Skip Navigation


Carcinogenesis Advance Access originally published online on May 5, 2006
Carcinogenesis 2006 27(9):1930-1937; doi:10.1093/carcin/bgl062
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/9/1930    most recent
bgl062v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Barroso, E.
Right arrow Articles by Ribas, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barroso, E.
Right arrow Articles by Ribas, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

FANCD2 associated with sporadic breast cancer risk

E. Barroso1, R.L. Milne2, L.P. Fernández1, P. Zamora3, J.I. Arias4, J. Benítez1,2 and G. Ribas1,*

1 Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Centre (CNIO) Madrid, Spain
2 National Genotyping Centre (CeGen), Human Cancer Genetics Programme, Spanish National Cancer Centre (CNIO) Madrid, Spain
3 Department of Oncology, La Paz Hospital Madrid, Spain
4 Unit of Surgery, Monte Naranco Hospital Oviedo, Spain

*To whom correspondence should be addressed at: Human Genetics Group, Human Cancer Genetics Program, C/Melchor Fdz Almagro, 3, E-28029, Madrid, Spain. Tel: +34 912246950; Email: gribas{at}cnio.es


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Several components of the Fanconi anaemia (FA) family of proteins allow the formation of the DNA repair complex foci formed by proteins such as BRCA1/2 and RAD51. Because the genes that participate in the DNA repair pathway have been described as low-penetrance breast cancer susceptibility genes, we postulated that variants in FA genes could also be associated with sporadic breast cancer risk. We studied seven SNPs in FANCA, FANCL and FANCD2 in a total of 897 consecutive and non-related sporadic breast cancer cases and 1033 unaffected controls from the Spanish population. We observed a statistically significant association with sporadic breast cancer for the variant rs2272125 (L1366L) located on FANCD2 (OR per allele = 1.35; 95% C.I. 1.09–1.67; P = 0.005). Both haplotype and diplotype analyses confirmed this association, where one haplotype and pooled diplotypes carrying it were associated with more than 4-fold risk (P = 0.007 and P = 0.006, respectively). Screening for potential causal variants in FANCD2 was performed, detecting one in the putative promoter region, which is located in a phylogenetically conserved motif with consensus binding sites for some transcriptional factors, suggesting a functional implication. Our data indicate that a relationship between FANCD2 and sporadic breast cancer risk may exist.

Abbreviations: FA, Fanconi anaemia; DSB, double-strand break; SNP, single nucleotide polymorphism. HR, homologous recombination; LD, linkage disequilibrium; MAF, minor allele frequency


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Breast cancer is one of the most common diseases among women in developed countries. Several breast cancer susceptibility genes have been identified, the most important being the high risk tumour suppressor genes BRCA1 (1) and BRCA2 (2), the latter also identified as FANCD1 (3,4). However, under the polygenic model of complex diseases, there are also low risk genes with variants that are common in the general population and that produce a mild susceptibility to the disease. Whether these variants interact together or with other environmental and lifestyle factors in an additive or multiplicative way, is not yet clear (5–8).

The BRCA1 and BRCA2 (FANCD1) proteins are implicated in several cellular mechanisms, the most relevant being double-strand breaks (DSBs) by homologous recombination (HR) (9,10). Both BRCA1 and BRCA2 interact with the RAD51 protein, a key component of HR DNA damage repair, to form the nuclear repair foci after DNA damage (11–14). Furthermore, the FA protein family has been identified as a component of this DSB HR repair machinery (15–17). To date, 12 complementation groups have been described, for which 11 responsible FA genes have been identified: FANC-A, -B, -C, -D1, -D2, -E, -F, -G, -J, -L and –M. Eight of these (FANC-A, -B, -C, -E, -F, -G, -L and -M) form a multi-subunit nuclear FA complex (18–20). Some of the FA proteins are structural proteins in the FA complex. For example, FANCA acts as dimer, FANCG plays an important role in the maintenance of the complex structure (21,20) and FANCF functions as a flexible adaptor protein which is required for the assembly of this complex (22). Other FA proteins have specific functions such as FANCL, a ubiquitin ligase (23,24), whose stability and function depends on FANCB (25,26). Another example is FANCD2, a monoubiquitinated protein, which is activated by FANCL and passes from isoform FANCD2-S to FANCD2-L (27,16). FANCD2 is located downstream of the FA complex and linked with it through FANCE (20). FANCJ, described previously as helicase BACH1/BRIP1 (28,29), and FANCM, a nuclease that may act as an engine that translocates the FA core complex along DNA, have been most recently identified (30).

The pathway activity that implicates FA proteins is initiated when DNA damage occurs, and ATM and ATR protein kinases function as DNA damage sensors (31,32). They phosphorylate FANCD2 which is required for the activation of an S-phase checkpoint (33,34) and for its appropriate monoubiquitination through the FA complex (35). They also phosphorylate several proteins in the FA complex (36) which are necessary for the activation of FANCL ubiquitin ligase activity. After monoubiquitination of FANCD2-S, FANCD2-L translocates to the nuclear repair complex foci, helped by BRCA1 in its formation and accumulation (37), where BRCA2 and RAD51 are also located (27). Thus, the monoubiquitination of FANCD2 is important because it allows its chromatin-associated foci assembly (38,39), either in response to DNA damage (40) during the S-phase of the normal cell cycle (41,42), or in replicative stress (43). It has therefore been proposed that FANCD2 could mediate the BRCA2–RAD51 interaction and also influence the speed and efficiency of DNA repair (17,40,44) although FANCG may also be implicated in this process (45). However, an alternative model proposes that FANCD2 functions in a DNA repair sub-pathway that is independent of DNA cross-over and sister chromatid exchange, where RAD51 focus formation depends on BRCA2, but not FANCD2 (46). It has been demonstrated that FA proteins are not essential for HR repair machinery. Furthermore, FA cells have a mild defect in the single-strand annealing pathway, which is also involved in DSB repair. Thus, FA proteins could work in other DNA damage repair pathways of HR (47).

