Genetic implications of CHST6 gene mutations and their corneal microstructural changes in macular corneal dystrophy patients

Abstract

Purpose: To collectively investigate the carbohydrate sulfotransferase 6 (CHST6) mutation spectrum and corneal morphological alterations of macular corneal dystrophy (MCD) patients using in vivo confocal microscopy (IVCM), histochemistry, immunohistochemistry, and further ascertaining the immunophenotype using an enzyme-linked immunosorbent assay (ELISA).

Methods: Sanger sequencing-based CHST6 gene screening was performed for 112 study participants (MCD patients, n = 68; family members, n = 44). Twenty-seven MCD patients underwent IVCM analyses, and corneal buttons were analyzed with histochemistry Alcian blue (AB) staining and immunohistochemistry anti-keratan sulfate (KS) monoclonal antibody, 5D4MoAb. An ELISA was used to determine serum KS levels. Quantitative analysis of the central corneal thickness (CCT), epithelial cell thickness, epithelial cell count, and stromal keratocyte cell count was performed using a one-way ANOVA.

Results: Eighteen distinct CHST6 mutations, including one novel (p.L129V), were identified. MCD patients with predominant immunophenotype IA (n = 15) harboring major p.Q182Rfs199 deletion, p.194_R196delinsRC (delins), and open reading frame (ORF) mutations displayed AB positivity corresponding to loss of Bowman's layer, interlamellar glycosaminoglycan (GAG) depositions, and faint KS expression (5D4-MoAb) only in stromal keratocytes. Notably, IVCM imaging revealed BL loss due to confluent clumps of hyper-reflective, granular deposits together with scar tissue seen only in this group. Eight patients (with missense mutations) displayed immunophenotype I with positive GAG deposits and negative KS expression. Patients with immunophenotype II (n = 4) with no mutations showed both positive GAG deposits and KS expression. A quantitative analysis revealed a statistically significant decrease in CCT (p-value < 0.001), epithelial cell thickness, epithelial cell count, and stromal keratocyte cell count among the patients with truncation mutations compared to the control group.

Conclusions: In this current study, the combinational findings of MCD-related corneal morphological alterations, immunophenotypes, and mutation spectrum are presented first, which indicated a severe phenotype in patients identified with truncation (deletion, delins, and deletion of ORF) mutations. However, additional studies with a larger number of patients would help highlight these findings and reinforce the possible correlation between genotypes and immunophenotypes in MCD pathogenesis.

Figure 1

Slit-lamp and IVCM images of MCD patients with different CHST6 mutation types. Slit-lamp and sequential in vivo confocal images of a healthy subject (A-control) and selected MCD patients (B–F), representing different corneal layers. Marked corneal layers are the suprabasal epithelium, BE, Bowman’s layer (BL), region immediately before anterior stroma, anterior stroma, middle stroma, posterior stroma, and endothelium. A slit-lamp and sequential IVCM analysis of the control eye showed normal cellular morphology of the different corneal layers. Suprabasal epithelium, BE of MCD patients - B, C, and F show scattered deposits and slightly altered nerve fibers (NF); D with cluster of deposits and loss of NF; and patient E (MCD patient with ORF deletion) shows clumps of deposits with scar tissue (S) and loss of NF. BL and region next to the anterior stroma of patients B and F show deposits along with loss of NF; Patients C, and D show confluent, clumps of deposits with scar tissue (S); Patient E showed clumps of highly reflective homogenous granular deposits with the rupture of basement membrane and loss of BL resulting in scar tissue. The endotheliums of Patients B and F show scattered deposits; Patients C, D, and E showed deposits along with polymegathism. Homogenous reflective material with dark striae (St)-like images was observed throughout the stroma (for patients B-F). Striae-like observations were not clearly visible due to the highly light-reflective clumps of deposits (E). Clinical and IVCM analysis across the corneal thickness of MCD patients 17 (C), 29 (D), and 60–1 (E) carrying fs deletion (del), deletion-insertion (delins), and deletion of ORF mutations, respectively, revealed severe scars in the BL and anterior stroma due to highly light-reflective clumps of deposits presenting severe corneal morphological alterations.

Figure 2

Representative images of corneal sections for histochemistry (HC-AB staining) and immunohistochemistry (IHC-5D4 MoAb immunoreactivity) analysis of normal (control) and MCD patients with different mutation types. Images on the left side represent AB-stained corneal sections, and the right side represents immunostained sections. A: The corneal layers marked in the control (cadaver) show normal morphology. B–F represent images of unrelated MCD patients (Patients 7, 17, 29, 60–1, and 67) harboring different CHST6 mutations. Major changes were observed for the patients harboring deletion (del), deletion-insertion (delins), and deletion of ORF mutations with severe morphological alterations in the BL and anterior stroma due to abnormal, amorphous, finely granulated GAG deposits (*) with altered collagen lamellae and altered keratocyte (K) cell shape. IHC also showed the presence of KS only in the stroma, with a smaller number of stromal keratocytes. F represents the images for the MCD patient with no coding region CHST6 mutation, showing a normal corneal morphology like the control (A). In the IHC images, red represents PI staining, and green indicates FITC-labeled anti-KS antibodies. Ep, epithelium; BL, Bowman’s layer; BM, basement membrane; S, stroma; DM, Descemet’s membrane; EN, endothelium; K, keratocytes.

Figure 3

Comparison of the central corneal thickness of MCD patients and the control group. Scatter plots show significantly reduced central corneal thickness in patients with truncation mutations compared to the control group. Quantitative analysis among the groups was performed using a one-way ANOVA. A p-value of less than 0.001 (***) was considered statistically significant (ns, not significant).

Figure 4

Comparison of corneal epithelial thickness and corneal epithelial cell count among MCD patients and the control group. A: Scatter plot shows a significantly reduced corneal epithelial thickness observed in the patients with truncation mutations compared to the control group. B: A significantly reduced count of corneal epithelial cells was observed in the patients with truncation mutations compared to the control group. Quantification among the groups was performed using a one-way ANOVA. A p-value of less than 0.001 (***) and a p-value of less than 0.05 (*) were considered statistically significant. (ns-not significant).

Figure 5

Comparison of stromal keratocyte cell counts among MCD patients and the control group. The scatter plot shows a significantly reduced count of corneal stromal cells in the patients with truncation mutations compared to the control group. Quantification among the groups was performed using a one-way ANOVA. A p-value of less than 0.001 (***) and a p-value of less than 0.05 (*) were considered statistically significant. (ns-not significant).