Research Article Prevalence of Catalase (-21 A/T) Gene ...downloads.hindawi.com/journals/bmri/2014/894237.pdf · decreased catalase activity can be in uenced by the disease itself

Research ArticlePrevalence of Catalase (-21 AT) Gene Variant inSouth Indian (Tamil) Population

A Lourdhu Mary K Nithya W Isabel and T Angeline

PG amp Research Department of Zoology and Biotechnology Lady Doak College Madurai Tamil Nadu 625 002 India

Correspondence should be addressed to A Lourdhu Mary lourdhubrittogmailcom

Received 28 February 2014 Revised 5 June 2014 Accepted 5 June 2014 Published 26 June 2014

Academic Editor Swaran J S Flora

Copyright copy 2014 A Lourdhu Mary 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

Catalase an endogenous antioxidant enzyme is responsible for regulating reactive species levels Several epidemiologic studies havesuggested that single nucleotide polymorphism in catalase gene may be associated with many diseases The genotype of CAT (-21AT) point mutation in promoter region of catalase gene was determined by polymerase chain based restriction fragment lengthpolymorphism analysis in the DNA of 100 healthy volunteersThe frequency of CAT (-21 AT) gene polymorphismAA AT and TTgenotypes was found to be 7 23 and 70 percent respectivelyThemutant ldquoTrdquo allele frequency was found to be 082 among the southIndian (Tamil) population Chi square analysis showed that the study population lies within the Hardy-Weinberg equilibriumThewild type genotype (AA) was found to be very low (7) and the mutant genotype (ATTT) was found to be more prevalent (93)among the south Indian population This suggests that the high prevalence of mutant genotype may increase the susceptibility tooxidative stress associated diseases

1 Introduction

Excessive oxidative stress has been implicated in the patho-genesis of several diseases In humans the antioxidantenzymes pathway is available to protect against the toxicitycaused by oxidative stress Many of the antioxidant genesare known to be polymorphic which may alter the enzymeactivity [1]

Catalase is an antioxidant enzyme that plays a major rolein controlling hydrogen peroxide concentration in humancells It decomposes H

2O2into H

2O and O

2 thereby pro-

tecting the cells from oxidative stress It has been suggestedthat functional polymorphism in the gene encoding catalaseenzyme affects the enzyme activity thereby reducing theprotection against oxidative stress [2] Apart from the decom-position of H

2O2 catalase inactivates many environmental

mutagens It also prevents chromosomal aberration causedby hypoxanthinexanthine oxidase in Chinese hamster cells[3] Several studies have reported on the alterations in catalaseactivity in hypertension [4] cancer [5] diabetes nephropathy[6 7] and other diseases accompanied by oxidative stress

Catalase is a homotetramer of 220ndash230 kDa mass havingfour heme groups in its structure [8 9] The CAT gene is

localized on chromosome 11p 1331 consisting of 13 exonsand 12 introns CAT (-21 AT) variant represents the A to Tsubstitution in the promoter region which is located insidethe promoter region just proximal to the start site [8] Themutant allele frequency may vary based on the differencesin the study populationrsquos race and ethnicity and also on theunderlying characters

This study which has been focused on the genetic variant-21 AT of the catalase enzyme was the first kind regardingthe prevalence of catalase gene variant in healthy individualsamong south Indian Tamil population The objective was tofind out the prevalence of mutant allele frequency of theevaluated catalase (-21 AT) gene polymorphism in healthyindividuals among the south Indian population

2 Materials and Methods

21 Study Design A total of 100 healthy controls with nohistory of cardiovascular disease diabetes hypertensioncancer or any infectious diseases and belonged to southIndian Tamil ethnicity were included for the study Thesamples from the volunteers were collected into EDTA coated

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 894237 4 pageshttpdxdoiorg1011552014894237

2 BioMed Research International

Lane 6 molecular weight marker (100bp)

250bp

Lane 1ndash5 PCR product (250bp)

Figure 1 PCR analysis of the catalase gene

tubes and the informed consent was obtained The presentstudy was approved by the ethical committee and biosafetycommittee of the institution

22 DNA Extraction Genomic DNA was extracted from thefrozen blood by phenol-chloroform method [10] For DNAextraction 500120583L of blood was used and the isolated DNAdissolved in TE was stored at minus20∘C The quality of the DNAwas checked in 07 percent agarose (Hi-Media Mumbai) gelelectrophoresis and quantified using UV spectrophotometry(Hitachi Japan)

