by rupture of blood vessels. A subset of the mutant adults shows cardiac dilation and a phenotype consistent with dilated cardiomyopathy. We further demonstrated that the homozygous embryos show increased sensitivity to cardiovascular stress, which results in a greater percentage of embryos with intracranial hemorrhages. The mutant embryos also exhibited increased vascular permeability as demonstrated by nano-particle injection into the circulation of 4-day post fertilization embryos. Conclusions: Our results suggest that the function of COL22A1 is important in maintaining vascular integrity and that mutations in COL22A1 are a potential one genetic cause for intracranial aneurysms in humans.

scholarly journals Comprehensive Analysis of LIN28A in Chinese Patients with Early Onset Parkinson’s Disease

2021 ◽  
Author(s):  
Xiaojing Gu ◽  
Yanbing Hou ◽  
Yongping Chen ◽  
Ruwei Ou ◽  
Bei Cao ◽  
...  
Keyword(s):  
Early Onset ◽  
Chinese Patient ◽  
Chinese Patients ◽  
Loss Of Function ◽  
Developmental Defects ◽  
Functional Studies ◽  
Whole Exome ◽  
Increased Risk ◽  
Related Phenotype ◽  
Early Onset Parkinson’S Disease

Abstract A loss-of-function variant in Lin-28 Homolog A gene (LIN28A p.R192G, rs558060339) has been identified in two East Asian ancestry patients with early-onset PD (EOPD). Functional studies revealed that such variant could lead to developmental defects and PD-related phenotype, and the phenotypes could be rescued after correction of the variant. The aim of the study was to screen the variants of LIN28A in Chinese patients with EOPD. A total of 682 EOPD patients were sequenced with whole exome sequencing and the coding and flanking region of LIN28A were analyzed. We identified a rare coding variant-p.P182L of LIN28A in a Chinese patient with EOPD. Moreover, we also found a 3’-UTR polymorphism(rs4659441) to be associated with an increased risk for PD. However, our rare variant burden analysis did not support a role for LIN28A as a major causal gene for PD.

Kindler's Syndrome with Recurrent Neutropenia: Report of Two Cases from Saudi Arabia

2020 ◽  
Author(s):  
Yousef Binamer ◽  
Muzamil A. Chisti
Keyword(s):  
Autosomal Recessive ◽  
Common Mechanism ◽  
Sequencing Analysis ◽  
Loss Of Function ◽  
Skin Atrophy ◽  
Whole Exome ◽  
Infancy And Childhood ◽  
Genetics And Genomics ◽  
New Feature ◽  
Generation Sequencing

AbstractKindler syndrome (KS) is a rare photosensitivity disorder with autosomal recessive mode of inheritance. It is characterized by acral blistering in infancy and childhood, progressive poikiloderma, skin atrophy, abnormal photosensitivity, and gingival fragility. Besides these major features, many minor presentations have also been reported in the literature. We are reporting two cases with atypical features of the syndrome and a new feature of recurrent neutropenia. Whole exome sequencing analysis was done using next-generation sequencing which detected a homozygous loss-of-function (LOF) variant of FERMT1 in both patients. The variant is classified as a pathogenic variant as per the American College of Medical Genetics and Genomics guidelines. Homozygous LOF variants of FERMT1 are a common mechanism of KS and as such confirm the diagnosis of KS in our patients even though the presentation was atypical.

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