Mediators of Inammation . //downloads.hindawi.com/journals/mi/2013/748435.pdfMediators of Inammation...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 748435 7 pageshttpdxdoiorg1011552013748435

Clinical StudyAssociation of CFH and CFB Gene Polymorphisms withRetinopathy in Type 2 Diabetic Patients

Jun Wang1 Ming Ming Yang2 Yan Bo Li1 Guo Dong Liu1 Yan Teng2 and Xiao Min Liu1

1 Department of Endocrinology First Affiliated Hospital of Harbin Medical University 23 Post Road Nangang RegionHarbin Heilongjiang 150001 China

2 EyeHospital First AffiliatedHospital HarbinMedical University 23 Post Road Nangang Region Harbin Heilongjiang 150001 China

Correspondence should be addressed to Yan Teng tengyan2005gmailcom and Xiao Min Liu liuxiaomin 1957163com

Received 13 April 2013 Revised 2 June 2013 Accepted 10 June 2013

Academic Editor Katarzyna Zorena

Copyright copy 2013 Jun Wang et alThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives The complement system is a key component of innate immunity and has been implicated in the pathogenesis ofdiabetic retinopathy (DR) This study aimed at investigating whether polymorphisms of two genes in the complement pathwaycomplement factor H (CFH) and complement factor B (CFB) are associated with DRMethods 552 well-defined subjects with type2 diabetes consisting of 277 DR patients and 275 diabetic controls were recruited Four Tag-SNPs rs1048709 rs537160 rs4151657and rs2072633 inCFB and rs800292 (I62V) inCFHwere examinedusingTaqManGenotypingAssaysResultsTherewere significantincreases in the frequencies ofA allele andAAgenotype for rs1048709 inDRpatients comparedwith diabetic controls (119875corr = 0035OR = 142 119875corr = 002 OR = 227 resp) meanwhile significant decreases in the frequencies of A allele and AA genotype forrs800292 were observed in DR patients compared with diabetic controls (119875corr = 004 OR = 072 119875corr = 0015 OR = 051 resp)Joint effect of these two loci was also identified Moreover rs800292AA genotype was found to be related with delayed progressionto DR Conclusions CFH-rs800292 and CFB-rs1048709 are associated with the presence of DR which strengthens the concept thatcomplement system plays an important role in the pathogenesis of DR

1 Introduction

The prevalence of diabetes has been reaching epidemic pro-portions at an alarming rate currently Diabetic retinopathy(DR) is the most common microvascular complication ofdiabetes and is a leading cause of blindness worldwide char-acterized by increased vascular permeability tissue ischemiaand neovascularization [1 2] To date many environmentaland clinical factors have been proposed to affect the develop-ment of DR such as alteration of glucose metabolism poorglycemic control and prolonged duration of diabetes [3 4]In addition an important conceptual consideration is thatDR can manifest in individuals with genetic predispositionthrough the existence of familial aggregation of severe DRamong the siblings and family member of diabetic patients[5ndash7] The pathogenesis of DR is complex and has multifac-torial causes Several molecules and metabolic pathways likeoxidative stress upregulation of growth factors activationof protein kinase C (PKC) pathway and so forth have been

implicated in the pathogenesis of DR [8ndash10] Recent researchinsights describing DR as a retinal disease associated withinflammation have drawn special attention and garneredgreat research interests and the evidence comes from theobservation of typical features such as tissue edema increasedleukostasis upregulation of inflammatory mediators andcomplement activation [11ndash13] In view of such inferencesmany inflammatory molecules are being investigated as atarget for a possible remedy in DR

The complement system is a key component of innateimmunity which can be divided into the classic lectin andalternative pathways and is involved in modulating variousimmune and inflammatory responses [14 15] Under normalconditions the complement system is continuously active ata low level and is tightly regulated by complement regulatorsDisruption in the balance of complement activation andregulation will result in harmful effects and can contributeto various inflammatory diseases such as age-related mac-ular degeneration (AMD) systemic lupus erythematosus

2 Mediators of Inflammation

(SLE) rheumatoid arthritis (RA) and Alzheimerrsquos disease[16ndash20] Increasing evidence from in vitro and in vivo studiessuggests a pathogenetic role of the complement system inthe development of diabetic angiopathy In these studiesincreased expression of several complement factors namelycomplement factor H (CFH) complement factor B (CFB)component 3 (C3) and component 5 (C5) has been observedin the vitreous of DR patients [21ndash23] In addition geneticvariants in the CFH and CFB genes have been also shownto be associated with a range of inflammatory diseases [24ndash27] Among the various polymorphisms in the CFH geneI62V (rs800292) was actively investigated and showed strongassociation with AMD a disorder shares many pathophys-iological features in common with DR and both appearto involve the inflammation and complement activationMoreover functional analyses revealed that rs800292 wasassociated with levels of complement proteins in serumand this functional variant was also found to affect theprotein-binding affinity with C3b and subsequently reducedthe activation of complement alternative pathway CFB anopponent of CFH involved in the alternative pathway withthe same binding site of C3b also contributes to regulate theactivation of complement cascade and the genetic impact ofCFB on DR is of interest [28]

