|Molecular Vision 2006;
Received 13 October 2005 | Accepted 8 March 2006 | Published 10 March 2006
CHST6 mutations in North American subjects with macular corneal dystrophy: a comprehensive molecular genetic review
Gordon K. Klintworth,1,2 Clayton F.
Smith,2 Brandy L. Bowling2
Departments of 1Pathology and 2Ophthalmology, Duke University Medical Center, Durham, NC
Correspondence to: Gordon K. Klintworth, MD, PhD, Duke University Medical Center, Box 3802, Durham, NC, 27710; Phone: (919) 684-3550; FAX: (919) 684-9225; email: Klint001@mc.duke.edu
Purpose: To evaluate mutations in the carbohydrate sulfotransferase-6 (CHST6) gene in American subjects with macular corneal dystrophy (MCD).
Methods: We analyzed CHST6 in 57 patients from 31 families with MCD from the United States, 57 carriers (parents or children), and 27 unaffected blood relatives of affected subjects. We compared the observed nucleotide sequences with those found by numerous investigators in other populations with MCD and in controls.
Results: In 24 families, the corneal disorder could be explained by mutations in the coding region of CHST6 or in the region upstream of this gene in both the maternal and paternal chromosome. In most instances of MCD a homozygous or heterozygous missense mutation in exon 3 of CHST6 was found. Six cases resulted from a deletion upstream of CHST6.
Conclusions: Nucleotide changes within the coding region of CHST6 are predicted to alter the encoded protein significantly within evolutionary conserved parts of the encoded sulfotransferase. Our findings support the hypothesis that CHST6 mutations are cardinal to the pathogenesis of MCD. Moreover, the observation that some cases of MCD cannot be explained by mutations in CHST6 suggests that MCD may result from other subtle changes in CHST6 or from genetic heterogeneity.
Macular corneal dystrophy (MCD; OMIM 217800), a rare inherited disorder first described in 1890 by Groenouw , has been identified throughout the world. MCD is most prevalent in India [2-4] and Saudi Arabia . In some countries, this disease accounts for a high percentage (10-75%) of the corneal dystrophies requiring keratoplasty [6,7]. Clinically, MCD is characterized by a cloudiness of the cornea and irregularly shaped superficial opacities of both eyes that progressively extend through the entire thickness of the central and peripheral corneal stroma. The corneal stroma is thinner than normal [8-10]. In 1938, its autosomal recessive mode of inheritance became appreciated . More than two decades later Jones and Zimmerman [12,13] differentiated MCD histopathologically from the other two major stromal corneal dystrophies known as granular and lattice corneal dystrophy. Histopathologically, MCD is typified by an intracellular storage of glycosaminoglycans (GAGs) within keratocytes and the corneal endothelium combined with an extracellular deposition of similar material in the corneal stroma and Descemet's membrane [5,14]. Guttae are common on Descemet's membrane . In 1964, because of similarities to the systemic mucopolysaccharidoses, Klintworth and Vogel  suggested MCD might be a mucopolysaccharidosis localized to the cornea. A metabolic defect in keratan sulfate (KS), the major corneal GAG , was suspected because of the histochemical attributes of the corneal accumulations and that the cornea was the only site of overt abnormalities. Cell culture studies disclosed differences from the systemic mucopolysaccharidoses , and analyses of sulfated GAGs produced by organ cultures of corneas with MCD led to the discovery that corneal tissue with MCD does not synthesize KS  or normal KS containing proteoglycans (PGs) because of defective sulfation [20,21]. Immunochemical studies using an antibody that recognizes antigenic KS (AgKS) disclosed heterogeneity among cases of MCD based on the reactivity of corneal tissue with the antibody . Subsequently this led to the recognition of three immunophenotypes that are clinically and histopathologically indistinguishable from each other [5,22-24]. Most often neither the serum nor the corneal tissue contain AgKS (MCD type I), but sometimes AgKS is absent in the corneal stroma and the serum but can be detected in the keratocytes (MCD type IA). A third immunophenotype (MCD type II) is characterized by the presence of AgKS in corneal tissue and detectable serum levels of AgKS that are often present in normal amounts. MCD type I corneas have been found to synthesize an abnormal KS-PG with lactosaminoglycan side chains that lack sulfate. In sharp contrast, an MCD type II cornea produced a normal ratio of KS-PG to dermatan sulfate-PG, but the net synthesis of PGs was below normal . However, the chondroitin/dermatan sulfate side chains on decorin were sulfated. Subsequently, fluorochrome-assisted carbohydrate electrophoresis disclosed that the KS chain size within the cornea and cartilage in MCD type I was reduced and chain sulfation was absent . In a cornea with MCD type II, the sulfation of N-acetylglucosamine and galactose was significantly reduced and the chain size was also reduced, but to a lesser degree than in MCD type I .
