a systematic analysis of the association studies between

©IndianAcademyofSciences

RESEARCH ARTICLE

A systematic analysis of the association studies between CASP8 D302Hpolymorphisms and breast cancer risk

YINLIANG ZHANG1, WEI LI1,2, YI HONG1,2, GUOYING WU1,2, KAN HE1,2∗ and DAHAI LIU1,2∗

1School of Life Sciences, Anhui University, Hefei City, Anhui 230601, People’s Republic of China2Center for Stem Cell and Translational Medicine, Anhui University, Hefei City, Anhui 230601, People’s Republic of

China

Abstract

Caspase 8 (CASP8) is a regulator of apoptosis, whose genetic variation has been reported to be associated with the riskof various cancers. Especially, the single-nucleotide polymorphism (SNP) rs1045485, which generates the substitutionD302H in CASP8, is likely to be associated with breast cancer. Several previous studies have reported the associationof CASP8 D302H polymorphism with breast cancer; however, the results are inconsistent. To validate the associationbetween CASP8 D302H polymorphism and breast cancer risk, we performed an updated meta-analysis of 18 studiesincluding 27,807 cases and 32,332 controls. We tested the overall association between this SNP and breast cancersusceptibility and stratified subgroups based on countries where cases are from. We confirmed a significant correlationbetween CASP8 D302H polymorphism and the reduced breast cancer susceptibility in population from UK, Germanyand Poland, but no significant association was observed in other countries, such as Finland or USA. Our findingsindicate the relationship of SNP CASP8 D302H and breast cancer would not be universal but only be sensitive in someparticular European countries. The genetic difference for diverse countries may be useful in individual and precisionmedicine or health.

[He K., Li W., Zhang Y., Hong Y., Wu G. and Liu D. 2017 A systematic analysis of the association studies between CASP8 D302Hpolymorphisms and breast cancer risk. J. Genet. 96, 283–289]

Introduction

CASP8 encodes a member of the cysteine–aspartic acidprotease (caspase) family, which is a cysteine peptidasethat can activate various cellular proteases or proteinsleading to apoptosis through the Fas cell surface deathreceptor (FAS)/FAS ligand (FASLG)-mediated apopto-sis pathway. CASP8 is located on chromosome 2q33-34,harbouring 10 exons that span ∼30 kb, in which therewere at least 168 single-nucleotide polymorphisms (SNPs),mostly rare or noncoding. Several studies have evalu-ated the associations between some of these CASP8 SNPsand risk of various cancers (Pittman et al. 2008; Ramuset al. 2008; Couch et al. 2009; Lubahn et al. 2010). Twofunctional SNPs, rs3834129 named as six-nucleotide inser-tion/deletion (−652 6N ins/del) in the promoter regionand rs1045485 named as D302H in the coding region of

*For correspondence. E-mail: Kan He, [email protected];Dahai Liu, [email protected] Zhang and Wei Li contributed equally to this work.

Keywords. breast cancer; caspase 8; rs1045485 polymorphism; meta-analysis.

CASP8, are both thought to be important in cancer aeti-ology. The SNP rs3834129 is considered to be associatedwith susceptibility to multiple cancers, while the effect ofSNP rs1045485 is mainly associated with the risk of breastcancer (Sun et al. 2007).

Previous studies on the association between rs1045485polymorphism and breast cancer indicate an inconsistentresult. Several studies observed a significant association(MacPherson et al. 2004a; Cox et al. 2007; Long et al.2013), while some other studies showed no associationor even no polymorphism (Guan et al. 2014; Michailidouet al. 2015). Recent meta-analysis study pooled the resultfrom four case–control studies, including 18,791 breastcancer cases and 20,318 controls of Caucasians (Sergen-tanis and Economopoulos 2010). Another meta-analysisstudy including cases of various cancer diseases showedthat rs1045485 was found to be only associated with breast

Journal of Genetics, DOI 10.1007/s12041-017-0774-y, Vol. 96, No. 2, June 2017 283

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Yinliang Zhang et al.

cancer risk (Ji et al. 2014). Thus, we conducted an updatedmeta-analysis by adding the latest data, avoiding sam-ple overlapping and stratifying subgroups with the aimof gaining a more reliable evaluation of the associationbetween rs1045485 polymorphism and breast cancer sus-ceptibility. Here, we performed a large-scale meta-analysisincluding 60,139 individuals (27,807 cases and 32,332 con-trols) fromEuropean countries,USAandAustralia, aimedto find the exact relationship between SNP rs1045485 andbreast cancer risk across different countries.

Materials and methods

Eligibility of relevant studies

All original articles published inEnglish that examined theassociation of the rs1045485 polymorphism with breastcancer (published before June 2015) were considered forour meta-analysis. The PubMed and Web of Science weresearched to identify appropriate studies. The followingcombinations of terms were used in our database searches:(‘breast cancer’) and (‘rs1045485’ or ‘CASP8 D302H’).Further, the searches were supplemented by referencescited in other papers. The flowchart of our analysis isshown in figure 1.

