Rapid genotyping for most common TGFBI mutations with real-time polymerase chain reaction.

Recent studies of the corneal dystrophies (CDs) have shown that most cases of granular CD, Avellino CD, and lattice CD type I are caused by mutations in the human transforming growth factor beta-induced (TGFBI) gene. The aim of this study was to develop a rapid diagnostic assay to detect mutations in the TGFBI gene. Sixty-six patients from 64 families with TGFBI-associated CD were studied. A primer probe set was designed to examine the genome from exons 4 and 12 of the TGFBI gene in order to identify mutant and wild-type alleles. A region spanning the mutations was amplified by the polymerase chain reaction (PCR) in a commercial cycler. Mutations were then identified by melting curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. Using this system, we clearly distinguished each CD genotype (homozygous and heterozygous 418G-->A, heterozygous 417C-->T, heterozygous 1710C-->T, and wild-type) of all the patients by means of the clearly distinct melting peaks at different temperatures. One thermal cycling took approximately 54 min, and all results were completely in concordance with the genotypes determined by conventional DNA sequencing. Thus, the technique is accurate and can be used for routine clinical diagnosis. We expect that our new method will help in making precise diagnoses of patients with atypical CDs and aid the revision of the clinical classification of inherited corneal diseases based on the genetic pathogenesis.