Scholarly Works - Cambridge Institute for Medical Research


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  • ItemPublished versionOpen Access
    C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction.
    (Springer Science and Business Media LLC, 2018-02-19) Lautenschläger, Janin; Stephens, Amberley D; Fusco, Giuliana; Ströhl, Florian; Curry, Nathan; Zacharopoulou, Maria; Michel, Claire H; Laine, Romain; Nespovitaya, Nadezhda; Fantham, Marcus; Pinotsi, Dorothea; Zago, Wagner; Fraser, Paul; Tandon, Anurag; St George-Hyslop, Peter; Rees, Eric; Phillips, Jonathan J; De Simone, Alfonso; Kaminski, Clemens F; Schierle, Gabriele S Kaminski; Stephens, Amberley D [0000-0002-7303-6392]; Ströhl, Florian [0000-0002-2603-0780]; St George-Hyslop, Peter [0000-0003-0796-7209]; Phillips, Jonathan J [0000-0002-5361-9582]; Kaminski, Clemens F [0000-0002-5194-0962]; Schierle, Gabriele S Kaminski [0000-0002-1843-2202]
    Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.
  • ItemAccepted versionOpen Access
    Autophagy as a promoter of longevity: insights from model organisms.
    (Springer Science and Business Media LLC, 2018-09) Hansen, Malene; Rubinsztein, David C; Walker, David W; Rubinsztein, David C [0000-0001-5002-5263]
    Autophagy is a conserved process that catabolizes intracellular components to maintain energy homeostasis and to protect cells against stress. Autophagy has crucial roles during development and disease, and evidence accumulated over the past decade indicates that autophagy also has a direct role in modulating ageing. In particular, elegant studies using yeasts, worms, flies and mice have demonstrated a broad requirement for autophagy-related genes in the lifespan extension observed in a number of conserved longevity paradigms. Moreover, several new and interesting concepts relevant to autophagy and its role in modulating longevity have emerged. First, select tissues may require or benefit from autophagy activation in longevity paradigms, as tissue-specific overexpression of single autophagy genes is sufficient to extend lifespan. Second, selective types of autophagy may be crucial for longevity by specifically targeting dysfunctional cellular components and preventing their accumulation. And third, autophagy can influence organismal health and ageing even non-cell autonomously, and thus, autophagy stimulation in select tissues can have beneficial, systemic effects on lifespan. Understanding these mechanisms will be important for the development of approaches to improve human healthspan that are based on the modulation of autophagy.
  • ItemPublished versionOpen Access
    The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes.
    (Cold Spring Harbor Laboratory Press, 2018-06) Robbez-Masson, Luisa; Tie, Christopher HC; Conde, Lucia; Tunbak, Hale; Husovsky, Connor; Tchasovnikarova, Iva A; Timms, Richard T; Herrero, Javier; Lehner, Paul J; Rowe, Helen M; Tchasovnikarova, Iva [0000-0002-0477-0956]; Timms, Richard [0000-0001-7275-597X]; Lehner, Paul [0000-0001-9383-1054]
    Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of a SVA retrotransposon reporter. By using naïve vs. primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. While the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages less than 5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon non-coding DNA.
  • ItemAccepted versionOpen Access
    Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.
    (Proceedings of the National Academy of Sciences, 2018-05-08) Wang, Eddie CY; Pjechova, Mariana; Nightingale, Katie; Vlahava, Virginia-Maria; Patel, Mihil; Ruckova, Eva; Forbes, Simone K; Nobre, Luis; Antrobus, Robin; Roberts, Dawn; Fielding, Ceri A; Seirafian, Sepehr; Davies, James; Murrell, Isa; Lau, Betty; Wilkie, Gavin S; Suárez, Nicolás M; Stanton, Richard J; Vojtesek, Borivoj; Davison, Andrew; Lehner, Paul J; Weekes, Michael P; Wilkinson, Gavin WG; Tomasec, Peter; Wang, Eddie CY [0000-0002-2243-4964]; Stanton, Richard J [0000-0002-6799-1182]; Davison, Andrew [0000-0002-4991-9128]
    CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.
  • ItemPublished versionOpen Access
    An interactive genome browser of association results from the UK10K cohorts project.
