University of, Interfaith Medical Center-Brooklyn (N01-HB-97085); Coronary Artery Risk in Young Adults (CARDIA): University of Alabama at Birmingham (N01-HC-48047)
September 2011. 2011

Of America
J Paul Geriatric Medicine
; Sticht
C-T Liu
M K Garnaas
A Tin
A Kottgen
N Franceschini
University of, Interfaith Medical Center-Brooklyn (N01-HB-97085); Coronary Artery Risk in Young Adults (CARDIA): University of Alabama at Birmingham (N01-HC-48047)

PLoS Genetics | www.plosgenetics.org
Baltimore, Maryland; University of Texas Houston; University of California Davis; New England Medical Center; Kaiser Foundation Research Institute; University of California Irvine; Boston University; Johns191002264September 2011. 2011Received January 31, 2011; Accepted July 5, 2011;United States of America, 14 Medical College of Georgia, Augusta, Georgia, United States of America, 15 University of California San Francisco, San Francisco, California, United States of America, 16 Cardiovascular Health Research Unit, United States of America, 19 United States of America, 31 General Internal Medicine, University of California San Francisco, San Francisco, California, United States of America, 32 Loyola University, Maywood, Illinois, United States of America, 33 Division of Kidney, Urologic, and Hematologic Diseases, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, United States of America, 37 National Heart, Blood, and Lung Institute's Framingham Heart Study and the Center for Population Studies, Framingham, Massachusetts, United States of America, Editor: This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The following nine parent studies have contributed parent study data, ancillary study data, and DNA samples through the Massachusetts Institute of Technology -Broad Institute (N01-HC-65226) to create this genotype/phenotype data base for wide dissemination to the biomedical research community: University of Mississippi (N01-HB-73005), St. Luke's Hospital-New York (N01-HB-73006), Alta Bates-Herrick Hospital (N01-HB-97051), Columbia University (N01-HB-97058), St. Jude's Children's Research Hospital (N01-HB-97066), Research Foundation of the State University of New York-Albany (N01-HB-97068, N01-HB-97069), New England Research Institute (N01-HB-97073) Data analyses for the HANDLS study utilized the high-performance computational capabilities of the Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD. (http://biowulf.nih.gov). 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. * . These authors contributed equally to this work. " These authors were joint senior authors on this work.
Chronic kidney disease (CKD) is an increasing global public health concern, particularly among populations of African ancestry. We performed an interrogation of known renal loci, genome-wide association (GWA), and IBC candidate-gene SNP association analyses in African Americans from the CARe Renal Consortium. In up to 8,110 participants, we performed metaanalyses of GWA and IBC array data for estimated glomerular filtration rate (eGFR), CKD (eGFR ,60 mL/min/1.73 m 2 ), urinary albumin-to-creatinine ratio (UACR), and microalbuminuria (UACR .30 mg/g) and interrogated the 250 kb flanking region around 24 SNPs previously identified in European Ancestry renal GWAS analyses. Findings were replicated in up to 4,358 African Americans. To assess function, individually identified genes were knocked down in zebrafish embryos by morpholino antisense oligonucleotides. Expression of kidney-specific genes was assessed by in situ hybridization, and glomerular filtration was evaluated by dextran clearance. Overall, 23 of 24 previously identified SNPs had directionconsistent associations with eGFR in African Americans, 2 of which achieved nominal significance (UMOD, PIP5K1B). Interrogation of the flanking regions uncovered 24 new index SNPs in African Americans, 12 of which were replicated (UMOD, ANXA9, GCKR, TFDP2, DAB2, VEGFA, ATXN2, GATM, SLC22A2, TMEM60, SLC6A13, and BCAS3). In addition, we identified 3 suggestive loci at DOK6 (p-value = 5.3610 27 ) and FNDC1 (p-value = 3.0610 27 ) for UACR, and KCNQ1 with eGFR (p = 3.6610 26 ). Morpholino knockdown of kcnq1 in the zebrafish resulted in abnormal kidney development and filtration capacity. We identified several SNPs in association with eGFR in African Ancestry individuals, as well as 3 suggestive loci for UACR and eGFR. Functional genetic studies support a role for kcnq1 in glomerular development in zebrafish. , N01-HC-95100); Framingham Heart Study (FHS): Boston University (N01-HC-25195, R01HL092577-01A1, RO1 HL076784, R01 AG028321); Jackson Heart Study (JHS): Jackson State University (N01-HC-95170), University of Mississippi (N01-HC-95171), Tougaloo College (N01-HC-95172); Multi-Ethnic Study of Atherosclerosis (MESA): University of Washington (N01-HC-95159), Regents of the University of California (N01-HC-95160), Columbia University (N01-HC-95161), Johns Hopkins University (N01-HC-95162, N01-HC-95168), University of Minnesota (N01-HC-95163), Northwestern University (N01-HC-95164), Wake Forest University (N01-HC-95165), University of Vermont (N01-HC-95166), New England Medical Center (N01-HC-95167), Harbor-UCLA Research and Education Institute (N01-HC-95169), Cedars-Sinai Medical Center (R01-HL-071205), University of Virginia (subcontract to R01-HL-071205); Sleep Heart Health Study (SHHS): Johns Hopkins University ((WG). These authors contributed equally to this work." These authors were joint senior authors on this work.

Introduction

Chronic kidney disease (CKD) affects approximately 15% of U.S adults [1]. Due in part to increasing rates of diabetes and obesity, the prevalence of CKD continues to rise [1]. Marked variability in the incidence of CKD suggests that factors other than diabetes and hypertension contribute to its etiology [2]. Recently, we identified 16 genomic loci associated with estimated glomerular filtration rate (eGFR), a primary measure of CKD, using genome-wide association studies (GWAS) in a combined sample of 67,093 European ancestry individuals from the CKDGen consortium [3,4]. However, these loci only account for 1.4% of the eGFR variation, suggesting that additional loci remain to be identified [5].

African American ethnicity is a well-established risk factor for CKD, and rates of end-stage renal disease (ESRD) are up to 4fold higher among African Americans as compared to European Americans [6]. Several prior studies, including the FIND consortium, have performed linkage analysis of diabetic ESRD [7][8][9]. Recent genome-wide admixture mapping studies identified genetic variation in the regions of MYH9 and APOL1 on chromosome 22 that may explain up to 70% of the differences in ESRD rates between European and African Americans [10][11][12]. While this finding has great implications for ESRD, recent evidence also suggests that African Americans progress faster from moderately decreased kidney function to ESRD, spending less time in the recognized earlier stages of CKD [13,14]. The identification of additional risk factors for CKD, including genetic loci in association with eGFR, may help to advance our understanding of the underpinnings of CKD in African Americans.

Thus, the goal of this study was to uncover loci for kidney traits in African ancestry participants in the Candidate-gene Association Resource (CARe) Consortium. CARe is a consortium of 9 studies which form a combined population of approximately 40,000 African and European Americans genotyped on the IBC array [15] and approximately 8,000 African American participants genotyped using a GWAS platform. We aimed to interrogate known regions previously associated with eGFR in European Ancestry populations [3,4], as well as to perform discovery analyses in African ancestry populations. In order to gain further insight into the functional implications of our associations, genes at or near loci of interest were knocked down by morpholino injection in zebrafish, and kidney gene expression and function were investigated using in situ hybridization and glomerular filtration assays.

Results

Study sample characteristics are shown in Table 1 and Table  S1. Overall, 7,382 participants of African ancestry had genomewide SNP data and phenotype information on eGFR and CKD; and 5,569 had data on UACR and albuminuria. For the IBC chip analysis, 8,110 participants of African ancestry had data on eGFR and CKD and 5,995 had data on UACR and albuminuria.

Interrogation of Known Renal Function Loci in African American Participants

Genome-wide association analyses were conducted for eGFR, CKD, UACR, and albuminuria. Quantile-quantile and Manhattan plots are displayed in Figure S1 and Figure S2, respectively; lambdas ranged from 1.0 to 1.02.

We examined previously published loci in association with eGFR in participants of European Ancestry [3,4,16] in our African American GWAS (Table 2). In 23 of 24 SNPs, the directions of the beta coefficients for eGFR were identical (p-value = 1.4*10 26 ) in CKDGen and CARe (rs6420094 at SLC34A1 was the only exception), even though only 2 of the SNPs achieved nominal significance (rs4293393 at the UMOD locus [p = 0.01] and rs4744712 at the PIP5K1B locus [p = 0.003]). We further interrogated the 250 kb flanking regions around each of these 24 SNP to identify the top SNP in CARe; statistical

Author Summary

Chronic kidney disease (CKD) is an increasing global public health problem and disproportionately affects populations of African ancestry. Many studies have shown that genetic variants are associated with the development of CKD; however, similar studies are lacking in African ancestry populations. The CARe consortium consists of more than 8,000 individuals of African ancestry; genome-wide association analysis for renal-related phenotypes was conducted. In cross-ethnicity analyses, we found that 23 of 24 previously identified SNPs in European ancestry populations have the same effect direction in our samples of African ancestry. We also identified 3 suggestive genetic variants associated with measurement of kidney function. We then tested these genes in zebrafish knockdown models and demonstrated that kcnq1 is involved in kidney development in zebrafish. These results highlight the similarity of genetic variants across ethnicities and show that cross-species modeling in zebrafish is feasible for genes associated with chronic human disease.

significance was determined based on a locus-specific Bonferroni correction (see statistical methods for more detail). Of the 24 SNPs with the lowest p-value in each of these regions identified in CARe African Americans, we were able to replicate 12 loci (UMOD, ANXA9, GCKR, TFDP2, DAB2, VEGFA, ATXN2, GATM, SLC22A2, TMEM60, SLC6A13, and BCAS3) in independent samples of 4358 participants (Table 3, Figure S3). We also interrogated the CARe CKD GWAS results at the chromosome 22 MYH9/APOL1 locus ( Figure S4); however, none of the G1 haplotype SNPs in APOL1 was present in our GWAS dataset. The lowest p-value was observed for rs739097 (MAF 0.36, p = 0.00179), which was in weak LD with the previously described rs4821480 in MYH9 (r2 0.03, D9 0.38 as determined by Hapmap Release 22 YRI phase 2) [10].

Genome-Wide Association Analyses for Renal Indices

We performed discovery analyses using GWAS and the IBC chip array; p-value thresholds for discovery were p,5.0*10 28 for GWAS and p,2.0*10 26 for the IBC array [17]. We observed no genome-wide signals; instead, we carried forward 8 SNPs from GWAS that had a p-value,5*10 26 in Stage 1 for replication; results for these SNPs are presented in Table 4.

Replication was performed in 4,358 participants of African ancestry for eGFR and 2,110 for UACR. Characteristics of the replication samples are shown in Table 1. Results from discovery, replication, and the combined GWAS analyses in African Americans are presented in Table 4 (imputation scores can be found in Table S2). Of the 8 SNPs from GWAS carried forward to replication, the combined Stage 1 + Stage 2 p-value was 5.3*10 27 for the association between UACR and rs4555246 in DOK6 ( Figure 1a) and the combined p-value was 2.9*10 27 for association between UACR and rs2880072 in FNDC1 (Figure 1b). Due to modest power of our replication set and the possibility that loci relevant for CKD may be similar across ethnic groups, we also attempted replication of the association between UACR and these two loci in the CKDGen consortium, a large consortium of participants of European ancestry (n = 31,580 with UACR [18]). The beta coefficient for rs4555246 in DOK6 in the CKDGen data was direction-consistent, although the p-value for this SNP was non-significant (p = 0.44). Recognizing that regions, but not necessarily specific tagging SNPs, may replicate across ethnicities, we interrogated the 250 kb flanking region of rs4555246 (n = 31 independent SNPs). The SNP with the lowest p-value was rs11151530 (MAF 0.17, p = 0.008), which did not meet the   Bonferroni-corrected threshold of p = 0.0016 (0.05/31). For FNDC1, the beta coefficient for the lead SNP was directionconsistent in CKDGen (beta = 20.0196), although the p-value was not significant (p = 0.053). Interrogating the 250 kb flanking region (n = 7 independent SNPs) revealed that rs7758822 had the lowest p-value at p = 0.002 (MAF 0.12), which met the Bonferroni-corrected threshold of p = 0.007 (0.05/7).

