A specific haplotype of single nucleotide polymorphisms on chromosome 19q13.2–3 encompassing the gene RAI is indicative of post-menopausal breast cancer before age 55

Bjørn A. Nexø1,5, Ulla Vogel2, Anja Olsen3, Tina Ketelsen1, Zuzanna Bukowy1,6, Birthe L. Thomsen3, Håkan Wallin2, Kim Overvad4 and Anne Tjønneland3

1 Institute of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark
2 National Institute of Occupational Health, DK-2100 Copenhagen O, Denmark
3 Institute of Epidemiology, The Danish Cancer Society, DK-2100 Copenhagen, Denmark
4 Department of Clinical Epidemiology, Aalborg Hospital and Aarhus University Hospital and Department of Epidemiology and Social Medicine, University of Aarhus, DK-8000 Aarhus C, Denmark

5 To whom correspondence should be addressed Email: nexo{at}humgen.au.dk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The new genetic information, in particular the greatly increased density of markers in the chromosomal maps, may permit analysis of the importance of genes in the development of disease exclusively from molecular epidemiological studies. Motivated by our previous results on the same region in relation to basal cell carcinoma we have investigated the occurrence of post-menopausal breast cancer in relation to a number of single nucleotide polymorphisms in the chromosomal region 19q13.2–3. A case–control study including 425 human cases and a similar number of controls was nested in a population-based prospective investigation encompassing 24 697 Danish post-menopausal women (aged 50–64 at inclusion) living in Copenhagen or Aarhus. We combined three markers located together in or near the gene RAI into a high-risk haplotype. Compared with all other haplotypes, those who were homozygous had a rate ratio of 1.64 (95% CI 1.17–2.29, P <= 0.004) for development of breast cancer. When we further focused on those persons with post-menopausal breast cancer before age 55 the rate ratio increased to 9.5 (95% CI 2.21–40.79, P <= 0.003). The likely explanation for our results is a common recessive genetic variant located in or near the gene RAI, which, when homozygous, conveys an increased risk of breast cancer. Presumably it is identical to the genetic variant previously observed in the same region that increases the risk of basal cell carcinoma before age 50.

Abbreviations: HRT, hormone replacement therapy; RR, rate ratio; SNP, single nucleotide polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A new page is being turned in the century-old book chronicling the discussions on the relative importance of inheritance and environment for human health. The emergence of genome sequence data and the advent of databases listing common genetic variants among humans for the first time allow systematic forays into the role of specific genome regions for common disease. Therefore the emphasis of the debate of nature versus nurture will probably swing towards nature, in casu genetics, in the coming years.

Cancer causation is a case in point. Excepting the relatively rare familial cancers, the prevailing opinion has been that most persons were alike and cancer risk was determined by different choices in lifestyle, work and other exposures. The rest was chance. This was justifiable in as much as such exposures could be studied; the genetics could not. A major problem was the relative weakness of family-based studies for analysing conditions caused by multiple genes (1,2). However, the new genetic information, in particular the greatly increased density of markers in the chromosomal maps, may now permit the analysis of the importance of genes in the development of disease exclusively from molecular epidemiological studies, using linkage disequilibrium to associate markers and disease in the population. In the present study we emphasize the potential of molecular epidemiology in relation to breast cancer.

We have previously reported that specific alleles of single nucleotide polymorphisms (SNPs) on chromosome 19q13.2–3 were associated with occurrence of basal cell carcinoma in two case–control studies of Caucasians (36). The association was strongest for cancers before age 50. In a search along the chromosome the strongest association was found in and around the gene RAI. Importantly, combining two markers in RAI or combining RAI with markers in other genes into haplotypes strengthened the association greatly.

