A Case of Korean Patient with Macular Corneal Dystrophy Associated with Novel Mutation in the CHST6 Gene
A 59 year-old female with a family history of MCD presented to our clinic with progressive loss of vision over a 40-year period. Her visual acuity was finger count/50 cm OU. Slit lamp examination revealed diffusely hazy corneas with bilateral opacities. There were multiple irregular grayish-white, dense, poorly delineated spots in the stroma. Because her parents died at an early age, her family history was unclear. Of note, her only son complained of foggy vision and was 37 years old. His slit lamp examination revealed a similar, but less severe appearing corneal exam compared to that of his mother ().\nPenetrating keratoplasty was performed on the right eye. A 7.75 mm donor button was sutured into a 7.5 mm host bed. Histopathologic examinations were conducted on the excised corneal buttons obtained from the procedure. Genomic DNA was extracted from the leukocytes of her peripheral blood via standard procedures. One year post-operatively, that patient had a best-corrected visual acuity of 20 / 100 OD and a clear cornea graft.\nInformed consent for both the clinical examinations and DNA analyses was obtained from the patient and her son in accordance with the Declaration of Helsinki. The study was approved by the institutional review board at the Catholic University of Korea, St. Mary's Hospital.\nHematoxylin and eosin (HE), alcian blue, periodic acid-Schiff (PAS), colloidal iron, and Masson's trichrome stains were performed on the specimen using standard techniques. For transmission electron microscopy, the corneal specimen was immersed immediately in a fixative solution containing 3% glutaraldehyde with 0.2 M sodium cacodylate at a pH of 7.4. After overnight fixation, the fixative solution was removed and replaced with a phosphate buffer, followed by 1% osmium tetroxide buffered with sodium cacodylate. After one hour, the osmium was replaced with increasing concentrations of ethanol through propylene oxide, and the tissue was embedded in the epoxy. The embedded tissue was then sectioned with an ultra-microtome into 1 µm-thick sections, and stained with toluidine blue. The area to be observed was placed under a light microscope, ultra-sectioned from 60 to 100 nm in thickness, double stained with uranyl acetate and lead citrate, and examined via transmission electron microscope (JEM 1010; JEOL, Tokyo, Japan).\nThe unique exon involving the coding region, exon 3, was amplified via polymerase chain reaction (PCR) with the previously described primers []. Thermal cycling was conducted via the following protocol. The cycling program began with an initial denaturing step of 5 minutes at 95℃ followed by 33 cycles of 94℃ for 30 seconds, 53℃ to 57℃ for 30 seconds, and 72℃ for 45 seconds with a final extension step at 72℃ for 10 minutes. The PCR products were purified and sequenced directly on both strands using an automatic DNA sequencer (ABIPrism 377XL; Applied Biosystems, Foster City, CA, USA). The nucleotide sequences were compared with the published cDNA sequence of CHST6 (NM_021615) [].\nLight microscopy showed a normal epithelium and Bowman's membrane. HE staining revealed faintly basophilic deposits between the stromal lamellae, and within keratocytes and endothelial cells. These deposits were positive to alcian blue, PAS, and colloidal iron stain, but negative to Masson's trichrome stain (). Electron microscopy revealed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles harbored dense osmophilic whorls. Similar vacuoles were also present in the interstromal lamellae and endothelial cells ().\nAnalysis of the entire CHST6 coding region revealed distinct genetic defects. One missense mutation was identified in a homozygous state (p.Arg205Trp [c.613C>T]), which had not been previously reported. Samples from the patient's son also showed the same missense mutation (p.Arg205Trp [c.613C>T]) ().