    (Oxford University Press (OUP), 2015-12-15) Geihs, Matthias; Yan, Ying; Walter, Klaudia; Huang, Jie; Memari, Yasin; Min, Josine L; Mead, Daniel; UK10K Consortium; Hubbard, Tim J; Timpson, Nicholas J; Down, Thomas A; Soranzo, Nicole; Soranzo, Nicole [0000-0003-1095-3852]
    UNLABELLED: High-throughput sequencing technologies survey genetic variation at genome scale and are increasingly used to study the contribution of rare and low-frequency genetic variants to human traits. As part of the Cohorts arm of the UK10K project, genetic variants called from low-read depth (average 7×) whole genome sequencing of 3621 cohort individuals were analysed for statistical associations with 64 different phenotypic traits of biomedical importance. Here, we describe a novel genome browser based on the Biodalliance platform developed to provide interactive access to the association results of the project. AVAILABILITY AND IMPLEMENTATION: The browser is available at Source code for the Biodalliance platform is available under a BSD license from, and for the LD-display plugin and backend from
  • ItemPublished versionOpen Access
    Clinical and molecular consequences of disease-associated de novo mutations in SATB2.
    (Elsevier BV, 2017-08) Bengani, Hemant; Handley, Mark; Alvi, Mohsan; Ibitoye, Rita; Lees, Melissa; Lynch, Sally Ann; Lam, Wayne; Fannemel, Madeleine; Nordgren, Ann; Malmgren, H; Kvarnung, M; Mehta, Sarju; McKee, Shane; Whiteford, Margo; Stewart, Fiona; Connell, Fiona; Clayton-Smith, Jill; Mansour, Sahar; Mohammed, Shehla; Fryer, Alan; Morton, Jenny; UK10K Consortium; Grozeva, Detelina; Asam, Tara; Moore, David; Sifrim, Alejandro; McRae, Jeremy; Hurles, Matthew E; Firth, Helen V; Raymond, F Lucy; Kini, Usha; Nellåker, Christoffer; Ddd Study; FitzPatrick, David R; Alvi, Mohsan [0000-0003-4331-7078]; Ibitoye, Rita [0000-0002-2887-2068]; Lynch, Sally Ann [0000-0003-0287-4134]; Nordgren, Ann [0000-0003-3338-8382]; Malmgren, H [0000-0003-3285-4281]; McKee, Shane [0000-0003-0446-3435]; Whiteford, Margo [0000-0002-1772-0106]; Connell, Fiona [0000-0002-0444-8203]; Mohammed, Shehla [0000-0001-6629-4118]; Sifrim, Alejandro [0000-0001-9308-5741]; McRae, Jeremy [0000-0001-8247-4020]; Kini, Usha [0000-0003-2652-3355]; FitzPatrick, David R [0000-0001-7609-3504]
    PURPOSE: To characterize features associated with de novo mutations affecting SATB2 function in individuals ascertained on the basis of intellectual disability. METHODS: Twenty previously unreported individuals with 19 different SATB2 mutations (11 loss-of-function and 8 missense variants) were studied. Fibroblasts were used to measure mutant protein production. Subcellular localization and mobility of wild-type and mutant SATB2 were assessed using fluorescently tagged protein. RESULTS: Recurrent clinical features included neurodevelopmental impairment (19/19), absent/near absent speech (16/19), normal somatic growth (17/19), cleft palate (9/19), drooling (12/19), and dental anomalies (8/19). Six of eight missense variants clustered in the first CUT domain. Sibling recurrence due to gonadal mosaicism was seen in one family. A nonsense mutation in the last exon resulted in production of a truncated protein retaining all three DNA-binding domains. SATB2 nuclear mobility was mutation-dependent; p.Arg389Cys in CUT1 increased mobility and both p.Gly515Ser in CUT2 and p.Gln566Lys between CUT2 and HOX reduced mobility. The clinical features in individuals with missense variants were indistinguishable from those with loss of function. CONCLUSION: SATB2 haploinsufficiency is a common cause of syndromic intellectual disability. When mutant SATB2 protein is produced, the protein appears functionally inactive with a disrupted pattern of chromatin or matrix association.Genet Med advance online publication 02 February 2017.
  • ItemPublished versionOpen Access
    Chemically-defined albumin-free differentiation of human pluripotent stem cells to endothelial progenitor cells.
