Abstract
Purpose:
We have previously found reduced levels of mRNA and protein of two antioxidant enzymes, GSTM1 and GSTM5, in AMD retinas relative to age-matched controls. Herein, we correlate serum RNA quantity to genomic copy number of both GSTM1 and GSTM5 in a study population to determine whether homozygous or heterozygous deletions (-/-, +/-) could explain the reduction of mRNA levels.
Methods:
Cheek swab DNA and serum RNA samples were collected from 50 AMD and 48 control patients. TaqMan gene copy number assays were conducted in technical quadruplicate for relative quantification of copy number variation in AMD vs. control patients. TaqMan gene expression assays were conducted in technical triplicate for relative quantification (RT-PCR).
Results:
GSTM1 and GSTM5 copy numbers between AMD vs. controls revealed no significant differences. GSTM5 copy number was two in all but one sample, and GSTM1 copy number ranged from 0-4. Homozygous deletions were not correlated with AMD status (mean copy number: AMD 1.21 vs. control 1.40, p = 0.31). A subgroup analysis of heterozygous deletions with a single GSTM1 copy number did not correlate with AMD status, (number of samples: AMD 6, control 8, p = 0.76). Relative quantification of serum GSTM1 mRNA was not significantly different between AMD vs. controls, (fold change AMD:Control, 0.91, p = 0.76). A subgroup analysis comparing homozygous presence of genomic GSTM1 did not show a significant difference in serum GSTM1 mRNA levels between AMD vs. controls, (Fold Change AMD:Control, 0.93, p = 0.80). Likewise, comparing relative quantification of serum GSTM1 mRNA to genomic copy number(s) 1-4 did not show any significant difference in AMD (r2 = 0.15) or controls (r2 = 0.12). Homozygous genomic deletions lacked any detectable GSTM1 RNA quantity. GSTM5 mRNA relative quantification did not change with AMD status, (fold change 0.97, p = 0.89).
Conclusions:
The reduced levels of both mRNA and protein of GSTM1 and GSTM5 in AMD retinas does not appear to be due to genomic deletion in this second study population. Genomic heterozygous or homozygous present GSTM1 (+/-, +/+, copy number 3-4) does not alter serum levels of the mRNA to any significance. The evidence presented suggests that the reduced amount of these antioxidants in AMD retinas may be due to another form of expression repression that is tissue specific. Diminished levels of these enzymes in AMD retinas may increase susceptibility to oxidative stress.
Keywords: 412 age-related macular degeneration •
533 gene/expression •
634 oxidation/oxidative or free radical damage