A Functional SNP in the CYP1B1 Promoter Region Is Strongly Associated With Primary Congenital Glaucoma

Abstract

Purpose: : Primary congenital glaucoma (PCG), an autosomal recessive disorder of the eye is largely ascibed to mutations in the human cytochrome P450 gene (CYP1B1) with a high degree of allelic heterogeneity. The prevalence of mutations in the coding regions of CYP1B1 and other candidate genes such as MYOC and FOXC1 among patients in India (~45%) and elsewhere does not explain their overall genetic involvement in PCG. The present study explored the role of CYP1B1 promoter in PCG.

Methods: : An extended cohort of clinically well characterized PCG cases (n=300) and ethnically matched normal controls (n=200) were studied. The CYP1B1 promoter was screened by resequencing a 486 bp upstream region containing the basal promoter with a TATA-like Box and an initiator motif and two SP1 binding sites. Promoter assay was carried out using Dual Luciferase Assay System in TM3 cell line derived from the trabecular meshwork and luciferase activity was measured using a luminometer. Possible transcription factor binding sites for the promoter variant was done using the MatInspector software. A haplotype analysis was also undertaken with the observed variants amongst cases and controls using the Haploview software.

Results: : Three PCG-associated variants were observed in the promoter, of which, a SNP within the DNA response element (DRE) and SP1 binding site was significantly associated with PCG cases harboring CYP1B1 mutations (p=5.01x10^-4). The presence of the risk allele in the haplotype was associated with significant risk amongst the cases (p=1.06x10^-7). Luciferase assay revealed a significant reduction in the promoter activity in individuals with the homozygous risk genotype suggesting that this variant could regulate CYP1B1 levels in vivo. Further bioinformatic analysis suggested two transcription factors (Nuclear Factor Y and Pbx-1) that could be possibly binding to this variant.

Conclusions: : These results underscore the potential role of the CYP1B1 promoter and certain uncharacterized gene(s), in PCG pathogenesis.

Keywords: genetics • intraocular pressure • trabecular meshwork