OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: EDWIN M. STONE, MD, PhD Novel Mutations in the CHST6 Associated With Macular Corneal Dystrophy in Southern India

John F. Warren, MD; Anthony J. Aldave, MD; M. Srinivasan, MD; Eugene J. Thonar, PhD; Abha B. Kumar, MD; Vicky Cevallos, BS; John P. Whitcher, MD, MPH; Todd P. Margolis, MD, PhD

Objective: To further characterize the role of the car- Glu274Lys), 2 compound heterozygous missense muta- bohydrate sulfotransferase (CHST6) gene in macular cor- tions (Arg93His and Ala206Thr), 5 homozygous dele- neal dystrophy (MCD) through identification of caus- tion mutations (delCG707-708, delC890, delA1237, ative mutations in a cohort of affected patients from del1748-1770, and delORF), and 2 homozygous replace- southern India. ment mutations (ACCTAC 1273 GGT, and GCG 1304 AT). One patient with type II MCD was heterozygous for Methods: Genomic DNA was extracted from buccal epi- the C890 deletion mutation, whereas 4 possessed no thelium of 75 patients (51 families) with MCD, 33 un- CHST6 coding region mutations. affected relatives, and 48 healthy volunteers. The cod- ing region of the CHST6 gene was evaluated by means Conclusion: A variety of previously unreported muta- of polymerase chain reaction amplification and direct se- tions in the coding region of the CHST6 gene are associ- quencing. Subtyping of MCD into types I and II was per- ated with type I MCD in a cohort of patients in southern formed by measuring serum levels of antigenic keratan India. sulfate. Clinical Relevance: An improved understanding of Results: Seventy patients were classified as having type the genetic basis of MCD allows for earlier, more accu- I MCD, and 5 patients as having type II MCD. Analysis rate diagnosis of affected individuals, and may provide of the CHST6 coding region in patients with type I MCD the foundation for the development of novel disease identified 11 homozygous missense mutations (Leu22Arg, treatments. His42Tyr, Arg50Cys, Arg50Leu, Ser53Leu, Arg97Pro, Cys102Tyr, Arg127Cys, Arg205Gln, His249Pro, and Arch Ophthalmol. 2003;121:1608-1612

ACULAR CORNEAL dys- the basis of analysis of antigenic KS trophy (MCD) is an (AgKS) levels in serum and immunohis- autosomal recessive tochemical evaluation of corneal buttons disorder of corneal obtained at the time of penetrating kera- keratan sulfate (KS) toplasty. Affected patients with unde- From the Francis I. Proctor metabolism that is characterized by bilat- tectable serum levels of AgKS are classi- M 1-4 Foundation and the Department eral progressive corneal clouding. A su- fied as having type I MCD, whereas those of Ophthalmology, University perficial stromal haze develops in the first with low or reference levels are classified of California–San Francisco to second decade of life, and is followed as having type II MCD.5 Type IA MCD is (Drs Warren, Aldave, Kumar, Whitcher, and Margolis and by an accumulation of irregular, focal, distinguished from type I MCD by the Ms Cevallos); the Cornea and gray-white corneal stromal deposits. These presence or absence, respectively, of External Disease Service, deposits involve the anterior corneal highly sulfated AgKS within corneal Aravind Eye Hospital, Madurai, stroma centrally and the deep stroma pe- keratocytes in individuals who have no India (Dr Srinivasan); and the ripherally. Affected patients typically ex- detectable amounts of this form of AgKS Departments of Biochemistry, perience an early-onset, progressive de- in serum.6,7 Orthopedic Surgery, and cline in vision, often leading to penetrating Recently, investigators have linked Internal Medicine, Rush keratoplasty. Histopathologic findings in MCD to mutations in a newly recognized Medical College, Chicago, Ill MCD demonstrate abnormal accumula- carbohydrate sulfotransferase gene (Dr Thonar). Dr Aldave is now 8 with the Jules Stein Eye tions of glycosaminoglycans in the extra- (CHST6) on 16 (16q22). Institute, University of cellular stroma, keratocytes, and endothe- The CHST6 gene product, corneal California–Los Angeles. The lial cells. N-acetylglucosamine-6-sulfotransferase authors have no relevant Macular corneal dystrophy is com- (C-GlcNac6ST), is thought to be impor- financial interest in this article. monly divided into types I, IA, and II, on tant in producing sulfated KS, the pre-

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 netic analysis was performed. In addition, a portion of serum from each blood sample was sent to Rush Medical College, Chi- cago, Ill, for quantification of AgKS levels in serum.

