Association of death receptor 4 haplotype 626C–683C with an increased breast cancer risk

Bernd Frank 1, *, Kari Hemminki 1, 2, Kalai S. Shanmugam 1, Alfons Meindl 3, Rüdiger Klaes 4, Rita K. Schmutzler 5, Barbara Wappenschmidt 5, Michael Untch 6, Peter Bugert 7, Claus R. Bartram 4 and Barbara Burwinkel 1

1 Division of Molecular Genetic Epidemiology, German Cancer Research Center, Heidelberg, Germany, 2 Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden, 3 Department of Gynaecology and Obstetrics, Klinikum rechts der Isar at the Technical University, Munich, Germany, 4 Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 5 Department of Gynaecology and Obstetrics, Division of Molecular Gynaeco-Oncology, Clinical Center University of Cologne, Germany, 6 Department of Gynaecology and Obstetrics at the Ludwig-Maximilians-University, Munich, Germany and 7 Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessia, Faculty of Clinical Medicine, University of Heidelberg, Mannheim, Germany

* To whom correspondence should be addressed. Tel: +49 6221 421802; Fax: +49 6221 421810; Email: b.frank{at}dkfz.de


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Dysregulation of apoptosis plays a crucial role in carcinogenesis. Tumour necrosis factor-related apoptosis-inducing ligand stimulates the extrinsic apoptotic pathway by binding to death receptor 4 (DR4). Thus, genetic alterations within the candidate tumour suppressor gene DR4 would be expected to provoke a deficient apoptotic signalling thereby facilitating the development of cancer. The DR4 variants Thr209Arg and Glu228Ala were genotyped in a series of 521 breast cancer cases and 1100 control subjects from Germany, determining their impact on breast cancer risk. Neither Thr209Arg (626C>G) nor Glu228Ala (683A>C) alone were significantly associated with breast cancer risk [odds ratio (OR) = 0.84, 95% confidence interval (CI) = 0.65–1.08, P = 0.18 and OR = 0.89, 95% CI = 0.72–1.12, P = 0.30]. However, haplotype analysis revealed a 3.5-fold risk for carriers of the 626C–683C haplotype (OR = 3.52, 95% CI = 1.45–8.52, P = 0.003).

Abbreviations: DR4, death receptor 4; SNP, single nucleotide polymorphism; TNF, tumour necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Apoptosis represents a physiological mechanism that eliminates damaged cells from an organism, thus controlling cell numbers and tissue size, and sustaining homeostasis. Suppression of apoptosis implicates deregulated cell proliferation and predisposition to cancer. The transmembrane death receptors of the tumour necrosis factor (TNF) receptor superfamily mediate the activation of the central forces of regulated cell death—the caspases—triggering cell dissolution (1,2). The proapoptotic death receptor 4 (DR4, TNFRSF10A, TRAILR-1) has been characterized as the first DR to efficiently bind the TNF-related apoptosis-inducing ligand (TRAIL) (3). DR4, located on chromosome 8p21–22, encodes 486 amino acids which form two extracellular cysteine-rich, ligand-binding pseudorepeats (50s and 90s loops), a single transmembrane helix and a cytoplasmic death domain which provokes apoptosis upon TRAIL binding (46). Allelic losses of 8p21–22 (710) and a decreased expression of DR4 mRNA in breast cancer cell lines and tumours have been shown to facilitate the inhibition of tumour suppression, thus very likely contributing to malignant phenotypes (5,11). DR4 mutations have been described in different human cancers, such as lung, head and neck cancer, non-Hodgkin's lymphoma as well as in breast cancer and breast cancer cell lines (5,12,13), which points to DR4 as an attractive candidate tumour suppressor gene. Several genotyping studies have addressed the association of DR4 Thr209Arg in different types of cancer, but the results have been controversial (12,14,15).

