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Although intraocular pressure (IOP) is the most definitive cause of glaucoma, a subtype of open angle glaucoma (OAG) termed normal tension glaucoma (NTG), which occurs in spite of normal IOP, accounts for a large part of glaucoma cases, especially in Japan. To find common genetic variants contributing to NTG in Japanese patients, we conducted a genome-wide association study (GWAS). We performed the first screening for 531,009 autosomal SNPs with a discovery cohort of 286 cases and 557 controls, and then a second screening for the top 30 suggestive loci in an independent cohort of 183 cases and 514 controls. Our findings identified a significantly associated SNP; rs523096 [combined p-value = 7.40× 10−8, odds ratio (OR) = 2.00 with 95% confidence interval (CI) 1.55–2.58] located 10 kbp upstream of CDKN2B on chromosome 9p21. Moreover, analysis of another independent case-control set successfully replicated the results of the screening studies (combined values of all 3 stages p = 4.96 × 10−11, OR = 2.13 with 95% CI 1.69–2.68). The SNPs near rs523096 were recently reported to be associated with OAG associated with elevated IOP in primary open-angle glaucoma (POAG), the predominant subtype of glaucoma in Caucasian populations. Our results revealed that the 9p21 locus is also associated with NTG in Japanese. In addition, we identified SNPs more strongly associated with NTG.
Citation: Takamoto M, Kaburaki T, Mabuchi A, Araie M, Amano S, Aihara M, et al. (2012) Common Variants on Chromosome 9p21 Are Associated with Normal Tension Glaucoma. PLoS ONE 7(7): e40107. https://doi.org/10.1371/journal.pone.0040107
Editor: Ian A. Trounce, Centre for Eye Research, Australia
Received: March 23, 2012; Accepted: June 1, 2012; Published: July 5, 2012
Copyright: © 2012 Takamoto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was funded by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (20592033) to Dr. Kaburaki. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Glaucoma is a neurodegenerative disease that leads to progressive loss of retinal ganglion cells [1], causing irreversible visual field defects [2], and one of the leading causes of irreversible blindness, affecting over 60 million people worldwide [3]. Presently, glaucoma is clinically defined by the existence of characteristic changes in the optic disc and corresponding visual field defects [4]. Elevation of intraocular pressure (IOP) is the only proven cause of glaucoma to date and lowering IOP is the only established therapy for reducing the rate of deterioration of the visual field in glaucoma patients [5]–[8].
Open-angle glaucoma (OAG) is the most prevalent type of glaucoma, and characterized by adult onset and chronic IOP-dependent progression. OAG associated with IOP consistently within a statistically normal range (≤21 mmHg) has been termed normal-tension glaucoma (NTG) [4].
NTG is thought to be included in the category of OAG. However, in addition to the obvious lower IOP than OAG in a classical sense, OAG is also associated with elevated IOP (≥22 mmHg) and primary open angle glaucoma (POAG), with several differences between NTG and POAG reported. Optic disc hemorrhage, an important and well-established negative prognostic factor for glaucoma, is much more frequently seen in NTG [9]–[12]. The majority of reported studies have noted small, but significant differences in the patterns of damage in the optic disc, retinal nerve fiber layer, and visual field [13]–[21]. Furthermore, immune aberrations [22]–[26], and abnormalities in systemic [27]–[31] and/or ocular circulation [32]–[34] are reportedly more likely to be encountered in NTG than POAG. Although the exact etiology of glaucomatous optic neuropathy is unknown, neuronal factors independent of IOP or factors to define hypersensitivity to IOP rather than elevating IOP have been suggested to underlie the etiology of NTG. Interestingly, there are differences among affected populations regarding the prevalence of NTG. In Caucasians and African-Americans, the prevalence ratios of NTG and POAG are approximately 1.0 [35]–[39], while those are about 3 in Koreans [40] and greater than 10 in Japanese [41].
Although familial aggregation of POAG has been reported [42], that has not been well investigated for NTG. Linkage analysis of a British family with NTG revealed optineurin as a gene causative of NTG [43], [44]. However, that mutation is very rare in other populations including Japanese [45]–[47]. In other candidate gene approaches, possible associations of apolipoprotein E [48]–[50], OPA1 [51]–[53], and toll-like receptor 4 [54] with NTG have been investigated, though the results were inconsistent. Recently, a genome-wide association study (GWAS) of 305 Japanese NTG patients and 355 controls revealed that rs3213787 in S1 RNA binding domain on chromosome 2 was significantly associated with NTG [55], [56].
Two GWASs for POAG in Caucasians have been reported, in which 3 genetic loci, caveolin 1 (CAV1) and caveolin 2 (CAV2) on 7q31 [57], TMCO1 on1q24, and cyclin-dependent kinase inhibitor 2B antisense RNA (CDKN2BAS) on 9p21 [58], were identified as susceptibility loci, while another GWAS of Japanese POAG patients provided only suggestive results [59].
In an attempt to identify genetic factors contributing to NTG, we conducted a GWAS for NTG in Japan, where a high prevalence rate of NTG has been observed. In the present study, a total of 620 definitively diagnosed NTG patients and 1258 controls were investigated. After GWAS in our discovery cohort, a second screen was performed for the top 30 suggestive loci. Thereafter, replication analysis was performed for an SNP found to be significantly associated in the first and second screening sets.
First and Second Screenings, and Replication Study
In the first screening, we performed a GWAS using discovery cohort of 286 cases and 557 controls, each of which had passed the sample quality control (QC) criteria. We applied an SNP QC [minor allele frequency (MAF) ≥0.05, call rates ≥0.98, Hardy-Weinberg equilibrium p-value ≥0.001 in controls, and visual cluster removal] and selected 531,009 SNPs for the first screening. We generated a quantile-quantile plot to inspect possible population stratification effects and obtained the genomic inflation factor (λ) of 1.046, indicating no population substructure (Figure S1). However, none of the SNPs reached genome-wide significance in the first screening. The resulting Manhattan plot is shown in Figure 1.
Negative common logarithm of p-value plots for genome-wide association study in the first screening. The X-axis indicates chromosomes and their positions, and the Y-axis indicates the negative common logarithm of the association p-value of 531,009 SNPs. No SNP showed a genome-wide significance at p = 9.416 × 10−8 (Bonferroni's correction).