IBC Chip Analyses for Renal Function Indices

In European Americans, we confirmed several known loci for eGFR/CKD [3,4,16]; no novel genome-wide significant associations (defined as p,2.0*10 26 ) were identified (Table S3). In African Americans, three loci for eGFR/CKD were brought forward to replication (Table 4). Of these, we observed nominal replication for rs7111394 in the KCNQ1 gene (Figure 2a, Stage 1 + Stage 2 p-value = 3.6*10 26 ). This SNP was not identified in the European American analysis as it was monomorphic in this population. Thus, we interrogated the 250 kb flanking region around this SNP (93 independent SNPs) in the CARe IBC European ancestry participants. The SNP with the lowest p-value was rs81204 (Figure 2b, MAF 0.16, p-value = 0.00036), which exceeded the corrected regional-specific threshold of 0.000538 (0.05/93).

Functional Studies of New Loci for Renal Function

To further understand the impact of the three new loci on kidney function and to bolster confidence in the sub-genome-wide statistical associations that we observed, we performed morpholino knockdown of kcnq1, dok6, and fndc1 in zebrafish embryos (see methods).

In situ hybridization for well-established renal markers was used to assess specific anatomic regions of the kidney during development. Kcnq1 knockdown caused abnormalities in glomerular gene expression in the majority of injected embryos, as shown by the global kidney marker pax2a at 48 hours post fertilization (hpf) (see Table 5, Figure 3F). Assessment of the podocyte markers wt1a at 24 hpf and nephrin at 48 hpf revealed similar, glomerularspecific effects. In contrast, the tubular markers slc20a1a and slc12a3 showed no significant changes. Analysis of glomerular architecture at 120 hpf by electron microscopy did not demonstrate significant differences between control and kcnq1 morphant embryos ( Figure S5), possibly due to diminished morpholino efficacy at this later stage. Knockdown of dok6 and fndc1 did not result in generalized edema or significant developmental abnormalities of the kidney (Table 5, Figure S6).

To determine whether differences in gene expression resulted in altered kidney function, we evaluated glomerular filtration in kcnq1 morphant embryos by assessing the kidney's capacity to retain fluorescent dextran. Fluorescently labeled high-molecular weight dextran has been used in zebrafish to directly visualize functional glomerular integrity [19]. Control or kcnq1 morphant embryos were equally loaded with rhodamine-labeled 10,000 MW dextran by injection into the cardiac sinus venosus at 48 hpf ( Figure S7A, S7D). Dextran clearance was assessed by overall fluorescence in the embryo at 72 and 96 hpf ( Figure S7B, S7C, S7E, S7F) and presence of red fluorescence in the green fluorescent tubules of cdh17:GFP reporter embryos. Kcnq1 morphant embryos exhibited decreased fluorescence by 96 hpf, indicative of increased dextran clearance compared to control embryos ( Figure 4C, 4F). Time course analysis confirmed equal loading and progressive loss of fluorescence over 48 hours in kcnq1 morphants. These results suggest that loss of kcnq1 causes decreased glomerular retention of macromolecules. The majority of embryos with increased loss of dextran fluorescence also exhibited generalized edema ( Figure 4A, 4B, 4D, 4E, 4G), which has been previously linked to kidney dysfunction in zebrafish [20,21].

Cross-Trait Associations

rs4555246 in DOK6 was associated with albuminuria (OR = 1.20, p = 0.001) but not with eGFR (p = 0.23) or CKD (p = 0.92; Table  S4). Similarly, rs2880072 in FNDC1 was associated with albuminuria (OR = 0.83, p = 4.9*10 25 ) but not with eGFR or CKD. Although rs7111394 in KCNQ1 was associated with eGFR, it was not significantly associated with CKD (direction consistent OR 1.07, p = 0.36), UACR, or albuminuria. These findings underscore the specificity of the genetic underpinnings of eGFR as compared to albuminuria.

Stratification by Hypertension and Diabetes Status

All replicating loci were stratified by hypertension and diabetes status (Table S5). We observed nominal significance for nearly all loci in the non-diabetes and non-hypertension strata, and many loci retained statistical significance in the diseased strata despite smaller sample sizes.

Discussion

In more than 8,000 African Americans, for 24 known renal susceptibility loci identified in European ancestry consortia for eGFR and CKD, we have identified the most significant SNP in African Americans, of which several showed evidence of confirmation or replication. In addition, we performed discovery analyses using GWAS and a candidate-gene based array, and uncovered 3 suggestive loci, including KCNQ1 in association with eGFR and DOK6 and FNDC1 in association with UACR. Finally, we show that loss of function of kcnq1 leads to abnormalities in glomerular gene expression and function during zebrafish development.

Despite having one of the largest GWAS datasets in African Americans, both our discovery and replication samples were of relatively modest size. Thus, we have focused primarily on interrogating regions previously identified in well-powered European ancestry meta-analyses for renal function in order to reduce our genome-wide penalty for multiple testing. While we have uncovered 3 suggestive loci, the strength of the statistical significance is only suggestive. Because of this, we have corroborated our findings with functional data from zebrafish, providing compelling evidence for the role of KCNQ1 in renal abnormalities.

Several prior genome-wide studies using GWAS or admixture approaches have identified loci for renal function in participants of European [3,4,16] and African ancestry [10,11]. These studies have identified loci for eGFR, CKD, and non-diabetic ESRD; however, few novel discoveries and relatively limited replication have been made for renal function indices in African Americans. African ancestry populations have smaller LD blocks and hence more genetic diversity, thus offering the potential opportunity to fine map genomic regions and to identify novel genomic regions for indices of renal function.

The genes located closest to our three suggestive loci are KCNQ1 in association with eGFR, as well as FNDC1 and DOK6 in association with UACR. KCNQ1 on chromosome 11 encodes for the potassium voltage-gated channel, KQT-like subfamily, member 1 (KvLQT1). The lead SNP identified among the African ancestry participants is located in an intron of KCNQ1 and is flanked by two recombination hotspots. The kcnq1 protein is abundantly expressed in the brush border membrane of renal proximal tubule cells, where it interacts with other K + channels (KCNE) to mediate net K + secretion. Loss of Kcnq1 in the mouse leads to impaired Na+ absorption with increased glucose load, however, developmental abnormalities have not been reported [22]. Variants in KCNQ1 have been associated with type 2 diabetes [23] and beta cell function [24], due to its role as a potassium channel in the pancreatic beta cells. A recent study of individuals  with diabetes from Japan identified variants in the KCNQ1 gene in association with diabetic nephropathy [25]. Our lead SNP is not in LD with this signal in either European or African ancestry individuals. Importantly, we observe a strong and significant association with KCNQ1 among participants without diabetes, and our functional work suggests an independent association of kcnq1 in renal development. Thus, it is unlikely that our observed associations are solely due to the association of KCNQ1 with diabetes. The DOK6 gene on chromosome 18 encodes a member of a family of intracellular adaptor proteins that have a role in the assembly of multimolecular signaling complexes. It is expressed in the human kidney as well as the ureteric buds of the murine developing kidney [26]. Although a direct role in renal disease has not been described so far, DOK6 interacts with Ret [26], the ablation of which resulted in kidney agenesis in model systems [27]. The lead SNP we identified is located in an intron of a very circumscribed region of the gene. DOK6 has been recently found in a GWAS for osteoporosis [28], although our lead SNP is not in LD with this variant.

The fibronectin type III domain containing 1 (FNDC1) gene on chromosome 6 is also expressed in the kidney; the lead SNP is located nearly 100 kb downstream of the gene. Little is known about the function of this gene to date; previous studies found that FNDC1 may mediate G protein signaling and have a role in hypoxia-induced apocytosis of cultured ventricular cardiomyocytes [29,30].

Zebrafish have been extensively used to study principal pathways of kidney development and function [31,32]. More recently, adult models of kidney injury have been developed [33]. For example, targeted knockdown of a prolyl 4-hydroxylase resulted in kidney dysfunction with edema and changes in podocytes and Bowman's capsule [34]. In addition, a zebrafish model of human nephrotic syndrome was generated by plce1 knockdown after positional cloning of this gene in affected siblings, similarly resulting in cardiac edema and functional abnormalities [35]. Here, we use morpholino knockdown to demonstrate that loss of kcnq1 leads to changes in global morphology and gene expression abnormalities during zebrafish kidney development. Furthermore, in vivo fluorescence-based functional analysis of zebrafish glomerular filtration capacity demonstrated decreased retention of macromolecules, as previously demonstrated for other genes affecting glomerular integrity [19,20]. Interestingly, KCNQ1 was identified in association with eGFR, and knockdown of kcnq1 in zebrafish predominantly causes glomerular gene expression and filtration defects. These results suggest that genes associated with polygenic chronic conditions can produce developmental phenotypes when knocked down in vivo.

Our analytic approach was complemented by a large-scale candidate gene analysis using the IBC SNP Chip array [15] and replication of our findings in an additional 4358 African American individuals. By using participants from predominantly populationbased cohorts, we were able to study disease initiation. An important focus of our multi-ethnic samples allowed us to explore allelic heterogeneity, and support the notion that genomic risk regions are observed across ethnicities. Our replication was derived from in silico samples, which allowed for the adjustment for principle components where necessary. Finally, we were able to corroborate our results in the zebrafish, a model organism for studying vertebrate kidney development.

While the advantage of the zebrafish model is to rapidly assess gene function during development, the role of genes in aging and chronic disease cannot be modeled by transient morpholino knockdown. eGFR was estimated using the MDRD study equation, as obtaining gold-standard measures of GFR in large population-based samples is not feasible. Serum creatinine and albuminuria were measured at a single-point in time, which may misclassify certain individuals and bias our results toward the null. Even with imputation, the coverage of the genome in African Americans is not comprehensive, thus limiting our statistical power. With respect to the chromosome 22 MYH9/APOL1 locus, neither the E1 haplotype in MYH9 nor the G1 haplotype in APOL1 were in our GWAS dataset, limiting our ability to examine this region in association with more modest CKD phenotypes. Finally, our power was low for CKD to detect SNPs with odds ratios of 1.2, the largest odds ratio detected in other GWAS for CKD [4].

We identified several SNPs in association with eGFR in African Ancestry individuals, as well as 3 suggestive loci for UACR and eGFR. Functional genomic studies support a role for kcnq1 in glomerular development and function in zebrafish.

Methods

Renal Function Indices

Serum creatinine was measured as described in Text S1. Serum creatinine was calibrated to NHANES in all studies (including replication cohorts) to account for between-laboratory variation as previously described [3,36,37]. Glomerular filtration rate was calculated based on serum creatinine (eGFR) with the Modification of Diet in Renal Disease (MDRD) equation [38]. We defined chronic kidney disease (CKD) as eGFR ,60 ml/min/1.73 m 2 in accordance with the National Kidney Foundation guidelines; CKD was based on a single serum creatinine measurement as described in Text S1. Urinary albumin to creatinine ratio (UACR, mg/g) was computed as described in Text S1; microalbuminuria was defined as UACR .17 mg/g [men] and .25 mg/g [women].

Covariate Definitions

We defined diabetes as fasting glucose $126 mg/dl, self-report, or pharmacologic treatment. Similarly, hypertension was defined as systolic blood pressure $140 mm Hg, diastolic blood pressure $90 mm Hg, or pharmacologic treatment.

Genotyping Platforms for Genome-Wide Genotype and Imputation

For the present study, the CARe consortium genotyped the IBC SNP chip [15] in 23767 European Americans and 8110 African Americans, as well as the Affymetrix 6.0 chip in 7382 African Americans. The IBC array contains nearly 50,000 SNPs across 2,000 loci. SNPs were selected using a tagging approach among populations represented in HapMap and the SeattleSNPs project. The array was designed to focus on candidate loci related to cardiovascular disease and its risk factors. More details can be found in the design paper [15]. Table S1 details the genotyping that was conducted. For the CARe study cohorts, quality control and imputation were conducted centrally using MACH 1.0.16 (http://www.sph.umich.edu/csg/abecasis/MaCH/). Imputation results were filtered using thresholds RSQ_HAT value of 0.3 and minor allele frequency 0.01. Fractional counts between 0 and 2 were coded for the imputed genotypes in order to estimate the number of copies of a pre-specified allele. For European samples, the CEU population from HapMap 2 (2.54 million SNPs) was used as the reference panel.