    (Elsevier BV, 2015-07) Bao, Xiaoping; Lian, Xiaojun; Dunn, Kaitlin K; Shi, Mengxuan; Han, Tianxiao; Qian, Tongcheng; Bhute, Vijesh J; Canfield, Scott G; Palecek, Sean P; Bhute, Vijesh [0000-0001-9441-7963]
    Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors are important for vascular research and therapeutic revascularization. Here, we report a completely defined endothelial progenitor differentiation platform that uses a minimalistic medium consisting of Dulbecco's modified eagle medium and ascorbic acid, lacking of albumin and growth factors. Following hPSC treatment with a GSK-3β inhibitor and culture in this medium, this protocol generates more than 30% multipotent CD34+ CD31+ endothelial progenitors that can be purified to >95% CD34+ cells via magnetic activated cell sorting (MACS). These CD34+ progenitors are capable of differentiating into endothelial cells in serum-free inductive media. These hPSC-derived endothelial cells express key endothelial markers including CD31, VE-cadherin, and von Willebrand factor (vWF), exhibit endothelial-specific phenotypes and functions including tube formation and acetylated low-density lipoprotein (Ac-LDL) uptake. This fully defined platform should facilitate production of proliferative, xeno-free endothelial progenitor cells for both research and clinical applications.
  • ItemAccepted versionOpen Access
    Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions.
    (Springer Science and Business Media LLC, 2016) Bao, Xiaoping; Lian, Xiaojun; Hacker, Timothy A; Schmuck, Eric G; Qian, Tongcheng; Bhute, Vijesh J; Han, Tianxiao; Shi, Mengxuan; Drowley, Lauren; Plowright, Alleyn; Wang, Qing-Dong; Goumans, Marie-Jose; Palecek, Sean P; Bhute, Vijesh [0000-0001-9441-7963]
    The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies.
  • ItemPublished versionOpen Access
    The Poly (ADP-Ribose) Polymerase Inhibitor Veliparib and Radiation Cause Significant Cell Line Dependent Metabolic Changes in Breast Cancer Cells.
    (Springer Science and Business Media LLC, 2016-11-04) Bhute, Vijesh J; Ma, Yan; Bao, Xiaoping; Palecek, Sean P; Bhute, Vijesh [0000-0001-9441-7963]
    Breast tumors are characterized into subtypes based on their surface marker expression, which affects their prognosis and treatment. Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in clinical trials, both as single agents and in combination with other chemotherapeutics, in several subtypes of breast cancer patients. Here, we used NMR-based metabolomics to probe cell line-specific effects of the PARP inhibitor Veliparib and radiation on metabolism in three breast cancer cell lines. Our data reveal several cell line-independent metabolic changes upon PARP inhibition. Pathway enrichment and topology analysis identified that nitrogen metabolism, glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism were enriched after PARP inhibition in all three breast cancer cell lines. Many metabolic changes due to radiation and PARP inhibition were cell line-dependent, highlighting the need to understand how these treatments affect cancer cell response via changes in metabolism. Finally, both PARP inhibition and radiation induced a similar metabolic responses in BRCA-mutant HCC1937 cells, but not in MCF7 and MDAMB231 cells, suggesting that radiation and PARP inhibition share similar interactions with metabolic pathways in BRCA mutant cells. Our study emphasizes the importance of differences in metabolic responses to cancer treatments in different subtypes of cancers.
  • ItemAccepted versionOpen Access
    Sucrose Nonfermenting-Related Kinase Enzyme-Mediated Rho-Associated Kinase Signaling is Responsible for Cardiac Function.
