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S. Yoshida, Y. Yamaji, A. Yoshida, Y. Noda, Y. Kumano, T. Ishibashi; Rapid Genotyping System for Common TGFBI Mutations With Real–Time PCR . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5548.
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© ARVO (1962-2015); The Authors (2016-present)
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 exon 4 and 12 of the TGFBI gene to identify mutant and wild type alleles. A region spanning the mutations was amplified by PCR using the LightCycler (Roche Diagnostics). The 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) by the clearly distinct melting peaks at apparently different temperatures. It took approximately 40 min for one thermal cycling, and all results were 100% in concordance with the genotypes determined by conventional DNA sequencing.
We have succeeded in developing a rapid method to detect the most common mutations in the TGFBI gene. The technique is accurate and can be used for routine clinical diagnosis. We expect that our new method will help in making precise diagnosis of patients with atypical CDs, and help in the revision of the clinical classification of inherited corneal diseases based on genetic pathogenesis.
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