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Carcinogenesis Advance Access originally published online on January 3, 2008
Carcinogenesis 2008 29(2):351-355; doi:10.1093/carcin/bgm290
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

SNPs in ultraconserved elements and familial breast cancer risk

Rongxi Yang1,2,*, Bernd Frank1,2, Kari Hemminki2,3, Claus R. Bartram4, Barbara Wappenschmidt5,6, Christian Sutter4, Marion Kiechle7, Peter Bugert8, Rita K. Schmutzler5,6, Norbert Arnold9, Bernhard H.F. Weber10, Dieter Niederacher11, Alfons Meindl7 and Barbara Burwinkel1,2

1 Helmholtz-University Group Molecular Epidemiology
2 Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany
3 Center for Family and Community Medicine, Karolinska Institute, 69120 Huddinge, Sweden
4 Institute of Human Genetics, University of Heidelberg, 69120 Heidelberg, Germany
5 Division of Molecular Gynaeco-Oncology, Department of Gynaecology and Obstetrics, Clinical Center University of Cologne, 50931 Köln, Germany
6 Center of Molecular Medicine Cologne (CMMC), University Hospital of Cologne, 50931 Köln, Germany
7 Department of Gynaecology and Obstetrics, Klinikum rechts der Isar, Technical University of Munich, 81675 Munich, Germany
8 Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessen, University of Heidelberg, Medical Faculty of Mannheim, 68167 Mannheim, Germany
9 Division of Oncology, Department of Gynaecology and Obstetrics, University Hospital Schleswig-Holstein, 24105 Kiel, Germany
10 Institute of Human Genetics, University of Regensburg, 93053 Regensburg, Germany
11 Division of Molecular Genetics, Department of Gynaecology and Obstetrics, Clinical Center University of Düsseldorf, 40225 Düsseldorf, Germany

* To whom correspondence should be addressed. Tel: +49 6221 421461; Fax: +49 6221 421464; Email: r.yang{at}dkfz.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Ultraconserved elements (UCEs) are segments of >200 bp length showing absolute sequence identity between orthologous regions of human, rat and mouse genomes. The selection factors acting on these UCEs are still unknown. Recent studies have shown that UCEs function as long-range enhancers of flanking genes or are involved in splicing when overlapping with exons. The depletion of UCEs among copy number variation as well as the significant under-representation of single-nucleotide polymorphisms (SNPs) within UCEs have also revealed their evolutional and functional importance indicating their potential impact on disease, such as cancer. In the present study, we investigated the influence of six SNPs within UCEs on familial breast cancer risk. Two out of six SNPs showed an association with familial breast cancer risk. Whereas rs9572903 showed only a borderline significant association, the frequency of the rare [G] allele of rs2056116 was higher in cases than in controls indicating an increased familial breast cancer risk ([G] versus [A]: odds ratio (OR) = 1.18, 95% confidence interval (CI) 1.06–1.30, P = 0.0020; [GG] versus [AA]: OR = 1.41, 95% CI 1.15–1.74, P = 0.0011). Interestingly, comparing with the older age group, the ORs were increased in woman younger than 50 years of age ([G] versus [A]: OR = 1.27, 95% CI 1.11–1.45, P = 0.0005; [GG] versus [AA]: OR = 1.60, 95% CI 1.22–2.10, P = 0.0007) pointing to an age- or hormone-related effect. This is the first study indicating that SNPs in UCEs might be associated with cancer risk.

Abbreviations: BC, breast cancer; CI, confidence interval; HWE, Hardy–Weinberg equilibrium; OR, odds ratio; SNP, single-nucleotide polymorphism; UCE, ultraconserved element


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Ultraconserved elements (UCEs) are the most extreme representatives of conserved non-coding sequences, which have a length of >200 bp showing 100% sequence identity with no insertions or deletions among orthologous regions of human, rat and mouse genomes (1). The reason for their extreme conservation remains a mystery. The gene sequence conservation rate in evolution has been shown to be correlated with its biological characteristics and importance (2–8). Analogous, it has been proposed that the UCEs evolving very slowly compared with selectively neutral sequences are functionally important (1,9–11). Another hypothesis is that the high conservational UCEs exist due to their locations in genomic regions of low mutation rates. However, several studies have indicated that UCEs are likely to be functional and not mutation cold regions (9–12). Whereas a minority of UCEs overlap with exons (so-called exonic UCEs), the majority of UCEs is located in introns or intergenic regions. The non-exonic—either intronic or intergenic—elements tend to congregate in clusters near transcription factors and developmental genes, whereas the exonic elements are enriched in genes involved in RNA binding and regulation of splicing (1) and might be critical for homeostatic maintenance of splicing factor expression levels (13,14).

