Akira Meguro

Genome-wide association studies identify IL23R - IL12RB2 and IL10 as Behçet's disease susceptibility loci

Behçets disease is a chronic systemic inflammatory disorder characterized by four major manifestations: recurrent ocular symptoms, oral and genital ulcers and skin lesions. We conducted a genome-wide association study in a Japanese cohort including 612 individuals with Behçets disease and 740 unaffected individuals (controls). We identified two suggestive associations on chromosomes 1p31.3 (IL23R-IL12RB2, rs12119179, P = 2.7 × 10−8) and 1q32.1 (IL10, rs1554286, P = 8.0 × 10−8). A meta-analysis of these two loci with results from additional Turkish and Korean cohorts showed genome-wide significant associations (rs1495965 in IL23R-IL12RB2, P = 1.9 × 10−11, odds ratio = 1.35; rs1800871 in IL10, P = 1.0 × 10−14, odds ratio = 1.45).

Genome-wide association analysis identifies new susceptibility loci for Behcet's disease and epistasis between HLA-B*51 and ERAP1.

Individuals with Behçets disease suffer from episodic inflammation often affecting the orogenital mucosa, skin and eyes. To discover new susceptibility loci for Behçets disease, we performed a genome-wide association study (GWAS) of 779,465 SNPs with imputed genotypes in 1,209 Turkish individuals with Behçets disease and 1,278 controls. We identified new associations at CCR1, STAT4 and KLRC4. Additionally, two SNPs in ERAP1, encoding ERAP1 p.Asp575Asn and p.Arg725Gln alterations, recessively conferred disease risk. These findings were replicated in 1,468 independent Turkish and/or 1,352 Japanese samples (combined meta-analysis P < 2 × 10−9). We also found evidence for interaction between HLA-B*51 and ERAP1 (P = 9 × 10−4). The CCR1 and STAT4 variants were associated with gene expression differences. Three risk loci shared with ankylosing spondylitis and psoriasis (the MHC class I region, ERAP1 and IL23R and the MHC class I–ERAP1 interaction), as well as two loci shared with inflammatory bowel disease (IL23R and IL10) implicate shared pathogenic pathways in the spondyloarthritides and Behçets disease.

Genetics of Behçet disease inside and outside the MHC

Background Behçet disease (BD) is a rare, chronic, systemic, inflammatory disorder characterised by recurrent ocular, genital and skin lesions. Although its aetiology is still uncertain, an intricate interplay between the environment (eg, viruses) and the host seems to initiate and/or perpetuate the disease, although the mechanism remains speculative. Since the identification of HLA-B*5101 (and more recently of MICA) as a susceptibility locus for BD, the identification of additional genetic locus/loci, whether inside, or perhaps more importantly outside the MHC has clearly stalled. Objective To carry out a genome-wide association study (GWAS) of BD. Methods 300 Japanese patients with BD and an equal number of controls were recruited. The samples were screened using a dense panel of 23 465 microsatellites (MS) covering the entire genome. Results The six best (of a total of 147) positively associated MS with BD were identified. Of these six, two were located within the human leucocyte antigen (HLA) class I region itself. Although one of these was clearly reminiscent of the association with HLA-B, the second, not in linkage disequilibrium with the former, was in the telomeric side of the class I region and remained to be formally identified. HLA genotyping and haplotype analysis conclusively led to the deciphering of a dual, independent, contribution of two HLA alleles to the pathogenesis of BD: HLA-B*5101 and HLA-A*26. Conclusions This GWAS highlights the premier genetic susceptibility locus for BD as the major histocompatibility complex itself, wherein reside two independent loci: HLA-B and HLA-A.

Targeted resequencing implicates the familial Mediterranean fever gene MEFV and the toll-like receptor 4 gene TLR4 in Behçet disease

