Research Article Study of Cysteine-Rich Protein 61 Genetic ...downloads.hindawi.com/journals/gri/2015/754872.pdf · Research Article Study of Cysteine-Rich Protein 61 Genetic Polymorphism

Research ArticleStudy of Cysteine-Rich Protein 61 Genetic Polymorphism inPredisposition to Fracture Nonunion A Case Control

Sabir Ali1 Syed Rizwan Hussain1 Ajai Singh1 Vineet Kumar1 Shah Walliullah1

Nazia Rizvi1 Manish Yadav1 Mohammad Kaleem Ahmad2 and Abbas Ali Mahdi2

1Department of Orthopaedic Surgery King Georgersquos Medical University Lucknow Uttar Pradesh 226 018 India2Molecular Cell Biology Lab Biochemistry King Georgersquos Medical University Lucknow Uttar Pradesh 226 003 India

Correspondence should be addressed to Ajai Singh as29762gmailcom

Received 9 October 2015 Accepted 17 November 2015

Academic Editor Francine Durocher

Copyright copy 2015 Sabir Ali et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Many factors are responsible for this impaired healing especially in long bones but a possible genetic predispositionfor the development of this complication remains unknown till now In the present study we aim to examine the CYR61 genepolymorphism in fracture nonunion patients and the correlation with clinical findings Materials and Methods We performedSNP analysis of the CYR61 gene in 250 fracture nonunion patients and 250 healthy subjects were genotyped in this hospital-based case control study and 56 cases were further evaluated for mRNA expression of CYR61 by real-time quantitative reverse-transcription PCR Results CYR61 gene TT TG and GG genotype frequencies of total fracture nonunion cases were 416 492and 920 and 544 392 and 640 in healthy controlsHeterozygousTGgenotypewas found statistically significant in fracturenonunion cases compared with that in controls whereas homozygous mutant GG genotype was not found significant Moreoverwe found that TG + GG genotypes were significantly different in serum expression of CYR61 mRNA when compared with cases(TT genotypes) Conclusions Our result signifies that genotype of CYR61 affects the mRNA expression and acts as a risk factor thatcould synergistically increase the susceptibility of a patient to develop fracture nonunion

1 Introduction

Fractures are a common orthopaedic problem mostly inlong bones Of all long bones shaft of tibia is one of thecommonest bones to get fractured with a relatively higherincidence of impaired healing at the fracture site due tolesser soft tissue coverage being a subcutaneous bone onanterior aspect [1 2] The mentioned reasons account for ahigh rate of tibial nonunions amounting for 2ndash10 of all tibialfractures leading to significant patientmorbidity [3ndash6] Apartfrom the reasons mentioned other factors contributing tofracture nonunion are as follows soft tissue damage inad-equate mechanical stability open fractures administrationof pharmacological agents such as NSAIDs smoking andso forth [7 8] Adequacy of vascular supply to the fracturesite is an essential prerequisite for healing process whereasinadequate vascularity results in delayednonunion [9]

Fracture shaft of tibia still frequently develops nonuniondespite all optimizedmultifactorial conditionsThis tendency

of fracture may reflect the role of some major geneticcomponents involved in the process of bone regenerationand fracture repair There are studies which support geneticvariability as one of the significant associations contributingto the process of bone regeneration as well as fracture healingrate [10 11] Although associations with genetic variability aremuch talked about their potential role in predisposition tofracture impairment still remains unknown and needs to beelucidated

The CYR61 (cysteine-rich protein 61) gene is located onchromosome 1p22 This is an important molecule which wasshown to participate in a number of key cellular processesincluding cell differentiation adhesion migration prolifer-ation wound healing and angiogenesis [12ndash14] The CYR61gene is key signalling molecules involved in angiogenesisthat is prerequisite for the initial process of fracture healingMany times due to improper angiogenesis at the fracturesite impaired healing may occur As per our knowledgepresent study is the first to suggest the possible effect of a

Hindawi Publishing CorporationGenetics Research InternationalVolume 2015 Article ID 754872 5 pageshttpdxdoiorg1011552015754872

2 Genetics Research International

functional polymorphism (promoter region rs3753793) inthe CYR61 gene on expression of CYR61 mRNA in nonuniontibial fracture cases