The absence or paucity of sulfate in KS and KS-containing PGs in corneas with MCD suggested that patients with MCD were deficient in a carbohydrate sulfotransferase that catalyzes the transfer of sulfate groups from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to KS in a manner comparable to which sulfate esters are put on other endogenous and exogenous substrates (PAPS + R-OH ->PAP + R-OSO3). An analysis of the serum in patients with MCD type I disclosed normal levels of enzymatic activity for sulfating at least one of the two sugars present in KS and an enzyme deficient for sulfating N-acetylglucosamine (GlcNAc) was thought to be present . Because the serum KS is considered to be derived from cartilage, the discovery of undetectable levels of AgKS in the serum of individuals with MCD type I suggested that the KS sulfotransferase deficiency was not restricted to the cornea. Direct evidence of cartilage involvement was obtained later in cartilage from the nose and ear in MCD type I [26,28]. Cartilage lacks abnormal accumulations comparable to those in the cornea, but the chondrocytes and extracellular matrix do not contain AgKS and the KS content of cartilage is at least 800 times lower than normal .
Using families from Iceland, where MCD was the most frequent indication for penetrating keratoplasty , the gene for MCD type I was mapped to chromosome 16 (16q22) and linkage data hinted that MCD type II was also at this locus . Fine mapping refined the location of the MCD gene. Two candidate genes (TAT and LCAT) in that part of chromosome 16 were excluded [30-32].
A sulfotransferase suspected of being defective in MCD would belong to the galactose/N-acetylgalactosamine/N-acetylglucosamine 6-O-sulfotransferase family of carbohydrate sulfotransferases. This family of enzymes catalyzes 6-O-sulfation on the 6-hydroxyl of GlcNAc, galactose (GAL), or N-acetylgalactosamine (GalNAc) . All known sulfotransferases contain highly conserved regions , including a 5'-PSB (phosphosulfate-binding) and a 3'-PB (3'-phosphate binding domain) . The carbohydrate sulfotransferases in different species possess marked sequence similarities at the amino acid level particularly in their catalytic domains and, with the exception of CHST3 (also known as GST-0), the coding sequences of the open reading frames (ORFs) for all CHST genes are within a single exon . The galactose/N-acetylgalactosamine/N-acetylglucosamine 6-O-sulfotransferases have been designated as belonging to the GST family , but this term is not favored because they can be confused with glutathione transferases.
One potential candidate for the MCD disease gene was CHST1 (also known as GST-1) that encodes for KS 6-O-sulfotransferase, but it could be excluded because its gene had been mapped to human chromosome 11 (11p11.1-11.2) . Further research on carbohydrate sulfotransferases resulted in finding a cluster of three carbohydrate sulfotransferases with apparent tissue restrictions on human chromosome 16 . CHST4, which encodes high endothelial cell GlcNAc 6-O-sulfotransferase, was mapped to human chromosome 16 (16q23.1-23.2) . Shortly thereafter, two more highly homologous carbohydrate sulfotransferases genes were identified in the same region by independent investigators [33,36]. These genes were CHST5 (also known as GST-4α and intestinal GST)  and CHST6 (also designated GST-4β) [36,39].
Because the gene responsible for MCD was suspected of encoding for a carbohydrate sulfotransferase and had been fine mapped to the same region as CHST5 and CHST6, these genes became logical suspects for the MCD disease gene. In 2000, Akama et al.  discovered mutations in CHST6 in MCD and also found insertional or deletional defects in the region between CHST5 and CHST6 in some cases. Subsequently, several laboratories confirmed these observations [2-4,39-52].
This report documents an analysis of CHST6 in 57 patients from 31 families from the United States with MCD, and reviews these findings with all previously documented mutations.