Genome-wide association studies (GWAS) are a powerful means of identifying genes with disease-associated common variants, but they are not well-suited to detecting genes with disease-associated rare and low-frequency variants. In the current study of Behçet disease (BD), nonsynonymous variants (NSVs) identified by deep exonic resequencing of 10 genes found by GWAS (IL10, IL23R, CCR1, STAT4, KLRK1, KLRC1, KLRC2, KLRC3, KLRC4, and ERAP1) and 11 genes selected for their role in innate immunity (IL1B, IL1R1, IL1RN, NLRP3, MEFV, TNFRSF1A, PSTPIP1, CASP1, PYCARD, NOD2, and TLR4) were evaluated for BD association. A differential distribution of the rare and low-frequency NSVs of a gene in 2,461 BD cases compared with 2,458 controls indicated their collective association with disease. By stringent criteria requiring at least a single burden test with study-wide significance and a corroborating test with at least nominal significance, rare and low-frequency NSVs in one GWAS-identified gene, IL23R (P = 6.9 × 10−5), and one gene involved in innate immunity, TLR4 (P = 8.0 × 10−4), were associated with BD. In addition, damaging or rare damaging NOD2 variants were nominally significant across all three burden tests applied (P = 0.0063–0.045). Furthermore, carriage of the familial Mediterranean fever gene (MEFV) mutation Met694Val, which is known to cause recessively inherited familial Mediterranean fever, conferred BD risk in the Turkish population (OR, 2.65; P = 1.8 × 10−12). The disease-associated NSVs in MEFV and TLR4 implicate innate immune and bacterial sensing mechanisms in BD pathogenesis.

Genome-wide association study identifies GIMAP as a novel susceptibility locus for Behçet's disease

Objectives To identify non-major histocompatibility complex susceptible genes that might contribute to Behçets disease (BD). Methods We performed a genome-wide association study using DNA samples from a Korean population consisting of 379 BD patients and 800 controls. A replication study was performed in a Japanese population (363 BD patients and 272 controls). To evaluate the functional implication of the target single nucleotide polymorphisms (SNP), gene expression levels in peripheral T cells, allele-specific modulation of promoter activity and biological effect of mRNA knockdown were investigated. Results We found a novel association of BD to the GIMAP locus, mapped to chromosome 7q36.1 (rs1608157, p=6.01×10−8 in a minor allele dominant model; rs11769828, allele based p=1.60×10−6). A fine mapping study identified an association with four additional SNP: rs1522596 (OR=1.45, p=7.70×10−6) in GIMAP4; rs10266069 (OR=1.32, p=2.67×10−4) and rs10256482 (OR=1.27, p=5.27×10−4) in GIMAP2; and rs2286900 (OR=1.61, p=3.53×10−5) in GIMAP1 areas. Replication study using DNA samples from the Japanese population validated the significant association between BD and the GIMAP locus. The GIMAP4 promoter construct plasmid with the minor allele of rs1608157 displayed significantly lower activity than one with the major allele. Moreover, CD4 T cells from BD patients showed a lower level of GIMAP4 mRNA, and GIMAP4 knockdown was protective against Fas-mediated apoptosis. Conclusions These results suggest that a GIMAP cluster is a novel susceptibility locus for BD, which is involved in T-cell survival, and T-cell aberration can contribute to the development of BD.

Genetic Variants on Chromosome 1q41 Influence Ocular Axial Length and High Myopia

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, β = −0.16 mm per minor allele, Pmeta = 2.69×10−10). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) = 0.75, 95% CI: 0.68–0.84, Pmeta = 4.38×10−7) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.

Association of Major Histocompatibility Complex Class I Chain-Related Gene A and HLA-B Alleles with Behcet's Disease in Turkey

PurposeBehçets disease (BD) is known to be associated with HLA-B*51 in many different ethnic groups. Recently, the major histocompatibility complex class I chain-related gene A (MICA), located near the HLA-B gene, has been proposed as a candidate gene for BD susceptibility in several ethnic groups. To compare the relative contribution of MICA polymorphisms and HLA-B*51 to BD in different ethnic groups, we studied MICA polymorphisms in Turkish BD patients.MethodsThirty-three Turkish BD patients and 65 healthy controls were enrolled for analysis of polymorphisms in the extracellular domains of MICA.ResultsThe phenotype frequencies of MICA*009 were significantly higher in BD patients (75.8%) than in controls (29.2%) (P = 0.000015). HLA-B*51 was also significantly more frequent in BD patients (81.8%) than in controls (29.2%) (P = 0.0000007). A strong association existed between MICA*009 and HLA-B*51. To assess the confounding effect of MICA*009 on HLA-B*51, we performed a stratification analysis that showed that BD was distinctly associated only with HLA-B*51.ConclusionOur results indicate that the major susceptibility gene for BD is HLA-B*51 and that the association between MICA*009 and BD arises from a strong linkage disequilibrium with HLA-B*51. However, we suggest that MICA*009 likely elicits an immune effect secondary to BD. Jpn J Ophthalmol 2007;51:431–436

