March 2007
Volume 48, Issue 3
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Cornea  |   March 2007
Phenotypic Investigation of Cell Junction–Related Proteins in Gelatinous Drop-Like Corneal Dystrophy
Author Affiliations
  • Maho Takaoka
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Kyoto, Japan; the
  • Takahiro Nakamura
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Kyoto, Japan; the
    Research Center for Regenerative Medicine, Doshisha University, Kyoto, Japan; the
  • Yuriko Ban
    Department of Ophthalmology, Nantan General Hospital, Kyoto, Japan.
  • Shigeru Kinoshita
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Kyoto, Japan; the
Investigative Ophthalmology & Visual Science March 2007, Vol.48, 1095-1101. doi:10.1167/iovs.06-0740
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      Maho Takaoka, Takahiro Nakamura, Yuriko Ban, Shigeru Kinoshita; Phenotypic Investigation of Cell Junction–Related Proteins in Gelatinous Drop-Like Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2007;48(3):1095-1101. doi: 10.1167/iovs.06-0740.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To identify the molecules involved in the pathogenesis of gelatinous drop-like corneal dystrophy (GDLD) by using immunohistochemical analysis of the expression of tight junction (TJ)-, desmosome-, and basement membrane (BM)–related proteins in human corneas with GDLD.

methods. Mutation analysis was performed on samples from three Japanese women with GDLD. Four corneal buttons from these patients were examined histopathologically by Congo red staining and immunohistochemical analysis for the expression of TJ-related proteins (ZO-1, occludin, and claudin-1), desmosome components (desmoplakin), BM-related proteins (integrins α6, β4, α3, and β1; laminin-5; and collagens IV and VII), amyloid P component, and lactoferrin.

results. Mutation analysis revealed that all three women had the Q118X mutation on M1S1. There were accumulations, primarily beneath the epithelium, of Congo-red–positive deposits with birefringence under polarized light. The BM was abnormally thickened and demonstrated a bandlike area. Immunofluorescence analysis revealed that neither ZO-1 nor occludin was expressed in the TJ areas of surface epithelial cells; there was no expression of claudin-1 or desmoplakin in the epithelial surface layer of GDLD corneas. Integrins α6, β4, α3, and β1 was expressed along the serrated surface of the BM, laminin-5 and collagens IV and VII were widely expressed throughout the BM, and lactoferrin was expressed in the amyloid deposits and the thickened BM.

conclusions. This is the first demonstration of the unique expression patterns of the major cell-junction–related proteins in GDLD corneas. The results show that in corneas with the Q118X mutation, there is a disturbance in cell-to-cell and cell-to-substrate junctions. These findings are an important step toward elucidating the pathogenesis of GDLD.

