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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 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.

The RPE65 protein appears late during the retinal development. To study the basis for this regulation, the rat RPE65 cDNA was sequenced and the mRNA subsequently quantitated at various stages by competitive RT-PCR. RPE65 mRNA was detected as early as E18 (36 copies/ng of whole eye total RNA). It gradually accumulates up to P12 (27000 copies/ng) at which point it reaches a steady state level. This increase is interrupted for 3 days (P2-P4) during which the levels of mRNA remain stable. This timing and rate of accumulation parallels that of rat and mouse opsin mRNA and suggests that common factors may control the activation of genes in photoreceptors and retinal pigment epithelium cells.