To identify significantly associated SNPs, we carried out the second screening using an independent set of 183 NTG cases and 514 controls. We genotyped 30 SNPs on the top 30 loci shown to be the most significant in the first screening. The results of association analysis in the second screening are shown in Table S2. We observed a significant association at a single SNP, rs523096 on chromosome 9p21 (p = 4.55 × 10–4, OR = 2.15 with 95% CI 1.39–3.32 in the second screening). The combined results of the first and 2nd screenings showed genome-wide significance (rs523096, combined p = 7.40×10–8, OR = 2.00 with 95% CI 1.55–2.58).
In the replication study, we performed genotyping of rs523096 for the replication cohort composed of 151 cases and 187 controls independent of the first and second screening, and the same significant association was obtained (p = 1.63 × 10–4). Thus, we confirmed the association of chromosome 9p21 with NTG. Finally, the combined p-value of all 3 stages was 4.96× 10–11 (OR = 2.13 with 95% CI 1.69–2.68), which indicated a definite association (Table 1).
Dense Association Mapping of 9p21
We further analyzed SNPs on chromosome 9p21 using the second screening case-control samples in dense association mapping. The 87-kbp region for dense association mapping was defined to cover the strong linkage disequilibrium (LD) region, including the associated SNPs in the first screening (Figure 2). The association plots on 9p21 in the first screening data, the region for the dense association mapping, and the LD plot calculated from the Japanese specimens of HapMap III using Haploview 4.2 are shown in Figure 2. A total of 29 SNPs (28 SNPs newly genotyped and rs523096) were entered into the analysis (Table S3). The association was reproduced in SNPs with a high LD with rs523096 in the second screening set. In dense association mapping, rs643319 showed the lowest p-value (p = 2.57× 10−5, OR = 1.78 with 95% CI 1.36–2.33). In addition, 10 SNPs around these 2 SNPs showed p-values lower than or nearly equal to that of rs523096. Combined with the results of the first GWAS data set, a total of 6 SNPs including the representative SNP rs523096 showed p-values markedly below the genome-wide significance of p = 9.416 × 10−8 (Table S3). Imputation analysis in the region showed no evidence of SNPs with a p-value extremely lower than those of rs523096, rs643319, or other SNPs (Figure S2).
Upper panel shows plots of negative common logarithm of association p-values for 9p21 locus. Each dot represents an SNP from the first screening and the colors of the dots represent the extent of linkage disequilibrium with rs523096 (purple diamond). Shaded area in the middle panel of the gene structures indicates the 87-kbp region, defined as the dense association mapping region. Lower panel shows an LD plot (D-prime) of Japanese specimens from HapMap III data.
The results of haplotype analysis are shown in Table 2. One haplotype carrying the G allele of rs523096 was most significantly associated and shown to be protective against the disease.
These results showed that multiple SNPs in this region are strongly associated with NTG.
Evaluation of POAG-associated SNPs
Results of the first screening of SNPs alternative for POAG-associated SNPs (rs17588172 for rs1052990, and rs6969706 for rs4236601 of the CAV1 and CAV2 regions on 7q31 [57], and rs4657477 for rs4656461 and rs7518099 of theTMCO1 region on 1q24 [58]) are shown in Table S4. The frequencies of the alleles are also shown, which were consistent with those reported as risk alleles for POAG [57], [58]. We were unable to obtain replicated results for these 3 SNPs, though the minor allele frequencies of rs4657477 and rs6969706 were so low that evaluation of an association was difficult. In addition, the effects of both rs4657477 on 1q24 and rs17588172 on 7q31 appeared to be opposite, as compared to results presented in previous reports.
The association between 6 SNPs (rs547984, rs540782, rs693421, rs2499601, rs7081455, and rs7961953) and Japanese POAG suggested in the previous GWAS [59] was not demonstrated in our first screening data (Table S5). The statistical powers for these SNPs indicated sufficient values (0.789 to 0.933, Table S5).
In the present study, we found a significant association of rs523096, which is located 10 kbp upstream of cyclin-dependent kinase inhibitor 2B (CDKN2B) and in the intron of CDKN2BAS (also known as ANRIL) on 9p21, with Japanese NTG. This locus is one of 3 genetic loci recently reported to be associated with POAG in Caucasian GWASs. In one of those studies, rs4977756 located 60 kbp upstream of CDKN2B, was most significantly associated [58]. Although rs4977756 was not included in our analysis, rs10965224, which was shown to be in absolute LD with rs4977756 (r2 = 1 in Japanese in Tokyo (JPT) of HapMap III), exhibited a relatively weak association (p = 0.013, OR = 1.45 in discovery cohort, Table S3). Two other groups have reported an association between Caucasian POAG and rs1063192 in the 3′ UTR of CDKN2B [60], [61]. Rs1063192 was originally reported as a lead SNP in a GWAS of normal variations of vertical cup to disc ratio (VCDR) in a general population [62]. In our data obtained with dense association mapping, the p-value for rs1063192 was 0.038, with an OR of 1.41. For these 2 SNPs, our results for Japanese NTG were similar in regard to the ORs of those for Caucasian POAG cohorts (1.39 for rs4977756 [58], and 1.32 and 1.37 for rs1063192 [60], [61]). Rs523096, which had an association identified in the present study, and the SNPs in absolute LD (rs2069418, rs573687, rs518394, rs564398, and rs7865618) in Japanese HapMap specimens showed high ORs (highest = 2.20 with 95% CI 1.40–3.46 for rs2069418) in dense association mapping. We identified multiple SNPs with more significant associations as compared to SNPs previously reported in Caucasians, though we were unable to identify a single SNP as the primary variant of this region using haplotype and imputation analyses. Furthermore, a high frequency of the risk allele of rs523096 (or low frequency of the protective allele) in Japanese as compared to that in Caucasians (0.907 vs. 0.551 in HapMap data) may be an explanation for the high prevalence rate of NTG in Japanese [41].
Consistently, a very recent report on the study of Japanese NTG by other independent GWAS group demonstrated rs523096, rs518394, rs564398 and rs7865618 were significant association SNPs with NTG and rs523096 was the most significant one [63]. In our study, selection criteria were more restricted to select severer and younger patients, and combining with all stages, stronger association of rs523096 with NTG was observed, and more SNPs on the CDKN2BAS region were demonstrated as genome-wide significance and some were more significant than reported SNPs [rs643319(p = 5.44×10−8), rs10965219(p = 2.38×10−8), rs10120688(p = 3.57×10−8)].
