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Yang Pan, Akiko Suga, Toru Noda, Inderjeet Kaur, Taraprasad Das, Subhabrata Chakrabarti, Robyn H Guymer, Margaret M DeAngelis, Paul N Baird, Takeshi Iwata; The ARMS2 insertion/deletion leads to systemic upregulation of secreted HTRA1 levels in the blood in AMD patients. Invest. Ophthalmol. Vis. Sci. 2021;62(8):253.
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AMD is a multi-factorial retinal disease, which affects millions of elderly people worldwide. We have previously demonstrated that the in/del sequence along with its downstream promoter region can significantly induce HTRA1 transcription in the transfected cell line 661W. Furthermore, our previous study of Htra1 transgenic mice driven by a CAG promoter showed a CNV-like phenotype after 12 months, while the use of a retinal pigment epithelial cell specific promoter by others has demonstrated polypoidal choroidal vasculopathy (PCV)-like phenotypes but not CNV. Therefore, we hypothesize that CNV is triggered by overexpression of HTRA1 in tissues outside of the eye. Here, we analyze the HTRA1 concentration in blood to correlate secretion of HTRA1 with the in/del in AMD.
Quantitative RT-PCR was performed to identify the HTRA1/Htra1 mRNA level across different human and mouse tissues, respectively. PCR and direct sequencing were performed to identify indel genotype in human samples from Japan, India, Australia, and the USA. To compare allelic or genotypic frequencies, the Chi-square test was used in additive models of each case group with controls. To identify the HTRA1 concentration in these blood samples (including serum and plasma), the ELISA was performed. The expression vector with the indel-HTRA1 was transfected into 661W, COS-7, and HEK-293 cells to observe HTRA1 secretion by ELISA. Moreover, ELISA was also performed in iPSCs derived from AMD patients.
HTRA1/Htra1 was ubiquitously expressed across different tissues in both human and mice. Overexpression of Htra1 in mice demonstrated increased Htra1 protein serum concentration. Comparison of AMD patients with controls showed a significantly increased HTRA1 concentration in blood of AMD patients. Moreover, the increased secretion of HTRA1 protein was observed in the in/del-HTRA1 transfected cells and iPSCs derived from AMD patients. Furthermore, the HTRA1 protein blood level rises progressively with age in controls but remains high in AMD patients.
The in/del genotype is associated with AMD leading to an increase of HTRA1 secretion. The HTRA1 blood level is significantly higher in AMD patients with GA or CNV than age-matched controls, strongly suggesting that AMD is triggered by systemic increases of HTRA1 reaching the retinal choroidal capillaries through the bloodstream.
This is a 2021 ARVO Annual Meeting abstract.
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