Therefore the purpose of the present study was to testa possible association of CFH variant rs800292 (I62V) andfour common variants of CFB gene by tag SNP selection withsusceptibility to DR Moreover since the clinical features andcauses of DR are variable we also evaluated the genotype-phenotype correlations to identify factors associated withprognosis and risk stratification

2 Methods

21 Study Design and Subjects The study involved 552 unre-lated individuals with type 2 diabetes mellitus (DM) witha defined ophthalmologic status who were recruited fromthe First Affiliated Hospital of Harbin Medical UniversityHarbin China Diagnosis of type 2 diabetes was based onWorld Health Organization criteria [29] The study wasapproved by the Bioethics Committee of the Harbin MedicalUniversity All the procedures were conducted according tothe tenets of the Declaration of Helsinki Informed consentwas obtained from all study subjects after explanation of thenature of the study

Ocular examination was performed by independentophthalmologist using fundus ophthalmoscopy after pupildilatation The stage of DR was determined according tothe Early Treatment Diabetic Retinopathy Study (ETDRS)criteria [30] Of this group 277 patients with type 2 diabeteswere diagnosed with DR 171 (617) with nonproliferativeDR (NPDR) and 106 (383) with proliferative DR (PDR)The control group consisted of 275 subjects without DR butwith type 2 diabetes duration of more than 10 years All sub-jects underwent a detailed ophthalmologic examination andclinical information collection including corrected visualacuity fundoscopic examination age gender duration ofdiabetes andDR bodymass index (BMI)HbA1c level smok-ing status presence of hypertension and hyperlipidemia and

treatment details People with any of the following situationswere excluded from the study peripheral vascular diseasesacute infection systemic inflammation diseases or any otherocular disorders such as AMD glaucoma or branch retinalvenous occlusion Overt diabetic nephropathy patients wereexcluded the exclusion criteria were as follows microalbu-min creatinine ratio gt30mgg and urinary microalbuminlevel gt300mgd [31]

22 DNA Extraction and Genotyping Genomic DNA wasextracted from peripheral blood with the QIAamp Bloodkit (Qiagen Hilden Germany) according to the supplierrsquosinstructions One CFH variant rs800292 (I62V) and fourtagging-SNPs (rs1048709 rs537160 rs4151657 and rs2072633)captured 100 of alleles in the CFB locus with MAF largerthan 01 and a mean 1199032 of 10 were selected All the SNPswere genotyped byTaqManSNPGenotypingAssays (AppliedBiosystems Inc Foster City CA USA) in the Light Cycler480 Genotyping Master (Roche Diagnostics Inc MannheinGermany) according to manufacturersrsquo protocols The PCRamplifications were performed with the thermal cyclingconditions of 94∘C for 10min followed by 40 cycles of 94∘Cfor 15 s and 60∘C for 15min The genotypes were read byPrism 7000 SDS software (version 11 ABI)

23 Statistical Analysis Hardy-Weinberg equilibrium(HWE) for genotype frequencies of the SNPs was testedby 1205942 test Allelic and genotypic frequencies betweenDR and DM were compared by 1205942 test or Fisher exacttest Dominant and recessive models were also applied toinvestigate the disease association with regard to the minorallele (rs800292[A] rs1048709[A] rs537160[A] rs4151657[C]and rs2072633[G]) Stratified analysis based on DR severity(NPDR and PDR) was also performed Logistic regressionanalysis was applied to adjust the association of these SNPswith age and gender The Student 119905 test and 1205942 test wereused to compare continuous clinical data and categoricalvariables respectively Pairwise linkage disequilibrium (LD1198631015840) between polymorphisms and EM-based haplotype

association analysis was performed by Haploview (ver 42)Odds ratios (OR) and 95 confidence intervals (CI) werealso calculated 119875 lt 005 was considered as statisticallysignificant P values were corrected by Bonferroni test formultiple comparisons (119899 = total number of SNPs) Thecorrection for multiple testing in the haplotype analysis wasperformed by permutation testing

3 Results

31 Study Group Comparison The demographic and clinicalcharacterization of the study subjects is presented in Table 1DM control group had a longer duration of type 2 diabetescompared to DR group (119875 lt 0001) Additionally DRgroup had a higher proportion of hyperlipidemia and higherprevalence of insulin therapy than the DM controls (119875 lt 001and 119875 lt 0001 resp) There was no significant difference inage gender HbA1c level BMI proportion of hypertensionsmoking status and family history of diabetes

Mediators of Inflammation 3

Table 1 Demographic and clinical characteristics of the study subjects

Characteristic DR (119899 = 277) DM (119899 = 275) 119875 valueAge (years) 555 plusmn 140 563 plusmn 76 044Gender (FM) 144133 153122 039Duration of diabetes (years) 136 plusmn 71 181 plusmn 67 lt0001Duration of DR (years) 50 plusmn 42 noneGaps between diabetes and DR onset (years) 83 plusmn 67 noneHbA1C () 82 plusmn 17 79 plusmn 19 009BMI (kgm2) 237 plusmn 46 241 plusmn 44 046Hypertension () 718 669 021Hyperlipidemia () 321 233 lt001Smoking () 134 160 038Insulin therapy () 451 262 lt0001Family history of diabetes () 256 215 025All 119875 values were compared by 1205942 or student 119905-test 119875 lt 005 as statistically significantDR diabetic retinopathy DM diabetes mellitus HbA1c glycosylated hemoglobin BMI body mass index