23 PCR Analysis PCR analysis was carried out using a ther-mal cycler (Eppendorf Master Cycler Germany) Approxi-mately 120 ng of genomic DNA was incubated in a total reac-tion mixture of 50120583L containing both the forward primer 51015840-AATCAGAAGGCAGTCCTCCC-31015840 and the reverse primer51015840-TCGGGGAGCACAGAGTGTAC-31015840 (sim70 picomoles)(GenScript Corp USA) 200120583M deoxynucleotide triphos-phate 10X PCR buffer of pH-83 containing 15mM MgCl

2

and 5 units of Taq DNA polymerase (New England BiolabsBeverly) [11] DNA was initially denatured at 95∘C for 4minprior to amplification A 250 bp of catalase genewas amplifiedusing the following conditions with 30 cycles consisting of1min denaturation at 94∘C 40 sec annealing at 61∘C and1min extension at 72∘C The final extension included 5minsat 72∘C and the PCR products were confirmed by ethidiumbromide stained 12 percent agarose gel and viewed in theUV-transilluminator (Figure 1)

24 Restriction Enzyme Analysis Restriction digestion wasperformed in a total volume of 10 120583L consisting of 5 120583Lamplicon 1 120583L NE buffer (50mM potassium acetate 20mMTris-acetate 10mM magnesium acetate and 1mM dithio-threitol pH-79 at 25∘C) and 10U of Hinf 1 enzyme (Fer-mentas Life Sciences Germany) [11] Samples were incubated

1 2 3 4 5 76


250bp

Lane 3 and 7 variant genotype (TT)-250bp

177bp

73bp

250bp Lane 4 and 5 heterozygous genotype (AT)- 177bp and 73bp

Lane 2 and 6 wild genotype (AA)-177bp and 73bp

Figure 2 Restriction bandpattern of catalase (-21AT) gene variant

Table 1 Catalase gene -21 AT polymorphism-genotype and allelefrequency in healthy controls

GenotypeAllele Healthy controls (119899 = 100)Genotypes (-21 AT)

AA 7AT 23TT 70

AllelesA 018T 082

for 6-7 hrs at 37∘C and the digested PCR products wereresolved in 2 percent agarose gel electrophoresis stainedwith ethidium bromide and separated bands were observedusing gel documentation system (Figure 2) The A rarr Tmutation at nucleotide 250 bp abolishes a Hinf 1 restrictionsite (T allele) which otherwise forms 177 and 73 bp fragments(A allele) when treated with Hinf 1 The catalase gene (-21)polymorphism abolishes a Hinf 1 site and so there will not beany cleavage of the PCR products (250 bp) and if there is nomutation (normal genotype-AA) the band pattern in the gelwill show the cleavage of the fragment into 177 bp and 73 bpIn the case of heterozygous genotype (AT) all the three typesof fragments (250 bp 177 bp and 73 bp) will be observed inthe gel

25 Sequencing of the CAT Gene The PCR product obtainedwas subjected to DNA sequencing which was carried outby Sangerrsquos sequencing method (Synergy scientific servicesChennai) [12] The DNA sequencing was done to checkand confirm whether the amplified product was CAT genesequence or not The obtained sequence was then subjectedto the BLASTN [13] analysis to study the homology sequenceof the amplified product

BioMed Research International 3

Table 2 Prevalence of the catalase gene -21 AT polymorphism in healthy controls

Country Ethnic population Number of subjects Genotype frequency T allele frequencyAA AT TT

India South Indian(present study) 100 7 23 70 082

Brazil [24] Brazilian 124 184 424 392 0604Finland [11] Finnish 245 18 48 34 058Brazil [25] Brazilian 135 22 39 39 057Czech Republic [15] Czechs 180 37 48 15 040China [5] Chinese 848 32 56 12 040China [2] Chinese 386 54 38 8 0272New Zealand [16] Europeans 190 48 42 10 031

3 Results and Discussion

The -21 AT (rs7943316) polymorphism in CAT gene gains itsimportancemainly by its position being close to transcriptionstart site or at sites where the transcription factors bind [14]In the present study one hundred healthy volunteers wereanalysed for SNP in catalase -21 AT gene polymorphism Ithas been observed that only 7 of the individuals have AAgenotype and 70 of individuals have TT genotype and theremaining 23 of individuals have AT heterozygous mutantgenotype (Table 1) The highest TT genotype (70) of -21 AT catalase gene polymorphism was observed amongsouth Indians and the lowest TT genotype (8) was observedamong Chinese population [2]

