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I. Garcia, J. Etxebarria, J. Soria, N. Gonzalez, I. Martinez, I. Rodriguez, T. Suarez-Cortes, A. Acera; Molecular Diagnosis of Limbal Deficiency by RT-PCR: Detection of Mucin MUC5AC Transcript in Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2381.
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To report a method to diagnose limbal deficiency (LD) based on the detection of MUC5AC transcript by Retrotranscription - Real-Time-Polymerase Chain Reaction (RT-PCR) in corneal epithelium obtained by impression cytology (IC).
This study was performed in 13 patients (14 eyes) clinically diagnosed with LD (all samples were collected in accordance with the Declaration of Helsinki). One 8 mm diameter circle of cellulose acetate filter paper was applied on the corneal and conjunctival epithelium surface of each patient respectively. The presence of goblet cells determined by PAS-hematoxylin stain was comparatively evaluated with the presence of the MUC5AC transcript in cornea by RT-PCR. For that purpose, total RNA purified from impression cytology samples was retrotranscripted and a specific region of the MUC5AC transcript was amplified using a custom primer design. The coupled amplification of the transcript of interest in conjunctival epithelium was used as a positive PCR control for each corneal sample. Amplification products were analyzed both assessing their Crossing Thresholds and running them on agarose-TAE gels.
Our study included 14 corneal samples coupled with their respective conjunctival samples. Amplification was detected in 10/14 corneal epithelium samples analyzed by RT-PCR, confirming the clinical diagnosis. In contrast, only 5/14 samples clinically diagnosed with limbal deficiency were confirmed by the conventional IC stain. On the other hand, 2 samples clinically diagnosed with limbal deficiency were found to be negative both by PAS-hematoxylin and by RT-PCR. Finally, there were 3 samples in which impression cytology was not available; one amplified MUC5AC transcript by RT-PCR, whereas the other two did not.
The correlation between clinical and molecular diagnosis of MUC5AC, is higher (100%) than the correlation found between the clinical and conventional impression cytology diagnosis (45%). The molecular methodology developed in this study is more sensitive (due to the amplification reaction), and more specific (due to the primer design) than the conventional PAS-hematoxylin stain. Finally, 4 of the patients clinically diagnosed with limbal deficiency were not confirmed by any of the two procedures employed in the present study. All these findings suggest that the novel methodology can constitute a valuable tool of help in clinical diagnosis.
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