32 Association Analysis All genotype frequencies of the fiveselected SNPs followed the Hardy-Weinberg equilibrium inall subjects Regarding CFH rs800292 there was a significantdecrease in the frequencies of A allele and AA homozygosityin DR patients compared with DM controls (119875corr = 004 OR= 072 95 CI = 057ndash092 119875corr = 0015 OR = 051 95 CI= 033ndash080 resp) indicating a protective effect Meanwhilesignificant association was also detected at CFB rs1048709where there was a significant increase in the frequencies ofA allele and AA homozygosity in DR patients comparedwith DM controls (119875corr = 0035 OR = 142 95 CI =110ndash183 119875corr = 002 OR = 227 95 CI = 128ndash404resp) No significant differences in the genotypic or allelicfrequencies were observed for other three SNPs betweenDR and DM after multiple testing correction (Table 2) Inaddition logistic regression analysis was used to assess therole of gene polymorphisms in DR after adjustment for agegender duration of diabetes hyperlipidemia and insulintherapy the results showed that the association did not alterbetweenDR and all the 5 SNPs after adjusting for these factors(data not shown)

33 Linkage Disequilibrium andHaplotype Association Analy-sis Pairwise LD analysis was performed across theCFB locusby using these 4 SNPs and one haplotype block was detectedincluding 3 SNPs in CFB (rs537160 rs4151657 and rs2072633Figure 1) A protective haplotype GTG defined by these 3SNPs was identified and conferred a 145-fold significantlydecreased risk ofDR but the statistical significancewould notremain after correction for (119875 = 0028 permutation119875 = 011Table 3)

34 Genotype-Phenotype Correlation Analysis Among the277DRpatients 171 (617)wereNPDRand 106 (383)werePDR Further stratification comparison was also performedon the clinical severity in terms of DR scale Between NPDRand PDR groups no significant differences were detected inthe allelic and genotypic frequencies of all 5 SNPs (data notshown) Given the significance of CFH rs800292 and CFB

CFB complement factor B preproprotein

rs10

4870

9

rs53

7160

rs41

5165

7

rs20

7263

3

1 2 3 4

66

4832

25

12

40

Block 1 (3kb)

32022 k 32023 k 32024 k 32025 k 32026 k 32027 k

NM_0017101

Figure 1 Pairwise LD among three SNPs in CFB gene Linkagedisequilibrium was measured by the 1198631015840 statistic using the datafrom all subjects A 1198631015840 value of 100 indicates a complete LDbetween 2 markers and a1198631015840 value of 0 indicates a complete linkageequilibrium Haplotype version 42 software was used

rs1048709 in this study as well as the observation of recessiveeffect correlations of their genotype group (homozygousminor allele versusmajor allele carriers) with clinical featureswere evaluated The results demonstrated that DR patientscarrying protective rs800292AA genotype would present alonger gap (years) between diabetes and DR onset comparedwith that in patients carrying AG + GG (119 plusmn 52 versus


Table 2 Genotype and allele frequencies of CFH and CFB polymorphisms in DR patients and DM controls

SNP ID Designation Allele distribution () 119875 value Odds ratio Genotype distribution () 119875 value Odds ratioDR (119899 = 554) DM (119899 = 550) (119875corr) (95 CI) DR (119899 = 277) DM (119899 = 275) (119875corr) (95 CI)

CFH

rs800292 G gt A A 208 (375) 250 (455) 0008 (004) 072(057ndash092) AA 38 (137) 65 (236) 015lowast 077

(055ndash110)

Exon2 (I62V) G 346 (625) 300 (544) AG 132 (477) 120 (436) 0003dagger (0015) 051(033ndash080)

GG 107 (386) 90 (327)CFB


(097ndash190)

Exon3 (R150R) G 357 (644) 396 (720) AG 117 (422) 116 (422) 0004dagger (002) 227(128ndash404)

GG 120 (433) 140 (509)

rs537160 G gt A A 268 (484) 266 (484) 10 10(079ndash127) AA 62 (224) 60 (218) 088lowast 097

(066ndash143)

IVS7 G 286 (516) 284 (516) AG 144 (520) 146 (531) 087dagger 103(069ndash155)

GG 71 (256) 69 (251)

rs4151657 T gt C C 149 (269) 138 (251) 049 110(084ndash144) CC 21 (76) 14 (51) 079lowast 105

(075ndash146)

IVS10 T 405 (731) 412 (749) CT 107 (386) 110 (400) 023dagger 153(076ndash307)

TT 149 (538) 151 (549)

rs2072633 A gt G G 330 (596) 311 (565) 031 113(089ndash144) GG 93 (336) 86 (313) 023lowast 132

(084ndash207)

IVS17 A 224 (404) 239 (435) AG 144 (520) 139 (505) 056dagger 111(078ndash159)

AA 40 (144) 50 (182)Data analysis was performed by 1205942 testlowast119875 value for dominant modeldagger119875 value for recessive model

Table 3 Haplotype analysis of CFB Polymorphisms between DR and DM

Haplotype Frequency Frequency119875 119875corr Odds ratio 95 CI

DR DMATA 0482 0482 0482 099 NS mdashGCG 0260 0269 0251 049 NS mdashGTG 0157 0133 0182 0028 NS 069 (050ndash096)GTA 0099 0114 0084 0095 NS mdash119875corr association analysis results from permutation test (iterations 10000)

