Association study of genetic variants on chromosome 7q31 with susceptibility to normal tension glaucoma in a Japanese population.

The caveolin 1 to caveolin 2 (CAV1-CAV2) gene region on chromosome 7q31 has been reported to be associated with susceptibility to primary open angle glaucoma (POAG) and normal tension glaucoma (NTG) in previous studies. We investigated whether genetic variants in the CAV1-CAV2 region are associated with NTG in Japanese patients. Two hundred and ninety-two Japanese patients with NTG and 352 Japanese healthy controls were recruited. We genotyped three single-nucleotide polymorphisms; that is, rs1052990, rs4236601, and rs7795356, in the CAV1-CAV2 gene region and assessed the allelic diversity among cases and controls. The frequency of the minor allele (G) of rs1052990 was significantly decreased in NTG cases compared with controls (P=0.014, OR=0.71), whereas NTG or POAG cases had a significantly higher frequency of the allele than controls in previous studies. Conversely, rs7795356 did not show any significant association with NTG cases, and rs4236601 was monomorphic in the Japanese study population. Our findings did not correspond with previous positive results, suggesting that CAV1-CAV2 variants studied in the present study are not important risk factors for NTG susceptibility in all populations. Further studies are needed to elucidate the possible contribution of the CAV1-CAV2 region to the development of glaucoma.

Partial protection against SIV challenge by vaccination of adenovirus and MVA vectors in rhesus monkeys.

This study explores the effect of priming rhesus monkeys with an Ad5/35 vector expressing simian immunodeficiency virus (SIV) gag and gp120, and then boosting the animals with an modified vaccinia virus Ankara (MVA) vector encoding the same antigens after a 2-month interval. The animals were intravenously challenged with 100 TCID50 of highly pathogenic SIVmac239 virus 2 months after the booster vaccination. The priming vaccination induced robust SIV-specific cell-mediated and humoral immune responses, and boosting further enhanced the cellular immunity. Vaccination reduced peak and long-term viral loads by 1-2 logs for a period of >6 months, as reflected by a reduction in both the SIV RNA and DNA levels. Of considerable interest, the immunized monkeys did not suffer from loss of CD4 T cells, particularly central memory CD4 T cells. These results demonstrate that prophylactic vaccination with Ad5/35 followed by MVA reduces viral replication and prevents CD4 T-cell loss, and that these effects may decrease the likelihood of disease progression.

Investigation of the association between the GLC3A locus and normal tension glaucoma in Japanese patients by microsatellite analysis.

PURPOSE: To investigate whether the GLC3A locus harboring the CYP1B1 gene is associated with normal tension glaucoma (NTG) in Japanese patients. MATERIALS AND METHODS: One hundred forty-two Japanese patients with NTG and 101 Japanese healthy controls were recruited. Patients exhibiting a comparatively early onset were selected as this suggests that genetic factors may show stronger involvement. Genotyping and assessment of allelic diversity was performed on 13 highly polymorphic microsatellite markers in and around the GLC3A locus. RESULTS: There were decreased frequencies of the 444 allele of D2S0416i and the 258 allele of D2S0425i in cases compared to controls (P = 0.022 and P = 0.034, respectively). However, this statistical significance disappeared when corrected (Pc > 0.05). We did not find any significant association between the remaining 11 microsatellite markers, including D2S177, which may be associated with CYP1B1, and NTG (P > 0.05). CONCLUSIONS: Our study showed no association between the GLCA3 locus and NTG, suggesting that the CYP1B1 gene, which is reportedly involved in a range of glaucoma phenotypes, may not be an associated factor in the pathogenesis of NTG.

New susceptibility locus for high myopia is linked to the uromodulin-like 1 (UMODL1) gene region on chromosome 21q22.3.

PURPOSE: To ascertain and define the position of a potential disease susceptibility gene around D21S0083i prioritized during our previous whole genome case-control association analysis with 27,158 microsatellite markers, in Japanese high-myopia patients. METHODS: 520 high myopic patients and 520 healthy controls were genotyped using 39 SNPs distributed around D21S0083i on chromosome 21q22.3. RESULTS: Only 1 SNP (rs2839471) of 39 SNPs was significant after correction for multiple testing (allele T: P=0.00027, Pc=0.01, OR=1.684). The SNP (rs2839471) did not reside in haplotype blocks constructed by the pair-wise linkage disequilibrium between the SNPs. CONCLUSIONS: The SNP (rs2839471) is suggested to be located in the frequent recombinant region within UMODL1. Together this region might play a critical role for susceptibility to high myopia, and warrants further confirming studies and investigations as to the mechanisms by which UMODL1 may contribute to myopia.

