Macular corneal dystrophy: mutational spectrum in German patients,novel mutations and therapeutic options

INTRODUCTION: The objective of this study was to investigate genotype-phenotype correlations, the consequences for surgical treatment, and the therapeutical options in patients with macular corneal dystrophy (MCD). MATERIAL AND METHODS: We investigated MCD genotype by using polymerase chain reaction followed by direct sequencing in one family and four patients with MCD. Results were confirmed by restriction analysis. Clinical phenotypes, histopathological findings, and therapeutical proceedings of each patient were reported and compared with the molecular genetic results. RESULTS: Five mutations, four missense mutations, and one frameshift mutation, from which three were novel, and one single-nucleotide polymorphism, were identified within the coding region of the CHST6 gene. In three patients, two with a homozygous mutation within the start codon (Met1Leu) and one with a heterozygous mutation (Leu200Arg) and a polymorphism (Arg162Gly), with irregular corneal surface and recurrent erosions a phototherapeutic keratectomy lead to a transient success. An additional fitting of rigid gas permeable contact lenses in one patient could further improve irregular astigmatism. In two patients, one with a frameshift mutation (1734_1735delTG; Arg211Gln) and one with two compound heterozygous mutations (Leu200Arg; Leu173Phe) and an additional polymorphism (Arg162Gly) a penetrating keratoplasty improved BCVA without any recurrence of the opacities within the follow-up time. DISCUSSION: Different genotypes imply several phenotypes, which influence therapeutical proceedings in MCD patients. Our study shows the wide range of diagnostic findings and therapeutical options in patients suffering from macular corneal dystrophy depending on the genotype.     

An unusual presentation of macular corneal dystrophy associated with uniparental isodisomy and a novel Leu173Pro mutation

PURPOSE: To report an unusual phenotype of macular corneal dystrophy (MCDC1) associated with a novel CHST6 mutation transmitted via maternal isodisomy. METHODS: Slit lamp examination of the patient and his parents was performed. DNA was collected from each individual for amplification and sequencing of the CHST6 coding region, as well as exons 4 and 12 of TGFBI. Serum antigenic keratan sulfate (AgKS) levels were measured for confirmation of the diagnosis and subtyping of MCDC1. Quantitative real-time PCR (qPCR) was performed to differentiate between homozygous and hemizygous sequence variants. Genotyping at 12 single nucleotide polymorphisms (SNPs) within and surrounding CHST6 was performed to determine the pattern of inheritance of mutations identified in CHST6. RESULTS: Examination of the proband revealed bilateral, discrete, axially distributed, gray-white deposits at the level of Bowman's layer, with diffuse fine corneal stromal haze. Screening of TGFBI exons 4 and 12 in the proband did not reveal any allelic variants. However, screening of CHST6 in the proband demonstrated a novel homozygous missense mutation involving a highly conserved amino acid (c.518T > C; Leu173Pro) and undetectable serum AgKS levels in the proband confirmed the diagnosis of type I MCDC1. Quantitative PCR confirmed that both copies of CHST6 were present in the patient, excluding the possibility that the mutation was present in the hemizygous state. The results of genotyping were consistent with maternal isodisomy, as the patient was homozygous for an allele possessed by his mother at each SNP, two of which were informative and demonstrated nonpaternal inheritance. CONCLUSION: A phenotypically unusual variant of MCDC1 was found to be associated with the novel Leu173Pro mutation in CHST6, transmitted via uniparental isodisomy, a previously unreported pattern of inheritance in the corneal dystrophies.     