Cyclin-dependent kinase inhibitor 2A (CDKN2A) encoding p16INK4a and p14ARF, CDKN2B encoding p15INK4b, and non-protein coding CDKN2BAS are located on the 9p21 locus. The cyclin-dependent kinase inhibitors p16INK4a and p15INK4b function as cell growth regulators to control cell cycle G1 progression, and are well-known tumor suppressor genes [64]. Germline mutations on 9p21 have been reported to be associated with familial predisposition to melanoma [65] and homozygous deletion on 9p21 is frequently detected in a wide variety of tumors [66]. Also, the expression of CDKN2B was found to be dramatically induced by transforming growth factor beta (TGF-β) [67], suggesting its role in TGF-β induced growth inhibition. TGF-β is also known to be involved in programmed cell death in the developing retina and optic nerve [68], [69], and has also been suggested to play an important role in glaucoma [69]–[71]. In fact, in the region of associated SNPs, a regulatory domain interacting with TGF-β-mediated proteins (Smad, sp1, miz-1, and myc) has been reported [72], [73] and rs2069418, which showed the highest OR in dense association mapping, is located in the Smad binding region [72], [73]. The products of CDKN2A regulate the Rb and p53 pathways, which induce cell cycle arrest, apoptosis, and senescence [74], and are reported to be involved in retinal ganglion cell apoptosis [75]. CDKN2BAS is a large antisense non-coding gene that overlays the CDKN2B gene in an antisense strand. Although the function of CDKN2BAS is not well elucidated, recent GWASs of several common diseases (coronary artery disease [76], type 2 diabetes [76], aortic aneurysm [77], intra-cranial aneurysm [76], endometriosis [78], glioma [79], [80]) have revealed associations with this long non-coding region. Also, CDKN2BAS has been suggested to influence the expressions of CDKN2B and CDKN2A [81], [82].
Measurement of IOP using Goldmann applanation tonometery is known to be influenced by central corneal thickness (CCT) with a thinner CCT giving a lower value [83]. In the present study, diagnosis of NTG was based on IOP≤21 mmHg without regard to CCT and we can not rule out the possibility that POAG eyes with thin CCT were misclassified as NTG. However, it was also reported that CCT in Japanese patients with NTG showed no significant difference from that in POAG patients or normal subjects [41], [84], thus we believe that such misclassification occurred in an extremely small portion of our cases.
In conclusion, we identified a susceptibility locus related to Japanese NTG. The locus on 9p21 is considered to be common in Caucasian POAG and Japanese NTG patients, while the SNPs on this locus showed stronger associations with Japanese NTG than with previously reported POAG-associated SNPs in Caucasians. Considering the population diversity of prevalence rates and sensitivity to IOP, our results are important for elucidating the pathogenesis of OAG, and support a hypothesis stating that the effect of 9p21 is responsible for the neuronal factor rather than high IOP in glaucomatous optic neuropathy, since a common genetic factor may be involved irrespective of baseline IOP. Further investigation is needed to identify the disease causative variant and reveal how genes on the locus play a role in the etiology of glaucomatous optic neuropathy.
Materials and Methods
Ethics Statement
The study protocol was approved by the Research Ethics Committees of Graduate School of Medicine, The University of Tokyo, and all participants provided written informed consent after an explanation of the nature and possible consequences of the study.
All participants in the present study were of Japanese ancestry. A total of 620 NTG patients registered at The University of Tokyo Hospital, Tajimi Municipal Hospital, Iwase Tajimi Clinic, Yamanashi University Hospital, Shirato Eye Clinic, Tokyo Metropolitan Police Hospital, Saitama Red Cross hospital, or Showa General Hospital were enrolled.
All affected subjects fulfilled the following diagnostic criteria: (1) existence of glaucomatous optic disc change, judged by glaucoma specialists, (2) existence of visual field loss consistent with a neuronal lesion [visual field loss evaluated using a Humphrey visual field analyzer (Zeiss-Humphrey) central 30–2 full threshold, Swedish Interactive Thresholding Algorithm Standard (SITA-S) Central 30-2 program, or SITA-S 24-2, according to Anderson and Patella's criteria [85]], (3) untreated IOP with a level consistently ≤21 mmHg during the follow-up period, (4) spherical equivalent refraction between −8.0 and 5.0 diopter, and (5) no apparent neurosurgical or otorhinological abnormalities that might cause glaucomatous changes in the optic disc or visual field defect. In addition the selection criteria were modified based on subject age, as follows: (1) no modification if the patient was diagnosed below the age of 50 years, (2) –5.00 dB of or worse in mean deviation measured by Humphrey visual field analyzer in at least one eye if the disease was diagnosed when the patient was between the ages of 50 and 65 years, (3) −10.00 dB or worse in at least one eye if the disease was diagnosed when the patient was older than 65 years. We divided these subjects into 3 stages (first screening, second screening, replication study). The clinical manifestations of the NTG cases in each stage are shown in Table S1.
Control samples for the first screening were comprised of 578 Japanese healthy volunteers from the Departments of Human Genetics [86] and Neuropsychiatry [87] of The University of Tokyo Hospital. Their data were obtained using the CEL files of Affymetrix Genome-Wide Human SNP array 6.0. Of these, 557 samples fulfilled the sample QC threshold described below and were used for association analysis. Control samples in the second screening and replication study were healthy volunteers from Tajimi Municipal Hospital and the Department of Human Genetics, The University of Tokyo. Control subjects did not undergo an ophthalmic examination. All DNA samples used in this study were derived from individuals after they had given written informed consent.
SNP Genotyping and Data Cleaning in First Screening
In the first screening, we genotyped 310 Japanese patients with NTG using Affymetrix Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA), according to the manufacturer's instructions. After exclusion of 24 cases and 19 control samples with QC call rates <95%, contrast QC <0.4, or a difference of contrast QC of Sty and that of Nsp>2, the remaining 286 case samples and 558 control samples were recalled using Birdseed version 2 software (Affymetrix). Next, identity by descent (IBD) was estimated using plink v1.07 [88] and 1 of a pair of control samples with pi-hat >0.125 was excluded from the first screening set. The pi-hat values for the other pairs were <0.125, showing no sample duplication or existence of unexpected relatives.
