Identification of Novel Mutations in the Carbohydrate Sulfotransferase Gene

Identiﬁcation of Novel Mutations in the Carbohydrate Sulfotransferase Gene (CHST6) Causing Macular Corneal Dystrophy

Mohamed F. El-Ashry,1,2,3 Mai M. Abd El-Aziz,1 Simon Wilkins,4 Michael E. Cheetham,4 Susan E. Wilkie,1 Alison J. Hardcastle,1 Stephanie Halford,1 Ahmed Y. Bayoumi,2 Linda A. Ficker,3 Stephen Tuft,3 Shomi S. Bhattacharya,1 and Neil D. Ebenezer1

PURPOSE. Macular corneal dystrophy (MCD) is a rare corneal phies that require penetrating keratoplasty, depending on the dystrophy that is characterized by abnormal deposits in the population.3,4 The disease becomes evident in the first decade corneal stroma, keratocytes, Descemet’s membrane, and endo- of life, defined by fine, diffuse, superficial clouding in the thelium, accompanied by progressive clouding. It has been central stroma that extends to the periphery and usually in- classified into three immunophenotypes—MCD types I, IA, and volves the entire thickness of the cornea by the second decade II—according to the serum level of sulfated keratan sulfate (KS) of life. These opacities are more superficial and prominent in and immunoreactivity of the corneal tissue. Recently, muta- the central cornea and are deeper and more discrete in the tions in a new carbohydrate sulfotransferase gene (CHST6) periphery. encoding corneal glucosamine N-acetyl-6-sulfotransferase (C- MCD has been classified into three immunophenotypes—I, GlcNac-6-ST) have been identified as the cause of MCD. Muta- IA, and II—based on measurement of the serum level of sul- tion screening of the CHST6 gene has been undertaken to fated keratan sulfate (KS) by enzyme-linked immunosorbent identify the underlying mutations in five unrelated British fam- assay (ELISA)5 and an immunohistochemical evaluation of the ilies with MCD. corneal tissue.6 In MCD type I, neither the cornea nor the ETHODS serum contains appreciable levels of sulfated KS, whereas in M . DNA was extracted from venous blood obtained 7 from all participants, and the coding region of CHST6 was MCD type II, there is detectable KS in the cornea and serum. amplified by polymerase chain reaction (PCR). The PCR prod- The third subtype, IA, in which sulfated KS is absent in the cornea and the serum but can be detected in the keratocytes, ucts were analyzed by direct sequencing and restriction en- 8 zyme digestion. Enzyme-linked immunosorbent assay (ELISA) has been identified in subjects from Saudi Arabia. All three was performed to assess the presence of KS in serum from the immunohistochemical subtypes have the same clinical pheno- probands of MCD-affected families participating in the study. type. Histologically, MCD is characterized by accumulation of RESULTS. Six novel missense mutations—four homozygous and glycosaminoglycans between the stromal lamellae, underneath two compound heterozygous—were identified in the CHST6 the epithelium, and within the keratocytes and endothelial gene. The ELISA showed that the disease in all patients partic- cells.9,10 An abnormality in the metabolism of KS has been ipating in the study was of MCD type I, including the sub- implicated in the pathogenesis of MCD11 and has been attrib- type IA. uted to an error in a specific sulfotransferase involved in the CONCLUSIONS. These novel mutations are thought to result in sulfation of KS.12–14 loss of corneal sulfotransferase function, which would account MCD types I and II had previously been linked to chromo- for the MCD phenotype. (Invest Ophthalmol Vis Sci. 2002;43: some 16 (16q22).15–17 Recently, mutations in a new carbohy- 377–382) drate sulfotransferase gene (CHST6) encoding corneal glucosamine N-acetyl-6-sulfotransferase (C-GlcNac-6-ST) have acular corneal dystrophy (MCD, On-line Mendelian Inher- been identified as the cause of MCD.18 A number of missense Mitance in Man (OMIM) 217800; provided in the public mutations were described in patients with MCD type I18–20 or domain by the National Center for Biotechnology Information, type IA.20 In MCD type II, deletions and/or rearrangements in Bethesda, MD, and available at http://www3.ncbi.nlm.nih.gov/ the upstream region of CHST618 and recently a missense mu- omim) is an autosomal recessive disorder characterized by tation20 were reported. bilateral progressive stromal clouding and central corneal thin- In our study, mutation screening of CHST6 in five unrelated ning.1,2 MCD accounts for 10% to 75% of the corneal dystro- British families with MCD and with no clinically apparent systemic manifestations, revealed four homozygous and two compound heterozygous mutations in CHST6, all of the mis- From the Departments of 1Molecular Genetics and 4Pathology, sense type. All the mutations involve the substitution of amino Institute of Ophthalmology, London, United Kingdom; the 2Depart- acids highly conserved across related carbohydrate sulfotrans- ment of Ophthalmology, Tanta University Hospitals, Tanta, Egypt; and ferases and/or represent the nonconservative substitution of 3Moorfields Eye Hospital, London, United Kingdom. nonpolar for polar residues and are thought to result in loss of Supported by a grant from Tanta University, Tanta, Egypt. CHST6 gene function. Submitted for publication June 14, 2001; revised September 7, 2001; accepted October 2, 2001. Commercial relationships policy: N MATERIALS AND METHODS The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Clinical Diagnosis ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Mohamed F. El-Ashry, Department of Mo- The study had the approval of Moorfields Eye Hospital Ethics Commit- lecular Genetics, Institute of Ophthalmology, 11-43 Bath Street, Lon- tee and conformed to the tenets of the Declaration of Helsinki. In- don EC1V 9EL, UK; [email protected]. formed consent was obtained from all participants for clinical and