Confirmation of TBK1 duplication in normal tension glaucoma

Recently, we discovered chromosome 12q14 duplications in patients with normal tension glaucoma (NTG) (Fingert et al., 2011). Three different but overlapping chromosome 12q14 duplications that all spanned the TBK1, XPOT, and RASSF3 genes were identified. The duplication of TBK1 was judged to be the most likely cause of NTG in these patients because: 1) TBK1 associates with the product of another NTG gene, optineurin (Morton et al., 2008); 2) Duplication of TBK1 leads to increased transcription of TBK1 (Fingert et al., 2011); and 3) TBK1 is specifically expressed in cells affected by glaucoma pathophysiology (retinal ganglion cells and their axons) (Fingert et al., 2011). Finally, the population-based segment of our previous study identified chromosome 12q14 duplications that span TBK1 in 2 (1.3%) of 152 NTG patients from Iowa (Fingert et al., 2011) suggesting that copy number variations of TBK1 may be responsible for a fraction of all NTG cases. Here we report the first replication study to investigate the role of copy number variations (CNVs) of TBK1 in NTG pathogenesis. The study was approved by local Institutional Review Boards and informed consent was obtained from all study participants. We tested NTG patients and ethnically matched normal control subjects from Japan. NTG patients from New York and North Carolina were also studied. Subjects were tested for duplication of TBK1 using a quantitative PCR assay and microarray analysis of SNPs at chromosome 12q14. Patients were examined by fellowship-trained glaucoma specialists and received complete ophthalmic examinations including gonioscopy, standardized computerized Humphrey (Zeiss, San Leonardo, Ca.) SITA visual field testing, and stereoscopic optic nerve examination. Subjects were diagnosed with open-angle glaucoma when optic nerve damage and corresponding visual field defects were detected in at least one eye as previously described (Alward et al., 1998) and NTG was diagnosed when the maximum untreated IOP was ≤21 mmHg in both eyes. The study dataset consisted of 252 NTG patients and 202 controls from Japan, 29 NTG patients from North Carolina, and 28 NTG patients from New York. DNA samples from each subject were tested for duplication of the TBK1 gene using a quantitative PCR assay (TaqMan Copy Number Assay, Applied BioSystems, Carlsbad, CA) as previously described (Fingert et al., 2011). Briefly, a segment of the TBK1 gene was PCR amplified in triplicate for each DNA sample, as was a control amplicon from a different gene on a different chromosome. Experiments were conducted using a 7900HT PCR machine and data was analyzed to detect CNVs using CopyCaller software (Applied BioSystems, Carlsbad, CA) with default settings. Subjects that exhibited a CNV that spanned TBK1 were retested to confirm its presence. One of 252 (0.40%) unrelated Japanese NTG subjects (patient GGJ-414) was found to carry a TBK1 duplication using quantitative PCR. The duplication in patient GGJ-414 was confirmed using microarray analysis (Affymetrix microarrays, Santa, Clara, CA) as previously described (Fingert et al., 2011). No duplications were detected in the Japanese control subjects or in any of the patients from North Carolina or New York. Further analysis of the genetic data (Partek software package, St. Louis, MO) showed that patient GGJ-414 carried a chromosome 12q14 duplication that extended from 64,802,839 to 65,098,981 bps (human genome build hg19). This 300 kbp duplication encompassed the TBK1 gene as well as XPOT and RASSF3 and is similar in extent to the duplication previously reported in Caucasian NTG subject GGA-1159-1 (Fingert et al., 2011). Patient GGJ-414 (subject II-4 in Figure 1) is a Japanese woman who was diagnosed with NTG at age 42 and has a strong family of disease. Representative disc photos from family members are shown in Figure 2. Other members of this family were tested for the presence of the chromosome 12q14 duplication using the quantitative PCR assay described above. Patient GGJ-414 (Figure 1, subject II-4) has a sister with NTG (Figure 1, subject II-3) and two sons that were diagnosed as NTG suspects, based on large cup-to-disc ratios (Figure 1, subjects III-2 and III-3), also carried the TBK1 duplication. The maximum recorded untreated intraocular pressure for patient GGJ-414 was 18 mm Hg OD and 17 mm Hg OS and central corneal thickness was 521 microns OD and 528 microns OS. One family member (subject II-1, Figure 1) that carried the TBK1 duplication had not been diagnosed with glaucoma when he was last examined at 45 years of age. At this examination, subject II-1 had large cup to disc ratios (0.75 OD and 0.70 OS) and had abnormal glaucoma hemifield test OD. Subject II-1 has not been examined in nine years and lacks further information on glaucoma status. Figure 1 Japanese NTG pedigree Figure 2 Disc Photos Over the last 15 years, family-based linkage studies have identified several genes (i.e. myocilin and optineurin) that are capable of causing POAG with little influence from other genes or environmental factors. These single gene or Mendelian forms of glaucoma represent approximately 5% of POAG cases. The vast majority of glaucoma-causing mutations in myocilin and optineurin are missense and nonsense sequence changes. More recently, we reported that duplication of a segment of chromosome 12q14 that encompasses the TBK1 gene is associated with NTG. Our study first detected a chromosome 12q14 duplication in a large African American pedigree with NTG and later identified two different but overlapping chromosome 12q14 duplications in 2 (1.3%) of 152 unrelated Caucasian NTG patients (Fingert et al., 2011). This is the first confirmation that chromosome 12q14 duplications which encompass the TBK1 gene are associated with NTG. Although it remains possible that other neighboring genes in and around the chromosome 12q14 duplication have a role in the pathogenesis of NTG, there is strong data to suggest that it is duplication of TBK1 that leads to this form of NTG (Fingert et al., 2011). The discovery and confirmation that another gene, TBK1, leads to glaucoma potentially represents a significant advance in glaucoma genetics. TBK1 encodes a kinase that influences gene expression in the NF-κB signaling pathway. Two other NTG genes, optineurin (Rezaie et al., 2002) and toll-like receptor 4 (Zareparsi et al., 2005), also participate in NF-κB signaling. Together these data strongly suggest that this biological pathway has an important role in the pathogenesis of NTG in at least a subset of patients, perhaps via its influence on key cellular processes including apoptosis. Furthermore, duplication of TBK1 is associated with 0.40% to 1.3% of NTG in Caucasians, African Americans (Fingert et al., 2011), and Japanese, suggesting that this defect may have a role in NTG pathogenesis in many ethnicities.