We analyzed CHST6 in 141 individuals (57 affected patients, 57 carriers [parents or children], and 27 normal blood related family members) from 31 families with MCD. In all instances, the diagnosis of MCD was made on a combination of the characteristic clinical features together with the typical histopathologic findings in the corneal tissue obtained following a penetrating keratoplasty in one or both eyes. All patients were born in the United states and some ancestral lines could be traced to England, Ireland, Germany, Holland, Northern Ireland, Scotland, Switzerland, and Norway. Nonmolecular genetic studies have been previously reported on some of these cases [14,16,18,19,53-56]. This study was approved by the Internal Review Board of Duke University and conformed to the tenets of the declaration of Helsinki. Written informed consent was obtained from all participants. To evaluate the CHST6 gene in 11 deceased or unavailable patients from five families with MCD, we analyzed the DNA of surviving siblings, parents, and children to establish which mutations had been transmitted to the carriers. We also analyzed DNA from 50 control specimens from the United States and DNA from 10 normal spouses who married into these families.
Determination of MCD immunophenotypes
Prior to molecular genetic studies, the immunophenotypes had been determined in 32 of the 57 subjects with MCD by ascertaining the presence or absence of sulfated epitopes in KS in the corneal tissue and/or serum. The reactivity of formalin-fixed paraffin-embedded corneal tissue sections with the 5D4 anti-KS monoclonal antibody was evaluated immunohistochemically at a dilution of 1:5,000 using the avidin-biotin immunoperoxidase complex method . For negative controls, diluted normal mouse serum was used. Serum AgKS levels were measured in 22 patients with MCD with an ELISA using an anti-KS monoclonal antibody (5D4; ICN Biomedical Inc., Costa Mesa, CA) directed against a highly sulfated epitope present on long KS chains . These immunochemical studies were assessed in a masked fashion without clinical information or knowledge of the serum levels of AgKS.
The coding region and upstream region of CHST6 in 57 patients, 57 carriers (parents or children), and 27 unaffected blood relatives from 31 families with MCD were screened for mutations. In 46 patients with MCD, CHST6 was analyzed on their own DNA, and it was done indirectly in 11 other affected subjects by studying the DNA of carriers related to them. Ten spouses of patients or their family members were also screened. DNA was isolated from blood relatives of 11 patients who were no longer available for study. DNA was extracted from peripheral blood leukocytes using the PuregeneTM Blood Kit (Gentra Systems, Minneapolis, MN) or from buccal samples using the PuregeneTM Buccal Cell Kit (Gentra Systems) as previously described by Liu et al. . DNA was quantitated using the NanoDrop® ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
Analyses with insufficient DNA
In cases when insufficient DNA was available for a complete analysis of CHST6, samples of genomic DNA were amplified using a protocol for the whole genome amplification (WGA) kit GenomiPhi (GE Healthcare, Piscataway, NJ, formerly Amersham Biosciences) [58-61].
Molecular analysis of the coding region of CHST6
The coding region of CHST6 was amplified in three overlapping fragments from genomic DNA by the polymerase chain reaction (PCR) using the same three pairs of primers as Akama et al.  (primers 17 and 19, 18 and 21, 20 and 22; Figure 1, Table 1). Each PCR was carried out in a 50 μl reaction mixture using the components of the Qiagen Taq DNA Polymerase Kit (Qiagen, Valencia, CA). Each reaction consisted of 1X PCR Buffer containing 1.5 mM MgCl2, 1X Q solution, 0.2 mM dNTPs, 0.2 μM of each primer, 1 unit of Taq DNA polymerase (Qiagen), and 200-800 ng of genomic DNA. Amplification was performed in a PTC-225 PeltierThermal Cycler (MJ Research, Waltham, MA). PCR conditions for all three amplicons of the coding region were as follows: 2 min at 95 °C and 31 cycles of 1 min at 95 °C, 1 min at 60 °C, and 1 min at 72 °C, followed by a 4 °C hold cycle.
The resulting amplified PCR products were purified using QIAquick® PCR Purification Kit (Qiagen) and then sequenced on both strands using a fluorescent Big Dye Terminator Cycle Sequencing system (Applied Biosystems, Foster City, CA) combined with an ABI 377 PRISM DNA Sequencing instrument (Applied Biosystems). The sequences were then aligned to the CHST6 cDNA using the SeqWeb Sequence Analysis web-based program (Accelrys, San Diego, CA) to seek changes in the nucleotide and amino acid sequences of exon 3 in comparison to the published cDNA sequence of CHST6 (GenBank accession number NM_021615) and reported mutations. To help evaluate the significance of identified CHST6 nucleotide changes in patients with MCD, we compared them with the sequences found in other genes encoding the related family of carbohydrate sulfotransferases and with the protein sequences of conserved domains in the Conserved Domain Database (CDD) within the Entrez system NCBI Conserved Domain Database .
