- Open Access
Bruch’s membrane abnormalities in PRDM5-related brittle cornea syndrome
© Porter et al. 2016
Received: 27 March 2015
Accepted: 19 October 2015
Published: 11 November 2015
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’s membrane.
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.
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.
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.
Brittle cornea syndrome (BCS) is an autosomal recessive connective tissue disorder predominantly affecting the cornea, skin and joints [1–4]. Extreme corneal thinning (220–450 μm) (normal range 520–560 μm) is the hallmark of the condition and affected individuals are at high risk of corneal rupture, leading to irreversible blindness [1, 2]. Other ocular features include blue sclerae, keratoconus, keratoglobus and high myopia. Extra-ocular manifestations include deafness, joint hypermobility, skin hyperelasticity, arachnodactyly, and developmental dysplasia of the hip . BCS has been described in patients in the absence of extra-ocular features  and diagnosis prior to ocular rupture is possible in the presence of a high index of clinical suspicion.
BCS results from mutations in one of two genes; ZNF469, encoding zinc finger protein 469 (BCS type 1 [MIM 229200])  and PRDM5, encoding PR domain-containing protein 5 (BCS type 2 [MIM 614161]) . Both ZNF469 and PRDM5 proteins are suggested to act on a common pathway regulating extracellular matrix (ECM) gene expression [2–5]. A previous study using chromatin immunoprecipitation (ChIP) - sequencing has shown a direct role for PRDM5 in the regulation of collagen genes .
A role for PRDM5 in bone development  and corneal development and maintenance  has been suggested. However, the exact localization of the protein in the human eye has not been described. We performed immunohistochemistry (IHC) in human eyes and found that PRDM5 localizes both to the corneal epithelium and the retina. Aiming to gain insights into a role for this protein in the retina, we examined the deposition of ECM proteins in the retinas of BCS patients with a PRDM5 Δ9–14 mutation using IHC, and found ECM abnormalities within Bruch’s membrane. We also report abnormal expression of ECM components in fibroblasts from a BCS patient with high axial myopia and choroidal neovascularization carrying a novel p.Glu134* mutation in PRDM5, a patient who had not sustained a corneal rupture. These data suggest that ECM abnormalities in PRDM5-related disease are more widespread than previously reported, and suggest a role for PRDM5 in the retina.
Participants and clinical evaluation
Informed written consent was obtained and investigations conducted in accordance with the principles of the Declaration of Helsinki, with Local Ethics Committee approval (NHS Research Ethics Committee reference 06/Q1406/52). Patients with BCS P1 and P2, with PRDM5 Δ9–14; and P3, with PRDM5 p.Arg590* have been previously described . Diagnosis of BCS in P4, with PRDM5 p.Glu134*, was based on clinical examination and confirmed by mutation analysis of ZNF469 and PRDM5. Detailed ophthalmic examinations included anterior segment examination by slit lamp biomicroscopy, measurement of corneal thickness using pachymetry, retinoscopy, color photography, fluorescein angiography, and optical coherence tomography (OCT). A systemic workup including full blood count, coagulation screen and renal function analyses was performed.
Clinical samples used for study
Functional consequence of mutation
IHC – human eye
WB – skin fibroblasts
IF – skin fibroblasts
Δ 9–14 exons
Shortened, internally deleted protein product
Δ 9–14 exons
Shortened, internally deleted protein product
Truncated protein product
Control post-mortem eye #1
Control post-mortem eye #2
Control skin fibroblasts #1
Control skin fibroblasts #2
The open reading frames of ZNF469 and PRDM5 were sequenced as described [3, 4]. Variants identified in PRDM5 were checked against control data sets including dbSNP (Build 137) (http://www.ncbi.nlm.nih.gov/SNP), the 1000 Genomes Project (May 2012 release) (http://browser.1000genomes.org/index.html), and the NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS).
Fibroblast cell lysis and preparation of nuclear extracts was performed according to Schnitzler GR . Total protein content was quantified using a BioRad protein quantification BCA assay (BioRad Laboratories). Skin fibroblasts nuclear extracts were subjected to standard SDS-PAGE using a custom-made antibody PRDM5 Ab2 [6, 8] at a concentration of 1 μg/ml, and GAPDH at a concentration of 2 μg/ml (Santa Cruz sc-47724) on equal amounts of nuclear fraction protein. Membranes were blocked with TBST (0.1 % Tween 20) containing 5 % non-fat dry milk, and incubated with primary antibodies overnight. Visualization was performed with an enhanced chemiluminescence western blotting kit (Cell Signalling Technologies #7003).