When BLAST N analysis was performed with the DNAsequence of PCR product (250 bp) 98 percent homologywas found between the CAT gene and the submitted DNAsequence The ldquoTrdquo allele frequency observed in the presentstudywas found to be very high (082) when compared to thatof Finnish population (058) [11] Czechs (04) [15] Europeans(031) [16] and Chinese (04 02) [2 5] (Table 2) A studyconducted among Indian population with diabetes mellitushas reported that 80 of the patients were of TT genotypeand 20were ofAT genotype AAgenotypewas not observedamong patients and healthy individuals were not included intheir study The study also found lower activity of scavengerenzymes in patients with diabetes mellitus [9] Howeverdecreased catalase activity can be influenced by the diseaseitself and not by gene polymorphism

Catalase is one of the major enzyme components of celldefense against oxidative stress and it has been hypothesizedthat the polymorphism of -21 AT CAT reduces the antiox-idant capacity and may serve as a risk factor for oxidativestress associated diseases The association between SOD1-251 AG CAT-21 AT and GPX1-198CT antioxidant genepolymorphisms in the risk of cataract was reported amongChinese population and was found to have no significantassociation inCAT-21AT andGPX1-198CTpolymorphismsbetween the controls and patients However SOD1-251 AGpolymorphism was found to be associated with an increasedrisk of cataract [2]

Experimental and clinical studies conducted amongessential hypertension and cerebral stroke patients suggested

that the polymorphism -21 AT CAT was associated withincreased risk and was found to play an important role inpathogenesis [17] Similar study conducted among Russianpopulation reported that there was an association betweenpolymorphism -21 AT of the catalase gene and bronchialasthma The study also reported that cigarette smokingand fruits and vegetables intake have potentially inversemodifying influences on the risk of asthma [18] In contrastfew studies reported that the catalase gene polymorphismwasnot related to the risk of cardiovascular diseases among type2 diabetes mellitus in Finnish population [11] and also amongtype 1 diabetes mellitus patients in Czech population [15]

Even though catalase was not essential for some celltypes under normal conditions it was reported to play animportant role in the adaptive response to oxidative stress[19 20] Earlier studies reported that the catalase activity wasfound to be higher among cancer patients However it hasbeen reported that an adaptive increase of catalase activitymay not be sufficient for inactivation of reactive oxygenspecies [21] Decreased catalase gene expression due to thepresence of mutant allele may further decrease the activityof catalase enzyme The presence of insufficient antioxidantenzyme system may increase the susceptibility to oxidativestress-associated diseases among south Indian population

Apart from -21 AT polymorphism of the catalase geneanother polymorphism (-262CT) in the promoter regionhas been shown to influence transcription factor bindingand correlates well with blood catalase levels [22] Therehas been a well-established association between the type 1diabetic neuropathy patients and the latter polymorphism inthe Russian population [23]

In conclusion the present study demonstrates that CAT(-21 AT) SNP is more prevalent among the south Indianpopulation Further study should be conducted to find outwhether there is alteration in catalase activity in the individ-uals with mutant genotype for CAT (-21 AT) polymorphismamong healthy individuals and also need to be correlatedwith any disease associated with oxidative stress such ascancer diabetes and coronary artery diseases The otherpolymorphisms within the same gene should also be studiedfor better understanding of the role of catalase gene in themediation of oxidative stress response


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Financial assistance by Science and Engineering ResearchBoard (SERB) New Delhi in the form of Young ScientistScheme (Fast Track) to Dr T Angeline is greatly acknowl-edged

References

[1] J E Klaunig L M Kamendulis and B A Hocevar ldquoOxidativestress and oxidative damage in carcinogenesisrdquo ToxicologicPathology vol 38 no 1 pp 96ndash109 2010

[2] Y Zhang L Zhang D Sun Z Li L Wang and P LiuldquoGenetic polymorphisms of superoxide dismutases catalaseand glutathione peroxidase in age-related cataractrdquo MolecularVision vol 17 pp 2325ndash2332 2011

[3] Y Kono and I Fridovich ldquoSuperoxide radical inhibits catalaserdquoThe Journal of Biological Chemistry vol 257 no 10 pp 5751ndash5754 1982

[4] X F Zhou J Cui A L Destefano et al ldquoPolymorphisms in thepromoter region of catalase gene and essential hypertensionrdquoDisease Markers vol 21 no 1 pp 3ndash7 2005

[5] D Chang Z L Hu L Zhang et al ldquoAssociation of catalasegenotype with oxidative stress in the predication of colorectalcancer modification by epidemiological factorsrdquo Biomedicaland Environmental Sciences vol 25 no 2 pp 156ndash162 2012

[6] A Ceriello A Morocutti F Mercuri et al ldquoDefective intracel-lular antioxidant enzyme production in type 1 diabetic patientswith nephropathyrdquoDiabetes vol 49 no 12 pp 2170ndash2177 2000

