The Transcription Factors PBX1 and GATA1 are Regulated by the Mutation Profiles of CYP1B1 in Primary Congenital Glaucoma

Abstract

Purpose : The CYP1B1 promoter has putative binding sites for transcription factors like PBX1 and GATA1 that regulate patterning during development. Earlier, we demonstrated that the binding of these transcription factors were affected due to a functional polymorphism (rs2567206) in the CYP1B1 promoter in primary congenital glaucoma (PCG). We now aimed at understanding the involvement of PBX1 and GATA1, in the background of CYP1B1 mutation profiles in PCG.

Methods : In a large PCG cohort (n=432), CYP1B1 mutations were observed in 168 (38.90%) cases and the rest (n=264) were devoid of mutations. In order to understand the involvement of PBX1 and GATA1, their expression profiles were analyzed in PCG cases having different combinations of CYP1B1 mutations in the coding region, in presence (n=60) or absence of the promoter variant (n=60), and those that were devoid of these variations (n=60). Age, gender and ethnically matched congenital cataract cases without any signs or symptoms of glaucoma were enrolled as controls (n=60). RNA was extracted by standard kits (Qiagen) and reverse transcribed (Biorad iScript). The relative expressions of these target genes (validated in triplicates) were analyzed by Real Time PCR (ABI 7900HT) using Power SYBR green chemistry (Applied Biosystems) with GAPDH as an endogenous control. The relative expressions were quantified by the 2^-ΔΔC_T method and expressed as fold changes (F_C).

Results : The PBX1 and GATA1 had a relatively higher down regulation in PCG cases harboring CYP1B1 mutations and the promoter variant (F_C= -4.08 to -5.28) than those with either of these variations (F_C=-1.38 to -1.02). The fold changes were higher in cases that harbored homozygous mutations (F_C=-5.28 to -5.03) compared to those with heterozygous changes (F_C=-1.80 to -1.18). A closer scrutiny indicated that cases with a termination codon (nonsense, deletions, frameshifts) exhibited significantly lower fold changes in GATA1 and PBX1 (p<0.01) than those harboring missense mutations. The expression profiles of these genes in cases without any variations were similar to those of the controls (p>0.05).

Conclusions : The present data indicated that developmental genes like PBX1 and GATA1 were regulated by the mutation profiles of CYP1B1 in presence of the promoter variant in PCG. It further emphasized the role of downstream regulators of CYP1B1 in PCG pathogenesis.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.