Yuichi Tokuda

Three susceptible loci associated with primary open-angle glaucoma identified by genome-wide association study in a Japanese population

Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel–Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese.

Background To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated. Methods and Principal Findings We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10−10). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients. Conclusions and Significance In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.

Association between prostaglandin E receptor 3 polymorphisms and Stevens-Johnson syndrome identified by means of a genome-wide association study

BACKGROUND Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucosa. They often affect the ocular surface and can result in permanent visual dysfunction. OBJECTIVES We sought to discover genetic markers for SJS/TEN susceptibility. METHODS We performed a genome-wide association study with 60 patients and 300 control subjects. We applied stringent filter and visual assessments for selecting single nucleotide polymorphisms (SNPs) and a high false discovery rate threshold. We fine-mapped the region where a candidate SNP was found and confirmed the results by means of sequencing. We evaluated the function of agonist-activated prostaglandin E receptor 3 (EP3), the gene for which contained several SNPs, in regulating cytokine production in human conjunctival epithelial (CE) cells. The expression levels of EP3 in the CE cells from patients and control subjects were also compared. RESULTS We identified 3 SNPs that passed the false discovery rate threshold. One (rs17131450) was close to the EP3 gene. Therefore we analyzed the EP3 region in detail and identified 5 other SNPs. We confirmed the association between SJS/TEN and all 6 SNPs. Activated EP3 was expressed in control CE cells, and it suppressed polyI:C-stimulated cytokine production, suggesting that EP3 might help prevent ocular surface inflammation. Concordantly, the EP3 levels were much lower in the CE cells of the patients than in those of the control subjects. CONCLUSION We demonstrated, using both genetic and functional analyses, that EP3 could be a key player in the pathogenesis of SJS/TEN accompanied by ocular complications.

Novel common variants and susceptible haplotype for exfoliation glaucoma specific to Asian population

The common variants in lysyl oxidase-like 1 gene (LOXL1) are associated with exfoliation glaucoma (XFG) patients developed through exfoliation syndrome (XFS). However, the risk allele of a variant in LOXL1 has been found to be inverted between Asian and Caucasian populations. Therefore, we newly performed a genome-wide association study using 201 XFS/XFG and 697 controls in Japanese, and identified 34 genome-wide significant single-nucleotide polymorphisms (SNPs) distributing in not only LOXL1 but also TBC1D21 and PML at the 15q24.1 locus. These SNPs were confirmed by an independent population consisted of 121 XFS/XFG and 263 controls in Japanese. Moreover, further analyses revealed a unique haplotype structure only from the combination of TBC1D21 and LOXL1 variants showing a high XFS/XFG susceptibility specific for the Asian population. Although there still should be other gene(s) in the other region(s) contributing to the disease process, these results suggested that the combination of newly discovered variants in these genes might be useful for precise XFG risk assessment, as well as for elucidating the molecular mechanism of XFG pathogenesis through XFS.

An approach to predict the risk of glaucoma development by integrating different attribute data

Primary open-angle glaucoma (POAG) is one of the major causes of blindness worldwide and considered to be influenced by inherited and environmental factors. Recently, we demonstrated a genome-wide association study for the susceptibility to POAG by comparing patients and controls. In addition, the serum cytokine levels, which are affected by environmental and postnatal factors, could be also obtained in patients as well as in controls, simultaneously. Here, in order to predict the effective diagnosis of POAG, we developed an “integration approach” using different attribute data which were integrated simply with several machine learning methods and random sampling. Two data sets were prepared for this study. The one is the “training data set”, which consisted of 42 POAG and 42 controls. The other is the “test data set” consisted of 73 POAG and 52 controls. We first examined for genotype and cytokine data using the training data set with general machine learning methods. After the integration approach was applied, we obtained the stable accuracy, using the support vector machine method with the radial basis function. Although our approach was based on well-known machine learning methods and a simple process, we demonstrated that the integration with two kinds of attributes, genotype and cytokines, was effective and helpful in diagnostic prediction of POAG.