This is the first case-control study to investigate the effect of the DR4 variant Glu228Ala (683A>C), along with Thr209Arg (626C>G), on familial breast cancer risk, revealing an increased risk for carriers of the 626C–683C haplotype.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Subjects
The breast cancer cases consisted of 521 unrelated German women (21–87 years of age; mean age 44.6) without BRCA1 and BRCA2 mutations. According to the German Consortium for Hereditary Breast and Ovarian Cancer they were classified into six categories based on family history: (i) families with two or more breast cancer cases including at least two cases with onset below the age of 50; (ii) families with at least one male breast cancer case; (iii) families with at least one breast cancer and one ovarian cancer case; (iv) families with at least two breast cancer cases comprising one case diagnosed before the age of 50; (v) families with at least two breast cancer cases diagnosed after the age of 50; and (vi) single cases of breast cancer diagnosed before the age of 35 (16). They were registered during the years 1997–2004 through the Institute of Human Genetics (Heidelberg, Germany), the Department of Gynaecology and Obstetrics (Cologne, Germany) and the Department of Medical Genetics (Munich, Germany). The control series included 1100 healthy and unrelated female blood donors (18–68 years of age, mean age 41.0) having the ethnic background of the breast cancer patients, registered by the Institute of Transfusion Medicine and Immunology (Mannheim, Germany). The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany).

Single nucleotide polymorphism selection
In order to validate annotated DR4 single nucleotide polymorphisms (SNPs; dbSNP database, http://www.ncbi.nlm.nih.gov/) and to select positive controls for subsequent TaqMan allelic discrimination assays, genomic DNA of 20 randomly chosen German breast cancer patients were used for sequencing. This analysis comprised three non-synonymous coding DR4 SNPs (Thr33Ile, Thr209Arg and Glu228Ala; Table I), Thr209Arg and Glu228Ala being confirmed. Since the DR4 variant His141Arg has been shown to cosegregate with Thr209Arg (12), we abandoned further investigation.


View this table:
[in this window]
[in a new window]
 
Table I. Allele frequencies of sequenced DR4 single nucleotide polymorphisms (SNPs) using DNA of 20 breast cancer patients, primer sequences for SNP verification and TaqMan probes for allelic discrimination

 
Genotyping
Both DR4 Thr209Arg and Glu228Ala were genotyped using TaqMan allelic discrimination assays. The design of TaqMan probes as well as primers was done with the Applied Biosystems Assay-by-Design service (Table I). As it has been shown that even rare polymorphisms in the vicinity of the SNP to be analysed might falsify TaqMan results, the SNP assays were validated by sequencing >4% of samples, attaining a concordance rate of 100% (17). Genotype distributions were consistent with the Hardy–Weinberg equilibrium. PCR amplification and sequencing were performed as previously described (18).

Statistical analysis
Calculations of Hardy–Weinberg equilibrium, genotype-specific odds ratios (ORs) and 95% confidence intervals (95% CIs) were carried out using a tool offered by the Institute of Human Genetics, Technical University Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Age-adjusted ORs and corresponding 95% CIs were computed by means of unconditional logistic regression using the Statistical Analysis System software (Version 8.2.; SAS Institute Inc., Cary, NC). P-values were calculated using two-sided {chi}2-tests. Haplotypes were determined using the PHASE 2 software created by Matthew Stephens (http://archimedes.well.ox.ac.uk/pise/PHASE.html) (19).


