The rare ERBB2 variant Ile654Val is associated with an increased familial breast cancer risk

Bernd Frank1,*, Kari Hemminki1,2, Michael Wirtenberger1, Justo Lorenzo Bermejo1, Peter Bugert3, Rüdiger Klaes4, Rita K. Schmutzler5, Barbara Wappenschmidt5, Claus R. Bartram4 and Barbara Burwinkel1

1 Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany, 2 Department of Biosciences at Novum, Karolinska Institute, Huddinge, Sweden, 3 Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessia, Faculty of Clinical Medicine, University of Heidelberg, Mannheim, Germany, 4 Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany and 5 Division of Molecular Gynaeco-Oncology, Department of Gynaecology and Obstetrics, Clinical Center, University of Cologne, Cologne, 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
 Material and methods
 Results
 Discussion
 References
 
Overexpression of the proto-oncogene ERBB2 (HER2/NEU) has been observed in 20–30% of breast cancers involving poor prognosis. Genetic alterations within ERBB2 have been shown to induce carcinogenesis and metastasis. We investigated eight annotated single nucleotide polymorphisms for occurrence in familial breast cancer samples. The confirmed variants Ile654Val, Ile655Val and Ala1170Pro were analysed in subsequent epidemiological studies on familial breast cancer risk. While Ala1170Pro resides within a C-terminally located regulatory domain, the two adjacent polymorphisms Ile654Val and Ile655Val are part of the transmembrane domain. A case–control study analysing a cohort of 348 German familial breast cancer cases and 960 corresponding controls showed no significant association of either Ile655Val (OR = 1.05, 95% CI = 0.82–1.34, P = 0.728) or Ala1170Pro (OR = 0.94, 95% CI = 0.74–1.20, P = 0.632) with familial breast cancer risk. Differences in haplotype frequencies between cases and controls could also not be detected. The ERBB2 variant Ile654Val, however, revealed an increased risk for carriers of the heterozygous Val654 allele (OR = 2.56, 95% CI = 1.08–6.08, P = 0.028). The rare Val654 variant is linked with the more frequent Val655, resulting in two consecutive valine instead of two isoleucine residues within the transmembrane domain. Computational analyses suggest that the Val654–Val655 allele provokes receptor dimerization and activation, thus stimulating kinase activity and cell transformation. We hypothesize that ERBB2 Val654 represents an oncogenic variant which might, in addition, influence clinical outcome and predict a worse prognosis.

Abbreviations: EGFR, epidermal growth factor receptor; HWE, Hardy–Weinberg equilibrium; MAPK, mitogen-activated protein kinase; 95% CI, 95% confidence intervals; OR, odds ratio; SNPs, single nucleotide polymorphisms.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Breast cancer is the most frequent malignancy among women in Western countries with 1 out of 12 women suffering from the disease during her life (1). According to a twin study, breast cancer is the result of both genetic (one-third) and environmental factors, leading to an amassing of mutations in essential genes (2). The genes involved in the pathogenesis of the hereditary forms of breast cancer, which constitute <5% of all breast cancer, include BRCA1, BRCA2, P53 and PTEN (3,4). Germline mutations of the high penetrance genes BRCA1 and BRCA2, for example, account for ~15–20% of familial breast cancer (4). Breast cancer susceptibility is likely to be influenced by genetic variations in low penetrance genes which may have a considerable impact on the aetiology of the disease (5,6). As a member of the epidermal growth factor receptor (EGFR) gene family (ERBB1ERBB4), the proto-oncogene ERBB2 (also designated HER2 or NEU) represents a major player in physiological processes like cell growth, differentiation and tissue development, as well as in carcinogenesis and metastasis (7,8). ERBB2 is located on chromosome 17q21 and encodes a transmembrane glycoprotein exhibiting tyrosine kinase activity (913). Activation of ERBB2 requires homo- or heterodimerization with other family members, followed by autophosphorylation and downstream signalling events (1416). Overexpression of EGFRs due to gene amplification is associated with a variety of solid tumour malignancies (1721). In 20–30% of breast cancers ERBB2 overexpression occurs, resulting in ligand-independent activation and more aggressive growth behaviour and reduced responses to chemotherapy and hormonal therapy, as well as a poor prognosis (18,22). Structural ERBB2 alterations have been implicated in carcinogenesis because they are frequently detected in breast cancer and other solid tumours (23). Single nucleotide polymorphisms (SNPs) residing in regulatory or functionally relevant gene regions might have an effect on protein function. Many genotyping studies have addressed the association between ERBB2 Ile655Val and breast cancer risk, but the results have remained controversial (2434).