We focused our study on the hypothesis that genes involved in the mechanism of DNA damage repair, acting similarly to tumour suppressors, would act as low susceptibility genes in the development of sporadic breast cancer, as proposed previously (48). In this study, we analysed seven genetic variants in three FA genes: two variants in FANCA, three in FANCL, and two in FANCD2. The first two genes encode proteins in the FA complex and the latter encodes a protein downstream of the FA complex.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Subjects
This case–control study included a total of 897 consecutive and non-related sporadic breast cancer cases and 1033 healthy controls. The case samples had a mean age of diagnosis of 50 years (range 23–90 years) (Table I). They were collected from seven Spanish public hospitals: 258 (29%) from Hospital Monte Naranco, in Oviedo; 164 (18%) from Fundación Jiménez Díaz, 132 (15%) from Hospital La Paz and 100 (11%) from Hospital Clínico San Carlos, all in Madrid; 115 (13%) from Hospital San Pau, in Barcelona; 100 (11%) from Hospital de Salamanca, in Salamanca; 28 (3%) from Hospital de Valencia, in Valencia. Controls (between 22 and 85 years of age) (Table I) were unaffected Spanish women collected from six centres: 462 (45%) from the Menopause Research Centre at the Instituto Palacios; 287 (28%) from the National Blood Transfusion Centre; 221 (21%) from Fundación Jiménez Díaz; 63 (6%) from Hospital Clínico San Carlos, all in Madrid.


View this table:
[in this window]
[in a new window]

  		Table I Age categories of control and case samples

 
Extraction of genomic DNA
Genomic DNA from cases and controls was isolated from peripheral blood lymphocytes using the MagNA Pure LC Instrument (Roche Molecular Biochemicals, Germany) following manufacturer's instructions (49), and a stock solution of DNA at 50 ng/µl was obtained, measured using the PicoGreen dsDNA Quantitation Kit (Molecular Probes, Eugene, OR).

SNP selection
Three public databases available via internet were used to collect information about single nucleotide polymorphisms (SNPs) in the three genes (FANCA, FANCL and FANCD2): NCBI (http://www.ncbi.nlm.nih.gov), Ensembl (http://www.ensembl.org) and HapMap (http://www.hapmap.org). SNPs were considered if they had a minor allele frequency (MAF) greater than or equal to 10%, the exception being putative coding SNPs with MAF ≥ 5%. Information on linkage disequilibrium (LD) (blocks within chromosomes that are more probably linked and segregate together than expected at random), was used to select representative SNPs in high LD blocks. Haploview v3.11 (50) was used to select tag-SNPs (a subset of SNPs that represent variation in a gene or chromosomal region) where possible. We selected and successfully genotyped two SNPs on the 3' downstream region of FANCA (rs7195066 and rs2239359, spaced 13 Kb apart), three located on introns 1, 7 and 8 of FANCL (rs848289, rs1011314 and rs3771224, respectively, spaced over 68 Kb) and two in FANCD2 (rs722509 and rs2272125, spaced 32 Kb apart), one on intron 19 and the other a synonymous coding change (L1366L) on exon 42. Gene structures and SNP positions are shown in Figure 1A.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 (A) Gene structure and SNP location. Schematic diagram of the FANCA, FANCL and FANCD2 genes. Grey boxes represent exons and solid lines represent untranslated and intron regions, with 5' upstream and 3'-UTR regions noted. Fine and arrowed lines represent gene lengths and solid arrows represent 5'–3' gene orientation. Functional motifs and assayed SNP locations are noted. (B) LD blocks located in the FANCD2 gene region. Schematic diagram of FANCD2 and surrounding genes located in the same LD block. Grey boxes represent genes. Solid lines represent LD blocks obtained by the Confidence Intervals Method or the 4 Gamete Rule.

 
Genotyping assays
Genotyping was carried out using the TaqMan platform. SNP assays were designed using Applied Biosystems Assay-by-design and Assay-on-Demand probes (Applied Biosystems, Foster City, CA, USA) (primer and probe information is provided in Supplementary Table I). PCR amplifications were carried out following the manufacturer's standardized protocol. The genotype of each sample was automatically assigned by measuring allele-specific fluorescence in the ABI Prism 7900HT Detection System, using the SDSv2.1 (Sequence Detection Software) for allele discrimination, by Applied Biosystems. Three non-DNA and nine duplicate samples were included in each plate of 384 samples used, as an internal control.

Statistical analysis
Deviations from Hardy–Weinberg equilibrium were tested using the genhwi command in STATAv8.0 (51), applying Pearson's Chi-squared test or Fisher's Exact Test where appropriate. Differences in the MAFs between cases and controls were tested using the Chi-squared test. In order to assess associations between genotypes, haplotypes (combination of variants along a chromosome) and diplotypes (haplotype combinations in individuals), and breast cancer risk, several analysis were performed. PHASEv2.0 software (52,53) was used to impute haplotypes and compare their frequency distributions in cases and controls. Unconditional logistic regression was applied using STATAv8.0 for the analysis of genotypes, haplotypes and diplotypes, the most likely imputed haplotype being assumed for the latter two. Odds ratios (OR), their 95% confidence intervals (CI) and Wald statistic P-values were estimated, using the most frequent genotype, haplotype and diplotype, respectively, among controls as reference. In the analysis of genotypes, dominant, recessive and multiplicative (single parameter) codominant models were assessed against the two parameter codominant model via the likelihood ratio test, and where the latter was not the best fit (nominal P-value greater than 0.05), the best fitting model was determined by parsimony. Age was included as a categorical variable (<40, 40–44, 45–49, 50–54, 55–59, 60–64, ≥65) (Table I). Analysis of haplotypes was repeated using the haplo.stats library implemented in R which compares haplotype frequencies in cases and controls in an unbiased way by including haplotype uncertainty in the estimation of ORs. For all statistical tests, results were considered significant for two-sided P-values less than 0.05.

