This article is protected by copyright. All rights reserved. 1

Association of Angiotensin Type 2 Receptor Gene

Polymorphisms with Ureteropelvic Junction Obstruction in

Brazilian Patients1

Running title: AGTR2 gene and UPJO

Debora M. Miranda1,2; Augusto Cesar dos Santos Junior1,2; Helena C.

Sarubi1; Luciana Bastos-Rodrigues1; Daniela Valadão Rosa1; Izabella S.

Freitas1; Luiz Armando De Marco1; Eduardo A. Oliveira1,2; Ana Cristina

Simões e Silva1,2.

1INCT / MM - Instituto Nacional de Ciência e Tecnologia em Medicina

Molecular, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte,

MG, Brazil.

2Unidade de Nefrologia Pediátrica, Departamento de Pediatria, Laboratório

Interdisciplinar de Investigação Médica, Faculdade de Medicina, UFMG, Belo

Horizonte, MG, Brazil

Correspondence to Ana Cristina Simões e Silva, MD, PhD

Laboratório Interdisciplinar de Investigação Médica

Av. Alfredo Balena, 190, 2o andar, sala 281, Belo Horizonte, MG, Brazil,

30130-100.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/nep.12308

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Phone: +55-31-34098073, FAX: + 55-31-34099750

E-mail: acssilva@hotmail.com

Abstract

Background and aims: The angiotensin type 2 (AT2) receptor takes part in

the process of ureteric bud during kidney development. Therefore, the gene

encoding AT2 receptor, the AGTR2 gene located in the X chromosome, is a

potential candidate for genetic association with Congenital Anomalies of the

Kidney and Urinary Tract (CAKUT). This study aimed to investigate if AGTR2

gene polymorphisms are associated with CAKUT in general or even with

common phenotypes of CAKUT in a Brazilian sample of paediatric patients.

Methods: We analysed 290 paediatric patients with CAKUT and 262 healthy

controls from the same geographic area. TaqMan single-nucleotide

polymorphism (SNP) genotyping assays for AGTR2 gene at rs1403543,

rs3736556, rs35474657, rs5193 and rs5194 were performed. The sample was

in Hardy Weinberg Equilibrium for all five SNPs. Results: The presence of

CAKUT in general was not significantly associated with the SNPs included in

this study. However, when patients were segregated according to major

phenotypes, the diagnosis of Ureteropelvic Junction Obstruction (UPJO) was

significantly associated with AGTR2 gene polymorphisms at rs3736556 and at

rs5194. On the other hand, the diagnoses of vesicoureteral reflux and of

multicystic dysplastic kidney were not associated with AGTR2 gene

polymorphisms. Conclusion: Our results support that the AGTR2 gene may

contribute to the pathogenesis of UPJO and the genetic origin of CAKUT

could vary according to phenotype expression. Acc

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Keywords: Angiotensin type 2 receptor (AGTR2) gene, Ureteropelvic

Junction Obstruction (UPJO), Congenital Anomalies of the Kidney and Urinary

Tract (CAKUT), Renin Angiotensin System.

Introduction

The genetic origins of congenital anomalies of the kidney and urinary

tract (CAKUT) have not been well defined, but the concomitant presence of

diverse urinary tract alterations and the familial pattern of inheritance with

variable penetrance have been observed in many cases1,2. Therefore, a

common genetic background for these anomalies has been speculated and

genes responsible for kidney and urinary tract morphogenesis have been

frequently associated with CAKUT3,4.

The critical role of the renin-angiotensin system (RAS) in kidney and

renal collecting system morphogenesis is evident from occurrence of diverse

forms of CAKUT in animals or humans as a result of RAS gene mutations5. In

this regard, Yosypiv et al.6 showed that Angiotensin II (Ang II) induces the

expression of glial-derived neurotrophic factor (GDNF)/Ret/Wnt11 pathway

genes, promotes cell proliferation preferentially in ureteric bud tip cells and

stimulates branching. The same research group also showed that Ang II type

2 (AT2) receptor is highly expressed in the ureteric bud epithelia during

metanephric development and the pharmacological antagonism of this

receptor at early stages of kidney development down-regulates

GDNF/Ret/Wnt11 pathway gene expression, decreases proliferation, and

induces apoptosis of the ureteric bud cells7. Acc

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The AT2 receptor is a 323-residue of the G-protein-coupled family

highly expressed in the fetal kidney, diminishing after the neonatal period8.

