Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells.

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

Diquafosol promotes corneal epithelial healing via intracellular calcium-mediated ERK activation.

Diquafosol is known as a purinergic P2Y2 receptor (P2Y2R) agonist that stimulates water and mucin secretion from conjunctival epithelial cells and goblet cells, leading to tear film stability in dry eye. However, its effect on corneal epithelial healing has not yet been elucidated. The aim of the present study was to evaluate the effect of diquafosol on corneal epithelial healing in vivo and on P2Y2R-related downstream signaling pathways in vitro. We administered 3% diquafosol ophthalmic solution on 3 mm-diameter epithelial defects made in rat corneas and assessed the wound closure over time. Corneal epithelial healing was significantly accelerated in diquafosol-treated eyes compared to control eyes at 12 and 24 h. During wound healing, P2Y2R staining appeared stronger in the re-epithelized margin near the wound defect. To evaluate whether diquafosol stimulates epidermal growth factor receptor/extracellular-signal-regulated kinase (EGFR/ERK)-related cell proliferation and migration, simian virus 40-transfected human corneal epithelial (THCE) cells were used for in vitro experiments. Cell proliferation was accelerated by diquafosol at concentrations from 20 to 200 µM during 48 h, but inhibited at concentrations over 2000 µM. The intracellular calcium ([Ca(2+)]i) elevation was measured in diquafosol (100 µM)-stimulated cells using Fluo-4/AM ([Ca(2+)]i indicator). [Ca(2+)]i elevation was observed in diquafosol-stimulated cells regardless of the presence of calcium in media, and suramin pretreatment inhibited the calcium response. The effect of diquafosol on phosphorylation of EGFR, ERK and Akt, and cell migration was determined by western blotting and in vitro cell migration assay. Diquafosol induced phosphorylation of EGFR at 2 min post-stimulation, and phosphorylation of ERK at 5 min post-stimulation. Phosphorylation of ERK was attenuated in cells pretreated with suramin or BAPTA/AM ([Ca(2+)]i chelator), and partially with AG1478 (EGFR inhibitor). Likewise, diquafosol-treated cells showed acceleration of gap closure in cell migration assay, which was inhibited by suramin, BAPTA/AM, AG1478, and U0126 (MEK inhibitor). These studies demonstrate that diquafosol is effective in promoting corneal epithelial wound healing and that this effect may result from ERK-stimulated cell proliferation and migration via P2Y2R-mediated [Ca(2+)]i elevation.

Proinflammatory gene polymorphisms are potentially associated with Korean non-Sjogren dry eye patients.

PURPOSE: To determine whether proinflammatory cytokine genes were potential susceptibility candidate genes for Korean patients with non-Sjogren dry eye, we investigated the association of the interleukin 1 beta (IL1B), interleukin 6 (IL6), and interleukin 6 receptor (IL6R) variations with this disease in Korean patients. METHODS: Genomic DNA was extracted from blood samples of unrelated non-Sjogren dry eye patients and healthy control individuals who visited the Eye Center and Health Promotion Center of St. Mary's Hospital in Seoul, Korea. For screening genetic variations in proinflammatory cytokine genes, the 511 (rs16944) and 31 (rs1143627) positions in the promoter region of IL1B, rs1143634 in exon 5 of IL1B, rs1800795 of the IL6 promoter, and Asp358Ala (rs8192284) of IL6R were genotyped using the polymerase chain reaction, restriction fragment length polymorphisms, and direct sequencing. RESULTS: Among the polymorphisms, rs1143634 (F105F) in exon 5 of IL1B was significantly different between the patient and control groups. The frequency of the C/T genotype in dry eye patients was decreased relative to that of the control subjects (10.4% versus 3.9%, p=0.043, OR=3.337). For the IL6R gene, the genotypic and allelic distribution of rs8192284 was different between the dry eye patients and the controls: CC genotype (p=0.017, OR=2.12) and C allele (OR=1.26). CONCLUSIONS: This is the first report of genetic variation screening of proinflammatory cytokine genes in Korean non-Sjogren dry eye patients. It is suggested that rs1143634 of IL1B and rs8192284 of IL6R act as susceptibility variations in Korean non-Sjogren dry eye patients.

