In vitro amyloid aggregate forming ability of TGFBI mutants that cause corneal dystrophies.

Purpose: We investigated the in vitro amyloid aggregation ability of TGFBI (transforming growth factor beta-induced) mutants causing corneal dystrophies (CDs).

Methods: Peripheral blood samples were collected from 42 unrelated Chinese CD patients and 185 healthy subjects for mutation screening in all TGFBI coding exons and flanking introns. The expression vector pCMV6_TGFBI containing wild-type, Arg-124, or Arg-555 mutations was transfected to HEK293 cells. Cell-free media was incubated with amyloid-beta (Aβ) (1-40) peptides with or without a chemical osmolyte, trimethylamine N-oxide (TMAO), for different time intervals. After ultracentrifugation, protein aggregates were analyzed by denatured gel electrophoresis. The effect of TMAO on chemical and morphological properties of Aβ aggregation was examined.

Results: TGFBI sequencing analysis showed c.Arg124Cys in all 6 lattice CD patients, c.Arg555Glu in all 11 granular CD type 1 patients, and c.Arg124His in 22 of 25 granular CD type 2 patients. Double heterozygosity (c.307-308delCT and c.Arg124His) was detected in one GCD2 patient. After transfection, cell-free media containing Arg-124 TGFBIp led to Aβ aggregation within 12 hours, whereas wild-type and Arg-555 mutant displayed aggregation after 24 hours. Western blot and Congo red binding assays showed that TMAO dose-dependently suppressed Arg-124-induced Aβ aggregation. Transmission electron microscopy showed that TMAO reduced the fibrillar aggregates caused by Aβ and c.124R > H mutated TGFBIp.

Conclusions: TGFBI sequence heterogeneity was observed in Chinese CD patients. TMAO reduced amyloid aggregation caused by Arg-124 mutants, which suggests a potential chemical-based treatment for CDs.