Analysis of genomic DNA sequence upstream of the coding region of CHST6
Genomic DNA was screened for rearrangements upstream of the coding region of CHST6 according to the method of Akama et al.  using the same conditions as described. The primer pairs were the same except that primer number 2' was replaced by primer number 2, and primer number 6' was changed to primer number 6 (Figure 1, Table 1). HotStarTaq® DNA polymerase (QIAGEN) was used and the annealing temperatures were adjusted to 58 °C and 62 °C for the primer pair number 8 and number 14 and the primer pair number 2 and number 6, respectively. In our experience the changing of primers number 2' and number 6' to number 2 and number 6 yielded more consistent results, perhaps because the original primers were subject to potential mispairing due to the high degree of sequence similarity of CHST5 and CHST6 in this location. All PCR amplicons were electrophoresed on 2% agarose gels, and the gels were documented using the BioChemi Image Acquisition and Analysis Software (UVP BioImaging Systems, Upland, CA). All amplicons indicating upstream DNA rearrangements were sequenced to confirm their nucleotide order. Because two of the resulting amplicons with our substituted primer pairs extended into less homologous areas, their sequences could be more readily verified.
Evaluation of splice regions
In cases of MCD that could not be explained on the basis of mutations in either the coding region of CHST6 or upstream of CHST6, possible splice site mutations were evaluated by performing PCR. This involved using primer pairs that covered the exons and their splice sites (exon 1 [primer number 7 and 8], exon 2 [number 9 and 10], exon 3 [primers number 11 and 12, and number 13 and 14] and exon 4 [primers number 15 and 16]). The resulting amplicons were sequenced to determine if any nucleotide changes were present (Figure 1, Table 1).
Determination of chromosomal location of mutations
Two approaches were used to determine whether detected mutations were on the same or different chromosomes. When possible, genomic DNA from the parents, children, and siblings were analyzed. Parents, children, and siblings of those affected with a heterozygous CHST6 mutation were all presumed to be carriers of the MCD disease gene. When specimens of DNA were not available from appropriate family members, we determined whether multiple mutations were on the same or different chromosomes by digesting PCR products with Surveyor nuclease (Transgenomic, Inc., Omaha, NE), which cleaves double-stranded DNA at mismatched nucleotides . Amplicons containing the mutations were heat denatured and mixed with amplicons from controls. After annealing, the heteroduplexes were digested with Surveyor nuclease, and the products were sized on an agarose gel.
The most frequent abnormalities found in this study were single base changes in the coding region of CHST6 that altered a coded amino acid. We identified 23 such examples in exon 3 of CHST6 (Table 2, Table 3).
Single nucleotide polymorphisms
We identified four single nucleotide polymorphisms (SNPs) that did not affect an amino acid (c.258A>C [Ala86Ala], c.294C>G [Ser98Ser], c.465G>A [Arg155Arg], and c.681C>T [Gly227Gly]) and which were presumably insignificant as known mutations could account for MCD in the affected individuals. In 14 individuals with MCD from five families (family 7, 13, 15, 22, and 26), c.484C>G (Arg162Gly) was associated with c.599T>G (Leu200Arg), but c.484C>G (Arg162Gly) was clearly not pathogenic. Arg162Gly was found in 11 blood relatives of persons with MCD and six of 50 American controls were heterozygous for this nucleotide change. Moreover, an unaffected 25-year-old son of a person with MCD in family 26 was homozygous for this change. He had no vision difficulties and a visual acuity of 20/15 when last examined at 23 years of age. The unaffected parent was heterozygous for Arg162Gly and has no other nucleotide changes in CHST6. Another unaffected 36-year-old sister of a patient with MCD in family 28 was also homozygous for this change. Her 61-year-old mother was heterozygous for Arg162Gly and carried Cys246Trp on the other chromosome, but had no corneal disease. An unaffected daughter of a patient in family 7 was homozygous for Arg162Gly and heterozygous for Leu200Arg. Her unaffected parent was heterozygous for Arg162Gly and has no other nucleotide changes in CHST6.
