Mami Ishihara

Microsatellite polymorphism between the tumor necrosis factor and HLA-B genes in Behçet's disease

Behçets disease is associated with the HLA-B51 antigen. However, it has not yet been clarified if the HLA-B51 gene itself is the susceptibility gene related to this disease or if it is some other non-HLA gene in linkage disequilibrium with HLA-B51. Therefore, we screened one of the HSP70 genes, HUM70t (HSP70-Hom), around the class III region and the microsatellite sequence located between the HLA-B and TNF genes for genetic polymorphism in BD. A comparison between patients with BD and healthy controls revealed no significant difference in the frequency of the HUM70t polymorphism. In the microsatellite sequence, Tau-a, in the region between the HLA-B and TNF genes, the frequency of 14 repetitions of GT was increased significantly and that of 11 repetitions was decreased significantly in the patient group. Further, the allelic distributions of the B51 antigen-associated microsatellite polymorphism differed significantly between patients and healthy controls, and in the B51 antigen-negative subjects, analysis of the microsatellite polymorphism also revealed a significant difference in the haplotype frequency between the patient and control groups. These results suggest that the HLA-B51 gene may not be the primary locus responsible for BD, and implicate some other gene(s) located between the TNF and HLA-B genes.

Humoral immune response directed against LEDGF in patients with VKH

Vogt-Koyanagi-Harada disease is an autoimmune systemic disorder. In Vogt-Koyanagi-Harada disease, inflammatory disorders occur in multiple organs containing melanocytes, including uvea (resulting in acute bilateral panuveitis), skin (resulting in vitiligo and alopecia), central nervous system (resulting in meningitis) and inner ears (resulting in hearing loss and tinnitus). These inflammatory aspects are attributed to the destruction of melanocytes through immunological mechanisms. Studies have been carried out to elucidate the exact etiology and target autoantigen in Vogt-Koyanagi-Harada disease, but much remains to be investigated. Identification of target autoantigen is important to understand the etiology of autoimmune diseases, and for development of antigen-specific immuno-modulation therapy. To identify the target autoantigens in Vogt-Koyanagi-Harada disease, we made use of an immunoscreening of a bovine uveal cDNA expression library with serum samples obtained from patients with Vogt-Koyanagi-Harada disease. We identified an immunoreactive cDNA clone that encodes bovine lens epithelium derived growth factor. mRNA of human lens epithelium derived growth factor was determined by reverse transcription-polymerase chain reaction and it was expressed in human uvea, retina and melanocytes. Immunoglobulin G (IgG) autoantibodies were quantitated in an enzyme-linked immunosorbent assay, using recombinant human lens epithelium derived growth factor. The prevalence of IgG anti-lens epithelium derived growth factor autoantibodies in patients with Vogt-Koyanagi-Harada disease was significantly higher than that in healthy controls (66.7% versus 21.6%, P<0.001). On the other hand, the prevalence of the autoantibody in patients with panuveitis of other etiology, Behçets disease and sarcoidosis, was almost same as that in healthy controls. These results suggest that the humoral immune response agonist lens epithelium derived growth factor is not a mere secondary phenomena caused by uveal tissue damage.

Clinical features of sarcoidosis in relation to HLA distribution and HLA-DRB3 genotyping by PCR-RFLP.

BACKGROUND--Susceptibility to the development of sarcoidosis has been demonstrated to be associated with HLA-DR5, -DR6, and -DR8 encoded by the DRB1 gene. However, involvement of the DRB3 (HLA-DR52) gene in the development of sarcoidosis remains unclear. METHODS--HLA-DRB3 genotyping was performed using the PCR-RFLP method and the clinical features of the patients with and without the DR3, 5, 6, 8 group antigens were compared. RESULTS--HLA-DRB3 genotyping indicated an association between DRB3*0101 and sarcoidosis. The DR8 haplotype lacking the DRB3 gene has been found to be increased significantly in sarcoidosis, suggesting that the HLA-DRB3 gene is not a primary determinant of predisposition to sarcoidosis. The association of DRB3*0101 with sarcoidosis is attributable to linkage disequilibrium with DR5- and DR6-associated alleles. There were significant decreases in the DR3, 5, 6, 8 group (DR5, DR6, or DR8) antigen frequencies in patients with retinal perivasculitis, high intraocular pressure (or secondary glaucoma), and optic nerve and/or macular lesion. Correlations were observed among the DR3, 5, 6, 8 group antigens, early onset sarcoidosis and disease with fewer intraocular lesions. CONCLUSION--This established a molecular basis for some of the clinical heterogeneity observed in sarcoidosis.

