Functional Polymorphisms in the Vascular Endothelial Growth Factor Gene Are Associated with Development of End-Stage Renal Disease in Males
Kent Doi*,,
Eisei Noiri*,
Akihide Nakao*,
Toshiro Fujita*,
Shuzo Kobayashi and
Katsushi Tokunaga
* Departments of Nephrology and Endocrinology and Human genetics, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and Department of Nephrology and Kidney and Dialysis Center, Shonan Kamakura General Hospital, Kanagawa, Japan
Address correspondence to: Dr. Eisei Noiri, Department of Nephrology & Endocrinology, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8655, Japan. Phone: +81-3-3815-5411; Fax: +81-3-5814-8696; E-mail: noiri-tky{at}umin.ac.jp
Received for publication January 23, 2005.
Accepted for publication November 23, 2005.
This study elucidates the genetic role of vascular endothelialgrowth factor (VEGF) as a predisposing factor for progressionof chronic kidney disease. Single-nucleotide polymorphisms weregenotyped and haplotype structures were determined in the 3'untranslated region (UTR) of VEGF gene, and the distributionof each haplotype in male patients with ESRD (n = 101) and healthymale control subjects (n = 189) was examined. The 936C/T and1451C/T polymorphisms in the 3' UTR were in nearly absolutelinkage disequilibrium, and haplotype analysis demonstratedthat they were the primary responsible single-nucleotide polymorphisms.The distribution of the 936CC-1451CC genotype was significantlymore frequent among patients with ESRD than among the age-matchedhealthy control subjects. In addition to case-control associationstudy, the 936CC-1451CC genotype was also associated with significantlyhigher plasma VEGF levels in healthy individuals, but a significantassociation was found only in males, not in females. We alsoexamined the effect of the 936C-1451C haplotype on mRNA stability.Consistent with the results of plasma VEGF levels, mRNA carrying936C-1451C haplotype showed higher stability. The 936CC-1451CCgenotype in the 3' UTR showed not only susceptibility for ESRDbut also higher plasma VEGF levels and mRNA stability, indicatingthe contribution of VEGF to chronic kidney disease progression,especially in males.
Chronic kidney disease (CKD) is classifiable as a multifactorialdisease because the accumulation and combination of geneticfactors and environmental factors influence the onset and developmentof ESRD. Although CKD results from various pathogeneses, includingglomerulonephritis, diabetes, hypertension, and urologic disorders,the most predictive factor for progression to ESRD is not thecause of glomerular injury but the degree of proteinuria andtubulointerstitial damage (1). Presumably, certain common pathwaysto ESRD exist, and both genetic and environmental factors seemto be responsible for these common pathways.
The kidney is one of the most highly vascularized organs andhas two important microvasculatures of glomerular and peritubularcapillaries. These two microvasculatures are responsible forthe common pathways to ESRD because the injury of glomerularcapillary causes proteinuria and the decrease of peritubularcapillary results in chronic hypoxia followed by fibrosis inthe tubulointerstitium. As described above, both proteinuriaand decreased peritubular capillary are putatively importantpredictive factors for progression to ESRD (1). Vascular endothelialgrowth factor (VEGF) is a main regulator of blood vessel growth.It plays a critical role in promoting endothelial survival andmaintaining these microvasculatures (2). However, controversyremains as to whether VEGF has vasoprotective or atherosclerogeniceffects because VEGF induces chemotaxis and activation of macrophages(3), and administration of VEGF enhanced atherosclerotic plaqueprogression (4).