Pennacchio et al. (15) have characterized the in vivo enhancer activity of a large set of non-exonic conserved elements among these UCEs identical in human, mouse and rat. They reported 45% of the 167 tested extremely conserved elements to function as tissue-specific enhancers of gene expression at embryonic day 11.5 in mice.

A recent study has pointed out that mammalian UCEs are strongly depleted among segmental duplications and copy number variants, which indicates that these duplications and deletions are eliminated at the cellular or organismal level through lethality, segregation distortion or lowered fitness and lead to the copy dose sensitivity of UCEs (16). Interestingly, most of the UCEs that have been found in segmental duplications and copy number variants are located exonic, indicating that this group of UCEs might be different from the non-exonic UCEs subset along with the above-mentioned findings. Derti et al. have speculated that UCEs are involved in the maintenance of the genomic integrity and deviations in UCE copy number or sequence would be eliminated from the population. They noticed that the participating of UCEs in copy counting can accommodate their activities, especially their enhancer-like function, which can participate in pairing-mediated phenomena (16–18). If a somatic loss of an UCE results in cellular lethality, the presence of that UCE may be considered as a protection of individuals from disease, such as cancer.

Due to the extremely high degree of conservation and the proposed functions of UCEs discussed above, we tested, for the first time, the hypothesis if polymorphisms within UCEs might be associated with an altered cancer risk. Bejerano et al. (1) has observed only six validated single-nucleotide polymorphisms (SNPs) in 481 examined UCEs, indicating a strong under representation of SNPs within UCEs.

In the present study, we investigated if SNPs in UCEs are associated with familial breast cancer (BC) risk using a large study population.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Study population
Genotyping was performed on genomic DNA of BRCA1/2 mutation-negative index patients from 1214 German BC families, among a subset of 668 high-risk BC cases (A1 group: families with two or more cases of BC including at least two cases with onset under the age of 50 years; B group: families with one or more cases of breast), and 2084 unrelated healthy German woman. The BC cases comprised unrelated women who had been tested BRCA1/2 mutation negative by applying the denaturing high-performance liquid chromatography method on all exons, followed by direct sequencing of conspicuous exons (19). The BC samples were collected during the years 1997–2007 by six centres of the German Consortium for Hereditary Breast and Ovarian Cancer (centres of Heidelberg, Würzburg, Cologne, Kiel, Düsseldorf and Munich, see authors’ affiliations). Index patients were first diagnosed with BC and then referred to a family registry. All BC patients gave informed consent.

The control population included healthy and unrelated female blood donors collected by the German Red Cross Blood Service of Baden-Württemberg—Hessen and Institute of Transfusion Medicine and Immunology (Mannheim), sharing a similar ethnic background with the BC patients. The age distribution in controls and cases was similar (controls: mean age 44.6 years, median age 43 years; cases: mean age 46.1 years, median age 46 years). According to the German guidelines for blood donation, all blood donors were examined by a standard questionnaire and gave their informed consent. They were randomly selected during the years 2004–2007 for this study and no further inclusion criteria were applied during recruitment. The study was approved by the Ethics Committee of the University of Heidelberg (Heidelberg, Germany).

Genotyping
The six SNPs described by Bejerano et al. (1), rs1861100, rs2056116, rs1538101, rs7092999, rs9572903 and rs7143938, were analysed using TaqMan allelic discrimination assays according to earlier descriptions (20). These SNPs are located in the UCE region of uc.53, uc.140, uc.252, uc.295, uc.353 and uc.374, respectively. Sequences of primers and probes are available upon request. The SNP assays were validated by re-genotyping 10% of all samples.