The role of the single nucleotide polymorphism (SNP)involving CYR61 gene in fracture healing if proved mayopen new horizons for innovations in this field with anaddition to our armamentarium to deal with complicationsassociated with fracture healing The association if estab-lished will add genetic predisposition as a new risk factor forimpaired bone healing It can also in the future be used as animportant prognostic tool for early identification of patientsat risk of impaired fracture healing

This study is probably the first of its kind carried out innorth Indian population to evaluate the association betweenCYR61 gene polymorphism ((T rarr G) gene polymorphism)and fracture nonunion in shaft of tibia

2 Materials and Methods

21 Samples Collection A total of 250 fracture shafts oftibia with nonunion cases and 250 healthy subjects wereselected for the study The study was carried out in theDepartment of Orthopaedic Surgery (OPD) King GeorgersquosMedical University LucknowThe Institutional ReviewBoardand Ethics Committee of King Georgersquos Medical UniversityLucknow approved this study and it was carried out duringJanuary 2011 to December 2014 Before enrolment eachsubjectrsquos written informed consent was obtained in responseto a fully written and verbal explanation of the nature of study

22 Inclusion and Exclusion Criteria Cases were defined assubjects of either sex within age group of 18ndash45 years withfracture nonunion shaft of tibia defined as bone healing withRadiographic Union Score for Tibial Fractures (RUST) lt7 by the end of 6th month along with clinical indicationof nonunion like pain abnormal mobility with presence ofno transmitted movements at site of fracture [15] Controlswere defined as otherwise normal subjects of similar agegroup Exclusion criteria included children and patientswith a known systemic inflammatory disease osteoporosisand other metabolic bone diseases pathological fracturesand subsequent nonunions and hypertrophic and infectednonunions [16]

23 Genomic DNA Isolation Venous blood was collectedin 05M EDTA vial and stored at minus80∘C Genomic DNAextraction for molecular genetic studies was performedusing the commercially available extraction kit (BangaloreGenei India) and was stored at minus80∘C DNA concentrationwas measured with a Nanodrop ND-100 Spectrophotometer(Thermo Fisher Scientific Inc Wilmington DE)

24 Analysis of CYR61 Gene (T rarr G) Polymorphism byPCR Amplification The CYR61 (T rarr G) gene polymor-phism (rs3753793) was analysed by the polymerase chainreaction (PCR) followed by restriction fragment length poly-morphism (RFLP) Genomic DNA was amplified (Applied

100 bp50bp

104 bp80bp

Figure 1 3 agarose gel analysis of CYR61 (T rarr G) genepolymorphism Lane 1 50 bp ladder Lanes 2 6 and 7 TT genotype104 bp Lanes 3 and 4 TG genotype 104 80 and 24 bp Lane 5 GGgenotype 80 24 bp

Biosystems Veriti Singapore) using the following PCR con-ditions 95∘C for 6min 36 cycles at 95∘C for 55 s 57∘C for45min 72∘C for 1min and finally 72∘C for 10min Theprimers used for amplification of the CYR61 (T rarr G) genepolymorphismwere as follows forward primer 51015840-CTTGCCTCT CAC CTT CGC TGT TAA-31015840 and reverse primer 51015840-GTC GTT TTG TTT GGT GAT GCG A-31015840 [17] Ampli-fication was performed with 25 120583L PCR reaction mixturecontaining 100 ng template DNA 10 pmol of each primerand 2x PCR master mixes (Fermentas Germany) Amplifi-cation success of samples was monitored by 3 agarose gelelectrophoresis Thereafter the PCR products were subjectedto digestion by KspA I enzyme (Fermentas Germany) toscreen for the CYR61 (T rarr G) gene polymorphism Theenzymatic mixture contained 1 120583L restriction enzyme 1 120583L10x buffer 6 120583L PCR products and 2 120583L distilled water themixture was incubated overnight at 37∘C for digestion Thedigested product was electrophoresed on 3 agarose gelelectrophoresis at 80 volts for one hour In cases with CYR61(T rarr G) gene polymorphism an undigested 104 bp bandshowed wild-type TT genotype while two bands of 80 and24 bp confirmed mutant GG genotype and three bands of104 80 and 24 bp were detected in the heterozygous TGgenotype [17] (Figure 1) Among the controls the genotypefrequency of the polymorphism was confirmed by Hardy-Weinberg equilibrium (119875 gt 005 shown in Table 2) Thequality control testing was performed usingGAPDH Also allthe RFLP genotyping was later confirmed using the sangersequencing method in subgroups (homozygous wild typeheterozygous and variant homozygous)