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TABLE 1. Mutations Identiﬁed within the CHST6 Gene in MCD-Affected Families

Type of Nucleotide Amino Acid Type of Amino Type of Family Nucleotide Change Change Change Acid Substitution Mutation

A 1012T3C Homozygous F107S Polar3nonpolar Nonconservative B [783C3Tϩ1291T3G] Heterozygous P31S Nonpolar3polar Non-conservative Heterozygous L200R Non-polar3acidic Non-conservative C [783C3Tϩ1291T3G] Heterozygous P31S Non-polar3polar Non-conservative Heterozygous L200R Non-polar3acidic Non-conservative D 1309C3T Homozygous A206V Non-polar3non-polar Conservative E 906C3T Homozygous P72S Non-polar3polar Non-conservative

molecular genetic study. Autosomal recessive MCD in our patients of tively. Similarly, DrdI, with recognition site 5Ј. . . GAC- white origin was diagnosed on the basis of pedigree structure and the NNNNˇNNGTC. . . 3Ј, was used to confirm the homozygous C1309T following clinical features: bilateral symmetrical superficial stromal mutation in family D. And finally, BanII, with recognition site 5Ј...G cloudiness studded by small, irregular, rounded, gray-white anterior (A/G) GC (T/C)ˇC...3Ј, was used to confirm the homozygous G905T stromal patches. The diagnosis was confirmed by histopathologic ex- change in family E. amination of corneal buttons from all patients after keratoplasty. The PCR products digested by HinfI, BstN I, and BanII restriction enzymes were analyzed by 6% nondenaturing polyacrylamide gel elec- Mutation Detection trophoresis (Protogel; National Diagnostics, Atlanta, GA) and were Genomic DNA was extracted from 10-mL blood samples obtained, visualized by staining with ethidium bromide. Products digested by according to standard protocols, from all subjects involved in the ApaI and Drd I were analyzed on 3% agarose gels (Bio-Rad, Herts, UK). study. The coding region of CHST6 was amplified by polymerase chain reaction (PCR). Each PCR was performed in a 50-␮L reaction mixture ␮ Assay of Sulfated KS in Serum containing genomic DNA (200 ng), primers (0.4 M each), MgCl2 (1.5–2 mM), deoxynucleoside triphosphates (dNTPs; 0.2 mM), 1ϫ PCR Inhibition ELISA for KS detection using the monoclonal antibody 5-D-4 buffer (containing 10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 0.1% (ICN Biochemicals Ltd., Basingstoke, UK) was performed to assess the gelatin) and Taq polymerase (0.5 U; Bioline, UK). Amplification reac- presence of KS in serum obtained from the probands of MCD-affected tions were performed under the following conditions: 3 minutes of families participating in the study. The method was performed as denaturation at 94°C followed by 35 cycles of denaturation at 94°C for 21,22 1 minute, annealing at 60 to 68°C for 1 minute, extension at 72°C for described previously, except that an antibody (Aggrecan; Sigma Ј 1 