Identification of novel mutations of the CHST6 gene in Vietnamese families affected with macular corneal dystrophy in two generations

PURPOSE: To report the clinical and genetic findings of Vietnamese families affected with macular corneal dystrophy (MCD) in 2 generations. METHODS: Two families, including 7 patients and 3 unaffected members, were examined clinically. Blood samples were collected. Fifty normal Vietnamese individuals were used as controls. Genomic DNA was extracted from leukocytes. Analysis of the carbohydrate sulfotransferase (CHST6) gene was performed using polymerase chain reaction and direct sequencing. RESULTS: The typical form of MCD was recognized in family B, in which sequencing of CHST6 gene revealed an nt 1067-1068ins(GGCCGTG) mutation (frameshift after 125V) homozygously in MCD patients and heterozygously in the unaffected members. Family N also showed clinical features of MCD, moderate in the mother but severe in the affected son. Sequencing revealed a single heterozygous Arg211Gln in the mother, compound heterozygous Arg211Gln+ Gln82Stop in the affected son, and heterozygous Arg211Gln mutation in the unaffected members. The identified mutations in these pedigrees were excluded from normal controls. CONCLUSIONS: The novel frameshift and compound heterozygous mutations might be responsible for MCD in the families studied. The phenotypic variation between affected parents and offspring was unclear. In family N, severe MCD phenotype seen in the affected son may be due the fact that he had an early stop codon mutation (Gln82Stop).     

Truncating mutations in the carbohydrate sulfotransferase 6 gene (CHST6) result in macular corneal dystrophy

PURPOSE: Identification of mutations in the CHST6 gene in 15 patients from 11 unrelated families affected with recessive macular corneal dystrophy (MCD). METHODS: Genomic DNA was extracted from peripheral blood leukocytes of the affected patients and their healthy family members, and the mutational status of the CHST6 gene was determined for each patient by a PCR-sequencing approach. Serum concentrations of antigenic keratan sulfate for each proband were determined by ELISA. RESULTS: ELISA indicated that all affected patients, except one, were of MCD type I or IA. Fourteen distinct mutations were identified within the CHST6 coding region: 2 nonsense, 2 frameshift, and 10 missense. Of these, 12 were novel, and a nonsense mutation in the homozygous state is reported for the first time. CONCLUSIONS: These molecular results in French patients with MCD combined with those reported in previous studies indicated CHST6 mutational heterogeneity. The characterization herein of nonsense mutations is in keeping with the fact that MCD results from loss of function of the CHST6 protein product.     

Different mutations in carbohydrate sulfotransferase 6 (CHST6) gene cause macular corneal dystrophy types I and II in a single sibship

PURPOSE: The aim of this study was to examine the carbohydrate sulfotransferase 6 (CHST6) gene for mutations in a sibship with both macular corneal dystrophy (MCD) types I and II. DESIGN: Clinically relevant laboratory investigation. METHODS: The coding region of the CHST6 gene was examined for mutations. RESULTS: In one sibling, MCD type I was due to a homozygous C1110T (Arg140end) mutation in CHST6. Two MCD type II individuals exhibited three heterozygous nucleotide changes: C1110T, G1360A (Gly223Asp), and G1685T (Gln331His). Analysis of the upstream region was performed on one individual with MCD type II, and no upstream deletion or substitution was found. CONCLUSIONS: These findings fit the haplotype analysis that we reported previously and indicate that the predicted protein that is encoded by CHST6 is more severely affected in the individual with MCD type I than in the siblings with MCD type II.     

A Case of Korean Patient with Macular Corneal Dystrophy Associated with Novel Mutation in the CHST6 Gene

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.     

斑状角膜营养不良一家系发现新的复合杂合突变

目的 明确一个中国斑状角膜营养不良(MCD)家系中致病基因及其突变的类型及组织病理学改变.方法 对行穿透性角膜移植术后获得的先证者角膜进行光镜和透射电子显微镜检查.收集此家系11个成员的外周血,提取DNA;合成特异性引物,利用聚合酶链式反应(PCR)扩增CHST6基因的编码区;PCR产物进行直接测序和限制性内切酶消化分析.选取100个无关健康个体作为对照.结果 组织特殊染色显示胶性铁染色阳性.透射电子显微镜下观察,可见滑面内质网扩张,胞质空泡化,有细颗粒物质.CHST6基因编码区进行突变检测发现,此家系3个患者CHST6基因第3个外显子存在一个复合杂合突变(Q298X和Y358H),其中第892位碱基C→T,导致第298位的谷氨酰胺变成终止密码子;第1072位碱基 T→C,导致第358位的酪氨酸被组氨酸取代.6个表型正常的个体均为杂合子,而在100个无血缘关系的健康个体中没有检测到突变.结论 确定了两个新的突变导致了该家系CHST6基因功能的丧失,引起MCD.该复合杂合突变在国内外为首次报道.     