Preferential activation of circulating CD8+ and gammadelta T cells in patients with active Behçet's disease and HLA-B51.

OBJECTIVE: To evaluate the activation status of circulating CD4+, CD8+, and gammadelta T cells in patients with active and inactive Behçet's disease (BD). METHODS: We studied 11 subjects with active BD, 28 with inactive BD, and 13 healthy controls. The expression of CD4, CD8, pan-gammadelta, Vdelta1, and Vdelta2 along with the early activation marker CD69 was analyzed by 3-color flow cytometry. RESULTS: Proportions of activated CD8+ and gammadelta T cells were significantly greater in patients with active BD than in those with inactive BD or healthy control subjects, but the proportion of activated CD4+ T cells did not differ among these 3 groups. In addition, significantly greater proportions of the Vdelta1+ and Vdelta2+ gammadelta T-cell subsets were activated in patients with active BD than in those with inactive BD or healthy controls; in active BD, the balance of activation between these subsets favored the Vdelta1+ T cells. No significant differences in these proportions were found between subjects with inactive BD and healthy controls. These findings were observed exclusively in patients with HLA-B51. A comparison of samples from 5 patients taken during active BD and after resolution of BD-related symptoms showed the proportions of activated CD8+ and gammadelta T cells dropped when the patients' BD became inactive. CONCLUSION: CD8+ and gammadelta T cells, rather than CD4+ T cells, were activated in vivo in patients with active BD and HLA-B51, but not in those with inactive BD, suggesting that these potentially cytotoxic T cells play a critical role in BD flares.

Re-evaluation of heterogeneity in HLA-B*510101 associated with Behçet's disease.

Behçet's disease (BD) is a chronic inflammatory disease characterized by oral aphthous ulcers, genital ulcers, uveitis and skin lesions. Etiology and pathogenesis of BD are not fully elucidated, but the association with human leukocyte antigen (HLA)-B51 or B*5101 has been repeatedly reported. Previous studies have shown that there are few sequence variations in the protein-coding region of B51, while there is a report on many variations in the 5'-flanking region and intron. In this study, HLA-B*5101 gene from 37 individuals including Japanese, Turkish, Jordanian and Iranian patients and healthy controls were fully sequenced to further clarify the B*5101 gene in association with BD. We found that all the patients and healthy controls carried B*510101 with no variation in the 5'-flanking region, exon and intron. However, seven polymorphisms were found in the 3'-flanking region. These polymorphisms composed of six haplotypes that were shared and stretched over the ethnic groups, suggesting that the susceptibility to BD was conferred by the B*510101 itself and not by any genes in linkage disequilibrium with B*510101. In addition, phylogenetic analyses of B*510101 showed that the 3'-flanking sequences followed an evolutional divergence differently from that of the other regions, implying that a unifying selection might operate to conserve B*510101.

Microsatellite analysis of the GLC1B locus on chromosome 2 points to NCK2 as a new candidate gene for normal tension glaucoma.

AIMS: The aim of this study was to investigate the association between normal tension glaucoma and the candidate disease locus glaucoma 1, open angle, B (GLC1B) on chromosome 2. There are many reports describing the results of association or linkage studies for primary open angle glaucoma (POAG), with GLC1B as one of the loci associated with normal or moderately elevated intraocular pressure. However, there are few reports about the association of genes or defined genomic regions with normal tension glaucoma, which is the leading type of glaucoma in Japan. The GLC1B locus is hypothesized to be a causative region for normal tension glaucoma. METHODS: Genomic DNA was extracted from whole blood of normal tension glaucoma (n = 143) and healthy controls (n = 103) of Japanese origin. RESULTS: Fifteen microsatellite markers within and/or near to the GLC1B locus were genotyped, and their association with normal tension glaucoma was analysed. Two markers D2S2264 and D2S176 had significant positive associations. CONCLUSION: The D2S176 marker had the strongest significant association and it is located 24 kb from the nearest gene NCK2, which now becomes an important new candidate gene for future studies of its association with normal tension glaucoma.

Ultrasound biomicroscopic measurement of anterior chamber biometry between before and after pupil dilation in children.