Screening for functional variants in the FANCD2 gene
Haploview v3.11 was used to graphically analyse the extent of LD in the FANCD2 region (Figure 1B). Selected sequences in FANCD2 (promoter region and nine exons) were further analysed by PCR, dHPLC and sequencing in samples carrying haplotype H2 in the FANCD2 gene. Primers in flanking exons, intron–exon boundaries and the promoter region were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (54), based on genomic information collected from NCBI (http://www.ncbi.nlm.nih.gov). PCR products, obtained under standardized conditions, were analysed by dHPLC using the WAVE DNA-fragment analysis system (Transgenomics Limited, Crewe, UK), and were determined by the WAVEMAKER software from Transgenomics (PCR primers and conditions, and dHPLC elution conditions are showed in Supplementary Table II). Those DNAs with heterozygous peaks detected by dHPLC analysis were purified using the High Pure PCR Product Purification Kit (Roche) and sequenced with the ABI Prism Big DyeTM System and ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Additional validation for the identified variants ss49839682, rs17032268, rs9811771 and rs7647987 in FANCD2 was carried out by dHPLC analysis (conditions shown in Supplementary Table II) in 95 case and 95 control non-H2 carriers. A sample of 551 case and 559 control non-H2 carriers were further genotyped using TaqMan probes (Supplementary Table I) in order to validate the results obtained for ss49839682 and rs9811771. FANCD2 variants were assessed for functional importance using PupaSNP (http://pupasnp.bioinfo.ochoa.fib.es), which synthesizes information from: Ensembl v25.34e, dbSNP 122, HapMap release #12, Match v2.2, Transfac Professional v8.3, Haploview v3.0 and PMUT (55). Transfac Professional v8.3 was also applied to screen for transcription factor consensus binding sites using rVista v2.0 (http://rvista.dcode.org) (56). Phylogenetic conservation analysis was performed using ECR Browser (http://ecrbrowser.dcode.org) (57). Phylogenetic sequence comparisons and construction of phylogenetic trees among mammal organisms were carried out using Tcoffee (http://igs-server.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi) (58) (see Figures 2A and B and Supplementary Figure 1).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Phylogenetic analyses of ss49839682 (A) and rs17032268 (B) sequence conservation in mammal organisms. Sequence comparison of surrounding variants and transcription factor consensus binding sites. Phylogenetic trees as measures of divergence are noted. Grey boxes represent highly conserved sequences. SNP sites and transcription factor binding sites are noted.

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Genotyping
Genotyping call rates were over 97%, and concordance rates for non-DNA and duplicate samples were 100%, for all SNPs genotyped (data are shown in Table II). All seven SNPs complied with Hardy–Weinberg equilibrium among controls.


View this table:
[in this window]
[in a new window]

  		Table II Statistical analysis of genotypes in FANCA, FANCL and FANCD2

 
Analysis of genotypes
A higher MAF was observed among cases than controls for rs2272125 in FANCD2 (0.13 versus 0.10, respectively, P = 0.005). The other six polymorphisms tested did not show any evidence of differences in the allelic frequencies (data not shown). Comparison of genotype frequencies suggested that the risk of developing sporadic breast cancer associated with rs2272125 followed a codominant model, with the estimated OR for rare CC homozygotes relative to common AA homozygotes (OR = 2.77; 95% CI = 1.08–7.13; P = 0.03) more than double that for heterozygotes (OR = 1.28; 95% CI = 1.02–1.62; P = 0.04). The best fitting model was a single parameter codominant one, with an associated multiplicative per-allele OR of 1.35 (95% CI = 1.09–1.67, P = 0.005). This association was consistently observed after adjusting for age (Table I) (OR = 1.32, P = 0.03). We did not find any other associations among the remaining SNPs in this study. Results for all genotyped SNPs are summarized in Table II.

The program Structure v.2.0 was applied to a set of 28 unlinked biallelic markers genotyped in a random subset of 163 cases and 180 controls, finding no evidence of population substructure, with an estimated posterior probability of approximately one for K (number of clusters) = 1, and minimum and maximum ancestry coefficients of 0.45 and 0.55, respectively for K = 2, consistent across independent runs (R.L. Milne unpublished data).

Analysis of haplotypes and diplotypes
We compared the imputed haplotypes and diplotypes formed by the SNPs in FANCD2 between cases and controls and the results are presented in Table III. The most common haplotype (H1) found in both cases and controls carried the more common alleles ‘T’ in rs722509 and ‘A’ in rs2272125. Comparison of haplotype frequencies between cases and controls suggested that one of the four haplotypes was associated with the risk of breast cancer. This haplotype (H2) carried the combination ‘TC’ and the estimated OR relative to H1 was 4.46 (95% CI = 1.51–13.21; P = 0.007) assuming the most likely haplotypes were actually observed, and 4.81 (95% CI = 1.63–14.18; P = 0.004) allowing for haplotype uncertainty [Table III (a)].


View this table:
[in this window]
[in a new window]

  		Table III Statistical analysis of haplotypes (a) and diplotypes (b) in the FANCD2 gene. Bold typeface indicate statistically significant haplotype and diplotype analises.

