Alternative mRNA Splicing and Promising Therapies in Cancer.

Cancer is among the leading causes of mortality worldwide. While considerable attention has been given to genetic and epigenetic sources of cancer-specific cellular activities, the role of alternative mRNA splicing has only recently received attention as a major contributor to cancer initiation and progression. The distribution of alternate mRNA splicing variants in cancer cells is different from their non-cancer counterparts, and cancer cells are more sensitive than non-cancer cells to drugs that target components of the splicing regulatory network. While many of the alternatively spliced mRNAs in cancer cells may represent "noise" from splicing dysregulation, certain recurring splicing variants have been shown to contribute to tumor progression. Some pathogenic splicing disruption events result from mutations in cis-acting splicing regulatory sequences in disease-associated genes, while others may result from shifts in balance among naturally occurring alternate splicing variants among mRNAs that participate in cell cycle progression and the regulation of apoptosis. This review provides examples of cancer-related alternate splicing events resulting from each step of mRNA processing and the promising therapies that may be used to address them.

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples.

BACKGROUND: BRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations.

Genetic anticipation in BRCA1/BRCA2 families after controlling for ascertainment bias and cohort effect.

BACKGROUND: Genetic anticipation, the earlier onset of disease in successive generations, has been reported in hereditary breast and ovarian cancer syndrome (HBOC), but little is known about its underlying mechanisms. Ascertainment bias has been suggested as a reason in previous studies. Likewise, cohort effect, which may be caused by environmental factors, can be misinterpreted as genetic anticipation. METHODS: The authors reviewed the pedigrees of 176 kindreds, segregating those with deleterious mutations in breast cancer genes 1 and 2 (BRCA1/BRCA2) who had at least 2 consecutive generations of the same cancer (breast or ovarian). By using mutation probabilities as analytical weights in weighted random-effect models, generational differences in the age at onset of breast/ovarian cancer were calculated. The analyses were further controlled for ascertainment bias by excluding probands and adjusting for birth-cohort effect in the anticipation models. RESULTS: The mean age at the onset of breast cancer for the probands' generation was 41.9 years, which was 6.8 years and 9.8 years earlier than the parents' and grandparents' generations, respectively. The anticipation effect for breast cancer remained significant after excluding the probands. There was a birth-cohort effect: patients who were born in 1930s and 1940s had breast cancer 5.0 years and 7.6 years earlier than patients who were born before 1920. The difference in breast cancer age of onset across generations was no longer significant after adjusting for birth-cohort effect. CONCLUSIONS: The observed anticipation effect was driven mainly by a decrease in age of onset across birth cohorts, underscoring the need for risk-reducing interventions that target changing environmental/lifestyle factors in BRCA1/BRCA2 carriers. Cancer 2016;122:1913-20. © 2016 American Cancer Society.

Inherited predisposition to breast cancer among African American women.

African Americans have a disproportionate burden of aggressive young-onset breast cancer. Genomic testing for inherited predisposition to breast cancer is increasingly common in clinical practice, but comprehensive mutation profiles remain unknown for most minority populations. We evaluated 289 patients who self-identified as African American with primary invasive breast cancer and with personal or family cancer history or tumor characteristics associated with high genetic risk for all classes of germline mutations in known breast cancer susceptibility genes using a validated targeted capture and multiplex sequencing approach. Sixty-eight damaging germline mutations were identified in 65 (22 %, 95 % CI 18-28 %) of the 289 subjects. Proportions of patients with unequivocally damaging mutations in a breast cancer gene were 26 % (47/180; 95 % confident interval [CI] 20-33 %) of those with breast cancer diagnosis before age 45; 25 % (26/103; 95 % CI 17-35 %) of those with triple-negative breast cancer (TNBC); 29 % (45/156; 95 % CI 22-37 %) of those with a first or second degree relative with breast cancer before age 60 or with ovarian cancer; and 57 % (4/7; 95 % CI 18-90 %) of those with both breast and ovarian cancer. Of patients with mutations, 80 % (52/65) carried mutations in BRCA1 and BRCA2 genes and 20 % (13/65) carried mutations in PALB2, CHEK2, BARD1, ATM, PTEN, or TP53. The mutational allelic spectrum was highly heterogeneous, with 57 different mutations in 65 patients. Of patients meeting selection criteria other than family history (i.e., with young age at diagnosis or TNBC), 48 % (64/133) had very limited information about the history of cancer in previous generations of their families. Mutations in BRCA1 and BRCA2 or another breast cancer gene occur in one in four African American breast cancer patients with early onset disease, family history of breast or ovarian cancer, or TNBC. Each of these criteria defines patients who would benefit from genomic testing and novel therapies targeting DNA repair pathways.