Novel CHST6 nonsense and missense mutations responsible for macular corneal dystrophy

PURPOSE: To identify the underlying mutations in two unrelated British families with macular corneal dystrophy (MCD) by screening the carbohydrate sulfotransferase (CHST6) gene. DESIGN: Case reports and results of DNA analysis. METHODS: Two subjects from two British families with MCD were studied. The genetic status of CHST6 was determined for all members of these MCD families. In addition, sulfated keratan sulfate (KS) assay from the probands was also undertaken. CHST6 gene was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by sequencing and restriction digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess KS presence in serum. RESULTS: Four compound heterozygous mutations were identified, three of which are novel. The ELISA showed that the probands were of MCD type I. CONCLUSIONS: These novel mutations are expected to result in loss of CHST6 function, which would account for the MCD phenotype.     

Keratoconus is not associated with mutations in COL8A1 and COL8A2

PURPOSE: To evaluate the suggested role of the COL8A1 and COL8A2 genes in the pathogenesis of the corneal ectatic disorders keratoconus and keratoglobus through mutation screening in affected patients. METHODS: DNA extraction, polymerase chain reaction amplification, and sequencing of COL8A1 and COL8A2 were performed in 50 unrelated keratoconus and 2 unrelated keratoglobus patients. RESULTS: No sequence variations were identified in COL8A1 and COL8A2 in the 2 patients with keratoglobus. Screening of COL8A1 in keratoconus patients revealed a previously identified single nucleotide polymorphism (SNP; c.1850C>T; Pro535Pro), in 1 patient. Screening of COL8A2 in keratoconus patients revealed 7 previously described SNPs: c.14G>A (Gly3Arg); c.112G>A (Ala35Ala); c.1012C>G (Leu335Leu); c.1308G>A (Arg434His); c.1492G>A (Gly495Gly); c.1512C>T (Thr502Met); and c.1765C>T (Pro586Pro). Four novel sequence variants were also identified, each in 1 affected patient: c.38_40dupCTG (Leu11dup), also identified in an unaffected relative of the affected proband, c.667G>A (Gly220Gly), c.1588G>A (Pro527Pro), and c.2026C>T (Val673Val). None of the 3 novel synonymous substitutions identified in COL8A2 was predicted to produce a splice acceptor site. CONCLUSIONS: The absence of pathogenic mutations in COL8A1 and COL8A2 in patients with keratoconus indicates that other genetic factors are involved in the pathogenesis of this corneal ectatic disorder.     

The usefulness of buccal swabs for mutation screening in patients with suspected corneal dystrophies

PURPOSE: To make ophthalmologists aware of the usefulness of buccal swabs for the collection of cells from which DNA may be extracted to be used in genetic screening of patients with known or suspected inherited ocular disorders, such as corneal dystrophies. DESIGN: Illustrative interventional case report. METHODS: Buccal epithelial swabs were collected from a 3-year-old boy with a presumed corneal dystrophy and his unaffected parents. Swabs were mailed to a laboratory where transforming growth factor-beta-induced gene (TGFBI) mutation screening was performed. MAIN OUTCOME MEASURES: Results of sequencing TGFBI exons 4 and 12. RESULTS: Transforming growth factor-beta-induced gene mutation screening in a child with bilateral anterior corneal stromal opacification revealed the following changes in exon 12: 1667T>C (Phe540Phe), 1684C>A (Ala546Asp), and 1699C>A (Pro551Gln). The child's father demonstrated the 1667T>C nucleotide substitution, but neither parent demonstrated the 1684T>C or the 1699C>A changes. CONCLUSIONS: Buccal swabs provide the ophthalmologist with a simple, inexpensive means of collecting and transporting cells from which DNA may be extracted to be used to either confirm or refute diagnoses solely on the basis of clinical features. In this case, the diagnosis of an atypical variant of lattice corneal dystrophy was made on the basis of the identification of previously reported mutations in TGFBI.     

