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Bruch's Membrane Abnormalities in PRDM5-Related Brittle Cornea

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Bruch's Membrane Abnormalities in PRDM5-Related Brittle Cornea Porter et al. Orphanet Journal of Rare Diseases (2015) 10:145 DOI 10.1186/s13023-015-0360-4 RESEARCH Open Access Bruch’s membrane abnormalities in PRDM5-related brittle cornea syndrome Louise F. Porter1,2,3, Roberto Gallego-Pinazo4, Catherine L. Keeling5, Martyna Kamieniorz5, Nicoletta Zoppi6, Marina Colombi6, Cecilia Giunta7, Richard Bonshek2,8, Forbes D. Manson1 and Graeme C. Black1,9* Abstract Background: Brittle cornea syndrome (BCS) is a rare, generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Recessive mutations in transcription factors ZNF469 and PRDM5 cause BCS. Both transcription factors are suggested to act on a common pathway regulating extracellular matrix genes, particularly fibrillar collagens. We identified bilateral myopic choroidal neovascularization as the presenting feature of BCS in a 26-year-old-woman carrying a novel PRDM5 mutation (p.Glu134*). We performed immunohistochemistry of anterior and posterior segment ocular tissues, as expression of PRDM5 in the eye has not been described, or the effects of PRDM5-associated disease on the retina, particularly the extracellular matrix composition of Bruch’smembrane. Methods: Immunohistochemistry using antibodies against PRDM5, collagens type I, III, and IV was performed on the eyes of two unaffected controls and two patients (both with Δ9-14 PRDM5). Expression of collagens, integrins, tenascin and fibronectin in skin fibroblasts of a BCS patient with a novel p.Glu134* PRDM5 mutation was assessed using immunofluorescence. Results: PRDM5 is expressed in the corneal epithelium and retina. We observe reduced expression of major components of Bruch’s membrane in the eyes of two BCS patients with a PRDM5 Δ9-14 mutation. Immunofluorescence performed on skin fibroblasts from a patient with p.Glu134* confirms the generalized nature of extracellular matrix abnormalities in BCS. Conclusions: PDRM5-related disease is known to affect the cornea, skin and joints. Here we demonstrate, to the best of our knowledge for the first time, that PRDM5 localizes not only in the human cornea, but is also widely expressed in the retina. Our findings suggest that ECM abnormalities in PRDM5-associated disease are more widespread than previously reported. Keywords: Brittle cornea syndrome, ZNF469, PRDM5, Corneal rupture, Bruch’s membrane, Choroidal neovascularization Background rupture, leading to irreversible blindness [1, 2]. Other ocu- Brittlecorneasyndrome(BCS)isanautosomalrecessive lar features include blue sclerae, keratoconus, keratoglobus connective tissue disorder predominantly affecting the cor- and high myopia. Extra-ocular manifestations include deaf- nea, skin and joints [1–4]. Extreme corneal thinning (220– ness, joint hypermobility, skin hyperelasticity, arachnodac- 450 μm) (normal range 520–560 μm) is the hallmark of the tyly, and developmental dysplasia of the hip [2]. BCS has condition and affected individuals are at high risk of corneal been described in patients in the absence of extra-ocular features [2] and diagnosis prior to ocular rupture is possible * Correspondence: in the presence of a high index of clinical suspicion. [email protected] 1 BCS results from mutations in one of two genes; Institute of Human Development, Centre for Genomic Medicine, Faculty of ZNF469, Medical and Human Sciences, University of Manchester, Manchester encoding zinc finger protein 469 (BCS type 1 Academic Health Science Centre (MAHSC), Saint Mary’s Hospital, Oxford [MIM 229200]) [3] and PRDM5, encoding PR domain- Road, Manchester M13 9WL, UK containing protein 5 (BCS type 2 [MIM 614161]) [4]. 9Centre for Genomic Medicine, Central Manchester University Hospitals, NHS Foundation Trust, MAHSC, Saint Mary’s Hospital, Oxford Road, Manchester Both ZNF469 and PRDM5 proteins are suggested to act M13 9WL, UK on a common pathway regulating extracellular matrix Full list of author information is available at the end of the article © 2016 Porter et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Porter et al. Orphanet Journal of Rare Diseases (2015) 10:145 Page 2 of 9 (ECM) gene expression [2–5]. A previous study using measurement of corneal thickness using pachymetry, reti- chromatin immunoprecipitation (ChIP) - sequencing has noscopy, color photography, fluorescein angiography, and shown a direct role for PRDM5 in the regulation of col- optical coherence tomography (OCT). A systemic