To include relevant studies in this meta-analysis, thefollowing criteria were used: (i) studies assessed link-age of rs1045485 polymorphism with breast cancer risk;(ii) female breast cancer patients / breast cancer casesshould be diagnosed explicitly; (iii) controls should beunrelated cancer-free individuals / case–control design;and (iv) reported in English. When multiple reports hadoverlapping sample populations, only the study withlargest sample size was retained.

Figure 1. The flowchart of our analysis.

The studies were excluded if: (i) data were reused onthe same polymorphism; (ii) control genotype distribu-tions were not in Hardy–Weinberg equilibrium (HWE);and (iii) incomplete reporting of genotype frequencies.

Data extraction

For each eligible study, the following information wasextracted: the first author, year of publication, ethnicityof participants, source of controls, number of genotypedcases/controls, method for quality control of genotypingresult. The data were primarily extracted from tables andsupplemented by significant information presented in textsand/or figures.

Statistical analysis

For each study, the HWEwas assessed in controls. The χ2

goodness of fit is used to test deviation fromHWE. Studieswere considered to deviate from HWE at P < 0.05 (GuoandThompson 1992). The inconsistency index, I 2 was cal-culated to evaluate the variation among studies owing toheterogeneity (0–25%, no heterogeneity; 25–50%, moder-ate heterogeneity; 50–75%, large heterogeneity; 75–100%,extreme heterogeneity) (Higgins et al. 2003). The datawerecombined using logistic regression with the fixed-effectspooling model if there was no or moderate heterogeneities(I 2 < 50%). Alternatively, the random effects model wasused (I 2 > 50%). Sensitivity analysis was performed byexcluding one study at a time to determine the corre-sponding magnitude of the weight of each study to thesummary results. The most biologically fit genetic modelwas selected according to the comprehensive effect of thegene using logistic regression. The association betweenrs1045485 polymorphism and breast cancer risk was eval-uated using the odds ratio (OR) and the 95% confidenceinterval (CI). Funnel plots used to observe the publica-tion bias were complemented with Egger’s regression andBegg’s rank correlation test (P > 0.10). The statisticalanalyses were performed using STATA ver. 11.2 (StataCorporation, Texas, USA).

Results and discussion

Study characteristics

Six articles including 22 studies were identified to meet theinclusion criteria, and the details are provided in table 1.We thoroughly reviewed these articles to detect overlap-ping samples. Shephard et al. (2009) used a staged-studydesign from three datasets from UK, Germany and Utah.Study of German dataset by Shephard et al. (2009) andFrank et al. (2005) consisted the same breast cancer casesand controls of German patients. Hence, we included onlythe study by Shephard et al. (2009) and Cox et al. (2007),which included data from 14 studies. Study of UK datasetby Shephard et al. (2009), study of Sheffield dataset byCox

284 Journal of Genetics, Vol. 96, No. 2, June 2017

A systematic analysis

Table

1.Cha

racteristics

ofstud

iesinclud

edin

thismeta-an

alysis.

Case

Con

trol

MAF

HWEPvalue

Reference

Region

Stud

yGG

CG

CC

GG

CG

CC

Case(%

)Con

trol

(%)

Cam

paet

al.(2011)

Europ

ean

dUSA

BPC3

6414

1539

110

8834

2345

197

21.8

24.1

0.004

Shepha

rdet

al.(2009)

UK

SBCS

896

292

17839

314

2727.1

31.2

0.708

German

yGC-H

BOC

275

764

815

260

2323.7

27.9

0.672

USA

UBCS

557

130

15338

7410

22.8

22.3

0.019

Sigu

rdsonet

al.(2007)

USA

RT

660

185

7802

232

2223.4

26.1

0.283

Cox

etal.(2007)

Australia

ABCFS/kC

onFaB

1117

307

2243

314

28

24.3

27.1

0.339

UK

BBC

440

135

843

514

215

25.9

29.1

0.407

German

yGENIC

A466

122

11464

137

1524.0

27.1

0.206

German

yHBCS

771

205

15745

246

1523.7

27.4

0.295

Finland

Helsink

i68

013

58

712

160

518

.319

.40.212

UK

ICR_F

BCS

772

238

1210

8235

231

25.6

28.3

0.706

Finland

Kuo

pio

374

703

349

800

17.0

18.6

0.033

USA

MayoClin

ic600

176

14603

201

2425.8

30.1

0.151

Poland

Poland

1590

430

251714

555

4523.5

27.9

0.993

UK

SEARCH

3117

827

663330

949

8123.9

25.5

0.164

UK

Sheffield

∗672

212

14675

265

2426.7

32.5

0.739

Sweden

SASB

AC

1164

328

201139

310

3724.3

25.8

0.005

Spain

CNIO

403

9714

417

137

824

.327

.20.387

USA

USR

T∗

578

158

7783

224

2023.1

25.7

0.398

Frank

etal.(2005)

German

yFrank

’s∗275

764

815

260

2323.7

27.9

0.672

MacPherson

etal.(2004b)

UK

Sheffield

∗718

221

15675

265

2426.3

32.5

0.739

UK

EastAng

lia14

6835

822

1591

450

4121

.825

.60.168

∗ Excludedfrom

meta-an

alysisforlargeov

erlapp

ingwithotherstud

y.