Gelatinous drop-like corneal dystrophy (GDLD), an uncommon, autosomal recessive disease, is characterized by bilateral corneal amyloidosis. It is not as rare in Japan (1 in 30,000) as in the rest of the world. 1 Subepithelial gelatinous deposits of amyloid material appear in the first decade of life and result in severe photophobia, tearing, and an ocular foreign body sensation. The deposits typically enlarge with increasing age; their accumulation within and beneath the corneal epithelium or within the anterior stroma eventually leads to severe bilateral vision loss by the third decade of life. Besides the typical appearance, GDLD corneas can demonstrate various phenotypes. These have been classified into four distinct subtypes: band keratopathy type, stromal opacity type, kumquatlike type, and typical mulberry type. 2  
Although mutation analysis has revealed mutations in many corneal dystrophies, little is known about the exact mechanisms that underlie these gene mutations and lead to corneal lesions. The M1S1 gene (also known as TACSTD2, TROP2, GA733-1, or EGP-2), implicated in GDLD 3 4 5 6 7 and putatively associated with the aggressiveness of colorectal cancer, 8 encodes a human cell surface glycoprotein that has been shown to accumulate at the cell-to-cell border, 4 suggesting that M1S1 gene mutations may disturb the cell-adhesion function. 
The high permeability of the corneal epithelium in many human GDLD corneas is demonstrated by abnormal fluorescein diffusion into the corneal stroma. We have reported slit lamp fluorophotometric findings that tight junctions (TJs) in GDLD corneas are functionally disturbed, as evidenced by higher fluorescein uptake. 9 10 Our electron microscopic study confirmed the abnormal penetration of horseradish peroxidase (HRP) through TJs and superficial desmosomes. 11 In addition, our scanning electron microscopic study showed that cell junctions in GDLD were morphologically disturbed. This disturbance was evidenced by abnormal gaps between surface epithelial cells, 9 disordered desmosomes in the superficial layer, and an abnormal basal epithelium with many spikelike projections protruding into the underlying amyloid–collagen tissue. 11 Others 12 have isolated lactoferrin, a major component of tear fluid, from GDLD amyloid deposits, suggesting that lactoferrin in amyloid deposits was the result of abnormal penetration through incomplete cell junctions. In combination, these earlier findings strongly suggest that in patients with GDLD, cell-junction–related proteins are not well-functioning or not in their original position. However, little is known about the precise molecules that make up the cell-junction–related proteins in GDLD. 
We report for the first time clinical, histologic, and immunohistochemical findings on GDLD corneas and identify the molecules comprising the cell-junction–related proteins in these patients. Our findings are a large step toward a further understanding of the pathogenesis of GDLD. 
Subjects and Methods
All experimental procedures were approved by the Institutional Review Board for Human Studies of Kyoto Prefectural University of Medicine. Prior informed consent was obtained from all patients in accordance with the tenets of the Declaration of Helsinki for research involving human subjects. 
Subjects
The study included four eyes of three GDLD patients. Case 1 involved a 65-year-old Japanese woman who had undergone two lamellar keratoplasties on both eyes. Her parents were first cousins. On slit lamp examination, her right eye demonstrated the typical mulberry pattern with translucent globular droplets; her left eye showed a mulberry pattern with massive whitish deposits (Figs. 1A 1B) . She underwent right and left lamellar keratoplasty on February 4 and December 16, 2005, respectively. Case 2 involved a 43-year-old Japanese woman. Her parents also were first cousins and her brother had already had a diagnosis of GDLD with the Q118X mutation in the M1S1 gene. She had undergone lamellar keratoplasty in the right eye and no prior operation in the left eye. Slit lamp examination demonstrated corneal stromal opacity in the right eye; her left eye manifested the typical mulberry-like appearance (Fig. 1C) . Deep lamellar keratoplasty of the left eye was performed on September 17, 2004. In case 3, a 58-year-old Japanese woman had undergone four keratoplasties in the right eye and five keratoplasties in the left. She had no family history of eye disorders. Clinical findings revealed subepithelial and stromal opacity in both corneas; thus, they were classified as stromal opacity type (Fig. 1D) . Penetrating keratoplasty in the left eye was performed on January 18, 2005. Three experts in corneal diseases observed these patients and performed the phenotype classification. 2 As control samples, we examined normal corneas obtained from human corneoscleral rims from the Northwest Lion Eye Bank (Seattle, WA). 
Mutation Analysis
We analyzed four mutations (Q118X, 632delA, Q207X, and S170X) in the M1S1 gene. Genomic DNA was extracted with a DNA extraction kit from peripheral leukocytes. Each exon of the M1S1 gene was amplified by polymerase chain reaction (PCR) using the appropriate primers. PCR products were purified and directly sequenced on an automated sequencer. DNA was digested with restriction enzymes to verify mutations in the M1S1 gene. 
Histopathology
Sectioned corneal buttons were immediately washed with phosphate-buffered saline (PBS) and embedded in optimal cutting temperature (OCT) compound. Frozen sections were sliced to a thickness of 8 μm, placed on gelatin-coated slides, air dried, and fixed in 100% acetone at 4°C. The sections were stained with hematoxylin, eosin, and Congo red, passed through a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%), and coverslipped with mounting medium. The sections were then viewed under polarized filters to confirm that the Congo red–positive deposits were amyloid materials. 
Immunofluorescence