80 plusmn 44 119875 lt 0001 Figure 2) and such difference wasnot observed for CFB rs1048709 (96 plusmn 56 versus 83 plusmn 45119875 = 011) No significant difference was detected in otherclinical features between different genotype groups (data notshown)

35 Joint-Effect Analysis Considering the biological rele-vance of CFH and CFB combined effects of rs800292 andrs1048709 were assessed and the corresponding ORs of DRfor each possible combination of the genotypes of the twoloci were estimated (Tables 4(a) and 4(b)) The ORs were

compared with the baseline genotype of the two genes Thefrequency of the homozygous risk genotypes at both loci was33-fold higher in DR (61) than in DM controls (18) (119875 =0002 Table 4(a)) A joint OR of 567 in individuals with bothhomozygous risk alleles was observed (Figure 3)

4 Discussion

In this study we investigated the association of complementgenes in type 2 diabetes patients with DR Our resultsdemonstrated that CFH rs800292 (I62V) and CFB rs1048709


Table 4 Interaction snalysis between CFH-rs800292 and CFB-rs1048709

(a)

Genotype distribution CFH rs800292

CFB rs1048709 DM (119899 = 275) DR (119899 = 277)AA AG GG AA AG GG

GG 35 (127) 53 (193) 52 (189) 21 (76) 57 (206) 42 (152)AG 27 (98) 56 (204) 33 (120) 13 (47) 56 (202) 48 (173)AA 3 (11) 11 (40) 5 (18) 4 (14) 19 (69) 17 (61)

(b)

Joint odds ratios and 95 confidence CFH rs800292CFB rs1048709 AA AG GGGG 100 (Ref) 179 (093ndash346) 135 (068ndash265)AG 080 (034ndash189) 167 (087ndash321) 242 (120ndash488)AA 222 (045ndash1092) 288 (165ndash722) 567 (182ndash1762)

0

2

4

6

8

10

12

14

16

18

AA

Gap

s bet

wee

n di

abet

es an

d D

R on

set (

year

s)

119 plusmn 52

AG + GG

80 plusmn 44

Figure 2 Comparison of gaps between diabetes and DR onset(years) in two genotype groups for CFH rs800292 in DR patients(119875 lt 0001)

(R150R) were significantly associated with the susceptibilityof developing DR Moreover since significant differenceshave been observed between DM and DR group (dura-tion of diabetes level of hyperlipidemia and percentage ofinsulin therapy) multivariate logistic regression analysis wasperformed to assess the genetic role of DR by consideringthese factors which showed that I62V and R150R remainsignificant when adjusted for all these factors implyingthat CFH and CFB polymorphisms are independent geneticfactors for susceptibility to DR Over recent years greatadvances have been made in understanding the geneticbackground of the disease more than 30 candidate genesinvolved in different metabolic mechanisms and functionalpathways have been reported while few of them were foundto have a strong association [32 33] Our findings provideadditional and convinced evidence for the involvement ofcomplement system in relation to DR To our knowledge the

0

1

2

3

4

5

6

GG

GG

AG

AG AAAACFB (rs1048709)

Odd

s rat

io

CFH

(rs8

0029

2)

AA

Ref

567

Figure 3 Two loci of CFH and CFB genotype-specific DR risk

genetic associations of variants inCFH andCFBwithDRhavenot been described previously

Activated complement is a ldquodouble-edged swordrdquo whichmight cause self-tissue damage especially for sensitive organslike the eyes The CFH gene is located in chromosome 1(1q32) which is a major soluble inhibitor of the alternativepathway for controlling complement activation [34] CFHrs800292 has been found to be associated with many inflam-matory and neovascular diseases and the change of rs800292G gtA nucleotide results in the synthesis of Isoleucine insteadof Valine This might leads to structural changes affectingthe ability of C3b binding and reducing the activation ofthe alternative pathway This subsequently causes excessiveactivation of the complement system to induce inflammatorydisorders [28] CFB gene is located tandemly in the majorhistocompatibility complex (MHC) class III region a clusteron chromosome 6p21 with respect to inflammation [35] Asmentioned above CFB is a competitor of CFH both involvedin the complement alternative pathway Conceivably much


like impaired CFH-mediated complement inhibition confersDR risk decreased complement activation by CFBmight alsoserve to affect DR Not surprisingly a joint effect of CFH andCFB risk homozygosity with an OR of 567 was identified inthis study In addition CFB polymorphisms were also foundto be associated with other inflammatory diseases suchas AMD lupus and atypical Hemolytic-uremic syndrome(aHUS) [26 27 36] Furthermore in vivo study has revealedthat human RPE cells can synthesize and express CFB andCFH and the level of CFB was increased in the vitreous ofPDR patients [37 38] These findings further strengthen theconcept that complement system especially the alternativepathway plays an important role in the pathogenesis ofDR Unfortunately the exactly pathogenic significance ofthe association of CFH and CFB polymorphisms with DRremains unclear in this study these variants in CFB repre-sent either synonymous substitutions (rs1048709 R150R) orintronic SNPs and there is no information on its biologicalfunctions currently One possible explanation is that thesepolymorphisms may be linked with an undiscovered butbiologically relevant structural variant in this region alterna-tively synonymous or intronic regulation could be involvedin gene transcription or tissue specificity of gene expressionFurther investigations of this region by extensive sequencingto uncover unknown variation are therefore requested

