SCIENTIFIC REPORT

Development of a DNA chip for the diagnosis of the most common corneal dystrophies caused by mutations in the ßigh3 gene

So Young Yoo¹, Tae-Im Kim², Sang Yup Lee³, Eung Kweon Kim², Ki Chang Keum⁴, Nae Choon Yoo⁵ and Won Min Yoo⁶

¹ Department of Chemical and Biomolecular Engineering (BK21 program), Korea Advanced Institute of Science and Technology, Daejeon, Korea; Medigenes, Seoul, Korea
² Corneal Dystrophy Research Institute, Department of Ophthalmology, Project Team of Nanobiomaterials for Cell-based Implants, Severance Hospital, Yonsei University, Seoul, Korea
³ Department of Chemical and Biomolecular Engineering (BK21 program), Centre for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Korea
⁴ Department of Radiation Oncology, Yonsei University College of Medicine, Seoul, Korea
⁵ Department of Oncology and Cancer Centre, Yonsei University College of Medicine, Seoul, Korea
⁶ Department of Plastic and Reconstructive Surgery, Yongdong Severance Hospital, Seoul, Korea

Correspondence to:
Correspondence to:
Professor S Y Lee
Department of Chemical & Biochemical Engineering, Korea Advanced Institute of Science and Technology (KAIST), 373–1 Guseong-dong, Yuseong-gu,Daejeon 305–701, Korea; leesy{at}kaist.ac.kr; Professor E K Kim, Department of Ophthalmology, Yousei University College of Medicine, Seodaemoongu Shinchondong 134, C PO Box 8044, Seoul, Korea; eungkkim{at}yumc.yonsei.ac.kr

Aim: To develop a diagnostic DNA chip to detect mutations in the ßigh3 gene causing the most common corneal dystrophies (CDs).

Methods: Samples from 98 people, including patients with ßigh3-associated CDs (ß-aCDs), were examined. Specific primer and probe sets were designed to examine exons 4 and 12 of the ßigh3 gene, in order to identify mutant and wild-type 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 ßigh3 gene in the patients’ genome showed that ß-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 ß-aCDs with a sensitivity of 100%. Mutational analysis of exons 4 and 12 of the ßigh3 gene, which are the mutational hot spots causing ß-aCDs, can be successfully performed with the DNA chip. Thus, this DNA chip-based method should allow a convenient, yet highly accurate, diagnosis of ß-aCDs, and can be further applied to diagnose other types of CDs.

Abbreviations: ACD, Avellino corneal dystrophy; ß-aCD, ßigh3-associated corneal dystrophy; CD, corneal dystrophy; FITC, fluorescein isothiocyanate; GCD, granular CD; LCD I, lattice CD type I; Ori, origin of replication; RBCD, Reis–Bucklers CD; SSPE, saline/sodium phosphate/EDTA

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