In the present investigation we have studied the same chromosomal region in relation to post-menopausal breast cancer in a large nested case–control study. We report that markers in and around RAI form a haplotype that, when homozygous, is strongly associated with occurrence of post-menopausal breast cancer before age 55. This haplotype is composed of alleles also associated with skin cancer before age 50.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study ‘Diet, Cancer and Health’ is a prospective cohort study, established with the primary aim of studying the aetiological role of diet on cancer risk. From December 1993 to May 1997, 79 729 women aged 50–64 years were invited to participate in the study and 29 875 accepted the invitation. Women eligible for invitation were born in Denmark, were living in the Copenhagen or Aarhus areas and were not at the time of invitation registered with a previous diagnosis of cancer (including non-melanoma skin cancer) in the Danish Cancer Registry.

‘Diet, Cancer and Health’ and the present sub-study were approved by the regional Ethical Committees on Human Studies in Copenhagen and Aarhus and by the Danish Data Protection Agency.

All cohort members attended one of two established study centres. Each participant filled in a food frequency questionnaire and a questionnaire concerning lifestyle, including use of hormone replacement therapy (HRT) and menopausal status. In the study centres a total of 30 ml of blood (non-fasting, collected in citrated and plain Venojects) was drawn from each participant. The samples were spun and divided into 6 x 1 ml tubes of plasma, 4 x 1 ml of serum, 2 x 1 ml of erythrocytes and 2 x 1 ml of ‘buffy coat’. All samples were processed and frozen within 2 h at -20°C. At the end of the day of collection all samples were stored in liquid nitrogen vapour (maximum -150°C).

Of the initial 29 875 women a total of 326 that were later reported to the Danish Cancer Registry with a cancer before the visit to the study clinic were excluded from the study. In addition, eight women were excluded from the study because they did not fill in the lifestyle questionnaire. Because the present analysis aimed at the sub-group of women who were post-menopausal at study entry, we further excluded 4844 supposedly pre-menopausal women, including 4798 women who had reported at least one menstruation no more than 12 months prior to entry and no use of HRT, nine women who gave a lifetime history of no menstruations and 37 women who did not answer the questions about current or previous use of HRT, leaving 24 697 post-menopausal women.

Cohort members were identified by a unique identification number, which was allocated to every Danish citizen by the Central Population Registry. All 24 697 post-menopausal cohort members were linked to The Central Population Registry for information on vital status and immigration. Information on cancer occurrence among cohort members was obtained through record linkage to the Danish Cancer Registry, which collects information on all inhabitants in Denmark who develop cancer (7). Linkage was performed by use of the personal identification number. Each cohort member was followed up for breast cancer occurrence from date of entry, i.e. date of visit to the study centre until the date of diagnosis of any cancer (except for non-melanoma skin cancer), date of death, date of emigration or 31 December 2000, whichever came first. A total of 434 women were diagnosed with incident breast cancer during the follow-up period.

Matching of cases and controls
Due to the large number of cohort members, it was not feasible to determine polymorphisms on them all. In order to optimize the utilization of the available cases, we used a nested case–control design. One control was selected for each of the 434 cases. The control was cancer-free ‘at the exact age at diagnosis of the case’ and was further matched on certainty of post-menopausal status (known/probably post-menopausal), use of HRT at inclusion into the cohort (current/former/never) and age at inclusion into the cohort (half-year intervals).

Of the 866 women (434 cases and 434 controls, including two that later became cases), nine cases were excluded due to lack of a blood sample. In addition, determination of the different polymorphisms failed for a varying number of persons (see Table II).


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Table II. Normalized linkage disequilibrium and P value for linkage disequilibrium different from 0 among controlsa

 
DNA purification
DNA was purified essentially as described (8) from lymphocytes donated by the persons in the cohort. However, RNase treatment was not included. Lymphocytes isolated from 10 ml of blood gave an average yield of 100 µg DNA.