Leu518Pro mutation of the beta ig-h3 gene causes lattice corneal dystrophy type I.

PURPOSE: To describe a Japanese family with lattice corneal dystrophy type I, which segregates with a novel mutation, Leu518Pro of the beta ig-h3 gene. METHODS: DNA was extracted from leukocytes in four members (three affected and one unaffected) of a Japanese family with lattice corneal dystrophy type I. Exon 12 of the beta ig-h3 gene was amplified and analyzed with a molecular biologic method. Clinical data were also collected. RESULTS: Three generations of this family have been positively diagnosed with lattice corneal dystrophy, indicating autosomal dominant inheritance. We found a heterozygous point mutation that segregates with the disease phenotype. It was a single base-pair transition (CTG to CCG, Leu to Pro). CONCLUSION: Although it is extremely rare compared with the Arg124Cys mutation of the beta ig-h3 gene, Leu518Pro mutation of the beta ig-h3 also causes lattice corneal dystrophy type I.     

Identification of novel mutations in the carbohydrate sulfotransferase gene (CHST6) causing macular corneal dystrophy

PURPOSE: Macular corneal dystrophy (MCD) is a rare corneal dystrophy that is characterized by abnormal deposits in the corneal stroma, keratocytes, Descemet's membrane, and endothelium, accompanied by progressive clouding. It has been classified into three immunophenotypes--MCD types I, IA, and II--according to the serum level of sulfated keratan sulfate (KS) and immunoreactivity of the corneal tissue. Recently, mutations in a new carbohydrate sulfotransferase gene (CHST6) encoding corneal glucosamine N-acetyl-6-sulfotransferase (C-GlcNac-6-ST) have been identified as the cause of MCD. Mutation screening of the CHST6 gene has been undertaken to identify the underlying mutations in five unrelated British families with MCD. METHODS: DNA was extracted from venous blood obtained from all participants, and the coding region of CHST6 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing and restriction enzyme digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess the presence of KS in serum from the probands of MCD-affected families participating in the study. RESULTS: Six novel missense mutations--four homozygous and two compound heterozygous--were identified in the CHST6 gene. The ELISA showed that the disease in all patients participating in the study was of MCD type I, including the subtype IA. CONCLUSIONS: These novel mutations are thought to result in loss of corneal sulfotransferase function, which would account for the MCD phenotype.     

Mutation analysis of the carbohydrate sulfotransferase gene in Vietnamese with macular corneal dystrophy

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Anterior basement membrane corneal dystrophy and pseudo-unilateral lattice corneal dystrophy in a patient with recurrent corneal erosions

PURPOSE: To report the utility of genetic testing in the diagnosis and management of patients with suspected corneal dystrophies. DESIGN: Case report. METHODS: A 58-year-old man with a history of recurrent corneal erosions was diagnosed with bilateral anterior basement membrane dystrophy and unilateral lattice corneal dystrophy. All 17 exons of the TGFBI gene were screened for mutations previously associated with lattice corneal dystrophy as well as novel coding region changes. RESULTS: No mutations were found in the 17 exons of the TGFBI gene. A nucleotide change in exon 6 (651C>G) did not result in a change in the encoded amino acid (Leu217Leu). CONCLUSIONS: In cases of suspected TGFBI corneal dystrophies, genetic testing is a useful tool to confirm the clinical diagnosis. In this case of suspected unilateral lattice corneal dystrophy, screening of the TGFBI gene ruled out the diagnosis, raising the possibility that the corneal changes were related to the coexistent anterior basement membrane dystrophy.     