For African American samples, a 1:1 combined HapMap 2 CEU+YRI reference panel was used. This panel includes SNPs that were present in both populations, as well as SNPs segregating in one panel and monomorphic and nonmissing in the other (2.74 million altogether). Since the African American samples were genotyped for both the Affymetrix 6.0 and IBC arrays, we were able to analyze imputation performance at non-genotyped SNPs. The use of the CEU+YRI panel resulted in an allelic concordance rate of ,95.6%, calculated as 1 -1/2*|imputed_dosage -chip_dosage| for imputation on the Affymetrix chip. This is similar to rates obtained from African ancestry participants imputed using HapMap 2 YRI individuals [39].

Statistical Methods for Discovery Stage

Trait creation details are described above (Renal Function Indices). Performed centrally but within each individual study, genome-wide association analyses and IBC chip analyses of natural log-transformed eGFR, UACR, CKD, and MA were conducted using linear and logistic regression with an additive genetic model. We adjusted for age, sex and study site (when applicable) and the first 10 principal components; relatedness was accounted for when necessary using linear mixed effect (LME) models for eGFR and UACR and logistic regression via generalized estimating equations (GEE) for CKD and MA. Additional details regarding the discovery cohorts are in Text S1.

Principal Components Analysis

Principal components were generated using EIGENSTRAT [40] within each study using the CARe African ancestry Affy6.0 genotype data. Two reference populations were included in the principal component analysis of African Americans: 1,178 European Americans from a multiple sclerosis GWA study (from Dr. Phil de Jager and colleagues), and 756 Nigerians from the Yoruba region from a hypertension GWA (provided by Dr. Richard Cooper and colleagues). Importantly, these two underwent extensively quality control procedures to remove population outliers using PCA. Ten principal components were generated for each study and used to adjust for population substructure.

Meta-Analysis

We performed fixed-effect meta-analyses of the IBC chip and genome-wide association data using the inverse-variance weighted approach in METAL (http://www.sph.umich.edu/csg/abecasis/ Metal/index.html). Genomic control correction was applied after calculating the inflation factor lambda (l) within each individual study and after the genome-wide association meta-analysis was performed.

The standard threshold of p,5610 28 for genome-wide significance in the genome-wide association and p,2.0610 26 in the IBC chip analyses was used. The rationale for the p-value threshold used for the IBC chip is based on an empiric test of the number of independent loci (,25,000) that appear on the IBC array [17]. We selected independent SNPs (pairwise r 2 ,0.2) at each locus for replication.

The R software (v2.9.0) was used for data management, statistical analyses and graphing.

Interrogation of CKDGen Loci in CARe

We developed a set of criteria to validate the lead SNPs and interrogate regions around each of the loci that were previously reported among European ancestry (EA) participants [3,4] in our African American (AA) CARe samples. For each lead SNP in EA, we looked-up the respective association result with eGFR in AA. To accommodate the difference of LD structure and possible allelic heterogeneity across different ethnicities, we then interrogated the 250 kb flanking region around each lead SNP to determine whether there exist other SNPs with stronger associations with the outcome. We used the following criteria to identify the top AA SNP: 1) the SNP with the smallest association p-value within the region; 2) MAF .0.03; 3) location of the AA lead SNP within the same recombination block of the lead EA SNP, where the recombination block was defined as a 20% recombination rate. The statistical significance of each identified SNP was evaluated using a region-specific Bonferroni correction. We determined the number of independent SNPs based on the variance inflation factor (VIF), which was calculated recursively within a sliding window with size 50 SNPs and pairwise r 2 value of 0.2 using PLINK.

Finally, each identified top SNP in AA was sent for replication in additional independent AA samples.

Interrogation of CARe Loci in CKDGen

Similar to the interrogation in AA for the EA lead loci, we also interrogated the newly identified loci from the CARe GWAS and the IBC chip in EA participants of the CKDGen consortium.

Stage 2 Replication Analysis

Replication analyses were performed using imputed in silico genome-wide association data; replication studies conducted the same association analyses as the Stage 1 phase. Details regarding the replication cohorts can be found in Text S1.

Replication was performed as follows: meta-analysis was conducted in the Stage 2 studies only, and then in the Stage 1 + Stage 2 studies combined. Replication was defined as a directionconsistent Stage 2 beta coefficient; replication p-values are thus represented as one-sided tests. SNPs were declared to replicate when the p-value in the Stage 1 + Stage 2 studies combined was smaller than the p-value in the Stage 1 alone.

Cross-Trait Analyses

The correlation between ln(UACR) and ln(eGFR) can range from non-significant to as high as 0.237 (p,0.001) in our study; therefore, we examined the cross-trait associations across albuminuria and eGFR phenotypes.

Functional Studies in Zebrafish

Zebrafish were maintained in accordance with established procedures and IACUC approval. At the one-cell stage, zebrafish embryos were injected with varying doses of morpholino antisense oligonucleotides (MO, GeneTools, Philomath OR). MO sequences are shown in Table S6. For in vivo observations, edema development was documented at 5-days post-fertilization.

In situ hybridization was performed according to established procedures (http://zfin.org/ZFIN/Methods/ThisseProtocol.html).

To visualize different regions of the kidney, we used pax2a (global kidney marker) [41], wt1a (podocyte marker) [41], nephrin (podocyte marker) [31], slc20a1a (proximal tubule) [42], and slc12a3 (distal tubule marker) [42]. The morphology of the expression pattern was independently scored by two investigators. Electron microscopy was performed as previously described [41].

Dextran clearance was assessed as described previously [20], 48 hours after morpholino injection, embryos were manually dechorionated, anesthetized in a 1:20 dilution of 4 mg/ml Tricaine in egg water and positioned on their back in a 1% agarose injection mold. An equal volume of tetramethylrhodamine dextran (10,000 MW; Invitrogen) was injected into the cardiac sinus venosus of each embryo, after which embryos were returned to fresh egg water. Embryos were imaged by fluorescence microscopy 6 hours post-injection (54 hpf) to demonstrate equal loading, then subsequently imaged at 72 and 96 hpf to evaluate dextran clearance. Confocal images were obtained from agarose embedded embryos using a Zeiss LSM500 microscope.  Figure S3 Regional association plots for all confirmed or replicating loci from the CKDGen loci interrogation; the blue notation represents the best SNP in whites with the p-value in African Americans, whereas red represents the lead SNP in African ancestry participants; the linkage disequilibrium shown uses YRI information from Hapmap2. (PDF) Figure S4 Regional association plot for the MYH9-APOL1 region in African ancestry participants. (TIF) Figure S5 Analysis of glomerular architecture after kcnq1 knockdown by electron microscopy does not reveal significant changes. (a-c) Glomerular architecture at 120 hpf after injection of control morpholino visualized by electron microscopy at 8000-, 15,000, and 50,000-fold magnification reveals endothelial capillaries (C) with basement membrane (BM), podocytes (P) and foot processes (FP) similar to mammalian glomerular ultrastructure.     Text S1 Study-specific methods. (DOC)

Supporting Information

Figure 1 .
1Regional association plots for DOK6 and FNDC1. For A) DOK6 and B) FNDC1, Stage 1 only. doi:10.1371/journal.pgen.1002264.g001

Figure 2 .
2Regional association plots for KCNQ1. For A) KCNQ1 in participants of African ancestry and B) in participants of European ancestry. doi:10.1371/journal.pgen.1002264.g002

Figure 3 .
3Kcnq1 knockdown in zebrafish embryos causes glomerular abnormalities. (A-E) Zebrafish embryos injected with control morpholino show normal glomerular and tubular morphology, as shown by in situ hybridization for the global kidney marker pax2a (A, inset showing lower-magnification image, with staining in both glomerulus and tubules), the podocyte markers wt1a (B) and nephrin (C), and the proximal and distal tubular markers slc20a1a (D) and slc12a3 (E). (F-J) Injection of the kcnq1 antisense morpholino, targeting the ATG site of the gene, at the onecell stage results in significant changes in glomerular gene expression (F-H). No changes were observed in the proximal tubule (I) or the distal tubule (J). doi:10.1371/journal.pgen.1002264.g003

Figure 4 .
4Tg(cdh17:GFP) embryos were injected with 200 uM control or kcnq1 morpholino (MO) at the 1-cell stage. Embryos were subsequently injected in the sinus venosus with 10,000 MW rhodamine dextran at 48 hpf. Dextran clearance was monitored over the next 48 hours by confocal microscopy. Compared to control MO-injected embryos (A-C), kcnq1 MO-injected embryos exhibit edema (D, E) and increased dextran clearance (F) 48 hours post-injection (hpi), suggestive of a filtration defect. At 5 dpf, 87% of kcnq1 morphant embryos (n = 38) develop edema (G). doi:10.1371/journal.pgen.1002264.g004

Figure S1
S1Quantile-quantile plots for genome-wide association for A) eGFR; B) CKD; c) UACR; 4) MA. (TIF) Figure S2 Manhattan plots for genome-wide association for A) eGFR; B) CKD; c) UACR; 4) MA. (TIF)

Figure S6
S6Dok6 and fndc1 knockdown in zebrafish embryos does not affect kidney development. (a-e) Uninjected control zebrafish embryos show normal glomerular and tubular morphology, as shown by in situ hybridization for the global kidney marker pax2a (a, inset showing lower-magnification image, with staining in both glomerulus and tubules), the podocyte markers wt1a (b) and nephrin (c), and the proximal and distal tubular markers slc20a1a (d) and slc12a3 (e). (f-o) Injection of dok6 (f-j) or fndc1 (k-o) morpholinos at the one-cell stage results in no significant changes in glomerular or tubular gene expression. (TIF) Figure S7 kcnq1 knockdown enhances loss of fluorescent dextran. Control and kcnq1 MO injected embryos were loaded with rhodamine-labeled dextran at 48 hpf. .80 embryos were analyzed for each group in 3 separate experiments (a,d) Fluorescence microscopy at 6 hpi reveals equal fluorescence loading between embryos. (b,e) At 72 hpf (24 hpi), fluorescence intensity in kcnq1 morphants is diminished. (c,f) At 96 hpf (48 hpi) control embryos retain their fluorescence, but kcnq1 morphants have significantly diminished fluorescence. (TIF)

Table 1 .
1Study sample characteristics in the CARE Renal Consortium.*Study

Sample Size
eGFRcrea/UACR

European
Ancestry1 %
Women %

Age
(years)

eGFRcrea
(ml/min/1.73 m 2 )
UACR (mg/g)1
MA (%)
CKD %

CARE African Ancestry Stage 1 Participants

ARIC
2786/1936
15.3(10.7,22.1)
63.1
53.3
99.9
2.69(0.85,9.94)
16.5
3.7

CARDIA
821/754
16.7(12.2,23.2)
61.1
39.4
111
4.28(3.09,7.40)
9.4
0.9

CHS**
728/426
20.6(12.4,32.7)
62.8
72.9
81.4
10.95(5.00,26.90)
31.9
18.4

JHS
2135/1246
15.7(11.8,21.1)
60.8
50
101
6.00(4.00,12.00)
16.5
4.2

MESA
1640/1633
18.8(11.5,29.7)
54.8
62.2
86.5
5.50(3.10, 13.10)
16.4
8.6

Total
8110/5995

CARE European Ancestry

ARIC
9581/7687
NA
53.5
54.3
90.3
3.94(2.04,7.69)
9.3
3.5

CARDIA
1331/1242
NA
53.2
40.7
99.2
4.42(3.30,6.90)
4.5
0.5

CHS
3938/2073
NA
56.1
72.8
76.4
10.20(5.50,22.70)
26.4
21

FHS
6624/6208
NA
53.6
48.8
94
4.50(2.60,9.57)
9.1
3.9

MESA
2293/2287
NA
52.3
62.7
82.4
4.70(3.10,8.60)
9.5
9.7

Total
23767/19497

Stage 2: Replication

GENOA
1217/1228
12.6(7.2,18.9)
71.7
63.2
88
6.13 (2.97, 19.73)
22.9
13

HANDLS
989/629
16.1(11.2,22.0)
55
48.4
121
5.26(3.17,15.96)
20.3
5.3

Health ABC
1139/253
22.4 (12.2, 32.6)
57.2
73.4
76.3
19.3(5.7, 96.5)
47
17.1

HUFS
1013/NA
19.7(14.3,27.0)
58.8
48.3
104.3
NA
NA
4.9

Total
4358/2110

Abbreviations: eGFRcrea: estimated glomerular filtration rate by serum creatinine, eGFRcys: estimated glomerular filtration rate by serum cystatin C, CKD: chronic kidney
disease, HTN: hypertension, DM: diabetes mellitus, NA: not available.
* Sample characteristics based on the larger eGFR sample.
** CHS only has data on the IBC platform.
1 Median, 25/75 th percentile.
doi:10.1371/journal.pgen.1002264.t001

