Proliferative Sickle Cell Retinopathy in SS and SC Hemoglobinopathies: Identification of New Candidate Genes
Authors: da Silva Costa Sueli Matilde , Ito Mirta Tomie , Souza Bruno Batista , Cruz Pedro Rodrigues Souza , Bapista Letícia de Carvalho , Rios Vinicius Mandolesi , Viturino Marina Gonçalves Monteiro , Pietro Stephanie Ospina , Ozelo Margareth C , Costa Fernando F. , Melo Monica Barbosa 
Published: October 2019

Abstract
Retinopathy is one of the major clinical manifestations of sickle cell disease. Its clinical aspects vary depending on the presence or absence of vaso-proliferation, classifying this complication as non-proliferative and proliferative sickle cell retinopathy (PSCR). Endothelial cell activation, injury and dysfunction are importantly involved with pathophysiological mechanisms of sickle cell disease and its complications such as vascular disease. In this study we compared the gene expression profile of endothelial progenitor cells from patients with sickle cell disease and proliferative retinopathy versus without retinopathy. Endothelial Colony-Forming Cells (ECFC) were isolated and cultured from peripheral blood samples of three HbSS patients (Group I) and six HbSC patients (Group II) with proliferative retinopathy as well as from three HbSS patients (Group III) and four HbSC patients (Group IV) without retinopathy. Ophthalmologic evaluation: fundus biomicroscopy, retinal mapping and retinography was performed in all patients. The isolated ECFC were characterized by cobblestone morphology and flow cytometry. RNA was extracted from the cells and transcriptome analysis was performed by next-generation sequencing (RNA-seq). Differentially expressed genes were identified through DESeq2 package. Comparative transcriptome analysis between groups I and III has identified 16 differentially expressed transcripts, 10 negatively regulated and 5 positively regulated. Among these genes, we highlight the most upregulated genes, CDCP1 and NR5A2.The expression of CDCP1 (log2FoldChange = 3,712 and padj value = 0.007) and NR5A2 (log2FoldChange = 3,021 and padj value = 0.019) genes was higher in HbSS patients with PSCR than in patients without retinopathy.On the other side, comparative transcriptome analysis between groups II and IV has identified 34 differentially expressed transcripts, 24 negatively regulated and 10 positively regulated. Among these genes, we highlight the most upregulated genes, ROBO1 and SLC38A5. Importantly, the expression of ROBO1 (log2FoldChange = 4,325 and padj value = 1,35E-11)and SLC38A5 (log2FoldChange = 3,364 and padj value = 1,59E-07)genes was significantly higher in HbSC patients with PSCR than in patients without retinopathy. CDCP1 has been proposed to play a role in regulation of cell differentiation and proliferation through interactions with extracellular matrix.NR5A2 is a constitutively active orphan nuclear receptor. Some of the pathways regulated by nuclear receptors includeangiogenesis, inflammation, and lipid metabolic dysregulation, mechanisms also important in the initiation and development of several retinal diseases.The ROBO1-mediated pathway has been proposed as a target for the treatment of several ocular neovascular diseases. The SLC38A5 gene is the principal mediator of glutamine transport in retinal Müller cells. Since glutamine metabolism is crucial for vessel formation, the blockage of SLC38A5 transporter deserves further attention as an alternative strategy to the inhibition of pathological angiogenesis. Our data suggest that specific pathways can be involved in PSCR of SS and SC hemoglobinopathies. Treatment for PSCR remains palliative, so therapeutic strategies blocking these pathways may contribute to the future development of novel therapies.

Disclosures
Ozelo: BioMarin: Honoraria, Speakers Bureau; Grifols: Honoraria; Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Shire: Honoraria, Research Funding, Speakers Bureau; Bioverativ: Honoraria, Research Funding.