PURPOSE: To evaluate anterior chamber biometry of the eyes of normal children using ultrasound biomicroscopy (UBM) and to evaluate the differences in biometry between children and adults, and before and after pupil dilation in children. METHODS: Anterior chamber depth (ACD) and trabecular-iris angle (TIA) were measured in 94 normal children and 15 normal adults using UBM. Before and after pupil dilation were measured in 42 children with emmetropic and hyperopic eyes. RESULTS: In 66 emmetropic children, ACD and TIA were 2.93+/-0.18 mm and 34.42+/-4.02 degrees, respectively. In 28 hyperopic children, ACD and TIA were 2.92+/-0.21 mm and 35.05+/-4.42 degrees, respectively. There was no significant difference in anterior chamber biometry associated with the refraction. ACD did not differ between children and adults, but TIA in children was wider than in adults. There was no significant difference in ACD or TIA before versus after pupil dilation in any case. CONCLUSIONS: Anterior chamber biometry in children showed no differences before and after pupil dilation. Also, there was no difference in ACD of children as compared to adults; however, TIA in children was significantly wider than in adults.

Spontaneous closure of a macular hole caused by a ruptured retinal arterial macroaneurysm.

PURPOSE: To report a spontaneous closure of a macular hole (MH) that was caused by a ruptured retinal arterial macroaneurysm (RAM). METHODS: Observational case report. Clinical examinations and optical coherence tomographic (OCT) evaluations of the retina of a 73-year-old woman who developed a MH secondary to a ruptured RAM. RESULTS: The first sign of a closure of the MH was the appearance of tissue bridging the MH in the OCT images. Later, OCT images showed a hyperreflective tissue, probably glial cells, that connected the bridging tissue to the RPE. Seven months after the first examination, the hyperreflective tissue was smaller and the shape of the foveal pit had recovered. CONCLUSIONS: A spontaneous closure of a MH caused by a ruptured RAM can occur and surgical intervention was not necessary. The tissue bridging over the MH and the hyperreflective tissue connecting the bridging tissue to the RPE most likely were involved in the spontaneous MH closure.

Mutation screening of the CARD15 gene in sarcoidosis.

CARD15 was first identified as a susceptibility gene for Crohn's disease. More recently, CARD15 mutations were shown to be associated with the pediatric granulomatous inflammatory diseases, Blau syndrome and early-onset sarcoidosis (EOS). The aim of the present study was to evaluate whether CARD15 variants also play a role in patients with ordinary sarcoidosis other than EOS. We enrolled 135 Japanese sarcoidosis patients with uveitis as well as 95 healthy individuals and performed mutation analysis by direct sequencing of CARD15 exon 4. Direct DNA sequencing in the sarcoidosis patients showed eight CARD15 variants, including five novel mutations (13402C>T, 13543C>T, 13775C>A, 13937G>A, and 14079C>T). Compared with healthy individuals, CARD15 mutations are not common in the Japanese patients with sarcoidosis. Based on the results, we examined the clinical manifestations in patients with sarcoidosis according to their CARD15 mutations. Sarcoidosis patients with these mutations have no specific clinical features with regard to course of the disease or disease severity. Our results indicate that in general, CARD15 mutations may not contribute to the risk of sarcoidosis.

Vertical distraction of a free vascularized osteocutaneous scapular flap in the reconstructed mandible for implant therapy.

This paper describes a case of vertical distraction osteogenesis of a free vascularized osteocutaneous scapular flap in the reconstructed mandible before implant therapy. The patient was a 67-year-old woman with squamous cell carcinoma of the right lower gingiva. She underwent segmental mandibulectomy for tumor ablation and reconstruction with an osteocutaneous scapular flap. The distraction protocol, clinical course and implant therapy are presented. Through this procedure, the bone height of the scapular graft increased by 10mm. Implants with adequate length could be placed in the distracted area. Two years after masticatory loading, the condition of these implants was stable. Vertical distraction osteogenesis of the scapular flap was considered effective when performed before implant therapy, to facilitate postoperative functional and esthetic restoration after tumor resection.

Lack of association of Toll-like receptor 9 gene polymorphism with Behçet's disease in Japanese patients.

Toll-like receptors (TLRs) play an important role in the induction of defense mechanisms of the innate and adaptive immune responses to microbial pathogens. Genetic polymorphisms within the TLR9 gene have been reported to be associated with a variety of inflammatory and infectious diseases. Behçet's disease (BD) is a chronic inflammatory disease, and the etiology of BD has yet to be fully elucidated. We investigated the potential association of the TLR9 gene with susceptibility to BD by analyzing the frequency of nine single nucleotide polymorphisms (SNPs) in a population of 200 Japanese BD patients and 102 randomized controls. Our results showed that SNPs in the TLR9 gene were not significantly associated with susceptibility to BD.