 
Analysis of diplotypes found that carrying H1 and H2 (diplotype D1–2) was associated with increased risk of breast cancer relative to D1–1 (OR 3.57; 95% CI = 1.17–10.90; P = 0.03). Moreover, only four women carried H2 in a diplotype other than D1–2, all in combination with H4 (D2–4) and all four were cases. Pooling all carriers of H2 (D1–2 and D2–4) confirmed that they were at increased risk (OR = 4.59; 95% CI = 1.54–13.63; P = 0.006) [Table III (b)].

Screening for putative causal mutations in FANCD2 risk haplotype carriers
rs2272125 is unlikely to directly cause the observed increased breast cancer risk because it is a synonymous coding SNP. Furthermore, we evaluated its potential implication in exon–intron splicing and found no exonic splicing enhancer (ESE) motifs in that region of FANCD2. The observed association might be due to a different causal variant in the same LD block as rs2272125, which extends over the whole region including FANCD2 (over 100kb, see Figure 1B). Because it was unclear where the causative variation could be located, we searched for putative causal variants in the region among the carriers of the risk haplotype H2 (D1–2 and D2–4). We studied the described functional regions in the FANCD2 protein by DNA sequencing: the phosphorylation motif, located in exon 9, and the monoubiquitination motif, located in exon 19 (Figure 1A). We did not detect any variation from the reference sequence used (NC_000004 [GenBank] ).

We also sought to characterize the region surrounding rs2272125, (from exon 38 to exon 43), as well as the promoter (1500 bp upstream of ATG) and 3'-UTR regions, the latter two as putative functional domains. Three variants were found in the putative promoter region: rs781871 (MAF not described), rs17032268 (NCBI MAF = 0.22) and a novel SNP ss49839682 (–544 bp). A transitional change A > G in intron 38, 13 bp from exon 38 (IV38+13), was identified in the public databases as rs9811771, with unknown MAF. Finally, four SNPs in the 3'-UTR region were detected, one (rs7647987) located before the polyA sequence (NCBI MAF = 0.12), and three others located after the polyA sequence: rs3826, rs11716842, and a novel polymorphism ss49839681 (+421 bp from the stop codon), all with unknown MAF (Table IV).


View this table:
[in this window]
[in a new window]

  		Table IV Variants found by dHPLC and sequencing in samples carrying haplotype H2.

 
All these variants detected in the H2 carriers were in high LD and, according to PHASEv2.0, the variant-haplotype VH2 (CTGGAG) was observed (see areas highlighted in grey in Table IV). Four individuals carried sections of VH2 and a fifth carried the entire haplotype, together representing 30% of the H2 carriers.

We genotyped four of these eight variants in 190 non-carriers of H2 (95 cases and 95 controls) in order to investigate whether any appeared accumulated in H2 carriers. These variants were: ss49839682, rs17032268, rs9811771 and rs7647987, chosen because our hypothesis was focused on detecting variants with functional implications that also shared the main part of VH2 (Table IV). Only two out of the four variants selected (ss49839682 and rs9811771) appeared to be more frequent in H2 carriers than non-carriers although neither was statistically significant (OR = 2.04; 95% CI = 0.73–5.66; P = 0.2; and OR = 1.68; 95% CI = 0.61–4.62; P = 0.3, respectively). We subsequently genotyped a sample of 1100 DNAs for these two SNPs, but did not observe statistical significance, although the tendency of the minor allele accumulating in H2 carriers was also observed (Table V).


View this table:
[in this window]
[in a new window]

  		Table V Observed allele frequencies for four putative functional SNPs, by H2 carrier status

 
Analysis of sequence conservation was performed for these four additional FANCD2 variants in several mammal organisms. Only ss49839682 and rs17032268 (and their surrounding regions) have been phylogenetically maintained in mammals. Furthermore, these two variants located in the promoter region contain several consensus motifs for transcriptional factors (Figure 2A and B, respectively). The variant rs9811771, located near the exon–intron splicing site, was only weakly conserved in Pan troglodites and Canis familiaris and rs7647987, located at the 3'polyA site, was not conserved at all (see Supplementary Figures 1A and B, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
Previous studies have demonstrated that several genes involved in DNA repair, other than BRCA1 or BRCA2, are related to breast cancer susceptibility (59–63). BRCA2 has been identified as FANCD1 (3), and several interactions between both BRCA1 and BRCA2 and FA proteins have been described (64,45,40,65). It is therefore plausible that alterations in other FA genes might be implicated in breast cancer risk (66). It is known that alterations in FA genes are linked to several types of cancer, included breast cancer, in the general (non-FA) population (67,68). For example, the absence of FANCF by aberrant promoter methylation (69,70), and BACH1/FANCJ mutations (71) have both been found in breast cancer cases. Furthermore, one study found that FANCD2 is more often expressed in high proliferation rate tissues, such as mammary glands (72), and mouse model studies have shown that a germline disruption of the murine Fancd2 gene results in ovarian and epithelial breast cancers (73,74). However, despite the evidence in the literature about the potential susceptibility to breast and other cancers associated with the FA family (75), there have been few studies investigating these associations (76).

In this study, we have investigated associations between genetic variants in three genes belonging to the FA family and sporadic breast cancer in the Spanish population. We genotyped seven SNPs in FANCA, FANCL and FANCD2. Whereas no associations with breast cancer risk were found for SNPs in FANCA and FANCL, we were able to identify an increased risk associated with the minor ‘C’ allele of rs2272125 in FANCD2 (OR = 1.35 per allele) (Table II). Both haplotype and diplotype analyses confirmed this association, with carriers of at least one copy of the H2 haplotype at an estimated greater than 4-fold risk of breast cancer (P = 0.007 and P = 0.006, respectively) (Table III).