Changes in the pattern of breast cancer burden among African American women: evidence based on 29 states and District of Columbia during 1998 to 2010.

PURPOSE: Assessment of breast cancer (BC) pattern in individual states with respect to ethnicity. METHODS: Population-based cancer registries from the Cancer Incidence in Five Continents databases (1998-2007) supplemented with Surveillance, Epidemiology, and End Results data from 2008 to 2010 were used. RESULTS: The age-specific burden showed a clear convergence of BC burden among African American (AA) and Caucasian American (CA) in most states. This was primarily because of a decrease in the BC rate among CA aged 50 years or older and an increase among AA of the same age group. The 2003-2007/1998-2002 rate ratio for CA was 0.91 (95% confidence interval [CI], 0.90-0.91) in the South, whereas it was 1.06 (95% CI, 1.04-1.08) for AA. This convergence was confirmed in states with available data for the period 2008 to 2010. The AA/CA rate ratio among women aged younger than 40 years was 0.99 (95% CI, 0.99-1.04) in the Northeast, 1.29 (95% CI, 1.25-1.33) in the South, and 1.10 (95% CI, 1.04-1.17) in the West. This pattern correlates with the estrogen receptor positive and progesterone receptor positive pattern. The strongest disparity in estrogen receptor negative was observed in Louisiana which with Detroit, have had the highest rates of estrogen receptor negative. CONCLUSIONS: The changes in postmenopausal hormone use and mammography screening might have played a role in the observed convergence.

Comprehensive annotation of splice junctions supports pervasive alternative splicing at the BRCA1 locus: a report from the ENIGMA consortium.

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Δ1Aq, Δ5, Δ5q, Δ8p, Δ9, Δ(9,10), Δ9_11, Δ11q, Δ13p and Δ14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.

Risk factors for pregnancy-associated breast cancer: a report from the Nigerian Breast Cancer Study.

PURPOSE: Little is known about risk factors for pregnancy-associated breast cancer (PABC), diagnosed during pregnancy or postpartum. METHODS: We enrolled 1715 premenopausal women from the Nigerian Breast Cancer Study from 1998 to 2011. Based on recency of last pregnancy from diagnosis, breast cancer cases were categorized as (1) PABC diagnosed 2 years or longer postpartum, (2) PABC diagnosed 3 to 5 years postpartum, or (3) non-PABC diagnosed more than 5 years postpartum. Controls were matched to cases on recency of last pregnancy. Multiple logistic regressions were performed comparing cases and controls within each group. RESULTS: Of the 718 cases, 152 (21.2%) had PABC 2 or more years postpartum, and 145 (20.2%) 3 to 5 years postpartum. Although not statistically significant, women with higher parity tend to have an elevated risk of PABC but reduced risk of non-PABC (p for heterogeneity = 0.097). Family history of breast cancer might be a strong predictor particularly for PABC 2 or more years postpartum (odds ratio, 3.28; 95% confidence interval, 1.05-10.3). Compared with non-PABC cases, PABC 2 or more years postpartum cases were more likely to carry BRCA1/2 mutations (P = .03). CONCLUSIONS: Parity may have different roles in the development of PABC versus other premenopausal breast cancer in Nigerian women. Prospective mothers with multiple births and a family history of breast cancer may have an elevated risk of breast cancer during their immediate postpartum period.