Immunohistochemical analysis of all primary antibodies was performed by using the indirect immunofluorescent technique. The corneal buttons were embedded, sliced, and rehydrated as in the histopathologic study. After several washings with PBS, the sections were incubated with 1% bovine serum albumin (BSA) at room temperature for 30 minutes to block nonspecific binding, incubated with the appropriate primary antibodies (Table 1)for 1 hour at room temperature, and washed three times in PBS for 15 minutes each. For negative control experiments, the equivalent serum was used. After staining with the primary antibodies, the sections were incubated at room temperature for 1 hour with suitable fluorescein-conjugated secondary antibodies. After several washes with PBS, they were coverslipped with antifade mounting medium containing propidium iodide (PI; Vector Laboratories Burlingame, CA). The slides were examined with a confocal microscope (Fluoview; Olympus, Tokyo, Japan). 
Immunohistochemistry
Lactoferrin was examined via the direct immunohistochemical method. Sections were incubated with 3% BSA at room temperature for 30 minutes to block nonspecific binding. The slides were then washed for 30 minutes in 0.2% hydrogen peroxide containing 0.1% sodium azide, incubated with HRP-conjugated antibody (Biogenesis, Poole, UK) or HRP-conjugated sheep IgG as a negative control (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes at room temperature, and washed three times in PBS for 10 minutes each. The sections were incubated at room temperature for 10 minutes with 0.02% diaminobenzidine (DAB) solution, dehydrated through an ethanol series (50%, 70%, 80%, 90%, 95%, and 100%), and coverslipped with mounting medium. 
Results
Mutation Analysis
Sequence analysis revealed that all three patients manifested the stop mutation at Q118X; no other mutations (632delA, Q207X, or S170X) were detected. Therefore, a diagnosis of GDLD attributable to the Q118X mutation was returned for all three patients (Fig. 1E)
Histopathology
Both corneas from case 1 are shown in Figure 2 . There were accumulations of Congo-red–positive deposits within or beneath the epithelium and within the anterior stroma, mainly in the central cornea (Figs. 2A 2D) . The left cornea exhibited more deposits than did the right (Figs. 2A 2D) . The deposits manifested different patterns depending on their position. Subepithelial deposits formed massive clumps that appeared to push up the epithelial layer to form mulberry-like patterns. Higher magnification of the area just beneath the epithelium revealed bandlike sections with many spikes vertical to the basement line or amyloid materials (Figs. 2B 2E) . The anterior stroma contained amyloid deposits along with a collagen layer, especially beneath the massive subepithelial deposits. All the deposits manifested birefringence under polarized light; thus, the deposits were identified as amyloid materials (Figs. 2C 2F)
Immunofluorescence and Immunohistochemistry
Neither ZO-1 nor occludin, present at the junctions of surface epithelial cells in normal corneas, was seen in the epithelial cells of GDLD corneas (Figs. 3A 3B 3C 3D) . We discerned no distinct differences between the subtypes of phenotypical classification. 2 Claudin-1 and desmoplakin, detected at intercellular junctions in normal and GDLD corneas, did not exist on the surface layer of our GDLD corneas (Figs. 3E 3F 3G 3H) . The fluorescence pattern of claudin-1 and desmoplakin appeared at the epithelial cell junctions, showing the surface layer of the epithelial cells to be abnormally flattened and thin (Figs. 3E 3F 3G 3H) . In normal corneas, the fluorescence of integrins was linear just under the epithelial layer, however, in the GDLD corneas, it was irregular and showed a linear, spikelike pattern beneath the epithelium (Figs. 4A 4B 4C 4D 4E 4F 4G 4H) . The expression patterns of laminin-5 and collagens IV and VII were also linear on normal corneas; on the GDLD corneas they extended over a wide portion of a bandlike area just beneath the spikelike pattern (Figs. 4I 4J 4K 4L 4M 4N) . Amyloid P was demonstrated in the same areas as the fluorescence of laminin-5 and collagen IV and VII (Figs. 4O 4P)
There was positive staining for lactoferrin in the areas of the amyloid deposits and the thickened BM, the staining pattern being similar to that of Congo red staining (Figs. 5A 5B) . The nuclei of epithelial cells also showed positive staining for lactoferrin (Fig. 5A) . Negative control slides showed no immunostaining for lactoferrin (Fig. 5C)
Discussion
To the best of our knowledge, this is the first immunohistochemical demonstration of the disturbance and abnormal localization pattern of cell-junction–related proteins in GDLD corneas with the Q118X mutation. Neither ZO-1 nor occludin was present in the TJ areas, claudin-1 and desmoplakin were not expressed in the surface layer of epithelial cell junctions, and BM-related proteins manifested an abnormal localization pattern, since the BM was abnormally thickened. 
Our results raise important questions regarding the anomalies in cell-junction–related proteins in patients with GDLD corneas, even though there is strangely no report that we could find about such abnormalities in other tissues. In all three of our patients, the M1S1 gene exhibited the Q118X mutation. Although the precise pathogenesis of GDLD is unknown because the M1S1 gene function remains to be clarified, there are several suggestions based on the following evidence. The M1S1 gene, cloned by Fornaro et al., 12 encodes a 35,709-Da type 1 transmembrane protein with a single-transmembrane domain. It is known to be expressed at high levels by the most human carcinomas. 13 14 15 16 It is also known as a calcium signal transducer 17 that regulates the growth of expressing tumor cells. 12 Its function is unclear, but several potential modification sites within the molecule have been suggested. M1S1 contains a phosphatidylinositol 4,5-bisphosphonate (PIP2)-binding site, 18 which regulates binding to other cytoplasmic molecules or to plasma membrane, 19 20 21 and its serine residue is phosphorylated by protein kinase C. 22 The nonsense or frameshift mutations cause loss of the PIP2-binding site, which may result in losing the adhesion function. Moreover, the TROP1 gene (also known as Ep-CAM, TACSTD1, KSA, or GA733-2), which is homologous to the M1S1 (TROP2) gene, has been shown to play a central role in cell adhesion function, to be expressed in most normal epithelial tissues on the basolateral surface, and to interact directly with the TJ protein claudin-7. 23 Furthermore, it has been reported that truncated M1S1 proteins do not accumulate at the cell membrane 3 and that normal M1S1 proteins accumulate at the cell–cell border. 4 These findings imply that the Q118X mutation affects the cell adhesion function and causes the disturbances of cell–cell or cell–substrate junctions. 