Despite being located in a coding region, the variant rs2272125 is of a synonymous nature and it is not located in any ESE motif. A likely explanation for this association is therefore that there is a causative functional change located in close proximity or in LD with this SNP. The region of high LD that includes rs2272125 is over 100 Kb and spans the entire FANCD2 gene. Applying two methods of LD inference, the Confidence Intervals Method (77) and the 4 Gametes Rule (78), it was found that this LD block contains FANCD2 and three other genes of unknown function. VHL, a gene close to FANCD2 which has been implicated in some cancers (79,80), was excluded because it was not included in the LD block according to the latter method (Figure 1B). We therefore focused on FANCD2, our hypothesis being that this gene could carry the putative causal variant.

In-depth analysis of several regions of the FANCD2 gene among H2 carriers allowed us to detect another eight variants, some of them described previously in the SNP databases, but others identified for the first time (ss49839682 and ss49839681) (Table IV). Four of these variants could have functional implications because of their location (promoter, exon–intron splicing site, and 3'-UTR regions). In addition, they seemed to form a variant-haplotype (VH2) within H2 carriers (Table IV). We therefore investigated how frequent these variants were in people not carrying the H2 haplotype. One of the two variants located in the likely promoter region fell in a strongly phylogenetically conserved motif with consensus binding sites for several transcriptional factors (Figure 2A), which made us speculate that it may have functional implications; however, we have been unable to validate statistically this hypothesis (Table V). It is also possible that other variants along FANCD2 or the immediate surrounding region could be responsible for the increased risk associated with this gene.

Another, less likely possibility, is that the observed association could be due to confounding by unmeasured factors, such as race. Although population stratification was tested for and not detected, this we done using only a subset of the sample and using only 28 markers. Population stratification cannot therefore be entirely discarded, although it appears unlikely to have influenced the results of this study. Alternatively, the observed association could be due to chance. Replication of these findings using samples from other populations is required to address these two concerns.

In conclusion, we found that a synonymous variant (rs2272125) and haplotype (H2) in the FANCD2 gene are associated with sporadic breast cancer. This is the first study to identify this association. We have also identified a putative functional variant (ss49839682) in the same high LD block, but were not able to conclude that this is causal. Further studies will be required to clarify the full implication of the FANCD2 protein in sporadic breast cancer risk.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material is available online at http://www.carcin.oupjournals.org/.


   Acknowledgments
 
This study was supported by the grant BFI2003-03852. EB and LPF were funded by the Comunidad Autónoma de Madrid and by the Spanish Ministry of Science and Technology (MCT), respectively. We would like to thank Álvaro Ruibal (University Hospital, Santiago de Compostela), and Santiago Palacios (Instituto Palacios, Madrid) for the use of samples of cases and controls. We would also like to thank Raquel Rodríguez-López for her contribution to the beginning of this work, and Fátima Mercadillo, Alicia Barroso, Emilio González, Jesús López, Victoria Fernández and Rocío Letón for their expert technical skills.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

