Research Article Genetic Variant of C-5434T REN Enhancer …downloads.hindawi.com/journals/ijhy/2015/486961.pdf · Research Article Genetic Variant of C-5434T REN Enhancer on Serum

Research ArticleGenetic Variant of C-5434T REN Enhancer onSerum Renin Levels and Binding Pattern of Signal Transducersand Activators Transcription 3

Imama Maslahah1 Mohammad Saifur Rohman2 Nashi Widodo3

Agustina Tri Endharti4 and Didik Utomo3

1Biomedical Sciences Faculty of Medicine University of Brawijaya Malang 65145 Indonesia2Department of Cardiology and Vascular Medicine Faculty of Medicine Brawijaya University Saiful Anwar General HospitalMalang 65145 Indonesia3Biology Department Faculty of Mathematics and Sciences University of Brawijaya Malang 65145 Indonesia4Parasitology Department Faculty of Medicine University of Brawijaya Malang 65145 Indonesia

Correspondence should be addressed to Mohammad Saifur Rohman ippoenkyahoocom

Received 3 February 2015 Revised 26 April 2015 Accepted 4 May 2015

Academic Editor Tomohiro Katsuya

Copyright copy 2015 Imama Maslahah et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The human renin gene has been widely known to be involved in essential hypertension (EH) pathogenesis Genetic variant C-5434T of REN enhancer contributed to renin gene transcription and serum renin regulation However the mechanism associatedwith the transcription level changes remains unknown and only a few reports exist that discussed serum renin levels on C-5434Tof REN Thus this study aims to investigate the relationship between genetic variant C-5434T of REN enhancer and serum reninlevels in Indonesian hypertensive patients SNP of C-5434T was genotyped in 56 hypertensive patients by using RFLP The datashowed that serum renin is slightly higher in hypertensive patients with the TT genotype (39 plusmn 103) than patients with the CCgenotype (33 plusmn 106) but the difference was not statistically significant (119901 = 035) Here we also present a docking approachfor predicting interaction between genetic variant -5434CT and STAT3 (Signal Transducers and Activators Transcription 3) thepredicted transcription factor that regulates renin gene enhancerThe results showed that STAT3-DNAallele Tmore favorably bindstoDNA than STAT3-DNA allele CThese data suggest that the presence of genetic variant C-5434T has changed the binding patternof STAT3 to REN enhancer This is likely to influence STAT3 activity to stimulate the expression of renin gene in producing renin

1 Introduction

Renin-angiotensin-aldosterone system (RAAS) plays a piv-otal role in blood pressure regulation Renin the key enzymeof the renin-angiotensin-aldosterone cascade plays a crucialrole in the regulation of bloodpressure andRENmaybe a can-didate gene for hypertension [1 2] Much progress has beenmade in elucidating the molecular mechanisms involved inREN expression [3] Previous study showed that the presenceof genetic variants within a distal enhancer region has beenreported to increase in vitro REN transcription [4]

A SNP C-5434T is one of the variants to be foundwithin distal enhancer region (nucleotides -5777 to -5312)

[4] We hypothesized that this variant can affect the basaltranscriptional activity in generating renin However inIndonesian population there is no report that investigatesthe relationship between genetic variant C-5434T of RENenhancer and serum renin levels

Transcription factors bound to the REN enhancer ele-ments can have the effect of increasing the REN tran-scription thus indicating that the transcription factor hasa crucial role in the regulation of gene transcription [5]One of the transcription factors that regulate transcriptionactivity in REN is STAT3 (Signal Transducers and Activa-tors Transcription 3) [6] REN enhancer has a motif DNAsequence for STAT3 binding in the STAT-binding element

Hindawi Publishing CorporationInternational Journal of HypertensionVolume 2015 Article ID 486961 5 pageshttpdxdoiorg1011552015486961