Detection of single nucleotide polymorphisms
The polymorphisms listed in Table I were typed using a variety of techniques. Briefly, XPD exon 23 was typed by PCR–RFLP (3). A 10% subset of the samples was typed twice with 98% identity. XPD exon 6 was typed by real-time PCR in an ABI 7700 (Applied Biosystems, Foster City, CA). Reactions (15 µl) contained mastermix, 100 nM probes, 600 nM primers and ~20–100 ng DNA. The probes were: C allele, 5'-FAM-CCC CAC TGC CGC TTC TAT GAG GT-TAMRA-3'; A allele, 5'-VIC-CCC CAC TGC CGA TTC TAT GAG GTT-TAMRA-3'. The primers were: forward, 5'-GTA CCA GCA TGA CAC CAG CCT-3'; reverse, 5'-TCC CTC CCT GAG CCC TG-3'. The reactions were run for 40 cycles of 15 s at 94°C, 60 s at 63°C. Controls were included in each run and 10% of the samples were retyped with identical results. XRCC1 exon 10 was typed in the ABI 7700 as described (6). XPD exon 10, RAI intron 1, ASE-1 exon 1 and ERCC1 exon 4 were typed on a LightcyclerTM (Roche, Geneva, Switzerland) as described (6). In general, if a SNP analysis failed on a sample, it was repeated first with the same amount of DNA. If it failed again it was repeated with a 4-fold increased amount of DNA. If this also failed, the determination of the sample was abandoned.


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Table I. The markers used, their sources of information and their currently estimated positions on chromosome 19

 
Statistical methods
Due to the sampling design, the breast cancer rate ratios (RRs) according to the polymorphisms were estimated using a conditional logistic regression analysis. Only discordant pairs contributed to estimation of the RRs, whereas all participants with successfully analysed blood samples (including controls for the nine cases without a blood sample) contributed to the calculation of allele frequencies. Tests and two-sided 95% confidence intervals were based on Wald's test on the log scale for the RRs. The analyses were performed using the procedure PHREG in the statistical software programme SAS on a UNIX platform.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have investigated the occurrence of post-menopausal breast cancer in relation to a number of SNPs in the chromosomal region 19q13.2–3 in 425 human cases and controls, nested in a population-based prospective investigation including 24 697 Danish women. Table I lists the SNPs in question, their source of information and their approximate positions on chromosome 19. While the relative positions of the SNPs are presumably final, the absolute chromosome positions still vary with the particular build of the map of chromosome 19. Table II shows the degree of linkage disequilibrium between the markers among the controls. The only apparent irregularity in the data is the absence of linkage disequilibrium between RAI intron 1 and ASE-1 exon 1, for which we have no explanation.

Table III lists the RRs of post-menopausal breast cancer in relation to the individual SNPs. None of the single SNPs showed a statistically significant association with post-menopausal breast cancer.


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Table III. Risk of breast cancer in relation to various markers on chromosome 19q13.2–3

 
In our studies of basal cell carcinoma we have found that combining neighbouring SNPs into haplotypes improved the definition of groups and increased the association with disease. Moreover, in those studies we found specific alleles of RAI, ERCC1 and ASE-1, namely RAI intron 1A, ASE1 exon 1G and ERCC1 exon 4A, to be relatively more common among persons with skin cancers (5,6); in the case of RAI intron 1, which showed the strongest association, it was also apparent that the sensitive allele was recessive. Individually, in the two groups of basal cell carcinoma RAI and ERCC1 had the highest association with cancer, respectively. Thus, in the further analysis we proceeded on the assumption that we were here dealing with a recessive mutation located in the region between RAI and ERCC1 and combined those participants that were homozygous for the risk haplotype, i.e. those with the genotype RAI intron 1AA, ASE-1 exon 1GG and ERCC1 exon 4AA, into one ‘high-risk’ group, which we compared with all other combinations of the three alleles (Table III). Analysed in this way the persons who were homozygous for the ‘high-risk’ haplotype with exclusively sensitive alleles had a RR for breast cancer of 1.64 (95% CI 1.17–2.29), indicating that the high-risk haplotype when homozygous conveyed an increased risk. The individual markers seemed to completely lose even marginal association with disease after adjustment for the high-risk haplotype.