The AT2 receptor is encoded by the AGTR2 gene, which is located in X

chromosome. The AGTR2 gene has two short non-coding exons, two introns

and three exons, which codify the complete protein9. AGTR2 gene is actively

transcribed at the onset of, and throughout, the embryonic development of the

kidney and urinary tract system, and is mostly inactivated by the time of

birth10. Nishimura et al.11 reported that AGTR2 mutant mice have lower

apoptosis around the ureteric bud and higher incidence of renal and urinary

tract anomalies than wild type animals, and, in a concurrent human study,

showed that AGTR2 genotype in Caucasians exhibited a significant

association with CAKUT. The general hypothesis is that abnormalities in the

expression of AT2 receptors hinder interaction between the ureteric bud and

metanephric blastema, and hamper normal development, resulting in

CAKUT5,12. It has been reported that A to G transition at the 1332 position in

AGTR2 gene is associated with abnormal splicing, producing shorter length

mRNA and thereby affecting AT2 receptor activity11. This polymorphism has

been inconsistently shown to be associated with CAKUT in Caucasians13,

Korean14 and Japanese subjects15.

However, only the A to G transition at the 1332 position in the AGTR2

gene has been investigated in human CAKUT samples with controversial

results11,14,16,17. In this context, we used an alternative experimental strategy

to evaluate the association between AGTR2 gene and CAKUT. We selected

three tag and one functional single-nucleotide polymorphisms (SNPs)

covering the entire AGTR2 gene and tested if any of these SNPs are Acc

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associated with the development of CAKUT or even with different phenotypes

of CAKUT in our sample of Brazilian paediatric patients.

Patients and methods

Case and control groups

Cases: This study included 290 patients with CAKUT, being 185 boys

and 85 girls from our Paediatric Nephrology Unit, which is consider a

reference centre in Brazil18,19. The estimated prevalence of CAKUT in the

Centre of Fetal Medicine of our institution is 17.7 per 1,000 live births18. At our

Centre of Fetal Medicine, all fetuses underwent a detailed ultrasonography

(US) aimed at detecting renal abnormalities and other malformations, as

previously detailed18. After birth, infants who present renal pelvic dilatation in

antenatal US or other renal alterations underwent systematic investigation for

urinary tract anomalies and have been prospectively followed up at the

Paediatric Nephrology Unit19. Renal pelvic dilatation (RPD) in antenatal US

was considered to be present if the maximum anteroposterior diameter of the

renal pelvis was equal to or greater than 5 mm at prenatal US performed after

28 weeks of gestation19. The systematic approach to infants with prenatally

detected RPD at our Unit included an US performed after the first week of

postnatal life (7 to 15 days) and a voiding cystourethrogram (VCUG) in a

selected subgroup of patients19. US scans, clinical examination (including

growth and blood pressure measurements), and laboratory reviews (including

urine culture and serum creatinine) were scheduled at 6-month intervals.

When   the  VCUG  was  normal   but   postnatal  US   showed  RPD≥10  mm,   renal  

scintilography was performed after the first month19. Clinical and image Acc

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findings were considered for the diagnosis of the phenotype expression of

CAKUT. The absence of any recognized malformation was classified as

idiopathic hydronephrosis. Ureteropelvic junction obstruction was defined as

the presence of moderate or severe hydronephrosis in US associated with an

obstructive pattern at renal dynamic scintigraphy19. Multicystic dysplastic

kidney (MCDK) was defined when cysts of various sizes without connection

between them were located in parenchyma of a structurally abnormal kidney

in which no renal pelvis could be demonstrated18. VUR was characterized by

the return of urine from bladder to renal pelvis and the diagnosis was

confirmed and graded by VCUG.

Controls: The control group included 262 healthy sex and age-matched

children from the same geographic area. All controls were normotensive and

had serum creatinine within normal range at the time of blood sampling.

Family history of any kind of renal disease, urinary tract malformation or

urinary tract infection automatically excluded the subject. Healthy status was

also determined through the medical history and either a parental report or

self-report to rule out the presence of chronic or acute diseases.