Lactoferrin, a major, 78.2-kDa component of tears, has attracted attention because it was found in GDLD amyloid deposits 24 and because lactoferrin fragments can form amyloid fibrils in vitro. 25 26 Although the source of lactoferrin in the amyloid deposits remains to be determined, it may originate from the tear film and penetrate disturbed cell junctions. Alternatively, its presence may be attributable to the overexpression of the lactoferrin gene in epithelial cells although a link between the lactoferrin- and M1S1 gene has been excluded. 24 We speculate that normally expressed lactoferrin enters the amyloid deposits by yet unknown means. Although the patients reported by others 24 27 had a clinical diagnosis of GDLD, those studies did not involve mutation analysis. Our study is the first to confirm the presence of lactoferrin within amyloid deposits in GDLD corneas with the Q118X mutation. 
Our results on GDLD corneas showed a lack of cell junction–related protein expression in TJ areas and the surface layer of epithelial cell junctions, and we identified lactoferrin in amyloid deposits and thickened BM. Together, these findings suggest that in GDLD corneas, the TJ- and surface-layer cell junction barriers may be compromised, thus allowing lactoferrin in the tear fluid to penetrate the loose barriers and flow into the BM and subepithelial area, resulting in the formation of amyloid deposits. 
Santagati et al. 28 reported that under cultured conditions, bovine corneal epithelial cells can produce lactoferrin. This finding raises the possibility that the lactoferrin we observed in the amyloid deposits originated not only from tear fluid, but also from corneal epithelial cells. Araki-Sasaki et al. 29 showed a significantly increased frequency of the lactoferrin polymorphism Glu561Asp in patients with secondary corneal amyloidosis. We did not address this question in the current investigation, and studies are under way to determine whether lactoferrin polymorphism is a key factor in the formation of amyloid deposits in GDLD. 
Lactoferrin immunostaining showed, unexpectedly, the existence of lactoferrin mainly in the nuclei of GDLD epithelial cells. We could not distinctly detect lactoferrin in the cytoplasm, and thus we were unable to isolate the mechanism behind the lactoferrin secretion from GDLD corneal epithelium. Most studies of the intracellular distribution of lactoferrin have shown it to be confined to the cytoplasm, but several investigators have shown lactoferrin localization to the nucleus. 30 31 32 33 34 35 Lactoferrin binds to DNA with high affinity 36 37 38 and is shown to be taken up by K562 human myelogenous leukemia cells and transported to the nucleus, where it is bound to DNA. 33 Moreover, lactoferrin is believed to play a role as a transcriptional regulator. 33 35 38 39 40 We did not investigate the mechanism behind lactoferrin localization in the nuclei of GDLD epithelial cells, yet such findings may be important for elucidating the pathogenesis of GDLD. To the best of our knowledge, this is the first report that suggests the existence of lactoferrin in the nuclei of corneal epithelial cells. 
In the mild form of GDLD (the right eye in case 1), there was moderate subepithelial amyloid accumulation. Just beneath the basal cells we noted a bandlike, thickened BM with many spikes. In contrast, in the severe form of GDLD (the left eye in case 1), massive accumulations of subepithelial amyloid deposits appeared to break the BM; however, the area just beneath the epithelium exhibited a bandlike pattern. Immunohistochemical investigation revealed that this area harbored amyloid P, laminin-5, and collagens IV and VII, a finding that coincides with our earlier high-resolution, scanning electron microscopic observations that collagen fibrils coexisted with amyloid deposits in GDLD. 9 Although others 41 42 had also noted this bandlike structure, the mechanism(s) underlying its formation were not discussed. We postulate that the accumulation of amyloid in BM areas rich in laminin-5 and collagen IV and VII leads to thickening of the BM and the formation of this bandlike structure. It is also possible that the thickening of the BM resulted from the chronic stress or some other reasons, but we were unable to determine when the thickening of the BM occurred or how it progressed because the corneal buttons had already undergone many changes. 
We also suggest that the many spikes protruding into the basement line of the BM may reflect differences in the rigidity of attachment between the corneal epithelium and the basement line. In other words, amyloid materials accumulated in a comparatively loose area within the BM, and the firmly attached deposits formed spikes. These spikes were prominent in integrins probably because integrins exist on the upper side of the BM, and the thickening of the underlying collagen part of the BM caused them to deform. The massive amyloid deposits in case 1 were primarily located between these bandlike structures and the stroma. The subepithelial area appeared to be very loose, as evidenced by the partial separation of the thickened BM from the stroma. Investigations are under way in our laboratory to determine whether lactoferrin passed through disturbed epithelial cell junctions and penetrated the compromised BM before entering into the subepithelial space and forming amyloid deposits. 
We present clinical, histologic, and immunohistochemical findings on corneas from patients with GDLD, focusing on the major component proteins of TJ complexes, desmosomes, and the BM. GDLD corneas with the Q118X mutation manifested severe TJ disturbances and the unique expression of desmosome- and BM-related proteins. Our findings are a large step toward a better understanding of the pathogenesis of GDLD. 
 
