HTRA1 (High Temperature Requirement A Serine Peptidase 1) Gene Is Transcriptionally Regulated by Insertion/Deletion Nucleotides Located at the 3′ End of the ARMS2 (Age-related Maculopathy Susceptibility 2) Gene in Patients with Age-related Macular Degeneration

Background: The biological function of insertion/deletion sequences associated with AMD has not been fully characterized. Results: The HTRA1 regulatory region contains an insertion/deletion sequence that is significantly up-regulated in retinal neuronal cell lines. Conclusion:HTRA1 expression is enhanced by a mutation in the insertion/deletion in the HTRA1 regulatory region. Significance: This is the characterization of the HTRA1 regulatory elements and the effect of insertion/deletion sequences associated with AMD. Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HtrA1 knock-out mice in which HtrA1 exon 1 was replaced with -galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HtrA1 and exhibits characteristics similar to those of wet AMD patients.