25 RNA Extraction and Analysis of Real-Time PCR To fur-ther detect the correlation between the CYR61 mRNA levelsand genotypes polymorphism in vivo fracture nonunionspecimens (whole blood) were obtained from 56 cases withdifferent genotypes Total RNA was isolated using the extrac-tion kit (Fermentas Germany) andwas stored atminus80∘C RNAconcentration was measured with a Nanodrop ND-100 Spec-trophotometer (Thermo Fisher Scientific Inc WilmingtonDE) An aliquot of the total RNA (15 120583L) from each samplewas reverse transcribed into single-strand cDNA using anoligo(dT) [18 19] The primers used for PCR amplificationwere 51015840-CCT GTC CGC TGC ACA CCA GC-31015840 and 51015840-GGA GAG CGC CAG CCT GGT CA-31015840 for CYR61 and51015840-GAA ATC CCA TCA CCA TCT TCC AGG-31015840 and 51015840-GAGCCCCAGCCTTCTCCATG-31015840 for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) The expression of

Genetics Research International 3

CYR61 relative to GAPDH RNA was determined using themethod as explained [20]

26 Sequence Analysis All samples selected for real-timePCR (119899 = 56) were also characterized by automatedsequencing The PCR product of each sample was firstpurified and then submitted in 25 120583L quantity with 10picomoles of appropriate primer The sequencing was per-formed by an automated direct DNA sequencing techniquewhich incorporates fluorescently labelled dideoxynucleotidesduring cycle sequencing and separates the resulting prod-ucts by capillary electrophoresis for detection on an ABI3730XL DNA Analyser (Applied Biosystems USA) Multiplealignment and sequence analysis were done using BLAST(Basic Local Alignment Search Tool) BioEdit FinchTVand AutoAssembler Software (Applied Biosystems USA)Sequences obtained were aligned using the BioEdit softwarewith normal sequences taken from Genbank and examinedfor the presence of polymorphism (Figure 3)

27 Statistical Analysis The significance of this study wasevaluated by Chi-square test Odds ratio (OR) was calculatedas an estimate of relative risk of having disease accordingto the relative frequency of different genotypes among thecases as well as the controls The association between thepolymorphism and fracture nonunion was estimated by oddsratios (ORs) and their 95 confidence intervals (CIs) whichwere calculated by unconditional logistic regression 119875 valuewas considered significant at lt005 The value was expressedin mean plusmn SD (Standard Deviation) By considering powerof 80 with minimum expected difference between the twomeans of 67 the sample size of 119899 = 86 was obtainedHowever in the present study we enrolled 250 cases as wellas controls

3 Results

In our study we recruited 250 fracture nonunion casesincluding 147 males and 103 females with age ranging from18 to 45 years The demographic and the baseline data werecompared as in Table 1 All the cases and controls weresuccessfully genotyped by PCR-RFLP The average CYR61(T rarr G) TT TG and GG genotype frequencies in totalfracture nonunion cases were 416 492 and 920 and544 392 and 640 in healthy controls respectivelyThe frequency of CYR61 (T rarr G) gene polymorphism andstatistical analysis of the cases and controls are shown inTable 2 The observed CYR61 (T rarr G) high expressionmutant G allele frequency was 338 in fracture nonunioncases and frequency was 260 in healthy controls (Table 2)

In this study among 250 cases and 250 controls wefound that TG genotype was present among 123 nonunioncases and 98 controls the GG genotype was present in 23fracture nonunion cases and 16 healthy controls and the TTgenotype was present among 104 fracture nonunion casesand 136 healthy controls (Table 2) The heterozygous TGgenotypes were more prevalent in fracture nonunion casescompared with healthy controls and difference between cases

Table 1 Demographic details of fracture nonunion patients andcontrols

Characteristics Cases (119899 = 250) Controls (119899 = 250) 119875 valueAge 3592 plusmn 494 3437 plusmn 521 0923Male 588 (119899 = 147) 644 (119899 = 161) 0578Female 412 (119899 = 103) 356 (119899 = 89) 0438Site of fracture left 107 (428) mdash mdashSite of fracture right 143 (572) mdash mdashHaemoglobin (Hb) 1015 plusmn 122 mdash mdashAlbumin 353 plusmn 025 mdash mdashFerritin 14254 plusmn 338 mdash mdash

