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Daisuke Iejima, Takeshi Iwata; Function analysis of HTRA1 regulatory element in Patients with Exudative Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2271.
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Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. ARMS2/HTRA1 mutation is known to major risk factor for AMD (De Wan et al., Science 2006). But, AMD pathogenic mechanism which derived from ARMS2/HTRA1 gene mutation is still unclear. Our previous studies suggest that the promoter sequence experiment showed that a great number of AMD patients had specific insertion/deletion (indel) mutation in 3.8 kb upstream of HTRA1 gene. 2-3-fold increase of promoter activity was observed in 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, of HtrA1 in vivo was shown to lead to choroidal neovascularization (CNV), similar to wet AMD patients (Nakayama, Iejima et al., IOVS 2014). So, we think that human HTRA1 expression is enhanced by AMD specific indel mutation in the promoter region of HTRA1 gene, and this enhanced HTRA1 may be concerned with induce retinal neovasucularization. But, this indel function is unclear. So, our study aim is to elucidate HTRA1 gene expression mechanism in indel mutation.
To elucidate the indel sequence binding protein, we designed both normal and indel mutant complementary DNA probes. Double strand DNA probe was designed based on the normal and mutant 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).
We detected indel specific binding protein using LC-MS/MS, and obtained candidate protein list. Especially, we focused on top-hit candidate transcription factor protein in this protein list, and this protein is General Transcription Factor IIi (GTF2I). Furthermore, we detected indel sequence binding GTF2I for western blot analysis using anti-GTF2I antibody.
Human HtrA1 expression is enhanced by AMD specific indel mutation in the promoter region of HtrA1 gene. Specific transcription factor, which likely to be involved in this enhancement was isolated and peptide sequence determined. Furthermore, we determined indel specific transcription factor: GTF2I.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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