Molecular Genetics and Functional Analysis Implicate CDKN2BAS1-CDKN2B Involvement in POAG Pathogenesis.

Sonika Rathi,Inderjeet Kaur,Harini V Gudiseva,Tarjani Vivek Dave,Sushma Vishwakarma,Maxwell Pistilli,Venkata R M Chavali,Joan M O’Brien,Lana Verkuil,Ian Danford

doi:10.3390/cells9091934

Abstract

The genes in the 9p21 locus (CDKN2B-AS1 & CDKN2B) are widely associated with Primary open-angle glaucoma (POAG). However, the functional importance of this locus in POAG pathogenesis is still unexplored. This study investigated the role of CDKN2BAS1-CDKN2B axis in POAG. We observed significant association of CDKN2B-AS1 SNP rs4977756 with POAG and its endophenotypic traits (vertical cup-disc ratio (p = 0.033) and central corneal thickness (p = 0.008)) by screening African American POAG cases (n = 1567) and controls (n = 1600). A luciferase reporter assay in Human embryonic kidney 293T (HEK293T) cells revealed that the region surrounding rs4977756 likely serves as a transcriptional repressor. siRNA-mediated knockdown of CDKN2B-AS1 in HEK293T cells and trabecular meshwork (TM) cells resulted in significantly increased expression of CDKN2B, which was also observed in human POAG ocular tissues. Pathway focused qRT-PCR gene expression analysis showed increased cellular senescence, TGFβ signaling and ECM deposition in TM cells after CDKN2B-AS1 suppression. In conclusion, we report that CDKN2B-AS1 may act as a regulator, and it could function by modulating the expression of CDKN2B. In addition, increase in CDKN2B levels due to CDKN2B-AS1 suppression may result in the senescence of trabecular meshwork cells leading to POAG pathogenesis.

Highlights

We observed a significant increase in the expression of CDKN2A (p = 0.011) and CDKN2B (p = 0.002) transcripts in Primary open-angle glaucoma (POAG) retina when compared to normal/control retina, further supporting their involvement in POAG pathogenesis (Figure 2a)
This study demonstrated that the CDKN2B-AS1 involved in mediating senescence, inflammation, 4
This study demonstrated that the CDKN2B-AS1 involved in mediating senescence, that the intronic region of CDKN2B-AS1 harboring the allele A at SNP rs4977756 was associated inflammation, and extracellular matrix (ECM) accumulation and may play an important role in the development of POAG

Summary

Introduction

Primary open-angle glaucoma (POAG) is the most common form of glaucoma, characterized by optic nerve (ON) degeneration and progressive loss of visual field leading to irreversible vision loss.POAG affects over 56 million people worldwide [1] and the number of patients diagnosed with POAG is expected to increase with increasing awareness and improvements to diagnostic technology [2].Cells 2020, 9, 1934; doi:10.3390/cells9091934 www.mdpi.com/journal/cellsPrevious studies have revealed a higher prevalence of POAG in African Americans compared to European Americans and other populations globally [3] and in the United States [4,5].POAG is a complex genetic disorder influenced by the interaction of multiple genes, risk factors and endophenotypic traits such as intraocular pressure (IOP), central corneal thickness (CCT), retinal fiber layer thickness (RNFL), and cup-to-disc ratio (CDR). Genome-wide association studies (GWAS) and candidate gene screening studies have identified the association of many genes including myocilin (MYOC), optineurin (OPTN), WD repeat domain 36 (WDR36), neurotrophic factor 4 (NTF4) and ankyrin repeat and SOCS box containing 10 (ASB10) with POAG and its endophenotypic traits [6,7,8,9,10,11,12] Variants in these POAG associated genes may impair the proper functioning of retinal ganglion cells (RGCs) or trabecular meshwork cells (TMs) leading to optic nerve cupping and ocular hypertension [13]

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Conclusion