Genetic characterization and susceptibility for sarcoidosis in Japanese patients: risk factors of BTNL2 gene polymorphisms and HLA class II alleles.

PURPOSE Sarcoidosis is a heterogeneous and multisystem granulomatous disorder. The etiology still is uncertain, but the disease currently is thought to be triggered by various genetic as well as environmental factors. Recently, an association between sarcoidosis and the butyrophilin-like 2 (BTNL2) gene located in close proximity to the HLA-DRB1 gene was reported. The purpose of our study was to verify the relationship between BTNL2 and HLA risk alleles for the susceptibility to sarcoidosis, and to assess whether the BTNL2 association is independent of the HLA risk alleles. METHODS In our study, 11 single nucleotide polymorphisms (rs28362677, rs2076533, rs2076530, rs2076529, rs2294881, rs3763304, rs2076523, rs28362682, rs3806156, rs9268499, rs3763317), including the functional rs2076530 (G > A) of the BTNL2 gene, and HLA-DRB1 and -DQB1 alleles, were genotyped in 237 Japanese patients diagnosed with sarcoidosis and 287 healthy Japanese control subjects. RESULTS In the patient group, the HLA-DRB1*08:03 (P = 6.15 × 10(-5), odds ratio [OR] = 2.43) and BTNL2 rs2076530_A (P = 6.90 × 10(-6), OR = 1.84) were associated with disease susceptibility. Upon stratification analysis in search for a synergistic effect given the extensive linkage disequilibrium between BTNL2 rs2076530_A and HLA-DRB1*08:03, our results suggested that the risk-bearing allele of these two loci interact negatively. No significant differences were observed in allele frequencies for alleles in patients with ocular and other systemic sarcoidosis. CONCLUSIONS Our studies implicated that the HLA-DRB1 allele is a major contributing genetic factor in the development of sarcoidosis in Japan. However, further studies are needed to verify how HLA or BTNL2 alleles confer the disease phenotype, severity of sarcoidosis.

HLA-C genotyping of patients with behçet's disease in the Japanese Population

Behçets disease has been known to be strongly associated with a particular HLA-B allele, B51. To address the possibility that the HLA-C gene, which is closely linked to HLA-B but has been poorly defined for allo-antigen specificity by the serologic method is involved in the susceptibility to Behçets disease, HLA-C genotyping was performed for 90 Japanese Behçets disease patients by the PCR-SSP method. The frequencies of HLA-Cw*14 and -Cw*15 were significantly higher in the patient with Behçets disease as compared to the controls (48.9% vs. 24.0%, p = 0.0005, and 17.8% vs. 7.3%, p = 0.0434, respectively). On the other hand, the frequencies of HLA-Cw*0304 and -Cw*01 were significantly decreased in the patient group as compared to the control group (7.8% vs. 25.0%, p = 0.0027, and 23.3% vs. 37.5%, p = 0.0398, respectively). The significantly higher HLA-Cw*14 and -Cw*15 alleles may tightly correlate with the B51 antigen, and hence may have increased as a result of a linkage disequilibrium with B51. Accordingly, the HLA-C allele frequencies were compared for the B51-positive or -negative patients and controls, but there was no HLA-C allele showing a significant difference between these patient and control groups. Conversely, analysis of the HLA-B allelic distribution in association with HLA-Cw*14 revealed that in the healthy controls, B44 and B51 were present at the frequencies of 57.1% and 35.7% of the HLC-Cw*14-positive individuals, respectively. In contrast, in the Cw*14-positive patients the frequency of B44 was merely 14.0% (p = 0.0001) and that of B51 was significantly high, amounting to 82.0% (p = 0.0001). These facts suggest that the pathogenic gene of Behçets disease is not the HLA-C gene (HLA-Cw*14 and/or HLA-Cw*15) but the HLA-B gene (HLA-B51) itself or a non-HLA gene residing in the centromeric side of the HLA-B gene rather than in the telomeric side around the HLA-C gene. This finding supports our previous mapping result, which located the susceptible gene between the TNF and HLA-B genes.