In the analysis of the splice sites, two previously reported SNPs were detected in the 3'-UTR region of exon 4 (c.3342A>T, rs424964 and c.3501G>A, rs10871313), which presumably are common variants in the general population. Some unaffected family members were homozygous for these SNPs. We have annotated nucleotide changes which are downstream from the coding region based on NM_021615 as it is considered the best estimate of the mRNA boundaries at this time (Personal communication, L. A. Pennacchio, DOE Joint Genome Institute, June, 2005).
Number of mutations in coding region
Although the coding region of CHST6 in MCD most often contained a single mutation, more than one nucleotide change on a single chromosome was detected in 11 families (families 6, 7, 9, 12, 13, 15, 22, 23, 26, 28, and 30). In family 20, some unaffected members contained two SNPs in the same chromosome, neither of which changed an amino acid. In three families (families 9, 12, and 30), the coding region of CHST6 contained two nucleotide changes within the same chromosome, both of which were consistent with disease-producing mutations (Ser131Leu plus Thr228Asp [family 30], Leu200Arg plus Arg334Cys [family 9], and His63Gln plus Arg114Cys [family 12]).
Insertions, deletions, frameshifts, and substitutions
Nucleotide deletions were found in the coding region of CHST6 in family 10 (c.51delG) and family 13 (c.740delG). An insertion was detected in family 23 (c.573_574insC), and family 3 had a complex mutation with a combination of a deletion plus an insertion at the same site (c.271_273delGCTinsA). Nucleotide insertions within exon 3 of CHST6 (c.573_574insC) caused a frameshift in family 23 (Ala192fs). Nucleotide deletions were found in the coding region of CHST6 in four families. Single nucleotide deletions resulted in frameshifts in family 10 (c.51delG, Gln18fs) and family 13 (c.740delG, Ala247fs). The entire coding region was deleted in family 5 and family 18. In family 5, exons 1 and 4 were amplifiable with PCR, but in family 18, none of the 4 exons were amplifiable. A deletion of 3 nucleotides and an insertion of 1 nucleotide (c.271_273delGCTinsA) in Family 3 resulted in a frameshift (Ala91fs). A nucleotide substitution (c.231G>A) in family 4 generated a heterozygous stop codon (Trp77X) which is predicted to encode a truncated sulfotransferase.
Mutations 5'-UTR upstream of CHST6
Four families (families 1, 11, 16, and 19) had deletions upstream of CHST6. In all cases these were 40 kb deletions from a region upstream of CHST5 to a homologous region upstream of CHST6 (Figure 2). In two of them, the deletions were heterozygous and each was associated with an additional mutation in the coding region of CHST6 on the other chromosome (Ser53Leu, Gln122Pro). One was found in a boy whose grandmother suffered from MCD due to homozygous Gln122 Pro mutation (family 1). A single copy of this mutation was inherited by the grandson, and the upstream deletion was inherited from his father. In family 16, there was a heterozygous upstream deletion and a heterozygous c.1A>T mutation (Met1?; Human Genome Variation Society discussions regarding the description of sequence variants), but it could not be determined whether or not they were on separate chromosomes. A homozygous upstream deletion was present in family 11.
Findings in splice regions
Splice site mutations were not found in the two cases of MCD from family 24 which could not be explained by mutations in the coding region of CHST6 or upstream of the gene.
Evaluation of maternal and paternal chromosomes
Because MCD is an autosomal recessive disorder, all affected individuals are expected to inherit a mutant gene from both the mother and the father. In 24 of the 31 families the MCD could be explained on the basis of two or more mutations in the CHST6 coding region and/or by an upstream deletion (Table 2, Table 3). Of the seven families that were not proven to have CHST6 mutations on both chromosomes, one family (family 23) was found to have two mutations. We were unable to establish whether they were or were not on the same chromosome because there was not enough DNA to amplify. In one other family (family 24), neither sequence analysis of the coding region of CHST6 nor a screening of the upstream region using the method of Akama et al.  disclosed mutations capable of explaining the genetic basis for MCD. In one other family (family 16), there was a heterozygous upstream deletion and a heterozygous coding region mutation, but it could not be determined if they were or were not on the same chromosome. In one family (family 27) there were no coding region mutations and there was insufficient DNA to determine whether or not upstream deletions or replacements were present. In three other families (families 29, 30, and 31), DNA could not be obtained from affected family members, and in each of these families, only one chromosome-carrying mutation could be detected among family members who were carriers.