We applied the following thresholds for QC in data cleaning: Hardy-Weinberg equilibrium with a p-value ≥0.001 for control samples, call rate for each SNP≥0.98, and MAF ≥0.05. All cluster plots for the SNPs showing a p-value <0.001 in association analyses were checked by visual inspection. SNPs with ambiguous genotype calls were excluded. A total of 531,009 SNPs on autosomal chromosomes passed the QC filters and were subjected to association analysis.
Although the 24 case samples noted above were excluded from the first screening by data cleaning, the quality of their DNA was adequate for SNP typing in the second screening and they were included in that screening.
Second Screening
The case and control sets for the second screening included 183 cases and 514 controls. To identify associated variants with high odds ratios (ORs), 30 SNPs were selected from the top list in the first screening.
Thirty SNPs were genotyped using the DigiTag2 method [89] and SNPs with a call rate >0.97 were adopted. Three of these 30 SNPs that could not pass the filter were genotyped again using a TaqMan assay (Applied Biosystems, Carlsbad, CA). The average call rate of the 30 SNPs was 0.997.
A DigiTag2 assay was performed as described by Nishida et al [89]. This assay enables determination of 96 or 32 SNPs genotypes at once. A multiplexed oligonucleotide ligation assay was performed after multiplex PCR, and a labeling reaction was achieved with two 5′ query probes and 1 common probe prepared for a single SNP site. The 5′ query probes had a sequence complementary to that of the 5′ sequence flanking the target SNP and each of the probes had an allele-specific sequence. Following this, a hybridization reaction was performed using a DNA microarray (NGK Insulators, Nagoya, Japan) with separated areas containing oligonucleotide probes for the SNPs. The genotype calls were determined using SNPStar software (version 0.0.0.8, Olympus, Tokyo, Japan) [89].
TaqMan assays were performed using a LightCycler 480 System II (Roche, Bazel, Switzerland), according to the manufacturer's protocol.
Replication Study
An independent set of 151 cases and 187 controls was included in the replication study. Rs523096, the significantly associated SNP in the screening studies, was genotyped using a TaqMan assay.
Dense Association Mapping of 9p21
For dense association mapping of 9p21, 33 SNPs on the 87-kbp region of 9p21 were planned to be newly genotyped for the second screening case-control set in addition to rs523096. To cover the strong LD region where associated SNPs in the first screening were distributed (53-kbp region between rs3217992 and rs10120688), the 87-kbp region was defined as the dense association mapping region.
In the 87-kbp region of dense association mapping, we extracted SNPs with an MAF >0.01 from Japanese specimens of HapMap phase III. SNPs in absolute LD (r2 = 1) with already selected SNPs were omitted, except for those with rs523096. All SNPs in absolute LD with rs523096 were included. In addition, an SNP previously reported to strongly affect CDKN2B expression [90] and 6 SNPs located just upstream of CDKN2B were selected. Of 33 SNPs, 32 were genotyped using the DigiTag2 method and 23 with call rates >0.97 were adopted. Four of 9 SNPs failed to be genotyped in DigiTag2 assays, while rs2069426 was genotyped using a TaqMan Assay. Finally, 29 SNPs were entered into the analysis. The average call rate of the 29 SNPs was 0.996 and Hardy-Weinberg equilibrium p-values for the controls were >0.05 for all adopted SNPs.
Evaluation of POAG-associated SNPs
We evaluated associations between NTG and SNPs on the other 2 loci found to be associated with POAG in previous GWASs [57], [58], as well as SNPs whose associations were suggested in the GWAS of Japanese POAG [59] using data obtained in our first screening.
The POAG-associated SNPs of rs1052990 and rs4236601 on 7q31 [57], and rs4656461 and rs7518099 on 1q24 [58] were not included in our first screening data, thus we extracted alternative SNPs that were in absolute LD (r2 = 1) with them in HapMap data from the SNPs in the first screening. As a result, rs17588172 for rs1052990, rs6969706 for rs4236601, and rs4657477 for rs4656461 and rs7518099 were selected as alternative SNPs. Because of the absence of polymorphism at rs4236601 in JPT of HapMap III, only rs4730748 was selected according to the LD in Chinese in Beijing (CHB).
Six SNPs (rs547984, rs540782, rs693421, rs2499601, rs7081455, and rs7961953) reported in a GWAS of Japanese POAG [59] were directly evaluated in our first screening data. For these, statistical powers were calculated after applying minor allele frequencies in our control samples for risk allele frequencies, odds ratios for reported ones [59], and 0.05 for alpha.
Statistical Analysis
In the genome-wide association analyses of the first and second screenings, as well as replication analysis we evaluated the association of each SNP using an allelic test. In the first screening, to ascertain whether the control samples were properly matched to the cases, the genomic inflation factor (λ) was estimated using the median chi-square value from the allelic test. The combined p-values of individual case-control sets were calculated based on the simply combined datasets. We set p = 1.667 × 10−3 as the significant p-value in the second screening only and p = 9.416 × 10−8 as the genome-wide significant value, according to Bonferroni's correction. Deviations from Hardy-Weinberg equilibrium were calculated using exact test. [91].
Genotype data from Japanese specimens of HapMap Phase II were used as reference panels in imputation analysis. Association and imputation analyses were performed as implemented in PLINK v1.07 [88]. Haplotype analysis was performed using Haploview 4.2. [92] LD blocks were defined using the method described by Gabriel et al [93]. Allelic association analysis of each haplotype was done using a one-sided chi-square test.
For evaluation of POAG-associated SNPs, Fisher's exact test was performed under an allelic model. Power analysis was performed using R version 2.12.1. Association plots are shown in the figures, where r2 was calculated for Japanese in Tokyo and Chinese in Beijing in HapMap Phase II.
Supporting Information
Figure S1.
Quantile-quantile plots of 531,009 SNPs in first screening data. X-axis indicates expected p-values and Y-axis indicates observed p-values. Obtained genomic inflation factor = 1.046.
Figure S2.
