CHST6 Mutations And Histopathologic Study In Macular Corneal Dystrophy

Objective: Macular corneal dystrophy (MCD) is a rare, autosomal recessive disease. In 1980, for the first time Groenouw described this disease and it is also called Groenouw type II corneal dystrophy, a type of corneal stromal dystrophy. Clinically, MCD is characterized by a cloudiness of the cornea with irregularly shaped off-white opacities in both eyes, that progressively extend to the central and peripheral corneal stroma. The clinical manifestations are progressive loss of vision, photophobia and discomfort of eyes. Eventually, keratoplasty is necessary. In 2000, Akama et al first discovered mutations in a carbohydrate sulfotransferase gene (CHST6) in MCD patients which is located on chromosome 16q22. CHST6 gene encodes corneal N-acetyl glucosamine-6-sulfotransferase (C-GlcNAc6ST), an enzyme that transfers sulfate to position 6 of GlcNAc residues and participates in the biosynthesis of keratan sulphate(KS) proteoglycan in the cornea. The enzyme activity is reduced due to CHST6 gene mutation which affects the synthesis of glycosamino- glycans such as keratan sulphate. The accummulation of glycosaminoglycans in the cornea damage the keratocytes and the fibers which eventually lead to the corneal opacity. After Akama, other scientists also discovered CHST6 gene mutation in sufferers of macular corneal dystrophy in their countries. There were many studies on this disease in our country, but most of them were limited to case reports and histopathologic studies, a few to gene mutations researches. This report documents an analysis of CHST6 in three patients from a family with macular corneal dystrophy in the northeast of China. We collected some peripheral venous blood, extracted DNA and sequenced CHST6 gene sequences with positive and negative two-way sequencing. In this study, we further discuss CHST6 mutation analysis and histopathological study in macular corneal dystrophy. Methods: Three patients of a family with macular corneal dystrophy from the northeast of China were studied, comprising of two males and one female. Peripheral blood (2ml) was collected from each participant and genomic DNA was extracted from their blood. CHST6 has 4 exons, and the coding region is located on the third exon. The coding region of CHST6 was amplified by the polymerase chain reaction (PCR), followed by the gene sequencing. Half of the cornea was taken after the partial penetrating keratoplasty, placed in 4% formaldehyde solution and fixed. Then paraffin embedding was carried out and the tissue was cut into slices which were stained with Alcian Blue (AB) and Alcian Blue-periodic acid schiff (AB-PAS). Another half of cornea was fixed in 2.5% glutaraldehyde, and observed under electron microscopy after epoxy resins embedding and staining.Results: We found a common mutation point after the two-way gene sequencing: c.61₆2insG, the original c.62T>A, altering the open reading frame. Histopatho- logically, keratocytes and elastic fibers stained blue with AB. With AB-PAS, corneal epithelium was thinner than normal, and underneath it many red and blue macular tissues were located. Under transmission electron microscopy, we found some round opacities under the corneal epithelium.Conclusions: We have strong evidences to suggest that there is a mutation in c.61₆2insG gene which causes these patients to suffer from macular corneal dystrophy. It was unreported previously in other articles.