title: Novel molecular diagnostic system of limbal stem cell deficiency based on MUC5AC transcript detection in corneal epithelium by PCR-reverse dot blot.
abstract: OBJECTIVE
  To evaluate the sensitivity and specificity of a PCR-strip system based on reverse dot blot for detection of MUC5AC mRNA in corneal epithelium samples from patients with limbal stem cell deficiency (LSCD), and to determine the correlation with clinical diagnosis.
  
  
  METHODS
  We obtained 87 corneal impression cytology (IC) samples from 55 subjects (37 patients clinically diagnosed with LSCD and 18 control subjects). Total RNA was extracted from each IC sample and retrotranscribed to cDNA. MUC5AC mRNA transcript was amplified by a customized RT-PCR assay and detected in PCR strips based on reverse dot blot hybridization and in agarose gels. Conjunctival IC samples were used as positive controls.
  
  
  RESULTS
  Forty-four of 45 corneal IC samples obtained from patients clinically diagnosed with LSCD were positive for MUC5AC, whereas 34 of 42 corneal ICs from healthy subjects were negative for MUC5AC. Four healthy corneas were found MUC5AC positive, and four rendered inconclusive results. A correlation of 91.4% (P < 0.001) between molecular testing and clinical diagnosis was found. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR-strip system were 98%, 89%, 92%, and 97%, respectively.
  
  
  CONCLUSIONS
  Corneal epithelium MUC5AC transcript detection by reverse dot blot PCR-strip is a highly sensitive technique for LSCD diagnosis. The test results strongly correlate with clinical diagnosis of characterized LSCD cases. The PCR-strip system may be used for early detection, and for the detection of mild cases of LSCD, and constitutes an objective clinical tool for the monitoring of treatments and surgical decisions.
authors.affiliation: Bioftalmik, Derio, Vizcaya, Spain.
authors.forename: Iker
authors.initials: I
authors.lastname: García
authors.forename: Jaime
authors.initials: J
authors.lastname: Etxebarria
authors.forename: Jesús
authors.initials: J
authors.lastname: Merayo-Lloves
authors.forename: Josep
authors.initials: J
authors.lastname: Torras
authors.forename: Ana
authors.initials: A
authors.lastname: Boto-de-Los-Bueis
authors.forename: David
authors.initials: D
authors.lastname: Díaz-Valle
authors.forename: Rosalía
authors.initials: R
authors.lastname: Méndez-Fernández
authors.forename: Arantxa
authors.initials: A
authors.lastname: Acera
authors.forename: Tatiana
authors.initials: T
authors.lastname: Suárez-Cortés
mesh_terms: D000328:Adult; D003316:Corneal Diseases / Q000175:diagnosis* / Q000235:genetics; D003941:Diagnostic Techniques, Ophthalmological; D019573:Epithelium, Corneal / Q000473:pathology* / Q000502:physiology; D005260:Female; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D006801:Humans; D016850:Limbus Corneae / Q000473:pathology* / Q000502:physiology; D008297:Male; D055271:Mucin 5AC / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013234:Stem Cells / Q000473:pathology* / Q000502:physiology
chemical_list: C524678:MUC5AC protein, human; D055271:Mucin 5AC; D012333:RNA, Messenger
keywords: MUC5AC; PCR-strip; cornea; impression cytology; limbal stem cell deficiency; reverse dot blot