Association plots of genotyped and imputed SNPs in the dense association mapping region. Genotyped SNPs are shown in square and imputed SNPs in circle. The colors of the dots represent the extent of linkage disequilibrium with rs643319 (a purple diamond). Although in the screening stages, we used trend test, here allelic p-values of genotyped and imputed SNPs in the dense association mapping region spanning 87-kbp are depicted. Genes and those structures are also depicted in the lower part.
Table S1.
Clinical manifestations of NTG cases in each set.
Table S2.
Summarized results of second screening of top 30 SNPs.
Table S3.
Results of dense association mapping.
Table S4.
Data from first screening of SNPs on POAG-associated loci.
Table S5.
Results for 6 SNPs previously reported as candidate SNPs for POAG.
The authors would like to sincerely thank all study participants and research staff that have contributed to this research.
Author Contributions
Conceived and designed the experiments: MT TK AM M. Araie SA JN KT M. Aihara AT AI FM KK SS NY HK FN. Contributed reagents/materials/analysis tools: MT TK AM M. Araie SA M. Aihara AT AI FM KK SS NY HK FN JN KT TS YK. Wrote the paper: MT TK AM M. Araie KT.
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House Speaker Paul Ryan, R-Wisconsin, speaks with House Conference Chair Cathy McMorris Rodgers, R-Washington, during a news conference at the U.S. Capitol on Wednesday.Ryan and House Republicans have proposed a woefully inadequate health care plan.
Ryan insisted that he felt "very good" about the bill's prospects but acknowledged that House leadership was "making fine-tuning improvements to the bill to reflect people's concerns".
"For six years, Republicans have campaigned over and over and over again saying, 'If only you'd give us the House, we'll repeal Obamacare, '" he said.
Lawmakers plan to have the healthcare legislation on the House floor this Thursday, Ryan said. This is going to be great for people.
"We have a president who is rolling up his sleeves ..."
President Trump said Friday he and GOP lawmakers made made "certain changes" to the bill. But it does not rise and fall, like the ACA's subsidies, so a person pays only a fixed percentage of their income on their health insurance premiums.
In a Facebook post Saturday night, Rep. Brian Fitzpatrick, R-Pa., said he couldn't vote for the bill, stressing a need "to take our time and to get this right".
Moderate Republicans have also expressed concerns about the bill, and their worries are often not the same as that of conservatives.
But Edwards and other demonstrators said they feel congressional Republicans have let them down by not moving more aggressively on a conservative approach to health care reform and other issues.
Meadows, a North Carolina Republican, told C-Span's "Newsmakers" the changes being considered for the Medicaid program would not go far enough, if they left it up to states to decide whether to put in place a work requirement. They have in the House plan bailouts for insurance companies.
"If there's any indication that some of this is not going to happen, I think there will be negative impact on the markets", Cantor said, reiterating that Congress must work with Trump to get these two major GOP agenda items across the finish line. "I believe that the real negotiation begins when we stop them". But I will say that NIH is something that's particularly popular in Congress.
Ryan spoke on "Fox News Sunday", Price appeared on ABC's "This Week", Collins was on NBC's "Meet the Press" and Cruz spoke on CBS' "Face the Nation".


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Welcome to our scrapbook layout gallery. Pull up a chair and browse through our thousands of layout ideas - both traditional scrapbooking and digi. You'll be amazed and inspired! We'd love to have you submit your layouts to our Gallery: click Submit To Gallery to get started.
Gallery Home > Traditional > KaiserCraft > Cosmo Cricket > November THEME Challenge
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Healthcare, Technical, and Industrial Furniture
Cramer LLC is the leading provider of task chairs and stools for workers in challenging environments. Their mission is to improve the health and productivity of workers in unique environments. Kay-Twelve.com is an official dealer for Cramer. Contact us to request a personalized quote or to discover more about Cramer.


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Re: Editing is good Barbara.
Posted By: Barbara <firstname.lastname@example.com>
Date: Thursday, 20 May 2004, at 8:34 a.m.
no no no!! martin, you must not keep your fish with blue gravel....get rid of it!..even planting the thought....; )
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A branch of the Essex Federation serving the local community in Sandon, Essex.
Meetings are held in the Village Hall on the 2nd Thursday in the month at 2.00p.m.
Visitors are always welcome.
Programme for July 2017 - December 2017
News item - Milson WI (New Zealand) August 2017 letter (new)
Note - We are on Facebook - see www.facebook.com/sandonwisandon


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|More Details
The quantity of an item has been updated based on available inventory at the selected store.


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New to PoetrySoup? Introduce yourself here. Tell us something about yourself.
Can't find a poem you've read before? Looking for a poem for a special person or an occasion? Ask other member for help.
Ways to improve your poetry. Post your techniques, tips, and creative ideas how to write better.
For poets who want unrestricted constructive criticism. This is NOT a vanity workshop. If you do not want your poem seriously critiqued, do not post here. Constructive criticism only. PLEASE Only Post One Poem a Day!!!
Ask PoetrySoup Members how to do something or find something on PoetrySoup.
Below are poems written by poet
Asha Graham. Click the Next or Previous links below the poem to navigate between poems. Remember, Poetrysoup is an environment of encouragement and growth. Thank you.
List of ALL
Graham poems
Asha Graham
The poem(s) are below...
Bite my lips so I won't cry,
The lump in my throat gets bigger as you lie,
As I 'soak it up' you seemed satisfied,
I'm at a loss for words; tongue tied.
There's no longer a vibrancy between me and you,
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What hurts more than your lying'
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But it'll never amount to the nights I've cried.
Copyright © Asha Graham | Year Posted 2011


text:
Central Penn College introduction to health
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Info concerning Central Penn College introduction to health, and registering for elective studies. Which college degrees give you the best chances of finding employment after graduation? Nursing is one of the fastest-growing job areas, and for good reason. As the population ages, medical care will continue to expand rapidly. Concentrating on Central Penn College introduction to health, for example, you may gain an advantage in job recruiting on-campus. Check with the public health department for course registration deadlines, and healthcare program requirements. Please use the form above to request admissions info for Central Penn College.
The Department of Health and Human Services (HHS) is the United States government's principal agency for protecting the health of all Americans and providing essential human services, especially for those who are least able to help themselves. Agencies of HHS conduct health and social science research, work to prevent disease outbreaks, assure food and drug safety, and provide health insurance.