TGGG TTSerum mRNA expression

002040608

112141618

Fold

chan

ge

Figure 2 CYR61 gene transcript in fracture nonunion patientsdetected by real-time quantitative reverse-transcript PCR Thefrequency distributions of the TT TG and GG genotypes were 2528 and 3 respectivelyThe fold changes were 116 for TT (plusmn054) and0855 for TGGG (plusmn035) which were standardised against GAPDH

and controls was statistically significant (OR = 164 95 CI =113ndash237 and 119875 = 0010) whereas homozygous GG genotypewas not significant The frequency of mutant G allele inCYR61 (T rarr G) was statistically significant in fracturenonunion cases compared with that in healthy controls (OR= 145 95 CI = 110ndash190 and 119875 = 0008) suggesting thatindividual G allele was associated with fracture nonunioncases (Table 2)

Further the association of CYR61 gene polymorphismwith CYR61 mRNA expression in 56 fracture nonunion caseswas analysed The effect of these three genotypes on CYR61mRNA expression was evaluated by real-time quantitativereverse-transcription PCR CYR61 quantification showedthat homozygous wild type of the TT genotype was withsignificantly increased mRNA levels when compared withhomozygousheterozygous mutant of the GTGG genotype(119875 = 001 119905 = 25521 CI = minus054460 to minus006540 Figure 2)

4 Discussion

This study was carried out on north Indian populationwith the aim of investigating and assessing whether a singlenucleotide polymorphism (SNP) in the promoter region ofthe CYR61 gene is associated with fracture nonunion risk inshaft of tibia or not

As is well known reasons for fracture nonunion inshaft of tibia are multifactorial and even with favourableconditions nonunion is relatively common in shaft of tibia


Table 2 Genotype and allele frequencies of CYR61 (T rarr G) gene polymorphism in fracture nonunion patients and healthy controls

CYR61 (T rarr G) genotyping Cases (119899 = 250) Controls (119899 = 250) 119875 value Odds ratio 95 CI Chi-squareTT 104 (416) 136 (544) mdash mdash mdash mdashTG 123 (492) 98 (392) 0010lowast 164 113ndash237 650GG 23 (920) 16 (640) 0099 188 094ndash373 270TG + GG 146 (584) 114 (456) 0005lowast 167 117ndash238 770T 331 (662) 370 (740) mdash mdash mdash mdashG 169 (338) 130 (260) 0008lowast 145 110ndash190 688lowastSignificant value

G T T T A C A T A T A C C C

(a)

G KT T T A C A T A A C C C

(b)

G GT T T A C A T A A C C C

(c)

Figure 3 Chromatograms of three cases showing (arrow) the three genotypes of the single nucleotide polymorphism found in CYR61 gene(a) Genotype TT homozygous wild type (b) Genotype TG heterozygous (K = TG) (c) Genotype GG homozygous mutant

This observation forces us to widen our vision to look intoa possible genetic factor responsible for impaired fracturehealing On observing various genotypes we concluded thatTT genotype was significantly associated with increased rateof fracture healing progression compared with GG and TGgenotypes Further analysis revealed that fracture nonunioncases with homozygous wild type of the TT genotype hadsignificantly increased mRNA levels contrary to homozy-gousheterozygous variants of the GG and TG genotypesFrom above observation it can be inferred that fracturehealing outcome therefore is possibly attributed to geneticvariations among patients Manigrasso and OrsquoConnor intheir study established significant contribution of geneticvariability in the process of bone regeneration and in theirexperiment onmice strains they also demonstrated the effectof genetic variability on the length of each stage of fracturehealing and the overall healing rate [10]

Another study reported a genetic polymorphism at func-tional promoter fragment of the human CYR61 gene onCYR61 mRNA expression in cases of nonunion fracture shafttibia [21] The above-mentioned effect is probably due to thefact that this polymorphism may affect the transcriptionalactivity of CYR61 gene As per our knowledge presentstudy is the first to suggest the possible effect of functionalpolymorphism in the CYR61 gene on expression of CYR61mRNA in nonunion cases The possible explanation for thisaltered expressionmay be the polymorphism in the promoterregion of CYR6 whichmay affect the transcriptional activityHowever further studies are needed to better explore theregulatory mechanisms of genetic variants that further affectmRNA expression