Journal of Genetics, Vol. 96, No. 2, June 2017 285


et al. (2007) and MacPherson et al. (2004a) were found toshare common sample sources of north-European originand resident in the Sheffield area. Therefore, only the studybyShephard et al. (2009), which had the largest sample sizeand latest data was used in our meta-analysis. Studies bySigurdson et al. (2007) and Cox et al. (2007) were found toshare common sample sources of US radiologic technol-ogists, and the study by Sigurdson et al. (2007) was usedin our meta-analysis because of the slightly larger sam-ple size. Finally, 18 studies including 27,807 breast cancercases and 32,332 controls were used in our meta-analysis.We checkedHWEof each studyanddivided them into sub-groups based on the controls that are deviated fromHWE.

Heterogeneity and model

All heterogeneity statistic I 2 values were found to be<25% in the present study, which indicated that theappropriate pooling model should have fixed effects. Fur-ther, using a suitable underlying genetic model in geneticassociation studies is crucial for combining data bio-logically rather than statistically. Previous studies listedthe association under different genetic models, but with-out a best fit model, here we carefully selected themost likely genetic model for representing the associ-ation between CASP8 D302H and breast cancer. The

estimated OR(CC vs GG), OR(CG vs GG) and OR(CC vs CG)

values were 0.73 (95% CI: 0.64–0.83), 0.90 (95% CI: 0.86–0.94) and0.81 (95%CI: 0.71–0.93), respectively.Accordingto the methodology for genetic model selection developedby Thakkinstian et al. (2005), the genetic model was mostlikely to be codominant. After the sensitivity analysis, noindividual study was found to affect the overall resultsrobustly, which implied the magnitude of the summaryevaluation.

Gene effect

Theoverall results of the genetic analysis indicated a signif-icant association between CASP8 D302H and a reducedbreast cancer risk (OR = 0.89, 95% CI: 0.86–0.92) (fig-ures 2 and 3).

To test whether the studies whose controls were not inHWEaffect thepooling result,we stratified two subgroups.Significant association between breast cancer risk andCASP8 D302H are shown in both the subgroups whoseHWE P values were >0.05 (OR = 0.87, 95% CI: 0.84–0.91) and<0.05 (OR = 0.91, 95%CI: 0.86–0.96) (figure 2),indicating that if the studies are in HWE, those could beneglected in this study. To gather asmuch data aswe could,we included the studies no matter if its controls were inHWE.

Figure 2. Stratified analysis based on HWE for the association between rs1045485 polymorphism and breast cancer risk using acodominant genetic model.



Figure 3. Stratified analysis based on the country of sources for the association between rs1045485 polymorphism and breast cancerrisk using a codominant genetic model.

We then performed the stratified analysis according tothe cases from different countries. Significant associationwas observed in the studies of UK (OR = 0.89, 95%CI: 0.84–0.95), Germany (OR = 0.85, 95% CI: 0.75–0.97) and Poland (OR = 0.82, 95%CI: 0.72–0.93). Campaet al. (2011) performed amultiethnicity case–control studywithin the National Cancer Institute’s Breast and ProstateCancer Cohort Consortium (BPC3), including Euro-peandescent, Latino,African-American,Asian-American

(mostly of Japanese origin) and Native Hawaiian. There-fore, we identified it as multiethnicity, not included inUSA or any European countries. Result shows significantassociation in this study. However, in Australia, Finland,Spain, Sweden andUSA, there is no significant associationbetween CASP8 D302H and breast cancer risk (figure 3).No heterogeneity was found between groups or betweenstudies. Hence, the association between CASP8 D302Hand breast cancer risk may be country-variable.