Figure 1.
 
Slit lamp examination of the right (A) and left (B) eye in case 1, the left eye in case 2 (C), and the left eye in case 3 (D). Note the typical mulberry opacity in both eyes in case 1 (A, B) and the left eye in case 2 (C). Corneal opacity was evident in the left eye in case 3 (D). (E) The sequence of the M1S1 gene around the Q118X mutation. All patients had this mutation.
Figure 1.
 
Slit lamp examination of the right (A) and left (B) eye in case 1, the left eye in case 2 (C), and the left eye in case 3 (D). Note the typical mulberry opacity in both eyes in case 1 (A, B) and the left eye in case 2 (C). Corneal opacity was evident in the left eye in case 3 (D). (E) The sequence of the M1S1 gene around the Q118X mutation. All patients had this mutation.
Table 1.
 
Primary Antibodies
Table 1.
 
Primary Antibodies
Antibodies Categories Source Dilution
ZO-1 Rabbit polyclonal Zymed, San Francisco, CA 1:25
Occludin Goat monoclonal Santa Cruz Biotechnology, Santa Cruz, CA 1:50
Claudin-1 Rabbit polyclonal Zymed 1:10
Desmoplakin Mouse monoclonal Progen, Queensland, Australia 1:1
Integrin α6 Mouse monoclonal Chemicon, Temecula, CA 1:200
Integrin β4 Mouse monoclonal Chemicon 1:500
Integrin α3 Mouse monoclonal Chemicon 1:200
Integrin β1 Mouse monoclonal Chemicon 1:500
Laminin-5 Mouse monoclonal Chemicon 1:100
Collagen IV Mouse monoclonal Chemicon 1:200
Collagen VII Mouse monoclonal Chemicon 1:100
Serum amyloid P component Rabbit polyclonal Dako Cytomation, Kyoto, Japan 1:200
Figure 2.
 