Hidden dangers: a cryptic exon disrupts BRCA2 mRNA.

The first mutation that disrupts BRCA2 mRNA by including a novel, cryptic exon is reported in this issue. The mutation lies deep within an intron and would not have been detected by conventional screening methods. In the future, more mutations may be discovered by direct mRNA analysis.

Towards an Oncology Database (ONCOD) Using a Warehousing Approach.

Here we report a model data mart developed upon a warehousing system focusing on oncology data to explore optimized system architecture to support enhanced data integration and application capacity.

Recurrent BRCA1 and BRCA2 mutations in breast cancer patients of African ancestry.

Recurrent mutations constituted nearly three quarters of all BRCA1 mutations and almost half of all BRCA2 mutations identified in the first cohort of the Nigerian Breast Cancer Study. To further characterize breast/ovarian cancer risks associated with BRCA1/BRCA2 mutations in the African diaspora, we genotyped recurrent mutations among Nigerian, African American, and Barbadian breast cancer patients. A replication cohort of 356 Nigerian breast cancer patients was genotyped for 12 recurrent BRCA1/2 mutant alleles (Y101X, 1742insG, 4241delTG, M1775R, 4359insC, C64Y, 1623delTTAAA, Q1090X, and 943ins10 from BRCA1, and 1538delAAGA, 2630del11, and 9045delGAAA from BRCA2) by means of SNaPshot followed by direct sequencing or by direct sequencing alone. In addition, 260 African Americans and 118 Barbadians were genotyped for six of the recurrent BRCA1 mutations by SNaPshot assay. Of all the BRCA1/2 recurrent mutations we identified in the first cohort, six were identified in 11 patients in the replication study. These mutation carriers constitute 3.1 % [95 % Confidence Interval (CI) 1.6-5.5 %] of the replication cohort. By comparison, 6.9 % (95 % CI 4.7-9.7 %) of the discovery cohort carried BRCA1/2 recurrent mutations. For the subset of recurrent mutations we tested in breast cancer cases from Barbados or the United States, only two 943ins10 carriers were identified in African Americans. Nigerian breast cancer patients from Ibadan carry a broad and unique spectrum of BRCA1/2 mutations. Our data suggest that BRCA1/2 mutation testing limited to recurrent mutations is not sufficient to understand the BRCA1/2-associated breast cancer risk in African populations in the diaspora. As the cost of Sanger sequencing is considerably reduced, deploying innovative technologies such as high throughput DNA sequencing of BRCA1/2 and other cancer susceptibility genes will be essential for identifying high-risk individuals and families to reduce the burden of aggressive early onset breast cancer in low-resource settings.

High prevalence of BRCA1 and BRCA2 mutations in unselected Nigerian breast cancer patients.

Inherited mutations in the BRCA1 and BRCA2 genes are the strongest genetic predictors of breast cancer and are the primary causes of familial breast/ovarian cancer syndrome. The frequency, spectrum and penetrance of mutant BRCA1/BRCA2 alleles have been determined for several populations, but little information is available for populations of African ancestry, who suffer a disproportionate burden of early onset breast cancer. We have performed complete sequence analysis of all BRCA1 and BRCA2 exons and intron-exon boundaries for 434 Nigerian breast cancer patients from the University College Hospital in Ibadan, Nigeria. In contrast to previous suggestions that BRCA1/BRCA2 mutation frequencies are low or undetectable in African American populations, we find that Nigerian breast cancer patients have an exceptionally high frequency of BRCA1 and BRCA2 mutations (7.1 and 3.9%, respectively). Sixteen different BRCA1 mutations were detected, seven of which have never been reported previously, while thirteen different BRCA2 mutations were seen, six of which were previously unreported. Thus, our data support enrichment for genetic risk factors in this relatively young cohort. To improve breast cancer outcomes, we suggest that family-based models of risk assessment and genetic counseling coupled with interventions to reduce breast cancer risk should be broadly disseminated in Nigeria and other underserved and understudied populations.