TGFBI and CHST6 gene analysis in Chinese stromal corneal dystrophies

AIM: To investigate whether mutations in TGFBI gene or CHST6 gene correlated with stromal corneal dystrophies (CD) in 8 Chinese probands. METHODS: Eight unrelated patients with stromal corneal dystrophies were recruited in this study; all affected members were assessed by completely ophthalmologic examinations. Genomic DNA was extracted from peripheral leukocytes, 17 exons of TGFBI gene and the exon of CHST6 gene were amplified by polymerase chain reaction (PCR), sequenced directly and compared with the reference database. RESULTS: Three heterozygous mutations in TGFBI gene were identified in six patients: c. 370C>T (p.Arg124Cys) was found in exon 4 of TGFBI gene in three members, c. 371G>A (p.Arg124His) was found in one patient; c. 1663C>T (p.Arg555Trp) was found in exon 12 in other two members. In addition,four polymorphisms with the nucleotide changes rs1442, rs1054124, rs4669, and rs35151677 were found in TGFBI gene. Mutations were not identified in the rest of 2 affected individuals in TGFBI gene or CHST6 gene. CONCLUSION: Within these patients, R124C, R124H and R555W mutations were co-segregated with the disease phenotypes and were specific mutations for lattice corneal dystrophy type I (LCD I), Avellino corneal dystrophy (ACD, GCDⅡ), granular corneal dystrophy type I (GCD I), respectively. Our study highlights the prevalence of codon 124 and codon 555 mutations in the TGFBI gene among the Chinese stromal corneal dystrophies patients.     

Leber先天性黑矇六家系临床表现和遗传分析

目的:观察分析Leber先天性黑矇（LCA）致病基因类型及临床表型特征。方法:回顾性临床研究。基因检测确诊的LCA患者6例及其家系成员18名纳入研究。患者分别来自6个无血缘关系家系。采集所有受检者外周静脉血,提取全基因组DNA。应用包含463个致病基因的遗传眼病捕获芯片进行靶向捕获富集高通量测序,对 TUL...     

Macular corneal dystrophy and associated corneal thinning

Purpose

To identify the molecular genetic cause of macular corneal dystrophy (MCD) in four probands, and characterize phenotypic similarities between MCD and keratoconus.

Methods

We performed ophthalmological examination, Scheimpflug imaging (Pentacam, Oculus Inc.), histopathological examination of excised corneal buttons, and direct sequencing of the CHST6 coding region.

Results

Pentacam measurements were taken in six eyes of three probands. All showed diffuse corneal thinning with paracentral steepening of the anterior corneal surface that was graded as keratoconus by the integrated software, but without associated ectasia of the posterior corneal surface or regional thinning. Homozygous or compound heterozygous CHST6 mutations were identified in all cases, including two novel mutations, c.13C>T; p.(Arg5Cys) and c.289C>T; p.(Arg97Cys).

Discussion

Localized elevation of the anterior corneal curvature can occur in MCD in the absence of other features of keratoconus. The identification of a further two Czech probands with the compound allele c.[484C>G; 599T>G] supports the enrichment of this allele in the study population.     

结晶样视网膜色素变性中与CYP4V2基因相关的致病突变

目的：使用Sanger测序法鉴定2个中国结晶样视网膜色素变性(BCD)家系中CYP4V2基因的突变位点。

方法：收集2019-01/09临床诊断为BCD患者的临床相关资料。采集患者、患者家系成员的外周血,提取DNA,利用Sanger测序法鉴定突变位点。

结果：共收集2个BCD先证者,先证者均表现为渐进性视力下降,眼底均可见典型的结晶样物质沉积。测序发现先证者1及其患病的哥哥,妹妹均在CYP4V2基因上存在c.802-8_810del17insGC的纯合突变。而先证者2则在CYP4V2基因上存在c.219T>A(p.F73L)、c.802-8_810del17insGC杂合突变。