Association with Corneal Remodeling Related Genes, ALDH3A1, LOX, and SPARC Genes Variations in Korean Keratoconus Patients.

PURPOSE: To determine whether the cornea remodeling-related genes aldehyde dehydrogenase 3A1 (ALDH3A1), lysyl oxidase (LOX), and secreted protein acidic and rich in cysteine (SPARC) were potential susceptibility candidate genes for keratoconus in Korean patients, we investigated the associations of single nucleotide polymorphisms (SNPs) in these three genes in Korean patients with keratoconus. METHODS: Genomic DNA was extracted from blood samples of unrelated patients with keratoconus and healthy control individuals. For screening of genetic variations, all exons from the entire coding regions of the ALDH3A1, LOX, and SPARC genes were directly sequenced to determine the presence of mutations. Control individuals were selected from the general population without keratoconus. RESULTS: In this study, we detected nine SNPs in ALDH3A1, four SNPs in LOX, and 18 SNPs in SPARC. rs116992290, IVS3-62c>t, rs116962241, and rs2228100 in ALDH3A1 and rs2956540 and rs1800449 in LOX were significantly different between patient and control groups. In the SPARC gene, the distribution of the *G allele of EX10+225 T>G (p = 0.018; odds ratio, 1.869) was strongly associated with the risk of keratoconus in the Korean population. In haplotype analysis, C-G of rs2956540-rs2288393 in LOX(p = 0.046) and C-C-G and G-G-G of rs60610024-rs2228100-rs57555435 (p = 0.021 and p < 0.001), G-A of IVS3-62 a>g - rs116962241 in ALDH3A1(p = 0.048) predisposed significantly to keratoconus. After cross-validation consistency and permutation tests, two locus model was the best SNP variations interaction pattern. CONCLUSIONS: Our results suggested that genetic variations in ALDH3A1, LOX, and SPARC genes were associated with a predisposition for keratoconus in Korean individuals. Moreover, variations in ALDH3A1 and LOX may serve as strong biomarkers for keratoconus.

Ocular surface inflammation induces de novo expression of substance P in the trigeminal primary afferents with large cell bodies.

We evaluated the changes in substance P (SP)-expressing trigeminal neurons (TNs) innervating the cornea following ocular surface inflammation. Ocular surface inflammation was induced in Sprague-Dawley rats using 0.1% benzalkonium chloride (BAK). The corneal staining score, corneal epithelial apoptosis, conjunctival goblet cells, and density of corneal subbasal nerve plexus (SNP) were assessed, and the mRNA levels of SP, interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α were measured in corneas and ipsilateral trigeminal ganglia (TG). SP-immunoreactivity (IR) was measured in corneal intraepithelial nerves and TNs. The cell size of corneal TNs in the TG was calculated. All parameters were observed immediately (BAK group), at 1 week (1 w group), and 2 months (2 m group) after 2 weeks of BAK application. BAK caused an increase in the corneal staining score and the number of apoptotic cells, loss of conjunctival goblet cells, reduced density of corneal SNP, and upregulated expression of SP and inflammatory cytokines in both the cornea and TG in the BAK group but those changes were not observed in the 2 m group. On the other hand, SP-IR% and mean cell size of corneal TNs increased significantly in the BAK, 1 w, and 2 m groups, compared to the control. Our data suggest that following ocular surface inflammation, large-sized corneal TNs which normally do not express SP, expressed it and this phenotype switching lasted even after the inflammation disappeared. Long-lasting phenotypic switch, as well as changes in the expression level of certain molecules should be addressed in future studies on the mechanism of corneal neuropathic pain.

Marked infiltration of neutrophils at the upper palpebral conjunctiva in patients with chronic graft-versus-host disease.