Corneal samples (case 1) were stained with hematoxylin, eosin, and Congo red. Right (A-C) and left (DF) eyes. (A, D, insets) Slit lamp photographs. Note the Congo red–positive accumulations within or beneath the epithelium and within the anterior stroma. In the center of the cornea, Congo red–positive deposits accumulated to form gelatinous droplike masses (A, D). Higher magnification revealed bandlike structures with a spikelike pattern just beneath the epithelium (B, E). The deposits demonstrated birefringence under polarized light (C, F). Scale bars: 100 μm.
Figure 2.
 
Corneal samples (case 1) were stained with hematoxylin, eosin, and Congo red. Right (A-C) and left (DF) eyes. (A, D, insets) Slit lamp photographs. Note the Congo red–positive accumulations within or beneath the epithelium and within the anterior stroma. In the center of the cornea, Congo red–positive deposits accumulated to form gelatinous droplike masses (A, D). Higher magnification revealed bandlike structures with a spikelike pattern just beneath the epithelium (B, E). The deposits demonstrated birefringence under polarized light (C, F). Scale bars: 100 μm.
Figure 3.
 
Immunohistochemical analysis of TJ- and desmosome-related protein expression in the right eye (case 1). ZO-1 (A, B), occludin (C, D), claudin-1 (E, F), and desmoplakin (G, H) on normal (A, C, E, G) and GDLD corneas (B, D, F, H). Arrows: upper part of the stroma. On normal corneas, ZO-1 and occludin were present at all junctions of surface epithelial cells (arrowheads). There was no ZO-1 and occludin fluorescence on GDLD corneas at those positions. (EH), fluorescence pattern of claudin-1 and desmoplakin on the basolateral surface of epithelial cells. On normal corneas, these patterns were present at almost all epithelial cell junctions, including the surface layer (arrowheads). There was no such expression on the surface layer of the epithelium on GDLD corneas. Scale bar, 50 μm.
Figure 3.
 
Immunohistochemical analysis of TJ- and desmosome-related protein expression in the right eye (case 1). ZO-1 (A, B), occludin (C, D), claudin-1 (E, F), and desmoplakin (G, H) on normal (A, C, E, G) and GDLD corneas (B, D, F, H). Arrows: upper part of the stroma. On normal corneas, ZO-1 and occludin were present at all junctions of surface epithelial cells (arrowheads). There was no ZO-1 and occludin fluorescence on GDLD corneas at those positions. (EH), fluorescence pattern of claudin-1 and desmoplakin on the basolateral surface of epithelial cells. On normal corneas, these patterns were present at almost all epithelial cell junctions, including the surface layer (arrowheads). There was no such expression on the surface layer of the epithelium on GDLD corneas. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical analysis of basement membrane-related proteins and amyloid P in the right eye (case 1). Integrin α6 (A, B), β4 (C, D), α3 (E, F), β1 (G, H), laminin-5 (I, J), collagen IV (K, L), collagen VII (M, N), and amyloid P (O, P) on normal (A, C, E, G, I, K, M, O) and GDLD corneas (B, D, F, H, J, L, N, P). Arrows: upper portion of the stroma. The fluorescence patterns of integrins on normal corneas were linear (A, C, E, G). However, on the GDLD cornea they were irregular with many spikes (B, D, F, H). Laminin-5 and collagen IV and VII patterns were also linear on the normal cornea (I, K, M). On the GDLD cornea, the fluorescence patterns (J, L, N) and the pattern of amyloid P (P) were bandlike. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical analysis of basement membrane-related proteins and amyloid P in the right eye (case 1). Integrin α6 (A, B), β4 (C, D), α3 (E, F), β1 (G, H), laminin-5 (I, J), collagen IV (K, L), collagen VII (M, N), and amyloid P (O, P) on normal (A, C, E, G, I, K, M, O) and GDLD corneas (B, D, F, H, J, L, N, P). Arrows: upper portion of the stroma. The fluorescence patterns of integrins on normal corneas were linear (A, C, E, G). However, on the GDLD cornea they were irregular with many spikes (B, D, F, H). Laminin-5 and collagen IV and VII patterns were also linear on the normal cornea (I, K, M). On the GDLD cornea, the fluorescence patterns (J, L, N) and the pattern of amyloid P (P) were bandlike. Scale bar, 50 μm.
Figure 5.
 