In the present study we observed a statistically significanteffect of the G allele of CYR61 for developing fracturenonunion suggesting that the polymorphism may affect the


progression of bony union as the G allele is associated withdecreased mRNA level of CYR61 gene which may be oneof the causes of fracture nonunion The basic concept of thepresent study is supported by study of Tao et al (2013) inwhich they observed that the G allele of rs3753793 (TGthornGG)had significantly lower risk of prostate cancer by downregu-lating the mRNA expression of CYR61 when compared withthe TT genotype [17] Thus this study proves that G allele ofCYR61 may affect the mRNA expression Further apart frompolymorphic study expressional study by Hadjiargyrou et al(2000) showed upregulation of CYR61 mRNA and proteinexpression after fracture promoted healing by inducing theangiogenesis process which is prerequisite for any fracturehealing [21] Lienau et al also found an upregulation of theCYR61 protein expression during the early phase of healingespecially in the chondrogenesis process [22]

In conclusion we provide the first evidence supportingthe genetic effect of CYR61 gene polymorphism on risk offracture nonunion in north Indian population In this studythe author also supposed that the homozygousheterozygousmutant G allele may affect the optimum angiogenesis processat the initial phase of healing which leads to increasedfracture nonunion risk followed by decrease in the mRNAexpression of CYR61 in related bone fracture nonunion casesThe author also realised further multicentric studies withlarge sample size to strengthen the obtained results

Conflict of Interests

The authors have no conflict of interests in this paper

References

[1] L K Reed and M A Mormino ldquoDistal tibia nonunionsrdquo Footand Ankle Clinics vol 13 no 4 pp 725ndash735 2008

[2] M Patel J J McCarthy and J Herzenberg ldquoTibial NonunionrdquoMedscape Reference 2011

[3] D Marsh ldquoConcepts of fracture union delayed union andnonunionrdquo Clinical Orthopaedics and Related Research no 355supplement pp S22ndashS30 1998

[4] A Praemer S Furner and D P Rice Musculoskeletal Condi-tions in the United States American Academy of OrthopaedicSurgeons Park Ridge Ill USA 1992

[5] A Sarmiento L M Gersten P A Sobol J A Shankwiler andC T Vangsness ldquoTibial shaft fractures treated with functionalbraces Experience with 780 fracturesrdquo The Journal of Bone ampJoint SurgerymdashBritish Volume vol 71 no 4 pp 602ndash609 1989

[6] L S Phieffer and J A Goulet ldquoDelayed unions of the tibiardquoTheJournal of Bone amp Joint SurgerymdashAmerican Volume vol 88 no1 pp 206ndash216 2006

[7] M R Brinker ldquoFrom nonunions evaluation and treatmentrdquo inSkeletal Trauma Basic Science Management and Reconstruc-tion B D Browner J P Jupiter A M Levine and P G TraftonEds pp 507ndash604 Saunders Philadelphia Pa USA 3rd edition2003

[8] P V Giannoudis D A MacDonald S J Matthews R MSmith A J Furlong and P De Boer ldquoNonunion of the femoraldiaphysis The influence of reaming and non-steroidal anti-inflammatory drugsrdquo The Journal of Bone amp Joint SurgerymdashBritish Volume vol 82 no 5 pp 655ndash658 2000

[9] D B Hausman M DiGirolamo T J Bartness G J Hausmanand R J Martin ldquoThe biology of white adipocyte proliferationrdquoObesity Reviews vol 2 no 4 pp 239ndash254 2001

[10] M B Manigrasso and J P OrsquoConnor ldquoComparison of fracturehealing among different inbred mouse strainsrdquo Calcified TissueInternational vol 82 no 6 pp 465ndash474 2008

[11] K J Jepsen B Hu S M Tommasini et al ldquoGenetic random-ization reveals functional relationships among morphologicand tissue-quality traits that contribute to bone strength andfragilityrdquo Mammalian Genome vol 18 no 6-7 pp 492ndash5072007

[12] P Bork ldquoThe modular architecture of a new family of growthregulators related to connective tissue growth factorrdquo FEBSLetters vol 327 no 2 pp 125ndash130 1993