In the human kidney, VEGF expression has been demonstrated inthe podocytes (5,6), tubular cells (7), and mesangial cellsin kidneys of patients with glomerulonephritis (8); VEGF receptorshave been detected in the endothelium of glomeruli and peritubularcapillaries (7). Furthermore, VEGF is expressed in inflammatorycells such as monocytes (9,10) and lymphocytes (11), which areresponsible for fibrosis in the tubulointerstitium of kidney.Various hormones, growth factors, cytokines, and specific stimulisuch as hypoxia regulate VEGF expression. Individual diversityof VEGF expression levels has also been observed (12). The VEGFgene was reported to be polymorphic, especially in the promoterregion, 5' untranslated region (UTR), and 3' UTR (13–15).The polymorphisms in these regions were reported to be associatedwith VEGF levels (14,15) and diseases such as acute renal allograftrejection (16), diabetic retinopathy (17), breast cancer (18),rheumatoid arthritis (19), and sarcoidosis (20). Our study elucidatesthe genetic role of VEGF polymorphisms as a predisposing factorfor progression of CKD. We determined the haplotype structuresin 3' UTR of the VEGF gene and compared the distribution ofeach haplotype in patients with ESRD and healthy control subjects.We also examined the association of VEGF polymorphisms withplasma VEGF levels and mRNA stability to identify the functionalpolymorphisms that potentially influence VEGF levels.
Study Population
This study enrolled 101 male patients who had ESRD and weretreated with hemodialysis at five dialysis centers in the Tokyoarea. Patients who started dialysis therapy under the age of50 (40.3 ± 11.1 yr [mean ± SD]) were investigatedbecause they showed rapid progression of kidney disease to ESRDfrom the onset. The causes of ESRD were chronic glomerulonephritis(n = 70), diabetic nephropathy (n = 14), nephrosclerosis (n= 8), and miscellaneous other conditions (n = 9). Patients withpolycystic kidney disease were also excluded. The control groupconsisted of 189 unrelated healthy male individuals. There wasno statistical difference between the patients and the controlsubjects with respect to age (51.7 ± 10.3 versus 51.9± 6.0 yr). All individuals in the control group werehealthy and showed no urinary abnormality, renal dysfunction,or hyperglycemia; they reported no use of medication. GenomicDNA from the healthy control subjects were obtained from a publicgene bank, Health Science Research Resources Bank (Osaka, Japan).This study was approved by the Ethics Committee for Human GenomeStudy of The University of Tokyo. Informed consent was obtainedfrom each individual at the time of recruitment.
Genotyping of Polymorphisms
Haplotype structures in the 3' UTR have not been revealed precisely.Therefore, we performed direct sequencing for the entire 3'UTR in 16 unrelated individuals and found five polymorphisms,numbered from the previous report as 936C/T, 1451C/T, 1612A/G,1725G/A, and 2295T/C (14). Two had already been reported byRenner et al. (14) (936C/T and 1612A/G), whereas the other threewere located in the database. Finally, we examined the 936C/T,1451C/T, 1612A/G, and 1725G/A polymorphisms for haplotype analysis.A single-nucleotide polymorphism (SNP) typing system using fluorescencecapillary electrophoresis–single-strand conformation polymorphism(CE-SSCP) analysis that we established recently (21) was usedfor the 936C/T polymorphism. Genotyping for 1451C/T, 1725G/A,and 1612A/G was performed by direct sequencing because thesethree polymorphic sites were included in a single PCR fragment.
Measurement of Plasma VEGF Levels
Plasma VEGF levels were determined in 32 healthy males and 31healthy premenopausal females. Venous blood was collected withEDTA and centrifuged immediately at 3000 x g for 10 min. Plasmawas stored at –30°C. Measurement was performed usinga commercially available enzyme immunoassay kit (human VEGFQuantikine; R&D Systems, Minneapolis, MN). This kit recognizedboth human unbound isoforms of VEGF121 and VEGF165 in equimolaramounts.