结论： 中国BCD患者中最常见的c.802-8_810del17insGC突变在先证者1家系中为纯合突变,是其家系的致病突变。而先证者2则携带了中国BCD患者最常见的c.802-8_810del17insGC杂合突变,同时先证者2还携带c.219T>A(p.F73L)错义突变,突变均影响了CYP4V2基因的正常编码,进而导致疾病。     

Homozygous and heterozygous retinal phenotypes in families harbouring IMPG2 mutations

Introduction: Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) have been shown to underlie recessive childhood-onset rod-cone dystrophy with early macular involvement in several families. In other families, heterozygous IMPG2 mutations have been associated with dominant vitelliform macular dystrophy. To date, the retinal phenotype of heterozygotes from families with recessive IMPG2-related retinal dystrophy has not been assessed. This study documents the genotypes and phenotypes observed in both homozygotes and available heterozygotes from additional families with IMPG2-related recessive rod-cone dystrophy.

Methods: Retrospective case series (2016–2018).

Results: Four families were identified. All were first-cousin marriages and had no known relation to each other. Individuals with biallelic pathogenic variants (7 individuals) had childhood-onset rod-cone dystrophy. Families 1 and 2 harboured the same novel homozygous mutation c.189dup;p.Gln64Thrfs*9 (5 individuals, 4–17 years old). Family 3 harboured the novel homozygous mutation c.533 + 4_533 + 7del;p.? (1 individual, 17 years old), and Family 4 harboured the previously reported homozygous mutation c.3262C>T;p.Arg1088* (1 individual, 45 years old). The 3 available carriers were genetically confirmed (both parents from Family 1 and the father from Family 3) and had macular focal retinal pigment epithelium thickening by optical coherence tomography (OCT). The father from Family 3 also had unilateral sectoral pigmentary retinopathy.

Conclusions: Childhood-onset recessive rod-cone dystrophy with early macular involvement should prompt examination of the parents for macular focal retinal pigment epithelium thickening on OCT. If present the possibility of biallelic IMPG2 mutations in the proband should be considered. Young affected relatives of the proband can show multimodal imaging abnormalities before they are overtly symptomatic.     

Mutations in the CHST6 gene in patients with macular corneal dystrophy: immunohistochemical evidence of heterogeneity

PURPOSE: Macular corneal dystrophy (MCD) is an autosomal recessive disorder leading to severe visual impairment. The carbohydrate sulfotransferase 6 (CHST6) gene, which encodes the corneal N-acetylglucosamine 6-O-sulfotransferase on 16q22 has been identified as a causative gene for MCD. The purpose of this study was to identify mutations in CHST6 in Japanese patients with MCD and evaluate them by means of immunohistochemistry. METHODS: CHST6 was screened in 7 patients and 45 healthy control subjects. Genomic DNA was isolated, and the open reading frame (ORF) of CHST6 was amplified by polymerase chain reaction (PCR). PCR products were analyzed by direct sequencing and restriction enzyme digestion. Immunohistochemistry with a monoclonal anti-keratan sulfate (KS) antibody was performed on corneas of four patients from three families. RESULTS: Three novel mutations (P204Q, R205L, and R177H) and two previously reported mutations (R211W and A217T) were identified in the ORF of CHST6. P204Q, R205L, and R211W were found to be homozygous and R177H and A217T compound heterozygous with R211W on another allele. Immunohistochemistry revealed that R205L homozygous cornea had negative reactivity against the anti-KS antibody, representing type I MCD, and that R211W homozygous and R211W/A217T compound heterozygous corneas had negative or very weak reactivity in the stroma with antibody positive deposits, which were distinct from any previously reported types. CONCLUSIONS: Two mutations (homozygoous R211W and compound heterozygous R211W/A217T) should be subclassified immunohistochemically into new phenotypes of MCD. This heterogeneity could provide further insights into the pathogenesis of MCD.