Ethical issues

The study followed the ethics guidelines of the Declaration of Helsinki

and was approved by the local Ethics Committee. Informed consent was

obtained from all subjects.

Sample collection and single-nucleotide genotyping

Peripheral blood was collected in EDTA vacuum tubes from all participating

individuals: cases and controls. Genomic DNA extraction was performed

following the high-salt method of Lahiri and Nurnberger20. Hapmap database Acc

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and literature were used to select tag and functional (rs35474657, MAF =

0.002 in Caucasians) SNPs covering the AGTR2 gene. A sample of 50ng of

DNA was used for TaqMan SNP genotyping assays (rs1403543, rs3736556,

rs35474657, rs5193 and rs5194), according to standard protocol (Applied

Biosystem, Foster City, CA). Genotyping was performed by real-time

polymerase chain reaction (PCR) using the allelic discrimination mode (7500

Real Time PCR, Applied Biosystems). Retypes of 20% of the whole sample

were performed for quality control. The sample was in Hardy Weinberg

Equilibrium (HWE) for all five SNPs.

Genomic ancestry analysis or control for population stratification

Individual DNA was independently genotyped for a set of 40 bi-allelic short

insertion/deletion polymorphisms (indels) for genomic ancestry analysis21.

Amplicons were then sized using an ABI 3130 DNA sequencer (Applied

Biosystems) and analysed using the Gene Mapper Software (version 3.7).

European, African and Amerindian individuals of the HGPD-CEPH Human

Genome Diversity Cell Line Panel22 were used as parental populations. To

estimate the proportion of European, African and Amerindian bio-geographical

ancestry at each individual the STRUCTURE software version 2.3 was used

(available at http://pritch.bsd.uchicago.edu/structure.html). Graphic was

performed using GraphPad Prism version 4.00 for Windows (GraphPad

software, San Diego, California, USA).

Statistical Analysis

Allele and haplotype association analysis were performed using

UNPHASED v.3.1.2 software23. The inter-marker linkage disequilibrium (LD)

were  expressed  by  D’  and  the  haplotype  block  structure  were  calculated  and  Acc

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visualized using the HAPLOVIEW v.3.32 software

(http://www.broad.mit.edu/mpg/haploview) according to the algorithm

described by Gabriel et al.24. We performed 1,000 permutations in each test

as an estimative of the global significance of results for all haplotype analysis

and to validate the expectation-maximization values. All tests were two tailed,

and p level for significance was set at 0.05.

Results

This study consisted of a sample of 552 individuals. The case group

had 290 patients (185 boys and 105 girls) with mean age of 6.4 (interquartile

range, 2.7-8.5), recruited at our Paediatric Nephrology Unit. The control group

included 262 healthy individuals (169 boys and 93 girls) with mean age of 6.1

years old (interquartile range, 2.6-8.2). The phenotypes detected in case

group were idiopathic hydronephrosis (n=70, 24.1%), VUR (n=62, 21.4%),

UPJO (n=55, 19%), MCDK (n=49, 16.9%), posterior urethral valve (n=15,

5.2%), ureterocele (n=10, 3.5%), primary megaureter (n=9, 3.1%), unilateral

hypo-dysplasia (n=9, 3.1%), ureterovesical junction obstruction (n=5, 1.8%)

and others.

No association was found between the case group and the SNPs of the

AGTR2 gene in our sample (Table 1). The functional SNP rs35474657 was

not identified in our case group. The markers were in high linkage

disequilibrium (LD) and pairwise LD as shown in Figure 1. However, when we

evaluated separately each of the most representative phenotypes of CAKUT

(VUR, UPJO and MCDK) in our sample, a significant association between

UPJO and this gene was identified at rs3736556 and at rs5194 (Table 2). On Acc

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the other hand, associations between the AGTR2 gene and VUR or MCDK

were not found.

Since the frequency of the alleles for the AGTR2 gene seems to differ

between African Americans and Europeans, the possibility of ethnic

stratification was ruled out with ethnical study. For this purpose, we genotyped

all samples for 40 polymorphic indels loci, which form a powerful ancestry

informative test battery21. The gene location and minor allele frequencies

(MAF) in polymorphic sites of AGTR2 gene is shown in Table 3. The

differences in the proportions of genomic ancestry between the two groups

were not significant.