    (Ovid Technologies (Wolters Kluwer Health), 2016-12) Cossette, Stephanie M; Bhute, Vijesh J; Bao, Xiaoping; Harmann, Leanne M; Horswill, Mark A; Sinha, Indranil; Gastonguay, Adam; Pooya, Shabnam; Bordas, Michelle; Kumar, Suresh N; Mirza, Shama P; Palecek, Sean P; Strande, Jennifer L; Ramchandran, Ramani; Bhute, Vijesh [0000-0001-9441-7963]
    BACKGROUND: Cardiac metabolism is critical for the functioning of the heart, and disturbance in this homeostasis is likely to influence cardiac disorders or cardiomyopathy. Our laboratory has previously shown that SNRK (sucrose nonfermenting related kinase) enzyme, which belongs to the AMPK (adenosine monophosphate-activated kinase) family, was essential for cardiac metabolism in mammals. Snrk global homozygous knockout (KO) mice die at postnatal day 0, and conditional deletion of Snrk in cardiomyocytes (Snrk cmcKO) leads to cardiac failure and death by 8 to 10 months. METHODS AND RESULTS: We performed additional cardiac functional studies using echocardiography and identified further cardiac functional deficits in Snrk cmcKO mice. Nuclear magnetic resonance-based metabolomics analysis identified key metabolic pathway deficits in SNRK knockdown cardiomyocytes in vitro. Specifically, metabolites involved in lipid metabolism and oxidative phosphorylation are altered, and perturbations in these pathways can result in cardiac function deficits and heart failure. A phosphopeptide-based proteomic screen identified ROCK (Rho-associated kinase) as a putative substrate for SNRK, and mass spec-based fragment analysis confirmed key amino acid residues on ROCK that are phosphorylated by SNRK. Western blot analysis on heart lysates from Snrk cmcKO adult mice and SNRK knockdown cardiomyocytes showed increased ROCK activity. In addition, in vivo inhibition of ROCK partially rescued the in vivo Snrk cmcKO cardiac function deficits. CONCLUSIONS: Collectively, our data suggest that SNRK in cardiomyocytes is responsible for maintaining cardiac metabolic homeostasis, which is mediated in part by ROCK, and alteration of this homeostasis influences cardiac function in the adult heart.
  • ItemPublished versionOpen Access
    Metabolomics Identifies Metabolic Markers of Maturation in Human Pluripotent Stem Cell-Derived Cardiomyocytes.
    (Ivyspring International Publisher, 2017) Bhute, Vijesh J; Bao, Xiaoping; Dunn, Kaitlin K; Knutson, Kylie R; McCurry, Eric C; Jin, Gyuhyung; Lee, Wei-Hua; Lewis, Sarah; Ikeda, Akihiro; Palecek, Sean P; Bhute, Vijesh [0000-0001-9441-7963]
    Cardiovascular disease is a leading cause of death worldwide. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) hold immense clinical potential and recent studies have enabled generation of virtually pure hPSC-CMs with high efficiency in chemically defined and xeno-free conditions. Despite these advances, hPSC-CMs exhibit an immature phenotype and are arrhythmogenic in vivo, necessitating development of strategies to mature these cells. hPSC-CMs undergo significant metabolic alterations during differentiation and maturation. A detailed analysis of the metabolic changes accompanying maturation of hPSC-CMs may prove useful in identifying new strategies to expedite hPSC-CM maturation and also may provide biomarkers for testing or validating hPSC-CM maturation. In this study we identified global metabolic changes which take place during long-term culture and maturation of hPSC-CMs derived from three different hPSC lines. We have identified several metabolic pathways, including phospholipid metabolism and pantothenate and Coenzyme A metabolism, which showed significant enrichment upon maturation in addition to fatty acid oxidation and metabolism. We also identified increases in glycerophosphocholine and the glycerophosphocholine:phosphocholine ratio as potential metabolic biomarkers of maturation. These biomarkers were also affected in a similar manner during murine heart development in vivo. These results support that hPSC-CM maturation is associated with extensive metabolic changes in metabolic network utilization and understanding the roles of these metabolic changes has the potential to develop novel approaches to monitor and expedite hPSC-CM maturation.
  • ItemPublished versionOpen Access
    Human pluripotent stem cell-derived epicardial progenitors can differentiate to endocardial-like endothelial cells.
    (Wiley, 2017-06) Bao, Xiaoping; Bhute, Vijesh J; Han, Tianxiao; Qian, Tongcheng; Lian, Xiaojun; Palecek, Sean P; Bhute, Vijesh [0000-0001-9441-7963]
    During heart development, epicardial progenitors contribute various cardiac lineages including smooth muscle cells, cardiac fibroblasts, and endothelial cells. However, their specific contribution to the human endothelium has not yet been resolved, at least in part due to the inability to expand and maintain human primary or pluripotent stem cell (hPSC)-derived epicardial cells. Here we first generated CDH5-2A-eGFP knock-in hPSC lines and differentiated them into self-renewing WT1+ epicardial cells, which gave rise to endothelial cells upon VEGF treatment in vitro. In addition, we found that the percentage of endothelial cells correlated with WT1 expression in a WT1-2A-eGFP reporter line. The resulting endothelial cells displayed many endocardium-like endothelial cell properties, including high expression levels of endocardial-specific markers, nutrient transporters and well-organized tight junctions. These findings suggest that human epicardial progenitors may have the capacity to form endocardial endothelium during development and have implications for heart regeneration and cardiac tissue engineering.
