Molecular characterization of CHST6 in Egyptian families with macular corneal dystrophy reveals recurrent and novel variants

BackgroundMacular corneal dystrophy (MCD), a rare autosomal recessive disorder, is caused by pathogenic mutations in the carbohydrate sulfotransferase 6 gene (CHST6) and is characterized by bilateral progressive stromal clouding and vision loss. Corneal transplantation is often necessary. This study aimed to identify disease-causing mutations in a Han-Chinese MCD patient.MethodsA 37-year-old female diagnosed with MCD was recruited. The clinical materials were observed and described, and peripheral blood sample was extracted. Whole exome sequencing (WES) and Sanger sequencing were used to reveal genetic defects. The pathogenicity of identified mutations was assessed using in silico analysis.ResultsThe patient had typical features of MCD, including decreased vision, multiple irregular gray-white corneal opacities, and corneal thinning. A novel nonsense mutation c.544C>T (p.Gln182Ter) and a validated missense mutation c.631C>G (p.Arg211Gly) were identified in the CHST6 gene coding region, both classified as “pathogenic” following the American College of Medical Genetics and Genomics standards and guidelines.ConclusionsThis study reports a Han-Chinese MCD patient with a novel nonsense mutation c.544C>T (p.Gln182Ter) and a recurrent missense mutation c.631C>G (p.Arg211Gly), which expand the spectrum of genetic mutations. The results of this study extend genotype-phenotype correlations between the CHST6 gene mutations and MCD clinical findings, contributing to a more accurate diagnosis and the development of potential gene-targeted MCD therapies.KeywordsCarbohydrate sulfotransferase 6 gene (CHST6); compound heterozygous mutations; Han Chinese family; macular corneal dystrophy (MCD)

Novel mutations in the carbohydrate sulfotransferase gene (CHST6) in American patients with macular corneal dystrophy

To report a novel mutation within the CHST6 gene, as well as describe light and electron microscopic features of a case of macular corneal dystrophy. A 59-year old woman with macular corneal dystrophy in both eyes who had decreased visual acuity underwent penetrating keratoplasty. Further studies including light and electron microscopy, as well as DNA analysis were performed. Light microscopy of the cornea revealed glycosaminoglycan deposits in the keratocytes and endothelial cells, as well as extracellularly within the stroma. All samples stained positively with alcian blue, colloidal iron, and periodic acid-Schiff. Electron microscopy showed keratocytes distended by membrane-bound intracytoplasmic vacuoles containing electron-dense fibrillogranular material. These vacuoles were present in the endothelial cells and between stromal lamellae. Some of the vacuoles contained dense osmophilic whorls. A novel homozygous mutation (c.613 C>T [p.Arg205Trp]) was identified within the whole coding region of CHST6. A novel CHST6 mutation was detected in a Korean macular corneal dystrophy patient.

Background/Objectives: In this study, we aim to evaluate in vivo confocal microscopy (IVCM) findings of corneal stromal dystrophies (CSDs) including granular, macular and lattice corneal dystrophy that can be used for differential diagnosis and monitoring recurrences after surgical interventions. Methods: Patients diagnosed with CSD who were followed-up in the cornea and ocular surface unit were included in this study. IVCM was performed using the Heidelberg Retina Tomograph 3, Rostock Cornea Module (Heidelberg Engineering, Germany) and anterior segment optical coherence tomography (AS-OCT) imaging was performed using the Spectralis OCT (Heidelberg Engineering, Germany). The morphological structure, size and location of deposits, epithelial involvement and presence of inflammatory and dentritic cells were compared among the three stromal dystrophies. Results: A total of 72 eyes from 36 participants were included in this study. Twelve patients (33.33%) had granular corneal dystrophy (GCD), ten (27.77%) had macular corneal dystrophy (MCD) and fourteen (38.88%) had lattice corneal dystrophy (LCD). In GCD, highly reflective deposits varying in size (20 µm-300 µm) were observed. In MCD, diffuse hyperreflective stroma with dark striae, dentritic cells around deposits and abnormal keratocytes were observed. In LCD, there were branching, lattice-like and granular deposits with epithelial cell disruption in some of the eyes. In MCD, the central corneal thickness was thinner (449.44 ± 65.45 µm) compared to GCD and LCD (565.16 ± 49.62 µm and 569.91 ± 39.32 µm p < 0.001). Recurrence was observed in five patients following penetrating keratoplasty. Conclusions: IVCM is a valuable tool for distinguishing CSD subtypes and monitoring recurrence following surgical interventions.