PURPOSE: Neutrophils may be involved in the local pathophysiology of chronic graft-versus-host disease (cGVHD). We evaluated neutrophil infiltration in cGVHD using conjunctival impression cytology (IC) and its clinical correlation with ocular surface status and neutrophil enzyme levels in tears. METHODS: This cross-sectional observational study included 76 patients with cGVHD. The ocular surface was assessed for the tear break-up time, Schirmer I test, corneal and conjunctival staining score, meiboscore, and the ocular surface disease index questionnaire. Conjunctival IC was performed at the temporal, superior bulbar, and upper palpebral conjunctiva, and the number of neutrophils (cells/high power field [HPF]) was calculated. Neutrophil elastase (NE), myeloperoxidase (MPO), and matrix metalloperoxidase-8 and -9 levels in tear washes were measured in 20 patients. RESULTS: The number of neutrophils was significantly greater at the upper palpebral conjunctiva (median [range], 16.5 [0 to 147] cells/HPF) than at the temporal and superior bulbar conjunctiva (0 [0 to 70] and 0 [0 to 105] cells/HPF; P < 0.0001). The number of neutrophils at the upper palpebral conjunctiva showed moderate correlations with the corneal staining score and the NE and MPO levels in tears (r = 0.668, 0.553, and 0.563, respectively; P < 0.0001, P = 0.014, and 0.012). CONCLUSIONS: Our results suggest that neutrophils at the upper palpebral conjunctiva associate with the clinical manifestations and inflammatory status of the ocular surface in cGVHD. Conjunctival neutrophils should be addressed when assessing the inflammatory activity of ocular cGVHD and exploring its pathogenesis.

Association of -31T>C and -511 C>T polymorphisms in the interleukin 1 beta (IL1B) promoter in Korean keratoconus patients.

PURPOSE: To investigate the genetic association between unrelated Korean keratoconus patients and interleukin 1 alpha (IL1A), interleukin 1 beta (IL1B), and IL1 receptor antagonist (IL1RN) gene polymorphisms. METHODS: We investigated the association between IL1A (rs1800587, rs2071376, and rs17561), IL1B (rs1143627, rs16944, rs1143634, and rs1143633), and IL1RN (rs419598, rs423904, rs424078, and rs315952, variable number tandem repeat [VNTR]) polymorphisms in 100 unrelated Korean keratoconus patients. One hundred control individuals without any corneal disease were selected from the general population. Polymerase chain reaction (PCR) - restriction fragment length polymorphism (RFLP) analysis and direct sequencing were used to screen for genetic variations in the IL1 gene cluster. Haplotypes for the IL1 gene cluster were constructed using Haploview version 4.0. RESULTS: We analyzed a total of 12 polymorphic sites in the IL1 gene cluster. Among them, the -511 (rs16944) and -31 (rs1143627) positions in the promoter region of IL1B were significantly different between patient and control groups. The C allele of rs16944 (-511C>T, p=0.022, odds ratio of risk [OR]=1.46, 95% confidence intervals [CI] 0.94<2.27) and the T allele of rs1143627 (-31T>C, p=0.025, OR=1.43, 95% CI 0.92<2.22) were associated with a significantly increased risk of keratoconus in Korean patients. Linkage of the two alleles, -31*C and -511*T, was associated with an increased risk for keratoconus with OR=2.38 (p=0.012, 95% CI=1.116-5.046). The *C/*A genotype of rs2071376 in IL1A intron 6 was significantly different between the keratoconus patients and control subjects (p=0.034, OR=0.59, 95% CI 0.32<1.11). Other polymorphisms did not show an association with keratoconus risk. CONCLUSIONS: This is the first report of IL1 gene cluster mutation screening in Korean keratoconus patients. Significant differences in allelic frequency of IL1B between keratoconus patients and the control group suggest that IL1B polymorphisms may play a role in the susceptibility of unrelated Koreans to develop keratoconus.

The COL1A1 gene and high myopia susceptibility in Japanese.