workup lagen genes [6]. including full blood count, coagulation screen and renal A role for PRDM5 in bone development [6] and cor- function analyses was performed. neal development and maintenance [4] has been sug- gested. However, the exact localization of the protein in Clinical samples the human eye has not been described. We performed BCS-affected ocular tissue was obtained from the Depart- immunohistochemistry (IHC) in human eyes and found ment of Histopathology, Manchester Royal Infirmary. Hu- that PRDM5 localizes both to the corneal epithelium man ocular tissue samples from control individuals were and the retina. Aiming to gain insights into a role for obtained from the Manchester Eye Bank (Table 1). In- this protein in the retina, we examined the deposition of formed written consent and ethics committee approval ECM proteins in the retinas of BCS patients with a was granted (14/NW/1495). PRDM5 Δ9–14 mutation using IHC, and found ECM abnormalities within Bruch’s membrane. We also report abnormal expression of ECM components in fibroblasts Genetic analysis ZNF469 PRDM5 from a BCS patient with high axial myopia and choroidal The open reading frames of and were se- PRDM5 neovascularization carrying a novel p.Glu134* mutation quenced as described [3, 4]. Variants identified in in PRDM5, a patient who had not sustained a corneal were checked against control data sets including dbSNP rupture. These data suggest that ECM abnormalities in (Build 137) (http://www.ncbi.nlm.nih.gov/SNP), the 1000 PRDM5-related disease are more widespread than previ- Genomes Project (May 2012 release) (http://browser. ously reported, and suggest a role for PRDM5 in the 1000genomes.org/index.html), and the NHLBI Exome Se- retina. quencing Project (http://evs.gs.washington.edu/EVS). Methods Immunoblotting Participants and clinical evaluation Fibroblast cell lysis and preparation of nuclear extracts Informed written consent was obtained and investiga- was performed according to Schnitzler GR [7]. Total tions conducted in accordance with the principles of the protein content was quantified using a BioRad protein Declaration of Helsinki, with Local Ethics Committee quantification BCA assay (BioRad Laboratories). Skin fi- approval (NHS Research Ethics Committee reference broblasts nuclear extracts were subjected to standard 06/Q1406/52). Patients with BCS P1 and P2, with SDS-PAGE using a custom-made antibody PRDM5 Ab2 PRDM5 Δ9–14; and P3, with PRDM5 p.Arg590* have [6, 8] at a concentration of 1 μg/ml, and GAPDH at a been previously described [4]. Diagnosis of BCS in P4, concentration of 2 μg/ml (Santa Cruz sc-47724) on with PRDM5 p.Glu134*, was based on clinical examin- equal amounts of nuclear fraction protein. Membranes ation and confirmed by mutation analysis of ZNF469 were blocked with TBST (0.1 % Tween 20) containing and PRDM5. Detailed ophthalmic examinations included 5 % non-fat dry milk, and incubated with primary anti- anterior segment examination by slit lamp biomicroscopy, bodies overnight. Visualization was performed with an Table 1 Clinical samples used for study Clinical samples Age Sex Pathology PRDM5 Functional consequence of mutation Application Reference mutation IHC – human eye WB – skin fibroblasts IF – skin fibroblasts P1 10 M BCS Δ 9–14 exons Shortened, internally deleted protein product IHC/WB [4] P2 21 F BCS Δ 9–14 exons Shortened, internally deleted protein product IHC/Q-PCR/WB [4] P3 8 M BCS Arg590* Truncated protein product Q-PCR/WB [4] P4 26 F BCS p.Glu134* Presumed null Case report/IF Control post-mortem eye #1 70 F None Wild-type IHC Control post-mortem eye #2 58 M None Wild-type IHC Control skin fibroblasts #1 12 M None Wild-type IF/WB [4] Control skin fibroblasts #2 19 F None Wild-type IF [4] IHC Immunohistochemistry, WB Western blotting, IF Immunofluorescence Porter et al. Orphanet Journal of Rare Diseases (2015) 10:145 Page 3 of 9 enhanced chemiluminescence western blotting kit (Cell [9, 10]. For analysis of integrins, cells were fixed in 3 % Signalling Technologies #7003). paraformaldehyde and 60 mM sucrose, and perme- abilized in 0.5 % Triton X-100. Cells were reacted for Histology and immunohistochemistry 1 h at room temperature with 1 μg/ml anti-α5β1and Histological analysis was carried out in accordance with anti-α2β1 integrin monoclonal antibodies. Cells were standard diagnostic protocols. 4 μm paraffin-embedded subsequently incubated with 10 μg/ml FITC- or TRITC- slides were stained with hematoxylin and eosin and elas- conjugated secondary antibodies. IF signals were acquired tin with van
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