[7] A D Hodgkinson T Bartlett P J Oates B A Millwardand A G Demaine ldquoThe response of antioxidant genes tohyperglycemia is abnormal in patients with type 1 diabetes anddiabetic nephropathyrdquoDiabetes vol 52 no 3 pp 846ndash851 2003

[8] F Quan R G Korneluk M B Tropak and R A GravelldquoIsolation and characterization of the human catalase generdquoNucleic Acids Research vol 14 no 13 pp 5321ndash5335 1986

[9] K Dhanapal N Selvan and V Dhananjeyan ldquoA study oncatalase activity and its genetic polymorphism in diabetesmellitus patientsrdquo Journal of Biological Sciences vol 10 no 7pp 653ndash657 2010

[10] V M Iranpur and A K Esmailizadeh Rapid Extraction ofHigh Quality DNA from Whole Blood Stored at 4∘C for LongPeriod Department of Animal Science Faculty of AgricultureShahrekord University Shahrekord Iran 2010

[11] O Ukkola P H Erkkila M J Savolainen and Y A KesaniemildquoLack of association between polymorphisms of catalasecopper-zinc superoxide dismutase (SOD) extracellular SODand endothelial nitric oxide synthase genes and macroangiopa-thy in patients with type 2 diabetes mellitusrdquo Journal of InternalMedicine vol 249 no 5 pp 451ndash459 2001

[12] F Sanger and A R Coulson ldquoA rapid method for determiningsequences in DNA by primed synthesis with DNA polymeraserdquoJournal of Molecular Biology vol 94 no 3 pp 441ndash448 1975

[13] DWMount SequenceampGenomeAnalysis Cold SpringHarborLaboratory Press 2nd edition 2004

[14] N M Panduru E Mota M Mota D Cimponeriu C Ser-afinceanu and D M Cheta ldquoPolymorphism of catalase genepromoter in Romanian patients with diabetic kidney diseaseand type 1 diabetesrdquoRomanian Journal of InternalMedicine vol48 no 1 pp 81ndash88 2010

[15] M Flekac J Skrha J Hilgertova Z Lacinova and MJarolimkova ldquoGene polymorphisms of superoxide dismutasesand catalase in diabetes mellitusrdquo BMCMedical Genetics vol 9article 30 2008

[16] R P Young R Hopkins P N Black et al ldquoFunctional variantsof antioxidant genes in smokers with COPD and in those withnormal lung functionrdquoThorax vol 61 no 5 pp 394ndash399 2006

[17] E K VialykhM A Solodilova O Y Bushueva I V BulgakovaandA V Polonikov ldquoCatalase gene polymorphism is associatedwith increased risk of cerebral stroke in hypertensive patientsrdquoZhurnal Nevrologii i Psihiatrii imeni SS Korsakova vol 112 no8 pp 3ndash7 2012

[18] A V Polonikov V P IvanovM A SolodilovaM A Kozhuhovand V I Panfilov ldquoTobacco smoking fruit and vegetable intakemodify association between -21AgtT polymorphism of catalasegene and risk of bronchial asthmardquo Journal of Asthma vol 46no 3 pp 217ndash224 2009

[19] J M Mates C Perez-Gomez and I N de Castro ldquoAntioxidantenzymes and human diseasesrdquoClinical Biochemistry vol 32 no8 pp 595ndash603 1999

[20] P Venditti P Masullo S Di Meo and C Agnisola ldquoProtectionagainst ischamia-reperfusion induced oxidative stress by vita-min E treatmentrdquo Archives of Physiology and Biochemistry vol107 no 1 pp 27ndash34 1999

[21] D Hristozov V Gadjeva T Vlaykova and G Dimitrov ldquoEval-uation of oxidative stress in patients with cancerrdquo Archives ofPhysiology and Biochemistry vol 109 no 4 pp 331ndash336 2001

[22] L Forsberg L Lyrenas U de Faire and R MorgensternldquoA common functional C-T substitution polymorphism inthe promoter region of the human catalase gene influencestranscription factor binding reporter gene transcription andis correlated to blood catalase levelsrdquo Free Radical Biology andMedicine vol 30 no 5 pp 500ndash505 2001

[23] D A Chistiakov E V Zotova K V Savostanov et alldquoThe 262TgtC promoter polymorphism of the catalase gene isassociated with diabetic neuropathy in type 1 diabetic Russianpatientsrdquo Diabetes and Metabolism vol 32 no 1 pp 63ndash682006