The collagen type Iota alpha Iota (COL1A1) gene encodes the extracellular matrix component, collagen, and resides in candidate MYP5 for high myopia on the chromosome 17q22-q23.3. This locus has recently been implicated in playing an important role in the pathogenesis of experimental myopia. We investigated the association of disruptions of COL1A1 gene with high myopia by analyzing the frequency of ten SNPs in a Japanese population of 330 subjects with high myopia of -9.25 D or less and 330 randomized controls without high myopia. Two SNPs (rs2075555 and rs2269336) were significantly associated with high myopia (P < 0.05, Pc < 0.1). Two different haplotype blocks in COL1A1 were observed by the pair-wise linkage disequilibrium between the SNPs. The frequency of GGC/GGC diplotype constructed by the three SNPs (rs2075555, rs2269336, rs1107946) was significantly high (OR = 1.6) and associated with high myopia (P = 0.028, Pc< 0.084). Together our results provide the first evidence for COL1A1 as a gene associated with high myopia.

The Blockade of IL6 Counterparts the Osmolar Stress-Induced Apoptosis in Human Conjunctival Epithelial Cells.

To determine the effect of hyperosmolarity on cell survival/apoptosis of conjunctival epithelial cells and evaluate the possible role of IL6, Wong-Kilbourne derivative of Chang conjunctival cell line (WKD) was used in this study. Confluent cells were incubated under different osmolarity (290 mOsm and 500 mOsm) with or without neutralizing IL6 antibody (50 ng/mL). The expression of IL6 level was measured in the supernatant of each conditioned medium. Cell viability/apoptosis assay was performed using Annexin V/Propidium Iodide (PI) and Cell Counting Kit-8 (CCK-8). Western blot was conducted to measure the abundance of apoptotic markers and IL6 related downstream signaling pathway. The concentration of IL6 showed time-dependent increase in cells treated with 500 mOsm. Although apoptosis of WKD cell is increased in treated 500 mOsm for 24 h, apoptosis reduced in WKD cell treated 500 mOsm with anti-IL6 for 24 h. Anti-IL6 inhibited the activation of JAK-STAT signaling pathway, which was induced by hyperosmolarity. Hyperosmolar condition induced apoptosis in conjunctival epithelial cells, along with increase of IL6 production. IL6 neutralizing antibody inhibited apoptosis and JAK-STAT signaling in hyperosmolar condition. These findings suggested that IL6 may be involved in apoptotic change and in hyperosmolarity.

Tear Osmolarity and Ocular Surface Parameters as Diagnostic Markers of Ocular Graft-Versus-Host Disease.

PURPOSE: To evaluate the diagnostic value of tear osmolarity and several ocular surface parameters in screening for ocular surface alterations in ocular graft-vs-host disease (GVHD) patients. DESIGN: Case-control study. METHODS: Sixty-three patients with ocular GVHD and 74 healthy participants were screened for ocular surface changes using the Ocular Surface Disease Index (OSDI), tear osmolarity, Schirmer test, tear break-up time (TBUT), and fluorescein corneal staining. The severity of ocular GVHD was diagnosed according to the National Institutes of Health (NIH) grading system. The diagnostic sensitivity and specificity and cutoff values were determined for each ocular parameter using a receiver operating characteristic (ROC) curve and area under the curve (AUC) analysis. Significance was defined at P < .05. RESULTS: The tear osmolarity, corneal staining score, and OSDI score gradually increased as the severity of ocular GVHD increased, and Schirmer value gradually decreased as the GVHD grade increased in severity. The Schirmer test showed greatest diagnostic sensitivity and specificity for ocular GVHD (92.1% sensitivity, 85.7% specificity, cutoff = 9 mm), followed by the TBUT (87.3% sensitivity, 75.0% specificity, cutoff = 6 s), tear osmolarity (98.4% sensitivity, 60.7% specificity, cutoff = 311 mOsm/L), corneal staining score (66.7% sensitivity, 82.1% specificity, cutoff = 2), and OSDI score (77.8% sensitivity, 66.1% specificity, cutoff = 20.8). CONCLUSIONS: Multiple diagnostic modalities should be used to detect ocular surface changes in GVHD patients. The severity of ocular GVHD can be effectively monitored using tear osmolarity; however, additional studies are required.