2 International Journal of Hypertension

Table 1 Baseline characteristics of the patients

Variable REN C-5434T119901 valuelowast

CC (119899 = 16) CT (119899 = 31) TT (119899 = 9)Age (years) 57 plusmn 673 59 plusmn 877 58 plusmn 698 057Gender (MF) 79 1417 72 019SBP (mmHg) 138 plusmn 1543 142 plusmn 1887 140 plusmn 1054 078DBP (mmHg) 89 plusmn 1062 84 plusmn 1115 87 plusmn 666 021Weight (kg) 65 plusmn 1320 66 plusmn 1124 67 plusmn 1054 087Height (cm) 157 plusmn 768 158 plusmn 820 161 plusmn 855 045BMI (kgm2) 27 plusmn 478 26 plusmn 368 26 plusmn 298 097Urea (mgdL) 29 plusmn 918 29 plusmn 1593 28 plusmn 867 095Creatinine (mgdL) 14 plusmn 127 09 plusmn 022 1 plusmn 026 013Blood glucose (mgdL) 98 plusmn 2832 95 plusmn 961 91 plusmn 969 058Smoking 115 031 18 022Cholesterol (mgdL) 182 plusmn 5986 185 plusmn 3838 181 plusmn 3587 096SBP systolic blood pressure DBP diastolic blood pressure BMI body mass index lowast119901 value le 005 significantly different between groups

(SBE) 51015840-TT(N5)AA-31015840 STAT3 is thought to be the major

transcription factor in genetic variant -5434 RENTherefore this study was designed to investigate the

possible role of the genetic variant C-5434T in regulating theexpression of REN and confirmed by serum renin levels inhypertensive patients in Indonesia

2 Materials and Methods

21 Subjects Fifty six patients with hypertension at theOutpatient Clinic of Dr Saiful Anwar General HospitalMalang Indonesia were enrolled in this study Patients withany form of secondary hypertension overt renal insufficiency(serum creatinine gt15mgdL) pregnancy and estrogen andcorticosteroid therapy were excluded The age gender BMI(kgm2) SBP DBP smoking status and normal laboratoryvalues for physiological homeostasis were required

22 Detection of Polymorphism GenomicDNA sampleswereidentified by means of a PCR followed by a RFLP ThePCR product was amplified to 376 bp using 51015840CGTAGTGCC-ATTTTTAGGAAC31015840 and 51015840TTTCTACTTACCAAATGG-CGTC31015840 The RFLP products were incubated at 65∘C for 5hours The presence of renin gene polymorphism resulted ina loss of theMaeII restriction site (51015840-ACGT-31015840)The digestedfragments were separated on a 15 agarose gel

23 Renin Levels Serum samples of all patients werescreened using indirect ELISAwith the following renin (A-1)sc-137252 as first antibodies and rabbit anti-human IgG-HRP(sc-2769) as secondary antibodies

24 Starting Structure of DNA and STAT3 A double-stranded 18 bp DNA was built using 3D-DART providedby HADDOCK (httphaddockscienceuunlservices3DDART) [7] We generated 3D structural models of DNAfrom sequences of REN enhancer (51015840-AGTTTTACTAGA-ACGTAG-31015840) for allele C and (51015840-AGTTTTACTAGAATG-TAG-31015840) for allele T The homology model of STAT3 human

was constructed using USF Chimera The structure fromprotein structure database under the PDB ID number 1BG1was chosen as a template for modeling

25 Docking Procedure STAT3 and DNA were docked usingthe docking program HADDOCK (httphaddockscienceuunlservicesHADDOCK) [7 8] HADDOCK was runusing its default but with additional information about activesite of DNA (T5 T6 A7 C8 T9 10A G11 A12 A13 andC14T14) and a list of amino acids that might be involved ininteractions with the DNA (M331 H332 K340 T341 V343Q344 R382 E415 R417 R423 I431 V432 S465 N466 andI467) The best 40 complex structures were selected on thebasis of HADDOCK score Then the complex of dockingresult was analyzed using NUCPLOT to know the aminoacids and nucleotides responsible for the interaction

26 Analysis Baseline characteristics and serum renin levelsfinding between 3 groups (CC CT and TT) were comparedby one-way ANOVA test for parametric and Chi-square testfor nonparametric analysis For all tests a 119901 value le005was considered statistically significant Statistical analysiswas performed with SPSS 160 For DNA-protein dockingwe analyzed descriptively binding pattern and protein-DNAcontacts

27 Ethics The study was approved by the local committeeon medical research ethics Written informed consent wasobtained from all study participants