Heterozygous mutations were detected in exon 3 of CHST6 in 18 families (families 1, 3, 4, 6, 7, 9, 10, 13, 15, 16, 17, 19, 20, 22, 23, 25, 26, and 28) and almost all were in association with another heterozygous mutation in the coding region of CHST6 on the other chromosome. The three exceptions had an upstream deletion (families 1, 16, and 19) on the other chromosome. In 14 instances, two or more independent heterozygous mutations were present. Aside from having two heterozygous nucleotide changes in CHST6 (c.484C>G, Arg162Gly and c.599T>G, Leu200Arg), family 9 had another homozygous missense mutation (c.1000C>T, Arg334Cys). Family 23 had two heterozygous mutations, but it was not possible to establish whether they were or were not on the same chromosome because of insufficient DNA. In family 1, one subject (case 1) had a homozygous c.365A>C (Gln122Pro) mutation, whereas an affected sibling (case 2) had a heterozygous c.365A>C(Gln12Pro) mutation together with a deletion upstream of CHST6 on the other chromosome 16.
Homozygous mutations were detected in the coding region of CHST6 in nine families (families 1, 2, 5, 8, 9, 12, 14, 18, and 21), and in two of them (families 5 and 18) a major portion of CHST6 that included the ORF was deleted. Another family (family 11) had a homozygous deletion upstream from CHST6. One family (family 9) had a homozygous c.1000C>T (Arg334Cys) mutation, but also two heterozygous mutations (c.484C>G, Arg162Gly; c.599T>G, Leu200Arg). In another family (family 1), one affected individual was homozygous for a single mutation (c. 365A>C, Gln122Pro), while another affected individual was heterozygous for this mutation, but also had a deletion upstream of CHST6 on the other chromosome. Whereas homozygous mutations are expected in the offspring of inbred matings, we were only able to establish consanguinity in two families with extensive genealogical analyses.
Results related to the MCD type
Most subjects studied had MCD type I (26 patients from 13 families). Six affected individuals with MCD type II were from four families (families 24, 27, 30, and 31). The c.231G>A mutation that generated a stop codon (Trp77X) was found in three subjects with MCD type 1. Affected subjects in two families (families 24 and 27) with MCD type II lacked mutations in the coding region of CHST6, and those in family 24 also had no deletion or insertion upstream of this gene. In family 27, there was insufficient DNA to determine whether or not there were upstream deletions or insertions. DNA was not available in two subjects with MCD type II but one allele in these cases could be determined by analyzing DNA from children of the subjects. The nucleotide changes in CHST6 in each of these carriers were c.392C>T (Ser131Leu), c681C>T (Gly227Gly), c.682A>G, 683C>A (Thr228Asp; family 30), and c.607G>A (Asp203Asn; family 31). In 14 families (19 affecteds and 23 carriers), the MCD immunophenotype was not determined.
Eighteen of the mutations found in this investigation have apparently not been reported before (c.51delG [Gln18fs], c.137T>C [Leu46Pro], c.189C>G [His63Gln], c.217G>C [Ala73Pro], c.231G>A [Trp77X], c.274G>C [Val92Leu], c.340C>T [Arg114Cys], c.392C>T [Ser131Leu], c.529C>T [Arg177Cys], c.573_574insC [Ala192fs], c.607G>A [Asp203Asn], c.682A>G, 683C>A [Thr228Asp], c.738C>G [Cys246Trp], c.740delG [Arg247fs], c.744C>G [Ser248Arg], c.815G>A [Arg272His], c.1046G>A [Cys349Tyr], and c.1047C>G [Cys349Trp]), but 12 have been previously documented. They include c.1A>T (Met1?) , c.91C>T (Pro31Ser)  c.158C>T (Ser53Leu) [2,3], c.271_273delGCTinsA (Ala91fs) , c.277C>A (Arg93Ser) , c.363C>G (Phe121Leu) , c.365A>C (Gln122Pro) , c.599T>G (Leu200Arg) [41-43,51,52], c.827T>C (Leu276Pro) , c.1000C>T (Arg334Cys) , upstream deletions [33,52], and a deletion of the coding region [2,33]. Together with past reported genomic DNA analyses, this brings the number of identified CHST6 mutations in MCD to 124 (Table 4). This vast number underscores the marked allelic heterogeneity in CHST6 that has been previously documented in subjects with MCD in Britain , France , Iceland , India [2-4], Italy , Japan [33,47], Saudi Arabia , United States [43,46,50], and Vietnam [45,49]. The vast majority of subjects with MCD have missense and nonsense mutations in CHST6 that involve a single nucleotide change that is predicted to alter a conserved amino acid [2-4,33,39-45,47-52]. That these nucleotide changes are disease related is supported by the fact that they have not been detected in analyses of CHST6 in numerous control subjects in different parts of the world [2-4,33,40-45,47,49,52]. In an earlier study of Icelandic individuals, c.383C>T (Ala128Val) was detected in four of 50 healthy controls. However, it was most likely a missense mutation occurring in carriers, because it was associated with MCD when present in a homozygous state .