DNA PREPARATION

After receiving informed consent from each study participant, a blood sample and buccal epithelial swab were obtained. Ap- proximately 3 mL of peripheral blood was drawn from each sub- ject by means of standard phlebotomy. In addition, samples of buccal mucosal epithelium were obtained by twirling a cytol- ogy brush (CytoSoft brush CP-5B; Medical Packaging Corpo- ration, Camarillo, Calif) vigorously against the buccal epithe- lium. Genomic DNA was prepared from the buccal epithelial cells and/or blood leukocytes using a spin protocol (QIAamp DNA Mini Kit; Qiagen Inc, Valencia, Calif).

POLYMERASE CHAIN REACTION AMPLIFICATION

The CHST6 coding region was amplified by means of polymer- Slit-beam photograph of a representative study subject. Sclerotic scatter ase chain reaction (PCR) using primers designed to create demonstrates the highly characteristic, diffusely distributed, irregular, focal, gray-white corneal stromal deposits of macular corneal dystrophy. 3 overlapping amplicons. The oligonucleotide primers used were identical to those reported by Akama et al,8 with the exception of the middle coding region reverse primer (5Ј-TCCGTGGGTGATGTTATGGAT-3Ј). Each reaction was dominant glycosaminoglycan expressed in the adult cor- performed in a 50-µL mixture containing 25 µL of PCR pre- nea. Lack of activity of this enzyme is thought to result mix (100mM Tris hydrochloride [pH, 8.3], 100mM potas- in the production of unsulfated KS, leading to a loss of sium chloride, 400µM of each deoxynucleotide phosphate, and transparency in the corneas of affected patients. Muta- proprietary concentrations of magnesium chloride and PCR en- tions within the coding region of CHST6 have been found hancer) (FailSafe PCR 2ϫPreMix D; Epicentre, Madison, Wis), to be associated with type I MCD, whereas deletions and 0.2µM of each primer, 1.5 U of DNA polymerase (AmpliTaq; rearrangements in the upstream regulatory region have (Applied Biosystems, Foster City, Calif), and approximately 100 been identified in patients with type II MCD. Despite the ng of genomic DNA. Thermal cycling was performed with variety of mutations reported, all of the gene defects are the following program: initial denaturation for 3 minutes at associated with a common disease phenotype.8-11 96°C, 35 cycles of 96°C for 30 seconds, 57°C for 30 seconds, and 72°C for 45 seconds, and final extension for 5 minutes at In this study, we sought to further characterize the 72°C (GeneAmp PCR System 9700; Applied Biosystems). role of the CHST6 gene in MCD through the identifica- tion of causative mutations in a large cohort of affected DNA SEQUENCING patients in southern India. The relatively high preva- lence of MCD in southern India is probably a result of Amplified DNA was column purified (QIAquick PCR purifi- the high frequency of consanguineous marriage within cation kit; Qiagen Inc) and sequenced directly according to the this population. Because many patients with MCD have protocols accompanying the cycle sequencing kit (BigDye Ter- been carefully followed up for years at the Aravind Eye minator kit; Applied Biosystems). A genetic analyzer (ABI Prism Hospital (AEH), Madurai, India, we were able to enroll 310; Applied Biosystems) was used to collect and analyze the a large series of patients to undergo molecular genetic sequence data. Nucleotide sequences were compared with the published CHST6 complementary DNA sequence. analysis.

