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Aarika Menees, Surendra Dasari, Ahmet Dogan, Sanjay Patel, Keith Baratz, Jason Theis, Julie Vrana, Diva Salomao; Mass Spectrometric - Based Proteomic Analysis of TGFBI Corneal Dystrophies. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4558.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the composition of corneal deposits at a proteomic level in patients with granular dystrophy type I and lattice dystrophy.
In a retrospective study, we identified archived penetrating keratoplasty specimens collected between January, 1904, and December, 2011, consisting of formalin-fixed paraffin-embedded tissue from patients with granular dystrophy type I (GD) or lattice dystrophy (LD). The protein deposits of interest, and the surrounding normal stroma, were dissected with laser micro-dissection (LMD). Resulting proteins were denatured with sonication and digested into peptides by using trypsin. The peptides were subjected to liquid chromatography-based tandem mass spectrometry (LC-MS/MS). Resulting spectra (MS/MS) were identified with MyriMatch search engine configured to search for known mutations in the TGFBI gene associated with corneal dystrophies. Independently, the MS/MS were searched with DirecTag-TagRecon software configured to look for unanticipated mutations. The peptide identifications were filtered with IDPicker software to a false discovery rate of 2%.
We analyzed 9 specimens from 7 patients; four patients were female (5 specimens), and three were male (4 specimens) with ages at the time of penetrating keratoplasty ranging from 29 to 80 years. Four patients had granular dystrophy type I (6 specimens), and 3 patients had lattice dystrophy (3 specimens), with the diagnoses confirmed by histopathology and histochemical stains. For all cases, we detected a higher concentration of TGFBI in the corneal deposits compared to the adjacent normal stroma dissected from the same specimen and compared to a normal control cornea. The lattice dystrophy cases showed amyloid deposits that were rich in apolipoprotein E and serum amyloid P-component protein in addition to TGFBI. The granular dystrophy type I specimens showed calcium binding proteins of the S100 family. We detected a known Arg124His mutation in TGFBI gene in one case of GD. We also detected four novel mutations in the TGFBI protein (Cys85Gly/Gly87Ala, Asp397Ala, His451Asp, and Ala481Cys) that have strong MS/MS evidence but need Sanger sequencing validation.
LMD-LC-MS/MS technique represents a new method to profile the protein content of corneal dystrophy deposits and to detect novel mutations. Our proteomic findings support a distinct pathogeneses for granular and lattice dystrophies.
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