Lactoferrin was positively stained in the areas of amyloid deposits and in the thickened basement membrane; the staining pattern being similar to that of Congo red staining (A, B). The nuclei of epithelial cells also showed positive staining for lactoferrin (A). The negative control using HRP-conjugated IgG is also shown (C). Scale bar, 50 μm.
Figure 5.
 
Lactoferrin was positively stained in the areas of amyloid deposits and in the thickened basement membrane; the staining pattern being similar to that of Congo red staining (A, B). The nuclei of epithelial cells also showed positive staining for lactoferrin (A). The negative control using HRP-conjugated IgG is also shown (C). Scale bar, 50 μm.
The authors thank Kaoru Araki-Sasaki and Yasuhiro Osakabe for advice and the gift of anti-lactoferrin antibody, Mina Kojo for assisting with patient care, and Ursula Petralia and John Bush for editing the manuscript. 
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Figure 1.
 
Slit lamp examination of the right (A) and left (B) eye in case 1, the left eye in case 2 (C), and the left eye in case 3 (D). Note the typical mulberry opacity in both eyes in case 1 (A, B) and the left eye in case 2 (C). Corneal opacity was evident in the left eye in case 3 (D). (E) The sequence of the M1S1 gene around the Q118X mutation. All patients had this mutation.
Figure 1.
 
Slit lamp examination of the right (A) and left (B) eye in case 1, the left eye in case 2 (C), and the left eye in case 3 (D). Note the typical mulberry opacity in both eyes in case 1 (A, B) and the left eye in case 2 (C). Corneal opacity was evident in the left eye in case 3 (D). (E) The sequence of the M1S1 gene around the Q118X mutation. All patients had this mutation.
Figure 2.
 
Corneal samples (case 1) were stained with hematoxylin, eosin, and Congo red. Right (A-C) and left (DF) eyes. (A, D, insets) Slit lamp photographs. Note the Congo red–positive accumulations within or beneath the epithelium and within the anterior stroma. In the center of the cornea, Congo red–positive deposits accumulated to form gelatinous droplike masses (A, D). Higher magnification revealed bandlike structures with a spikelike pattern just beneath the epithelium (B, E). The deposits demonstrated birefringence under polarized light (C, F). Scale bars: 100 μm.
Figure 2.
 
Corneal samples (case 1) were stained with hematoxylin, eosin, and Congo red. Right (A-C) and left (DF) eyes. (A, D, insets) Slit lamp photographs. Note the Congo red–positive accumulations within or beneath the epithelium and within the anterior stroma. In the center of the cornea, Congo red–positive deposits accumulated to form gelatinous droplike masses (A, D). Higher magnification revealed bandlike structures with a spikelike pattern just beneath the epithelium (B, E). The deposits demonstrated birefringence under polarized light (C, F). Scale bars: 100 μm.
Figure 3.
 
Immunohistochemical analysis of TJ- and desmosome-related protein expression in the right eye (case 1). ZO-1 (A, B), occludin (C, D), claudin-1 (E, F), and desmoplakin (G, H) on normal (A, C, E, G) and GDLD corneas (B, D, F, H). Arrows: upper part of the stroma. On normal corneas, ZO-1 and occludin were present at all junctions of surface epithelial cells (arrowheads). There was no ZO-1 and occludin fluorescence on GDLD corneas at those positions. (EH), fluorescence pattern of claudin-1 and desmoplakin on the basolateral surface of epithelial cells. On normal corneas, these patterns were present at almost all epithelial cell junctions, including the surface layer (arrowheads). There was no such expression on the surface layer of the epithelium on GDLD corneas. Scale bar, 50 μm.
Figure 3.
 
