Comparison of two phenotypically distinct lattice corneal dystrophies caused by mutations in the transforming growth factor beta induced (TGFBI) gene

Abstract

Purpose In this study, we investigated whether the phenotypic difference observed between two lattice corneal dystrophy type 1 (LCD type 1) cases caused by either a single A546D substitution or a A546D/P551Q double substitution in TGFBIp, can be ascribed to (I) a difference in the proteomes of corneal amyloid deposits, (II) altered proteolysis of TGFBIp or (III) structural changes of TGFBIp introduced by the P551Q amino acid substitution. Experimental design Amyloid deposits were isolated from the corneas of two siblings with LCD type 1 resulting from A546D/P551Q mutations in TGFBI using laser capture microdissection and a subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteolytic processing of TGFBIp was addressed by counting peptide spectra. Lastly, to study the possible effect of the P551Q substitution, recombinant FAS1-4 domain variants were subjected to in vitro stability assays. Results The amyloid proteomes and TGFBIp processing of the two A546D/P551Q LCD type 1 cases were similar to each other as well as to the A546D amyloid proteome previously reported by us. The stability assays revealed a minor destabilization of the FAS1-4 domain upon the addition of the P551Q mutation, moreover, it resulted in different accessibility to tryptic cleavage sites between the A546D and A546D/P551Q mutant FAS1-4 domain variants. Conclusion The difference in A546D and A546D/P551Q LCD type 1 phenotypes cannot be ascribed to altered corneal amyloid composition or altered in vivo proteolytic processing of TGFBIp. Instead, a small difference in thermodynamic stability introduced by the P551Q mutation most likely causes structural changes of TGFBIp.

Variant lattice corneal dystrophy associated with compound heterozygous mutations in theTGFBIgene

“…Indirect in vitro evidence suggests that the presence of valine-valine at positions 112–113 may play an important role in amyloid fibril formation, yet the exact role of Val113 in TGFBI folding remains to be elucidated 21. Missense mutations in the fourth FAS1 domain lead to dystrophic corneal deposition by altering TGFBI structure, stability and subsequent protein processing or by affecting TGFBI turnover and fibrillation rates, causing increased aggregation 2. Specifically, a proteolytic cleavage site has been shown to be between the wild-type Arg557 and Leu558 residues in the core of the fourth FAS1 domain 22.…”

“…To the best of our knowledge, only six families have been reported with affected individuals who are heterozygous for two different TGFBI mutations (table 1). 2 10–16 23 However, in five of the six families, one of the two mutations has not been reported separately, and thus an affected phenotype has been established for both of the identified mutations in only one of the six families 14. In the family reported by Yamada et al , the dystrophic corneal deposits in individuals with both the p.(Arg124His) and p.(Asn544Ser) mutations consisted of a combination of the stellate deposits associated with the p.(Arg124His) mutation and the lattice lines associated with the p.(Asn544Ser) mutation.…”

“…To date, more than 50 different autosomal dominant coding region mutations have been identified in TGFBI , with the vast majority of the affected individuals demonstrating a single pathogenic coding region mutation 2 6. However, in populations in which TGFBI mutations are relatively common, such as Korea where 1 in every 875 individuals carries the mutation associated with GCD type II, individuals who are homozygous for the mutant allele demonstrate an earlier onset and more severe manifestation of the affected phenotype 7.…”

Insight into the Protein Composition of Immunoglobulin Light Chain Deposits of Eyelid, Orbital and Conjunctival Amyloidosis

“…Procuring of amyloid deposits was performed as previously described [30]. Shortly, 8 µm thick tissue sections of formaldehyde fixed and paraffin embedded tissue were cut and placed centrally on non-charged glass slides, followed by deparaffinization, staining with Congo red, and dehydration using standard histological procedures.…”

Early Events in the Amyloid Formation of the A546T Mutant of Transforming Growth Factor β-Induced Protein in Corneal Dystrophies Compared to the Nonfibrillating R555W and R555Q Mutants

“…15–17 The fibril core region extends from the final part of α4 to the first part of β3. The R555W and R555Q mutations are located on the surface of the protein (Figure 1B,C), while the A546T mutation is more buried inside the protein structure (Figure 1D).…”