Macular corneal dystrophy (MCD) is an autosomal recessive disease characterized by corneal opacities and caused by mutations in a carbohydrate sulfotransferase gene, known as CHST6. MCD type I patients show missense mutations in the CHST6-coding region, and MCD type II patients show a large deletion and replacement in the upstream region of CHST6. The objective of this study was to identify the genetic defect in CHST6 gene causing MCD in Italian families. We investigated MCD genotype by using polymerase chain reaction followed by direct sequencing, and results were confirmed by restriction analysis. An enzyme-linked immunosorbent assay was performed to assess the presence of sulfated keratan sulfate in the serum of MCD patients. Biochemical analysis revealed a MCD type I phenotype in two families and a type II phenotype in another family. Two novel missense mutations and a polymorphism in the coding region of CHST6 gene were identified in patients with MCD type I. In one MCD II family, a homozygous deletion in the upstream region of CHST6 gene was found.

To further characterize the role of the carbohydrate sulfotransferase (CHST6) gene in macular corneal dystrophy (MCD) through identification of causative mutations in a cohort of affected patients from southern India. Genomic DNA was extracted from buccal epithelium of 75 patients (51 families) with MCD, 33 unaffected relatives, and 48 healthy volunteers. The coding region of the CHST6 gene was evaluated by means of polymerase chain reaction amplification and direct sequencing. Subtyping of MCD into types I and II was performed by measuring serum levels of antigenic keratan sulfate. Seventy patients were classified as having type I MCD, and 5 patients as having type II MCD. Analysis of the CHST6 coding region in patients with type I MCD identified 11 homozygous missense mutations (Leu22Arg, His42Tyr, Arg50Cys, Arg50Leu, Ser53Leu, Arg97Pro, Cys102Tyr, Arg127Cys, Arg205Gln, His249Pro, and Glu274Lys), 2 compound heterozygous missense mutations (Arg93His and Ala206Thr), 5 homozygous deletion mutations (delCG707-708, delC890, delA1237, del1748-1770, and delORF), and 2 homozygous replacement mutations (ACCTAC 1273 GGT, and GCG 1304 AT). One patient with type II MCD was heterozygous for the C890 deletion mutation, whereas 4 possessed no CHST6 coding region mutations. A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I MCD in a cohort of patients in southern India. An improved understanding of the genetic basis of MCD allows for earlier, more accurate diagnosis of affected individuals, and may provide the foundation for the development of novel disease treatments.

Characterization of In Vivo Biomechanical Properties in Macular Corneal Dystrophy

Purpose Macular corneal dystrophy is a rare autosomal recessive eye disease primarily affecting the corneal stroma. Abnormal deposits have been observed intra‐ and extracellularly in the stromal layer. In addition to the stromal keratocytes and corneal lamellae, deposits are also present in the basal epithelial cells, endothelial cells and Descemet's membrane. Misfolded proteins have a tendency to gather into aggregating deposits. We studied the interaction of molecular chaperones and proteasomal clearance in macular dystrophy human samples and in human corneal HCE‐2 epithelial cells in this disease.Methods Seven cases of macular dystrophy and 5 normal human corneal buttons were collected during corneal transplantation. Fluorescent immunohistochemistry on human donor corneal button was used to verify the spatial distribution pattern of selected Hsp70, SQSTM1/p62 and ubiquitin molecules, respectively. The expression level of these proteins were analysed in HCE‐2 cells using western blotting and transmission electron microscopy, respectively.Results Highly elevated Hsp70, SQSTM1/p62 and ubiquitin protein conjugates were observed in intracellular space of the epithelial cells in macular dystrophies. All the studied proteins were also highly elevated under proteasome inhibition in human corneal epithelial HCE‐2 cells in cell cultures.Conclusion We report a novel proteostatic regulatory mechanism that connects the molecular chaperone and proteasomal clearance system with the pathogenesis of macular corneal dystrophy.

Purpose:The aim was to determine the frequency and describe the main histopathologic features of corneal stromal dystrophy in Saudi Arabia.Methods:A single-center, retrospective analysis of 193 corneal specimens diagnosed with stromal dystrophy. All samples were retrieved from the Histopathology Department at King Khaled Eye Specialist Hospital over a 10-year period (2002 to December 31, 2011). Cases of stromal dystrophy undergoing keratoplasty were included in the study. Routine histopathologic stains and specific stains were used to determine a diagnosis. The corresponding demographic data and basic clinical/surgical information were collected via chart review.Results:The study sample was comprised of 193 eyes. The final diagnoses were macular corneal dystrophy (MCD) in 180 (93.26%) eyes, granular corneal dystrophy (GCD) in 9 (4.66%) and lattice corneal dystrophy (LCD) in 4 (2.07%) eyes. The mean age at presentation was 27.03 years for MCD, 26.33 years for GCD and 53.75 years for LCD. The interval between diagnosis and surgical intervention was not statistically different between the macular and granular groups (P = 0.141). There was a positive family history for the MCD (37.22%) and GCD (44.44%) groups. All eyes underwent penetrating keratoplasty (PKP) except 10 MCD cases that underwent lamellar keratoplasty. Diffuse stromal deposits were present in 87.2% of MCD corneas and 66.67% of GCD corneas. Seventeen eyes with MCD were misdiagnosed as GCD. None of the LCD cases were clinically identified since all of these cases were diagnosed as corneal scarring. In eyes with MCD that underwent PKP, there was diffuse stromal involvement (in 87.22% eyes) and changes in Descemet's membrane (in 53.5% eyes).Conclusion:This pathological study suggested that MCD was the most common corneal stromal dystrophy that required keratoplasty in Saudi Arabia. Patient with MCD and GCD presented at a significantly younger age than LCD. The clinical diagnosis of MCD is not achieved in all cases likely due to a more severe phenotype in the Saudi population or the presence of corneal scarring that is associated with previous trachoma, which obscures the classical appearance of LCD. We believe that PKP is first-line surgical treatment, especially for MCD because it involves all corneal layers. However, deep stromal involvement and changes in Descemet's membrane in MCD should be considered when selecting the surgical procedure.