In our studies of basal cell carcinoma we found that the strongest increase in cancer risk was seen among participants with onset before age 50 (3,5). Therefore, we stratified the data into three groups, based on age of diagnosis (<=55, 55–60 and >60 years) and again investigated the association of the more interesting SNPs with post-menopausal breast cancer (Table IV). In the youngest age group, all three SNPs showed a strengthened relation to the rate of breast cancer, however, only the association with RAI intron 1 reached statistical significance, with RRs of 0.32 (95% CI 0.12–0.84) for RAI intron 1AG and 0.19 (95% CI 0.02–1.80) for RAI intron 1GG. However, persons in the youngest age group who were homozygous for the ‘high-risk’ haplotype had a considerably increased risk of post-menopausal breast cancer (RR 9.50, 95% CI 2.21–40.79, P <= 0.003). We could not with certainty establish any association between single SNPs or the ‘high-risk’ genotype in the older age groups. Raising the upper age limit in the younger group to 60 years diluted the effect, which seems confined to younger women, but it was still significant (RR 1.84, 95% CI 1.13–2.99, P = 0.01).


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Table IV. Risk of breast cancer at different ages according to genotypes of ERCC1 exon 4, ASE-1 exon 1 and RAI intron 1

 
To investigate to what degree the increased risk of the single SNPs could be accounted for by the ‘high-risk’ genotype, we adjusted all analyses for the genotype (Tables III and IV). After adjustment, no association between the SNPs under investigation and post-menopausal breast cancer could be proved, indicating that most or all of the association was due to the ‘high-risk’ genotype. We also tested whether the distribution of haplotypes made from the three markers were different between cases and controls, if we abandoned assumptions about which alleles were sensitive and about recessiveness versus dominance, using a Monte-Carlo method (Arlequin; 10). The level of significance obtained was 0.031.

Finally, we subdivided the group of persons which were not homozygous for the high-risk haplotype into three groups: those that were definitely not carriers of the high-risk haplotype, those that might be heterozygous for the high-risk haplotype and those that were definitely heterozygous for the high-risk haplotype. We combined this with information about age at diagnosis (Table V). This further increased the RR for being high-risk homozygous in the total group as well as among those that got breast cancer before age 55. Importantly, the group of definite heterozygotes for the high-risk haplotype did not show an increased RR.


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Table V. Risk of breast cancer at different ages in relation to the presence of the high-risk haplotype in homozygote or heterozygote form

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This paper demonstrates a strong association of post-menopausal breast cancer below the age of 55 with a combination of alleles of SNPs located in the region 19q13.2–3. The sampling design used is a simple associative analysis, which hinges heavily upon the presence of linkage disequilibrium in the region of investigation. Luckily, analysis of the association of the different markers showed the presence of such a disequilibrium in the present population (Table II).

We assume we are dealing with a single causative gene variant, but this is unlikely to be any of the investigated SNPs, as a combination of SNPs would not then be expected to improve the results. More likely, a haplotype located on the relevant part of chromosome 19 combining the responsible variant and the markers has tended to flow relatively unchanged through the generations during the emergence of the present Danish population. It remains a limitation in the present study that we do not know the causative variation.

It is important to emphasize that we are presumably tracking a common gene variant which, when homozygous, increases the risk of cancer. In this respect it behaves like a tumour suppressor gene. This is in contrast to the BRCA genes, where a set of relatively rare dominant gene mutations causes cancer (11). It is, however, qualitatively consistent with recent epidemiological analysis of familial influences on breast cancer (12,13).

