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Amyloid deposits with Arg124 mutated TGFBI protein have been identified in autosomal dominant blinding corneal dystrophies. We assessed in vitro the mechanisms determining TGFBI protein amyloid transformation involving mutations of Arg124. Eight peptides synthesized following the TGFBI protein sequence, centered on codon Arg124 holding the previously reported amyloidogenic mutations and the respective controls were studied. Cys124 and His124 mutated peptide preparations contained significantly higher amounts of amyloid than the native peptide. Blocking the SH group of Cys124 and deleting the first four NH2-terminal amino acids including Val112-Val113 resulted in a decrease in amyloid fibril formation while deletion of the nine CONH2-terminal residues increased amyloid fibril concentration. Fourrier transformed-infrared spectroscopy analysis of the different peptide solutions showed an increase in beta-pleated sheet structures in those with enhanced amyloid yielding. We designed a peptide (BB1) likely to counteract the role of Val112-Val113 in amyloid fibril formation. Incubation of Cys124 peptide with BB1 indeed resulted in a 35% inhibition of amyloid fibril formation. Our results are in keeping with the clinical observations of Arg124 mutation-linked amyloidosis and show the importance of Val112-Val113, disulfide and hydrogen bonding in increasing the beta-pleated conformation and amyloid formation. These findings shed new light on the molecular mechanisms of TGFBI protein amyloidogenesis and encourage further research on the use of specifically designed peptides as putative therapeutic agents for these disabling diseases.

Purpose: To assess the morphologic differences of three types of lattice corneal dystrophies (LCDs) from histologic, immunohistochemical, and ultrastructural studies., Methods: Corneas from three patients, one LCD1, one His626Arg-LCD, and one LCD3A were processed for Congo red, betaig-h3(541-564) antibodies immunostaining, and electron transmission microscopy studies. Control tissues were submitted to identical analyses and consisted of one cornea from a patient not having LCD and one skin biopsy from the patient suffering from LCD1., Results: The three corneas displayed birefringent congophilic deposits under polarized light, confirming their amyloid nature. The deposits differed regarding their shape and location in each of the corneas. A strong immunoreactivity for betaig-h3 was shown in the LCD1 and His626Arg-LCD deposits, which was faint for the LCD3A deposits. Ultrastructural analysis confirmed the dissimilarity of the deposits among the different types of LCD. No amyloid deposits were observed in the skin from the LCD1 patient, whereas immunostaining showed the presence of high amounts of betaig-h3., Conclusion: Our results show that betaig-h3 is involved in amyloid deposition in all the LCDs included in the study (LCD1, His626Arg-LCD, and LCD3A). These three forms of LCD, clinically different, were also distinguishable histologically, confirming that they belong to distinctive groups of LCDs. The absence of amyloid deposition in skin from the LCD1 patient supports cornea-specific amyloid formation. In light of the present clinical, histologic, and ultrastructural data, His626Arg and related LCDs constitute a separate group of LCD that could be considered as of intermediate type on clinical grounds.

BetaIGH3 protein has been recently involved in the pathogenesis of blinding corneal diseases, some of which have characteristic amyloid corneal deposits. The 124 codon of the betaig-h3 gene seems to be crucial for the amyloidogenicity of the protein product. We presently report an in vitro system that reproducibly forms amyloid fibrils from betaIGH3((110-131)) derived peptides. We also assessed the differences in fibril formation of two 22-amino acid peptides centered on the 124 residue: the native form and the Arg124Cys peptide (mutation linked to lattice corneal amyloid dystrophy type 1). After dialysis of Arg124Cys peptide against PBS 1/15 M pH 7.4 for 72 hours, Congo red staining and electron microscopy demonstrated the presence of abundant material fulfilling the criteria of amyloid. Quantitative analysis with thioflavine T fluorescence studies confirmed the high capacity of Arg124Cys peptide to form amyloid fibrils when compared to the native form., (Copyright 2000 Academic Press.)

Purpose: To screen the BIGH3 gene in three unrelated families with lattice corneal dystrophy (LCD), two of which disclosed a particular phenotype., Methods: Genomic DNA was extracted from peripheral leukocytes of the affected patients and their family members. The entire coding sequence of the BIGH3 gene was screened for mutations by means of transcript analysis on total RNA isolated from peripheral leukocytes by reverse transcription-polymerase chain reaction performed with primers designed for this study. Each mutation was confirmed at the genomic level, by using published primers., Results: One family that had a typical form of LCD, had the described R124C mutation in the BIGH3 gene. Two families with atypical forms of LCD were negative for the previously known mutations of the gene. Direct sequencing of the BIGH3 mRNA in the latter two families allowed us to identify two mutations located in exon 14. They consist of a 9-bp insertion at position 18851886 and one missense mutation at position 1877 of the BIGH3 gene. Three new polymorphisms were also observed., Conclusions: Two mutations different from those linked to LCD have been found in clinically distinguishable forms of this disease, intermediate between LCDs types I and IIIA. The DNA segment comprising both alterations normally encodes for a highly conserved region of the fourth internal domain of the Betaig-h3 protein, suggesting that this region may be of functional and/or structural importance. The identification of new mutations by screening of the complete BIGH3 gene and the comparative analysis of the induced modifications in betaig-h3 protein should shed light in the understanding of the molecular mechanisms underlying LCDs resulting from mutations in the BIGH3 gene, and may help to explain their phenotypic heterogeneity.