The present case–control study has investigated, for the first time, the effects of Ile654Val (1960A->G) and Ala1170Pro (3508G->C), along with Ile655Val (1963A->G), on familial breast cancer risk and revealed an increased risk for heterozygous carriers of the rare Ile654Val variant.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
SNP verification
A randomly chosen set of 23 German familial breast cancer cases was initially screened for annotated ERBB2 SNPs (dbSNP database, NCBI) by DNA sequencing. This initial analysis comprised three synonymous (Ser442Ser, Ser457Ser and Lys1177Lys) as well as five non-synonymous polymorphisms (Trp452Cys, Ile654Val, Ile655Val, Pro927Arg and Ala1170Pro) covering the entire coding region of ERBB2 (Table I). The confirmed SNPs Ile654Val, Ile655Val and Ala1170Pro were chosen for further analyses using a large cohort of familial breast cancer cases.


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Table I. Allele frequencies of sequenced ERBB2 SNPs using 23 breast cancer samples, primer sequences for SNP verification and TaqMan probes for allelic discrimination

 
Study population
The familial breast cancer cases consisted of 348 unrelated German women (21–80 years of age) lacking BRCA1 and BRCA2 mutations and were categorized into six classes based on family history: (A1) families with two or more breast cancer cases including two cases with onset below the age of 50; (A2) families with at least one male breast cancer case; (B) families with at least one breast cancer and one ovarian cancer case; (C) families with at least two breast cancer cases comprising one case diagnosed before the age of 50; (D) families with at least two breast cancer cases comprising one case diagnosed after the age of 50; (E) single cases of breast cancer diagnosed before the age of 35 (35). They were collected during the years 1997–2003 through the Institute of Human Genetics (Heidelberg, Germany) and the Department of Gynaecology and Obstetrics (Cologne, Germany). The control sample included 960 healthy and unrelated blood donors (18–67 years of age) collected by the Institute of Transfusion Medicine and Immunology (Mannheim, Germany) sharing the same ethnic background as the breast cancer cases. The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany).

PCR amplification
Standard PCR amplification was performed using 10 ng genomic DNA, 1x PCR buffer, 1.5 mM MgCl2, 0.11 µM dNTP mixture (Invitrogen, Paisley, UK), 0.15 µM each primer (MWG Biotech AG, Ebersberg, Germany) and 0.4 U PlatinumTaq DNA polymerase (Invitrogen) in an 11 µl reaction volume. Primers for detecting ERBB2 SNPs were established on the basis of NT_010755 and NM_004448 (NCBI; Table I). Thermal cycling was carried out in GeneAmp PCR System 9700 thermocyclers (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation for 2 min at 94°C followed by four cycles of 30 s at 94°C, 20 s at 64°C and 30 s at 72°C; three cycles of 30 s at 94°C, 20 s at 62°C and 30 s at 72°C; four cycles of 30 s at 94°C, 20 s at 60°C and 30 s at 72°C; five cycles of 25 s at 94°C, 20 s at 58°C and 35 s at 72°C; 32 cycles of 25 s at 94°C, 20 s at 56°C, 30 s at 72°C; a final extension step for 4 min at 72°C.

Sequencing
Sequencing reactions were performed using a BigDye® Terminator Cycle Sequencing Kit (Applied Biosystems) in a 10 µl volume containing pre-treated PCR product (30 min at 37°C and 15 min at 85°C with 0.75 µl of ExoSapIT®; Amersham Biosciences, Uppsala, Sweden) and sequencing primer (Table I) under the following PCR conditions: 96°C for 2 min prior to 27 cycles of 96°C for 30 s, 54°C for 10 s and 60°C for 4 min. Sequencing products were precipitated with 2-propanol, washed with 75% ethanol, resuspended in 25 µl of H2O and finally loaded onto an ABI Prism 3100 Genetic analyser (Applied Biosystems). Primary sequencing data were analysed by means of Sequence Analysis software provided by Applied Biosystems and DNAStar Lasergene software (DNAStar Inc., Madison, WI).

SNP genotyping
ERBB2 Pro1170Ala was tested using TaqMan allelic discrimination. Design of the TaqMan probes and primers was carried out using the Assay-by-Design service (Applied Biosystems; Table I). Due to the close vicinity of Ile654Val and Ile655Val, design of the TaqMan probes failed. Thus, we had to fall back upon sequencing for the analysis of these two SNPs. TaqMan assays were performed in a reaction volume of 10 µl comprising 5 ng genomic DNA, 50 nM each probe, 225 nM each primer and 1x Universal Master Mix under the following amplification conditions: 2 min at 50°C, 10 min at 95°C and 35–45 cycles at 92°C for 15 s and 60°C for 1 min. Amplification products were measured and analysed with an ABI Prism 7900HT sequence detection system and SDS software (version 1.2). About 12% of the genotyping results were confirmed by sequencing.

