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Light and Electron Microscopic Studies, Gene Mutation Analysis of Case of Macular Corneal Dystrophy Department of Ophthalmology, St. Mary’s Hospital, College of Medicine, The Catholic University of Korea Sung Kun Chung, MD, PhD, You Kyung Lee, MD, Dong Jin Chang, MD, The authors have no financial interest in the subject matter of this poster. Purpose • To report and describe a novel mutation within the CHST6 gene via light and electron microscopic study in a case of macular corneal dystrophy after penetrating keratoplasty in Korea. Materials • Case – A 59 year-old female – presented with progressive loss of vision over a period of 50 years. – family history of macular corneal dystrophy. – underwent penetrating keratoplasty of the right eye. • Light microscopy and transmission electron microscopy were conducted on the removed corneal specimens. – Hematoxylin and eosin(HE), Alcian blue, periodic acidSchiff (PAS), Colloidal iron, and Masson-Trichrome stains were performed on the specimen. • genomic DNA was obtained from peripheral blood samples. Materials The squares indicate males and the circles indicate females. A shadowing image indicates a person affected with macular corneal dystrophy. The arrow indicates the case patient Results Comparison between light microscopic appearances (upper line: x100, lower line: x400) Positive finding for glycosaminoglycan was indicated with arrows (B,C,D). A, Hematoxylin and Eosin stain. B, Alcian blue stain. C, Colloidal iron stain. D, Periodic acid-Schiff stain E, Masson-Trichrome stain Results Transmission electron microscopic findings. A, Keratocyte distended by membrane-bound intracytoplasmic vacuoles containing electron dense fibrillogranular material (black arrow). Vacuole containing dense fibrillogranular material in the interstromal lamellae (white arrow). Asterisk indicates relatively normal keratocyte (Scale bar: 2㎛). B, Keratocyte with vacuoles containing dense osmiophilic whirls (Scale bar: 0.5㎛). Results Direct sequencing analysis of the coding region of CHST6. Sequence of the coding region of CHST6 revealed a change of the nucleotide at codon 205 (CGG→TGG). This missense mutation has not been previously reported. Results Slit lamp photograph of case approximately 1 year after penetrating keratoplasty shows no complications. Conclusions • Macular corneal dystrophy (MCD), an autosomal-recessive disease, is the least common of the classic stromal dystrophies. • We describe a case of macular corneal dystrophy with histopathological findings. We also report a novel missense mutation within the CHST6 gene (p.Arg205Trp) in Korea. • The p.Arg205Trp mutation is a new missense mutation which changes arginine to tryptophan, inducing the substitution of a neutral amino acid to a basic amino acid. This mutation could also affect protein function. Conclusions • Light microscopy presents abnormal deposits of glycosaminoglycans in Bowman’s histiocytes, keratocytes, between the stromal lamellae, Descemet’s membrane, and endothelium. These glycosaminoglycans stain positively with Alcian blue, Colloidal iron and periodic acid-Schiff (PAS). • Electron microscopy revealed that these deposits correspond to electro-lucent fibrillogranular material visible within membranebound intracytoplasmic vacuoles. Such abnormalities have been shown to be the sequelae of an error in glycosaminoglycans metabolism within the cornea, in particular the proteoglycan keratan sulfate, resulting in its abnormal intra and extracellular deposition. Conclusions • The CHST6 protein is a sulfotransferase, a carbohydrate sulfotransferase of the semi-methyl-D/N-acetylgalactose/Nacetyl glucosamine-6-O-sulfotransferase family, which can catalyze the phosphorylation of 6-hydroxy-6-O of Nacetyl glucosamine, galactose, and N-acetyl galactosamine. • The variations in the coding region of CHST6 may reduce the activity of the enzyme or cause it to be lost, resulting in a low sulfated form or non-sulfated form of keratan sulfate. Due to the loss of its soluble properties, non-sulfated keratan sulfate cannot be completely metabolized, inducing the deposition of sediment in the corneal stroma.