The defect of SFRP2 modulates an influx of extracellular calcium in B lymphocytes

BackgroundIn the Wnt pathway, the secreted frizzled-related protein 2 (SFRP2) is thought to act as one of the several competitive inhibitors of Wnt. However, the precise role of SFRP2 is still poorly understood especially in B lymphocytes. Here, we investigated the function of SFRP2, comparing the SFRP2 defective as well as normal B lymphocytes in mice.ResultsWe demonstrated that calcium influx from extracellular to intracellular space in splenic B cells was clearly affected by the defect of SFRP2. In addition, the phosphorylation of phospholipase Cγ2 was observed to be reduced in SFRP2 defective splenic B cells with B cell receptor stimulation.ConclusionsSFRP2 is suggested to modulate the influx from extracellular calcium in the B cell receptor signaling pathway.

Stronger Association of CDKN2B-AS1 Variants in Female Normal-Tension Glaucoma Patients in a Japanese Population

We read with great interest the article by Ng et al. entitled ‘‘Genetic Association at the 9p21 Glaucoma Locus Contributes to Sex Bias in Normal-Tension Glaucoma,’’ that was recently published in Investigative Ophthalmology & Visual Science. As the 9p21 locus was identified by a genome-wide association study (GWAS) to be associated with primary open-angle glaucoma (POAG), and the association was found to be significantly stronger among the normal-tension glaucoma (NTG) subgroup (for review see Ref. 2), Ng et al. extended the analysis to investigate the influence of sex difference on the genetic association at the 9p21 locus. They carefully analyzed the genotype data of 13 variants linked with clinical information and clearly presented the results that: (1) the sex bias was present within advanced NTG cases, (2) the strongest associated variant in all POAG cases showed stronger association in females than in males with a statistically significant difference in female-to-male odds ratio (OR) comparison, and (3) the significant association was observed only in females under the NTG to high-tension glaucoma (HTG) subanalysis along with a significant difference in OR comparison. Although they used a large reliable number of samples and obtained robust results, they honestly pointed out the limitation of the study as the analyses were performed without replication using a cohort derived from a single ethnic background (i.e., Australian of European descent). Therefore, we reanalyzed our two GWAS datasets using our Japanese population in an attempt to assess their findings. We ended up with 975 healthy controls and 1244 POAG cases composed of 545 HTG and 699 NTG patients (HTG and NTG were denoted as HPG and NPG, respectively, in our previous article) for the analysis (Table 1). Among the 13 variants at the 9p21 locus that Ng et al. analyzed, we were able to directly compare the genotype data of three variants on CDKN2B-AS1 from our previous datasets. With regard to the female-to-male analyses, we indeed observed a stronger association for all of the variants in the female patients (Table 2). Especially rs7049105, a variant in Japanese NTG patients considered to be influenced by sex, because the OR of NTG to control showed a significant heterogeneity (P1⁄40.0427) between the female and male groups (Table 2). Overall, these results suggested that although the allele frequencies of the variants slightly differs between the populations, the stronger association of the female NTG patients seen in the Australian population seemed to be replicated in our Japanese female NTG patients. In the normal clinical setting, the female NTG patients who we encounter tend to be elderly and thin, and often possess other clinical features such as low blood pressure, myopia, migraine, neck stiffness, and so on. Not only identifying the genetic variants responsible for the disease onset by casecontrol association studies, but also carefully linking these phenotypes to each identified variant (as Ng et al. performed for the sex difference), should greatly contribute to the elucidation of the complex etiology of glaucoma.

Efficient and reliable establishment of lymphoblastoid cell lines by Epstein-Barr virus transformation from a limited amount of peripheral blood

Lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus (EBV) serve as an unlimited resource of human genomic DNA. The protocol that is widely used to establish LCLs involves peripheral blood mononuclear cell isolation by density gradient centrifugation, however, that method requires as much as 5 ml of peripheral blood. In this study, in order to provide a more simple and efficient method for the generation of LCLs, we developed a new protocol using hemolytic reaction to enrich white blood cells for EBV transformation and found that the hemolytic protocol successfully generated LCLs from a small volume (i.e., 0.1 ml) of peripheral blood. To assess the quality of genomic DNA extracted from LCLs established by the hemolytic protocol (LCL-hemolytic), we performed single nucleotide polymorphism (SNP) microarray genotyping using the GeneChip® 100 K Array Set (Affymetrix, Inc.). The concordances of the SNP genotyping resulting from genomic DNA from LCL-hemolytic (99.92%) were found to be as good as the technical replicate (99.90%), and Kappa statistics results confirmed the reliability. The findings of this study reveal that the hemolytic protocol is a simple and reliable method for the generation of LCLs, even from a small volume of peripheral blood.