Thrombospondin 1 missense alleles induce extracellular matrix protein aggregation and TM dysfunction in congenital glaucoma
Haojie Fu, … , Janey L. Wiggs, Robert J. D’Amato
Haojie Fu, … , Janey L. Wiggs, Robert J. D’Amato
Published December 1, 2022
Citation Information: J Clin Invest. 2022;132(23):e156967. https://doi.org/10.1172/JCI156967.
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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. Thbs1R1034C-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.

Authors

Haojie Fu, Owen M. Siggs, Lachlan S.W. Knight, Sandra E. Staffieri, Jonathan B. Ruddle, Amy E. Birsner, Edward Ryan Collantes, Jamie E. Craig, Janey L. Wiggs, Robert J. D’Amato

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Figure 3

Thbs1R1034C-mutant mice exhibit elevated IOP, increased outflow resistance, and RGC loss.

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Thbs1R1034C-mutant mice exhibit elevated IOP, increased outflow resista...
(A) IOP measurements in Thbs1R1034C homozygous and heterozygous mutant mice. Compared with age-matched WT controls, both homozygous (P = 0.0016) and heterozygous (P = 0.007) mutant mice showed elevated IOP. Heterozygous, n = 14; homozygous, n = 18; controls, n = 24. Data represent the mean ± SEM. P values were determined by repeated-measures 1-way ANOVA followed by Bonferroni’s correction. (B) In vivo measurement of aqueous humor outflow facility (reciprocal of outflow resistance) in 4-month-old mice. Thbs1R1034C-mutant mice showed a significant reduction of outflow facility compared with WT controls. Heterozygous, n = 9; heterozygous controls, n = 9; homozygous, n = 9; homozygous controls, n = 9. ***P ≤ 0.01, by 2-tailed Student’s t test followed by Bonferroni’s correction. The box boundaries extend from the 25th to the 75th percentiles, the line within the boxes are the mean values, and the whiskers indicate the minimum and maximum values. (C) RGC density was determined by BRN3A staining on retinal whole mounts. RGC counts from 6 central and 12 peripheral AOI (AOI = 0.04 mm2) were averaged for each eye. Scale bar: 1.0 mm. (D) Representative AOI images from C. Scale bar: 50 μm; insets, ×0.32. (E) Compared with WT mice, there was a significant reduction of RGC density in both the central and peripheral retinas of homozygous (15.9% and 22.2%, respectively, n = 8) and heterozygous (5.3% and 10.1%, respectively, n = 8) animals. Data represent the mean ± SEM. **P = 0.007 and ****P ≤ 0.0001, by 2-way ANOVA.