METHODS AgKS QUANTIFICATION

After study approval was obtained from the institutional re- An epitope present on long KS chains was quantified using a view board at the AEH and the University of California–San well-characterized enzyme-linked immunosorbent assay that 12-14 Francisco (H7177-18489-01), a computerized medical record uses the 1/20/5-D-4 monoclonal antibody. Levels of AgKS search was performed to identify all patients with MCD seen in serum are reported here as equivalents of a standard of AgKS 12-14 at AEH between January 1, 1990, and January 1, 2000. The di- highly purified from human costal cartilage. agnosis of MCD was based on the distinctive clinical features (Figure), and in most cases was confirmed by results of his- topathologic examination of the excised corneal buttons. All RESULTS cases were consistent with an autosomal recessive inheritance pattern. Families with affected patients older than 20 years were A total of 75 affected patients, representing 51 different contacted by telephone or letter and asked to participate in the families, were enrolled in the study. On the basis of un- study. Unrelated, unaffected, healthy volunteers were re- detectable levels of serum AgKS, 70 of the affected pa- cruited to serve as control subjects. All study subjects re- turned to AEH for slitlamp examination and collection of blood tients (47 families) were classified as having type I MCD. samples and buccal mucosal swabs. Family members were con- The other 5 affected patients (4 families) had serum lev- sidered unaffected if they were older than 20 years and with- els of AgKS that were low or within the reference range out clinical evidence of MCD. All samples were transported to (103-210 ng/mL) and were classified as having type II University of California–San Francisco, where molecular ge- MCD. Levels of AgKS within the reference range were

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Table 1. Missense Mutations of the CHST6 Gene Associated With Type I MCD in Southern India

DNA Change Amino Acid Substitution R-Group Change No. of Patients No. of Families T757G Leu22Arg Nonpolar to basic 1 1 C816T His42Tyr Basic to polar 2 1 C840T Arg50Cys Basic to polar 3 2 G841T Arg50Leu Basic to nonpolar 3 2 C850T Ser53Leu Polar to nonpolar 7 4 C850T heterozygous Ser53Leu Polar to nonpolar 2 1 G982C Arg97Pro Basic to nonpolar 1 1 G997A Cys102Tyr Polar to polar 3 2 C1071T Arg127Cys Basic to polar 5 3 G1306A Arg205Gln Basic to polar 2 2 A1438C His249Pro Basic to nonpolar 1 1 G1512A Glu274Lys Acidic to basic 1 1 G970A; G1308A Arg93His; Ala206Thr Basic to basic; nonpolar to polar 2 1

Abbreviations: CHST6, carbohydrate sulfotransferase gene; MCD, macular corneal dystrophy.

tation (heterozygous for Ser53Leu). One patient with type Table 2. Deletion and Insertion Mutations II MCD was heterozygous for the frameshift mutation in the Coding Region of the CHST6 Gene delC890. No coding region mutations were identified in Associated With Type I MCD in Southern India the other 4 patients with type II MCD. Most (24/31 [77%]) of the unaffected relatives were No. of No. of DNA Change Protein Change Patients Families identified as heterozygous for a mutation detected in their affected relatives. Parents or children were heterozy- Replacement 194 Asn Leu Arg 196; 62 ACCTAC 1273 GGT 194 Arg Cys 195 gous in 11 (92%) of 12 cases. The 1 exception was the Replacement Major 1 1 father of a patient with a homozygous point mutation GCG 1304 AT (C1071T). Paternity testing was not performed. Sib- Deletion CG707/708 Major 1 1 lings were heterozygous in 12 (80%) of 15 cases and sec- Deletion C890 Major 9 6 ond-degree relatives in 1 (25%) of 4 cases. As antici- Deletion A1237 Major 16 12 pated, the son of a patient with a presumed deletion of Deletion 1748-1770 Major 1 1 Deletion of open Absent 3 2 the open reading frame had a wild-type genotype, and reading frame therefore could not be positively classified. Of the 48 con- trols, one was discovered to be heterozygous for a pre- Abbreviations: CHST6, carbohydrate sulfotransferase gene; MCD, macular sumed disease-causing mutation (C1071T). corneal dystrophy. We performed multiple alignment analysis (Clustal W, version 1.8)15,16 to compare the amino acid sequence of the CHST6 gene product, C-GlcNAc6ST, with that of 5 detected in the serum of all 33 unaffected relatives and closely related members of the sulfotransferase enzyme fam- 48 controls (range, 134-515 ng/mL). ily.17-21 Of the missense mutations identified in this study, Analysis of the CHST6 coding region identified 20 11 (85%) of 13 were found to occur at highly conserved distinct genetic defects (Table 1 and Table 2). Eleven sites. Examination of the R groups of the predicted amino homozygous missense mutations were identified in 29 acid substitutions revealed that 11 (85%) of 13 would re- patients from 20 different families (Leu22Arg, His42Tyr, sult in a change in polarity or charge (Table 1). Arg50Cys, Arg50Leu, Ser53Leu, Arg97Pro, Cys102Tyr, Arg127Cys, Arg205Gln, His249Pro, and Glu274Lys). Three homozygous frameshift mutations (delCG707- 708, delC890, and delA1237) were identified in 26 pa- COMMENT tients from 19 families, and 2 homozygous replacement mutations (ACCTAC1273GGT and GCG1304AT) were detected in 7 patients from 3 families. Both affected in- In this study of 51 families with MCD, 20 different type dividuals in 1 family were found to be compound het- I disease-associated CHST6 coding region mutations were erozygotes (Arg93His and Ala206Thr). A 23-nucleotide identified. These included 13 point mutations, 5 dele- deletion (del1748-1770) was identified in a family with tions, and 2 replacements (deletion-insertion). This rep- 1 affected individual. Three patients from 2 unrelated fami- resents the largest cohort of patients with MCD to un- lies were presumed to have a deletion of the entire CHST6 dergo mutational analysis, and results in a doubling of open reading frame on the basis of failure of PCR am- the number of gene defects associated with type I MCD. plification. In these patients, none of the CHST6 coding Furthermore, 17 of the mutations identified in our co- region primer pairs produced a detectable amplicon, hort were novel (C1071T,10 G1512A,8 and deletion of the whereas normal amplification of other genomic sites was open reading frame8 have been previously reported). The observed. Two patients (same family) with type I MCD open reading frame deletions discovered in 2 of our fami- were found to possess only 1 CHST6 coding region mu- lies may also be novel gene defects; however, more ac-