Table 2 .
2Interrogation of known loci in EA in AA for the trait eGFRcrea; best SNP at each locus is shown below; SNP ID in bold from Kottgen et al, Nature Genetics 2009, and Nature Genetics 2010.SNP ID in EA
Chr

Genes Within
60 kb

Coded Allele
Frequency
(allele)

Beta coefficient
in EA relative
to coded allele

Coded allele
frequency for
the lead SNPs
in EA in
AA (allele)

Beta coefficient
in AA for the
lead SNP
in EA

p-value in
AA for lead
SNP in EA

rs17319721
4
SHROOM3
0.43 (A)
20.013
0.22 (A)
20.004
0.44

rs10109414
8
STC1
0.42 (T)
20.008
0.34 (T)
20.005
0.30

rs4293393*
16
UMOD
0.82 (A)
20.016
0.81 (A)
20.013
0.01

rs267734
1
ANXA9
0.8 (T)
20.010
0.96 (T)
20.016
0.13

rs1260326
2
GCKR
0.41 (T)
0.009
0.16 (T)
0.007
0.24

rs13538
2
ALMS1
0.77 (A)
20.009
0.48 (A)
20.003
0.57

rs347685
3
TFDP2
0.72 (A)
20.009
0.75 (A)
20.007
0.13

rs11959928
5
DAB2
0.44 (A)
20.009
0.30 (A)
20.002
0.75

rs6420094
5
SLC34A1
0.66 (A)
0.011
0.83 (A)
20.002
0.83

rs881858
6
VEGFA
0.72 (A)
20.011
0.36 (A)
20.003
0.50

rs10224210
7
PRKAG2
0.73 (T)
0.010
0.92 (T)
0.019
0.06

rs4744712
9
PIP5K1B
0.39 (A)
20.008
0.42 (A)
20.015
0.0003

rs653178
12
ATXN2
0.51 (T)
0.003
0.92 (T)
0.001
0.87

rs626277
13
DACH1
0.60 (A)
20.009
0.34 (A)
20.003
0.50

rs1394125
15
UBE2Q2
0.35 (A)
20.009
0.35 (A)
20.007
0.15

rs12460876
19
SLC7A9
0.61 (T)
20.008
0.72 (T)
20.010
0.03

rs10794720
10
WDR37
0.08 (T)
20.014
0.21 (T)
20.007
0.18

rs491567
15
WDR72
0.78 (A)
20.009
0.45 (A)
20.001
0.87

rs2453533
15
SPATA5L1;GATM
0.38 (A)
20.013
0.84 (A)
20.008
0.18

rs7422339
2
CPS1
0.32 (A)
20.009
0.32 (A)
20.005
0.38

rs2279463**
6
SLC22A2
0.88 (A)
0.013
N/A
N/A
N/A

rs6465825
7
TMEM60
0.61 (T)
0.008
0.51 (T)
0.000
0.92

rs10774021
12
SLC6A13
0.64 (T)
20.008
0.50 (T)
20.005
0.23

rs9895661
17
BCAS3
0.81 (T)
0.011
0.53 (T)
0.008
0.05

* Lead SNP rs12917707 in EA not present in AA dataset; therefore used rs4293393, which is in perfect LD in EA (r2 = 1.0).
** rs2279463 not present in AA dataset.
doi:10.1371/journal.pgen.1002264.t002

Table 3 .Table 4 .
34Interrogation of known loci in EA in AA for the trait eGFRcrea; best SNP at each locus is shown below; SNP ID in bold from Kottgen et al, Nature Genetics 2009, and Nature Genetics 2010. Stage 1 and Stage 2 results from loci in African Americans from GWAS (p,5.0*10E-06): SNP association with renal traits.SNP ID
in EA
Chr

Best SNP
ID in AA in
the Region

Coded
Allele
Frequency
in AA
(allele)

Beta
coefficient
in AA for
best SNP

P-value
in AA

Number of
independent
(typed) SNPs
interrogated
in CARe AA

Bonferroni
p-value
threshhold
(0.05/P)

Replication
beta
coefficient

Replication
p-value

Stage
1+Stage
2 beta

Stage
1+Stage 2
p-value

rs17319721 4
rs4371638
0.19 (T)
20.020
0.003
28
0.002
20.002
0.42
20.015
8.8E-03

rs10109414 8
rs192841
0.79 (A)
20.016
0.007
29
0.002
0.012
0.91
20.008
1.3E-01

rs4293393
16
rs4293393
0.81 (A)
20.013
0.014
14
0.004
20.017
0.02
20.014
1.5E-03

rs267734
1
rs3738479
0.39 (A)
0.013
0.005
19
0.003
0.012
0.04
0.013
9.0E-04

rs1260326
2
rs13022873
0.81 (A)
0.013
0.017
17
0.003
0.007
0.21
0.011
1.3E-02

rs13538
2
rs7600291
0.58 (C)
0.011
0.011
19
0.003
20.011
0.93
0.005
1.6E-01

rs347685
3
rs6781340
0.41 (T)
20.014
0.002
21
0.002
20.014
0.03
20.014
2.1E-04

rs11959928 5
rs3822460
0.83 (T)
20.013
0.020
11
0.005
20.010
0.13
20.012
1.0E-02

rs6420094
5
rs10463065
0.95 (C)
0.032
0.004
20
0.003
20.001
0.52
0.023
1.6E-02

rs881858
6
rs1750571
0.07 (A)
0.023
0.005
31
0.002
0.027
0.02
0.024
5.0E-04

rs10224210 7
rs6464167
0.42 (A)
0.015
0.021
20
0.003
0.004
0.33
0.010
3.5E-02

rs4744712
9
rs1556751
0.59 (A)
0.016
0.0002
65
0.001
0.006
0.19
0.013
2.6E-04

rs653178
12
rs12302645
0.94 (A)
20.018
0.050
10
0.005
20.014
0.16
20.017
3.0E-02

rs626277
13
rs9318029
0.13(T)
0.016
0.046
7
0.007
0.002
0.42
0.011
8.5E-02

rs1394125
15
rs2454472
0.84 (A)
20.020
0.0009
28
0.002
20.007
0.25
20.016
1.4E-03

rs12460876 19
rs3795058
0.43 (C)
20.012
0.004
30
0.002
0.009
0.88
20.007
6.7E-02

rs10794720 10
rs2805575
0.20 (C)
0.013
0.01
45
0.001
20.016
0.97
0.005
2.3E-01

rs491567
15
rs16966247
0.17 (C)
20.022
0.0002
35
0.001
20.004
0.31
20.017
6.0E-04

rs2453533
15
rs1153859
0.52 (T)
20.014
0.001
14
0.004
20.012
0.04
20.013
2.2E-04

rs7422339
2
rs957749
0.79 (A)
20.011
0.04
13
0.004
0.007
0.82
20.006
2.0E-01

rs2279463
6
rs3798156
0.12 (T)
20.027
7.8E-05
22
0.002
20.032
0.003
20.028
1.2E-06

rs6465825
7
rs6973213
0.20 (A)
20.019
2.79E-04 25
0.002
20.008
0.19
20.017
2.75E-04

rs10774021 12
rs485514
0.91 (T)
0.019
0.03
20
0.003
0.013
0.17
0.017
1.6E-02

rs9895661
17
rs11650989
0.22 (A)
0.016
0.002
20
0.003
0.013
0.06
0.016
5.8E-04

doi:10.1371/journal.pgen.1002264.t003

Trait
SNP ID
Chr

Genes In
or Nearby

SNP
function

Coded Allele
Frequency#
Beta

Stage 1
P-value

Replication
beta for
lead trait

Stage 2
p-value

Stage
1+Stage
2 beta

Stage
1+Stage
2 p-value

GWAS

UACR
rs4555246
18
DOK6
Intron
0.77 (A)
0.156
8.74E-07
0.078
0.10
0.140
5.33E-07

UACR
rs13213851
6
HACE1
N/A
0.68 (A)
20.130
1.86E-06
0.102
0.97
20.084
5.25E-04

UACR
rs2880072
6
FNDC1
N/A
0.66 (A)
20.128
1.99E-06
20.097
0.04
20.122
2.98E-07

MA
rs1009840
6
SGK1
Intron
0.19 (A)
0.363
2.62E-07
0.034
0.36
0.246
1.21E-05

eGFRcrea
rs6581768
12
DYRK2
N/A
0.87 (A)
0.035
3.00E-07
0.002
0.43
0.029
2.62E-06

eGFRcrea
rs7784820
7
GNAT3
N/A
0.70 (A)
0.023
5.30E-07
20.008
0.87
0.014
2.33E-04

CKD
rs12575381
11
B3GAT1
N/A
0.18 (A)
0.521
9.97E-07
20.127
0.90
0.179
1.26E-02

CKD
rs6428106
1
RGS1
downstream 0.17(T)
0.549
2.95E-07
0.144
0.07
0.334
3.70E-06

IBC

eGFRcrea
rs7111394
11
KCNQ1
Intron
0.83 (T)
20.025
4.67E-06
20.012
0.10
20.0213
3.61E-06

eGFRcys
rs6865647
5
PDE4D
Intron
0.74 (A)
0.108
1.491E-07
20.007
0.74
0.0169
6.87E-02

CKD
rs4648006
4
NFKB1
Intron
0.13 (T)
0.433
1.06E-05
20.140
0.89
0.1886
1.07E-02

doi:10.1371/journal.pgen.1002264.t004

Table 5 .
5Number of observed abnormalities/number of embryos examined at 200 uM injection (kcnq1) and 400 uM injection (dok6 and fndc1).Global Renal
Marker pax2a

Podocyte
Marker wt1a

Podocyte
Marker nephrin

Proximal Tubular
Marker slc20a1a

Distal Tubular
Marker slc12a3

Control MO
2/49 (4.1%)
3/63 (4.8%)
2/47 (4.3%)
45/142 (31%)
0/62 (0%)

kcnq1

Splice
45/68 (66%)
30/60 (50%)
35/50 (70%)
54/143 (38%)
0/35 (0%)

ATG
46/68 (68%)
33/78 (42%)
29/46 (63%)
65/157 (41%)
0/41 (0%)

Chi-Sqr p-value*
,0.0001
,0.0001
,0.0001
0.34
1

Chi-Sqr p-value**
,0.0001
,0.0001
,0.0001
0.11
1

Uninjected control
0/38 (0%)
0/31 (0%)
0/60 (0%)
1/16 (6%)
0/18 (0%)

dok6

ATG
5/54 (9%)
3/28 (11%)
1/50 (2%)
5/32 (16%)
2/42 (5%)

Chi-Sqr p-value**
0.14
0.2
0.93
0.65
0.88

fndc1

Splice
4/39 (10%)
1/30 (3%)
0/51 (0%)
8/31 (26%)
0/35 (0%)

Chi-Sqr p-value*
0.13
0.99
1
0.22
1

*Control or Uninjected versus splice MO.
**Control or Uninjected versus ATG MO.
doi:10.1371/journal.pgen.1002264.t005

Table S1
S1Genotyping and imputation platforms. (DOC)

Table S2
S2Genome-wide significant loci: SNP imputation quality* in Discovery and Replication cohorts. (DOC)

Table S3
S3Known loci for eGFRcrea and eGFRcys among participants of European Ancestry present on the IBC chip. (DOC)Table S4 Cross-trait associations for novel loci from Stage 1+Stage 2 in participants of African ancestry. (DOC) Table S5 Diabetes and Hypertension stratified analyses, Stage 1 data. (DOC)

Table S6
S6Morpholino sequences. (DOC)
PLoS Genetics | www.plosgenetics.org
September 2011 | Volume 7 | Issue 9 | e1002264
AcknowledgmentsThe authors wish to acknowledge the support of the National Heart, Lung, and Blood Institute and the contributions of the research institutions, study investigators, field staff, and study participants in creating this resource for biomedical research. The authors wish to thank Andrea Calhoun and Dr. Susan Hagen at the Electron Microscopy Core Facility at Beth Israel Deaconess Medical Center for expert assistance with electron microscopy and Drs. Dirk Hentschel and Helmut Rennke at Brigham and Women's Hospital for advice on electron microscopy images. We thank Dr. Iain
Prevalence of chronic kidney disease in the United States. J Coresh, E Selvin, L A Stevens, J Manzi, J W Kusek, JAMA. 298Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, et al. (2007) Prevalence of chronic kidney disease in the United States. JAMA 298: 2038-2047.