Discussion

Normal morphogenesis of the kidney, ureteral bud differentiation,

ureteropelvic junction formation, and bladder development are regulated by

complex epithelial-mesenchymal signalling events25. Failure of these

signalling events at specified times results in diverse phenotypes of CAKUT26.

Murine knockout models have examined the role of various signalling

molecules in genitourinary organogenesis6,11,27. In this regard, animal studies

have reinforced two possible roles for AGTR2 gene in ureteral development.

One is the regulation of apoptosis of the undifferentiated mesenchymal cells

surrounding the developing ureter27. A second role is the inhibition of ectopic

ureteral budding11. Indeed, the AT2 receptor activation has been required for

normal apoptosis of the mesenchymal cells that surrounded the developing

ureter11. When apoptosis of the undifferentiated mesenchymal cells was

halted or otherwise delayed as a result of failure to activate the AT2 receptor, Acc

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diverse patterns of CAKUT were seen17.

Studies of AGTR2 gene in patients with CAKUT showed controversial

results. Nishimura et al.11 observed an increased A–G transition in AGTR2

gene in male Caucasian American and German patients with MCDK and

UPJO, while Hiraoka et al.15 reported that there is no evidence for this AGTR2

gene derangement in human urinary tract anomalies in Japanese patients.

The frequency of the G allele in AGTR2 gene was higher in CAKUT patients

than in the general population in Italian16 and Korean children14, while no

differences were found in a more recent study from Greece13.   Nishimura’s  

subjects were selected through strict inclusion criteria, namely male

UPJO/MCDK patients11. In contrast, the Japanese15, the Italian16, the

Korean14 and the Greece13 CAKUT samples included male and female

patients with various phenotypes. The Korean study reported that the A–G

transition in intron 1 of the AGTR2 gene is associated with UPJO/MCDK14,

whereas, in the Greece study, the same transition of the AGTR2 gene was

not associated with the development of the CAKUT13. However, the number

of CAKUT patients in these previous studies seemed to be low to reach

definitive conclusions and may not allow the detection of associations

between AGTR2 gene and specific CAKUT phenotypes. In addition, only the

A–G transition at 1332 position of the AGTR2 gene was tested in previous

studies11,13,14,16. In this context, our study extended previous findings by

increasing the sample size (290 cases and 262 controls) and by testing five

SNPs that cover the entire AGTR2 gene. We found a significant association of

AGTR2 gene at rs3736556 and at rs5194 with UPJO. No associations were

detected with MCDK and VUR. Acc

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Findings obtained in animal studies corroborate the association between

AGTR2 gene and UPJO found in the present study. In general, the inhibition

of endogenous AT2 receptor signalling or the knockout of AGTR2 gene

resulted in impaired ureteric bud branching11. Indeed, AT2 receptor plays an

important role in the expansion of the ampulla, subsequent branching, and

directional bud elongation25. We might speculate that the alteration of these

morphogenetic pathways may contribute to the pathogenesis of UPJO. It

should be pointed, however, that the penetrance of AGTR2 gene in mice is

reported as 21% in males and 5% in females11, and the anatomical spectrum

of CAKUT in null mutants for the AGTR2 gene is impressively wide,

documenting that a common gene abnormality can result in different CAKUT

phenotypes5. It should be also mentioned that AGTR2 gene interacts with

other genetic pathways3. For example, given that bone morphogenic protein 4

(BMP4) inhibits UB branching27, decreased UB branching in AT2 receptor

knockout mice may be mediated, in part, by enhanced BMP4 signalling. In

this regard, Song and co-workers detected an increased expression of BMP4

gene in metanephroi of AT2 receptor knockout mice, suggesting that the

stimulatory effects of endogenous AT2 receptor on ureteric bud branching are

mediated, at least in part, via down-regulation of BMP47. Accordingly, our

research group studying the same CAKUT sample recently found an

association between BMP4 gene polymorphisms and UPJO29. Taken

together, these findings support that the expression of the ureteric bud

morphogenetic program genes and ureteric bud growth depend on complex

network of regulatory pathways and signalling effectors. Acc

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The functional effect of the AGTR2 gene in the origin of UPJO is still