Other MCD-causing mutations are nucleotide insertions or deletions in the coding region of CHST6 that cause frameshift changes and a few deletions or substitutions upstream of CHST6. The latter is a consequence of the marked sequence similarity of nucleotide sequences within the tandem CHST5 and CHST6 genes and the adjacent regions. It predisposes the region to anomalous chromosomal crossovers that lead to deletions and replacements within the involved chromosomes (Figure 2). Such defects in the promoter region were detected in six subjects with MCD in the present study and in 12 previously documented cases [33,52]. Fifty-five of the documented CHST6 mutations have been detected in more than one family with MCD; some of these families may be related to each other as a common ancestor has not been excluded yet.
Our detection of four SNPs in CHST6 that did not affect an amino acid (c.258A>C [Ala86Ala], c.294C>G [Ser98Ser], c.465G>A [Arg155Arg], and c.681C>T [Gly227Gly]) together with other reported SNPs brings the number of identified SNPs in CHST6 to 88. SNPs outside of the coding region could affect gene expression by altering or activating splice sites or by affecting the stability or translational efficiency of the transcript , but in individuals in whom we found these SNPs the MCD could be explained by other mutations. Although SNPs in the 3'-UTR have been reported to affect expression  and mRNA stability , the ones we detected (c.3342A>T and c.3501G>A) were also homozygous in some unaffected family members, indicating that they are not involved in the pathogenesis of MCD.
As illustrated in Figure 3, mutations found in MCD have been in highly conserved parts of carbohydrate 6-O-sulfotransferases, suggesting that the altered amino acids impair the normal functioning of the encoded enzyme and are pathogenic. Seventeen mutations involve the domains necessary for the interaction between the sulfotransferase and the sulfate donor PAPS . Six of these mutations are situated within the 5'-PSB (5'-phosphosulfate-binding) domain that interacts with the 5'-phosphate group of PAPS , and 11 mutations involve the 3'-phosphate-binding domain that interacts with the 3'-phosphate of PAPS. There are also four mutations in the hydrophobic site, but none in any of the four glycosylation sites.
All SNPs in the coding region of CHST6 that alter a codon are presumed to be of pathogenic significance because a conserved amino acid is altered. However, at least one SNP (c.484C>G), which is predicted to change arginine to glycine at codon 162, does not seem to alter the encoded sulfotransferase significantly. This SNP has been previously reported in 6.2% of an American control sample  and in 9.4% of Italian controls . We have also observed it in 12.0% of an American control sample and in 16.2% of an Icelandic control population (unpublished). Moreover, Abbruzzese et al.  suggested that this amino acid change does not affect enzyme activity since it is not conserved in mouse intestinal GlcNAc 6-O-sulfotransferase which has been shown to have the same enzymatic activity as human corneal GlcNAc 6-O-sulfotransferase .
Aside from the marked allelic heterogeneity of CHST6 in MCD, it is noteworthy that 10 families (families 6, 9, 12, 13, 15, 22, 23, 26, 28, and 30) with MCD in the present study had two SNPs in the coding region of CHST6 in a single chromosome. In addition to our study, other cis SNP combinations have included c.6G>A; 7C>A (Trp2X and Leu3Met) , c.166G>A; 167T>G; 500C>T (Val56Arg and Ser167Phe) , c.213G>T; 214C>T (Glu71Asp and Pro72Ser) , c.293C>G; 294C>G (Ser98Trp) , c.293C>T; 294C>G (Ser98Leu) , c.494G>C; 495C>T (Cys165Ser) [4,44], c.668G>A; 993G>T (Gly223Asp and Gln331His) , and c.484C>G; 599T>G (Arg162Gly and Leu200Arg) [43,52]. Together, these observations suggest that this part of human chromosome 16 may be prone to mutations that may not be readily corrected by the usual DNA repair mechanisms.