  • ItemPublished versionOpen Access
    ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps.
    (International Union of Crystallography (IUCr), 2018-06-01) Croll, Tristan Ian; Croll, Tristan [0000-0002-3514-8377]
    This paper introduces ISOLDE, a new software package designed to provide an intuitive environment for high-fidelity interactive remodelling/refinement of macromolecular models into electron-density maps. ISOLDE combines interactive molecular-dynamics flexible fitting with modern molecular-graphics visualization and established structural biology libraries to provide an immersive interface wherein the model constantly acts to maintain physically realistic conformations as the user interacts with it by directly tugging atoms with a mouse or haptic interface or applying/removing restraints. In addition, common validation tasks are accelerated and visualized in real time. Using the recently described 3.8 Å resolution cryo-EM structure of the eukaryotic minichromosome maintenance (MCM) helicase complex as a case study, it is demonstrated how ISOLDE can be used alongside other modern refinement tools to avoid common pitfalls of low-resolution modelling and improve the quality of the final model. A detailed analysis of changes between the initial and final model provides a somewhat sobering insight into the dangers of relying on a small number of validation metrics to judge the quality of a low-resolution model.
  • ItemPublished versionOpen Access
    Loss of function NFKB1 variants are the most common monogenic cause of CVID in Europeans.
    (Elsevier, 2018-10) Tuijnenburg, Paul; Lango Allen, Hana; Burns, Siobhan O; Greene, Daniel; Jansen, Machiel H; Staples, Emily; Stephens, Jonathan; Carss, Keren J; Biasci, Daniele; Baxendale, Helen; Thomas, Moira; Chandra, Anita; Kiani-Alikhan, Sorena; Longhurst, Hilary J; Seneviratne, Suranjith L; Oksenhendler, Eric; Simeoni, Ilenia; de Bree, Godelieve J; Tool, Anton TJ; van Leeuwen, Ester MM; Ebberink, Eduard HTM; Meijer, Alexander B; Tuna, Salih; Whitehorn, Deborah; Brown, Matthew; Turro, Ernest; Thrasher, Adrian J; Smith, Kenneth GC; Thaventhiran, James E; Kuijpers, Taco W; NIHR-BioResource – Rare Diseases Consortium; Lango Allen, Hana [0000-0002-7803-8688]; Biasci, Daniele [0000-0003-3148-8152]; Thaventhiran, James [0000-0001-8616-074X]
    BACKGROUND: The genetic etiology of primary immunodeficiency disease (PID) carries prognostic information. OBJECTIVE: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR-BioResource - Rare Disease cohort. METHODS: In the predominantly European study population of principally sporadic unrelated PID cases (n=846), a novel Bayesian method identified NFKB1 as one most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n=390) in the cohort. Amino-acid substitutions predicted to be pathogenic were assessed by analysis of structural protein data. Immunophenotyping, immunoblotting and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype co-segregation analyses. RESULTS: Both sporadic and familial cases demonstrated evidence of the non-infective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%) and autoimmune disease (48%), features prior studies correlate with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B lymphocyte differentiation. Detailed assessment of B lymphocyte numbers, phenotype and function identifies the presence of a raised CD21lowB cell population: combined with identification of the disease-causing variant, this distinguishes between healthy individuals, asymptomatic carriers and clinically affected cases. CONCLUSION: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID that results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.
  • ItemPublished versionOpen Access
    Prevalence and clinical challenges among adults with primary immunodeficiency and recombination-activating gene deficiency.