Trends in diabetes, high cholesterol, and hypertension in chronic kidney disease among. C S Fox, P Muntner, Diabetes Care. 31Fox CS, Muntner P (2008) Trends in diabetes, high cholesterol, and hypertension in chronic kidney disease among U.S. adults: 1988-1994 to 1999-2004. Diabetes Care 31: 1337-1342.

Multiple loci associated with indices of renal function and chronic kidney disease. A Kottgen, N L Glazer, A Dehghan, S J Hwang, R Katz, Nat Genet. 41Kottgen A, Glazer NL, Dehghan A, Hwang SJ, Katz R, et al. (2009) Multiple loci associated with indices of renal function and chronic kidney disease. Nat Genet 41: 712-717.

New loci associated with kidney function and chronic kidney disease. A Kottgen, C Pattaro, C A Boger, C Fuchsberger, M Olden, Nat Genet. 42Kottgen A, Pattaro C, Boger CA, Fuchsberger C, Olden M, et al. (2010) New loci associated with kidney function and chronic kidney disease. Nat Genet 42: 376-384.

Genomewide Linkage Analysis to Serum Creatinine, GFR, and Creatinine Clearance in a Community-Based Population: The Framingham Heart Study. C S Fox, Q Yang, L A Cupples, C Y Guo, M G Larson, J Am Soc Nephrol. 15Fox CS, Yang Q, Cupples LA, Guo CY, Larson MG, et al. (2004) Genomewide Linkage Analysis to Serum Creatinine, GFR, and Creatinine Clearance in a Community-Based Population: The Framingham Heart Study. J Am Soc Nephrol 15: 2457-2461.

Renal Data System, USRDS 2008 Annual Data Report, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases. U S , Bethesda, MDU.S. Renal Data System, USRDS 2008 Annual Data Report, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2008.

Genomewide Linkage Scan for Diabetic Renal Failure and Albuminuria: The FIND Study. R P IgoJr, S K Iyengar, S B Nicholas, K A Goddard, C D Langefeld, Am J Nephrol. 33Igo RP, Jr., Iyengar SK, Nicholas SB, Goddard KA, Langefeld CD, et al. (2011) Genomewide Linkage Scan for Diabetic Renal Failure and Albuminuria: The FIND Study. Am J Nephrol 33: 381-389.

Genome-wide scan for estimated glomerular filtration rate in multi-ethnic diabetic populations: the Family Investigation of Nephropathy and Diabetes (FIND). J R Schelling, H E Abboud, S B Nicholas, M V Pahl, J R Sedor, Diabetes. 57Schelling JR, Abboud HE, Nicholas SB, Pahl MV, Sedor JR, et al. (2008) Genome-wide scan for estimated glomerular filtration rate in multi-ethnic diabetic populations: the Family Investigation of Nephropathy and Diabetes (FIND). Diabetes 57: 235-243.

Genome-wide scans for diabetic nephropathy and albuminuria in multiethnic populations: the family investigation of nephropathy and diabetes (FIND). S K Iyengar, H E Abboud, K A Goddard, M F Saad, S G Adler, Diabetes. 56Iyengar SK, Abboud HE, Goddard KA, Saad MF, Adler SG, et al. (2007) Genome-wide scans for diabetic nephropathy and albuminuria in multiethnic populations: the family investigation of nephropathy and diabetes (FIND). Diabetes 56: 1577-1585.

MYH9 is associated with nondiabetic end-stage renal disease in African Americans. W H Kao, M J Klag, L A Meoni, D Reich, Y Berthier-Schaad, Nat Genet. 40Kao WH, Klag MJ, Meoni LA, Reich D, Berthier-Schaad Y, et al. (2008) MYH9 is associated with nondiabetic end-stage renal disease in African Americans. Nat Genet 40: 1185-1192.

MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis. J B Kopp, M W Smith, G W Nelson, R C Johnson, B I Freedman, Nat Genet. 40Kopp JB, Smith MW, Nelson GW, Johnson RC, Freedman BI, et al. (2008) MYH9 is a major-effect risk gene for focal segmental glomerulosclerosis. Nat Genet 40: 1175-1184.

Association of trypanolytic ApoL1 variants with kidney disease in African Americans. G Genovese, D J Friedman, M D Ross, L Lecordier, P Uzureau, Science. 329Genovese G, Friedman DJ, Ross MD, Lecordier L, Uzureau P, et al. (2010) Association of trypanolytic ApoL1 variants with kidney disease in African Americans. Science 329: 841-845.

Racial differences in the progression from chronic renal insufficiency to end-stage renal disease in the United States. C Y Hsu, F Lin, E Vittinghoff, M G Shlipak, J Am Soc Nephrol. 14Hsu CY, Lin F, Vittinghoff E, Shlipak MG (2003) Racial differences in the progression from chronic renal insufficiency to end-stage renal disease in the United States. J Am Soc Nephrol 14: 2902-2907.

Predictors of the progression of renal disease in the Modification of Diet in Renal Disease Study. L G Hunsicker, S Adler, A Caggiula, B K England, T Greene, Kidney Int. 51Hunsicker LG, Adler S, Caggiula A, England BK, Greene T, et al. (1997) Predictors of the progression of renal disease in the Modification of Diet in Renal Disease Study. Kidney Int 51: 1908-1919.

Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies. B J Keating, S Tischfield, S S Murray, T Bhangale, T S Price, 10.1371/journal.pone.0003583PLoS ONE. 33583Keating BJ, Tischfield S, Murray SS, Bhangale T, Price TS, et al. (2008) Concept, design and implementation of a cardiovascular gene-centric 50 k SNP array for large-scale genomic association studies. PLoS ONE 3: e3583. doi:10.1371/journal.pone.0003583.

Genetic loci influencing kidney function and chronic kidney disease. J C Chambers, W Zhang, G M Lord, H P Van Der, D A Lawlor, Nat Genet. 42Chambers JC, Zhang W, Lord GM, van der HP, Lawlor DA, et al. (2010) Genetic loci influencing kidney function and chronic kidney disease. Nat Genet 42: 373-375.

Genetic association analysis highlights new loci that modulate hematological trait variation in Caucasians and African Americans. K S Lo, J G Wilson, L A Lange, A R Folsom, G Galarneau, Hum Genet. 129Lo KS, Wilson JG, Lange LA, Folsom AR, Galarneau G, et al. (2011) Genetic association analysis highlights new loci that modulate hematological trait variation in Caucasians and African Americans. Hum Genet 129: 307-317.

CUBN Is a Gene Locus for Albuminuria. C A Boger, M H Chen, A Tin, M Olden, A Kottgen, J Am Soc Nephrol. Boger CA, Chen MH, Tin A, Olden M, Kottgen A, et al. (2011) CUBN Is a Gene Locus for Albuminuria. J Am Soc Nephrol.

Cofilin-1 inactivation leads to proteinuria-studies in zebrafish, mice and humans. S Ashworth, B Teng, J Kaufeld, E Miller, I Tossidou, 10.1371/journal.pone.0012626PLoS ONE. 512626Ashworth S, Teng B, Kaufeld J, Miller E, Tossidou I, et al. (2010) Cofilin-1 inactivation leads to proteinuria-studies in zebrafish, mice and humans. PLoS ONE 5: e12626. doi:10.1371/journal.pone.0012626.

Rapid screening of glomerular slit diaphragm integrity in larval zebrafish. D M Hentschel, M Mengel, L Boehme, F Liebsch, C Albertin, Am J Physiol Renal Physiol. 293Hentschel DM, Mengel M, Boehme L, Liebsch F, Albertin C, et al. (2007) Rapid screening of glomerular slit diaphragm integrity in larval zebrafish. Am J Physiol Renal Physiol 293: F1746-F1750.

A model organism approach: defining the role of Neph proteins as regulators of neuron and kidney morphogenesis. E Neumann-Haefelin, A Kramer-Zucker, K Slanchev, B Hartleben, F Noutsou, Hum Mol Genet. 19Neumann-Haefelin E, Kramer-Zucker A, Slanchev K, Hartleben B, Noutsou F, et al. (2010) A model organism approach: defining the role of Neph proteins as regulators of neuron and kidney morphogenesis. Hum Mol Genet 19: 2347-2359.

KCNQ1-dependent transport in renal and gastrointestinal epithelia. V Vallon, F Grahammer, H Volkl, C D Sandu, K Richter, Proc Natl Acad Sci U S A. 102Vallon V, Grahammer F, Volkl H, Sandu CD, Richter K, et al. (2005) KCNQ1- dependent transport in renal and gastrointestinal epithelia. Proc Natl Acad Sci U S A 102: 17864-17869.

SNPs in KCNQ1 are associated with susceptibility to type 2 diabetes in East Asian and European populations. H Unoki, A Takahashi, T Kawaguchi, K Hara, M Horikoshi, Nat Genet. 40Unoki H, Takahashi A, Kawaguchi T, Hara K, Horikoshi M, et al. (2008) SNPs in KCNQ1 are associated with susceptibility to type 2 diabetes in East Asian and European populations. Nat Genet 40: 1098-1102.

Variations in KCNQ1 are associated with type 2 diabetes and beta cell function in a Chinese population. C Hu, C Wang, R Zhang, X Ma, J Wang, Diabetologia. 52Hu C, Wang C, Zhang R, Ma X, Wang J, et al. (2009) Variations in KCNQ1 are associated with type 2 diabetes and beta cell function in a Chinese population. Diabetologia 52: 1322-1325.

A single nucleotide polymorphism in KCNQ1 is associated with susceptibility to diabetic nephropathy in japanese subjects with type 2 diabetes. T Ohshige, Y Tanaka, S Araki, T Babazono, M Toyoda, Diabetes Care. 33Ohshige T, Tanaka Y, Araki S, Babazono T, Toyoda M, et al. (2010) A single nucleotide polymorphism in KCNQ1 is associated with susceptibility to diabetic nephropathy in japanese subjects with type 2 diabetes. Diabetes Care 33: 842-846.

Dok-6, a Novel p62 Dok family member, promotes Ret-mediated neurite outgrowth. R J Crowder, H Enomoto, M Yang, E M Johnson, Jr Milbrandt, J , J Biol Chem. 279Crowder RJ, Enomoto H, Yang M, Johnson EM, Jr., Milbrandt J (2004) Dok-6, a Novel p62 Dok family member, promotes Ret-mediated neurite outgrowth. J Biol Chem 279: 42072-42081.

Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. A Schuchardt, D 'agati, V Larsson-Blomberg, L Costantini, F Pachnis, V , Nature. 367Schuchardt A, D'Agati V, Larsson-Blomberg L, Costantini F, Pachnis V (1994) Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367: 380-383.

An integration of genome-wide association study and gene expression profiling to prioritize the discovery of novel susceptibility Loci for osteoporosis-related traits. Y H Hsu, M C Zillikens, S G Wilson, C R Farber, S Demissie, 10.1371/journal.pgen.1000977PLoS Genet. 61000977Hsu YH, Zillikens MC, Wilson SG, Farber CR, Demissie S, et al. (2010) An integration of genome-wide association study and gene expression profiling to prioritize the discovery of novel susceptibility Loci for osteoporosis-related traits. PLoS Genet 6: e1000977. doi:10.1371/journal.pgen.1000977.

Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gbetagamma. M Sato, M J Cismowski, E Toyota, A V Smrcka, P A Lucchesi, Proc Natl Acad Sci U S A. 103Sato M, Cismowski MJ, Toyota E, Smrcka AV, Lucchesi PA, et al. (2006) Identification of a receptor-independent activator of G protein signaling (AGS8) in ischemic heart and its interaction with Gbetagamma. Proc Natl Acad Sci U S A 103: 797-802.

