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

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 5

Thbs1R1034C-mutant mice form aberrant ECM deposition in TM.

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Thbs1R1034C-mutant mice form aberrant ECM deposition in TM. (A) Abnorma...
(A) Abnormal ECM deposition was evident in 2-month-old Thbs1R1034C-mutant mice (homozygous and heterozygous) compared with WT controls, primarily in the JCT adjacent to SC. In mutant mice, a dense fibrogranular material was present in the ECM but was not evident in WT mice, which exhibited organized collagen fibers. Scale bar: 1 μm; enlarged insets, ×5. (B) Gold particle labeling was combined with electron microscopy to localize THBS1 protein in TM at the ultrastructural level. Gold particles labeled aggregated THBS1R1034C deposits (small black dots). Scale bars: 0.125 μm. (C) Loss of cellularity was evident in the JCT cells of mutant mice, presumably due to the aggregation of THBS1R1034C. Scale bar: 1 μm. GV, giant vacuole.