Ancestry-shift refinement mapping of the C6orf97-ESR1 breast cancer susceptibility locus.

We used an approach that we term ancestry-shift refinement mapping to investigate an association, originally discovered in a GWAS of a Chinese population, between rs2046210[T] and breast cancer susceptibility. The locus is on 6q25.1 in proximity to the C6orf97 and estrogen receptor alpha (ESR1) genes. We identified a panel of SNPs that are correlated with rs2046210 in Chinese, but not necessarily so in other ancestral populations, and genotyped them in breast cancer case:control samples of Asian, European, and African origin, a total of 10,176 cases and 13,286 controls. We found that rs2046210[T] does not confer substantial risk of breast cancer in Europeans and Africans (OR = 1.04, P = 0.099, and OR = 0.98, P = 0.77, respectively). Rather, in those ancestries, an association signal arises from a group of less common SNPs typified by rs9397435. The rs9397435[G] allele was found to confer risk of breast cancer in European (OR = 1.15, P = 1.2 x 10(-3)), African (OR = 1.35, P = 0.014), and Asian (OR = 1.23, P = 2.9 x 10(-4)) population samples. Combined over all ancestries, the OR was 1.19 (P = 3.9 x 10(-7)), was without significant heterogeneity between ancestries (P(het) = 0.36) and the SNP fully accounted for the association signal in each ancestry. Haplotypes bearing rs9397435[G] are well tagged by rs2046210[T] only in Asians. The rs9397435[G] allele showed associations with both estrogen receptor positive and estrogen receptor negative breast cancer. Using early-draft data from the 1,000 Genomes project, we found that the risk allele of a novel SNP (rs77275268), which is closely correlated with rs9397435, disrupts a partially methylated CpG sequence within a known CTCF binding site. These studies demonstrate that shifting the analysis among ancestral populations can provide valuable resolution in association mapping.

Searching for large genomic rearrangements of the BRCA1 gene in a Nigerian population.

BRCA1/2 germline mutations predispose to breast and ovarian cancer. Large genomic rearrangements (LGRs) have widened the mutational spectrum of the BRCA1 gene, but the frequencies vary in different populations. In this study, we want to determine the spectrum of LGRs in BRCA1 gene in Nigerian breast cancer patients. The multiplex ligation-dependent probe amplification (MLPA) assay was used to screen BRCA1 rearrangements in 352 patients who previously tested negative for BRCA1 and BRCA2 point mutations and small insertions/deletions. Positive MLPA result was confirmed and located by long-range PCR. The breakpoints of the candidate rearrangement were characterized by sequencing. A novel deletion of BRCA1 exon 21 (c.5277 + 480_5332 + 672del) was detected in 1 out of 352 Nigerian breast cancer patients (0.3% occurrence frequency). Further analysis of breakpoints revealed that the deletion involves two Alu-elements: one AluSg in intron 20 and the AluY in intron 21. These data suggest that while BRCA1 genomic rearrangement exists, they do not contribute significantly to BRCA1-associated risk in the Nigerian population.

Mitotic expression of Spo13 alters M-phase progression and nucleolar localization of Cdc14 in budding yeast.

Spo13 is a key meiosis-specific regulator required for centromere cohesion and coorientation, and for progression through two nuclear divisions. We previously reported that it causes a G2/M arrest and may delay the transition from late anaphase to G1, when overexpressed in mitosis. Yet its mechanism of action has remained elusive. Here we show that Spo13, which is phosphorylated and stabilized at G2/M in a Cdk/Clb-dependent manner, acts at two stages during mitotic cell division. Spo13 provokes a G2/M arrest that is reversible and largely independent of the Mad2 spindle checkpoint. Since mRNAs whose induction requires Cdc14 activation are reduced, we propose that its anaphase delay results from inhibition of Cdc14 function. Indeed, the Spo13-induced anaphase delay correlates with Cdc14 phosphatase retention in the nucleolus and with cyclin B accumulation, which both impede anaphase exit. At the onset of arrest, Spo13 is primarily associated with the nucleolus, where Cdc14 accumulates. Significantly, overexpression of separase (Esp1), which promotes G2/M and anaphase progression, suppresses Spo13 effects in mitosis, arguing that Spo13 acts upstream or parallel to Esp1. Given that Spo13 overexpression reduces Pds1 and cyclin B degradation, our findings are consistent with a role for Spo13 in regulating APC, which controls both G2/M and anaphase. Similar effects of Spo13 during meiotic MI may prevent cell cycle exit and initiation of DNA replication prior to MII, thereby ensuring two successive chromosome segregation events without an intervening S phase.