3 Results

31 Baseline Characteristics The baseline characteristics ofhypertensive patients between 3 groups (CC CT and TT) aresummarized in Table 1 Statistical analysis showed that therewas no significant difference at each baseline

32 Analysis of Genetic Variation We amplified a 376 bpregion (-5547 to -5172) using PCR followed by a RFLP

International Journal of Hypertension 3

5998400

3998400

(a)

5998400

3998400

(b)

Figure 1The differences of binding pattern in STAT3-DNA allele C (a) and STAT3-DNA allele T (b) interactions STAT3 protein (dark green)with DNA (gold)

Table 2 Comparison of genetic variant REN C-5434T and serumrenin level

VariableREN C-5434T

119901 valuelowastCC(119899 = 16)

CT(119899 = 31)

TT(119899 = 9)

Reninconcentration(pgmL)

33 plusmn 1063 36 plusmn 777 39 plusmn 1646 035

lowast

119901 value le 005 significantly different between groups

Only one SNP was identified at position -5434 (CrarrT) Thegenotype frequencies in this population were 286 for theCC genotype 554 for the CT genotype and 16 for theTT genotypeThe allele frequencies were 56 for C allele and44 for T allele

33 Renin Serum Level and Genetic Variant C-5434T RENFifty six patients were divided into 3 groups according togenotyping result (CC CT andTT)Then renin levels of eachpatient was measured using indirect elisa method Statisticalanalysis (Table 2) showed that serum renin levels were higherin patients with TT genotype (39 plusmn 1646) than patients withCC genotype (33 plusmn 1063) and CT genotype (36 plusmn 777) butthe difference was not statistically significant (119901 = 035)

34 STAT3 Protein-DNA Docking A monomeric structureof STAT3 was used in this study This structure consists ofcoiled-coil domain DNA binding domain and SH2 domainIn order to directly study the interactions between STAT3 andDNA the monomeric structure of this protein was dockedonto its DNA consensus sequence The docking resultsshowed that DNA binding domain of STAT3 was directly incontact with the major groove of DNA allele T In contrastthere were changes in the conformation of the bindingpattern DNA binding domain only focused on the minorgroove of DNA allele C (Figure 1) Furthermore we analyzedin more detail the differences of complex docking usingNUCPLOT program The specific amino acid-nucleotidecontacts were analyzed (Table 3) Most of the amino acidsin complex STAT3 and DNA allele C bond were unfavorablecontacts but complex STAT3 and DNA allele T bond werefavorable contacts in Arg423 residue with guanine 29 Overall

analysis of the docking results showed that STAT3 morepreferentially binds to -5434T variant than to -5434C variant

4 Discussion

Renin-angiotensin system (RAS) plays a pivotal role in themaintenance of blood pressure [9] Molecular variants ofthe renin gene thought to be a genetic risk factor may beinvolved in the etiology of hypertension [10] In this studywe found the presence of SNP C-5434T genetic variant in ourpopulation Then we investigated the relationship betweengenetic variant C-5434T of REN enhancer and serum reninlevels in Indonesian hypertensive patients The data showedthat serum renin levels were higher in patients with TT thanpatients with CC and CT genotype but the difference was notstatistically significant

Contributions of renin in hypertension are associatedwith the presence of genetic variation in this gene Theexistence of a wide range of genetic variations affects theREN transcriptional activity in producing renin [11] Previousstudies which identified three variants of the renin genenamely SNP T 17int4G VNTR in intron 7 and missensemutation in exon 9 (G105A) in the Japanese populationshowed that the missense mutation in exon 9 affects theenzymatic function of renin [10] On the other hand theysuggested that -5312 -5434 and A BglI G polymorphisms ofrenin gene might play an important role in the occurrenceof arterial hypertension (AH) But a single analysis of the C-5434T found no significant association with the incidence ofAH [12]

Here we also present a docking approach for predictinginteraction between genetic variant -5434CT and STAT3Based on binding pattern (see Figure 1) we found that STAT3more preferentially binds to DNA in SBE sequence (in alleleT)

The protein that interacts on themajor groove of B-DNAespecially in binding sequence shows a more functionalgroup that identifies base pairs The major groove of DNAis rich in chemical information compared to minor grooveand is important for recognition by nucleotide sequencespecific binding protein [13 14] The difference in the STAT3binding in allele C (focused on the minor groove) suggeststhat STAT3 loses contact with the center of SBE sequencewhich is very likely not able to stimulate transcription ofREN Then we focused on contact residues analysis between