Four-digit allele genotyping of the HLA-A and HLA-B genes in Japanese patients with Behcet's disease by a PCR-SSOP-Luminex method.

The present study represents the first four-digit allele genotyping of HLA-A and -B in Japanese Behcet's disease (BD) patients and controls using a new genotyping method (named the PCR-SSOP-Luminex method) to determine the association of certain HLA-A or -B alleles with BD. Peripheral blood lymphocytes were collected from 180 Japanese BD patients and 170 healthy controls. The genotype frequency of HLA-B*5101 was significantly increased in the patients (61.7%) as compared with the controls (15.9%) (Pc = 1 x 10(-16), OR = 8.5). When we recalculated the phenotype frequencies after excluding the HLA-B*51-positive patients and controls to account for the effects of the linkage disequilibrium and the abundance of the HLA-B*51 allele, the frequencies of HLA-A*2602 and HLA-B*3901 had a weak association in the patient group without HLA-B*51 as compared with the control group without HLA-B*51 (A*2602; Pc = 0.130, OR = 4.3, B*3901; Pc = 0.099, OR = 3.5). This study confirmed on the basis of using a new and more accurate genotyping method that Japanese BD patients have a strong primary association with HLA-B*5101. The significant increase of HLA-A*2602 and B*3901 in the patient group without HLA-B*51 suggests that these two alleles might also have some secondary influence on the onset of BD.

Immunology and functional genomics of Behçet's disease.

Behçet's disease (BD) is a multisystemic inflammatory disorder. Although the cause and pathogenesis of BD are still unclear, there is evidence for genetic, immunologic and infectious factors at the onset or in the course of BD. This review focuses on the functional genomics and immunology of BD. HLA-B51 is the major disease susceptibility gene locus in BD. An increased number of gammadelta T cells in the peripheral blood and in the involved tissues have been reported. However, the T cells at the sites of inflammation appear to be a phenotypically distinct subset. There is also a significant gammadelta T cell proliferative response to mycobacterial 65-kDa heat shock protein peptides. Homologous peptides derived from the human 60-kDa heat shock protein were observed in BD patients. There is evidence that natural killer T cells may also play a role in BD.

Hyperkeratosis and leukocytosis in transgenic mice carrying MHC class I chain-related gene B (MICB).

Major histocompatibility complex (MHC) class I chain-related gene A and B (MICA and MICB) are located very close to HLA-B. MICA is reported to be strongly associated with Behçet's disease (BD), a multisysytemic inflammation disorder characterized by oral apthous ulcers, skin lesions and genital ulcers. These two molecules are highly conserved at the amino acid levels. To determine the function of MICB in vivo and the relationship between the expression of MICB and BD experimentally, we produced several transgenic mouse lines (termed CAG-MICB) expressing human MICB cDNA under a ubiquitous promoter. They exhibited a 50% increase in the number of white blood cells compared with their non-transgenic littermates, and also exhibited a 10-20% reduction in body weight compared with non-transgenic littermates. Exfoliation of the skin first appeared around 7 days after birth and disappeared after 2 weeks of age. This was repeatedly observed in the transgenic offspring of two independent CAG-MICB lines examined. Histopathological analysis of skin of young mice exhibiting skin abnormalities revealed hyperkeratosis of the epidermis and thickening of the granular layer with slight infiltration of inflammatory cells in the dermis without any vasculitis. Other remarkable abnormalities associated with BD have not been observed in the CAG-MICB lines. Furthermore, fluorescein angiography of eyes of the CAG-MICB lines was performed, but there were no marked changes of BD-related uveitis in the ocular fundus. These findings suggest that (i) MICB expression is related to temporary skin inflammation, and (ii) expression of MICB is not directly associated with BD.

Analysis of microsatellite polymorphism around the HLA-B locus in Iranian patients with Behçet's disease.