In the genotype-phenotype analysis our findings demon-strated a significant relationship between CFH rs800292 andduration (in years) between DM and DR onset the protectiveAA genotype showed association with delayed progression ofDR however it needs further corroboration by consideringfactors such as duration of diabetes and glycemic controlNevertheless the results not only extend the genetic spectrumof DR but also provide novel understanding for the geneticimpact on disease prognosis Stratification analysis by DRscale showed that there were no significant differences in theallelic and genotypic frequencies for all 5 SNPs between PDRand NPDR groups implying that genetic variations of CFHand CFBmight not be associated with DR severity

The strength of our study is that all patients and controlsare of the same ethnic origin All subjects were examined ina predetermined standardized order with strict diagnosticcriteria Moreover to our best knowledge this is the firstgenetic study to investigate the associations of complementfactor genes in DR patients However there are certainlimitations of this study which includes the relatively smallsample size thus nonsignificant SNPs in the CFBmight lackadequate power Secondly it is a retrospective case-controlstudy that thus lacked the details of follow-up informationFinally the SNPs selected in our study might not fully reflectthe disease risk of unexamined variants in the genes Furtherevaluation of these genes by direct sequencing to uncovermore variants will be beneficial to identify variants withrelevant function in DR

In summary this study first revealed that CFH and CFBpolymorphisms are associated with the development of DRas well as with delayed progression to DR in type 2 diabetesA joint effect between CFH rs800292 and CFB rs1048709conferring a significantly increased risk for DR was alsoidentified Further studies to replicate these candidate SNPs

in others ethnic groups and determine the biological roles ofthese polymorphisms in DR are worthwhile

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Jun Wang and Ming Ming Yang contributed equally to thiswork and should be considered co-first authors

Acknowledgment

The authors express their greatest gratitude to all the partici-pants in this study

References

[1] R N Frank ldquoDiabetic Retinopathyrdquo New England Journal ofMedicine vol 350 no 1 pp 48ndash58 2004

[2] S Resnikoff D Pascolini D Etyarsquoale et al ldquoGlobal data onvisual impairment in the year 2002rdquoBulletin of theWorldHealthOrganization vol 82 no 11 pp 844ndash851 2004

[3] R Klein B E K Klein S E Moss M D Davis and DL De Mets ldquoThe Wisconsin Epidemiologic Study of DiabeticRetinopathy III Prevalence and risk of diabetic retinopathywhen age at diagnosis is 30 or more yearsrdquo Archives of Ophthal-mology vol 102 no 4 pp 527ndash532 1984

[4] T J Orchard J S Dorman R E Maser et al ldquoPrevalenceof complications in IDDM by sex and duration PittsburghEpidemiology of Diabetes Complications Study IIrdquo Diabetesvol 39 no 9 pp 1116ndash1124 1990

[5] M Rema G Saravanan R Deepa and V Mohan ldquoFamilialclustering of diabetic retinopathy in South Indian Type 2diabetic patientsrdquoDiabetic Medicine vol 19 no 11 pp 910ndash9162002

[6] N H Arar B I Freedman S G Adler et al ldquoHeritabilityof the severity of diabetic retinopathy the FIND-Eye studyrdquoInvestigative Ophthalmology amp Visual Science vol 49 no 9 pp3839ndash3845 2008

[7] M Murea L Ma and B I Freedman ldquoGenetic and environ-mental factors associated with type 2 diabetes and diabeticvascular complicationsrdquo The Review of Diabetic Studies vol 9no 1 pp 6ndash22 2012

[8] S-I Yamagishi and T Matsui ldquoAdvanced glycation end prod-ucts (AGEs) oxidative stress and diabetic retinopathyrdquo CurrentPharmaceutical Biotechnology vol 12 no 3 pp 362ndash368 2011

[9] S Wang J K Park and E J Duh ldquoNovel targets against retinalangiogenesis in diabetic retinopathyrdquo Current Diabetes Reportsvol 12 no 4 pp 355ndash363 2012

[10] M I L Galvez ldquoProtein Kinase C inhibitors in the treatment ofdiabetic retinopathy Reviewrdquo Current Pharmaceutical Biotech-nology vol 12 no 3 pp 386ndash391 2011

[11] A M A El-Asrar ldquoRole of inflammation in the pathogenesis ofdiabetic retinopathyrdquo Middle East African Journal of Ophthal-mology vol 19 no 1 pp 70ndash74 2012

[12] T S Kern ldquoContributions of inflammatory processes to thedevelopment of the early stages of diabetic retinopathyrdquo Exper-imental Diabetes Research vol 2007 Article ID 95103 14 pages2007


[13] J Zhang C Gerhardinger and M Lorenzi ldquoEarly complementactivation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimentaldiabetic retinopathyrdquo Diabetes vol 51 no 12 pp 3499ndash35042002

[14] M J Walport ldquoComplement First of two partsrdquo New EnglandJournal of Medicine vol 344 no 14 pp 1058ndash1066 2001

[15] M J Walport ldquoComplement Second of two partsrdquo NewEngland Journal ofMedicine vol 344 no 15 pp 1140ndash1144 2001