    (Elsevier BV, 2018-06) Lawless, Dylan; Geier, Christoph B; Farmer, Jocelyn R; Lango Allen, Hana; Thwaites, Daniel; Atschekzei, Faranaz; Brown, Matthew; Buchbinder, David; Burns, Siobhan O; Butte, Manish J; Csomos, Krisztian; Deevi, Sri VV; Egner, William; Ehl, Stephan; Eibl, Martha M; Fadugba, Olajumoke; Foldvari, Zsofia; Green, Deanna M; Henrickson, Sarah E; Holland, Steven M; John, Tami; Klemann, Christian; Kuijpers, Taco W; Moreira, Fernando; Piller, Alexander; Rayner-Matthews, Paula; Romberg, Neil D; Sargur, Ravishankar; Schmidt, Reinhold E; Schröder, Claudia; Schuetz, Catharina; Sharapova, Svetlana O; Smith, Ken GC; Sogkas, Georgios; Speckmann, Carsten; Stirrups, Kathleen; Thrasher, Adrian J; Wolf, Hermann M; Notarangelo, Luigi D; Anwar, Rashida; Boyes, Joan; Ujhazi, Boglarka; NIHR BioResource–Rare Diseases Consortium; Thaventhiran, James; Walter, Jolan E; Savic, Sinisa; Lango Allen, Hana [0000-0002-7803-8688]; Smith, Kenneth [0000-0003-3829-4326]; Johnson, Kathleen [0000-0002-6823-3252]; Thaventhiran, James [0000-0001-8616-074X]
  • ItemPublished versionOpen Access
    An Optical Technique for Mapping Microviscosity Dynamics in Cellular Organelles
    (American Chemical Society (ACS), 2018-05-22) Chambers, Joseph; kubankova, Marketa; Huber, Roland; Lopez-Duarte, Ismael; Avezov, Edward; Bond, Peter; Marciniak, SJ; Kuimova, Marina; Chambers, Joseph [0000-0003-4675-0053]; Avezov, Edward [0000-0002-2894-0585]; Marciniak, Stefan [0000-0001-8472-7183]
    Microscopic viscosity (microviscosity) is a key determinant of diffusion in the cell and defines the rate of biological processes occurring at the nanoscale, including enzyme-driven metabolism and protein folding. Here we establish a Rotor-based Organelle Viscosity Imaging (ROVI) methodology that enables real-time quantitative mapping of cell microviscosity. This approach uses environment sensitive dyes termed molecular rotors, covalently linked to genetically encoded probes to provide compartment specific microviscosity measurements via fluorescence lifetime imaging (FLIM). ROVI visualised spatial and temporal dynamics of microviscosity with sub-organellar resolution, reporting on a microviscosity difference of nearly an order of magnitude between subcellular compartments. In the mitochondrial matrix, ROVI revealed several striking findings: a broad heterogeneity of microviscosity amongst individual mitochondria, unparalleled resilience to osmotic stress, and real-time changes in microviscosity during mitochondrial depolarisation. These findings demonstrate the use of ROVI to explore the biophysical mechanisms underlying cell biological processes.
  • ItemPublished versionOpen Access
    Structural brain abnormalities in a single gene disorder associated with epilepsy, language impairment and intellectual disability.
    (Elsevier BV, 2016) Bathelt, Joe; Astle, Duncan; Barnes, Jessica; Raymond, F Lucy; Baker, Kate; Astle, Duncan [0000-0002-7042-5392]; Raymond, Lucy [0000-0003-2652-3355]; Baker, Kate [0000-0003-2986-0584]
    Childhood speech and language deficits are highly prevalent and are a common feature of neurodevelopmental disorders. However, it is difficult to investigate the underlying causal pathways because many diagnostic groups have a heterogeneous aetiology. Studying disorders with a shared genetic cause and shared cognitive deficits can provide crucial insight into the cellular mechanisms and neural systems that give rise to those impairments. The current study investigated structural brain differences of individuals with mutations in ZDHHC9, which is associated with a specific neurodevelopmental phenotype including prominent speech and language impairments and intellectual disability. We used multiple structural neuroimaging methods to characterise neuroanatomy in this group, and observed bilateral reductions in cortical thickness in areas surrounding the temporo-parietal junction, parietal lobule, and inferior frontal lobe, and decreased microstructural integrity of cortical, subcortical-cortical, and interhemispheric white matter projections. These findings are compared to reports for other genetic groups and genetically heterogeneous disorders with a similar presentation. Overlap in the neuroanatomical phenotype suggests a common pathway that particularly affects the development of temporo-parietal and inferior frontal areas, and their connections.
  • ItemPublished versionOpen Access
    Patients with Griscelli syndrome and normal pigmentation identify RAB27A mutations that selectively disrupt MUNC13-4 binding.