Table 3 Protein-DNA contacts (hydrogen bonds) observed in STAT3-DNA allele Callele T

STAT3-DNA allele C STAT3-DNA allele TAcceptor Residue contact Description Acceptor Residue contact DescriptionT5 Gln344 NE2 Unfavorable contact T4 Gln344 NE2 Unfavorable contact

T6 His332 NE2Lys340 NZ Unfavorable contact T5 Gln344 N Unfavorable contact

A7 Lys340 NZ Unfavorable contact T6 Lys340 NZ Unfavorable contact

A13 Arg417 NH1 Unfavorable contact T14 Asp427 NAla428 N Unfavorable contact

C14 Arg423 NE Unfavorable contact T27 Thr433 OG1Gln469 NE2 Unfavorable contact

G15 Arg423 NH1 Unfavorable contact G29 Arg417NE NH2 Favorable contact

A28 Arg417 NH2 Unfavorable contact T30 Arg417 NH2 Unfavorable contact

T27Arg382 NH1

Val432Gln469 NE2

Unfavorable contact

STAT3 and DNA allele CT complexes Active residues ofSTAT3 are Arg417 and Arg423 [15] In this study we foundthat most of the amino acids in complex STAT3-DNA alleleC bond were unfavorable contacts In contrast STAT3-DNAallele T bond was a favorable contact in Arg423 with G29Interaction between arginine or lysine and guanine givesfavorable contacts [16] and the presence of arginine residuemediates the nuclear translocation of STAT3 [17] This resultmight explain why the renin level of REN in -5434T washigher compared to -5434C

However in our study the number of subjects examinedwas too small Moreover an adequate number of samples areneeded in further study to analyze the statistical significanceof the association between genotype and phenotype Besidesthat it is interesting to analyze transcription level of STAT3using in vitro studies to validate the in silico result

5 Conclusion

REN is considered as one of the target genes that are directlyregulated by STAT3 STAT3 more favorably binds to DNA inSBE sequence allele T than allele C Thus the presence ofgenetic variant C-5434T can change the binding pattern ofSTAT3 to REN enhancer This is likely to influence STAT3activity to stimulate transcriptional activity in producingrenin

Conflict of Interests

No potential conflict of interests relevant to this paper wasreported

Acknowledgments

The authors thank the hypertension project team (MifetikaL Arina M Jayarani F Aditya K Rusdianto Akhiyan HFrastiqa F D S Aprilia Shila S Amelia I and Ika Arum)and their families for excellent technical help and support

This paper was supported by Saiful Anwar General HospitalMalang Indonesia

References

[1] P A Doris ldquoHypertension genetics single nucleotide polymor-phisms and the common disease common variant hypothesisrdquoHypertension vol 39 no 2 pp 323ndash331 2002

[2] L C van Vark M Bertrand K M Akkerhuis et alldquoAngiotensin-converting enzyme inhibitors reduce mortality inhypertension a meta-analysis of randomized clinical trials ofrenin-angiotensin-aldosterone system inhibitors involving 158998 patientsrdquo European Heart Journal vol 33 no 16 pp 2088ndash2097 2012

[3] N Petrovic T A Black J R Fabian et al ldquoRole of proximalpromoter elements in regulation of renin gene transcriptionrdquoJournal of Biological Chemistry vol 271 no 37 pp 22499ndash22505 1996

[4] S Fuchs J Philippe S Germain et al ldquoFunctionality of twonew polymorphisms in the human renin gene enhancer regionrdquoJournal of Hypertension vol 20 no 12 pp 2391ndash2398 2002

[5] L Pan C A Jones S T Glenn and KW Gross ldquoIdentificationof a novel region in the proximal promoter of the mouse reningene critical for expressionrdquoTheAmerican Journal of PhysiologyRenal Physiology vol 286 no 6 pp F1107ndashF1115 2004

[6] H Castrop K Hocherl A Kurtz F Schweda V Todorov andC Wagner ldquoPhysiology of kidney reninrdquo Physiological Reviewsvol 90 no 2 pp 607ndash673 2010

