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. 2024 Apr 1;65(4):34.
doi: 10.1167/iovs.65.4.34.

Levels of the HtrA1 Protein in Serum and Vitreous Humor Are Independent of Genetic Risk for Age-Related Macular Degeneration at the 10q26 Locus

Brandi L Williams  1 , Moussa A Zouache  1 , Nathan A Seager  1 , Chris M Pappas  1 , Jin Liu  1 , Robert A Anstadt  1 , William C Hubbard  1 , Julie Thomas  1 , Jill L Hageman  1 , Jennifer Mohler  1 , Burt T Richards  1 , Gregory S Hageman  1
Affiliations

Levels of the HtrA1 Protein in Serum and Vitreous Humor Are Independent of Genetic Risk for Age-Related Macular Degeneration at the 10q26 Locus

Brandi L Williams et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: The purpose of this study was to determine if levels of the HtrA1 protein in serum or vitreous humor are influenced by genetic risk for age-related macular degeneration (AMD) at the 10q26 locus, age, sex, AMD status, and/or AMD disease severity, and, therefore, to determine the contribution of systemic and ocular HtrA1 to the AMD disease process.

Methods: A custom-made sandwich ELISA assay (SCTM ELISA) for detection of the HtrA1 protein was designed and compared with three commercial assays (R&D Systems, MyBiosource 1 and MyBiosource 2) using 65 serum samples. Concentrations of HtrA1 were thereafter determined in serum and vitreous samples collected from 248 individuals and 145 human donor eyes, respectively.

Results: The SCTM ELISA demonstrated high specificity, good recovery, and parallelism within its linear detection range and performed comparably to the R&D Systems assay. In contrast, we were unable to demonstrate the specificity of the two assays from MyBioSource using either recombinant or native HtrA1. Analyses of concentrations obtained using the validated SCTM assay revealed that genetic risk at the 10q26 locus, age, sex, or AMD status are not significantly associated with altered levels of the HtrA1 protein in serum or in vitreous humor (P > 0.05).

Conclusions: HtrA1 levels in serum and vitreous do not reflect the risk for AMD associated with the 10q26 locus or disease status. Localized alteration in HTRA1 expression in the retinal pigment epithelium, rather than systemic changes in HtrA1, is the most likely driver of elevated risk for developing AMD among individuals with risk variants at the 10q26 locus.

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Conflict of interest statement

Disclosure: B.L. Williams, University of Utah (P); M.A. Zouache, None; N.A. Seager, None; C.M. Pappas, University of Utah (P); J. Liu, None; R.A. Anstadt, None; W.C. Hubbard, None; J. Thomas, None; J.L. Hageman, is the spouse of G. S. Hageman; J. Mohler, None; B.T. Richards, University of Utah (P); G.S. Hageman, is a shareholder of Perceive Biotherapeutics, LLC (C, P), University of Utah (P), the Sharon Eccles Steele Center for Translational Medicine is partly funded through sponsored research agreements with Perceive Biotherapeutics, LLC

Figures

Figure 1.
Figure 1.
Linearity and parallelism of the SCTM and R&D ELISA assays using two recombinant HtrA1 standards. (A–D) Variation of HtrA1 detected as a function of HtrA1-S328A (A, C) and HtrA1-WT (B, D) standard input demonstrating linearity of the SCTM (A, B) and R&D (C, D) assays. (E, F) Parallelism of the two assays was demonstrated using serial dilutions of either recombinant HtrA1 protein in serum. (F) No significant differences in detected HtrA1 concentration were observed between the two assays within their respective linear range (LR).
Figure 2.
Figure 2.
Association between levels of HtrA1 in serum and age, sex, AMD status, AMD severity and Chr10 genotype. In (B, C, E, F) sample size, median concentration, and range between the first and last quartiles are also indicated. (A, B) No significant associations were observed between serum HtrA1 levels and (A) age or (B) sex. (C) Serum HtrA1 was significantly higher in patients with late AMD as compared to subjects with early/intermediate AMD (P = 0.03, with Bonferroni correction applied for comparison between the three independent groups no AMD, early/intermediate AMD, and late AMD). This difference was independent from genotype at the Chr10 locus. (D, E) Age (D) or AMD status (E) do not significantly influence serum HtrA1 levels when stratified by genotype group. (F) No association between Chr10 genotype and HtrA1 level in serum was detected.
Figure 3.
Figure 3.
Association between levels of HtrA1 in vitreous and age, sex, AMD status, and Chr10 genotype. In (B, C, E) sample size, median concentration and range between the first and last quartiles are also indicated. (A, B, C) No significant associations were observed between HtrA1 level in vitreous and (A) age, (B) sex, or (C) AMD status. (D) Age does not significantly alter HtrA1 levels in vitreous humour. (E) No association between Chr10 genotype and HtrA1 level in vitreous was detected.
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