Statistical analysis
Hardy–Weinberg equilibrium (HWE) test was undertaken using the chi-square ‘goodness-of-fit’ test by a tool from the Institute of Human Genetics, Technical University Munich, Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). Genotype-specific odds ratios (ORs), 95% confidence intervals (CIs) and P values were computed by unconditional logistic regression using SAS version 9.1 (SAS Institute, Cary, NC). Age, treated as a continuous variable was included in the regression as covariate. P values were calculated using two-sided chi-square test. The power ({alpha} = 0.05) was calculated using the power and sample size calculation software PS version 2.1.31 (http://www.mc.vanderbilt.edu/prevmed/ps/index.htm) (21). SNPs linked with rs2056116 and rs9572903 with r2 ≥ 0.8 and block definition were identified using Haploview version 3.32 (http://www.broad.mit.edu/mpg/haploview). The linked SNPs’ BC associations were further checked if they have being analysed in the Cancer Genetic Markers of Susceptibility genome-wide association study (CGEMS) (https://caintegrator.nci.nih.gov/cgems/browseSetup.do).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
The present case–control study was focussed on the potential impact of six validated SNPs within UCEs on familial BC risk. All SNPs lie in the intergenic regions. The genotype analysis of rs1538101, rs7092999, rs7143938, rs1861100, rs2056116 and rs9572903 (1) were performed on genomic DNA of BRCA1/2 mutation-negative index patients from 756 German BC families with the mean age of 46 years, and 736 unrelated German control individuals with the mean age of 48 years. As the two SNPs, rs2056116 and rs9572903, showed a significant association with familial BC, they were analysed on an enlarged samples set (in total 1214 familial BC cases/2084 controls; see Materials and Methods). Genotype distribution in controls was consistent with the HWE. The SNP assays were validated by re-genotyping 10% of all samples attaining concordance rates of >99.5% for all investigated SNPs.

Genotype frequencies of rs1538101, rs7092999, rs7143938 and rs1861100 were similar between familial BC cases and control samples, showing no association with familial BC (Table I). The SNP rs9572903 showed a borderline significant association between the rare G allele and the major A allele (OR = 1.15, 95% CI 1.00–1.32, P = 0.0480). Homozygous [GG] carriers had a 1.40-fold, but not significant, higher risk than homozygous wild-type [AA] carriers (Table I). Comparing the high-risk and non-high-risk familial subgroups, the ORs of rs9572903 in both [G] versus [A] alleles as well as [GG] versus [AA] genotypes had no obvious difference in these subgroups (Table II). The age stratification indicated a higher risk of rs9572903 rare [G] allele in the subgroup of older than 50 years ([G] versus [A], OR = 1.30, 95% CI 1.03–1.65, P = 0.0260, Table III). The OR of [GG] versus [AA] genotype was 1.55, though not significant. In the subgroup of younger than 50 years, rs9572903 showed no significant effect (Table III).


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  		Table I. Genotype frequencies of SNPs within UCEs in familial BC study population

 


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  		Table II. Genotype frequencies of SNPs within UCEs in high-risk/non-high-risk familial BC study population

 


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  		Table III. Age-related genotype frequencies of SNPs within UCEs in familial BC study population

 
The analysis of the polymorphism rs2056116 revealed a significant association with familial BC. The rare G variant allele was more frequent in cases than controls ([G] versus [A]: OR = 1.18, 95% CI 1.06–1.30, P = 0.0020). The risk effect was increased when comparing homozygous genotype carriers ([GG] versus [AA]: OR = 1.41, 95% CI 1.15–1.74, P = 0.0011, Table I). The chi-square test for trend indicated a dose-dependent association between the rare allele G of rs2056116 and an increased familial BC risk (Ptrend = 0.0025, Table I). In the high-risk familial BC subgroup, the respective ORs were increased ([G] versus [A], OR = 1.24, 95% CI 1.09–1.40, P = 0.0010; [GG] versus [AA], OR = 1.55, 95% CI 1.21–1.99, P = 0.0006; Ptrend = 0.0012, Table II). In contrast, the ORs in the non-high-risk familial BC group are lower and not significant ([G] versus [A], OR = 1.10, 95% CI 0.96–1.27, P = 0.1561; [GG] versus [AA], OR = 1.26, 95% CI 0.95–1.66, P = 0.1039; Ptrend = 0.1622, Table II).