[7] S J de Vries M van Dijk and A M J J Bonvin ldquoTheHADDOCKweb server for data-driven biomolecular dockingrdquoNature Protocols vol 5 no 5 pp 883ndash897 2010

[8] T A Wassenaar M van Dijk N Loureiro-Ferreira et alldquoWeNMR structural biology on the gridrdquo Journal of GridComputing vol 10 no 4 pp 743ndash767 2012

[9] M A Perazella and J F Setaro ldquoRenin-angiotensin-aldosteronesystem fundamental aspects and clinical implications in renaland cardiovascular disordersrdquo Journal of Nuclear Cardiologyvol 10 no 2 pp 184ndash196 2003


[10] B Hasimu T Nakayama Y Mizutani et al ldquoHaplotype analysisof the human renin gene and essential hypertensionrdquoHyperten-sion vol 41 no 2 pp 308ndash312 2003

[11] N Moore P Dicker J K OrsquoBrien et al ldquoRenin gene polymor-phisms and haplotypes blood pressure and responses to renin-angiotensin system inhibitionrdquo Hypertension vol 50 no 2 pp340ndash347 2007

[12] N M Chikhladze K F Samedova M A Sudomoina et alldquoContribution of CYP11B2 REN and AGT genes in geneticpredisposition to arterial hypertension associated with hyper-aldosteronismrdquo Kardiologiia vol 48 no 1 pp 37ndash42 2008

[13] H Lodish A Berk S L Zipursky et al Molecular CellBiology Section 105 Eukaryotic Transcription Activators andRepressors W H Freeman New York NY USA 4th edition2000

[14] J Watson T A Baker S P Bell A Gann M Levine and RLosick ldquoMaintenance of the genomerdquo in Molecular Biology ofthe Gene Part 2 Chapter 6 Pearson Benjamin Cummings SanFransisco Calif USA 2004

[15] S Becker B Groner and C W Muller ldquoThree-dimensionalstructure of the Stat3120573 homodimer bound toDNArdquoNature vol394 no 6689 pp 145ndash151 1998

[16] B Lustig and R L Jernigan ldquoConsistencies of individual DNAbase-amino acid interactions in structures and sequencesrdquoNucleic Acids Research vol 23 no 22 pp 4707ndash4711 1995

[17] J Ma T Zhang V Novotny-Diermayr A L C Tan and X CaoldquoAnovel sequence in the coiled-coil domain of Stat3 essential forits nuclear translocationrdquo Journal of Biological Chemistry vol278 no 31 pp 29252ndash29260 2003

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Table 1 Baseline characteristics of the patients

Variable REN C-5434T119901 valuelowast

CC (119899 = 16) CT (119899 = 31) TT (119899 = 9)Age (years) 57 plusmn 673 59 plusmn 877 58 plusmn 698 057Gender (MF) 79 1417 72 019SBP (mmHg) 138 plusmn 1543 142 plusmn 1887 140 plusmn 1054 078DBP (mmHg) 89 plusmn 1062 84 plusmn 1115 87 plusmn 666 021Weight (kg) 65 plusmn 1320 66 plusmn 1124 67 plusmn 1054 087Height (cm) 157 plusmn 768 158 plusmn 820 161 plusmn 855 045BMI (kgm2) 27 plusmn 478 26 plusmn 368 26 plusmn 298 097Urea (mgdL) 29 plusmn 918 29 plusmn 1593 28 plusmn 867 095Creatinine (mgdL) 14 plusmn 127 09 plusmn 022 1 plusmn 026 013Blood glucose (mgdL) 98 plusmn 2832 95 plusmn 961 91 plusmn 969 058Smoking 115 031 18 022Cholesterol (mgdL) 182 plusmn 5986 185 plusmn 3838 181 plusmn 3587 096SBP systolic blood pressure DBP diastolic blood pressure BMI body mass index lowast119901 value le 005 significantly different between groups

(SBE) 51015840-TT(N5)AA-31015840 STAT3 is thought to be the major

transcription factor in genetic variant -5434 RENTherefore this study was designed to investigate the

possible role of the genetic variant C-5434T in regulating theexpression of REN and confirmed by serum renin levels inhypertensive patients in Indonesia