minute, and a further extension step at 72°C for 5 minutes. Chemical Co., Poole, UK) was used to coat the plates and 2, 2 -azino- For amplification of the CHST6 coding region the following primer di-[3-ethylbenzthiazoline sulfonate 6] (ABTS; Roche Molecular Bio- pairs were used: for the 5Ј-coding region, CK71h-intrn (5Ј-GCCCCTA- chemicals, Lews, UK) was used as the substrate. The plates were read ACCGCTGCGCTCTC-3Ј) and CK71h-R1180 (5Ј-GGCTTGCACACGGC- at 410 nm with a reference wavelength of 490 nm on a flow plate CTCGCT-3Ј) designed by Akama et al.18 For the middle coding region, reader (ICN Biochemicals Ltd.). The ELISA was sensitive to KS concen- two pairs of primers were designed: CK71M-F1 (5Ј-GACATGGACGT- trations of 10 to 10,000 ng/mL. GTTTGATGC-3Ј) and CK71M-R1 (5Ј-GCACGATGCCGTTGTCAC-3Ј); CK71M-F2 (5Ј-GCTCAACCTACGCATCGTG-3Ј) and CK71M-R2 (5Ј-ATC- CGTGGGTGATGTTATGG-3Ј); and for the 3Ј-coding region the following primer pair was designed: CK71L-F (5Ј-GAGCCGCTGGCAGAAATC- 3Ј) and CK71L-R (5Ј-TGCACCATGCACTCTCCTC-3Ј). A mismatch primer pair was also designed, sulfohin-F (5Ј-CTGTGCGACATGGAC- GAGT-3Ј) and sulfohin-R (5Ј-CACCACGTGGCTGTAGGAG-3Ј) to confirm one of the homozygous mutations (in family A, F107S) which does not alter any known restriction enzyme site. The mismatch primer created an HinfI site in conjunction with the mutation. For direct sequencing, PCR products were purified (Qiaquick PCR purification kit; (Qiagen, Crawley, UK) and sequenced using an auto- matic fluorescence DNA sequencer (ABI Prism 373A; Perkin Elmer, Foster City, CA), according to the manufacturers’ instructions. Nucle- otide sequences were compared with the published cDNA sequence of CHST6 (GenBank accession number AF219990; GenBank is provided in the public domain by the National Center for Biotechnology Infor- mation, Bethesda, MD, and is available at http://www.ncbi.nlm.nih. gov/genbank), and mutations identified were excluded from 100 control chromosomes of white origin by restriction enzyme digestion. Restriction Enzymes Five enzymes were used to confirm the mutations identified. HinfI, Ј ˇ Ј FIGURE 1. DrdI restriction digest on 3% agarose gels. Lane 1: undi- with recognition site 5 ... GANTC. . . 3 , was used to study the gested PCR product (400 bp); lane 2: digested mutant allele (360 bp) homozygous T1012C and C906T in families A and E, respectively. from the proband of family D; lane 3: digested mutant and undigested ˇ ApaI, with recognition site 5Ј. . . GGGCC C...3Ј, and BstNI, with wild-type alleles from the proband’s mother (heterozygous); lanes 4 recognition site 5Ј...CCˇ(A/T)GG. . . 3Ј, also were used to confirm the and 5: unaffected proband’s sisters (homozygous) with undigested heterozygous changes C783T and T1291G in families B and C, respec- wild-type alleles; lane M: ␾X174 RF DNA HaeIII marker.

