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Daisuke Iejima, Toru Noda, Atsushi Mizota, Takeshi Iwata; Characterization of HtrA1 Promoter in Patients with Exudative Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):317.
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Age-related macular degeneration (AMD) is a leading cause of vision loss and blindness in the elderly. The dry form is more common and accounts for about 85~90% of AMD patients in US, while Japanese AMD patients predominantly progress to wet-form or polypoidal choroidal vasculopathy (PCV). Recent studies have shown HtrA1, a serine protease gene, as major risk factor for wet form AMD (DeWan et al., Science, 2006). Furthermore, we reported that Japanese typical wet form AMD patients show significant association with ARMS2/HtrA1 (rs10490924: p=4.1x10-14, OR=4.16) (Goto, Akahori et al., JOBDI, 2009). The purpose of study is to characterize promoter function of ARMS2 and HtrA1 genes in wet-form AMD.
Human peripheral blood was obtained from controls (cataract patient: CAT, 228 case) and Wet-AMD (226 case) patients. Genome DNA was extracted from peripheral blood samples using Magtration System 8Lx and 10Kb of promoter region containing ARMS2 gene was sequenced using ABI 3130 Genetic analyzer. ARMS2 and HtrA1 promoter activity was measured by luciferase assay (Dual-Glo Luciferase assay system, Promega, WI). Double strand DNA probe was designed based on the wild-type and mutant sequence and Electrophoresis Mobility Shift Assay (EMSA) (LightShift Chemiluminescent EMSA kit, Thermo Fisher Scientific, MA) 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, LCQ DECA XP plus, Thermo Fisher Scientific, MA).
The promoter sequence experiment showed that a great number of AMD patients had specific in/del mutation in 3.8 Kb upstream of HtrA1 gene. 2~3-fold increase of promoter activity was observed in in/del HtrA1 promoter compared to wild-type sequence. Furthermore, we detected in/del specific binding factors using EMSA and LC-MS/MS. These results suggest that Htra1 gene expression is influenced by transcription factor specifically binding to this region.
Human HtrA1 expression is enhanced by AMD specific in/del 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.
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