[16] M Aiyaz M K Lupton P Proitsi J F Powell and S Love-stone ldquoComplement activation as a biomarker for Alzheimerrsquosdiseaserdquo Immunobiology vol 217 no 2 pp 204ndash215 2012

[17] G Di Muzio C Perricone E Ballanti et al ldquoComplementsystem and rheumatoid arthritis relationships with autoanti-bodies serological clinical features and anti-TNF treatmentrdquoInternational Journal of Immunopathology and Pharmacologyvol 24 no 2 pp 357ndash366 2011

[18] B W Ornstein J P Atkinson and P Densen ldquoThe com-plement system in pediatric systemic lupus erythematosusatypical hemolytic uremic syndrome and complocentric mem-branoglomerulopathiesrdquoCurrentOpinion inRheumatology vol24 no 5 pp 522ndash529 2012

[19] D Ricklin G Hajishengallis K Yang and J D LambrisldquoComplement a key system for immune surveillance andhomeostasisrdquo Nature Immunology vol 11 no 9 pp 785ndash7972010

[20] E Wagner and M M Frank ldquoTherapeutic potential of comple-ment modulationrdquoNature Reviews Drug Discovery vol 9 no 1pp 43ndash56 2010

[21] DMuramatsu YWakabayashi Y Usui Y Okunuki T Kezukaand H Goto ldquoCorrelation of complement fragment C5a withinflammatory cytokines in the vitreous of patients with pro-liferative diabetic retinopathyrdquo Graefersquos Archive for Clinical andExperimental Ophthalmology vol 251 no 1 pp 15ndash17 2013

[22] B-B Gao X Chen N Timothy L P Aiello and E P FeenerldquoCharacterization of the vitreous proteome in diabetes withoutdiabetic retinopathy and diabetes with proliferative diabeticretinopathyrdquo Journal of Proteome Research vol 7 no 6 pp2516ndash2525 2008

[23] M Garcıa-Ramırez F Canals C Hernandez et al ldquoProteomicanalysis of human vitreous fluid by fluorescence-based differ-ence gel electrophoresis (DIGE) a new strategy for identifyingpotential candidates in the pathogenesis of proliferative diabeticretinopathyrdquo Diabetologia vol 50 no 6 pp 1294ndash1303 2007

[24] J Caprioli F Castelletti S Bucchioni et al ldquoComplement factorH mutations and gene polymorphisms in haemolytic uraemicsyndrome the C-257T the A2089G and the G2881T poly-morphisms are strongly associated with the diseaserdquo HumanMolecular Genetics vol 12 no 24 pp 3385ndash3395 2003

[25] X Liu P Zhao S Tang et al ldquoAssociation study of complementfactor H C2 CFB and C3 and age-related macular degenera-tion in a Han Chinese populationrdquo Retina vol 30 no 8 pp1177ndash1184 2010

[26] E Sanchez M E Comeau B I Freedman et al ldquoIdentificationof novel genetic susceptibility loci in African American lupuspatients in a candidate gene association studyrdquo Arthritis andRheumatism vol 63 no 11 pp 3493ndash3501 2011

[27] B Gold J E Merriam J Zernant et al ldquoVariation in factor B(BF) and complement component 2 (C2) genes is associatedwith age-related macular degenerationrdquo Nature Genetics vol38 no 4 pp 458ndash462 2006

[28] I C Pechtl D Kavanagh N Mcintosh C L Harris and P NBarlow ldquoDisease-associated N-terminal complement factor Hmutations perturb cofactor and decay-accelerating activitiesrdquoJournal of Biological Chemistry vol 286 no 13 pp 11082ndash110902011

[29] K G Alberti and P Z Zimmet ldquoDefinition diagnosis andclassification of diabetes mellitus and its complications Part1 diagnosis and classification of diabetes mellitus provisionalreport of aWHO consultationrdquoDiabetic Medicine vol 15 no 7pp 539ndash553 1998

[30] ldquoGrading diabetic retinopathy from stereoscopic color fundusphotographsmdashan extension of the modified Airlie House clas-sification ETDRS report number 10 Early Treatment DiabeticRetinopathy Study Research Grouprdquo Ophthalmology vol 98no 5 supplement pp 786ndash806 1991

[31] V Viswanathan P Tilak and S Kumpatla ldquoRisk factors asso-ciated with the development of overt nephropathy in type 2diabetes patients a 12 years observational studyrdquo The IndianJournal of Medical Research vol 136 no 1 pp 46ndash53 2012

[32] K M Warpeha and U Chakravarthy ldquoMolecular genetics ofmicrovascular disease in diabetic retinopathyrdquo Eye vol 17 no3 pp 305ndash311 2003

[33] K Uhlmann P Kovacs Y Boettcher H-P Hammes and RPaschke ldquoGenetics of diabetic retinopathyrdquo Experimental andClinical Endocrinology and Diabetes vol 114 no 6 pp 275ndash2942006

[34] M K Liszewski T C Farries D M Lublin I A Rooney and JP Atkinson ldquoControl of the complement systemrdquo Advances inImmunology vol 61 pp 201ndash283 1996

[35] R Horton L Wilming V Rand et al ldquoGene map of theextended human MHCrdquo Nature Reviews Genetics vol 5 no 12pp 889ndash899 2004