    (Elsevier BV, 2015-05) Cetica, Valentina; Hackmann, Yvonne; Grieve, Samantha; Sieni, Elena; Ciambotti, Benedetta; Coniglio, Maria Luisa; Pende, Daniela; Gilmour, Kimberly; Romagnoli, Paolo; Griffiths, Gillian M; Aricò, Maurizio; Griffiths, Gillian [0000-0003-0434-5842]
    BACKGROUND: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare and often fatal disorder characterized by defective cellular cytotoxicity and hyperinflammation, and the only cure known to date is hematopoietic stem cell transplantation. Mutations in RAB27A, LYST, and AP3B1 give rise to FHL associated with oculocutaneous albinism, and patients with FHL are usually only screened for mutations in these genes when albinism is observed. A number of patients with FHL and normal pigmentation remain without a genetic diagnosis. OBJECTIVE: We asked whether patients with FHL with immunodeficiency but with normal pigmentation might sometimes have mutations that affected cellular cytotoxicity without affecting pigmentation. METHODS: We carried out mutation analysis of RAB27A, LYST, and AP3B1 in patients with FHL with pigment dilution, as well as a cohort with no clinical evidence of pigment dilution but no mutations in the other known FHL-related genes (PRF1, STXBP2, and UNC13D). RESULTS: We identify patients with Griscelli syndrome type 2 with biallelic mutations in RAB27A in the absence of albinism. All 6 patients carried mutations at amino acids R141, Y159, or S163 of Rab27a that disrupt the interaction of Rab27a with Munc13-4, without impairing the interaction between melanophilin and Rab27a. CONCLUSION: These studies highlight the need for RAB27A sequencing in patients with FHL with normal pigmentation and identify a critical binding site for Munc13-4 on Rab27a, revealing the molecular basis of this interaction.
  • ItemPublished versionOpen Access
    Fas Ligand localizes to intraluminal vesicles within NK cell cytolytic granules and is enriched at the immune synapse.
    (Wiley, 2018-06) Lee, Jeansun; Dieckmann, Nele MG; Edgar, James R; Griffiths, Gillian M; Siegel, Richard M; Siegel, Richard M [0000-0001-5953-9893]
    INTRODUCTION: T cell and NK cell cytotoxicity can be mediated via the perforin/granzyme system and Fas Ligand (FasL, CD178). FasL is synthesized as a type II transmembrane protein that binds its cognate receptor Fas (CD95). Membrane-bound FasL is expressed on the plasma membrane of activated lymphocytes and is the main form of FasL with cytotoxic activity, but whether FasL is delivered to the immune synapse along with granzyme and perforin-containing granules is unclear. METHODS: We stably expressed FasL-fluorescent fusion proteins into human NK cells and examined the localization of FasL relative to other intracellular markers by confocal and immunoelectron microscopy, and examined the trafficking of FasL during formation of immune synapses with HLA-deficient B cells. RESULTS: FasL co-localized with CD63 more strongly than perforin or Lamp1+ in cytolytic granules. Electron microscopy revealed that FasL is enriched on intraluminal vesicles (ILVs) adjacent to the dense-core within cytolytic granules. In NK cells forming immune synapses with HLA-deficient B cells, a portion of FasL-containing granules re-localize toward the immune synapse, while a distinct pool of FasL remains at the distal pole of the cell. CONCLUSIONS: Localization of FasL to intra-luminal vesicles within cytolytic granules facilitates FasL trafficking to immune synapses and cytotoxic function in NK cells.
  • ItemAccepted versionOpen Access
    A new quantitative 3D approach to imaging of structural joint disease
    (Nature Publishing Group) Turmezei, TD; Treece, GM; Gee, AH; Houlden, R; Poole, KES; Turmezei, Tom [0000-0003-0365-8054]; Treece, Graham [0000-0003-0047-6845]; Poole, Kenneth [0000-0003-4546-7352]
    Imaging of joints with 2D radiography has not been able to detect therapeutic success in research trials while 3D imaging, used regularly in the clinic, has not been approved for this purpose. We present a new 3D approach to this challenge called joint space mapping (JSM) that measures joint space width in 3D from standard clinical computed tomography (CT) data, demonstrating its analysis steps, technical validation, and reproducibility. Using high resolution peripheral quantitative CT as gold standard, we show a marginal over-estimation in accuracy of +0.13 mm and precision of ±0.32 mm. Inter-operator reproducibility bias was near-zero at -0.03 mm with limits of agreement ±0.29 mm and a root mean square coefficient of variation 7.5%. In a technical advance, we present results from across the hip joint in 3D with optimum validation and reproducibility metrics shown at inner joint regions. We also show JSM versatility using different imaging data sets and discuss potential applications. This 3D mapping approach provides information with greater sensitivity than reported for current radiographic methods that could result in improved patient stratification and treatment monitoring.