unknown. Stankovic and co-workers17 found mRNA expression for AT2

receptors in ureter samples of children affected by CAKUT and this

expression was not affected by -1332A/G polymorphism. The detection of AT2

receptor expression after birth in human ureter tissue suggests a prolonged

role in CAKUT origin. Indeed, the asymmetry of the CAKUT phenotype is well

described3,4, which suggests the existence of modifying genes and/or

environmental factors. Changes in post-transcriptional and post-translational

events and also in protein expression may not be ruled out. This is in line with

results provided by the animal model of unilateral ureteral obstruction, which

mimic CAKUT associated renal injuries after ureteral obstruction, where the

expression of AT2 receptor was reported30.

We are aware of the limitations of our study. The existence of different

phenotypes, incomplete penetrance, variable aggregation, and clinical

presentation makes uniform data collection difficult and introduces selection

bias. Uncertainty in the clinical classification resulted in the aggregation of

different entities under the acronym CAKUT. On the other hand, there is

genetic support for the use of the term CAKUT, since mutations in a single

gene can have pleiotropic effects on the development of the kidney and

urinary tract2. Therefore, the heterogeneity of our CAKUT sample might be

responsible for the absence of association between AGTR2 gene and the

entire case group. One might also consider the possibility that the

heterogeneity of Brazilian population interfered with molecular studies.

However, despite the fact that Brazilian population is a result of a distinctive

mixture, our ancestral genetic data is quite similar from all previously studied Acc

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populations and seems perfectly comparable to HapMap studied populations.

Nevertheless, the large number of patients and controls of our study made

possible the evaluation of CAKUT phenotypes, as OJUP, VUR and MCKD,

separately. In addition, this study is the first one that selected five SNPs

covering the entire AGTR2 gene.

Finally, the data obtained in this study at least party contribute to the

understanding of CAKUT origins in Brazilian population. Data show that the

AGTR2 gene is associated UPJO, but not with MCKD and VUR. This gene

might have an essential role in the development of the ureter. However,

functional studies should be carried out to unravel the molecular pathways of

AGTR2 gene in CAKUT phenotypes.

Acknowledgments and Financial Support

This study was partially supported by CNPq (Conselho Nacional de

Desenvolvimento Científico e Tecnológico, Brazil), FAPEMIG (Fundação de

Amparo à Pesquisa do Estado de Minas Gerais, Brazil) and by the Grant

INCT-MM (Instituto Nacional de Ciência e Tecnologia – Medicina Molecular:

FAPEMIG: CBB-APQ-00075-09 / CNPq 573646/2008-2).

Statement of Interest – No conflict of interest

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17. Stanković   A,   Zivković   M,   Kostić   M,   Atanacković   J,   Krstić   Z,   Alavantić   D.  Expression profiling of the AT2R mRNA in affected tissue from children with CAKUT. Clin. Biochem. 2010; 43: 71-5.

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Table 1- Genotype and allele frequency of AGTR2 polymorphisms in case

(CAKUT patients) and control group

SNP name Cases (290) Controls (262) p-value Odds-R 95%CI nº % nº % rs1403543 / c.-95-29G>A GG 117 0.4366 72 0.4138 0.2010 1 1 - 1 GA 50 0.1866 58 0.3333 0.9049 1.119 0.63 – 1.98 AA 101 0.3769 44 0.2529 0.1542 1.413 0.89 - 2.24 G 197 0.5368 162 0.5645 0.4803 1 1 - 1 A 170 0.4632 125 0.4355 1.1180 0.82 - 1.53 rs3736556 / c.-36+55A>T AA 162 0.7751 138 0.6603 0.4311 1 1 - 1 AT 23 0.1100 48 0.2397 0.4472 0.7802 0.43 - 1.43 TT 24 0.1148 23 0.1100 0.7910 0.8889 0.48 – 1.65 A 239 0.8328 271 0.7787 0.0884 1 1 - 1 T 48 0.1672 77 0.2213 0.7068 0.47 – 1.06 rs35474657 / c.971G>A GG 290 1 262 1 1 1 1 - 1 G 393 1 431 1 1 1 1 - 1 rs5193 / c.*199G>T