Statistical analysis
Allele and genotype frequencies were both tested for Hardy–Weinberg equilibrium (HWE) and significance using {chi}2 tests. HWE was tested using a tool offered by the Institute of Human Genetics, TU Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/hwal.pl). Odds ratios (OR) (genotypic risks comparing genotype frequencies between control individuals and breast cancer patients) and 95% confidence intervals (95% CI) were calculated in the same manner. Logistic regression was used to calculate age-adjusted ORs.

Haplotype analysis
Haplotypes for ERBB2 Ile655Val and Ala1170Pro (Table III) were determined using PHASE 2 software by Matthew Stephens (36; http://archimedes.well.ox.ac.uk/pise/PHASE-simple.html).


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Table III. Haplotype distribution of ERBB2 Ile655Val (1963A > G) and Ala1170Pro (3508G > C) in German breast cancer patients and control individuals, odds ratios (with 1963A-3508G considered as 1), 95% CI and corresponding P-values

 

    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
DNA from 23 familial breast cancer patients (46 chromosomes) was amplified and sequenced for eight annotated coding ERBB2 polymorphisms (Table I). Among these, the adjacent Ile654Val and Ile655Val and the C-terminally located Ala1170Pro could be confirmed. To evaluate the impact of Ile654Val, Ile655Val and Ala1170Pro on breast cancer risk, we performed a case–control study using 348 German BRCA1 and BRCA2 mutation-negative familial breast cancer cases and 960 German control samples. While the relevance of Ile655Val has been controversially discussed many times (2434), there has never been a case–control study aimed at associating Ile654Val and Ala1170Pro with breast cancer risk.

Ile655Val and Ala1170Pro show no association with familial breast cancer risk
Concerning Ile655Val, genotype frequencies between breast cancer cases and control samples were similar (Table II), showing no significance with regard to breast cancer risk (OR = 1.05, 95% CI = 0.82–1.34, P = 0.73). The age-adjusted odds ratio was 1.02 (95% CI = 0.77–1.36, P = 0.88). Genotype frequencies among German controls were concordant with data of previously published case–control studies using British and German Caucasian individuals (26,29). Genotype distributions were consistent with the HWE (P = 0.42 for cases and P = 0.37 for controls). Ala1170Pro showed no association with familial breast cancer risk either (OR = 0.94, 95% CI = 0.74–1.2, P = 0.63), being consistent with the HWE (P = 0.94 for cases and P = 0.12 for controls). The age-adjusted odds ratio for Ala1170Pro was 0.87 (95% CI = 0.65–1.15, P = 0.32). Neither did the Ile655Val and Ala1170Pro genotypes exhibit significance with respect to breast cancer risk when stratified according to age at diagnosis, i.e. ≤40 or >40 (Table II). The distribution of haplotypes between cases and controls was similar. Comparing the most frequent haplotype among cases and controls (1963A–3508G) with all others showed no significance with regard to breast cancer risk (Table III).


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Table II. Genotype frequencies of ERBB2 polymorphisms in German breast cancer patients and controls, odds ratios (OR) with 95% confidence intervals (95% CI) and respective P-values

 
Val654 increases risk of familial breast cancer
The analysis of Ile654Val revealed a significant difference in genotype frequencies between breast cancer cases and controls (Table II), resulting in an increased risk of familial breast cancer with an odds ratio (OR) of 2.56 (95% CI = 1.08–6.08, P = 0.028; Table II). The age-adjusted odds ratio was 2.83 (95% CI = 1.01–7.92, P = 0.048). As this variant is very rare, the homozygous genotype GG (Val/Val) was not detected in either the cases or the controls. Stratification according to the age at diagnosis showed an association of Ile654Val with an increased breast cancer risk for women >40 years (OR = 2.86, 95% CI = 1.1–7.5, P = 0.025; Table II). The risk for women aged ≤40 years was increased (OR = 2.21), but not significant (95% CI = 0.61–8.04, P = 0.22). Genotype distributions were consistent with the HWE (P = 0.79 for cases and P = 0.86 for controls). The heterozygous genotype AG (Ile/Val) exclusively occurred in combination either with the more frequent heterozygous genotype AG (Ile/Val) or with the rare homozygous genotype GG (Val/Val) at position 655, indicating linkage between the rare Val654 and the more frequent Val655. Due to the rareness of Ile/Val at position 654, haplotype analysis between the cases and the controls was not performed.