The observed RR of 13 for breast cancer before age 55 is extremely high (14). To obtain an initial estimate of how many persons are affected by the presumed causative gene variant, we note that there is an excess of 120 - 83 = 37 high-risk genotypes among the cases relative to the controls (Table II). This is ~10% of the cancers observed. As the lifetime risk of getting breast cancer among women is ~10%, we arrive at the conclusion that of the order of 1% of the female population carries the causative gene variant in homozygous form. If the gene variant is in Hardy–Weinberg equilibrium this would mean that close to 20% of the population are heterozygous carriers. This calculation is obviously a crude approximation in several ways: we do not take into account that the persons may have spent different times in the cohort, that the age distributions in the cohort and in the general population are different, that there is no correction for the fact that we, by eliminating all persons with prior cancers from the cohort, may inadvertently have reduced the frequency of the ‘high-risk’ gene variant and that there is no correction for possible incomplete penetrance. What we can deduce from the numbers is that the causative gene variant may affect the same order of magnitude of persons as do the BRCA genes and that carriers are fairly common. It is not clear whether these carriers also have a somewhat increased risk of breast cancer, but in the present study no increased risk was observed among the definite heterozygotes for the high-risk haplotype, a group likely to include a high proportion of carriers.

The women in this study were all post-menopausal at diagnosis. However, we exclusively observed an effect of the high-risk haplotype in the age bracket 50–55, i.e. very shortly after most women become menopausal. In view of the year-long development of breast cancer we find it likely that many of the tumours initiated in the pre-menopausal period and persisted through menopause.

RAI is an acronym for RelA-associated inhibitor. RAI was cloned as an inhibitor of the transcription regulator NF-{kappa}B and thus is presumably involved in transcriptional control (15), most likely in the control of cell division and/or apoptosis. Because of its nuclear localization the product of RAI may be a last minute block of NK-{kappa}B activity forcing the apoptosis of damaged or unwanted cells. Increased activity of NF-{kappa}B has been associated with several cancers (16,17), which could reflect a suboptimal concentration or function of the RAI product. Also, the subunit RelA, which the product of RAI is presumed to inhibit, transforms cells in certain settings (18). Finally, it is noteworthy that NF-{kappa}B up-regulates transcription of the breast cancer gene BRCA2 (19).

Alternatively one may speculate on an effect via the nearby genes for nucleotide excision repair. Breast carcinogenesis is thought to involve endogenous oxidative stress, which mostly seems to be repaired by base excision. However, it has recently been discovered that one lesion caused by oxidative stress, namely cyclodeoxyadenosine, is repaired by nucleotide excision repair (20).

There is epidemiological evidence that the formation of skin and breast cancer may sometimes be related (21,22). It has repeatedly been reported that persons with skin cancer are at increased subsequent risk of getting other forms of cancer, including breast cancer. The overall effects observed are limited, typically with relative risks of 1.1–1.5, but larger after basal cell carcinomas occurring at an early age. Thus, the presumed causal variant in or near RAI may well account for the association.

It is remarkable that two cancer forms as different in aetiology and biology as basal cell carcinoma and breast cancer both seem to be affected by the same gene variant. Moreover, several recent studies have found markers near this region to be associated with the development of melanoma, glioma and lung cancer (2325). No attempt has yet been made to locate the gene(s) responsible, but it could be the same as for breast cancer. Thus, we are possibly dealing with a general mechanism affecting multiple cancer forms. Indeed, recent evidence suggests that lung cancer is also influenced by the here-described high-risk haplotype in homozygous form (U.Vogel et al., Two regions in chromosome 19q13.2–3 are associated with risk of lung cancer, manuscript in preparation).


    Notes
 
6Exchange student from the Department of Biology and Environmental Protection, University of Silesia, Katowice, Poland Back


    Acknowledgments
 
We gratefully acknowledge the expert technical assistance of Anne-Karin Jensen, Birgitte Korsholm, Maj-Britt Højgaard and Katja Boll. The Danish Cancer Society (grant DP00027), the Danish Ministry of Health, Research Centre for Environmental Health's Fund (‘Cemik’ and ‘Genetic variation in genes involved in oxidative stress as risk factors for breast cancer’), the Danish SUE program (Journal no. 9800647-67) and the Novo-Nordisk Foundation supported this paper.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 28, 2002; revised January 31, 2003; revised March 5, 2003; accepted March 6, 2003.