Activator of G protein signaling 8 (AGS8) is required for hypoxia-induced apoptosis of cardiomyocytes: role of G betagamma and connexin 43 (CX43). M Sato, Q Jiao, T Honda, R Kurotani, E Toyota, J Biol Chem. 284Sato M, Jiao Q, Honda T, Kurotani R, Toyota E, et al. (2009) Activator of G protein signaling 8 (AGS8) is required for hypoxia-induced apoptosis of cardiomyocytes: role of G betagamma and connexin 43 (CX43). J Biol Chem 284: 31431-31440.

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes. A G Kramer-Zucker, S Wiessner, A M Jensen, I A Drummond, Dev Biol. 285Kramer-Zucker AG, Wiessner S, Jensen AM, Drummond IA (2005) Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes. Dev Biol 285: 316-329.

Kidney development and disease in the zebrafish. I A Drummond, J Am Soc Nephrol. 16Drummond IA (2005) Kidney development and disease in the zebrafish. J Am Soc Nephrol 16: 299-304.

Acute renal failure in zebrafish: a novel system to study a complex disease. D M Hentschel, K M Park, L Cilenti, A S Zervos, I Drummond, Am J Physiol Renal Physiol. 288Hentschel DM, Park KM, Cilenti L, Zervos AS, Drummond I, et al. (2005) Acute renal failure in zebrafish: a novel system to study a complex disease. Am J Physiol Renal Physiol 288: F923-F929.

Deficiency of a transmembrane prolyl 4-hydroxylase in the zebrafish leads to basement membrane defects and compromised kidney function. J Hyvarinen, M Parikka, R Sormunen, M Ramet, K Tryggvason, J Biol Chem. Hyvarinen J, Parikka M, Sormunen R, Ramet M, Tryggvason K, et al. (2010) Deficiency of a transmembrane prolyl 4-hydroxylase in the zebrafish leads to basement membrane defects and compromised kidney function. J Biol Chem.

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. B Hinkes, R C Wiggins, R Gbadegesin, C N Vlangos, D Seelow, Nat Genet. 38Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow D, et al. (2006) Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet 38: 1397-1405.

Calibration and random variation of the serum creatinine assay as critical elements of using equations to estimate glomerular filtration rate. J Coresh, B C Astor, G Mcquillan, J Kusek, T Greene, Am J Kidney Dis. 39Coresh J, Astor BC, McQuillan G, Kusek J, Greene T, et al. (2002) Calibration and random variation of the serum creatinine assay as critical elements of using equations to estimate glomerular filtration rate. Am J Kidney Dis 39: 920-929.

Predictors of new-onset kidney disease in a community-based population. C S Fox, M G Larson, E P Leip, B Culleton, P W Wilson, JAMA. 291Fox CS, Larson MG, Leip EP, Culleton B, Wilson PW, et al. (2004) Predictors of new-onset kidney disease in a community-based population. JAMA 291: 844-850.

The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modification of Diet in Renal Disease Study Group. S Klahr, A S Levey, G J Beck, A W Caggiula, L Hunsicker, N Engl J Med. 330Klahr S, Levey AS, Beck GJ, Caggiula AW, Hunsicker L, et al. (1994) The effects of dietary protein restriction and blood-pressure control on the progression of chronic renal disease. Modification of Diet in Renal Disease Study Group. N Engl J Med 330: 877-884.

Genotypeimputation accuracy across worldwide human populations. L Huang, Y Li, A B Singleton, J A Hardy, G Abecasis, Am J Hum Genet. 84Huang L, Li Y, Singleton AB, Hardy JA, Abecasis G, et al. (2009) Genotype- imputation accuracy across worldwide human populations. Am J Hum Genet 84: 235-250.

Principal components analysis corrects for stratification in genome-wide association studies. A L Price, N J Patterson, R M Plenge, M E Weinblatt, N A Shadick, Nat Genet. 38Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, et al. (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38: 904-909.

Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. I A Drummond, A Majumdar, H Hentschel, M Elger, L Solnica-Krezel, Development. 125Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, et al. (1998) Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function. Development 125: 4655-4667.

The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. R A Wingert, R Selleck, J Yu, H D Song, Z Chen, 10.1371/journal.pgen.0030189PLoS Genet. 3189Wingert RA, Selleck R, Yu J, Song HD, Chen Z, et al. (2007) The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PLoS Genet 3: e189. doi:10.1371/journal.pgen.0030189.


Expression and prognostic significance of cox-2 and p-53 in hodgkin lymphomas: a retrospective study

Nagehan O Barisik
Suheyla Bozkurt
Mahmut Gumus
Isik Kaygusuz
Nimet Karadayi
Emine Bas
Mahmut Bayik
Tulay Tecimer
Expression and prognostic significance of cox-2 and p-53 in hodgkin lymphomas: a retrospective study
R E S E A R C H Open Access
Background: Cyclooxygenase (cox) is the rate-limiting enzyme, which catalyzes the conversion of arachidonic acid into prostaglandins and contributes to the inflammatory process. Cyclooxygenase-2 (cox-2), which is one of the two isoforms, plays a role in tumor progression and carcinogenesis. p53 contributes to apoptosis, DNA renewal and cell cycle. Studies concerning the relationship of cox-2 and p53 expressions and carcinogenesis are available, but the association between cox-2 and p53 in Hodgkin lymphoma (HL) is not exactly known. In our study, we examined the association of cox-2 and p53 expression, with age, stage, histopathological subtype, and survival in HL. We also examined correlation between cox-2 and p53 expression.Methods: Cox-2 and p53 expressions in Hodgkin-Reed Sternberg cells (HRS) were examined in 54 patients with HL depending on cox-2 expression, stained cases were classified as positive, and unstained cases as negative. Nuclear staining of HRS cells with p53 was evaluated as positive. The classifications of positivity were as follows: negative if<10%; (1+) if 10-25%; (2+) if 25-50%; (3+) if 50-75%, (4+) if >75%. Results: Cox-2 and p53 expressions were found in 49 (80%) and 29 (46%) patients, respectively. There were differences between histological subtypes according to cox-2 expression (p = 0.012). Mixed cellular (MC) and nodular sclerosing (NS) subtypes were seen most of the patients and cox-2 expression was evaluated mostly in the mixed cellular subtype. There were no statistically significant relationships between p53 and the histopathological subtypes; or between p53, cox-2 and the factors including stage, age and survival; or between p53 and cox-2 expression (p > 0.05). Conclusion: Considering the significant relationship between the cox-2 expression and the subtypes of HL, cox-2 expression is higher in MC and NS subtypes. However the difference between these two subtypes was not significant. This submission must be advocated by studies with large series

Introduction

Hodgkin lymphomas (HL) are malignancies derived from neoplastic Reed Sternberg (RS) cells which are found in the inflammatory media consist of plasma cells, eosinophils and histiocytes. The RS and its variant Hodgkin (H) cells form 1-3% of the whole mass [1]. It generates about 1% of the overall cancers and 30% of the lymphoid malignancies [1,2]. In the polymerase chain reaction (PCR) analyses performed to determine the function and histogenesis of Hodgkin-Reed Sternberg (HRS) cells, it is estimated that these cells are originated from the germinal center B cells and they have somatically mutated immunoglobulin (Ig) gene rearrangements. Not only the Ig expression levels of the HRS cells which are identified as B cells, but also their levels of transcription that is mediated by inactivation of Ig promoters, are disturbed. It is reported that HRS cells can block the apoptosis through an apoptotic mechanism which is not clearly understood, when the normal B cells go into apoptosis after loosing their capacity of Ig expression. It is suggested in the literature that Ebstein-Barr virus (EBV) infection and the nuclear factor kappa B (NFkB) released from HRS cells also act through this way [1,[3][4][5][6].

Although various factors for the etiology have been widely studied, it is estimated that EBV infection contributes in some of the cases [2,3]. It is reported in large series of epidemiologic studies that the risk of HL is lower in people using anti-inflammatory drugs, compared to the ones who do not use or who irregularly use nonsteroidal anti-inflammatory drugs (NSAIDs) [3].

The best known target of NSAIDs is the cyclooxygenase (cox) enzyme. Cox enzyme catalyzes the synthesis of prostaglandins (PGs) from arachidonic acid. Two isoforms of the cox gene have been identified. Cyclooxygenase-1(cox-1) is constitutively expressed on cell membranes in normal tissues and related to physiological functions, such as cytoprotection of the stomach and control of platelet aggregation: and cyclooxygenase-2 (cox-2) is cytoplasmic in location, not detectable in most of the normal tissues and induced by inflammatory and mitogenic stimuli. Furthermore expression of cox-2 is stimulated by oncogenes, growth factors, cytokines, tumor promoters and it has been shown to inhibit apoptosis [7,8]. In several tumors such as colon, esophagus, stomach, cervix and breast, cox-2 is found to be associated with carcinogenesis and tumor progression [7,[9][10][11][12][13][14].

The p53 gene located on the short arm of chromosome 17 has been described as a tumor suppressor gene producing a 53 kD nuclear binding protein [15,16]. It is thought that p53 has a role in regulation of normal cell cycle, apoptosis and in response to DNA damage [16]. It leads to selective growth and tumor formation by the replication of mutant p53 and deregulating cell cycle [17]. It is reported that mutant p53 acts by changing the genetic structure in lung, breast, pancreas and lymphoid malignancies [5,16,17]. It is declared in the previous studies that there might be p53 mutations and the antiapoptotic effect of p53, in the HRS cells [5,18,19].

The aim of our study is to determine the association between cox-2 and p53 expressions with the histological subtypes, age, survival and tumor dissemination, in HL cases.

Materials and methods

In this study, a total of 54 patients were enrolled who were diagnosed with HL in Dr. Lutfi Kirdar Kartal Education and Research Hospital and University of Marmara Medical School between 2000 and 2007.

The cases were classified according to age, survival, stage and the histological subtypes. Ann Arbor staging system was performed for classification, and staging results were obtained from the patients' files. All samples were fixed in formalin and embedded in paraffin. The histological slides of all of the patients were reviewed by two blinded pathologists and categorized according to the WHO classification of hematological malignancies into nodular lymphocyte predominant (NLP), nodular sclerosing (NS), lymphocyte-rich (LR), mixed cellular (MC) and lymphocyte depleted (LD) type [1]. After preparing the slides form the appropriate paraffin block, immunohistochemical assessment was performed for cox-2 and p53, for diagnostic evaluation.

Immunohistochemistry

Three μ thick sections were placed on 3-aminopropylethylene-covered slides and deparaffinized at 60°C for 1 hour. The slides were dipped in xylol for 15 minutes for deparaffinization, soaked with alcohols for 15 minutes and washed in distilled water. Afterwards, they were applied in pH 6 citrate buffer for antigen retrieval process in microwave oven for 20 minutes. Endogenous peroxidase activity was blocked by 10 minutes of incubation with 3% hidrogen peroxidase. To prevent nonspecific bindings, blockage was performed for 10 minutes (Dako Cytomation protein block serum-free, ref × 0909, lot 10016138, Denmark). Cox-2 was incubated with monoclonal mouse anti-human (Dako Cytomation EnVision Clone CX294, Code M3617, 1:100 dilution for 90 min, Denmark). They were incubated for 30 minutes with polyvalent HRP polymer (Spring Bioscience cat: DPE-125, Lot: 70918, California). P53 was incubated with monoclonal mouse anti-human (Dako Cytomatin En Vision Clone DO-7, Code N1581 ready-touse for 30 min, Denmark)

AEC chromogen system was applied to slides for 15 minutes (Spring Bioscience catalog: ASS-125 ready to use, California). Contrast staining was done with mayerhematoxylene and slides were covered with water-based cover material.

Evaluation of immunostaining

The cases, in which cytoplasmic cox-2 expressions were determined in their HRS cells, were accepted as positive. RS cells are large with variable amount of cytoplasm that is eosinophilic or amphophilic. The nuclei are large thick nuclear membranes. Also there can be two nuclei, symmetric with ''mirror image" appearance. On the other hand H cells mono nuclei or multinuclei and these cells have positive staining with CD15 or and CD30 antibodies in immunohistochemical staining. Nuclear staining of p53 was evaluated as follows: negative if <10%; (1+) if 10-25%; (2+) if 25-50%; (3+) if 50-75%; (4+) if >75% of HRS cells were positive [7].

Statistical analysis

Statistical analyzes were performed using SPSS 13.0. Chi-square test was used in the comparison of classified data as well as in evaluation of descriptive statistical methods (median range). Survival analyses were performed using the Kaplan-Meier method. Results were evaluated in 95% confidence interval and p < 0.05 was determined as the level of significance.