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FIGURE 2. Direct sequencing analysis of the coding region of CHST6.(A) Unaffected member of family A shows the wild-type allele; (B) the proband of family A shows a homozygous single-base pair transition (TTT3TCT, F107S); (C, E) unaffected members of families B and C show the wild-type allele; (D) the probands of families B and C show a heterozygous single-base pair transition (CCC3TCC, P31S); (F) the probands of families B and C show a heterozygous single-base pair transition (CTG3CGG, L200R); (G) unaffected member of family D shows the wild-type allele; (H) the proband of family D shows a homozygous single-base pair transition (GCC3GTC, A206V); (I) unaffected member of family E shows the wild-type allele; (J) the proband of family E shows two homozygous single-base pair transversion and transition (GAG3GAT, E71D and CCC3TCC, P72S).

RESULTS (Figs. 2G, 2H). In family E, two consecutive homozygous changes were identified. The first is a G905T transversion, Six novel missense mutations were identified within the coding located at the third nucleotide position of codon 71, which region of the CHST6 gene in five unrelated families with MCD changes the amino acid from glutamic acid to aspartic acid. type I (Table 1). None of these changes was detected in the The second change, a C906T transition located at the first control population of the same ethnic origin, as determined by nucleotide position of codon 72 leads to a proline-to-serine restriction enzyme digest on 100 chromosomes, confirming substitution in the same patient (Figs. 2I, 2J). Although the first that they are likely to be pathogenic mutations. In addition, mutation predicts a change in the amino acid within the same they were shown to segregate with the disease by sequencing group, the second alteration changes the amino acid residue to and restriction enzyme analyses (Fig. 1). a different anionic group. Four of these mutations are homozygous and were detected In families B and C two heterozygous changes were identi- in families A, D, and E. In family A, a T1012C transition at the fied, with a different mutation on each allele (i.e., compound second nucleotide position of codon 107 was identified, result- heterozygotic mutations), which could account for such auto- ing in a phenylalanine-to-serine substitution (Figs. 2A, 2B). In somal recessive inheritance. The first heterozygous change, a family D, a C1309T transition at the second nucleotide position C783T transition, occurred at the first nucleotide position of of codon 206 was found, with an alanine-to-valine substitution codon 31, predicting a change of amino acid proline to serine

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28 FIGURE 3. Alignment of human CHST6 and other carbohydrate sulfotransferases peptide sequences. A computer program was used for multiple alignment of amino acid sequences. The positions of mutated amino acids in patients with MCD reported in this study are shaded in gray. The 5Ј- and 3Ј-PB site motifs29,30 are boxed. Note that two of the mutations occur at highly conserved positions within the 3Ј-PB. The peptide sequences compared are I-GlcNAc6ST (intestinal N-acetylglucosamine-6-sulfotransferase),23 HecGlcNAc6ST (human high-endothelial-cell GlcNAc-6-sulfotransferase),24 GlcNAc6ST (human GlcNAc-6-sulfotransferase),25 Chon6ST (human chondroitin-6-sulfotransferase),26 KSG6ST (human keratan sulfate Gal-6-sulfotransferase).27

(Figs. 2C, 2D). The other change, a T1291G transversion, was residue type is not conserved across the carbohydrate sulfo- identiﬁed at the second nucleotide position of codon 200, transferases, all the mutations occur at positions where the changing the amino acid from leucine to arginine (Figs. 2E, 2F). residue type is highly conserved across all the carbohydrate To assess the signiﬁcance of these mutations, an alignment sulfotransferases. Furthermore, mutations F107S (family A), of the primary sequence of CHST6 with other published car- L200R (families B and C), and P72S (family E) are all noncon- bohydrate sulfotransferases,23–27 a computer program for si- servative changes involving substitution of a nonpolar residue multaneous alignment of many nucleotide or amino acid se- for a polar residue that may affect CHST6 enzyme activity. Two quences to detect or demonstrate homology between new of the substitutions are conservative changes, E71D sequences and existing families of sequences (Clustal W, ver. (acidic3acidic, family E) and A206V (nonpolar3nonpolar, 1.7; SGI, Mountain View, CA) was used28 (Fig. 3). With the family D). Of these, E71D occurs in tandem with the P72S exception of a P31S substitution (families B and C) that repre- substitution (which is more likely to be disease causing) and sents a nonconservative change but occurs at a position where thus may not itself have a major deleterious effect. It is not