Previously, Sergentanis and Economopoulos (2010)investigated four studies of association between CASP8D302H and breast cancer risk, the summary of each studywas pooled into ameta-analysis by using dominantmodel,but without distinguishing the countries where cases werefrom.Overall result indicates that theCASP8D302Hpoly-morphism may be associated with reduced breast cancerrisk in Caucasian population (OR = 0.87, 95% CI: 0.83–0.92). A case–control study in Han Chinese populationfound no polymorphism of CASP8 D302H (Guan et al.2014). A recent study in African-American populationshowed that CASP8 D302H were deviated from Hardy–Weinberg test and had a Minor Allele Frequency (MAF)< 5% in African-ancestry populations and it was notreplicated in all the previous studies of African-ancestrypopulations (Long et al. 2013). Therefore, the associationbetween CASP8 D302H and breast cancer risk may onlyoccur in some specific countries.While meta-analysis shed light on the trend of associ-

ation between SNP and disease risk, gene–environmentinteractions have the potential to reveal the biological pro-cesses leading to disease, identify the most relevant riskfactors, and improve the accuracy of epidemiological riskmodels. Travis et al. (2010) tested gene–environment inter-actions in a large prospective UK cohort, studying theeffects of 12 polymorphisms, including CASP8 D302H,in relation to 10 established environmental risk factors(age at menarche, parity, age at first birth, breastfeeding,menopausal status, age at menopause, use of hormonereplacement therapy, body-mass index, height and alco-hol consumption). Results showed that the risks of breastcancer associated with CASP8 D302H do not vary signifi-cantly withmost of the established environmental risk fac-tors, except for alcohol consumption. Replication studiesforCASP8D302Hand alcohol consumption are reported,suggesting CASP8 D302H is a complicated SNP andworth more investigation to explore the plausible biologi-cal mechanisms that can explain this association (Nickelset al. 2013; Fletcher and Dudbridge 2014). Some of theunderlying causes that can partly explain the differencesobserved by country may be as follows: weak association,statistical power issues in some countries, possible differ-ent distribution of breast cancer subtypes in the differentcountries, possible gene–environment interaction, etc.The publication bias was accessed using Begg’s (P =

0.495) and Egger’s (P = 0.517) tests. The funnel plot dis-played a symmetric shape (figure 4), indicating the absenceof a publication bias for both positive and negative ornonsignificant findings from published studies.In conclusion, CASP8 has long been considered as

a breast cancer susceptibility gene, at least 168 SNPshave been reported for CASP8, mostly rare or noncod-ing. Here, we provide a comprehensive meta-analysis onCASP8 D302H polymorphism, also known as rs1045485,including 27,807 cases and 32,332 controls fromEuropeancountries, USA and Australia. An overall trend indicates

Figure 4. Begg’s funnel plot displaying a symmetric shape. Thehorizontal axis represents the standard error of OR value, andthe vertical axis represents the OR value of each study.

a protective effect of the polymorphism. This is in accor-dance with previous ones, which had been performed onsmaller number of studies (Breast Cancer Association2006; Janssens et al. 2009). However, a recentGWAS studydetects no association between rs1045485 and breast can-cer risk in European women (Michailidou et al. 2015). Toaddress this problem, we gathered as many studies as wecould and divided them into subgroups by the countrieswhere cases were from.Stratified analysis implies the protective association

seems to pertain only to a part of Caucasian, who weremainly resident in UK, Germany and Poland. To the peo-ple who are from Finland, Spain, Sweden, Australia andUSA, no significant association is observed. In addition,rs1045485 lacks polymorphism in Chinese and African-American population, suggesting that the associationbetween CASP8 D302H and breast cancer risk may becountry-sensitive, the fact which may be due to the effectof gene–environment interaction (Long et al. 2013; Guanet al. 2014).Since, the early investigations were taken place in

populations of UK or Germany, many follow-up stud-ies focussed on these two countries too. Eventhough,we already excluded some large overlapping studies, themajority source data are still from UK or Germany. Onone hand, the lack of CASP8 D302H polymorphismin ethnicity other than Caucasians should be confirmedby genotyping in other ethnicity. On the other hand, inCaucasians, or in European descent, detailed investiga-tion should be performed to explain why the differencesoccurred in same ethnic group who lived in different coun-tries. The difference in the MAF and different LinkageDisequilibrium (LD) structure in these populations mayalso partly explain the difference between ethnic groupsfor the association between rs1045485 and breast can-cer risk. According to the study of Michailidou et al.(2015), increasing the size of the population can signifi-cantly improve the test results and obtain a deeper andclearer view of the function of CASP8 D302H.



Acknowledgements

We acknowledge financial support by the ScientificResearch Foundation and Academic and Technology Lead-ers Introduction Project, and 211 Project of Anhui University(10117700023, 02303203-32030081), and The Student ResearchTraining Programme of Anhui University (J18520131), andNat-ural Science Foundation Project of Anhui Province (1508085MH189, 1508085QC63) as well as The Education RevitalizationProject ofAnhui Province: StemCell andTranslationalMedicine(Y05201374).

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Received 6 June 2016, in revised form 22 August 2016; accepted 26 August 2016Unedited version published online: 29 August 2016

Final version published online: 17 June 2017

Corresponding editor: L. S. Shashidhara


a systematic analysis of the association studies between

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