    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
ERBB2 amplification and overexpression have been implicated in a variety of human cancers, particularly in breast cancer (18). Assuming that genetic variations in ERBB2 might affect individual breast cancer risk, we analysed the prevalence of eight coding ERBB2 SNPs in a set of 46 chromosomes from breast cancer patients and confirmed Ile654Val, Ile655Val and Ala1170Pro. The latter resides within a C-terminal, intracellular regulatory domain, whereas the adjacent polymorphisms Ile654Val and Ile655Val are part of the ERBB2 transmembrane domain (37). To evaluate the putative influence of these SNPs on breast cancer risk, we performed a case–control study testing familial German breast cancer cases and controls. Case–control studies using familial cases reveal a considerably enhanced power to detect rare alleles affecting breast cancer risk and, compared with studies utilizing unselected cases, a lower sample size is needed to obtain a similar power (38,39). For this reason, our study exclusively comprised familial breast cancer cases which lacked BRCA1 and BRCA2 mutations, thus ruling out the effects of these high penetrance genes (4). The Ile->Val polymorphism at codon 655 has been repeatedly tested for an association with breast cancer risk, showing either increased risk or no effect, probably depending on ethnic origin (30). According to our results, Ile655Val is not associated with familial breast cancer risk in German women (Table II). In both Chinese and Australian women diagnosed before the age of 45 and 40 years, respectively, homozygosity for Val655 has been associated with an increased risk of early onset breast cancer (24,33). Our data on early onset cases diagnosed at ≤40 years show no significant association, yet a trend towards an increased risk (OR = 1.58, 95% CI = 0.79–3.17, P = 0.19). The C-terminally located Ala1170Pro variant was not associated with breast cancer risk in the total or age-stratified samples (Table II). The Ile655Val and Ala1170Pro haplotypes showed no association with familial breast cancer risk either (Table III).

As the first study to investigate the influence of the rare ERBB2 variant Ile654Val on familial breast cancer risk, we found an increased risk for carriers of the heterozygous genotype (Ile/Val) with an OR of 2.56 (Table II). Stratification according to age of onset revealed a somewhat higher risk for women older than 40, with an OR of 2.86. The Ile654Val variant resides next to an N-terminal dimerization motif within the transmembrane domain, mediating dimerization of ERBB2 in the cell membrane (14). Val654 is linked to Val655, resulting in two consecutive valine instead of two isoleucine residues within the transmembrane domain. Both Ile654 and Ile655 are highly conserved among species (Figure 1). Using a computational exploration space of the transmembrane segments of an ERBB2 homodimer, Fleishman et al. characterized two stable conformations of the transmembrane domain (15; http://www.pnas.org/cgi/content/full/252640799/DC1/1). Based on this molecular switch model for the activation of ERBB2, we hypothesize that a substitution of Ile654 by a valine residue would stabilize the formation of active ERBB2 dimers, thus increasing autophosphorylation, tyrosine kinase activity and cell transformation. The exchange of two consecutive isoleucines for two valines in these positions of the transmembrane domain would have an even stronger effect on the formation of active dimers. This activation of the ERBB2 signal transduction pathway turns on the mitogen-activated protein kinase (MAPK) signalling pathway (40) with stress-activated protein kinase-2, a member of the MAPK family, playing a pivotal role in tumour angiogenesis and metastasis (41). Generally, isoleucine to valine changes are known to affect stabilization of hydrophobic protein domains, such as transmembrane domains, by altering hydrophobicity (42). Moreover, a point mutation in the transmembrane domain of the ERBB2 rat homolog NEU (Val664Glu) has been shown to mimic ligand induction thus causing constitutive dimerization and oncogenic activation (43).



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Fig. 1. Multiple sequence alignment of the transmembrane domains of EGFR family members. Bold letters indicate positions 654 and 655 of the human ERBB2 protein.

 
We hypothesize that ethnic differences in association of the Ile655Val SNP with breast cancer risk might be explained by the linkage between Val654 and Val655. Assuming that the Val654–Val655 allele basically accounts for the increased familial breast cancer risk, deviating risks of Ile655Val might be due to the different Val654/Val655 ratios in ethnic groups. Recently, Tommasi et al. have reported on ERBB2 expression in human breast cancer cell lines (37). They have detected the Val654–Val655 haplotype in MCF-7 cells, thus emphasizing the significance of our results.

In conclusion, our study showed an association of Ile654Val with familial breast cancer risk, suggesting Val654 as an oncogenic variant. As ligand-independent activation of ERBB2 results in an aggressive growth behaviour and a reduced response to chemotherapy and hormonal treatment (18,22), this rare ERBB2 variant Val654 might affect clinical outcome and predict a worse patient prognosis.


    Acknowledgments
 
The authors wish to thank Kerstin Wagner for support. The German breast cancer samples were collected within a project funded by the Deutsche Krebshilfe headed by Prof. C.R.Bartram (Heidelberg) and Prof. R.K.Schmutzler (Cologne).


    References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received September 28, 2004; revised November 10, 2004; accepted November 14, 2004.