[36] H Tawadrous T Maga J Sharma J Kupferman R J H Smithand M Schoeneman ldquoA novel mutation in the ComplementFactor B gene (CFB) and atypical hemolytic uremic syndromerdquoPediatric Nephrology vol 25 no 5 pp 947ndash951 2010

[37] M Chen J V Forrester and H Xu ldquoSynthesis of complementfactor H by retinal pigment epithelial cells is down-regulatedby oxidized photoreceptor outer segmentsrdquo Experimental EyeResearch vol 84 no 4 pp 635ndash645 2007

[38] M Chen E Muckersie M Robertson J V Forrester and HXu ldquoUp-regulation of complement factor B in retinal pigmentepithelial cells is accompanied by complement activation in theaged retinardquo Experimental Eye Research vol 87 no 6 pp 543ndash550 2008

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Evidence-Based Complementary and Alternative Medicine

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(SLE) rheumatoid arthritis (RA) and Alzheimerrsquos disease[16ndash20] Increasing evidence from in vitro and in vivo studiessuggests a pathogenetic role of the complement system inthe development of diabetic angiopathy In these studiesincreased expression of several complement factors namelycomplement factor H (CFH) complement factor B (CFB)component 3 (C3) and component 5 (C5) has been observedin the vitreous of DR patients [21ndash23] In addition geneticvariants in the CFH and CFB genes have been also shownto be associated with a range of inflammatory diseases [24ndash27] Among the various polymorphisms in the CFH geneI62V (rs800292) was actively investigated and showed strongassociation with AMD a disorder shares many pathophys-iological features in common with DR and both appearto involve the inflammation and complement activationMoreover functional analyses revealed that rs800292 wasassociated with levels of complement proteins in serumand this functional variant was also found to affect theprotein-binding affinity with C3b and subsequently reducedthe activation of complement alternative pathway CFB anopponent of CFH involved in the alternative pathway withthe same binding site of C3b also contributes to regulate theactivation of complement cascade and the genetic impact ofCFB on DR is of interest [28]

Therefore the purpose of the present study was to testa possible association of CFH variant rs800292 (I62V) andfour common variants of CFB gene by tag SNP selection withsusceptibility to DR Moreover since the clinical features andcauses of DR are variable we also evaluated the genotype-phenotype correlations to identify factors associated withprognosis and risk stratification

2 Methods

21 Study Design and Subjects The study involved 552 unre-lated individuals with type 2 diabetes mellitus (DM) witha defined ophthalmologic status who were recruited fromthe First Affiliated Hospital of Harbin Medical UniversityHarbin China Diagnosis of type 2 diabetes was based onWorld Health Organization criteria [29] The study wasapproved by the Bioethics Committee of the Harbin MedicalUniversity All the procedures were conducted according tothe tenets of the Declaration of Helsinki Informed consentwas obtained from all study subjects after explanation of thenature of the study

Ocular examination was performed by independentophthalmologist using fundus ophthalmoscopy after pupildilatation The stage of DR was determined according tothe Early Treatment Diabetic Retinopathy Study (ETDRS)criteria [30] Of this group 277 patients with type 2 diabeteswere diagnosed with DR 171 (617) with nonproliferativeDR (NPDR) and 106 (383) with proliferative DR (PDR)The control group consisted of 275 subjects without DR butwith type 2 diabetes duration of more than 10 years All sub-jects underwent a detailed ophthalmologic examination andclinical information collection including corrected visualacuity fundoscopic examination age gender duration ofdiabetes andDR bodymass index (BMI)HbA1c level smok-ing status presence of hypertension and hyperlipidemia and

treatment details People with any of the following situationswere excluded from the study peripheral vascular diseasesacute infection systemic inflammation diseases or any otherocular disorders such as AMD glaucoma or branch retinalvenous occlusion Overt diabetic nephropathy patients wereexcluded the exclusion criteria were as follows microalbu-min creatinine ratio gt30mgg and urinary microalbuminlevel gt300mgd [31]

22 DNA Extraction and Genotyping Genomic DNA wasextracted from peripheral blood with the QIAamp Bloodkit (Qiagen Hilden Germany) according to the supplierrsquosinstructions One CFH variant rs800292 (I62V) and fourtagging-SNPs (rs1048709 rs537160 rs4151657 and rs2072633)captured 100 of alleles in the CFB locus with MAF largerthan 01 and a mean 1199032 of 10 were selected All the SNPswere genotyped byTaqManSNPGenotypingAssays (AppliedBiosystems Inc Foster City CA USA) in the Light Cycler480 Genotyping Master (Roche Diagnostics Inc MannheinGermany) according to manufacturersrsquo protocols The PCRamplifications were performed with the thermal cyclingconditions of 94∘C for 10min followed by 40 cycles of 94∘Cfor 15 s and 60∘C for 15min The genotypes were read byPrism 7000 SDS software (version 11 ABI)

