Exploring the pathogenicity of THBS1 variants in glaucoma

Abstract

Purpose : We previously identified THBS1 missense alleles impacting the highly evolutionarily conserved Arginine at position 1034 (p.Arg1034) in primary congenital glaucoma (PCG), and showed abnormal accumulation of misfolded mutant THBS1 in trabecular meshwork extracellular matrix¹. Herein, we use a similar approach to investigate the pathogenicity of additional THBS1 variants in PCG, early-onset (EOG) and adult-onset glaucoma (AOG).

Methods : Using whole-exome or whole-genome sequencing, THBS1 missense alleles were identified in 7 PCG (age of diagnosis <3), 4 EOG (age of diagnosis 3 to 40) and 5 AOG cases (selected from UK Biobank data). All variants met minimal criteria for expected pathogenicity including minor allele frequency (MAF) <0.1% and suggested deleterious effects by in silico programs. The five AOG variants had at least nominal evidence for glaucoma association (p>0.05 and beta >3.0). For each variant, the force field program FoldX, and the protein structure program AlphaFold were used to predict the change in folding energy (ΔΔG) caused by the amino acid change. For selected mutants, we examined the variant impact on THBS1 accumulation in extracellular matrix (ECM) by using site-directed mutagenesis of human THBS1 expressing plasmids in transfected COS-7 cells, followed by immunofluorescence microscopy to evaluate THBS1 deposition in ECM.

Results : FoldX and AlphaFold predicted significant destabilization (ΔΔG>1.2) for 2/7 PCG variants, 2/4 EOG variants and 4/5 variants found in AOG cases, with the highest values for protein destabilization found in two PCG variants (ΔΔG=4.98, p.Gly1145Ala; ΔΔG=24.58 , p.Cys836Phe) and one AOG case (ΔΔG=12.54, p.Arg517His). In the ECM assay the two PCG variants with high ΔΔG also had the highest THBS1 deposition relative to wild type (100x). Of the remaining variants only one AOG variant (p. Pro622Gln) exhibited high THBS1 deposition (10x).

Conclusions : We showed that 8/16 THBS1 variants identified in glaucoma patients were predicted to destabilize protein structure. Of these, 3 caused high accumulation of THBS1 in extracellular matrix. We are currently creating knock-in mice to further test these variants for evidence of glaucoma. These results also demonstrate the value of in silico and in vitro tests to prioritize potential disease-causing variants for further functional studies.

Reference:
1. Fu et al. J Clin Invest. 2022 132(23):e156967.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.