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 curate mapping of these mutations will be required to Our cohort included only 5 patients classified as hav- make this determination. ing type II MCD. These patients had clinical findings con- Despite significant heterogeneity in the location and sistent with the diagnosis of MCD and levels of serum AgKS type of coding region mutations identified in our pa- that were low or within the reference range. Akama et al8 tient cohort, each was predicted to cause significant struc- discovered that their patients with type II MCD pos- tural or site-specific changes in the encoded protein, sessed upstream rearrangements or deletions that presum- C-GlcNAc6ST. Most of the deletion and insertion mu- ably resulted in altered promoter function and reduced cor- tations were found to cause a reading frame shift, result- nea cell-specific transcription of the CHST6 gene. They very ing in major changes in the primary protein structure. accurately mapped a deletion that appeared to have re- The amino acid substitutions (point mutations) were sulted from recombination between the highly similar se- shown to occur at highly conserved sites (11/13) and/or quences surrounding the CHST5 and CHST6 . Us- result in a significant change in polarity or charge (11/13). ing the same PCR primers, we failed to find the same An additional 1 of 13 would result in substitution of ty- changes in our patients with type II MCD. One of our pa- rosine for cysteine, with the resulting loss of a potential tients with type II MCD was found to be heterozygous for disulfide bonding site and insertion of a large cyclical R a type I disease-causing mutation (delC890). This is not group, likely to have profound consequences on pro- surprising because the type II phenotype was shown to be tein structure.22 dominant in compound heterozygotes (individuals pos- Consistent with a pattern of autosomal recessive sessing types I and II alleles).8 Our discovery of a family inheritance, all 68 of the individuals in our cohort with with type I MCD that included 2 affected individuals, both 2 CHST6 coding region mutations (66 homozygous and of whom carry only 1 type I allele (Ser53Leu heterozy- 2 compound heterozygous) were affected by corneal opaci- gotes), was surprising. We are continuing to investigate ties characteristic of MCD. These same 68 subjects were these families in an effort to better define their disease- found to have uniformly low (undetectable) levels of se- causing genetic defects. rum AgKS. The results of the measurement of AgKS lev- The use of buccal epithelial cells as a source of ge- els in serum, if viewed as a surrogate for C-GlcNAc6ST nomic DNA is a feature of this study worthy of special activity, indicate total (or near total) absence of enzy- mention. Although blood samples were obtained from matic activity in cases of type I MCD. The 24 relatives who all subjects for measuring serum levels of AgKS, all of were heterozygous for coding region mutations were clini- the genetic testing was performed on DNA extracted from cally and serologically indistinguishable from those with swabs of the buccal mucosa. There are a number of ad- 2 normal copies of the gene. These findings support our vantages to this approach. First, buccal swabbing is conclusion that the CHST6 gene defects identified in this quicker and easier to perform, and does not induce the patient cohort are responsible for causing corneal opaci- anxiety associated with needles and blood. Second, mu- fication by disrupting C-GlcNAc6ST function. cosal epithelium is a much more efficient source of ge- Recent reports have provided additional evidence nomic DNA than blood, as, in the latter, only the white to indicate that C-GlcNAc6ST plays an important role blood cell fraction is nucleated. Third, the