Aim: To develop a diagnostic DNA chip to detect mutations in
the bigh3 gene causing the most common corneal dystrophies
(CDs).
Methods: Samples from 98 people, including patients with
bigh3-associated CDs (b-aCDs), were examined. Specific
primer and probe sets were designed to examine exons 4
and 12 of the bigh3 gene, in order to identify mutant and wildtype
alleles. Mutations were then identified by hybridisation
signals of sequence-specific probes immobilised on the slide
glass.
Results: Direct sequencing of exons 4 and 12 of the bigh3 gene
in the patients’ genome showed that b-aCDs could be mainly
classified into five types: homozygotic Avellino corneal
dystrophy (ACD), heterozygotic ACD, heterozygotic lattice
CD I, heterozygotic Reis–Bucklers CD and heterozygotic
granular CD. Blind tests were performed by applying the
target DNA amplified from the genomic DNA isolated from the
peripheral blood of the participants onto a DNA chip. The
results obtained by DNA chip hybridisation matched well with
the direct DNA sequencing results.
Conclusions: The DNA chip developed in this study allowed
successful detection of b-aCDs with a sensitivity of 100%.
Mutational analysis of exons 4 and 12 of the bigh3 gene, which
are the mutational hot spots causing b-aCDs, can be
successfully performed with the DNA chip. Thus, this DNA
chip-based method should allow a convenient, yet highly
accurate, diagnosis of b-aCDs, and can be further applied to
diagnose other types of CDs.