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immediately apparent, however, why the A206V substitution formation of poorly or nonsulfated KS and causes corneal should be disease causing, although conservative substitutions opacity.38 More recently, it has been reported that mouse elsewhere in CHST6 have been reported to underlie MCD type intestinal GlcNac6-O-ST (mIGn6ST) has the same activity as I in Icelandic families.19 human corneal GlcNac6-O-ST (C-GlcNA6ST) suggesting that In the alignment, 5Ј- and 3Ј-phosphate-binding (PB) do- mIGn6ST is the orthologue of human C-GlcNA6ST and func- mains that interact with adenosine 3Ј-phosphate 5Ј-phospho- tions as a sulfotransferase to produce KS in the cornea. More- sulfate (PAPS), a sulfate donor for sulfotransferases, are boxed over, the amino acid substitutions in human C-GlcNA6ST re- (Fig. 3).29,30 These domains form part of the vital active site sulting from missense mutations in CHST6 found in patients region of the enzyme. Two of the new mutations identified in with MCD abolished the sulfotransferase activity by functional this study, L200R and the conservative substitution A206V, inactivation rather than protein degradation or mislocaliza- occur within the 3Ј-PB domain. tion.39 Inhibition ELISA performed on serum from the patients The homozygous mutations detected in our patients infers with MCD showed little or no inhibition of the monoclonal an essential role of C-GlcNA6ST, the CHST6 protein product, in antibody 5-D-4. Control experiments using increasing KS con- the production of normally functioning KS, whereas its inacti- centrations effectively inhibited 5-D-4 binding. These data from vation is responsible for the MCD phenotype. Furthermore, the our patients are consistent with MCD type I or type IA. heterozygous changes identified affected both alleles in two families, suggesting that these compound heterozygous mutations could also result in the MCD phenotype. DISCUSSION We conclude that mutated carbohydrate sulfotransferase could result in loss of function of the sulfotransferase enzyme In this study we collected five unrelated British families with required for proper sulfation of KS, an essential element for clinically and histopathologically diagnosed MCD and screened corneal transparency, leading to the MCD phenotype. the CHST6 gene, which is reported to be involved in the pathogenesis of this disorder by affecting the synthesis of KS. KS proteoglycans (lumican, keratocan, and mimecan) are Acknowledgments present in the cornea as the major class of proteoglycans and The authors thank John Dart, David Gartry, and Frank Larkin for help are thought to play an important role in corneal transpar- 31–33 with the clinical data; the genetic nurses, Catherin Willis and Catherine ency. The sulfate group of KS appears to be crucial for its Plant, for samples collection; Nigel Fullwood for advice and guidance; biological function, because the degree of sulfation of KS and all patients who participated in this study. increases during corneal development34,35 and the synthesis of unsulfated KS is observed in the corneas of patients with MCD.36,37 In view of the autosomal recessive inheritance of References the condition, MCD probably results from a deficiency in 10 1. Klintworth GK. Research into the pathogenesis of macular corneal sulfotransferase specific for proper sulfation of KS. dystrophy. Trans Ophthalmol Soc UK. 1980;100:186–194. We have identified six novel missense mutations in the 2. Donnenfeld ED, Cohen EJ, Ingraham HJ, Poleski SA, Goldsmith E, probands from all families participating in this study. The Laibson PR. Corneal thinning in macular corneal dystrophy. 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