mRNA Stability Analysis
Peripheral blood mononuclear cells (PBMC) were isolated usingPolymorphprep (Axis-Shield PoC AS, Oslo, Norway) from 10 healthyindividuals who were heterozygous for both 936C/T and 1451C/Tpolymorphisms. PBMC were cultured for 24 h in RPMI 1640 (Sigma-AldrichCorp., St. Louis, MO) supplemented with 10% FCS and 1% penicillin-streptomycinsolution. After stimulation with placenta growth factor (Sigma-AldrichCorp.) at a concentration of 10 ng/ml for 24 h, actinomycinD (Sigma-Aldrich Corp.) was added to inhibit transcription ofmRNA. The final concentration of actinomycin D was 5 µg/ml.Total RNA was isolated with Trizol total RNA isolation reagent(Invitrogen Corp., Carlsbad, CA) at 0, 2, and 4 h after addingactinomycin D and treated with DNase I to remove potential contaminationof DNA (DNA-free; Ambion Inc., Austin, TX). The mRNA then wasreverse-transcribed to cDNA with MLV reverse transcriptase (RiverTraAce; Toyobo Co. Ltd., Osaka, Japan).
Quantitative CE-SSCP analysis was performed to measure the ratioof cDNA carrying the 936C allele to total cDNA of the VEGF gene.We have already reported that our CE-SSCP system can estimateallele frequencies of SNP, including the VEGF 936C/T polymorphism,precisely with pooled DNA samples that contained an equal amountof DNA from 136 individuals (21). This system calculated thepeak heights of respective alleles using DNA analysis software(GeneScan; Applied Biosystems, Foster City, CA). Peaks thatrepresent two alleles were previously found not to show equalheight in heterozygotes (22). Therefore, 10 heterozygotes wereselected randomly, and the two peaks’ heights were measuredto calculate the relativity factor (23). After correction usingthe relativity factor, the relative ratio of the DNA amountwas calculated according to the relative heights of the peaks.This assay is capable of measuring all VEGF mRNA isoforms becauseoligonucleotide primers for the 936C/T polymorphism are locatedin exon 8.
Mixed DNA samples were used to validate the accuracy of estimationby quantitative CE-SSCP analysis. Genomic DNA from two homozygousindividuals, 936CC and 936TT of the 936C/T polymorphism, weremixed into tubes at various ratios (1:11, 2:10, 3:9, 4:8, 5:7,6:6, 7:5, 8:4, 9:3, 10:2, and 11:1). The final concentrationsof mixed DNA were adjusted to 10 ng/µl. Using mixed DNAsamples, the relative ratios of DNA carrying 936C allele wereestimated in comparison with the total. Correlation betweenthe estimated values and calculated values was determined usingPearson correlation coefficient.
Reverse Transcriptase–PCR for VEGF Isoforms
PCR amplifications were performed with cDNA samples from PBMCdescribed above to examine the contributions of VEGF isoformsin mRNA stability assay. Oligonucleotide primers were designedto amplify VEGF mRNA to identify unique VEGF isoforms as describedpreviously. The sense primer was located in exon 3, and theantisense primer was located in exon 8 (24). Products were analyzedusing agarose gel electrophoresis with ethidium bromide staining.
Statistical Analyses
The 2 test was used to compare frequencies of the SNP allelesin the study groups. Conformity of the genotype proportion toHardy-Weinberg equilibrium was examined in the patient groupand the control group. The frequencies of haplotypes were estimatedusing an expectation-maximization algorithm based on a maximum-likelihoodmethod (25). Permutation-based hypothesis testing (permutationtest) procedures were performed to examine associations of estimatedhaplotype frequencies with ESRD (26,27) The 2 statistics werederived from a series of simple 2 x 2 tables based on respectivehaplotypes’ frequencies versus all others combined betweencase and control groups. The null distribution of 2 test statisticsthen was approximated via a randomization test in which thecase/control status indicators were permuted randomly amongthe individuals in the sample; then the 2 statistics were recomputed.The P value of the permutation test was determined empiricallyusing the ratio of the data sets above the null 2 statisticsfor 10,000 permutations. These calculations were performed usinga commercial program (SNPAlyze Ver3.2 Pro; Dynacom Co. Ltd.,Chiba, Japan).
The Mann-Whitney U test was used to compare plasma VEGF levels,and the paired t test was used to compare the ratio of 936C/936Tin the mRNA stability assay (StatView-J5.0; SAS Institute Inc.,Cary, NC). P < 0.05 was considered significant.