Immunohistochemical analysis of TJ- and desmosome-related protein expression in the right eye (case 1). ZO-1 (A, B), occludin (C, D), claudin-1 (E, F), and desmoplakin (G, H) on normal (A, C, E, G) and GDLD corneas (B, D, F, H). Arrows: upper part of the stroma. On normal corneas, ZO-1 and occludin were present at all junctions of surface epithelial cells (arrowheads). There was no ZO-1 and occludin fluorescence on GDLD corneas at those positions. (EH), fluorescence pattern of claudin-1 and desmoplakin on the basolateral surface of epithelial cells. On normal corneas, these patterns were present at almost all epithelial cell junctions, including the surface layer (arrowheads). There was no such expression on the surface layer of the epithelium on GDLD corneas. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical analysis of basement membrane-related proteins and amyloid P in the right eye (case 1). Integrin α6 (A, B), β4 (C, D), α3 (E, F), β1 (G, H), laminin-5 (I, J), collagen IV (K, L), collagen VII (M, N), and amyloid P (O, P) on normal (A, C, E, G, I, K, M, O) and GDLD corneas (B, D, F, H, J, L, N, P). Arrows: upper portion of the stroma. The fluorescence patterns of integrins on normal corneas were linear (A, C, E, G). However, on the GDLD cornea they were irregular with many spikes (B, D, F, H). Laminin-5 and collagen IV and VII patterns were also linear on the normal cornea (I, K, M). On the GDLD cornea, the fluorescence patterns (J, L, N) and the pattern of amyloid P (P) were bandlike. Scale bar, 50 μm.
Figure 4.
 
Immunohistochemical analysis of basement membrane-related proteins and amyloid P in the right eye (case 1). Integrin α6 (A, B), β4 (C, D), α3 (E, F), β1 (G, H), laminin-5 (I, J), collagen IV (K, L), collagen VII (M, N), and amyloid P (O, P) on normal (A, C, E, G, I, K, M, O) and GDLD corneas (B, D, F, H, J, L, N, P). Arrows: upper portion of the stroma. The fluorescence patterns of integrins on normal corneas were linear (A, C, E, G). However, on the GDLD cornea they were irregular with many spikes (B, D, F, H). Laminin-5 and collagen IV and VII patterns were also linear on the normal cornea (I, K, M). On the GDLD cornea, the fluorescence patterns (J, L, N) and the pattern of amyloid P (P) were bandlike. Scale bar, 50 μm.
Figure 5.
 
Lactoferrin was positively stained in the areas of amyloid deposits and in the thickened basement membrane; the staining pattern being similar to that of Congo red staining (A, B). The nuclei of epithelial cells also showed positive staining for lactoferrin (A). The negative control using HRP-conjugated IgG is also shown (C). Scale bar, 50 μm.
Figure 5.
 
Lactoferrin was positively stained in the areas of amyloid deposits and in the thickened basement membrane; the staining pattern being similar to that of Congo red staining (A, B). The nuclei of epithelial cells also showed positive staining for lactoferrin (A). The negative control using HRP-conjugated IgG is also shown (C). Scale bar, 50 μm.
Table 1.
 
Primary Antibodies
Table 1.
 
Primary Antibodies
Antibodies Categories Source Dilution
ZO-1 Rabbit polyclonal Zymed, San Francisco, CA 1:25
Occludin Goat monoclonal Santa Cruz Biotechnology, Santa Cruz, CA 1:50
Claudin-1 Rabbit polyclonal Zymed 1:10
Desmoplakin Mouse monoclonal Progen, Queensland, Australia 1:1
Integrin α6 Mouse monoclonal Chemicon, Temecula, CA 1:200
Integrin β4 Mouse monoclonal Chemicon 1:500
Integrin α3 Mouse monoclonal Chemicon 1:200
Integrin β1 Mouse monoclonal Chemicon 1:500
Laminin-5 Mouse monoclonal Chemicon 1:100
Collagen IV Mouse monoclonal Chemicon 1:200
Collagen VII Mouse monoclonal Chemicon 1:100
Serum amyloid P component Rabbit polyclonal Dako Cytomation, Kyoto, Japan 1:200
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