Absence of an association between lumican promoter variants and high myopia in the Korean population.

PURPOSE: To determine the association of single nucleotide polymorphisms (SNPs) in the promoter region of the lumican (LUM) gene with high myopic Korean patients. METHODS: Genomic DNA samples were obtained from 128 unrelated Korean patients with high myopia who had refractive errors ≤ -9.25 and axial lengths ≥ 26.5 mm in both eyes, and 235 control subjects. We investigated two promoter SNPs of the LUM gene. RESULTS: For the rs3759222, the C/C genotype was less prevalent in the high myopia group compared to the control group (46.1% vs. 53.2%); however, there was no statistical significance (p = 0.068, OR = 0.754, 95% CI: 0.491-1.159). The "C" allele frequency in the high myopia group (68.0%) was slightly lower than the control group (72.6%), but this difference was not statistically significant (p = 0.061, OR = 0.810, 95% CI:0.582-1.126). For the rs3759223, the genotype frequencies of T/T, T/C, and C/C were 67.2%, 26.6%, and 6.2%, respectively, in the high myopia group and 64.7%, 30.6%, and 4.7 %, respectively, in the control group. The allele frequency of T was 80.5% in the high myopia group and 80.0% in the control group (p = 0.077, OR = 1.03, 95% CI: 0.703-1.508). There were no significant differences in the distribution of genotype and allele frequencies for the two promoter SNPs tested. CONCLUSIONS: The current study did not support an association between the promoter SNPs of the LUM gene with high myopia in the Korean population.

Three cases with unusual ophthalmic phenotypes of congenital aniridia.

OBJECTIVE: To report 3 cases with unusual ophthalmic phenotypes of congenital aniridia. DESIGN: Interventional case series. PARTICIPANTS: A 10-day-old infant with cloudy and large cornea in both eyes, 1 month-old male with bilateral corneal opacity, and 27-year-old male with low vision. METHODS: Complete ophthalmic examination and genetic evaluation. RESULTS: Case 1 was a neonate with concurrent presentation of congenital aniridia and glaucoma. Case 2 was diagnosed as congenital aniridia combined with Peters anomaly in both eyes. Case 3 had 2 unusual features of aniridia, which were asymmetric iris involvement and absence of limbal deficiency. CONCLUSIONS: It is important to perform thorough ophthalmologic evaluations in patients with congenital aniridia because of the possibilities of coexistence of other ocular abnormalities.

Correlations between tear cytokines, chemokines, and soluble receptors and clinical severity of dry eye disease.

PURPOSE: To determine cytokine and chemokine concentrations in the tears of patients with dry eye disease (DED) and analyze the possible relationships with the clinical severity of DED. METHODS: Patients were examined using the Ocular Surface Disease Index, corneal and conjunctival staining, tear breakup time, and impression cytology. They were divided into four groups according to the Dry Eye Workshop severity classification. Tears were collected from 133 patients with DED and 70 healthy controls. Concentrations of cytokines, chemokines, and soluble receptors in collected tear samples were analyzed using current technology with a Human Cytokine/Chemokine kit, a Human Cytokine/Chemokine Panel, and a Human Soluble Cytokine Receptor Panel. RESULTS: The levels of cytokines interleukin (IL)-1ß (P < 0.05), IL-6 (P < 0.001), IL-16 (P < 0.001), IL-33 (P < 0.05), G-CSF (P < 0.001), and transforming growth factor (TGF)-α (P < 0.05) were significantly higher in patients with DED, whereas those of cytokines IL-4 (P < 0.001), IL-12 (p40) (P < 0.001), IL-17A (P < 0.05), and interferon-γ (P < 0.001) were significantly lower. The levels of Fractalkine (chemokine [C-X3-C motif] ligand 1; CX3CL1), MCP-1 (chemokine [C-C motif] ligand 2; CCL2), MIP-1δ (chemokine [C-C motif] ligand 15; CCL15), and ENA-78 (chemokine [C-X-C motif] ligand 5; CXCL5) (P < 0.001, respectively) and soluble receptors, sIL-1RI (P < 0.05), soluble glycoprotein (sgp) 130 (P < 0.05), sIL-6R (P < 0.001), soluble epidermal growth factor receptor (P < 0.05), and soluble tumor necrosis factor receptor 2 (P < 0.001), were higher in patients with DED. There were significant correlations between these molecules and the clinical severity of DED. CONCLUSIONS: Fifteen molecules were elevated in the tears of patients with DED; four molecules were decreased. Although the levels of sIL-6R, sIL-6R, and sgp130 may be potential indicators of the homeostatic process, an increase in the levels of IL-6 and IL-1 ß are the earliest observable changes in patients with DED. Further study on the biomarkers in the pathogenesis of DED and treatment target modalities would be needed.