Histology and immunohistochemistry
Histological analysis was carried out in accordance with standard diagnostic protocols. 4 μm paraffin-embedded slides were stained with hematoxylin and eosin and elastin with van Gieson. Immunohistochemistry was performed using PRDM5 Ab2 [6, 8] and mouse monoclonal antibodies against collagen I (ab90395, Abcam); collagen III (ab6310, Abcam) and collagen IV (ab6311, Abcam). PRDM5 AB2 epitope is situated within the region corresponding to N- terminal amino acids 60–142. Staining was performed on a Ventana Benchmark XT Automated Immunostaining Module (Ventana Medical Systems) together with the XT ultraView Universal Red Alkaline Phosphatase detection system for all antibodies except PRDM5, where DAB was used as the chromogen. Antigen retrieval was performed separately using heat-induced antigen retrieval for PRDM5, and no pre-treatment for collagens I, III and IV. Primary antibodies were diluted in Dako REAL™ Antibody Diluent (Dako, Agilent Technologies, UK) to the indicated optimal dilutions of 3.5 μg/ml for PRDM5; 3 μg/ml for collagen I; 10 μg/ml for collagen III; 2.5 μg/ml for collagen IV. Sections of patient eye tissue were processed in parallel with the control tissue and were collected, sorted and fixed in an identical manner. Tissue section slides were masked for origin and scored for detection of cells showing nuclear PRDM5 staining subjectively by an independent human observer using a binary scale (positive or negative). Tissues were considered positive when >20 % of the cells displayed nuclear PRDM5 staining .
Cell culture and immunofluorescence (IF)
Polyclonal rabbit anti-fibronectin (FN) antibody, mouse anti-tenascin monoclonal antibody (clone BC-24), recognizing all the tenascins (TNs), and TRITC- conjugated rabbit anti-goat antibody were from Sigma Aldrich; mouse anti-α5β1 (clone JBS5) and anti-α2β1 (clone BHA.2) integrin monoclonal antibodies; and goat anti-type I collagen, anti-type III collagen and anti-type V collagen antibodies were from Millipore Chemicon Int. Inc. (Billerica, MA). FITC- and TRITC-conjugated goat anti-rabbit and anti-mouse secondary antibodies were from Calbiochem-Novabiochem Int. (San Diego, CA, USA). Antibody dilutions were: anti-tenascin and anti-α5β1: 2 μg/ml; anti-α2β1: 4 μg/ml; anti-FN and anti-type I collagen, 10 μg/ml; anti-type III and type V collagen: 20 μg/ml. Primary dermal fibroblast cultures were established from skin biopsies by routine procedures, maintained and harvested as described [9, 10]. 1.0 × 105 cells were grown for 48 h on glass coverslips, fixed in methanol and incubated with the specified antibodies as reported [9, 10]. For analysis of integrins, cells were fixed in 3 % paraformaldehyde and 60 mM sucrose, and permeabilized in 0.5 % Triton X-100. Cells were reacted for 1 h at room temperature with 1 μg/ml anti-α5β1 and anti-α2β1 integrin monoclonal antibodies. Cells were subsequently incubated with 10 μg/ml FITC- or TRITC-conjugated secondary antibodies. IF signals were acquired by a CCD black/white TV camera (SensiCam-PCO Computer Optics GmbH, Germany) mounted on a Zeiss fluorescence-Axiovert microscope, and digitalized by Image Pro Plus program (Media Cybernetics, Silver Spring, MD).
Extracted total RNA was reverse-transcribed into single-stranded cDNA using a High Capacity RNA-to-cDNA Kit (Life Technologies, Paisley, UK), according to the manufacturer’s instructions. RT-PCR and data analysis was performed as previously described . The assay numbers for the mRNA endogenous control (GAPDH) and target gene were: GAPDH (Hs02758991_g1*) and ITA8 (Hs00233321_m1*) (Life Technologies). Cycles to threshold (CT) values were determined for each sample and its matched control and relative mRNA expression levels determined by the 2−ΔΔCt method, providing the fold change value . Error bars representing 95 % confidence intervals around the mean are represented for all experiments. P-values were derived using the 2-tailed T-test with significance level set at 0.01 to compare results between mutant and wild-type cells. One-way ANOVA and Dunnett’s multiple comparison posttest using mean values and standard error were also performed on fold change means in all groups assessed.
PRDM5 mutations, functional consequences, and associated phenotypes
PRDM5 is expressed in the adult human cornea and retina
Structural abnormalities in Bruch’s membrane in BCS patients
Extracellular matrix abnormalities in skin fibroblasts from BCS patient P4 with the novel PRDM5 mutation p.Glu134*
PRDM5-related disease is known to affect the cornea, skin and joints [1–5]. Here we show that PRDM5 localizes not only in the human cornea, but is also widely expressed in the retina. PRDM5 expression has been reported to be predominantly nuclear, for example in intestinal crypts where stem cells reside, with cytoplasmic expression in some tissues, including colonic villi . We show both cytoplasmic and nuclear PRDM5 expression in the retina. We show reduced expression of major collagenous components of Bruch’s membrane  (Additional file 1: Figure S2) in two patients with a deletion of exons 9–14 of PRDM5 (Fig. 4). An association between PRDM5 and altered collagen expression has been shown in previous studies [4–6]. Here we show that PRDM5 mutations lead to notable differences in the expression of ECM proteins in Bruch’s membrane that may impinge on its structural integrity.