We have previously suggested that in a Japanese population the susceptible locus for Behçet's disease (BD) is HLA-B51 itself. To confirm this finding in another population, we performed HLA class I typing using the PCR-SSP method and analyzed eight polymorphic markers distributed within 1100 kb around the HLA-B gene using automated sequencer and subsequent automated fragment detection by fluorescent-based technology with the DNA samples of 84 Iranian patients with BD and 87 healthy ethnically matched controls. As a result, three microsatellite alleles (MICA-A6, MIB-348, C1-4-1-217) and HLA-B51 were found to be strongly associated with BD. Of these alleles HLA-B51 is the most strongly associated allele. There were no alleles that were increased in allele frequency at any microsatellite loci centromeric of MICA or telomeric of HLA-B51. Therefore, HLA-B51 was confirmed to be by far the most strongly associated gene with BD in an Iranian population.

Sequencing-based typing of HLA-B*51 alleles and the significant association of HLA-B*5101 and -B*5108 with Behçet's disease in Greek patients.

Behçet's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groups.Recently, HLA-B51 allele typing of Greek BD patients was performed to study the distribution of B*5101-B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101. As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele genotyping among 58 Greek patients with BD. After serological HLA typing, typing of HLA-B*51 alleles was performed using the polymerase chain reaction-sequencing-based typing (PCR-SBT) method. The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (75.9% of patients vs 22.0% of controls. In the genotyping of B51 alleles, 34 out of 44 B51-positive patients possessed B*5101, 13 out of the 44 carried B*5108. In contrast, all of the 9 B51-positive normal controls carried B*5101. This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease.

HLA-B*51 allele analysis by the PCR-SBT method and a strong association of HLA-B*5101 with Japanese patients with Behçet's disease.

Behçet's disease (BD) is known to be associated with human leukocyte antigen (HLA) B51 in many different ethnic groups. An increased incidence of HLA-B51 in the patient group has also been reported in a Japanese population. Recently, the B51 antigen has been identified to comprise 21 alleles, B*5101-B*5121. Further, not only HLA-B*5101 but also HLA-B*5108 were found to be relatively increased in the patient groups among Italian and Saudi Arabian populations. Therefore, we performed HLA-B*51 allele genotyping by the polymerase chain reaction-sequencing based typing (PCR-SBT) method in order to investigate whether there is any correlation of one particular B51-associated allele with Japanese BD. Ninety-six Japanese patients with BD and 132 healthy Japanese volunteers were enrolled in this study. As a result, the phenotype frequency of the B51 antigen was confirmed to be remarkably increased in the patient group as compared to the ethnically matched control group (59.4% in patients vs. 13.6% in controls; Pc=0.0000000000098, R.R.=9.3). In the B*51 allele genotyping, 56 out of 57 B51-positive patients were defined as B*5101 and the remaining one was B*5102. In contrast, all of 18 B51-positive normal controls were B*5101. None of the Japanese patients and healthy controls carried the HLA-B*5108 allele. This study revealed that B*51 allelic distribution in Japanese was different from those in Italian and Saudi Arabian populations, and that the significantly high incidence of the HLA-B51 antigen in the Japanese BD patient group was mostly caused by the significant increase of the HLA-B*5101 allele.

Stratification analysis of MICA triplet repeat polymorphisms and HLA antigens associated with ulcerative colitis in Japanese.

We previously reported a conserved haplotype of HLA B52-DR2 and a significantly high frequency of the major histocompatibility complex (MHC) class I chain-related gene A (MICA) transmembrane-short tandem repeat (TM-STR) 6 allele in Japanese patients with ulcerative colitis (UC). To examine the predominance of the MICA TM-STR 6 allele as a marker of the susceptibility to UC within the susceptible haplotype, the association of each allele with UC was estimated following stratification of the patients to control for any possible confounding effects of other alleles positively associated with UC. Sixty-four patients with UC and 236 unrelated healthy controls were included in this study. All subjects were Japanese. HLA-A, -B, -C, and -DR antigens were determined serologically. A triplet repeat polymorphism of the MICA was determined by direct sequencing. To control for the effect of linkage disequilibrium, Mantel-Haenszel weighed odds ratios were calculated. Significantly higher phenotype frequencies of B52, MICA TM-STR 6, and DR2 were observed in patients with UC. Linkage disequilibria among alleles associated with UC revealed that a B52 - MICA TM-STR 6 - DR2 haplotype was conserved in patients with UC, as in controls. When the association of HLA-B52 was estimated after patient stratification for the possible confounding effect of MICA TM-STR 6 or DR2, a strong significant association of B52 with UC was still observed. In contrast, no association with UC was observed for MICA TM-STR 6 or DR2, after stratification of the possible confounding effect of HLA-B52. These results imply that the significant increase in MICA TM-STR 6 in Japanese patients with UC is attributable to linkage disequilibrium with HLA-B52.