Remarkably, when stratifying for age <50 years and ≥50 years, the risk was higher in the younger age group (<50 years old) ([G] versus [A]: OR = 1.27, 95% CI 1.11–1.45, P = 0.0005; [GG] versus [AA]: OR = 1.60, 95% CI 1.22–2.10, P = 0.0007; Ptrend = 0.0006, investigating all cases and controls, Table III), whereas no association was observed in the older age group (≥50 years old) (Table III). In the high-risk familial BC group, the same age-related effect with even increased ORs was observed (<50 years: [G] versus [A]: OR = 1.33, 95% CI 1.14–1.55, P = 0.0004; [GG] versus [AA]: OR = 1.75, 95% CI 1.27–2.40, P = 0.0006; Ptrend = 0.0004; whereas ≥50 years: [G] versus [A]: OR = 1.10, 95% CI 0.86–1.42, P = 0.4470; [GG] versus [AA]: OR = 1.28, 95% CI 0.79–2.09, P = 0.3177; Ptrend = 0.4553, data not shown).

We intended to compare our results with findings from genome-wide studies. We analysed 200 kb flanking regions of rs2056116 and rs9572903 including the haplotype blocks of rs2056116 and rs9572903, respectively. Both regions do not include any gene. Neither of the SNPs, rs2056116 or rs9572903 itself, nor SNPs in linkage disequilibrium with rs2056116 (r2 ≥ 0.8) have been analysed in the Cancer Genetic Markers of Susceptibility genome-wide association study (CGEMS). Although two SNPs in linkage disequilibrium with rs9572903 (rs9572899 located 18.54 kb upstream; r2 = 0.938 and rs9572900 located 18.84 kb upstream; r2 = 0.938) had been analysed in CGEMS, they did not show any association with BC.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
BC is the most common cause of cancer-related death in women worldwide, and after lung cancer, the second most frequent cancer in the world (22). According to the polygenic model of inherited BC, unfavourable combinations of polymorphic genetic variants in low-penetrance susceptibility genes jointly contribute to the excess familial BC risk. Most of these susceptibility genes have not been discovered yet (23,24).

Our work is the first study focussing on the possible impact of SNPs in UCEs on familial BC risk. The strength of the present study is the large sample size of familial index cases resulting high statistical power. Furthermore, only BRCA1/2 mutation negative familial BC cases were included in the study to avoid the effects derived from mutations in these high-penetrance susceptibility genes (19). Given our sample size, we had a power of 80% ({alpha} = 0.05) to detect an OR of 1.25 for SNP rs2056116 and of 1.26 for SNP rs9572903, not considering the usage of familial cases. The power of an association study based on familial cases is even about two times higher compared to a study of unselected cases (25,26).

Out of six SNPs analysed, four showed no significant and SNP rs9572903 only a borderline significant association being not significant after Bonferroni adjustment. However, SNP rs2056116 showed a significant association with familial BC risk, remaining even significant after Bonferroni adjustment. Restricting our analysis to high-risk familial cases, the respective ORs of SNP rs2056116 were slightly increased. This is in agreement with previous studies that have shown stronger risk effects in the high-risk subgroup (27–30). The BC categories A1 and B (see Materials and Methods) were chosen as high-risk categories, since these categories are based on the most stringent family history inclusion criteria and have shown the highest BRCA1/2 mutation frequencies in a German study population (19).

Both SNPs, rs9572903 and rs2056116, affect intergenic UCEs (uc.353 and uc.140, respectively; 1). It has been discussed that this category of UCEs may function as long-range enhancers for upstream genes (12). These regulatory elements have the ability to modulate gene expression over very long distances (31,32). Indeed, Pennacchio et al. (15) have characterized the in vivo enhancer activity of a large set of non-exonic conserved elements, including the UCEs uc.353 and uc.140. They reported 45% of the tested extremely conserved elements, including UCEs uc.353 and uc.140, to function as tissue-specific enhancers of gene expression.

The nearest upstream and downstream genes of rs9572903 are dachshund homolog 1 (DACH1) and FLJ22624, respectively. Although the function of FLJ22624 has not been reported yet, DACH1 is involved in developmental processes (33). Furthermore, it associates with sine oculis homeobox homolog 6 (SIX6) to regulate proliferation by directly repressing cyclin-dependent kinase inhibitors and participates in the negative regulation of tumour growth factor-β signalling pathway by interacting with nuclear cofactor repressors and mothers against decapentaplegic homolog 4 (SMAD4) (34,35). Thus, it is not inconceivable that the SNP rs9572903 may influence DACH1 expression and in consequence the cancer risk. However, due to the borderline significance of the result, the association of this SNP by chance has also to be considered.