Translational integrity and continuity: personalized biomedical data integration.

Translational research data are generated in multiple research domains from the bedside to experimental laboratories. These data are typically stored in heterogeneous databases, held by segregated research domains, and described with inconsistent terminologies. Such inconsistency and fragmentation of data significantly impedes the efficiency of tracking and analyzing human-centered records. To address this problem, we have developed a data repository and management system named TraM (http://tram.uchicago.edu), based on a domain ontology integrated entity relationship model. The TraM system has the flexibility to recruit dynamically evolving domain concepts and the ability to support data integration for a broad range of translational research. The web-based application interfaces of TraM allow curators to improve data quality and provide robust and user-friendly cross-domain query functions. In its current stage, TraM relies on a semi-automated mechanism to standardize and restructure source data for data integration and thus does not support real-time data application.

Evidence for an ancient BRCA1 mutation in breast cancer patients of Yoruban ancestry.

BACKGROUND: BRCA1 recurrent mutations have rarely been assessed in non-founder populations. Still, identifying such mutations could be important for designing genetic testing strategies for high-risk breast/ovarian cancer families in non-founder populations. OBJECTIVE: To determine whether the recurrent BRCA1 Y101X mutation identified in Yoruban breast cancer patients represents a single historical mutation event, and determine the prevalence of this mutation in a hospital based cohort. METHODS: 365 breast cancer patients and 177 controls of Yoruban ancestry from Nigeria, unselected for age of onset or family history were screened for the BRCA1 Y101X mutation. The haplotypes on which the Y101X mutation occurred were characterized using microsatellite markers and single-nucleotide polymorphisms (SNPs). Phase ambiguity was resolved using allele-specific PCR. RESULTS: The BRCA1 Y101X mutation was detected in four Yoruban patients with no documented family history of breast cancer among a cohort of 365 (1.1, 95% C.I. = 0.43-2.78%) unrelated Yoruban breast cancer patients. This study reveals the four Y101X mutations occur on a single, rare haplotype. Further characterization in a patient of European ancestry with a strong family history of breast/ovarian cancer revealed the same Y101X mutation on the same haplotype as those in the Yoruban carriers. These observations suggest the Y101X mutations identified in the Yoruban patients may have originated from a single mutation event. CONCLUSIONS: BRCA1 Y101X is the first reported recurrent mutation occurring in patients of African ancestry for which prevalence has been determined. Identification of this mutation in a woman of European ancestry with strong family history of breast/ovarian suggests further that this mutation occurred once, probably many generations ago.

Aberrant RNA splicing and its functional consequences in cancer cells.

Among the myriad of alterations present in cancer cells are an abundance of aberrant mRNA transcripts. Whether abnormal gene transcription is a by-product of cellular transformation or whether it represents an inherent element that contributes to the properties of cancer cells is not yet clear. Here, we present growing evidence that in many cases, aberrant mRNA transcripts contribute to essential phenotypes associated with transformed cells, suggesting that alterations in the splicing machinery are common and functionally important for cancer development. The proteins encoded by these abnormal transcripts are often truncated or missing domains, thereby altering protein function or conferring new functions altogether. Thus, aberrant splicing regulation has genome-wide effects, potentially altering gene expression in many cancer-associated pathways.

Common variants on chromosome 5p12 confer susceptibility to estrogen receptor-positive breast cancer.