  1. Miki Y., Swensen J., Shattuck-Eidens D., et al. (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266:66–71.[Abstract/Free Full Text]
  2. Wooster R., Bignell G., Lancaster J., Swift S., Seal S., Mangion J., Collins N., Gregory S., Gumbs C., Micklem G. (1995) Identification of the breast cancer susceptibility gene BRCA2. Nature 378:789–792.[CrossRef][Medline]
  3. Howlett N.G., Taniguchi T., Olson S., et al. (2002) Biallelic inactivation of BRCA2 in Fanconi anemia. Science 297:606–609.[Abstract/Free Full Text]
  4. Stewart G. and Elledge S.J. (2002) The two faces of BRCA2, a FANCtastic discovery. Mol. Cell 10:2–4.[CrossRef][ISI][Medline]
  5. Risch N. (2001) The genetic epidemiology of cancer: interpreting family and twin studies and their implications for molecular genetic approaches. Cancer Epidemiol. Biomarkers Prev. 10:733–741.[Abstract/Free Full Text]
  6. Antoniou A.C., Pharoah P.D., McMullan G., Day N.E., Ponder B.A., Easton D. (2001) Evidence for further breast cancer susceptibility genes in addition to BRCA1 and BRCA2 in a population-based study. Genet. Epidemiol. 21:1–18.[CrossRef][ISI][Medline]
  7. Antoniou A.C. and Easton D.F. (2003) Polygenic inheritance of breast cancer: Implications for design of association studies. Genet. Epidemiol. 25:190–202.[CrossRef][ISI][Medline]
  8. Comings D.E., Gade-Andavolu R., Cone L.A., Muhleman D., MacMurray J.P. (2003) A multigene test for the risk of sporadic breast carcinoma. Cancer 97:2160–2170.[CrossRef][ISI][Medline]
  9. Liang F., Han M., Romanienko P.J., Jasin M. (1998) Homology-directed repair is a major double-strand break repair pathway in mammalian cells. Proc. Natl Acad. Sci. USA 95:5172–5177.[Abstract/Free Full Text]
  10. Moynahan M.E., Chiu J.W., Koller B.H., Jasin M. (1999) Brca1 controls homology-directed DNA repair. Mol. Cell 4:511–518.[CrossRef][ISI][Medline]
  11. Marmorstein L.Y., Ouchi T., Aaronson S.A. (1998) The BRCA2 gene product functionally interacts with p53 and RAD51. Proc. Natl Acad. Sci. USA 95:13869–13874.[Abstract/Free Full Text]
  12. Yuan S.S., Lee S.Y., Chen G., Song M., Tomlinson G.E., Lee E.Y. (1999) BRCA2 is required for ionizing radiation-induced assembly of Rad51 complex in vivo. Cancer Res. 59:3547–3551.[Abstract/Free Full Text]
  13. Moynahan M.E., Pierce A.J., Jasin M. (2001) BRCA2 is required for homology-directed repair of chromosomal breaks. Mol. Cell 7:263–272.[CrossRef][ISI][Medline]
  14. Davies A.A., Masson J.Y., McIlwraith M.J., Stasiak A.Z., Stasiak A., Venkitaraman A.R., West S.C. (2001) Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol. Cell 7:273–282.[CrossRef][ISI][Medline]
  15. Digweed M., Rothe S., Demuth I., Scholz R., Schindler D., Stumm M., Grompe M., Jordan A., Sperling K. (2002) Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia. Carcinogenesis 23:1121–1126.[Abstract/Free Full Text]
  16. D'Andrea A.D. and Grompe M. (2003) The Fanconi anaemia/BRCA pathway. Nat. Rev. Cancer 3:23–34.[CrossRef][ISI][Medline]
  17. Wang X. and D'Andrea A.D. (2004) The interplay of Fanconi anemia proteins in the DNA damage response. DNA Repair (Amst) 3:1063–1069.[Medline]
  18. Joenje H., Oostra A.B., Wijker M., et al. (1997) Evidence for at least eight Fanconi anemia genes. Am. J. Hum. Genet. 61:940–944.[ISI][Medline]
  19. Medhurst A.L., Huber P.A., Waisfisz Q., de Winter J.P., Mathew C.G. (2001) Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway. Hum. Mol. Genet. 10:423–429.[Abstract/Free Full Text]
  20. Gordon S.M. and Buchwald M. (2003) Fanconi anemia protein complex: mapping protein interactions in the yeast 2- and 3-hybrid systems. Blood 102:136–141.[Abstract/Free Full Text]
  21. Nakanishi K., Moran A., Hays T., Kuang Y., Fox E., Garneau D., de Oca R.M., Grompe M., D'Andrea A.D. (2001) Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9. Exp. Hematol. 29:842–849.[CrossRef][ISI][Medline]
  22. Leveille F., Blom E., Medhurst A.L., et al. (2004) The Fanconi anemia gene product FANCF is a flexible adaptor protein. J. Biol. Chem. 279:39421–39430.[Abstract/Free Full Text]
  23. Meetei A.R., de Winter J.P., Medhurst A.L., et al. (2003) A novel ubiquitin ligase is deficient in Fanconi anemia. Nat. Genet. 35:165–170.[CrossRef][ISI][Medline]
  24. Meetei A.R., Yan Z., Wang W. (2004) FANCL replaces BRCA1 as the likely ubiquitin ligase responsible for FANCD2 monoubiquitination. Cell Cycle 3:179–181.[ISI][Medline]
  25. Meetei A.R., Levitus M., Xue Y., et al. (2004) X-linked inheritance of Fanconi anemia complementation group B. Nat. Genet. 36:1219–1224.[CrossRef][ISI][Medline]
  26. Fei P., Yin J., Wang W. (2005) New advances in the DNA damage response network of Fanconi anemia and BRCA proteins. FAAP95 replaces BRCA2 as the true FANCB protein. Cell Cycle 4:80–86.[ISI][Medline]
  27. Garcia-Higuera I., Taniguchi T., Ganesan S., Meyn M.S., Timmers C., Hejna J., Grompe M., D'Andrea A.D. (2001) Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol. Cell 7:249–262.[CrossRef][ISI][Medline]
  28. Litman R., Peng M., Jin Z., Zhang F., Zhang J., Powell S., Andreassen P.R., Cantor S.B. (2005) BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ. Cancer Cell 8:255–265.[CrossRef][ISI][Medline]
  29. Bridge W.L., Vandenberg C.J., Franklin R.J., Hiom K. (2005) The BRIP1 helicase functions independently of BRCA1 in the Fanconi anemia pathway for DNA crosslink repair. Nat. Genet. 37:953–957.[CrossRef][ISI][Medline]