GG 101 0.8347 93 0.7686 0.6193 1 1 -1 GT 12 0.0992 19 0.1570 0.2806 1.5800 0.66 – 3.76 TT 8 0.0661 9 0.0744 0.6232 0.8185 0.30 – 2.21 G 137 0.8726 171 0.8407 0.5537 1 1 - 1 T 20 0.1274 30 0.1493 0.8321 0.45– 1.53 rs5194 / c.*205A>G AA 50 0.3704 22 0.3056 0.8704 1 1 - 1 AG 23 0.1704 22 0.3056 0.5083 1.2880 0.54 – 3.10 GG 62 0.4593 28 0.3889 0.7240 0.9743 0.50 – 1.91 A 81 0.4451 59 0.4758 0.5960 1 1 - 1 G 101 0.5549 65 0.5242 1.132 0.72 – 1.79 * After a 1000 permutation, corrected best p-value > 0.05

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Table 2 - Genotype and allele frequency of AGTR2 polymorphisms in patients

with ureteropelvic junction obstruction (UPJO) and controls

SNP name Patients (55) Controls (262) p-value Odds-R 95%CI nº % nº % rs1403543 / c.-95-29G>A GG 28 0.56 72 0.41 0.62 1 1 - 1 GA 6 0.12 58 0.33 0.41 0.62 0.19 – 1.90 AA 16 0.32 44 0.25 098 0.94 0.45 - 1.92 G 41 0.63 162 0.56 0.33 1 1 - 1 A 24 0.37 125 0.44 0.76 0.43 - 1.32 rs3736556 / c.-36+55A>T AA 36 0.92 138 0.66 0.02 1 1 - 1 AT 2 0.05 48 0.24 0.21 0.33 0.07 - 1.59 TT 1 0.03 23 0.11 0.07 0.17 0.02 – 1.28 A 48 0.94 271 0.78 0.01 1 1 - 1 T 3 0.06 77 0.22 0.22 0.07 – 0.73 rs35474657 / c.971G>A GG 55 1 262 1 1 1 1 - 1 G 71 1 431 1 1 1 1 - 1 rs5193 / c.*199G>T

GG 20 0.87 93 0.77 0.84 1 1 -1 GT 1 0.04 19 0.16 0.69 0.64 0.07 – 5.88 TT 2 0.09 9 0.07 0.94 1.03 0.21 – 5.15 G 26 0.90 171 0.84 0.51 1 1 - 1 T 3 0.10 30 0.15 0.66 0.19– 2.31 rs5194 / c.*205A>G AA 5 0.20 22 0.31 0.09 1 1 - 1 AG 3 0.12 22 0.31 0.98 0.33 0.33 – 11.96 GG 17 0.68 28 0.39 0.11 0.85 0.85 – 8.38 A 8 0.25 59 0.48 0.02 1 1 - 1 G 24 0.75 65 0.52 2.72 1.14 – 6.53

* After a 1000 permutation, corrected best p-value > 0.05

Table 3- Gene location and Minor allele frequencies (MAF) in polymorphic

sites of AGTR2 gene

Gene location

Caucasian African American Chinese CAKUT Brazilian sample

rs1403543 Intron 0.39 (G) 0.43 (A) 0.33 (G) 0.45 (A) rs3736556 Intron 0.23 (T) 0.07 (T) 0.18 (T) 0.20 (T) rs35474657 Intergenic

/Unknown 0.0 (A) 0.03 (A) - 0.0 (A)

rs5193 UTR  3’  Intragenic

0.31 (T) 0.0 (T) 0.16 (T) 0.14 (T)

rs5194 UTR 3 0.42 (A) 0.37(G) 0.36 (A) 0.46 (A)

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This article is protected by copyright. All rights reserved. 18

Figure legend Figure 1 : LD block structure across AGTR2 gene. The upper panel shows the location of five polymorphisms in AGTR2 gene and lower panel shows the output of Haploview (v.4.1.) LD plot, each square (with D´ values written within the box) represents a pairwise LD relationship between the two SNPs. Red squares indicate statistical significant LD between the pair of SNPs as measured by the D´ statistic. Darker colors of red indicate higher values of D´, up to a maximum of 1, and the white square indicate pairwise D´ values of < 1 with no statistically significant evidence of LD.

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