In addition to administering Medicare and Medicaid, which together provide health insurance to one in four Americans, HHS also oversees the National Institutes of Health, the Food and Drug Administration, and the Centers for Disease Control. The Secretary of Health and Human Services oversees a budget of approximately $700 billion and approximately 65,000 employees. The Department's programs are administered by 11 operating divisions, including 8 agencies in the U.S. Public Health Service and 3 human services agencies. Beyond major requirements within the public health department, you may consider Ozarka College health insurance as a complementary elective to your primary course of study.
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Your Diet After Liposuction
Posted July 18, 2017
Liposuction is a very popular procedure among patients that are looking for a more permanent body contouring solution. However, patients going through this procedure should note that while the results may be life changing it is very important that maintaining these results will rely on the adaptation of a healthy lifestyle. Before and after the … Read More


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Retail price
This product is on offer:
|Carpet ID
|Knotted by hand
|Also known as
|Kashan, Keszan
This carpet is hand knotted/woven; therefore, difference in shape, thickness, pattern and sizes can occur. This applies particularly to nomadic and village carpets.
|Knot density
approx 90 000 knots per m²
You can easily calculate the knot density yourself by counting the number of knots 1 cm down and 1 cm across, and then multiplying these numbers together. Then multiply the total by 10,000 to give the number of knots per m².
Some carpet sellers exaggerate the knot density, so to be sure that you are comparing like for like when it comes to price, it is worth checking the knot density yourself. Most of CarpetVista's carpets have a photograph of the reverse with a ruler, making it easy to examine the knot quality and count the exact knot density before choosing your carpet.
|Old 20-50 years
|Persia / Iran
|Thickness approx
|8 mm
Information about Keshan Patina carpet from Persia / Iran.
CarpetVista Patina is a series of carefully selected carpets. These carpets have a certain wear that does not reduce the impression of them, and is in fact part of their beautiful natural aging process. Common to these selected carpets is that they are at least 40 years old and have an elegance and harmony in both pattern and colour. We select them in order to preserve the great cultural heritage and to allow them to be discovered by a new generation.
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These older oriental carpets are unique works of art that deserve to be preserved and used by several generations to come.
Read more about restoration here...
This carpet is knotted in central Persia. The carpet with a high quality usually have medallion patterns with floral fields, but variations occur. The patterns from the Keshan carpet is often used as originals for machine manufactured carpets. The Keshan carpet is represented in most European courts.
The colors in the carpet will be perceived differently depending on the angle you view it from.


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Franchise Services of North America (FSNA) announced Feb. 17 that Adreca Holdings Corp., a subsidiary of Macquire Capital and has completed one of the scheduled acquisitions of additional on- and off-airport concessions from Hertz Global Holdings Inc.
The additional airport concessions are a result of the acquisition by Adreca of the Advantage Rent-A-Car brand from Hertz in December 2012 and is more fully described here.
As a result of the acquisition, Adreca opened new rental locations at Minneapolis-St. Paul International Airport (MSP) as an in-terminal operation, and has also moved pre-existing off-airport operations at Palm Beach International Airport (PBI), Jacksonville International Airport (JAX), Milwaukee General Mitchell International Airport (MKE) and Norfolk International Airport (ORF) to in-terminal operations.
"We continue to progress with the addition of these new offices and look forward to expand our footprint of in-terminal locations in new destinations to service our existing and new customers," said Thomas P. McDonnell, III, FSNA's CEO and chairman.
Adreca's acquisition of the remaining additional airports and certain other divested assets from Hertz is expected to close in stages continuing in the first and second quarters of 2013. Adreca will be Hertz's sole divestiture partner in conjunction with the dispositions required to be made by Hertz by the consent decree issued by the United States Federal Trade Commission.
As previously described in its first announcement in August, FSNA and Adreca have entered into an agreement to merge a wholly-owned subsidiary of the company with Adreca. FSNA and Adreca are parties to a Management Services Agreement pursuant to which FSNA provides Adreca with certain management services in respect of Advantage and the other assets divested by Hertz pending closing of the Merger.
Completion of this merger is subject to a number of conditions, including TSX Venture Exchange acceptance and approval by the company's shareholders. The merger cannot close until all required approvals are obtained. There can be no assurance that the transaction will be completed as proposed, or at all.
Related news: FSNA's 2012 Year-End Revenue Down from 2011
Read the latest
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Technology that allows cab drivers flexible access to a shared fleet is leveling the playing field for an 89-year-old taxi company.
As advanced safety technologies become even more pervasive on mainstream vehicles, are we creating "stupid drivers?"
In the dialogue surrounding new transportation paradigms, fleets have been off the public radar until recently. Here's what's going on, and a way for you to participate in the conversation.


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- Dependable Septic Pumping in Swartz Creek, MI
- Reliable Swartz Creek Septic Pumping Pros
- Free Estimates on Septic Pumping in Swartz Creek
Septic Pumping in Swartz Creek, MI
Septic pumping in Swartz Creek, MI is a crucial part of caring for your home's comfort and reliable functionality, as you will have to ensure that sludge does not accumulate to the point where your entire system gets backed up. You will find that septic tank pumping Swartz Creek, MI services can prevent your tank from overflowing and polluting the ground beneath the spill. If you want to avoid some very serious complications as well as expensive repair bills later on, it is absolutely imperative that you advantage of these services.
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It is crucial that you pay for professional Swartz Creek septic pumping services at least once every year so you don't encounter any unnecessary complications with your system. Allowing too much sludge to build up in your septic tank over time can lead to some big problems later on down the road, which is precisely why you will have to hire experienced Help You Services professionals for these pumping services. Our team of professionals will inspect your septic tank, measure the levels of sludge and scum in it, and check the pipes in the system as well as inspect the drain field to ensure that the liquid is seeping into the ground just as it should so there won't be any issues.
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Paleo diet increase testosterone
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text:
L-Arginine Alpha-Ketoglutarate (AAKG)
Maintaining a peak level of physical fitness requires a significant level of exertion from the body—a level that some struggle to reach without the aid of certain supplements. L-Arginine Alpha-Ketoglutarate, a chemical found in the body, is one such supplement that can help you push past your plateaus and reach your fitness goals [2].