[13] P Jay J L Berge-Lefranc C Marsollier C Mejean S Taviauxand P Berta ldquoThe human growth factor-inducible immediateearly gene CYR61 maps to chromosome 1prdquo Oncogene vol 14no 14 pp 1753ndash1757 1997

[14] B Perbal ldquoNOV (nephroblastoma overexpressed) and the CCNfamily of genes structural and functional issuesrdquo MolecularPathology vol 54 no 2 pp 57ndash79 2001

[15] D B Whelan M Bhandari D Stephen et al ldquoDevelopment ofthe radiographic union score for tibial fractures for the assess-ment of tibial fracture healing after intramedullary fixationrdquoJournal of Trauma and Acute Care Surgery vol 68 no 3 pp629ndash632 2010

[16] R Dimitriou I M Carr R M West A F Markham and PV Giannoudis ldquoGenetic predisposition to fracture non-uniona case control study of a preliminary single nucleotide poly-morphisms analysis of the BMPpathwayrdquo BMCMusculoskeletalDisorders vol 12 article 44 2011

[17] L Tao J Chen H Zhou et al ldquoA functional polymorphism inthe CYR61 (IGFBP10) gene is associated with prostate cancerriskrdquo Prostate Cancer and Prostatic Diseases vol 16 no 1 pp95ndash100 2013

[18] S R Hussain H Naqvi F Mahdi C Bansal and S G BabuldquoKIT proto-oncogene exon 8 deletions at codon 419 are highlyfrequent in acute myeloid leukaemia with inv(16) in Indianpopulationrdquo Molecular Biotechnology vol 54 no 2 pp 461ndash468 2013

[19] A M Babic M L Kireeva T V Kolesnikova and L F LauldquoCYR61 a product of a growth factor-inducible immediate earlygene promotes angiogenesis and tumor growthrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 95 no 11 pp 6355ndash6360 1998

[20] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCRand the 2minusΔΔ119862TmethodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[21] M Hadjiargyrou W Ahrens and C T Rubin ldquoTemporalexpression of the chondrogenic and angiogenic growth factorCYR61 during fracture repairrdquo Journal of Bone and MineralResearch vol 15 no 6 pp 1014ndash1023 2000

[22] J Lienau H Schell D R Epari et al ldquoCYR61 (CCN1) proteinexpression during fracture healing in an ovine tibial modeland its relation to the mechanical fixation stabilityrdquo Journal ofOrthopaedic Research vol 24 no 2 pp 254ndash262 2006

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Genetics Research International


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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


functional polymorphism (promoter region rs3753793) inthe CYR61 gene on expression of CYR61 mRNA in nonuniontibial fracture cases

The role of the single nucleotide polymorphism (SNP)involving CYR61 gene in fracture healing if proved mayopen new horizons for innovations in this field with anaddition to our armamentarium to deal with complicationsassociated with fracture healing The association if estab-lished will add genetic predisposition as a new risk factor forimpaired bone healing It can also in the future be used as animportant prognostic tool for early identification of patientsat risk of impaired fracture healing

This study is probably the first of its kind carried out innorth Indian population to evaluate the association betweenCYR61 gene polymorphism ((T rarr G) gene polymorphism)and fracture nonunion in shaft of tibia

2 Materials and Methods

21 Samples Collection A total of 250 fracture shafts oftibia with nonunion cases and 250 healthy subjects wereselected for the study The study was carried out in theDepartment of Orthopaedic Surgery (OPD) King GeorgersquosMedical University LucknowThe Institutional ReviewBoardand Ethics Committee of King Georgersquos Medical UniversityLucknow approved this study and it was carried out duringJanuary 2011 to December 2014 Before enrolment eachsubjectrsquos written informed consent was obtained in responseto a fully written and verbal explanation of the nature of study

22 Inclusion and Exclusion Criteria Cases were defined assubjects of either sex within age group of 18ndash45 years withfracture nonunion shaft of tibia defined as bone healing withRadiographic Union Score for Tibial Fractures (RUST) lt7 by the end of 6th month along with clinical indicationof nonunion like pain abnormal mobility with presence ofno transmitted movements at site of fracture [15] Controlswere defined as otherwise normal subjects of similar agegroup Exclusion criteria included children and patientswith a known systemic inflammatory disease osteoporosisand other metabolic bone diseases pathological fracturesand subsequent nonunions and hypertrophic and infectednonunions [16]