Heterozygous Avellino corneal dystrophy 9 years after photorefractive keratectomy: natural or laser-induced accelerated course?

PURPOSE: To report a photorefractive keratectomy (PRK)-treated patient who first developed stromal opacities with a typical granular pattern 9 years after surgery, which was confirmed as heterozygous Avellino dystrophy by DNA analysis. METHODS: A 37-year-old woman, who had undergone PRK 17 years ago, has been followed for glaucoma and corneal dystrophy. She had preoperative clear corneas in both eyes. RESULTS: Nine years after PRK, at the age of 29 years, 2-3 small, white, anterior stromal corneal opacities were first detected in both eyes. Seventeen years postoperatively, multiple anterior stromal corneal deposits with a dot, crumb, or snowflake appearance were noted to the same degree in both eyes. The intervening cornea was relatively clear. Her DNA was heterozygous for the R124H mutation. CONCLUSIONS: Slowly developing, mild corneal deposits occurred after PRK in this patient with heterozygote Avellino corneal dystrophy. Various clinical manifestations can occur after excimer laser refractive surgery in patients with Avellino corneal dystrophy.

VSX1 gene variants are associated with keratoconus in unrelated Korean patients.

Keratoconus is a bilateral ectatic disorder characterized by the central thinning of corneal tissue leading to visual impairment. To investigate the possibility of visual system homeobox 1 (VSXI) as a candidate susceptibility gene for Korean patients with keratoconus, we performed a mutation screening of the VSXI gene in 249 unrelated patients with keratoconus and 208 control subjects without the ocular disorder. We found two heterozygous novel missense mutations in exon 2: N151S and G160V. The G160V mutation was identified in 13 keratoconus patients (5.3%), and the N151S mutation was found in only one keratoconus patient (0.4%). We also detected three synonymous polymorphisms and four intragenic polymorphisms. The IVS1-11*a allele was associated with a significantly increased risk of keratoconus in Korean patients [3.6 vs. 0.5%, p = 0.001, odds ratio (OR) = 7.76, 95% confidence interval (CI) 1.989-30.241). Other polymorphisms did not show an association with keratoconus risk. Our data is the first reported VSX1 mutation screening in Korean keratoconus patients. We detected two novel missense mutations and one intragenic polymorphism in the VSX1 gene, which show a strong statistical association with unrelated keratoconus patients. Consequently, our study suggests that VSX1 gene variants seem to be significant genetic variants for keratoconus predisposition in unrelated Korean patients.

A single nucleotide polymorphism analysis of the LAMA1 gene in Japanese patients with high myopia.

Although a myopia susceptibility gene has not yet been elucidated, ten candidate regions (MYP1-MYP10) have been associated with myopia by linkage analysis employing large pedigrees. We report herein on the results of our analysis pertaining to polymorphisms of LAMA1 (alpha subunit of laminin), a promising candidate gene for high myopia present in the MYP2 region of Japanese subjects with high myopia. Three hundred and thirty Japanese subjects with high myopia at a level of greater than -9.25 D and ethnically and sex matched 330 normal controls without high myopia was enrolled in this study. The thirteen SNPs located on the LAMA1 gene were analyzed using PCR and SNP-specific fluorogenic probes. Two of the SNPs were monomorphic and none of the 11 SNPs showed statistically significant association with high myopia in the Japanese population. There is no convincing evidence to prove a connection between nucleotide sequence variations in LAMA1 and high myopia. The pairwise linkage disequilibrium (LD) mapping disclosed a strong value (D' > 0.8) and narrow ranged block within these SNPs.