[24] A L Miranda-Vilela A K Akimoto P C Alves et al ldquoDietarycarotenoid-rich pequi oil reduces plasma lipid peroxidationand DNA damage in runners and evidence for an associationwith MnSOD genetic variant -Val9Alardquo Genetics and MolecularResearch vol 8 no 4 pp 1481ndash1495 2009

[25] A L Miranda-Vilela P C Z Alves A K Akimoto et al ldquoGenepolymorphisms against DNA damage induced by hydrogenperoxide in leukocytes of healthy humans through cometassay a quasi-experimental studyrdquo Environmental Health vol9 article 21 2010

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology



250bp

Lane 1ndash5 PCR product (250bp)

Figure 1 PCR analysis of the catalase gene

tubes and the informed consent was obtained The presentstudy was approved by the ethical committee and biosafetycommittee of the institution

22 DNA Extraction Genomic DNA was extracted from thefrozen blood by phenol-chloroform method [10] For DNAextraction 500120583L of blood was used and the isolated DNAdissolved in TE was stored at minus20∘C The quality of the DNAwas checked in 07 percent agarose (Hi-Media Mumbai) gelelectrophoresis and quantified using UV spectrophotometry(Hitachi Japan)

23 PCR Analysis PCR analysis was carried out using a ther-mal cycler (Eppendorf Master Cycler Germany) Approxi-mately 120 ng of genomic DNA was incubated in a total reac-tion mixture of 50120583L containing both the forward primer 51015840-AATCAGAAGGCAGTCCTCCC-31015840 and the reverse primer51015840-TCGGGGAGCACAGAGTGTAC-31015840 (sim70 picomoles)(GenScript Corp USA) 200120583M deoxynucleotide triphos-phate 10X PCR buffer of pH-83 containing 15mM MgCl

2

and 5 units of Taq DNA polymerase (New England BiolabsBeverly) [11] DNA was initially denatured at 95∘C for 4minprior to amplification A 250 bp of catalase genewas amplifiedusing the following conditions with 30 cycles consisting of1min denaturation at 94∘C 40 sec annealing at 61∘C and1min extension at 72∘C The final extension included 5minsat 72∘C and the PCR products were confirmed by ethidiumbromide stained 12 percent agarose gel and viewed in theUV-transilluminator (Figure 1)

24 Restriction Enzyme Analysis Restriction digestion wasperformed in a total volume of 10 120583L consisting of 5 120583Lamplicon 1 120583L NE buffer (50mM potassium acetate 20mMTris-acetate 10mM magnesium acetate and 1mM dithio-threitol pH-79 at 25∘C) and 10U of Hinf 1 enzyme (Fer-mentas Life Sciences Germany) [11] Samples were incubated

1 2 3 4 5 76


250bp

Lane 3 and 7 variant genotype (TT)-250bp

177bp

73bp

250bp Lane 4 and 5 heterozygous genotype (AT)- 177bp and 73bp

Lane 2 and 6 wild genotype (AA)-177bp and 73bp

Figure 2 Restriction bandpattern of catalase (-21AT) gene variant

Table 1 Catalase gene -21 AT polymorphism-genotype and allelefrequency in healthy controls

GenotypeAllele Healthy controls (119899 = 100)Genotypes (-21 AT)

AA 7AT 23TT 70

AllelesA 018T 082

for 6-7 hrs at 37∘C and the digested PCR products wereresolved in 2 percent agarose gel electrophoresis stainedwith ethidium bromide and separated bands were observedusing gel documentation system (Figure 2) The A rarr Tmutation at nucleotide 250 bp abolishes a Hinf 1 restrictionsite (T allele) which otherwise forms 177 and 73 bp fragments(A allele) when treated with Hinf 1 The catalase gene (-21)polymorphism abolishes a Hinf 1 site and so there will not beany cleavage of the PCR products (250 bp) and if there is nomutation (normal genotype-AA) the band pattern in the gelwill show the cleavage of the fragment into 177 bp and 73 bpIn the case of heterozygous genotype (AT) all the three typesof fragments (250 bp 177 bp and 73 bp) will be observed inthe gel

25 Sequencing of the CAT Gene The PCR product obtainedwas subjected to DNA sequencing which was carried outby Sangerrsquos sequencing method (Synergy scientific servicesChennai) [12] The DNA sequencing was done to checkand confirm whether the amplified product was CAT genesequence or not The obtained sequence was then subjectedto the BLASTN [13] analysis to study the homology sequenceof the amplified product


















Acknowledgment


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















Acknowledgment


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgment


References

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Prevalence of Catalase (-21 A/T) Gene ...downloads.hindawi.com/journals/bmri/2014/894237.pdf · decreased catalase activity can be in uenced by the disease itself