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Members of the TNF receptor family are thought to play a pivotal role in carcinogenesis by regulating apoptotic events. Assuming that genetic variations in DR4 might affect individual breast cancer risk, we investigated the prevalence of annotated DR4 polymorphisms in a set of 20 DNA samples from breast cancer patients and confirmed Thr209Arg and Glu228Ala (Table I). Both SNPs reside next to the DR4 ligand-binding ectodomain and may affect TRAIL binding, and—as a result—apoptotic signalling (12). To evaluate their influence on breast cancer risk, a case–control study was performed using 521 German BRCA1 and BRCA2 mutation negative breast cancer cases and 1100 control samples. While several reports have controversially discussed the impact of the Thr209Arg variant on breast, bladder, lung, head and neck cancer risk (12,14,15), this is the first study to investigate the role of DR4 Glu228Ala and DR4 haplotypes on cancer predisposition. Thr209Arg showed no significance with regard to breast cancer risk (OR = 0.84, 95% CI = 0.65–1.08, P = 0.18; Table II). These results are in agreement with the previously published data using subjects from Sheffield, UK (15). Similarly, Glu228Ala did not show any association with breast cancer risk (OR = 0.89, 95% CI = 0.72–1.12, P = 0.30, Table II). Adjustment for age did not change the ORs of the respective SNPs (Thr209Arg: OR = 0.83, 95% CI = 0.64–1.07, P = 0.16; Glu228Ala: OR = 0.87, 95% CI = 0.69–1.08, P = 0.20). Neither variants exhibited a significant association with breast cancer risk after stratification according to age at diagnosis (<50; ≥50). Combining DR4 Thr209Arg and Glu228Ala for the haplotype analysis resulted in four distinct haplotypes (Table III). Investigation of the respective haplotypes for linkage to breast cancer showed no association of 626C–683A and 626G–683A with breast cancer risk (Table III). The common haplotype 626G–683C showed a decreased OR with a borderline significance (OR = 0.86, 95% CI = 0.71–1.04, P = 0.11, Table III; age-adjusted OR = 0.84, 95% CI = 0.70–1.02, P = 0.08). However, analysis of the rare haplotype 626C–683C revealed a significant difference in frequency when comparing breast cancer cases with control samples, resulting in an increased risk of breast cancer with an OR of 3.52 (95% CI = 1.45–8.56, P = 0.003, Table III; age-adjusted OR = 3.20, 95% CI = 1.31–7.83, P = 0.01). The observed association indicates that the combination of 626C and 683C might be functionally relevant by altering TRAIL binding or that 626C–683C might be in linkage disequilibrium with a functional variant residing in DR4 or in neighbouring genes. Since the haplotype 626C–683C is very rare, it represents a moderate amount of risk regarding familial breast cancer in general. Although the effect of 626–683C is high (OR = 3.52), it is based on small numbers (Table III). As a result, additional studies would be valuable to specify our risk assessment.


View this table:
[in this window]
[in a new window]
 
Table II. Genotype frequencies of DR4 polymorphisms in unrelated German BRCA1/2 mutation negative breast cancer patients and healthy unrelated female control subjects, odds ratios (ORs) with 95% confidence intervals (95% CIs) and respective P-values, which were calculated using two-sided {chi}2-test

 

View this table:
[in this window]
[in a new window]
 
Table III. Haplotype distributions of DR4 Thr209Arg (626C>G) and Glu228Ala (683A>C) in unrelated German BRCA1/2 mutation negative breast cancer patients and healthy unrelated female control individuals

 
In summary, this study showed a significant association between the DR4 haplotype 626C–683C and increased risk of developing breast cancer in a German population.


    Acknowledgments
 
The authors are grateful to Justo Lorenzo Bermejo for statistical advice and Michael Wirtenberger for support. The German breast cancer samples were collected within a project funded by the Deutsche Krebshilfe and coordinated by Prof. Rita K. Schmutzler.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 