We identified myopic choroidal neovascularization (CNV)  in a 26-year old lady with PRDM5-related BCS, suspected to have the disease due to the presence of corneal thinning and marfanoid features. High myopia is a significant risk factor for the development of both CNV and retinal detachment  and has been described in a number of patients with BCS . Patients with BCS may therefore benefit from daily monocular monitoring with an Amsler chart, to detect early metamorphopsia (visual distortion), with urgent referral to an ophthalmologist if metamorphopsia develops. CNV has been described in a number of connective tissue disorders including pseudoxanthoma elasticum , Beals-Hecht syndrome , Ehlers-Danlos syndrome , and osteogenesis imperfecta . Associations between connective tissue disease and CNV have however seldom been investigated at the histological and cellular levels. PRDM5 mutations may further contribute to weaknesses at the level of Bruch’s membrane caused by myopia, a hypothesis consistent with our immunohistochemical results, although IHC was only performed on two patients with a PRDM5 Δ9-14, which results in the production of a truncated protein product. Retinal basement membrane abnormalities have been also noted upon ultrastructural studies of retinas from patients with Alport syndrome, caused by mutations in different transcripts of the collagen IV gene .
We looked at expression of fibronectin, integrins and tenascins in dermal fibroblasts. Fibronectin is present in Bruch’s membrane (Additional file 1: Figure S2), and integrins α2β1 and α5β1 are the major integrin receptors for collagen and fibronectin, respectively. Integrin α8 is the major tenascin receptor  and homozygous mutations in tenascin X cause a subtype of Ehlers-Danlos syndrome [20, 21]. When present in dermal fibroblasts, the PRDM5 p.Glu134* mutation results in the absence of tenascin staining (Fig. 5). These findings are consistent with our previous study examining patient fibroblasts from patients with the Δ9–14 and p.Arg590* PRDM5 mutations . Here we confirm that expression of integrins α2β1, α5β1 is markedly reduced (Fig. 5). We also note reduced RNA expression levels of integrin α8 in two patients (Additional file 1: Figure S3). Integrin α8 is the major tenascin receptor . Tenascins are a family of four extracellular matrix proteins, tenascin X and C are major isoforms expressed in ocular tissues . The altered expression of collagen V in fibroblasts lacking PRDM5, together with the absence of tenascins, is reminiscent of a subtype of autosomal recessive Ehlers-Danlos syndrome characterized by tenascin X deficiency [20, 21]. Tenascin X is a large ECM glycoprotein abundantly expressed during development and in adult tissue strongly associated with ocular basement membranes (Bowman’s layer and Descemet’s membrane in particular) . Our data suggests that PRDM5 may play a role in the regulation of collagen, integrin and tenascin expression, proteins that participate in ocular basement membrane development including Bruch’s membrane [12, 22]. A role for PRDM5 as a direct activator of collagen genes has been reported with direct binding of PRDM5 in conjunction with RNA polymerase II shown in a ChIP-sequencing experiment performed on murine MC3T3 cells (6). This role is also supported by the observation of a significant downregulation of structural collagens in fibroblasts of patients with BCS2 (4). While it is also possible that the basement membrane structural abnormalities observed in Bruch’s membrane also involve the corneal endothelial and/or epithelial basement membranes, the presence of extreme corneal scarring and ECM changes linked to tissue remodelling precluded this analysis.
PDRM5-related disease is known to affect the cornea, skin and joints. Our study shows expression of PRDM5 in the human cornea and retina, and demonstrates downregulation of major structural components of Bruch’s membrane in the eyes of two patients with BCS type 2. These findings suggest that ECM abnormalities in PRDM5-associated disease are more widespread than previously reported.
The authors wish to thank Professor Anders Lund for his kind gift of anti-PRDM5 polyclonal antibody and Dr Panagiotis Sergouniotis and Professor Jan Keunen for critical review of the manuscript.
Funding and support
Louise Porter is supported by a National Institute of Health Research (NIHR) pre-doctoral fellowship (NIHR-BRF-2011-015). Professor Graeme Black and Dr Forbes Manson are partly supported by a grant from Action Medical Research (reference 1967). Catherine Keeling and Martyna Kamieniorz are funded by NIHR Comprehensive local research network (CLRN). The project was supported by NIHR Manchester Biomedical Research Centre.
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