Macular corneal dystrophy in a Chinese family related with novel mutations of CHST6

Abstract

Purpose: To identify mutations in the carbohydrate sulfotransferase gene (CHST6) for a Chinese family with macular corneal dystrophy (MCD) and to investigate the histopathological changes in the affected cornea.

Methods: A corneal button of the proband was obtained by penetrating keratoplasty. The half button and ultrathin sections from the other half button were examined with special stains under a light microscope (LM) and an electron microscope (EM) separately. Genomic DNA was extracted from peripheral blood of 11 family members, and the coding region of CHST6 was amplified by the polymerase chain reaction (PCR) method. The PCR products were analyzed by direct sequencing and restriction enzyme digestion.

Results: The positive reaction to colloidal iron stain (extracellular blue accumulations in the stroma) was detected under light microscopy. Transmission electron microscopy revealed the enlargement of smooth endoplasmic reticulum and the presence of intracytoplasmic vacuoles. The compound heterozygous mutations, c.892C>T and c.1072T>C, were identified in exon 3 of CHST6 in three patients. The two transversions resulted in the substitution of a stop codon for glutamine at codon 298 (p.Q298X) and a missense mutation at codon 358, tyrosine to histidine (p.Y358H). The six unaffected family individuals carried alternative heterozygous mutations. These two mutations were not detected in any of the 100 control subjects.

Conclusions: Those novel compound heterozygous mutations were thought to contribute to the loss of CHST6 function, which induced the abnormal metabolism of keratan sulfate (KS) that deposited in the corneal stroma. It could be proved by the observation of a positive stain reaction and the enlarged collagen fibers as well as hyperplastic fibroblasts under microscopes.

Figure 1

Pedigree of a Chinese family including three individuals affected with MCD. Open symbols indicate clinically unaffected members, filled symbols indicate clinically affected members, and the arrow indicates the proband.

Figure 2

A photograph of the right eye of the proband. It shows stromal cloudiness studded by irregular rounded gray-white opacities in the central and peripheral cornea.

Figure 3

Paraffin section of the proband’s cornea stained with Hale’s colloidal iron. Blue plaque deposits were found in the corneal stroma and under the endothelial layer. These deposits presented a significantly positive reaction to colloidal iron staining.

Figure 4

Transmission electron microscopy result of paraffin section of the proband’s cornea stained with Hale's colloidal iron. A: The picture shows that the collagen fibers in the corneal stroma are irregular, enlarged, and intensive (1,500X). B: The picture shows hyperplastic, active fibroblasts and intracytoplasmic vacuoles in the cornea (5,000X).

Figure 5

Direct sequencing analysis of the coding region of CHST6. A: Normal sequence at codon 298. B: Sequence of the coding region of CHST6 in the proband revealed a change of the nucleotide at codon 298 (CAG→TAG). C: Normal sequence at codon 358. D: Sequence of the coding region of CHST6 in the proband showed a substitution at codon 358 (TAC→CAC).

Figure 6

PvuII digestion of PCR products for the c.892C>T transition. PCR products in individuals with the c.892C>T transition were digested into 153 bp and 173 bp and into only 153 bp in individuals without the c.892C>T transition. A: Members of the Chinese family with MCD. PCR products from individuals II:2, III:1, III:2, III:4, III:6 and IV:1 with c.892C>T transition were digested into 153bp and 173bp, other members without c.892C>T transition only had a 153bp segment. B :Part of 100 control subjects. Control subjects without c.892C>T transition only had a 153bp segment. M indicates DNA ladder, U indicates undigested PCR product, and P indicates one patient with MCD in the Chinese family.

Figure 7

PmacI digestion of PCR products with for the c.1072T>C transition. PCR products in individuals with the c.1072T>C transition were digested into 337 bp and 357 bp and into only 357bp in individuals without the c.1072T>C transition. A: Members of the Chinese family with MCD. PCR products from individuals II:3, III:2, III:4, III:5, III:6 and IV:2 with c.1072T>C transition were digested into 337bp and 357bp, other members without c.1072T>C transition only had a 357bp segment. B: Part of 100 control subjects. Control subjects without c.1072T>C transition only had a 357bp segment. U indicates undigested PCR product, and P indicates one patient in the Chinese family with MCD.