  30. Meetei A.R., Medhurst A.L., Ling C., et al. (2005) A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M. Nat. Genet. 37:958–963.[CrossRef][ISI][Medline]
  31. Morrison C., Sonoda E., Takao N., Shinohara A., Yamamoto K., Takeda S. (2000) The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J. 19:463–471.[CrossRef][ISI][Medline]
  32. Cliby W.A., Roberts C.J., Cimprich K.A., Stringer C.M., Lamb J.R., Schreiber S.L., Friend S.H. (1998) Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints. EMBO J. 17:159–169.[CrossRef][ISI][Medline]
  33. Grompe M. (2002) FANCD2: a branch-point in DNA damage response? Nat. Med. 8:555–556.[CrossRef][ISI][Medline]
  34. Taniguchi T., Garcia-Higuera I., Xu B., Andreassen P.R., Gregory R.C., Kim S.T., Lane W.S., Kastan M.B., D'Andrea A.D. (2002) Convergence of the fanconi anemia and ataxia telangiectasia signaling pathways. Cell 109:459–472.[CrossRef][ISI][Medline]
  35. Andreassen P.R., D'Andrea A.D., Taniguchi T. (2004) ATR couples FANCD2 monoubiquitination to the DNA-damage response. Genes Dev. 18:1958–1963.[Abstract/Free Full Text]
  36. Venkitaraman A.R. (2004) Tracing the network connecting BRCA and Fanconi anaemia proteins. Nat. Rev. Cancer 4:266–276.[CrossRef][ISI][Medline]
  37. Vandenberg C.J., Gergely F., Ong C.Y., Pace P., Mallery D.L., Hiom K., Patel K.J. (2003) BRCA1-independent ubiquitination of FANCD2. Mol. Cell 12:247–254.[CrossRef][ISI][Medline]
  38. Montes de Oca R., Andreassen P.R., Margossian S.P., Gregory R.C., Taniguchi T., Wang X., Houghtaling S., Grompe M., D'Andrea A.D. (2005) Regulated interaction of the Fanconi anemia protein, FANCD2, with chromatin. Blood 105:1003–1009.[Abstract/Free Full Text]
  39. Park W.H., Margossian S., Horwitz A.A., Simons A.M., D'Andrea A.D., Parvin J.D. (2005) Direct DNA binding activity of the Fanconi anemia D2 protein. J. Biol. Chem. 280:23593–23598.[Abstract/Free Full Text]
  40. Hussain S., Wilson J.B., Medhurst A.L., et al. (2004) Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways. Hum. Mol. Genet. 13:1241–1248.[Abstract/Free Full Text]
  41. Taniguchi T., Garcia-Higuera I., Andreassen P.R., Gregory R.C., Grompe M., D'Andrea A.D. (2002) S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51. Blood 100:2414–2420.[Abstract/Free Full Text]
  42. Rothfuss A. and Grompe M. (2004) Repair kinetics of genomic interstrand DNA cross-links: evidence for DNA double-strand break-dependent activation of the Fanconi anemia/BRCA pathway. Mol. Cell. Biol. 24:123–134.[Abstract/Free Full Text]
  43. Howlett N.G., Taniguchi T., Durkin S.G., D'Andrea A.D., Glover T.W. (2005) The Fanconi anemia pathway is required for the DNA replication stress response and for the regulation of common fragile site stability. Hum. Mol. Genet. 14:693–701.[Abstract/Free Full Text]
  44. Wang X., Andreassen P.R., D'Andrea A.D. (2004) Functional interaction of monoubiquitinated FANCD2 and BRCA2/FANCD1 in chromatin. Mol. Cell. Biol. 24:5850–5862.[Abstract/Free Full Text]
  45. Hussain S., Witt E., Huber P.A., Medhurst A.L., Ashworth A., Mathew C.G. (2003) Direct interaction of the Fanconi anaemia protein FANCG with BRCA2/FANCD1. Hum. Mol. Genet. 12:2503–2510.[Abstract/Free Full Text]
  46. Ohashi A., Zdzienicka M.Z., Chen J., Couch F.J. (2005) Fanconi anemia complementation group D2 (FANCD2) functions independently of BRCA2- and RAD51-associated homologous recombination in response to DNA damage. J. Biol. Chem. 280:14877–14883.[Abstract/Free Full Text]
  47. Nakanishi K., Yang Y.G., Pierce A.J., Taniguchi T., Digweed M., D'Andrea A.D., Wang Z.Q., Jasin M. (2005) Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. Proc. Natl Acad. Sci. USA 102:1110–1115.[Abstract/Free Full Text]
  48. Kuschel B., Auranen A., McBride S., et al. (2002) Variants in DNA double-strand break repair genes and breast cancer susceptibility. Hum. Mol. Genet. 11:1399–1407.[Abstract/Free Full Text]
  49. Kessler H.H., Muhlbauer G., Stelzl E., Daghofer E., Santner B.I., Marth E. (2001) Fully automated nucleic acid extraction: MagNA Pure LC. Clin. Chem. 47:1124–1126.[Free Full Text]
  50. Barrett J.C., Fry B., Maller J., Daly M.J. (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–265.[Abstract/Free Full Text]
  51. StataCorp. (2003) Stata Statistical Software: Release 8.0 (TX: Stata Corporation, College Station).
  52. Stephens M., Smith N.J., Donnelly P. (2001) A new statistical method for haplotype reconstruction from population data. Am. J. Hum. Genet. 68:978–989.[CrossRef][ISI][Medline]
  53. Stephens M. and Donnelly P. (2003) A comparison of bayesian methods for haplotype reconstruction from population genotype data. Am. J. Hum. Genet. 73:1162–1169.[CrossRef][ISI][Medline]
  54. Rozen S. and Skaletsky H. (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132:365–386.[Medline]
  55. Conde L., Vaquerizas J.M., Santoyo J., Al-Shahrour F., Ruiz-Llorente S., Robledo M., Dopazo J. (2004) PupaSNP Finder: a web tool for finding SNPs with putative effect at transcriptional level. Nucleic Acids Res. 32:W242–W248.[Abstract/Free Full Text]
  56. Loots G.G. and Ovcharenko I. (2004) rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Res. 32:W217–W221.[Abstract/Free Full Text]
  57. Ovcharenko I., Nobrega M.A., Loots G.G., Stubbs L. (2004) ECR Browser: a tool for visualizing and accessing data from comparisons of multiple vertebrate genomes. Nucleic Acids Res. 32:W280–W286.[Abstract/Free Full Text]