Arginine Alpha Ketoglutarate (AAKG) and Nitric Oxide
L-Arginine Alpha-Ketoglutarate works by increasing the nitric oxide levels in your blood, resulting in dilation of the blood vessels as you workout [1]. For this reason, the supplement is very popular among body builders and those who enjoy extreme workouts, as dilation of blood vessels allows enhanced delivery of oxygen and nutrients to the muscles during exercise [2, 3]. As a result, many feel it improves weight training by providing muscles with greater strength.
L-Arginine Alpha Ketoglutarate also works throughout various body pathways to not only develop muscle, but also heal serious wounds [2].
L-Arginine Alpha-Ketoglutarate (AAKG) Benefits
L-Arginine Alpha Ketoglutarate provides many benefits to the health conscious. Not only does it help to promote increased blood flow to the working muscle, it also helps to reduce the excess ammonia that results from strenuous physical activity [2]. It has also been shown to boost the health of numerous organs including kidneys, liver, and the gastrointestinal tract [2].
There is also evidence that L-Arginine Alpha Ketoglutarate can help with bacterial infections, yeast infections, and eye problems [2]. Additionally, L-Arginine Alpha Ketoglutarate may help protect the heart from damage resulting from poor blood flow and muscle breakdown that comes with surgery or significant physical trauma [3].
L-Arginine Alpha-Ketoglutarate (AAKG) Dosage
The typically recommended dosage for L-Arginine Alpha Ketoglutarate is 2.6 grams, taken as many as three times per day [4]. It is considered safe for adults when utilized properly, and it is not known to have any significant side effects [2, 3]. There are no known supplement or food interactions with this product [3].
CAS Number:Â 16856-18-1
Synonyms:Â AAKG, Arginine AKG
- http://www.ncbi.nlm.nih.gov/pubmed/21813912
- http://www.webmd.com/vitamins-supplements/ingredientmono-144-alpha-ketoglutarate.aspx?activeingredientid=144&activeingredientname=alpha-ketoglutarate
- http://www.uofmhealth.org/health-library/hn-4452006
- http://www.uofmhealth.org/health-library/hn-4395009#hn-4395009-why-use


text:
Legal Status
02/14/1994 Issue
Document Issue Table of Contents
Agricultural Marketing Service
Uncategorized Document
Revision of Cottonseed Sampler License Procedures
Agriculture Department
- See Agricultural Marketing Service 1
- See Commodity Credit Corporation 1
- See Cooperative State Research, Education, and Extension Service 1
- See Farm Service Agency 1
Uncategorized Document
Celery Grown in Florida; Decreased Expenses and Assessment Rate
Cost Containment and Vulnerability
Foreign Donation of Agricultural Commodities
Heat-Processing Procedures, Cooking Instructions, and Cooling, Handling and Storage Requirements for Uncured Meat Patties
Milk in the Central Arizona Marketing Area; Extension of Time for Filing Exceptions on Proposed Amendments to Tentative Marketing Agreement and Order
Milk in the Georgia Marketing Area; Suspension of Certain Provisions of the Order
Milk in the Southwest Plains Marketing Area; Proposed Suspension of Certain Provisions of the Order
Procedures for Appealing Product Retentions
Centers for Disease Control and Prevention
Uncategorized Document
Workshop on Community Involvement in Public Health Service Activities and Research at Department of Energy Sites
Commerce Department
Uncategorized Document
Marine Mammals; Permits
New England Fishery Management Council; Public Meeting
Pacific Coast Groundfish Fishery; Public Hearings
Pacific Fishery Management Council; Meeting
United States-Canada Free-Trade Agreement, Article 1904 Binational Panel Reviews; Notice of Decision of Panel
Western Pacific Crustacean Fisheries
Commodity Credit Corporation
Uncategorized Document
1994 Marketing Quota and Price Support for Flue-Cured Tobacco
Cooperative State Research, Education, and Extension Service
Uncategorized Document
Special Research Grants Program, Aquaculture Research; Solicitation of Applications; Correction
Defense Department
Uncategorized Document
Availability for the Final Programmatic Environmental Impact Statement for Joint Training Exercise Roving Sands at Fort Bliss, Texas and New Mexico and White Sands Missile Range, New Mexico
Employment and Training Administration
Uncategorized Document
Electrode Corp. Chardon, OH; Dismissal of Application for Reconsideration
Energy Department
Uncategorized Document
2322117 Nova Scotia Limited; Errata to Determination of Exempt Wholesale Generator Status
EEA I, L.P.; Filing
El Paso Natural Gas Co., et al.; Natural Gas Certificate Filings
El Paso Natural Gas Co.; Request Under Blanket Authorization
Florida Gas Transmission Co.; Request Under Blanket Authorization
Gas Transport, Inc.; Application
KN Interstate Gas Transmission Co.; Technical Conference
Lawrence E. and Veronica P. Smith; Availability of Final Environmental Assessment
Mojave Pipeline Co.; Request Under Blanket Authorization
Montana Power Co.; Informal Settlement Conference
Natural Gas Pipeline Company of America; Intent To Prepare an Environmental Assessment for Proposed Amarillo Upgrade Project and Request for Comments
Natural Gas Pipeline Company of America; Technical Conference
Northern Natural Gas Co.; Proposed Changes in FERC Gas Tariff
Sho-Me Power Corp.; Availability of Draft Environmental Assessment
Western Gas Interstate Co.; Tariff Filing
Environmental Protection Agency
Uncategorized Document
Forum on State and Tribal Toxics Action (FOSTTA); Coordinating Committee and Projects; Open Meetings
Farm Service Agency
Uncategorized Document
1994 Marketing Quota and Price Support for Flue-Cured Tobacco
Federal Aviation Administration
Uncategorized Document
Airport Certification; Amendment of the Compliance Date for Signs Identifying Taxiing Routes; Final Rule DEPARTMENT OF TRANSPORTATION
Airworthiness Directives; Canadair Model CL-215-1A10 Series Airplanes
Airworthiness Directives; Jetstream Aircraft Limited Model 4101 Airplanes
Federal Communications Commission
Uncategorized Document
Cable Television Act of 1992
Federal Election Commission
Uncategorized Document
Federal Energy Regulatory Commission
Uncategorized Document
New York State Electric & Gas Corp., et al.; Electric Rate and Corporate Regulation Filings
Fish and Wildlife Service
Uncategorized Document
Letters of Authorization To Take Marine Mammals
Food and Drug Administration
Uncategorized Document
Regulations Review Program Under Executive Order 12866; Correction
Requirements for Accrediting Bodies of Mammography Facilities and Quality Standards and Certification Requirements for Mammography Facilities; Corrections
Health and Human Services Department
Uncategorized Document
Advisory Council for the Elimination of Tuberculosis; Meeting
Idaho National Engineering Laboratory Environmental Dose Reconstruction Project; Public Meetings
Medical Devices; Classification of Temporomandibular Joint Implants
Medicare Program; Standards for Quality of Water Used in Dialysis and Revised Guidelines on Reuse of Hemodialysis Filters for End-Stage Renal Disease Patients
National Committee on Vital and Health Statistics (NCVHS) subcommittee on Health Statistics for Minority and Other Special Populations: Meeting
Suspension Lifted; Laboratory Again Meets Minimum Standards to Engage in Urine Drug Testing for Federal Agencies
Housing and Urban Development Department
Uncategorized Document
Federal Property Suitable as Facilities to Assist the Homeless; Notice DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT
Interior Department
Uncategorized Document
Box Elder Resource Management Plan, Bear River Resource Area, UT
International Trade Administration
Uncategorized Document
Industrial Phosphoric Acid From Israel; Final Results of Antidumping Duty Changed Circumstances Review
Interstate Commerce Commission
Uncategorized Document
Illinois Central Railroad Company; Abandonment Exemption; in Perry and Randolph Counties, IL
Los Angeles County Metropolitan Transportation Authority Abandonment ExemptionBetween Arcadia and Los Angeles, CA, et al.