Results

Patients and follow-up

This study included 54 patients: the mean age at diagnosis was 37 years. In accordance with the histological subtypes, there were 26 (48.1%) cases of NS subtype, 24 (44.4%) cases of MC subtype, 3 (5.6%) cases of LR subtype, and 1 (1.9%) case of NLPHL. Data from clinical follow-up were obtained in 23 patients, up to 66 months. The mean follow-up was 39 (5-66) months.

Among 23 patients, there were 20 (90%) cases administered with ABVD, 1 (4.5%) case with ABVD+CMOPP and 1 (4.5%) case with ABVD+CBVP-K. Three (13%) cases were in stage I, 6 (26.1%) cases were in stage II, 10 (43.5%) cases were in stage III and 4 (17.4%) cases were in stage IV. 9 (39%) cases were administered with RT but 14 (60.9%) cases were not (table 1).

Cox-2 and p53 expression

Immunoreactivity with Cox-2 was seen in 49 (80%) of the 54 cases with HL ( figure 1). On the other hand p53 staining was seen in 26 (48%) of the 54 cases (figure 2). Significant relationship was determined between the cox-2 expression and the histological subtypes (p = 0.012). Considering the NS and MC subtypes, which generate the majority of the cases, MC subtype demonstrated more staining than NS subtype. However there is no statistically significant difference between NS and MC subtypes considering cox-2 expression (p = 0.61). All cases were stained with cox-2 in lymphocyte rich type which generates the minority of the cases. However no staining was detected in NLP type. No statistically significant results were derived from the evaluation of cox-2 expression according to their stages in 23 cases who had complete follow-up (p = 0.142). Evaluating the cox-2 expression in terms of age, 24 cases were <35 and 30 patients were >35 years old. There was no statistically significant relationship between the cox-2 expression and age (p = 0.367). The association of cox-2 expression with the histological subtypes, stage and age are presented on table 2. No significant relationship was detected between p53 and the histological subtypes, stage and age (p > 0.05) (table 3).

The association between cox-2 and p53 expression

The association between cox-2 expression and p53 is evaluated, but no statistically significant relationship was detected (p > 0.05) (table 4).

The association between cox-2 expression and survival

Five years survival was 87.7%, in the follow-up group. The median overall survival could not be achieved (figure 3).

The effects of p53 and cox-2 expressions on survival have been investigated as well, but statistically significant results were not obtained, due to the small number of the potentially follow-up group and death of only two of the patients.

Discussion

It is known that cox-2 acts in tumor mechanisms including tumor proliferation, differentiation, immunosupression, angiogenesis and inhibition of apoptosis, by synthesis of PGs [20]. In the previous studies it has been demonstrated that cox-2 expression increases in colon, stomach, esophagus, lung, over, cervix, liver, pancreas adenocarcinomas and brain tumors and it is reported as a negative prognostic parameter [12,13,[21][22][23][24]. Moreover, it is reported in epidemiologic and experimental studies that cox-2 inhibitors and NSAIDs prevent the carcinogenesis [9][10][11]13,14,20,21,25,26]. A great number of studies regarding the solid organ tumors are available, however, there are very little numbers of studies presented concerning the hematological malignancies in association with the cox-2 expression, and thus the relationship between the lymphoid malignancies and the cox-2 expression is not clearly understood.

Ohsava     CHL cases, respectively [7,27]. In our study, the cox-2 expression was determined in 49 (80%) patients among the 54 HL cases. It is emphasized that cox-2 increases the expression of angiogenic factors such as PGE2, as well as the expression of proangiogenic factors; and also that PGE2 is a strong vascular endothelial growth factor (VEGF) inducer and consequently cox-2 contributes to angiogenesis and increases the tumor proliferation [7,13,22].

The study of Ohsawa et al. on this point investigated the cox-2 expression in association with proliferation index and microvessel density, and demonstrated that Ki67 proliferation index and the microvessel density were 2 and 4 folds greater in cox-2 expressing group [7]. It is known that interleukin-1 (IL-1), interleukin-5 (IL-5), NFkB, granulocyte colony stimulating factor, tumor necrosis factor and many other cytokines are released from the HRS cells (1,28). These cytokines result in B symptoms such as fever, sweating and weight loss which are included within the negative prognostic parameters [1]. Hazar et al. evaluated the association of cox-2 with histological type, age, stage and the response to the therapy. They obtained a lesser response to the therapy in non-HL cases with cox-2 expression and reported cox-2 expression as a negative prognostic parameter [27]. In our study, the association of cox-2 with histological subtypes was investigated and a greater staining was determined in the MC subtype which generates the majority among the overall groups. Since the cytokines are known to induce the cox-2 expression, we can accept that MC type (except lymphocyte poor type) involves more HRS cells, and therefore releases more cytokines compared to the other types. However there is no statistically significant difference between NS and MC subtypes considering cox-2 expression (p = 0.61). With regard to the fact that negative prognostic features such as advanced stage and contiguity dissemination are more often observed in the MC type. In our study, the association of age and survival with cox-2 expression was observed, but no statistically significant relationships were obtained however higher level of cox-2 expression in MC type was noteworthy.

It is suggested that p53 plays an important role in cell-cycle control, maintenance of genomic stability, cell differentiation and programmed cell-death. The mutation or depletion of p53 oncogen is one of the most frequently reported lesions in various types of cancers [4,7,15,16,28,29].

Generally it is accepted that, most of the mutations of the p53 gene result in production of a mutant p53 protein. The half time of the mutant p53 protein is extended; therefore the cells tend to accumulate.

Reports about the abnormal accumulation of p53 in HRS cells are available [2,4,19]. Moreover, the p53 gene mutations may lead to the disturbance of p53 mediated transactivation and this evidence may considered to be the key factor of apoptosis resistance of the p53 gene mutation. However, the association between the high levels of the p53 protein and p53 mutations could not be determined in some of the studies [4]. In a study carried out by Rongen et al., the p53 gene was evaluated in 8 CHL cases and 6 (80%) of them demonstrated p53 gene mutations in their HRS cells [5].    [7,16,19]. Wang et al. evaluated the association of p53 with the clinical stage, EBV, bcl-2, retinoblastoma gene, p21, Ki67 and topoisomerase-II-alfa and detected p53 gene in 14 patients among the 62 CHL cases. They have detected p53 expression more frequently in stage I, but in a lesser frequency in the advanced stages such as stage III and IV. However, no significant relationship was found between the clinical stages, histological subtypes and the p53 [4]. In this study it is reported that the rate of p53 expression is small; therefore abnormal p53 protein expression may not be effective in the blockage of apoptosis mediated deaths of HRS cells [4]. In our study, p53 expression was detected in 26 (48%) of the 54 cases, but no association was determined with the histological subtype, the stage, age and survival.

Association of cox-2 and p53 was reported in the studies previously performed. Subbaramaiah et al., compared the cox-2 expression between the wild type (wt) p53, mutant p53 and p53 non-expressing mouse fibroblasts and discovered that cox-2 expression was markedly repressed by wt p53. They thought that this investigation may describe why these levels of cox-2 protein are not seen in normal epithelial cells and contrarily why mutant p53 increases the levels of cox-2 expression in malignant tissues. They reported that the cytokines in mutant p53 expressing cells, oncogenes and growth factors can increase cox-2 induction [18].

There are also studies available investigating the cox-2 and p53 expressions in ovarian adenocarcinoma and in head and neck tumors and a positive association between cox-2 and p53 was reported [17,20].

However, Ohsawa et al. did not determine an association between cox-2 and p53 expression [7]. In our study, the association between p53 and cox-2 is also investigated, but no statistically significant evidence was found (p = 0.663).

Consequently, evaluating the association between the histological subtypes and cox-2 expression, statistically significant association between histological subtypes and cox-2 was determined. However, considering both cox-2 and p53, significant association of cox-2 and p53 with age, stage and survival were not detected. Median follow-up was 36 months, in our study and it was below 5 years of follow-up. The statistically significant association between cox-2 and histological types is promising, nevertheless, for the corroboration of the data and in support of including the cox-2 in prognostic parameters, long term follow-up studies with large series are needed. We think that our study is elucidating in this aspect.

et al., Hsu et al., and Hazar et al. observed the cox-2 expressions in classical Hodgkin lymphoma (CHL) and detected cytoplasmic cox-2 expression in 7(70%) of 10 CHL cases, 7(70%) of 10 CHL cases 15 (48%) of 31

(cox-2 × 100) cox-2 exspresion in H/R-S cells.

Figure 2 (
2p53 × 100) p53 expression in H/R-S cells.

And Education Hospital Pathology Department/Istanbul-Turkey.2 Marmara University Pathology Department/Istanbul-Turkey.3 Kartal

Figure 3
3Association

Table 1
1Distrubution of ParametersN
%

Age (n = 54)
≤ 35 years
24
44,4

>35 years
30
55,6

Histological type (n = 54)
NS
26
48,1
MC
24
44,4

LR
3
5,6

NLP
1
1,9

Therapy (n = 22)
ABVD
20
91,0

ABVD+CMOPP
1
4,5

ABVD+ CBVP-K
1
4,5

Stage (n = 23)
1
3
13,0

2
6
26,1
3
10
43,5

4
4
17,4

Bulky (n = 23)
Yes
2
8,7

No
21
91,3

RT (n = 23)
Yes
9
39,1

No
14
60,9

Table 2
2The association of Cox-2 expression with the histological subtypes, stage and ageCOX-2 expression
(negative) n%

COX-2 expression
(positive) n%

p
value

Histologic
type

0.012*

LR
0(100)
3(100)

MC
1(4.2)
23(95.8)

NS
3(11.6)
23(88.4)

NLP
1(100)
0(0)
Stage
0.142

Stage I-II
2(22.2)
7(77.8)

StageIII-IV
0(0)
14(100)

Age
0.367

<35
1
23

>35
4
26

* Chi-square test p < 0.05

Table 3
3Histological type, stage, age and p 53 expression

P53 expression
(negative) n%

P53 expression
(positive)n %

p
value

Histologic
type

0.078

LR
3(100)
0(0)

MC
15(62.5)
9(37.5)

NS
10(38.5)
16(61.5)

NLP
0(0)
1(100)

Stage
0.669
StageI-II
3(33.3)
6(66.7)

StageIII-IV
7(50)
7(50)

Age
0.548

<35
11(45.7)
13(44.3)

>35
17(56.6)
13(53.4)

+ Chi-square test p < 0.05

Table 4 The
4The p53 expression in HRS cells were assessed and shown in 36 (72%) cases among the 50 CHL patients in the study of Gupta et al., in all of the 67 CHL patients in the study of Magio et al. and in 14 (42%) cases among the 33 CHL patients in the study of Ohsawa et al.association between cox-2 and p53
expression

P 53
expression

COX-2 expression
(negative) n%

COX-2 expression
(positive) n%

P
value

Negative
2(7.1)
26(92.9)
0.663

Positive
3(11.5)
23(88.5)

Total
5
49

+ Chi-square test p > 0.05

Authors' contributions NOB participated in conception and design. SB performed immunohistochemical staining. MG performed the statistical analysis. IK participated in acquisition of data. NK participated in acquisition of data. EB conceived of the study. MB participated in collecting of data. TT participated in writing of the manuscript. All authors read and approved the final version of the manuscript.Competing interestsThe authors declare that they have no competing interests.
Ioachim's HL: Medeiros Lymph Node Pathology. classical Hodgkin lymphoma Philadelphia: Lippincott Williams @ WilkinsMurphy J. Ioachim's HL: Medeiros Lymph Node Pathology. classical Hodgkin lymphoma Philadelphia: Lippincott Williams @ WilkinsMurphy J, McGough J , 4 2009, 306-324.

WHO classification of tumours of haematopoietic and lymphoid tissues Lyon. H Stein, ; Hodgkin Lymphoma, S H Iarc Presswerdlow, E Campo, N L Haris, E S Jaffe, S A Pileri, H Stein, Thielej, J W Vardiman, Stein H: Hodgkin lymphoma. WHO classification of tumours of haematopoietic and lymphoid tissues Lyon: IARC PresSwerdlow SH, Campo E, Haris NL, Jaffe ES, Pileri SA, Stein H, ThieleJ, Vardiman JW 2008, 321-334.