  58. Poirot O., Suhre K., Abergel C., O'Toole E., Notredame C. (2004) 3DCoffee{at}igs: a web server for combining sequences and structures into a multiple sequence alignment. Nucleic Acids Res. 32:W37–W40.[Abstract/Free Full Text]
  59. Easton D.F. (1994) Cancer risks in A-T heterozygotes. Int. J. Radiat. Biol. 66:S177–S182.[ISI][Medline]
  60. Thorstenson Y.R., Roxas A., Kroiss R., et al. (2003) Contributions of ATM mutations to familial breast and ovarian cancer. Cancer Res. 63:3325–3333.[Abstract/Free Full Text]
  61. Rodriguez-Lopez R., Osorio A., Sanchez-Pulido L., De La Hoya M., Barroso A., Caldes T., Benitez J. (2003) No mutations in the XRCC2 gene in BRCA1/2-negative high-risk breast cancer families. Int. J. Cancer 103:136–137.[CrossRef][ISI][Medline]
  62. Smith T.R., Miller M.S., Lohman K., Lange E.M., Case L.D., Mohrenweiser H.W., Hu J.J. (2003) Polymorphisms of XRCC1 and XRCC3 genes and susceptibility to breast cancer. Cancer Lett. 190:183–190.[CrossRef][ISI][Medline]
  63. Meijers-Heijboer H., van den Ouweland A., Klijn J., et al. (2002) Low-penetrance susceptibility to breast cancer due to CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations. Nat. Genet. 31:55–59.[CrossRef][ISI][Medline]
  64. Folias A., Matkovic M., Bruun D., Reid S., Hejna J., Grompe M., D'Andrea A., Moses R. (2002) BRCA1 interacts directly with the Fanconi anemia protein FANCA. Hum. Mol. Genet. 11:2591–2597.[Abstract/Free Full Text]
  65. Levran O., Attwooll C., Henry R.T., et al. (2005) The BRCA1-interacting helicase BRIP1 is deficient in Fanconi anemia. Nat. Genet. 37:931–933.[CrossRef][ISI][Medline]
  66. Venkitaraman A.R. (2002) Connecting Fanconi's anaemia to breast cancer predisposition. Lancet 360:1344–1345.[CrossRef][ISI][Medline]
  67. Colleu-Durel S., Guitton N., Nourgalieva K., Leveque J., Danic B., Chenal C. (2001) Genomic instability and breast cancer. Oncol. Rep. 8:1001–1005.[ISI][Medline]
  68. Alter B.P., Greene M.H., Velazquez I., Rosenberg P.S. (2003) Cancer in Fanconi anemia. Blood 101:2072.[Free Full Text]
  69. Van Der Heijden M.S., Brody J.R., Kern S.E. (2004) Functional screen of the fanconi anemia pathway in cancer cells by Fancd2 immunoblot. Cancer Biol. Ther. 3:534–537.[ISI][Medline]
  70. Turner N., Tutt A., Ashworth A. (2004) Hallmarks of ‘BRCAness’ in sporadic cancers. Nat. Rev. Cancer 4:814–819.[CrossRef][ISI][Medline]
  71. Lewis A.G., Flanagan J., Marsh A., Pupo G.M., Mann G., Spurdle A.B., Lindeman G.J., Visvader J.E., Brown M.A, Chenevix-Trench G. (2005) Mutation analysis of FANCD2, BRIP1/BACH1, LMO4 and SFN in familial breast cancer. Breast Cancer Res. 7:R1005–R1016.[CrossRef][ISI][Medline]
  72. Holzel M., van Diest P.J., Bier P., Wallisch M., Hoatlin M.E., Joenje H., de Winter J.P. (2003) FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia. J. Pathol. 201:198–203.[CrossRef][ISI][Medline]
  73. Houghtaling S., Timmers C., Noll M., Finegold M.J., Jones S.N., Meyn M.S., Grompe M. (2003) Epithelial cancer in Fanconi anemia complementation group D2 (Fancd2) knockout mice. Genes Dev. 17:2021–2035.[Abstract/Free Full Text]
  74. Houghtaling S., Granville L., Akkari Y., Torimaru Y., Olson S., Finegold M., Grompe M. (2005) Heterozygosity for p53 (Trp53+/–) accelerates epithelial tumor formation in fanconi anemia complementation group D2 (Fancd2) knockout mice. Cancer Res. 65:85–91.[Abstract/Free Full Text]
  75. Seal S., Barfoot R., Jayatilake H., et al. (2003) Evaluation of Fanconi Anemia genes in familial breast cancer predisposition. Cancer Res. 63:8596–8599.[Abstract/Free Full Text]
  76. Thompson E., Dragovic R.L., Stephenson S.A., Eccles D.M., Campbell I.G., Dobrovic A. (2005) A novel duplication polymorphism in the FANCA promoter and its association with breast and ovarian cancer. BMC Cancer 5:43.[CrossRef][Medline]
  77. Gabriel S.B., Schaffner S.F., Nguyen H., et al. (2002) The structure of haplotype blocks in the human genome. Science 296:2225–2229.[Abstract/Free Full Text]
  78. Wang N., Akey J.M., Zhang K., Chakraborty R., Jin L. (2002) Distribution of recombination crossovers and the origin of haplotype blocks: the interplay of population history, recombination, and mutation. Am. J. Hum. Genet. 71:1227–1234.[CrossRef][ISI][Medline]
  79. Kim W.Y. and Kaelin W.G. (2004) Role of VHL gene mutation in human cancer. J. Clin. Oncol. 22:4991–5004.[Abstract/Free Full Text]
  80. Otis C.N., Krebs P.A., Albuquerque A., Quezado M.M., San Juan X., Sobel M.E., Merino M.J. (2002) Loss of heterozygosity of p53, BRCA1, VHL, and estrogen receptor genes in breast carcinoma: correlation with related protein products and morphologic features. Int. J. Surg. Pathol. 10:237–245.[Abstract/Free Full Text]
Received November 29, 2005; revised March 21, 2006; accepted April 19, 2006.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. Berwick, J. M. Satagopan, L. Ben-Porat, A. Carlson, K. Mah, R. Henry, R. Diotti, K. Milton, K. Pujara, T. Landers, et al.
Genetic Heterogeneity among Fanconi Anemia Heterozygotes and Risk of Cancer
Cancer Res., October 1, 2007; 67(19): 9591 - 9596.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
27/9/1930    most recent
bgl062v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Barroso, E.
Right arrow Articles by Ribas, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Barroso, E.
Right arrow Articles by Ribas, G.
Social Bookmarking