risks of ex- in maintaining corneal transparency through the pro- posing investigators to blood-borne pathogens can be de- duction of properly sulfated KS. In vitro assays have con- creased. Finally, DNA is more stable on a buccal swab firmed that C-GlcNAc6ST catalyzes the transfer of sul- than in a tube of blood. Blood must be refrigerated until fate to position C-6 of GlcNAc.23-25 The C-GlcNAc6ST the time of DNA extraction, or frozen for long-term stor- activity is undetectable in extracts from corneas af- age. In contrast, DNA is readily isolated from buccal epi- fected by MCD,23 and is similarly abolished in cells trans- thelial cells even after storage for weeks to months at room fected with C-GlcNAc6ST containing MCD-associated temperature.29,30 amino acid substitution.24 There is heavy labeling of ab- The great advances made recently in the under- normal unsulfated KS in the deposits found in the stroma, standing of the genetic basis of the corneal dystrophies keratocytes, and endothelium of corneas with type I MCD provide clinicians with the ability to confirm or refute compared with healthy controls.26 Finally, analysis of KS diagnoses that previously were based primarily on phe- chain fine structure in corneas with MCD revealed sig- notypic criteria. By identifying the distinct mutations re- nificantly reduced chain size and chain sulfation com- sponsible for each form of corneal dystrophy, including pared with healthy tissue.27 macular dystrophy, we will be better able to predict the It is intriguing that such a large number and wide prognosis for affected patients and their offspring, and variety of novel mutations were discovered distributed properly counsel them. The identification of causative throughout the CHST6 coding sequence. This suggests mutations in genes such as CHST6 that result in dystro- 2 additional conclusions. First, in contrast to the BIGH3 phic corneal opacification is the first step toward under- gene, with its hot spots for corneal dystrophy– standing the role that such genes play in maintaining cor- associated amino acid substitution,28 the CHST6 gene does neal clarity. not demonstrate a highly conserved number of disease- causing mutations. Indeed, the CHST6-encoded sulfo- Submitted for publication October 15, 2002; final revision transferase does not seem particularly robust, with in- received June 9, 2003; accepted July 2, 2003. activation resulting from changes at a number of different This study was supported by the Heed Fellowship Foun- locations. Second, it appears that the southern Indian dation, Cleveland, Ohio (Dr Warren); the AOS Knapp Fel- population is genetically distinct from other popula- lowship Fund, Cleveland (Dr Aldave); the Peierls Founda- tions previously studied. tion, New York, NY; Research to Prevent Blindness, New

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©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 York; the Alta California Eye Research Foundation, Los Methods in Cartilage Research. Orlando, Fla: Academic Press Inc; 1990:170-172. Altos, Calif; and grants EY02162 and AG04736 from the 14. Thonar EJ, Manicourt DH. Noninvasive markers in osteoarthritis. In: Moskowitz R, ed. Osteoarthritis. 3rd ed. San Diego, Calif: Harcourt Brace & Co; 2001:293-313. National Institutes of Health, Bethesda, Md (Dr Thonar). 15. Higgins DG, Thompson JD, Gibson TJ. Using CLUSTAL for multiple sequence Corresponding author: John F. Warren, MD, Francis alignments. Methods Enzymol. 1996;266:383-402. I. Proctor Foundation, 95 Kirkham St, San Francisco, CA 16. Worley KC, McLeod MP. BCM search launcher: multiple sequence alignments. 94143-0944. Sequencing Center Web site. Available at: http://searchlauncher .bcm.tmc.edu/multi-align/multi-align.html. Accessed September 2002. 17. 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