Polymorphisms in VEGF Gene Were Associated with Development of ESRD, Especially in Males
The frequencies of genotypes in the patients with ESRD and thehealthy control subjects did not differ significantly from thoseexpected under Hardy-Weinberg equilibrium. Table 1 shows thatfour common haplotypes were presumed in the promoter-3' UTR.In the 3' UTR, four polymorphisms were genotyped: 936C/T and1451C/T polymorphisms were in nearly absolute linkage disequilibrium.The 936C allele almost exclusively co-occurred with the 1451Callele, and the 936T allele was found with the 1451T allele.A permutation test demonstrated that the frequency of one haplotype(936T-1451T-1612A-1725G) in 3' UTR significantly decreased inpatients with ESRD compared with healthy control subjects (Table 1).This haplotype was determined distinctly with these twopolymorphisms. Moreover, mutant carrier frequencies showed asignificant association with patients with ESRD only in thesetwo polymorphisms (936C/T: P = 0.013, odds ratio [OR] 2.02;95% confidence interval [CI] 1.15 to 3.53; 1451C/T: P = 0.0045,OR 2.24, 95% CI 1.27 to 3.94). The other polymorphisms showedno significant association in terms of the frequencies of genotypes,alleles, or mutant carriers. These results indicated that the936C/T and 1451C/T polymorphisms were the primary responsibleSNP.
Table 1. Estimated haplotype frequencies in 3' UTR of VEGF genea
Next we calculated the sample size required for case-controlassociation study as described previously (28). Consideringthat the frequency of the disease-susceptible allele (936C-1451C),which showed a recessive mode of inheritance, was approximately80% and morbidity of ESRD in Japan was 0.2%, we calculated thepenetrances for genotypes 936CC-1451CC, 936CT-1451CT, and 936TT-1451TTas 0.25, 0.1, and 0.1%, respectively. With these parameters,the sample size required for the power of 1 – above 0.8was calculated as 104 under the assumption that the number ofpatients and control subjects was equal. This indicated thatthe number of our samples provided virtually enough detectionpower in this study. Finally, the distribution of the 936CC-1451CCgenotype was shown to be significantly higher in patients withESRD compared with the age-matched control subjects by logisticanalysis (P = 0.0030; OR 2.37; 95% CI 1.34 to 4.18; Table 2).
Table 2. The distribution of the high-risk genotype in male patients with ESRD and age-matched male control subjectsa
Association of 936C/T and 1451C/T Polymorphisms with Plasma VEGF Levels
Plasma VEGF levels were measured in 32 healthy males and 31healthy premenopausal females. Those levels were significantlyhigher in the 936CC-1451CC individuals than the other genotypesin male samples (936CC-1451CC [n = 16]: median 44.5 pg/ml, range16.3 to 154.2 pg/ml; 936CT-1451CT [n = 11]/936TT-1451TT [n =5]: median 32.5 pg/ml; range 9.0 to 54.6 pg/ml; P = 0.026; Figure 1).However, no significant difference was found in female samples(936CC-1451CC [n = 17]: median 36.5 pg/ml; range 10.4 to 74.6pg/ml; 936CT-1451CT [n = 12]/936TT-1451TT [n = 2]: median 49.0pg/ml; range 20.8 to 80.8 pg/ml; P = 0.20; Figure 1). Furthermore,no significant association was shown for other polymorphismswith plasma VEGF levels (data not shown).
Figure 1. Association of the 936C/T and 1451C/T polymorphisms with plasma vascular endothelial growth factor (VEGF) levels. Plasma VEGF levels of 31 healthy female and 32 healthy male subjects were compared with the VEGF genotype. Boxes indicate the range of lower to upper quartile values; the lines inside the boxes indicate the median values. Error bars represent the 10th and 90th percentiles of data. CC, individuals with 936CC-1451CC, CT 936CT-1451CT, TT 936TT-1451TT.
