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  1. Article ; Online: A CRISPR/Cas9-generated mutation in the zebrafish orthologue of PPP2R3B causes idiopathic scoliosis

    Marian Seda / Berta Crespo / Michelangelo Corcelli / Daniel P. Osborn / Dagan Jenkins

    Scientific Reports, Vol 13, Iss 1, Pp 1-

    2023  Volume 17

    Abstract: Abstract Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic ... ...

    Abstract Abstract Idiopathic scoliosis (IS) is the deformation and/or abnormal curvature of the spine that develops progressively after birth. It is a very common condition, affecting approximately 4% of the general population, yet the genetic and mechanistic causes of IS are poorly understood. Here, we focus on PPP2R3B, which encodes a protein phosphatase 2A regulatory subunit. We found that PPP2R3B is expressed at sites of chondrogenesis within human foetuses, including the vertebrae. We also demonstrated prominent expression in myotome and muscle fibres in human foetuses, and zebrafish embryos and adolescents. As there is no rodent orthologue of PPP2R3B, we used CRIPSR/Cas9-mediated gene-editing to generate a series of frameshift mutations in zebrafish ppp2r3b. Adolescent zebrafish that were homozygous for this mutation exhibited a fully penetrant kyphoscoliosis phenotype which became progressively worse over time, mirroring IS in humans. These defects were associated with reduced mineralisation of vertebrae, resembling osteoporosis. Electron microscopy demonstrated abnormal mitochondria adjacent to muscle fibres. In summary, we report a novel zebrafish model of IS and reduced bone mineral density. In future, it will be necessary to delineate the aetiology of these defects in relation to bone, muscle, neuronal and ependymal cilia function.
    Keywords Medicine ; R ; Science ; Q
    Language English
    Publishing date 2023-04-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Analysis of transgenic zebrafish expressing the Lenz-Majewski syndrome gene PTDSS1 in skeletal cell lineages [version 1; peer review

    Marian Seda / Emma Peskett / Charalambos Demetriou / Dale Bryant / Gudrun E. Moore / Philip Stanier / Dagan Jenkins

    F1000Research, Vol

    2 approved]

    2019  Volume 8

    Abstract: Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo ... ...

    Abstract Background: Lenz-Majewski syndrome (LMS) is characterized by osteosclerosis and hyperostosis of skull, vertebrae and tubular bones as well as craniofacial, dental, cutaneous, and digit abnormalities. We previously found that LMS is caused by de novo dominant missense mutations in the PTDSS1 gene, which encodes phosphatidylserine synthase 1 (PSS1), an enzyme that catalyses the conversion of phosphatidylcholine to phosphatidylserine. The mutations causing LMS result in a gain-of-function, leading to increased enzyme activity and blocking end-product inhibition of PSS1. Methods: Here, we have used transpose-mediated transgenesis to attempt to stably express wild-type and mutant forms of human PTDSS1 ubiquitously or specifically in chondrocytes, osteoblasts or osteoclasts in zebrafish. Results: We report multiple genomic integration sites for each of 8 different transgenes. While we confirmed that the ubiquitously driven transgene constructs were functional in terms of driving gene expression following transient transfection in HeLa cells, and that all lines exhibited expression of a heart-specific cistron within the transgene, we failed to detect PTDSS1 gene expression at either the RNA or protein levels in zebrafish. All wild-type and mutant transgenic lines of zebrafish exhibited mild scoliosis with variable incomplete penetrance which was never observed in non-transgenic animals. Conclusions: Collectively the data suggest that the transgenes are silenced, that animals with integrations that escape silencing are not viable, or that other technical factors prevent transgene expression. In conclusion, the incomplete penetrance of the phenotype and the lack of a matched transgenic control model precludes further meaningful investigations of these transgenic lines.
    Keywords Medicine ; R ; Science ; Q
    Subject code 572
    Language English
    Publishing date 2019-03-01T00:00:00Z
    Publisher F1000 Research Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis

    Michael Taschner / Anna Lorentzen / André Mourão / Toby Collins / Grace M Freke / Dale Moulding / Jerome Basquin / Dagan Jenkins / Esben Lorentzen

    eLife, Vol

    2018  Volume 7

    Abstract: Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the Chlamydomonas IFT-B protein ... ...

