OPHTHALMIC MOLECULAR GENETICS SECTION EDITOR: EDWIN M. STONE, MD, PhD Novel Mutations in the CHST6 Gene 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 chromosome 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- (REPRINTED) ARCH OPHTHALMOL / VOL 121, NOV 2003 WWW.ARCHOPHTHALMOL.COM 1608 ©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 (REPRINTED) ARCH OPHTHALMOL / VOL 121, NOV 2003 WWW.ARCHOPHTHALMOL.COM 1609 ©2003 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Table 1.