Targeted Resequencing of CDKN2BAS Locus Identifies Variants Associated with Primary Open Angle Glaucoma

Abstract

 

Purpose:

 

The NEIGHBOR consortium genome-wide association study (GWAS) of 2517 primary open-angle glaucoma (POAG) cases and 2428 controls found CDKN2BAS to be strongly associated. We sought to identify possible functional variants responsible for the association.

 

Methods:

 

We identified risk and protective haplotypes in the CDKN2BAS region from the GWAS data. We selected 43 individuals: 23 cases (12 homozygous for risk and 11 for protective haplotypes), and 20 controls (9 homozygous for risk and 11 for protective haplotypes). We Sanger sequenced all 25 exons in CDKN2A, CDKN2B, and CDKN2BAS in these 43 individuals. This variant discovery experiment identified 7 single nucleotide polymorphisms (SNPs) with significantly different allele frequencies between cases and controls. These SNPs were not present on the Illumina Human660W BeadChip used in the NEIGHBOR GWAS, so we used MACH to impute their genotypes using the 1000 genomes as a reference panel. This software uses haplotypes calculated from the reference sequences to infer the genotypes of untyped markers.

 

Results:

 

We compared imputed genotypes for these 7 SNPs with genotypes experimentally determined by TaqMan assays on 600 individuals included in the GWAS. Inferred genotypes were highly accurate: concordance rates between experimentally determined and inputed genotypes range from 96.6% to 99.3%. We next conducted an association analysis between these 7 SNPs and POAG affection status. All 7 are highly significant, are located within the CDKN2BAS non-coding RNA, and have previously been associated with coronary artery disease (CAD), type II diabetes (T2D), or other disorders.

 

Conclusions:

 

CDKN2BAS modulates expression of the cyclin dependent kinase inhibitor CDKN2B by regulating histone methylation, and these variants may affect glaucoma risk by altering this activity. These SNPs are strong candidates for investigation in the pathobiology of glaucoma.  

 

Keywords: genetics • candidate gene analysis • transcription