We carried out a genome-wide association study of breast cancer predisposition with replication and refinement studies involving 6,145 cases and 33,016 controls and identified two SNPs (rs4415084 and rs10941679) on 5p12 that confer risk, preferentially for estrogen receptor (ER)-positive tumors (OR = 1.27, P = 2.5 x 10(-12) for rs10941679). The nearest gene, MRPS30, was previously implicated in apoptosis, ER-positive tumors and favorable prognosis. A recently reported signal in FGFR2 was also found to associate specifically with ER-positive breast cancer.

Genetic polymorphisms in uridine diphospho-glucuronosyltransferase 1A1 and breast cancer risk in Africans.

The UDP-glucuronosylatransferase 1A1 (UGT1A1) gene is involved in the metabolism of estrogen and detoxification of potential carcinogens. The number of TA repeats in the promoter region of UGT1A1 has been linked to breast cancer risk, but results varied by race. We performed a comprehensive assessment of genetic polymorphisms in the UGT1A1 gene, and examined these polymorphisms and TA repeats in relation to breast cancer risk in a case-control study in Nigeria. 512 breast cancer cases and 226 community controls were genotyped for UGT1A1. Compared with high-activity TA repeat genotypes, the odds ratios (OR) for low-activity and moderate-activity genotypes were 0.47 (95% confidence interval CI, 0.26-0.83) and 0.64 (95% CI, 0.39-1.06), respectively, in premenopausal women (P = 0.009 for trend), but no association was observed in postmenopausal women (P = 0.24). The effect of TA repeats was also differentiated by age: the OR was 0.39 (95% CI 0.21-0.71) for low-activity genotypes and 0.58 (95% CI 0.33-1.00) for moderate-activity genotypes in women <45 years old (P = 0.002 for trend), but no association was observed in women >or=45 years old (P = 0.15). Haplotype analysis showed that UGT1A1 haplotypes were highly diverse with blocked structures. We found a specific haplotype in block 2 that was significantly associated with a 2.1-fold elevated risk (95% CI 1.05-4.39; P = 0.04). In contrast with previous studies, we found low-activity TA repeat alleles were protective against breast cancer among premenopausal indigenous Africans, suggesting that the role of UGT1A1 in breast cancer development may vary by population, presumably due to different environmental and genetic modifier effects.

Estrogen receptor alpha, BRCA1, and FANCF promoter methylation occur in distinct subsets of sporadic breast cancers.

Estrogen receptor alpha (ER) and its ligand estrogen play vital roles in the development, progression and treatment of breast cancer. An increasing number of studies have also provided evidence linking disruption of the Fanconi anemia/BRCA cascade to breast cancer. Our objectives were to examine the methylation status and expression profiles of ER, correlate the findings with BRCA1 and FANCF methylation and map the critical CpGs for ER expression. We found that the CpG islands in the 5' region of the ER gene are methylated in 59 of 120 (49.2%) primary breast cancers, including 45 of 59 ER-negative tumors (76.3%, P < 0.00001). In addition, we observed a strong correlation between ER promoter and BRCA1 promoter methylation (odds ratio 3.12, 95% confidence interval 1.10-9.68, P = 0.02). In contrast, FANCF methylation was rare in breast tumors: one of 120 (0.8%). ER methylation was associated with high tumor grade (60.4% methylated vs. 39.6% unmethylated in grade 3 tumors, P = 0.04) and tumor subtype (P = 0.03). Though small in number, all tumors of the medullary subtype were ER methylated. In contrast, the lobular subtype had the least methylation (23.1% methylated vs. 76.9% unmethylated). After treatment of MDA-MB-231 cells with 5-aza-cytidine (5-aza-dC) and trichostatin, which resulted in re-expression of ER mRNA, we localized dramatic demethylation effects to CpG islands in positions +68, +165, +192, +195, +337, +341 and +405 from transcription start site of the ER promoter. These data suggest that unlike FANCF, both ER and BRCA1 are specifically targeted for methylation in sporadic breast cancers, a phenomenon that should be explored for development of novel diagnostic and therapeutic approaches.