Aspirin and the risk of Hodgkin's lymphoma in a populationbased case-control study. E T Chang, T Zheng, E G Weir, M Borowitz, R B Mann, D Spiegelman, N E Mueller, J Natl Cancer Inst. 96Chang ET, Zheng T, Weir EG, Borowitz M, Mann RB, Spiegelman D, Mueller NE: Aspirin and the risk of Hodgkin's lymphoma in a population- based case-control study. J Natl Cancer Inst 2004, 96:305-315.

Apoptosis and cell cyle-related genes and proteins in classical Hodgkin lymphoma. J Wang, C R Taylor, Appl Immunohistochemistry-Mol Morphology. 113Wang J, Taylor CR: Apoptosis and cell cyle-related genes and proteins in classical Hodgkin lymphoma. Appl Immunohistochemistry-Mol Morphology 2003, 11(3):206-213.

Mutation of the p53 gene is not a typical feature of Hodgkin and Reed-Sternberg Cells in Hodgkin's disease. M M Rongen, A Roers, R Küppers, K Rajewsky, M L Hansman, Neoplasia. 945Rongen MM, Roers A, Küppers R, Rajewsky K, Hansman ML: Mutation of the p53 gene is not a typical feature of Hodgkin and Reed-Sternberg Cells in Hodgkin's disease. Neoplasia 1999, 94(5):1755-1760.

BCL-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer prognosisi in patients treated with ABVD or equivalent regimes. G Z Rassidakis, J L Medeiros, T P Vassilakopoulos, S Viviani, V Bonfante, G Nadali, Blood. 1002Rassidakis GZ, Medeiros JL, Vassilakopoulos TP, Viviani S, Bonfante V, Nadali G, et al: BCL-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer prognosisi in patients treated with ABVD or equivalent regimes. Blood 2002, 100(2):3935-3941.

Expression of cyclooxcygenase-2 in Hodgkin's lymphoma:Its role in cell proliferation and angiogenesis. M Ohsawa, H Fukushima, Y Ikura, T Inoue, N Shıraı, Y Sugama, Leuk Lymphoma. 479Ohsawa M, Fukushima H, Ikura Y, Inoue T, Shıraı N, Sugama Y, et al: Expression of cyclooxcygenase-2 in Hodgkin's lymphoma:Its role in cell proliferation and angiogenesis. Leuk Lymphoma 2006, 47(9):1863-1871.

Mixeded message: modulation of inflamtion and immune responses by prostaglandins and trombaxanes. S Tilley, T M Coffman, B H Koller, J Clin Invest. 108Tilley S, Coffman TM, Koller BH: Mixeded message: modulation of inflamtion and immune responses by prostaglandins and trombaxanes. J Clin Invest 2001, 108:15-23.

The Cyclooxygenas -2 inhibitor celecoxib is a potent preventive and therapeutic agent in min mouse model of adenomatous polyposis. R F Jacoby, K Seibert, C E Cole, G Kelloff, R A Lubet, Cancer Res. 60Jacoby RF, Seibert K, Cole CE, Kelloff G, Lubet RA: The Cyclooxygenas -2 inhibitor celecoxib is a potent preventive and therapeutic agent in min mouse model of adenomatous polyposis. Cancer Res 2000, 60:5040-5044.

Aspirin and the risk of colorectal cancer in women. E Gıovannuccı, K M Egan, D J Hunter, M J Stampfer, G A Coldıtz, W C Willet, N Engl J Med. 333Gıovannuccı E, Egan KM, Hunter DJ, Stampfer MJ, Coldıtz GA, Willet WC, et al: Aspirin and the risk of colorectal cancer in women. N Engl J Med 1995, 333:609-614.

Cyclooxygenase -2 expression in human breast cancers and adjacent ductal carcinoma in situ. E Half, X M Tang, Gwyn K Sahin, A Wathen, K Sinicrope, F A , Cancer Res. 62Half E, Tang XM, Gwyn K, Sahin A, Wathen K, Sinicrope FA: Cyclooxygenase -2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Res 2002, 62:1676-1681.

Up-regulation of cyclooxygenase-2 in squamos carcinogenesis of the esophagus. A Shamma, H Yamamoto, Y Doki, J Okami, M Konda, Y Fujiwara, M Yano, Clin Cancer Res. 6Shamma A, Yamamoto H, Doki Y, Okami J, Konda M, Fujiwara Y, Yano M, et al: Up-regulation of cyclooxygenase-2 in squamos carcinogenesis of the esophagus. Clin Cancer Res 2000, 6:1229-1238.

Expression of cyclooxygenase-2 in adenocarcinomas of the uterin cercix and its relation to angiogenesis and tumor growth. J S Lee, Y D Choi, J H Lee, J H Nam, C Choi, M C Lee, Gynecol Oncol. 95Lee JS, Choi YD, Lee JH, Nam JH, Choi C, Lee MC, et al: Expression of cyclooxygenase-2 in adenocarcinomas of the uterin cercix and its relation to angiogenesis and tumor growth. Gynecol Oncol 2004, 95:523-529.

Effects of nonselective cyclooxygenase inhibition with low-dose ibuprofen on tumor growth, angiogenesis, metastasis and survival in a mouse model of colorectal cancer. M Yao, W Zhou, S Sangha, Albert A Chang, A J Liu, T C Wolfe, M M , Clin Cancer Res. 11Yao M, Zhou W, Sangha S, Albert A, Chang AJ, Liu TC, Wolfe MM: Effects of nonselective cyclooxygenase inhibition with low-dose ibuprofen on tumor growth, angiogenesis, metastasis and survival in a mouse model of colorectal cancer. Clin Cancer Res 2005, 11:1618-1628.

P53 mutation spectrum and load:the generation or hypotheses linking the exposure of endogenous or exogenous carcinogens to human cancer. P S Hussain, C C Haris, Mutat Res. 428Hussain PS, Haris CC: P53 mutation spectrum and load:the generation or hypotheses linking the exposure of endogenous or exogenous carcinogens to human cancer. Mutat Res 1999, 428:23-32.

P53expression in Reed -Sternberg cells of Hodgkin's disease. R K Gupta, A J Norton, I W Thompson, T A Lister, J G Bodmer, Br J Cancer. 66Gupta RK, Norton AJ, Thompson IW, Lister TA, Bodmer JG: P53expression in Reed -Sternberg cells of Hodgkin's disease. Br J Cancer 1992, 66:649-652.

Down-regulation of nitric oxide Synthase-2 and cyclooxygenase-2 pathways by p53 in squamous cell carcinoma. O Gallo, N Schiavone, L Papucci, I Sardi, L Magnelli, A Franchi, E Masini, S Capacioli, Am J Pathol. 163Gallo O, Schiavone N, Papucci L, Sardi I, Magnelli L, Franchi A, Masini E, Capacioli S: Down-regulation of nitric oxide Synthase-2 and cyclooxygenase-2 pathways by p53 in squamous cell carcinoma. Am J Pathol 2003, 163:723-732.

Inhibition of cyclooxygenase-2 gene expression by P53. K Subbaramaiah, N Altorki, W J Chung, J R Mestre, A Sampat, A J Dannenberg, J Biol Chem. 27416Subbaramaiah K, Altorki N, Chung WJ, Mestre JR, Sampat A, Dannenberg AJ: Inhibition of cyclooxygenase-2 gene expression by P53. J Biol Chem 1999, 274(16):10911-10915.

TP53 gene mutations in Hodgkin lymphoma are infrequent and not associated with absence of Ebstein -Barr virus. E Maggıo, E Stekelenburg, Berg Van Den, A Poppema, S , Int J Cancer. 94Maggıo E, Stekelenburg E, Berg Van Den A, Poppema S: TP53 gene mutations in Hodgkin lymphoma are infrequent and not associated with absence of Ebstein -Barr virus. Int J Cancer 2001, 94:60-66.

Expression of cylooxygenase-2 and its relationship to p53 accumulation in ovarian adenocarcinomas. K Shigemasa, X Tıan, L Gu, Y Shıroyama, N Nagaı, K Ohama, Int J Oncol. 22Shigemasa K, Tıan X, Gu L, Shıroyama Y, Nagaı N, Ohama K: Expression of cylooxygenase-2 and its relationship to p53 accumulation in ovarian adenocarcinomas. Int J Oncol 2003, 22:99-105.

Cylooxygenase-2 expression in squamous displasia and squamous cell carcinoma of the esophagus. H P Yu, S H Xu, L Liu, L Y Shi, X K Cai, W H Lu, B Lu, Cancer Lett. 198Yu HP, Xu SH, Liu L, Shi LY, Cai XK, Lu WH, Lu B, et al: Cylooxygenase-2 expression in squamous displasia and squamous cell carcinoma of the esophagus. Cancer Lett 2003, 198:193-201.

Reduced expression of cylooxygenase-2 proteins in hereditery nonpolyposis colorectal cancers relative to sporadic cancers. F A Sinicrope, M Lemoine, Xi L Lynch, P M Clearly, K R Shen, Y Frazier, M , Gastroenterol. 117Sinicrope FA, Lemoine M, XI L, Lynch PM, Clearly KR, Shen Y, Frazier M: Reduced expression of cylooxygenase-2 proteins in hereditery nonpolyposis colorectal cancers relative to sporadic cancers. Gastroenterol 1999, 117:350-358.

Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. H Sheng, J Shao, J D Morrow, R D Beauchamp, R N Dubois, Cancer Res. 58Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN: Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998, 58:362-366.

Up-regulation of cylooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. C E Eberhart, R J Coffey, A Radhika, F M Giardiello, S Ferrenbach, R N Dubois, Gastroenterol. 107Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach S, Dubois RN: Up-regulation of cylooxygenase-2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterol 1994, 107:1183-1188.

Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytocrome c-depent apoptotic pathway in human colon cancer cells. Y Sun, M Tang, E Half, M T Kuo, F A Sinicrope, Cancer Res. 62Sun Y, Tang M, Half E, Kuo MT, Sinicrope FA: Cyclooxygenase-2 overexpression reduces apoptotic susceptibility by inhibiting the cytocrome c-depent apoptotic pathway in human colon cancer cells. Cancer Res 2002, 62:6323-6328.

Risk of cancer in large cohort of nonaspirin NSAID users: a population-based study. H T Sorensen, S Friis, B Nergard, L Mellemkjaer, W J Blot, J K Mclaugglin, A Ekbom, J A Baron, Br J Cancer. 88Sorensen HT, Friis S, Nergard B, Mellemkjaer L, Blot WJ, McLaugglin JK, Ekbom A, Baron JA: Risk of cancer in large cohort of nonaspirin NSAID users: a population-based study. Br J Cancer 2003, 88:1687-1692.

Cyclooxygenase-2 (Cox-2) expression in lymphomas. B Hazar, M Ergin, E Seyrek, S Erdogan, I Tuncer, S Hakverdi, Leuk lymphoma. 45Hazar B, Ergin M, Seyrek E, Erdogan S, Tuncer I, Hakverdi S: Cyclooxygenase-2 (Cox-2) expression in lymphomas. Leuk lymphoma 2004, 45:1395-1399.

Expression of prostaglandin H synthase (Cyclooxygenase) in Hodgkin's mononuclear and Reed-Sternberg cells. S M Hsu, P Hsu, S S Lo, K K Wu, Am J Pathol. 133Hsu SM, Hsu P, Lo SS, Wu KK: Expression of prostaglandin H synthase (Cyclooxygenase) in Hodgkin's mononuclear and Reed-Sternberg cells. Am J Pathol 1988, 133:5-12.

The p53 tumour-supressor gene integrates numerous signals that control cell life and death. As when a highly connected node in the internet breaks down the disruption of p53 has severe consequences. B Vogelstein, D Lane, A J Levine, 10.1186/1746-1596-5-19Nature. 408Vogelstein B, Lane D, Levine AJ: The p53 tumour-supressor gene integrates numerous signals that control cell life and death. As when a highly connected node in the internet breaks down the disruption of p53 has severe consequences. Nature 2000, 408:307-310. doi:10.1186/1746-1596-5-19

Expression and prognostic significance of cox-2 and p-53 in hodgkin lymphomas: a retrospective study. Barisik, Diagnostic Pathology. 519Cite this article asCite this article as: Barisik et al.: Expression and prognostic significance of cox-2 and p-53 in hodgkin lymphomas: a retrospective study. Diagnostic Pathology 2010 5:19.