Seroprevalence of anti-Borrelia antibodies among patients with confirmed sarcoidosis in a region of Japan where Lyme borreliosis is endemic

Abstract · Background: Sarcoidosis is a multisystemic granulomatous disease of unknown etiology, while Lyme borreliosis is a multisystemic disorder caused by Borrelia burgdorferi. The purpose of this study is to evaluate the relationship between sarcoidosis and Lyme borreliosis in a region of Japan where Lyme borreliosis is endemic. · Methods: We determined the seroprevalence of anti-Borrelia burgdorferi antibodies as well as antibodies three Japanese Borrelia strains by enzyme-linked immunosorbent assay and dotblot assay using purified Borrelia-specific proteins in 46 patients with confirmed sarcoidosis and 150 controls (50 disease controls and 100 healthy controls) in Hokkaido, the affected region. · Results: Fifteen patients with sarcoidosis (32.6%) tested positive for Borrelia spirochete in both assays, compared with two disease controls (4.0%) and two healthy controls (2.0%). The seroprevalence of anti-Borrelia antibodies in patients with sarcoidosis was much higher in the affected region than in the region in our previous study where Lyme borreliosis is non-endemic. · Conclusion: In a region where Lyme borreliosis is endemic, Borrelia infection may be partially associated with sarcoidosis.

Genetic influences on sarcoidosis

To investigate the genetic influences underlying the development of sarcoidosis, HLA class II genotyping was performed in Japanese patients with sarcoidosis and healthy controls using the PCR-RFLP method. The frequencies of both DR52 group antigen-associated alleles (HLA-DRB1*11, -DRB1*12 and -DRB1*14) and DRB1*08 alleles were higher in the patient group, suggesting that the common, specific amino acid residue on the DRB1 molecule of these alleles may determine susceptibility to sarcoidosis. Alternatively, it is possible that another susceptibility gene, linked to these DRB1 alleles, exists within the MHC region. We screened the TNFA, TNFB, HSP70-1 and Hum70t genes around the class III region, as well as the HLA-DMA and -DMB genes in the class II region, for genetic polymorphism in sarcoidosis. None of these genes suggested a susceptibility to sarcoidosis. These studies support the thesis that one of the major genetic factors controlling the development of sarcoidosis is located within the DRB1 locus in the HLA class II region.

Mutation screening of the CARD15 gene in sarcoidosis

CARD15 was first identified as a susceptibility gene for Crohns 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.

A complete type patient with Behçet's disease associated with HLA-B*5102

Behqet’s disease (BD) is thought to be a multifactorial disease caused by some environmental agents under certain genetic factors. The strong association of BD with HLA-B51 has been observed in several ethnic groups, although it is not clear whether HLA-B51 itself or another non-HLA gene(s) around the HLA-B gene closely linked to HLA-B51 is primarily responsible for the predisposition to BD (Mizuki et al. 1992a,b). The HLA-B51 antigen is now known to be divided into four alleles, HLA-B*5101-B*5104, at the DNA level (Zemmour 8c Parham 1992) and each of them can be distinguished &om one another by serological typing, except B*5104. We reported the closer correlation of HLA-B*5101 with Japanese BD patients in the previous paper, where all the 46 B51-positive Japanese BD patients examined turned out to carry the HLA-B*5101 allele (Mizuki et al. 1993). In this paper, we report on one B51-positive patient who was definitely diagnosed as BD, and who exhibited the HLA-B*5102 allele in our expanded study.

Clinical Course before and after Cataract and Glaucoma Surgery under Systemic Infliximab Therapy in Patients with Behçet's Disease.

Purpose: Patients with Behçet’s disease often need intraocular surgeries for the treatment of secondary cataract or glaucoma. This study aims to report the clinical course before and after the intraocular surgeries of 5 patients who were systematically treated with infliximab. Methods: Retrospective case series. Results: Seven eyes of 5 male patients with Behçet’s disease, who underwent intraocular surgery while under systemic infliximab therapy at Yokohama City University Hospital from 2007 to 2009, were included in the study. The mean age at surgery was 44.2 years. Phacoemulsification was performed on 4 eyes, and trabeculectomy was done on the remaining 3 eyes. The mean duration since the onset of the ocular symptoms was 107 months. Control of the ocular attacks with the use of other systemic medications was difficult for all patients; however, the use of infliximab enabled adequate control of the attacks. The visual acuity status during the preoperative stage did not worsen during the postoperative period. No infectious complication was observed in all cases. Conclusions: Our results suggest that infliximab treatment does not complicate any subsequent intraocular surgery. Patients with Behçet’s disease in need of intraocular surgery can benefit from control of attacks with infliximab treatment.