Labor Department
Uncategorized Document
Determinations Regarding Eligibility To Apply for Worker Adjustment Assistance and NAFTA Transitional Adjustment Assistance
Investigations Regarding Certifications of Eligibility To Apply for Worker Adjustment Assistance
Koch Gathering Systems, Inc., (KGSI) Duncan, OK; Dismissal of Application for Reconsideration
Minimum Wages for Federal and Federally Assisted Construction; General Wage Determination Decisions
Petitions for Modification
Management and Budget Office
Uncategorized Document
Budget Rescissions and Deferrals; Notice OFFICE OF MANAGEMENT AND BUDGET
National Archives and Records Administration
Uncategorized Document
NARA Research Room Hours
National Archives at College Park, MD; Location of Records and Hours of Use
National Highway Traffic Safety Administration
Uncategorized Document
Federal Motor Vehicle Safety Standards; Air Brake Systems; Air Applied, Mechanically Held Brake Systems
National Oceanic and Atmospheric Administration
Uncategorized Document
Groundfish of the Gulf of Alaska
National Science Foundation
Uncategorized Document
Special Emphasis in Biological Sciences; Notice of Meetings
Special Emphasis Panel in Mathematical Sciences; Notice of Meetings
Nuclear Regulatory Commission
Uncategorized Document
Detroit Edison Co.; Environmental Assessment and Finding of No Significant Impact
Final Memorandum of Understanding Between the Nuclear Regulatory Commission and the State of Kansas
Indiana Michigan Power Co.; Donald C. Cook Nuclear Plant, Units 1 and 2; Environmental Assessment and Finding of No Significant Impact
Licensing Support System Advisory Review Panel; Renewal
Renewal of Charter for Nuclear Safety Research Review Committee
Tennessee Valley Authority; Browns Ferry Nuclear Plant Units 1 and 3
Resolution Trust Corporation
Uncategorized Document
Coastal Barrier Improvement Act; Property Availability; Parcel A/ North Ranch at Ken Caryl, Jefferson County, CO, et al.
Securities and Exchange Commission
Uncategorized Document
First Transamerica Life Insurance Company, et al.; Application for Exemption
Forms Under Review by the Office of Management and Budget
Lincoln Benefit Life Co., et al.; Application for Exemption
Self-Regulatory Organizations; Filing and Immediate Effectiveness of Proposed Rule Change by the Chicago Stock Exchange, Inc. to Extend a Credit Against Dues Given to Registered Market Makers for Trading the Chicago Stock Basket
Self-Regulatory Organizations; Filing and Order Granting Partial Temporary Accelerated Approval to Proposed Rule Change by American Stock Exchange, Inc. Relating to a Pilot Program for Execution of Odd- lot Market Orders
Self-Regulatory Organizations; Filing of Proposed Rule Change by the Boston Stock Exchange, Inc. Relating to the Insider Trading and Securities Fraud Enforcement Act of 1988
Self-Regulatory Organizations; Notice of Filing and Immediate Effectiveness of Proposed Rule Change by the Chicago Stock Exchange, Inc. to Establish a Temporary Policy Relating to the Automatic Execution Feature of the Midwest Automated Execution System.
Self-Regulatory Organizations; Notice of Filing of Proposed Rule Change by the Philadelphia Stock Exchange, Inc. Regarding Unbundling of PACE Orders
Self-Regulatory Organizations; Pacific Stock Exchange, Inc.; Order Approving Proposed Rule Change and Notice of Filing and Order Granting Accelerated Approval of Amendment No. 1 to the Proposed Rule Change Relating to Waiver of Maximum Bid/Ask Differentials in Individual Equity Options
Smith Barney Shearson Income Trust, et al.; Application for Exemption
Transamerica Occidental Life Insurance Co., et al.; Application for Exemption
State Department
Uncategorized Document
Property Claims Against the Government of Hungary
Transportation Department
Treasury Department
Uncategorized Document
Implementation of Public Law 103-159, Including the Brady Handgun Violence Prevention Act
Implementation of Public Law 103-159, Including the Brady Handgun Violence Prevention Act; Rule and Proposed Rule DEPARTMENT OF THE TREASURY
United States Information Agency
Uncategorized Document
Advisory Panel on Radio Marti and TV Marti; Meetings
Veterans Affairs Department
Uncategorized Document
Procedural Due Process and Appellate Rights
VA Acquisition Regulation: Loan Guaranty and Vocational Rehabilitation and Counseling Programs
Other Documents
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