23 Statistical Analysis Hardy-Weinberg equilibrium(HWE) for genotype frequencies of the SNPs was testedby 1205942 test Allelic and genotypic frequencies betweenDR and DM were compared by 1205942 test or Fisher exacttest Dominant and recessive models were also applied toinvestigate the disease association with regard to the minorallele (rs800292[A] rs1048709[A] rs537160[A] rs4151657[C]and rs2072633[G]) Stratified analysis based on DR severity(NPDR and PDR) was also performed Logistic regressionanalysis was applied to adjust the association of these SNPswith age and gender The Student 119905 test and 1205942 test wereused to compare continuous clinical data and categoricalvariables respectively Pairwise linkage disequilibrium (LD1198631015840) between polymorphisms and EM-based haplotype

association analysis was performed by Haploview (ver 42)Odds ratios (OR) and 95 confidence intervals (CI) werealso calculated 119875 lt 005 was considered as statisticallysignificant P values were corrected by Bonferroni test formultiple comparisons (119899 = total number of SNPs) Thecorrection for multiple testing in the haplotype analysis wasperformed by permutation testing

3 Results

31 Study Group Comparison The demographic and clinicalcharacterization of the study subjects is presented in Table 1DM control group had a longer duration of type 2 diabetescompared to DR group (119875 lt 0001) Additionally DRgroup had a higher proportion of hyperlipidemia and higherprevalence of insulin therapy than the DM controls (119875 lt 001and 119875 lt 0001 resp) There was no significant difference inage gender HbA1c level BMI proportion of hypertensionsmoking status and family history of diabetes








rs10

4870

9

rs53

7160

rs41

5165

7

rs20

7263

3

1 2 3 4

66

4832

25

12

40

Block 1 (3kb)

32022 k 32023 k 32024 k 32025 k 32026 k 32027 k

NM_0017101






CFH


(055ndash110)


GG 107 (386) 90 (327)CFB


(097ndash190)


GG 120 (433) 140 (509)


(066ndash143)


GG 71 (256) 69 (251)


(075ndash146)


TT 149 (538) 151 (549)


(084ndash207)









4 Discussion




(a)



GG 35 (127) 53 (193) 52 (189) 21 (76) 57 (206) 42 (152)AG 27 (98) 56 (204) 33 (120) 13 (47) 56 (202) 48 (173)AA 3 (11) 11 (40) 5 (18) 4 (14) 19 (69) 17 (61)

(b)


0

2

4

6

8

10

12

14

16

18

AA

Gap

s bet

wee

n di

abet

es an

d D

R on

set (

year

s)

119 plusmn 52

AG + GG

80 plusmn 44



0

1

2

3

4

5

6

GG

GG

AG


Odd

s rat

io

CFH

(rs8

0029

2)

AA

Ref

567














Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























rs10

4870

9

rs53

7160

rs41

5165

7

rs20

7263

3

1 2 3 4

66

4832

25

12

40

Block 1 (3kb)

32022 k 32023 k 32024 k 32025 k 32026 k 32027 k

NM_0017101






CFH


(055ndash110)


GG 107 (386) 90 (327)CFB


(097ndash190)


GG 120 (433) 140 (509)


(066ndash143)


GG 71 (256) 69 (251)


(075ndash146)


TT 149 (538) 151 (549)


(084ndash207)









4 Discussion




(a)



GG 35 (127) 53 (193) 52 (189) 21 (76) 57 (206) 42 (152)AG 27 (98) 56 (204) 33 (120) 13 (47) 56 (202) 48 (173)AA 3 (11) 11 (40) 5 (18) 4 (14) 19 (69) 17 (61)

(b)


0

2

4

6

8

10

12

14

16

18

AA

Gap

s bet

wee

n di

abet

es an

d D

R on

set (

year

s)

119 plusmn 52

AG + GG

80 plusmn 44



0

1

2

3

4

5

6

GG

GG

AG


Odd

s rat

io

CFH

(rs8

0029

2)

AA

Ref

567














Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















CFH


(055ndash110)


GG 107 (386) 90 (327)CFB


(097ndash190)


GG 120 (433) 140 (509)


(066ndash143)


GG 71 (256) 69 (251)


(075ndash146)


TT 149 (538) 151 (549)


(084ndash207)









4 Discussion




(a)



GG 35 (127) 53 (193) 52 (189) 21 (76) 57 (206) 42 (152)AG 27 (98) 56 (204) 33 (120) 13 (47) 56 (202) 48 (173)AA 3 (11) 11 (40) 5 (18) 4 (14) 19 (69) 17 (61)

(b)


0

2

4

6

8

10

12

14

16

18

AA

Gap

s bet

wee

n di

abet

es an

d D

R on

set (

year

s)

119 plusmn 52

AG + GG

80 plusmn 44



0

1

2

3

4

5

6

GG

GG

AG


Odd

s rat

io

CFH

(rs8

0029

2)

AA

Ref

567














Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)



GG 35 (127) 53 (193) 52 (189) 21 (76) 57 (206) 42 (152)AG 27 (98) 56 (204) 33 (120) 13 (47) 56 (202) 48 (173)AA 3 (11) 11 (40) 5 (18) 4 (14) 19 (69) 17 (61)

(b)


0

2

4

6

8

10

12

14

16

18

AA

Gap

s bet

wee

n di

abet

es an

d D

R on

set (

year

s)

119 plusmn 52

AG + GG

80 plusmn 44



0

1

2

3

4

5

6

GG

GG

AG


Odd

s rat

io

CFH

(rs8

0029

2)

AA

Ref

567














Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Acknowledgment


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Mediators of Inammation . //downloads.hindawi.com/journals/mi/2013/748435.pdfMediators of Inammation...

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