Association of 936C/T and 1451C/T Polymorphisms with VEGF mRNA Stability
Mixed DNA samples were used to validate the accuracy of estimationby quantitative CE-SSCP analysis. Figure 2 shows the calculatedratio by peak heights. The mixing ratio of 936C allele to thetotal was very highly correlated (r = 0.99). Using quantitativeCE-SSCP analysis, the influence of 936C/T and 1451C/T polymorphismson mRNA stability was examined on PMBC that were isolated from10 individuals who were heterogeneous for 936C/T and 1451C/Tpolymorphisms. After the inhibition of transcription by actinomycinD, the relative ratio of cDNA carrying the 936C allele increased(Figure 3). This result indicated that the transcript with 936C-1451Chaplotype was more stable than that with the 936T-1451T haplotype.
Figure 2. Validation by quantitative capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) analysis. Estimation of the ratio of genomic DNA with 936C by quantitative CE-SSCP analysis showed good correlation compared with the mixing ratio of two homozygous samples (936CC and 936TT; r = 0.99)
Figure 3. mRNA stability assay using quantitative CE-SSCP analysis. Transcription of peripheral blood mononuclear cells (PBMC) from 10 936C/T-1451C/T heterozygotes was inhibited by actinomycin D. The ratio of cDNA carrying the 936C allele increased, indicating that the transcript with 936C was more stable than 936T (P = 0.000018). •, ratio of cDNA with 936C with actinomycin D inhibition; , this ratio without inhibition.
We also performed reverse transcription–PCR to examineisoforms that were transcribed by isolated PBMC. Transcriptsof the unbound isoforms of VEGF121 and VEGF165 were dominantlyfound even after placenta growth factor and actinomycin D treatment(Figure 4). VEGF189 and VEGF206 isoforms were not detected clearly.
Figure 4. VEGF mRNA isoform expressed in PBMC. Reverse transcription–PCR for identifying each VEGF isoform was performed with PBMC. VEGF165 and VEGF121 expressions were found in nonstimulated PBMC and in stimulated PBMC that were examined in mRNA stability assay.
We performed a case-control association study including a totalnumber of 290 ESRD male patients and healthy male control subjectsand found significant associations of the 936C/T and 1451C/Tpolymorphism in 3' UTR of the VEGF gene. These polymorphismswere in nearly absolute linkage disequilibrium, and the 936C-1451Chaplotype was inferred as a risk haplotype for ESRD (Table 1).The distribution of the 936CC-1451CC genotype was demonstratedto be significantly higher in patients with ESRD compared withage-matched healthy control subject (Table 2). Therefore, weconcluded that the 936CC-1451CC genotype was associated withESRD. To our knowledge, this is the first such report, but threereports have shown significant associations of the 936C/T polymorphismwith certain diseases such as breast cancer (18), rheumatoidarthritis (19), and sarcoidosis (20).
In addition to the association with ESRD in case-control associationstudy, the 936CC-1451CC genotype showed significantly higherplasma VEGF levels and mRNA stability. These results indicatedthat the 936CC-1451CC individuals who show higher VEGF levelsthan the others might be susceptible to ESRD. VEGF seems toplay an important role in maintaining normal kidney vasculatureas a strong survival factor for endothelium. Ostendorf et al.(29) showed the blockade of capillary repair in a Thy-1 glomerulonephritismodel by administration of an aptamer that inhibits VEGF. Kanget al. (30,31) showed that VEGF preserved endothelium and attenuatedrenal dysfunction and histologic damage in experimental ratmodels of CKD. However, not only mice that lack one VEGF allelebut also mice that overexpress VEGF in renal podocyte developedglomerular disease (32). Moreover, VEGF has a chemotactic effectfor monocytes and macrophages and could amplify the inflammatoryreactions. Celletti et al. (4) demonstrated that administrationof VEGF to hyperlipidemic mice promoted progression of atherosclerosiswith increased monocytes in the aortic plaque. Analysis of patientswith type 1 diabetes revealed that plasma VEGF levels were higherin patients with nephropathy than in subjects of a normoalbuminuriagroup (33). These results indicate that VEGF has dual effects.Strict regulation of VEGF expression is crucial for the progressionof kidney diseases and atherosclerosis.