2 Materials and Methods

21 Subjects Fifty six patients with hypertension at theOutpatient Clinic of Dr Saiful Anwar General HospitalMalang Indonesia were enrolled in this study Patients withany form of secondary hypertension overt renal insufficiency(serum creatinine gt15mgdL) pregnancy and estrogen andcorticosteroid therapy were excluded The age gender BMI(kgm2) SBP DBP smoking status and normal laboratoryvalues for physiological homeostasis were required

22 Detection of Polymorphism GenomicDNA sampleswereidentified by means of a PCR followed by a RFLP ThePCR product was amplified to 376 bp using 51015840CGTAGTGCC-ATTTTTAGGAAC31015840 and 51015840TTTCTACTTACCAAATGG-CGTC31015840 The RFLP products were incubated at 65∘C for 5hours The presence of renin gene polymorphism resulted ina loss of theMaeII restriction site (51015840-ACGT-31015840)The digestedfragments were separated on a 15 agarose gel

23 Renin Levels Serum samples of all patients werescreened using indirect ELISAwith the following renin (A-1)sc-137252 as first antibodies and rabbit anti-human IgG-HRP(sc-2769) as secondary antibodies

24 Starting Structure of DNA and STAT3 A double-stranded 18 bp DNA was built using 3D-DART providedby HADDOCK (httphaddockscienceuunlservices3DDART) [7] We generated 3D structural models of DNAfrom sequences of REN enhancer (51015840-AGTTTTACTAGA-ACGTAG-31015840) for allele C and (51015840-AGTTTTACTAGAATG-TAG-31015840) for allele T The homology model of STAT3 human

was constructed using USF Chimera The structure fromprotein structure database under the PDB ID number 1BG1was chosen as a template for modeling

25 Docking Procedure STAT3 and DNA were docked usingthe docking program HADDOCK (httphaddockscienceuunlservicesHADDOCK) [7 8] HADDOCK was runusing its default but with additional information about activesite of DNA (T5 T6 A7 C8 T9 10A G11 A12 A13 andC14T14) and a list of amino acids that might be involved ininteractions with the DNA (M331 H332 K340 T341 V343Q344 R382 E415 R417 R423 I431 V432 S465 N466 andI467) The best 40 complex structures were selected on thebasis of HADDOCK score Then the complex of dockingresult was analyzed using NUCPLOT to know the aminoacids and nucleotides responsible for the interaction

26 Analysis Baseline characteristics and serum renin levelsfinding between 3 groups (CC CT and TT) were comparedby one-way ANOVA test for parametric and Chi-square testfor nonparametric analysis For all tests a 119901 value le005was considered statistically significant Statistical analysiswas performed with SPSS 160 For DNA-protein dockingwe analyzed descriptively binding pattern and protein-DNAcontacts

27 Ethics The study was approved by the local committeeon medical research ethics Written informed consent wasobtained from all study participants

3 Results

31 Baseline Characteristics The baseline characteristics ofhypertensive patients between 3 groups (CC CT and TT) aresummarized in Table 1 Statistical analysis showed that therewas no significant difference at each baseline

32 Analysis of Genetic Variation We amplified a 376 bpregion (-5547 to -5172) using PCR followed by a RFLP


5998400

3998400

(a)

5998400

3998400

(b)



VariableREN C-5434T

119901 valuelowastCC(119899 = 16)

CT(119899 = 31)

TT(119899 = 9)



lowast






4 Discussion














T27Arg382 NH1

Val432Gln469 NE2

Unfavorable contact



5 Conclusion




Acknowledgments



References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5998400

3998400

(a)

5998400

3998400

(b)



VariableREN C-5434T

119901 valuelowastCC(119899 = 16)

CT(119899 = 31)

TT(119899 = 9)



lowast






4 Discussion














T27Arg382 NH1

Val432Gln469 NE2

Unfavorable contact



5 Conclusion




Acknowledgments



References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























T27Arg382 NH1

Val432Gln469 NE2

Unfavorable contact



5 Conclusion




Acknowledgments



References
























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Genetic Variant of C-5434T REN Enhancer …downloads.hindawi.com/journals/ijhy/2015/486961.pdf · Research Article Genetic Variant of C-5434T REN Enhancer on Serum