a 59 year - old female with a family history of mcd presented to our clinic with progressive loss of vision over a 40-year period .
there were multiple irregular grayish - white , dense , poorly delineated spots in the stroma . because her parents died at an early age , her family history was unclear .
of note , her only son complained of foggy vision and was 37 years old .
his slit lamp examination revealed a similar , but less severe appearing corneal exam compared to that of his mother ( fig .
one year post - operatively , that patient had a best - corrected visual acuity of 20 / 100 od and a clear cornea graft .
informed consent for both the clinical examinations and dna analyses was obtained from the patient and her son in accordance with the declaration of helsinki .
the study was approved by the institutional review board at the catholic university of korea , st .
hematoxylin and eosin ( he ) , alcian blue , periodic acid - schiff ( pas ) , colloidal iron , and masson 's trichrome stains were performed on the specimen using standard techniques . for transmission electron microscopy
, the corneal specimen was immersed immediately in a fixative solution containing 3% glutaraldehyde with 0.2 m sodium cacodylate at a ph of 7.4 .
after overnight fixation , the fixative solution was removed and replaced with a phosphate buffer , followed by 1% osmium tetroxide buffered with sodium cacodylate .
after one hour , the osmium was replaced with increasing concentrations of ethanol through propylene oxide , and the tissue was embedded in the epoxy .
the embedded tissue was then sectioned with an ultra - microtome into 1 m - thick sections , and stained with toluidine blue .
the area to be observed was placed under a light microscope , ultra - sectioned from 60 to 100 nm in thickness , double stained with uranyl acetate and lead citrate , and examined via transmission electron microscope ( jem 1010 ; jeol , tokyo , japan ) .
the unique exon involving the coding region , exon 3 , was amplified via polymerase chain reaction ( pcr ) with the previously described primers .
the cycling program began with an initial denaturing step of 5 minutes at 95 followed by 33 cycles of 94 for 30 seconds , 53 to 57 for 30 seconds , and 72 for 45 seconds with a final extension step at 72 for 10 minutes .
the pcr products were purified and sequenced directly on both strands using an automatic dna sequencer ( abiprism 377xl ; applied biosystems , foster city , ca , usa ) .
the nucleotide sequences were compared with the published cdna sequence of chst6 ( nm_021615 ) .
he staining revealed faintly basophilic deposits between the stromal lamellae , and within keratocytes and endothelial cells .
these deposits were positive to alcian blue , pas , and colloidal iron stain , but negative to masson 's trichrome stain ( fig .
electron microscopy revealed keratocytes distended by membrane - bound intracytoplasmic vacuoles containing electron - dense fibrillogranular material .
one missense mutation was identified in a homozygous state ( p.arg205trp [ c.613c > t ] ) , which had not been previously reported .
samples from the patient 's son also showed the same missense mutation ( p.arg205trp [ c.613c > t ] ) ( fig .
hematoxylin and eosin ( he ) , alcian blue , periodic acid - schiff ( pas ) , colloidal iron , and masson 's trichrome stains were performed on the specimen using standard techniques . for transmission electron microscopy , the corneal specimen was immersed immediately in a fixative solution containing 3% glutaraldehyde with 0.2 m sodium cacodylate at a ph of 7.4 .
after overnight fixation , the fixative solution was removed and replaced with a phosphate buffer , followed by 1% osmium tetroxide buffered with sodium cacodylate .
after one hour , the osmium was replaced with increasing concentrations of ethanol through propylene oxide , and the tissue was embedded in the epoxy .
the embedded tissue was then sectioned with an ultra - microtome into 1 m - thick sections , and stained with toluidine blue .
the area to be observed was placed under a light microscope , ultra - sectioned from 60 to 100 nm in thickness , double stained with uranyl acetate and lead citrate , and examined via transmission electron microscope ( jem 1010 ; jeol , tokyo , japan ) .
the unique exon involving the coding region , exon 3 , was amplified via polymerase chain reaction ( pcr ) with the previously described primers .
the cycling program began with an initial denaturing step of 5 minutes at 95 followed by 33 cycles of 94 for 30 seconds , 53 to 57 for 30 seconds , and 72 for 45 seconds with a final extension step at 72 for 10 minutes .
the pcr products were purified and sequenced directly on both strands using an automatic dna sequencer ( abiprism 377xl ; applied biosystems , foster city , ca , usa ) .
the nucleotide sequences were compared with the published cdna sequence of chst6 ( nm_021615 ) .
he staining revealed faintly basophilic deposits between the stromal lamellae , and within keratocytes and endothelial cells .
these deposits were positive to alcian blue , pas , and colloidal iron stain , but negative to masson 's trichrome stain ( fig .
electron microscopy revealed keratocytes distended by membrane - bound intracytoplasmic vacuoles containing electron - dense fibrillogranular material .
one missense mutation was identified in a homozygous state ( p.arg205trp [ c.613c > t ] ) , which had not been previously reported .
samples from the patient 's son also showed the same missense mutation ( p.arg205trp [ c.613c > t ] ) ( fig .
in this study , we describe histopathological findings and a novel mutation in the chst6 gene ( p.arg205trp [ c.613c > t ] ) in a case of mcd .
light microscopy revealed abnormal deposits of glycosaminoglycans in bowman 's histiocytes and keratocytes , as well as between the stromal lamellae , descemet 's membrane , and endothelium .
electron microscopy revealed that these deposits corresponded to electron - lucent fibrillogranular material visible within membrane - bound intracytoplasmic vacuoles .
such abnormalities have been identified as sequelae of an error in glycosaminoglycans metabolism within the cornea . in particular , errors in proteoglycan keratan sulfate ( ks ) metabolism
the product of the chst6 gene , corneal n - acetylglucosamine-6-sulfotransferase ( c - glcnac6st ) , has been demonstrated to catalyze the sulfation of glcnac in ks . in normal corneal tissue
chst6 can transfer a sulfuric acid group from the 3'-adenosine 5'-phosphate acid to ks via competition with an endogenous or exogenous substrate .
the variations in the coding region of chst6 may reduce enzyme activity or cause it to be lost , resulting in a low sulfated form or non - sulfated form of ks . due to the loss of its soluble properties ,
non - sulfated ks can not be completely metabolized , inducing the deposition of sediment in the corneal stroma .
the mutation identified in this korean patient is different from those reported previously in asians ( japanese and chinese ) or whites ( american , british , and icelandic ) .
although the p.arg205gln mutation has been reported in a southern indian patient , the p.arg205trp mutation has not been reported previously .
the immunophenotype of mcd was not performed in our study since we could not test ks serum levels .
regardless of the type of macular corneal dystrophy , the patient showed indistinguishable clinical characteristics linked to the same responsible gene . in summary
, we identified a novel homozygous missense mutation in chst6 in a korean patient with macular corneal dystrophy .
this novel gene mutation expands the mutation spectrum of the chst6 gene and contributes to the study of molecular pathogenesis of corneal dystrophy .
further studies on these mutations would be helpful in further understanding the molecular mechanisms underlying macular corneal dystrophy .
 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 . 
Case Report
Histopathologic examination
DNA analysis
Results
Discussion