Abstract
Purpose :
Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. ARMS2/HTRA1 haplotype is well known as the major risk factor for AMD (De Wan et al., Science 2006). But, the molecular mechanism, which derived the disease onset by the ARMS2/HTRA1 haplotype is still unclear. Our previous studies suggest that the insertion/deletion (indel) sequence located 3.8 kb upstream of HTRA1 gene is functionally association with the disease onset. Approximately 2-3-fold increase of promoter activity was observed for indel HTRA1 promoter compared to control sequence (Iejima et al., JBC 2015). Furthermore, we created transgenic mice ubiquitously overexpressing mouse HtrA1 using the chicken actin promoter (CAG) leading to choroidal neovascularization (CNV) similar to wet AMD patients (Nakayama et al., IOVS 2014). These experimental results suggest that the human HTRA1 expression is enhanced by indel haplotype in the enhancer region of HTRA1 gene, and this enhanced HTRA1 may be related to the induction of retinal neovascularization.
Methods :
To elucidate the HTRA1 gene expression mechanism by the indel haplotype, transcriptional factors involved with this region was investigated. Double strand DNA probe was designed based for the normal and indel sequence and Electrophoresis Mobility Shift Assay (EMSA) was performed. The same probe was used to isolate binding transcription factors and to determine the peptide sequence using liquid chromatography-mass spectrometry (LC-MS/MS).
Results :
Transcriptional proteins specifically binding to the indel sequence was determined by LC-MS/MS. One of the top-hit candidate transcription factor protein is the General Transcription Factor IIi (GTF2I), which was detected by anti-GTF2I antibody against western blot of the EMSA assay. The SNPs associated with AMD is located in the linkage disequilibrium (LD) block, which expands from first exon of ARMS2 to the first exon of HTRA1 gene.
Conclusions :
Our data suggests that the haplotype in this region containing both gene is associated with the enhancement of human HTRA1 expression regulated by different transcription factors. Transcription factor GTF2I, which likely to be involved in the enhancement was isolated and peptide sequence determined by LC-MS/MS.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.