Development of CKD to ESRD seems to be influenced by many geneticand environmental factors. The contribution of the 936CC-1451CCgenotype might be significant but moderate, as shown in othermultifactorial diseases such as hypertension and diabetes (34,35).In other words, the 936CC-1451CC genotype would not engenderan all-or-nothing event. This study demonstrated the functionalsignificance of the 936CC-1451CC genotype using plasma of healthyindividuals and PBMC of the 936C/T-1451C/T heterozygous individualsto exclude influences of other factors that regulate VEGF production.We examined plasma of healthy individuals because plasma andserum VEGF levels would differ in patients with hypertension(36) and peripheral arterial occlusive disease (37). The influenceby VEGF polymorphisms might be inapparent using patient samples.We also examined mRNA stability using PBMC of heterozygotesand found a significant difference in the relative mRNA amount.Examining heterozygous samples is useful for detecting subtlebut significant differences because heterozygotes express virtuallyequal amounts of mRNA. Furthermore, possible modifications byexperimental procedures are excluded.
We found a significant association of the polymorphisms in 3'UTR with mRNA stability (Figure 3). Generally, the 3' UTR isknown to contain important sequences that contribute directlyand significantly to the longevity of mRNA transcripts (38),and many factors that bind to functional sequences within the3' UTR of VEGF have been reported (39–43). Although the936C/T and 1451C/T polymorphisms are not included directly inthe binding sequences of the known factors, the binding sitesof hypoxia-inducible RNA-protein (39) and heterogeneous nuclearribonucleoprotein L (41) are located close to the polymorphicsites. Thus, it is possible that unknown regulatory proteinsbind to these polymorphic sites.
Alternative splicing in VEGF gene generates four major isoforms:VEGF121, VEGF165, VEGF189, and VEGF206. The expression of unboundisoforms VEGF165 and VEGF121 have been demonstrated in podocytes,isolated glomeruli, and tubular cells (6,24). We examined theseisoforms of PBMC that were used for mRNA stability assay andshowed the VEGF165 and VEGF121 expression in PBMC (Figure 4).Those results indicated that alternative splicing in the VEGFgene in PBMC was similar to that in renal intrinsic cells. Moreover,all VEGF mRNA transcripts of respective isoforms and cells includingPBMC and the intrinsic renal cells such as podocytes, tubularcells, and mesangial cells contain the 936C/T and 1451C/T polymorphismsin 3' UTR because they have the same genome DNA sequence. Therefore,the 936CC-1451CC genotype potentially influences VEGF productionin all types of renal cells and contributes to progression toESRD. For instance, the 242C/T polymorphism in the CYBA geneencoding the p22 phox component is assumed to play an importantrole in NAD(P)H oxidase activity. Several reports have demonstratedthe functional significance of this genetic polymorphism byusing neutrophils that were isolated from healthy individuals(44), saphenous vein and internal mammary artery from patientswho underwent coronary artery bypass surgery (45), and vasodilatorfunction of epicardial arteries (46). These reports indicatedthat genetic polymorphisms can make an impact in the differenttypes of cells, although regulation of cell biology, includingdegeneration of mRNA, might differ according to cell type andcircumstances around cells. We demonstrated the contributionof the 936CC-1451CC genotype for mRNA stability by using isolatedPBMC. Further investigations are required to demonstrate whetherthe polymorphisms in VEGF gene can differently affect renalcells under physiologic and pathologic conditions. Recently,an inhibitory isoform of VEGF has been reported (47). This isoforminhibited proliferation and migration of endothelial cells.It was found in immortalized human podocytes (48). Antibodiesthat were used for plasma VEGF levels by enzyme immunoassayand oligonucleotide primers in this study were incapable ofdistinguishing this inhibitory isoform.
