Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma

Abstract

Glaucoma is a highly heritable disease that is a leading cause of blindness worldwide. Here, we identified heterozygous thrombospondin 1 ( THBS1 ) missense alleles altering p.Arg1034, a highly evolutionarily conserved amino acid, in 3 unrelated and ethnically diverse families affected by congenital glaucoma, a severe form of glaucoma affecting children. Thbs1 R1034C -mutant mice had elevated intraocular pressure (IOP), reduced ocular fluid outflow, and retinal ganglion cell loss. Histology revealed an abundant, abnormal extracellular accumulation of THBS1 with abnormal morphology of juxtacanalicular trabecular meshwork (TM), an ocular tissue critical for aqueous fluid outflow. Functional characterization showed that the THBS1 missense alleles found in affected individuals destabilized the THBS1 C-terminus, causing protein misfolding and extracellular aggregation. Analysis using a range of amino acid substitutions at position R1034 showed that the extent of aggregation was correlated with the change in protein-folding free energy caused by variations in amino acid structure. Extracellular matrix (ECM) proteins, especially fibronectin, which bind to THBS1, also accumulated within THBS1 deposits. These results show that missense variants altering THBS1 p.Arg1034 can cause elevated IOP through a mechanism involving impaired TM fluid outflow in association with accumulation of aggregated THBS1 in the ECM of juxtacanalicular meshwork with altered morphology.

Transcriptomic Analysis of the Ocular Posterior Segment Completes a Cell Atlas of the Human Eye

“…The compression then leads to dysfunction and eventual death of the RGCs which is the proximate cause of glaucomatous vision loss (7,21,24). To date, at least 13 genes have been associated with early onset or congenital forms of glaucoma (21,(25)(26)(27)(28)(29). In addition, large GWAS meta-analyses have associated 127 genomic loci with primary open angle glaucoma (POAG; the most common late onset form of glaucoma), and 112 loci with IOP (30,31).…”

Thrombospondin Mutations and Patients With Primary Congenital Glaucoma in a United States Population

“…One mutation Arg1034Cys was detected in 2 PCG pedigrees, whereas a different mutation at the same THBS1 codon, Arg1034Ser, was detected in a third pedigree. 14 This arginine amino acid at position 1034 in THBS1 seems to be a hot spot for disease-causing mutations (Fig. 1).…”

“…Mutations that cause PCG have been detected in cytochrome P450 1B1 (CYP1B1) 10 and latent transforming growth factor binding protein 2 (LTPB2), 11,12 tunica interna endothelial cell kinase (TEK), 13 and most recently thrombospondin 1 (THBS1). 14 While PCG caused by CYP1B1 and LTBP1 mutations has autosomal recessive inheritance, 10,11 glaucoma caused by TEK or THBS1 mutation seems to have an autosomal dominant inheritance with low penetrance. 13,14 Mutations in THBS1 were reported in 3 PCG pedigrees from European, Indian, and Sudanese ancestry.…”

“…14 While PCG caused by CYP1B1 and LTBP1 mutations has autosomal recessive inheritance, 10,11 glaucoma caused by TEK or THBS1 mutation seems to have an autosomal dominant inheritance with low penetrance. 13,14 Mutations in THBS1 were reported in 3 PCG pedigrees from European, Indian, and Sudanese ancestry. One mutation Arg1034Cys was detected in 2 PCG pedigrees, whereas a different mutation at the same THBS1 codon, Arg1034Ser, was detected in a third pedigree.…”

“…THBS1 mutations caused abnormal accumulation of THBS1 protein in trabecular meshwork extracellular matrix, reduced facility of outflow, and elevated IOP in transgenic mice. 14 The prevalence of THBS1 mutations in patients with PCG, however, has not yet been studied. Consequently, we sought to determine the role of THBS1 in a cohort of 20 unrelated patients with PCG that were recruited for study at the University of Iowa.…”