Lack of association with high myopia and the MYP2 locus in the Japanese population by high resolution microsatellite analysis on chromosome 18.

MYP2 was reported for a candidate locus associated with high grade myopia by linkage analysis, but no candidate gene has been detected. We report an association study in the Japanese population using 750 microsatellite markers on chromosome 18 that include MYP2 locus. 450 Japanese subjects with high myopia whose refractive error was greater than or equal to -9.25D in at least one eye and equal number of normal control subjects were recruited in this study. Three steps screening on the pooled DNA of patients and the pooled DNA of controls were performed in this study. A total of 722 microsatellite markers could be analyzed, and we obtained 4 positive markers. Then to avoid experimental errors and artifacts, we confirmed true allele frequency by individual genotyping using initial set of 450 patients and controls. Only marker D18S0301i showed statistically significance, and no marker showed statistically significance on the MYP2 locus. Near the marker D18S0301i, GALNT1 gene was located, but its relation to high myopia has remained to be identified.

Analysis of single nucleotide polymorphisms at 13 loci within the transforming growth factor-induced factor gene shows no association with high myopia in Japanese subjects.

A previous study in China first indicated that the transforming growth factor-induced factor (TGIF) is a probable candidate gene for high myopia. The purpose of our study was to investigate whether there are significant associations between high myopia and single nucleotide polymorphism (SNP) variants in the TGIF gene of Japanese subjects. Genomic DNA was collected from 330 Japanese subjects with high myopia and at a level refractive error was less than -9.25 Dsph and 330 randomized controls without high myopia. Thirteen SNPs were detected by polymerase chain reaction (PCR) and primer extension or by PCR and SNP-specific fluorogenic probes in all of the cases and controls. Thirteen SNPs were found within the TGIF genes of the cases and controls. Two of the SNPs were monomorphic and none of the 13 SNPs showed a significant result. The pairwise linkage disequilibrium (LD) mapping confirmed that these alleles have a comparatively strong LD index of >0.8 for D' and >0.4 for r(2). We found no statistical association between any of the 13 SNPs located on the TGIF gene and high myopia in Japanese subjects. Based on our study using Japanese subjects and the previous studies of TGIF gene polymorphism in Chinese and northern European subjects with myopia, there is no convincing evidence to prove a connection between nucleotide sequence variations in TGIF and high myopia.

Association of MICA polymorphism with HLA-B51 and disease severity in Korean patients with Behcet's disease.

The HLA-B51 allele is known to be associated with Behcet's disease (BD) in many ethnic group. However, it has not yet been clarified whether the HLA-B51 gene itself is the pathogenic gene related to BD or whether it is some other gene in linkage disequlibrium with HLA-B51. Recently, the Triplet repeat (GCT/AGC) polymorphism in transmembrane region of the MHC class I chain-related A (MICA) gene was identified. To investigate the association of MICA with BD, we studied the MICA polymorphism in 108 Korean BD patients and 204 healthy controls in relation to the presence of HLA-B51 and clinical manifestations. The triplet repeat polymorphism was determined by polymerase chain reaction (PCR)-denaturing polyacrylamide gel electrophoresis (PAGE). The phenotype frequency of the MICA*A6 allele (relative risk, RR=2.15, p=0.002) and HLA-B51(RR=1.87, p=0.022) were significantly increased in the Korean patients with BD. A strong linkage disequilibrium was observed between the MICA*A6 and HLA-B51 in both the patients with BD and control subjects. Stratification analysis showed that MICA*A6 homozygosity was strongly associated with BD in the HLA-B51-negative population, and HLA-B51 was also associated with MICA*A6-negative population. In conclusion, MICA*A6 rather than HLA-B51 was strongly associated with Korean patients with BD, and the MICA*A6 allele is a useful susceptibility marker of BD, especially in the HLA-B5-negative