CHST6 mutation screening and endoplasmatic reticulum stress in macular corneal dystrophy

Abstract

Macular corneal dystrophy (MCD) is an autosomal recessive disorder mainly caused by gene mutations of carbohydrate sulfotransferase (CHST6) leading to bilateral visual impairment. Because the mechanism underlying this degeneration remains poorly understood, we investigated molecular alterations and pathways that may be involved in MCD in this issue. Different mutation sites were screened by direct sequencing of the coding region of CHST6. In addition, we described morphological changes in MCD keratocytes by light microscopy and electron microscopy and determined the relationship between the development of this disease and the occurrence of apoptosis through flow cytometry, cell counting kit-8, colony formation assay and other experiments. Western blotting and quantitative real-time polymerase chain reaction were used to determine if endoplasmic reticulum (ER) stress was activated. We found 10 kinds of mutations among these families with 3 novel mutations included. The percentage of apoptotic keratocytes increased in MCD patients; furthermore, the expression of apoptosis related protein B-cell lymphoma-2 (Bcl-2) was down-regulated while Bcl-2 associated X protein was upregulated. Finally, ER stress was activated with the upregulation of glucose-regulated protein 78 and CCAAT-enhancer-binding protein homologous protein. Our clinical and in vitro results suggest that the CHST6 mutation associated with MCD is associated with apoptosis, and ER stress is probably involved in this apoptosis pathway.

Keywords: CHST6; apoptosis; endoplasmic reticulum stress; keratocytes; macular corneal dystrophy.

Figure 1. Slit-lamp photographs of patients from nine families with macular corneal dystrophy (MCD)

(A-I) Showing multiple, irregular gray-white hazes diffused on the central and peripheral cornea. Photograph of family 10 was missing.

Figure 2. Sequencing chromatograms of variations in CHST6 identified in this study

(A) Sequence chromatograms showing one novel homozygous missense mutation in family 1, c.382 G>A. (B-G, I-N) sequences of the open reading frame of CHST6 from heterozygous mutation families were subcloned into p3xFLAG-CMV10 vectors and directly sequenced for heterozygous mutation analysis. (H) One founder homozygous missense mutation in family 5, c.1072 T>C.

Figure 3. Pathological changes of MCD tissues observed by histologic and ultrastructural analysis

(A) Subepithelial collagen fibers showed irregularity with hyalin deposited, stained with hematoxylin and eosin. (B-C) PAS and Alcian blue stain showed abnormal accumulations in the subepithelial and upper layer of the stroma. (D) Ultrastructural analysis showed that a large number of electron dense deposits are deposited in the corneal stroma with activated rough ER. Arrows show the polysaccharide sulfate deposits. The asterisked triangle represents the rough ER. Triangles indicate the electron dense deposits.

Figure 4. The changes in proliferation and apoptosis in MCD keratocytes

(A) Keratocytes from patients were larger, longer and included more sick cells. (B) Characterization of apoptosis of MCD keratocytes analyzed by transmission electron microscope examination. Concentration of encapsulated cells, chromosome aggregation and apoptosis body formation are shown. (C) Flow cytometry results show that the percentage of apoptotic cells increased in the MT group. (D-E) Cell proliferation and clonogenic assays illustrated that keratocytes of MT had better cell viability and a stronger ability to form clones than WT. Statistical analysis between the two groups were analyzed by t-test, in which a p-value of less than 0.05 was considered statistically significant (^** p<0.01, ^***p<0.001).

Figure 5. Expression of regulation proteins of apoptosis and ER stress pathways in WT and MT keratocytes

(A) Expression of CHOP, an ER stress-associated transcription factor, and GRP78, an ER chaperone. (B) Expression of Bcl-2 family proteins in WT and MT keratocytes. Bcl-2 was downregulated, but Bax was upregulated. (C-F) Relative mRNA levels of CHOP, GRP78, Bcl-2 and Bax. Only Bcl-2 was decreased (* p<0.05, ^*** p<0.001).

Figure 6. Ten MCD families who participated in the study

Open and closed symbols indicate unaffected and affected individuals, respectively. Deceased family members are denoted by diagonal slashes, the arrow marks the proband in these families.