BackgroundMacular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters.MethodsA multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact.ResultsOphthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity.ConclusionWe identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0308-0) contains supplementary material, which is available to authorized users.

Corneal dystrophies are a group of non-inflammatory inherited disorders of the cornea. This review considers treatment options for epithelial-stromal and stromal corneal dystrophies: namely Reis–Bücklers, Thiel–Behnke, lattice, Avellino, granular, macular and Schnyder corneal dystrophies. Where there is visual reduction, treatment options may include either phototherapeutic keratectomy (PTK) or corneal transplantation. Due to the anterior location of the deposits in Reis-Bücklers and Thiel–Behnke dystrophies, PTK is considered the treatment of choice. For lattice, Avellino, granular and macular corneal dystrophies, PTK provides temporary visual improvement; however, with recurrences, repeat PTK or a corneal transplant would be needed. For Schnyder dystrophy, should treatment be required, PTK may be the preferred option due to the potential for recurrence of the disease in corneal transplantation. This review discusses the literature and evidence base for the treatment of corneal dystrophies in terms of visual outcomes and recurrence rate.

Mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal N-acetylglucosamine-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of macular corneal dystrophy (MCD) in various ethnicities. This study was conducted to examine the CHST6 gene in Vietnamese with MCD. Nineteen unrelated families, including 35 patients and 38 unaffected relatives were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals served as control subjects. Genomic DNA was extracted from leukocytes. Analysis of the CHST6 gene was performed with polymerase chain reaction and direct sequencing. Corneal buttons were studied histopathologically. A slit lamp examination revealed clinical features of MCD with gray-white opacities and stromal haze between. On histopathology, corneal sections showed positive staining with colloidal iron. Sequencing of the CHST6 gene revealed six homozygous and three compound heterozygous mutations. The homozygous mutations, including L59P, V66L, R211Q, W232X, Y268C, and 1067-1068ins(GGCCGTG) were detected, respectively, in two, one, eight, one, one, and two families. Compound heterozygous mutations R211Q/Q82X, S51L/Y268C, and Y268C/1067-1068ins(GGCCGTG) were identified, each in one family. A single heterozygous change at codon 76 (GTG-->ATG) was detected in family L, resulting in a valine-to-methionine substitution (V76M). None of these mutations was detected in the control group. Mutations identified in the CHST6 gene cosegregated with the disease phenotype in all but one family studied and thus caused MCD. Among these, the R211Q detected in 9 of 19 families may be the most common mutation in Vietnamese. These data also indicate that significant allelic heterogeneity exists for MCD.

Comparison of Penetrating Keratoplasty and Deep Lamellar Keratoplasty for Macular Corneal Dystrophy and Risk Factors of Recurrence

A case of a 54-year-old patient with macular corneal dystrophy who underwent a penetrating keratoplasty (PK) in the left eye and a deep anterior lamellar keratoplasty (DALK) in the right eye is described. The merits of PK versus DALK for visual rehabilitation in macular corneal dystrophy are discussed. After deep lamellar dissection in the left eye, the remaining bed of residual stroma, Descemet membrane, and endothelium was unacceptably hazy and thus converted to PK. The right eye had successful DALK because a relatively clear bed was noted after deep lamellar separation using the big-bubble technique. The spectacle-corrected visual acuities at 2-year follow-up are 6/6 OD and 6/9+ OS. There is mild residual haze in the right eye, although the visual acuity is better in this eye. Endothelial cell counts were satisfactory and not significantly different in both eyes. This case demonstrates that DALK with the big-bubble technique can be successfully carried out for visual rehabilitation in macular dystrophy. DALK should be considered in cases with no significant Descemet membrane and endothelial involvement because of the potential advantages offered by retaining the host endothelium.

Comparison of Deep Lamellar Keratoplasty and Penetrating Keratoplasty for Lattice and Macular Corneal Dystrophies