The nearest downstream gene of the highly significant BC-associated SNP rs2056116 is Ras-associated protein 28 (Rab28). The distance is 360 kb. Though, only a few studies have been published on Rab28 and most of them were on plants (36,37), in general, Rab proteins are small GTPases of the Ras superfamily that are well known in the regulation of exocytic and endocytic membrane trafficking and are also involved in the signalling cascades of gene expression, cell–cell adhesion, mitosis and apoptosis (38–41). In line with the importance of Rab GTPases in many cellular functions, the altered expression or mutation of Rab proteins such as Rab25, Rab5, Rab7 and Rab32 or their effectors have been associated with cancer (42–45). Hence, it is possible that Rab28 may play similar roles as other Rab proteins having an effect on tumour-related pathways. The nearest upstream gene of rs2056116 is the heparan sulphate D-glucosaminyl 3-O-sulfotransferase 1 (HS3ST1). The distance is 1580 kb. HS3ST1 is a heparan sulfate biosynthetic enzyme, playing key roles in generating heparan sulfate glycosaminoglycans fine structures (46). Heparan sulfate glycosaminoglycans are present at the cell extracellular matrix and modulate cell signalling, thereby regulating several aspects of cancer biology by interaction with a variety of proteins, such as growth factors and morphogens (47,48). As the UCE uc.140 affected by rs2056116 has shown to be a long-distance enhancer regulating the expression in the forming of limb (http://enhancer.lbl.gov/cgi-bin/imagedb.pl?form = presentation&show = 1&experiment_id = 259) and as its further downstream gene bagpipe homeobox homolog 1 (BAPX1) (distance 540 kb) is known to play an important role in limb formation (49), it is possible that this SNP affecting u.140 might influence the expression of Rab28 (distance 360 kb) or further downstream genes and in consequence the cancer risk. As the increased BC risk associated with rs2056116 predominantly affects premenopausal woman, this points to a possible undisclosed age- or hormone-related effect.

Our study focussed on the analysis of six SNPs described by Bejerano et al. (1). Recent studies have described further SNPs within UCEs (10,11). However, also these studies concluded that SNPs are still significantly underrepresented in UCEs. Especially low-frequent SNPs and mutations have been described, indicating that UCEs are still under strong ongoing selection—in fact, under stronger selection than coding regions (11). The authors concluded that UCEs are currently, as well as historically, strongly retained functional elements (11). Though, the selection factors acting on UCEs are still unknown.

The genotype distributions of all SNPs investigated in our study were in accordance with the HWE in controls, pointing against strong ongoing selection regarding these frequent SNPs. These results are in agreement with recent findings of Chen et al. (10) who also did not observe any HWE deviation regarding frequent SNPs. Moreover, these findings are in concordance with our observation of an only moderately increased BC risk mediated by rs2056116 (and eventually by rs9572903).

As our study focussed on the investigation of six rather frequent SNPs, it was biased against SNPs of low frequency where a stronger ongoing selection is more likely (11). Although some of these very rare mutations or SNPs are more likely to be already deleterious in earlier grasping disease, it would be interesting to investigate all SNPs and mutations within UCEs, especially SNPs and mutations located within or downstream of cancer-related genes, for a putative impact on cancer risk. Due to the rare frequency of most SNPs and mutations within UCEs, very large study populations are required.

In summary, we found the UCE located SNP rs2056116 to be associated with an increased familial BC risk. As predominantly premenopausal woman were affected, our results indicate that an increased cancer risk might contribute in part to the selection factors acting on UCEs.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Deutsche Krebshilfe (107054), supported by the Center of Molecular Medicine, Cologne to R.K.S.; Helmholtz society and the EU (LSHC-CT-2004-503465).


   Acknowledgments
 
The authors are grateful to Bowang Chen and Justo L. Bermejo for statistical analyses as well as to Sandrine Tchatchou for support.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received October 10, 2007; revised December 6, 2007; accepted December 10, 2007.


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