  1. Hengartner,M.O. (2000) The biochemistry of apoptosis. Nature, 407, 770–776.[CrossRef][ISI][Medline]
  2. Evan,G.I. and Vousden,K.H. (2001) Proliferation, cell cycle and apoptosis in cancer. Nature, 411, 342–348.[CrossRef][ISI][Medline]
  3. Pan,G., O'Rourke,K., Chinnaiyan,A.M., Gentz,R., Ebner,R., Ni,J. and Dixit,V.M. (1997) The receptor for the cytotoxic ligand TRAIL. Science, 276, 111–113.[Abstract/Free Full Text]
  4. Hymowitz,S.G., Christinger,H.W., Fuh,G., Ultsch,M., O'Connell,M., Kelley,R.F., Ashkenazi,A. and de Vos,A.M. (1999) Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Mol. Cell, 4, 563–571.[CrossRef][ISI][Medline]
  5. Seitz,S., Wassmuth,P., Fischer,J., Nothnagel,A., Jandrig,B., Schlag,P.M. and Scherneck,S. (2002) Mutation analysis and mRNA expression of trail-receptors in human breast cancer. Int. J. Cancer, 102, 117–128.[CrossRef][ISI][Medline]
  6. Ashkenazi,A. and Dixit,V.M. (1998) Death receptors: signaling and modulation. Science, 281, 1305–1308.[Abstract/Free Full Text]
  7. Emi,M., Fujiwara,Y., Nakajima,T., Tsuchiya,E., Tsuda,H., Hirohashi,S., Maeda,Y., Tsuruta,K., Miyaki,M. and Nakamura,Y. (1992) Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer. Cancer Res., 52, 5368–5372.[Abstract]
  8. Kagan,J., Stein,J., Babaian,R.J., Joe,Y.S., Pisters,L.L., Glassman,A.B., von Eschenbach,A.C. and Troncoso,P. (1995) Homozygous deletions at 8p22 and 8p21 in prostate cancer implicate these regions as the sites for candidate tumor suppressor genes. Oncogene, 11, 2121–2126.[ISI][Medline]
  9. Wistuba,I.I., Behrens,C., Virmani,A.K., Milchgrub,S., Syed,S., Lam,S., Mackay,B., Minna,J.D. and Gazdar,A.F. (1999) Allelic losses at chromosome 8p21-23 are early and frequent events in the pathogenesis of lung cancer. Cancer Res., 59, 1973–1979.[Abstract/Free Full Text]
  10. Yaremko,M.L., Kutza,C., Lyzak,J., Mick,R., Recant,W.M. and Westbrook,C.A. (1996) Loss of heterozygosity from the short arm of chromosome 8 is associated with invasive behavior in breast cancer. Genes Chromosomes Cancer, 16, 189–195.[CrossRef][ISI][Medline]
  11. Shin,M.S., Kim,H.S., Lee,S.H. et al. (2001) Mutations of tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2) genes in metastatic breast cancers. Cancer Res., 61, 4942–4946.[Abstract/Free Full Text]
  12. Fisher,M.J., Virmani,A.K., Wu,L., Aplenc,R., Harper,J.C., Powell,S.M., Rebbeck,T.R., Sidransky,D., Gazdar,A.F. and El-Deiry,W.S. (2001) Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer. Clin. Cancer Res., 7, 1688–1697.[Abstract/Free Full Text]
  13. Lee,S.H., Shin,M.S., Kim,H.S. et al. (2001) Somatic mutations of TRAIL-receptor 1 and TRAIL-receptor 2 genes in non-Hodgkin's lymphoma. Oncogene, 20, 399–403.[CrossRef][ISI][Medline]
  14. Hazra,A., Chamberlain,R.M., Grossman,H.B., Zhu,Y., Spitz,M.R. and Wu,X. (2003) Death receptor 4 and bladder cancer risk. Cancer Res., 63, 1157–1159.[Abstract/Free Full Text]
  15. MacPherson,G., Healey,C.S., Teare,M.D., Balasubramanian,S.P., Reed,M.W., Pharoah,P.D., Ponder,B.A., Meuth,M., Bhattacharyya,N.P. and Cox,A. (2004) Association of a common variant of the CASP8 gene with reduced risk of breast cancer. J. Natl. Cancer Inst., 96, 1866–1869.[Abstract/Free Full Text]
  16. Meindl,A. and the German Consortium for Hereditary Breast and Ovarian Cancer. (2002) Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 mutation profiles and frequencies for the German population. Int. J. Cancer, 97, 472–480.[CrossRef][ISI][Medline]
  17. Frank,B., Hemminki,K. and Burwinkel,B. (2005) A bias in genotyping the ERBB2 (Her2) Ile655Val variant. Carcinogenesis, Apr 28; [Epub ahead of print].
  18. Frank,B., Hemminki,K., Wirtenberger,M., Bermejo,J.L., Bugert,P., Klaes,R., Schmutzler,R.K., Wappenschmidt,B., Bartram,C.R. and Burwinkel,B. (2005) The rare ERBB2 variant Ile654Val is associated with an increased familial breast cancer risk. Carcinogenesis, 26, 643–647.[Abstract/Free Full Text]
  19. Stephens,M., Smith,N.J. and Donnelly,P. (2001) A new statistical method for haplotype reconstruction from population data. Am. J. Hum. Genet., 68, 978–989.[CrossRef][ISI][Medline]
Received April 25, 2005; revised June 6, 2005; accepted June 15, 2005.





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
26/11/1975    most recent
bgi164v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Frank, B.
Articles by Burwinkel, B.
PubMed
PubMed Citation
Articles by Frank, B.
Articles by Burwinkel, B.