One case of MCD in this study could not be explained by mutations in the coding region of CHST6, by major deletions or insertions in the upstream region, or by splice site mutations which create or destroy signals for exon-intron splicing . Others have also failed to detect mutations on one or both alleles of individuals with MCD [2-4,42,43]. A genetic explanation for these cases of MCD remains unknown, but several theoretical possibilities exist. It is conceivable that the relevant abnormality involves transcription factor binding sites (regulatory promoters) in the region immediately upstream of the minimal promoter that direct the expression of the gene or cis-acting distant genomic elements (enhancers, repressors, or insulators) located upstream or downstream of the transcription unit . In this regard, Hemmerich et al. , suggested that the closely proximated CHST5 and CHST6 genes may be regulated by common promoters and/or enhancers, and they drew attention to a conspicuous triplet of binding sites for the zinc-dependent Sp1 transcription factor upstream of CHST6, which has three contiguous Zn (II) fingermotifs believed to be metalloprotein structures that interact with DNA. The Sp1 binding sites are present in the 5'-regulatory sequences of numerous genes that encode carbohydrate-modifying enzymes . In other protein-coding genes, a 5'-promoter element contiguous with the transcription site (the minimal or core promoter) of the gene is necessary to splice the exons needed to produce the mRNA that encodes the appropriate protein . This is presumably also true for CHST6. Other yet to be identified regulatory elements of CHST6 may also play a role, and genes on other chromosomes may affect the gene expression as well. A defect may reside in the 5'-untranslated region (5'-UTR) of CHST6, which has not been fully analyzed, because this part of other genes contributes to the specificity and overall efficiency of translation initiation . Moreover, genetic heterogeneity remains to be excluded.
In their landmark publication, Akama et al.  provided evidence that MCD type II might be caused by genetic abnormalities upstream of CHST6. While a defect in the promoter region of CHST6 is expected to control corneal sulfotransferase activity and hence might cause the milder immunophenotype of MCD characterized by AgKS in the serum and cornea, other cases of MCD type II have not only had other mutations in the coding region of CHST6, but have also lacked evidence of a defect upstream of CHST6. A possible molecular explanation for the different immunophenotypes of MCD was provided by an unusual sibship that contained both MCD types I and II [30,50]. In that family, the sibling with the greater deficiency of AgKS (MCD type I) had homozygous c.418C>T (Arg140X) mutations in CHST6, which is predicted to generate a stop-codon and hence a truncated sulfotransferase. The individuals with the milder MCD type II in the sibship were heterozygous for c.418C>T and for cis c.993G>T and c.668G>A nucleotide changes in CHST6 and would presumably still have some residual enzymatic function albeit defective. It is noteworthy that the one family in the present study with a heterozygous stop codon in the coding region of CHST6 (family 4) had MCD type I. However, the molecular basis for the different immunophenotypes is clearly more complex as a molecular genetic study of MCD in Saudi Arabia found identical CHST6 mutations in families with MCD types I, IA, and II .
Despite overwhelming evidence implicating CHST6 in the pathogenesis of MCD, an understanding of the molecular events that lead to the characteristic lesions of MCD remain incomplete. The intracellular storage of GAGs may reflect a failure of transport of PGs with insufficiently sulfated lactosaminoglycans from the Golgi apparatus, but the characteristic extracellular deposits in Descemet's membrane remains to be explained. Also, a knockout of CHST6 might be expected to cause MCD, but mice and other nonprimates lack CHST6 and can be viewed as being equivalent to animals in which CHST6 has been deleted by genetic engineering yet they do not manifest MCD. However, the KS in corneas of mice are predominantly undersulfated and express a low level of reactivity with the 5D4 antibody used to differentiate MCD types I and II. The epitope of 5D4 is linear pentasulfated sequences of N-acetyl lactosamine disaccharides of KS PGs in which the GalNAC and Gal are sulfated. Hence, the 5D4 antibody reactivity in the mouse cornea is absent, but low sulfated KS is detectable with antibodies that react with lesser sulfated sequences of N-acetyl lactosamine disaccharides of KS (IB4 and 4D1) , and there is evidence that intestinal GlcNAc 6-O-sulfotransferase encoded by CHST5 in the mouse produces some sulfation of KS in the murine cornea .
We would like to thank the affected and unaffected individuals for participating in this study. Dr. Eugene J. Thonar kindly determined the serum antigenic keratan sulfate levels in 22 of the patients with MCD. This study was supported by a research grant from the National Eye Institute (R01-EY08249).
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