23 Genomic DNA Isolation Venous blood was collectedin 05M EDTA vial and stored at minus80∘C Genomic DNAextraction for molecular genetic studies was performedusing the commercially available extraction kit (BangaloreGenei India) and was stored at minus80∘C DNA concentrationwas measured with a Nanodrop ND-100 Spectrophotometer(Thermo Fisher Scientific Inc Wilmington DE)

24 Analysis of CYR61 Gene (T rarr G) Polymorphism byPCR Amplification The CYR61 (T rarr G) gene polymor-phism (rs3753793) was analysed by the polymerase chainreaction (PCR) followed by restriction fragment length poly-morphism (RFLP) Genomic DNA was amplified (Applied

100 bp50bp

104 bp80bp

Figure 1 3 agarose gel analysis of CYR61 (T rarr G) genepolymorphism Lane 1 50 bp ladder Lanes 2 6 and 7 TT genotype104 bp Lanes 3 and 4 TG genotype 104 80 and 24 bp Lane 5 GGgenotype 80 24 bp

Biosystems Veriti Singapore) using the following PCR con-ditions 95∘C for 6min 36 cycles at 95∘C for 55 s 57∘C for45min 72∘C for 1min and finally 72∘C for 10min Theprimers used for amplification of the CYR61 (T rarr G) genepolymorphismwere as follows forward primer 51015840-CTTGCCTCT CAC CTT CGC TGT TAA-31015840 and reverse primer 51015840-GTC GTT TTG TTT GGT GAT GCG A-31015840 [17] Ampli-fication was performed with 25 120583L PCR reaction mixturecontaining 100 ng template DNA 10 pmol of each primerand 2x PCR master mixes (Fermentas Germany) Amplifi-cation success of samples was monitored by 3 agarose gelelectrophoresis Thereafter the PCR products were subjectedto digestion by KspA I enzyme (Fermentas Germany) toscreen for the CYR61 (T rarr G) gene polymorphism Theenzymatic mixture contained 1 120583L restriction enzyme 1 120583L10x buffer 6 120583L PCR products and 2 120583L distilled water themixture was incubated overnight at 37∘C for digestion Thedigested product was electrophoresed on 3 agarose gelelectrophoresis at 80 volts for one hour In cases with CYR61(T rarr G) gene polymorphism an undigested 104 bp bandshowed wild-type TT genotype while two bands of 80 and24 bp confirmed mutant GG genotype and three bands of104 80 and 24 bp were detected in the heterozygous TGgenotype [17] (Figure 1) Among the controls the genotypefrequency of the polymorphism was confirmed by Hardy-Weinberg equilibrium (119875 gt 005 shown in Table 2) Thequality control testing was performed usingGAPDH Also allthe RFLP genotyping was later confirmed using the sangersequencing method in subgroups (homozygous wild typeheterozygous and variant homozygous)

25 RNA Extraction and Analysis of Real-Time PCR To fur-ther detect the correlation between the CYR61 mRNA levelsand genotypes polymorphism in vivo fracture nonunionspecimens (whole blood) were obtained from 56 cases withdifferent genotypes Total RNA was isolated using the extrac-tion kit (Fermentas Germany) andwas stored atminus80∘C RNAconcentration was measured with a Nanodrop ND-100 Spec-trophotometer (Thermo Fisher Scientific Inc WilmingtonDE) An aliquot of the total RNA (15 120583L) from each samplewas reverse transcribed into single-strand cDNA using anoligo(dT) [18 19] The primers used for PCR amplificationwere 51015840-CCT GTC CGC TGC ACA CCA GC-31015840 and 51015840-GGA GAG CGC CAG CCT GGT CA-31015840 for CYR61 and51015840-GAA ATC CCA TCA CCA TCT TCC AGG-31015840 and 51015840-GAGCCCCAGCCTTCTCCATG-31015840 for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) The expression of





3 Results






002040608

112141618

Fold

chan

ge




4 Discussion







(a)


(b)


(c)










References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





3 Results






002040608

112141618

Fold

chan

ge




4 Discussion







(a)


(b)


(c)










References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(a)


(b)


(c)










References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






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 Study of Cysteine-Rich Protein 61 Genetic ...downloads.hindawi.com/journals/gri/2015/754872.pdf · Research Article Study of Cysteine-Rich Protein 61 Genetic Polymorphism