The effect of VEGF genetic polymorphisms might be undetectablein females because VEGF transcription is regulated by estrogens,and this regulation was mediated by transcriptional activationof the estrogen receptor (49). Our preliminary data showed nosignificant association between ESRD female patients and youngfemale healthy individuals (data not shown). Therefore, we investigatedthe association of VEGF genetic polymorphisms only with maleindividuals. Further investigations are indispensable for confirmationof the association of the 936CC-1451CC genotype with ESRD infemales. In this study, significant associations of VEGF plasmalevels with the 936C/T and 1451C/T polymorphism were found onlyin males, not in females (Figure 1). With VEGF plasma levels,two previous reports also demonstrated significant associationsof the 936C/T polymorphism in healthy males (14) and healthypostmenopausal females (18). We found reproducible results inmales. In contrast, we found no significant association in premenopausalfemales, who differed from postmenopausal females in terms oftheir estrogen levels. Moreover, males are known to be vulnerableto progression of renal disease in experimental animal models(50–52) and in a meta-analysis of human clinical studies(53). Recently, Antus et al. (54) demonstrated protective effectsof estrogen on rat remnant kidney, and Kang et al. (55) introducedthe possibility that estrogen protects female rats in a remnantkidney model by regulating VEGF expression. Our results indicatethat VEGF regulation both by genetic polymorphisms and by sexhormones such as estrogens plays an important role in progressionto ESRD.
Potential limitation of this study is related to a reportedhigh 10-yr survival rate that exceeds 75% among the youngerincident dialysis patients in Japan (56,57). Because the averageduration of dialysis therapy of our patients with ESRD was approximately10 yr, our case-control study design cannot rule out potentialconfounding as a result of ascertainment bias in the case inwhich the 936CC-1451CC genotype is associated with a survivaladvantage in male patients with ESRD. Prospective cohort studiesare needed to confirm a direct association of the 936CC-1451CCgenotype with ESRD development.
Our case-control association study demonstrated a significantassociation of 936C/T and 1451C/T polymorphisms located in the3' UTR of the VEGF gene with ESRD in males. The 936C-1451C haplotypeshowed not only susceptibility to ESRD but also higher plasmaVEGF levels and mRNA stability, advocating that these functionalpolymorphisms contribute to the development to ESRD by regulatingVEGF production.
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Researchon Priority Areas (C) "Medical Genome Science" (K.T.); grant#14370315 from the Ministry of Education, Culture, Sports, Science,and Technology of Japan (E.N.); and grants from the Cell ScienceResearch Foundation (E.N.) and Takeda Medical Research Foundation(E.N.).
We are grateful to Dr. Jun Ohashi (University of Tokyo) forsuggestions about statistical analysis and to Drs. Yasushi Yukiyama,Hitoshi Miyake (Fujitsu Kawasaki Hospital), Tadahiro Nishi (NishiClinic), Takashi Ozawa (Kodaira Kitaguchi Clinic), HisakazuDegawa (Sinkoiwa Clinic Funabori), and Hiroshi Nonaka (NonakaClinic) for providing samples. We also thank Ms. Mami Haba,Ms. Rui Maeda, and Ms. Yoshimi Ishibashi (University of Tokyo)for technical assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Introduction
2 test was used to compare frequencies of the SNP alleles in the study groups. Conformity of the genotype proportion to Hardy-Weinberg equilibrium was examined in the patient group and the control group. The frequencies of haplotypes were estimated using an expectation-maximization algorithm based on a maximum-likelihood method (25). Permutation-based hypothesis testing (permutation test) procedures were performed to examine associations of estimated haplotype frequencies with ESRD (26,27) The 
above 0.8 was calculated as 104 under the assumption that the number of patients and control subjects was equal. This indicated that the number of our samples provided virtually enough detection power in this study. Finally, the distribution of the 936CC-1451CC genotype was shown to be significantly higher in patients with ESRD compared with the age-matched control subjects by logistic analysis (P = 0.0030; OR 2.37; 95% CI 1.34 to 4.18; Table 2). 
, this ratio without inhibition.