Association of TLR4 polymorphisms with Behçet's disease in a Korean population

OBJECTIVES HLA-B51 is strongly associated with Behçets disease (BD) in any ethnic background. We recently reported that another gene, Toll-like receptor-4 (TLR4) is also implicated in BD in a Japanese population. To confirm these results, we investigated polymorphisms in the TLR4 gene in Korean patients with BD. METHODS In this study, 119 patients with BD and 141 healthy controls were enrolled; every participant was a Korean. Nine single nucleotide polymorphisms previously detected in TLR4 by direct sequencing were analysed for an association with BD. RESULTS The most frequent haplotype, TAGCGGTAA, was significantly increased in HLA-B*51-positive BD patients (49.5%), compared with healthy control participants [32.3%; P = 0.029; odds ratio (OR) = 2.01; 95% CI 1.25-3.23]. This haplotype was also significantly increased in BD patients with arthritis (48.2%; P = 0.003; OR = 1.96; 95% CI 1.26-3.26). There were no significant differences in the allele and genotype frequencies of patients and controls for each single nucleotide polymorphism. CONCLUSIONS The haplotype of TLR4 may increase the risk for developing BD and the complication of arthritis in the Korean population.

Association of the toll-like receptor 4 gene polymorphisms with Behçet’s disease

Behcet’s disease (BD) is a multisystemic inflammatory disorder characterised by recurrent ocular symptoms, oral and genital ulcers, and skin lesions.1 2 The aetiology of BD remains unclear, but likely both genetic and environmental factors play an important part in BD development. We performed a whole-genome association analysis of BD using 23 465 microsatellite markers and ultimately found significant association for 147 markers (unpublished data). One of the 147 markers is located within 100 kb from the toll-like receptor (TLR) 4 gene on chromosome 9. Among the TLR family members, TLR4 is the receptor most exhaustively investigated and has been shown to recognise and interact with heat shock protein (HSP) and …