    Abstract Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the Chlamydomonas IFT-B protein IFT80, which reveals the architecture of two N-terminal β-propellers followed by an α-helical extension. The N-terminal β-propeller tethers IFT80 to the IFT-B complex via IFT38 whereas the second β-propeller and the C-terminal α-helical extension result in IFT80 homo-dimerization. Using CRISPR/Cas to create biallelic Ift80 frameshift mutations in IMCD3 mouse cells, we demonstrate that IFT80 is absolutely required for ciliogenesis. Structural mapping and rescue experiments reveal that human disease-causing missense mutations do not cluster within IFT80 and form functional IFT particles. Unlike missense mutant forms of IFT80, deletion of the C-terminal dimerization domain prevented rescue of ciliogenesis. Taken together our results may provide a first insight into higher order IFT complex formation likely required for IFT train formation.
    Keywords Chlamydomonas reinhardtii ; Cilium ; Intraflagelllar transport ; protein structure ; ciliopathies ; IFT80 ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
    Language English
    Publishing date 2018-04-01T00:00:00Z
    Publisher eLife Sciences Publications Ltd
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism

    Wai-Yee Lam / Clara Sze-Man Tang / Man-Ting So / Haibing Yue / Jacob Shujui Hsu / Patrick Ho-Yu Chung / John M. Nicholls / Fanny Yeung / Chun-Wai Davy Lee / Diem Ngoc Ngo / Pham Anh Hoa Nguyen / Hannah M. Mitchison / Dagan Jenkins / Christopher O'Callaghan / Maria-Mercè Garcia-Barceló / So-Lun Lee / Pak-Chung Sham / Vincent Chi-Hang Lui / Paul Kwong-Hang Tam

    EBioMedicine, Vol 71, Iss , Pp 103530- (2021)

    2021  

    Abstract: Background: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common ... ...

    Abstract Background: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined. Methods: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients’ liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. Findings: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]= 2.58 [1.15–6.07], adjusted p = 0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. Interpretation: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. Funding: The study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.
    Keywords Biliary atresia ; Whole exome sequencing ; Rare variants ; Cilia dysfunction ; Medicine ; R ; Medicine (General) ; R5-920
    Subject code 610 ; 616
    Language English
    Publishing date 2021-09-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  5. Article ; Online: Diverse species-specific phenotypic consequences of loss of function sorting nexin 14 mutations

    Dale Bryant / Marian Seda / Emma Peskett / Constance Maurer / Gideon Pomeranz / Marcus Ghosh / Thomas A. Hawkins / James Cleak / Sanchari Datta / Hanaa Hariri / Kaitlyn M. Eckert / Daniyal J. Jafree / Claire Walsh / Charalambos Demetriou / Miho Ishida / Cristina Alemán-Charlet / Letizia Vestito / Rimante Seselgyte / Jeffrey G. McDonald /
    Maria Bitner-Glindzicz / Myriam Hemberger / Jason Rihel / Lydia Teboul / W. Mike Henne / Dagan Jenkins / Gudrun E. Moore / Philip Stanier

    Scientific Reports, Vol 10, Iss 1, Pp 1-

    2020  Volume 11

    Abstract: Abstract Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and ... ...

    Abstract Abstract Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.
    Keywords Medicine ; R ; Science ; Q
    Subject code 572
    Language English
    Publishing date 2020-08-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

    Karsten Boldt / Jeroen van Reeuwijk / Qianhao Lu / Konstantinos Koutroumpas / Thanh-Minh T. Nguyen / Yves Texier / Sylvia E. C. van Beersum / Nicola Horn / Jason R. Willer / Dorus A. Mans / Gerard Dougherty / Ideke J. C. Lamers / Karlien L. M. Coene / Heleen H. Arts / Matthew J. Betts / Tina Beyer / Emine Bolat / Christian Johannes Gloeckner / Khatera Haidari /
    Lisette Hetterschijt / Daniela Iaconis / Dagan Jenkins / Franziska Klose / Barbara Knapp / Brooke Latour / Stef J. F. Letteboer / Carlo L. Marcelis / Dragana Mitic / Manuela Morleo / Machteld M. Oud / Moniek Riemersma / Susan Rix / Paulien A. Terhal / Grischa Toedt / Teunis J. P. van Dam / Erik de Vrieze / Yasmin Wissinger / Ka Man Wu / Gordana Apic / Philip L. Beales / Oliver E. Blacque / Toby J. Gibson / Martijn A. Huynen / Nicholas Katsanis / Hannie Kremer / Heymut Omran / Erwin van Wijk / Uwe Wolfrum / François Kepes / Erica E. Davis / Brunella Franco / Rachel H. Giles / Marius Ueffing / Robert B. Russell / Ronald Roepman / UK10K Rare Diseases Group

    Nature Communications, Vol 7, Iss 1, Pp 1-

    2016  Volume 13

    Abstract: Mutations in proteins that localize to primary cilia cause devastating diseases, yet the primary cilium is a poorly understood organelle. Here the authors use interaction proteomics to identify a network of human ciliary proteins that provides new ... ...

    Abstract Mutations in proteins that localize to primary cilia cause devastating diseases, yet the primary cilium is a poorly understood organelle. Here the authors use interaction proteomics to identify a network of human ciliary proteins that provides new insights into several biological processes and diseases.
    Keywords Science ; Q
    Language English
    Publishing date 2016-05-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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