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High-resolution methylation analysis of the BRCA1 promoter in ovarian tumors
Cancer Genetics and Cytogenetics 159 (2005) 114–122
High-resolution methylation analysis of the BRCA1 promoter in ovarian tumors Cathy B. Wilcox, Bora E. Baysal, Holly H. Gallion1, Mary A. Strange, Julie A. DeLoia* Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, Lab 420, 204 Craft Avenue, Pittsburgh, PA 15213 Received 27 August 2004; received in revised form 9 December 2004; accepted 13 December 2004
Abstract
Both hereditary and sporadic ovarian tumors frequently have decreased BRCA1 expression. One mechanism of downregulating BRCA1 expression is hypermethylation of the BRCA1 promoter. Studies have shown that the BRCA1 promoter is aberrantly hypermethylated in a subset of ovarian tumors, although the proportion varies widely between reports. High-resolution analysis of the BRCA1 promoter in ovarian cancer may provide information regarding the extent and heterogeneity of methylation and guide future studies using methylation-specific polymerase chain reaction (MSPCR). We screened 50 primary epithelial ovarian tumors for BRCA1 promoter hypermethylation using MS-PCR. The BRCA1 promoter was hypermethylated in 16% (8 of 50) of the tumors, including two stage IA tumors. Sequence analysis of the promoter revealed that methylation of the CpG island is both extensive and mosaic in the methylated samples. Two CpG dinucleotides in the BRCA1 promoter, within and adjacent to a Myb consensus binding site, were most frequently methylated in ovarian tumors. BRCA1 expression was significantly lower in methylated than in unmethylated samples. Our analysis of the BRCA1 promoter revealed preferential methylation of specific CpG sites in ovarian tumors. This finding could be exploited in the design of highly sensitive MS-PCR assays for direct assessment of tumor DNA and potentially for early detection of ovarian cancer in body fluids. 쑖 2005 Elsevier Inc. All rights reserved.
1. Introduction Ovarian cancer is the leading cause of death from gynecologic malignancies, with a lifetime risk of diagnosis of 1.7% and a mortality rate of 1.0% [1]. Approximately 5% of patients with ovarian cancer have a constitutional mutation in BRCA1, the breast cancer susceptibility gene [2–5], located at Homo sapiens (HSA) 17q21. The vast majority (90%) of ovarian cancer cases are sporadic, with no family history of disease. Somatic mutations in BRCA1 are rarely identified in these sporadic tumors [6–10]; however, decreased expression of BRCA1 in sporadic ovarian and breast tumors has been observed and may contribute to the pathoetiology of disease [11–14]. Aberrant promoter hypermethylation and transcriptional repression of tumor suppressor genes, including those encoding the proteins Rb [15], p16 [16], and E-cadherin [17], 1 Present address: Precision Therapeutics, Inc., 2516 Jane Street, Pittsburgh, PA 15203. * Corresponding author. Tel.: (412) 641-6070; fax: (412) 641-6156. E-mail address: [email protected] (J.A. DeLoia).
0165-4608/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2004.12.017
have been identified in many types of cancer [18,19]. The BRCA1 promoter contains a very large CpG island, a region with a high concentration of CpG dinucleotides, that is normally unmethylated but is hypermethylated in 10–31% of ovarian tumors [20–24]. Various methods have been used to analyze promoter methylation, including methylation-specific restriction digestion and methylation-specific polymerase chain reaction (MS-PCR) [25,26]. Both of these methods, although rapid, are prone to false positive results due to incomplete digestion and nonspecific primer binding, respectively. In addition, these methods provide information for only a small fraction of the potential methylation sites. A more detailed and reliable analysis of methylation is achieved by sequencing multiple clones of a promoter region following bisulfite conversion. Bisulfite conversion is a chemical reaction that converts unmethylated cytosine to uracil, while leaving methylated cytosine unaltered. Sequencing a promoter region after bisulfite treatment thus permits unambiguous detection of methylation status of every CpG site in the cloned region. Analyzing multiple clones provides a good estimation of the amount of methylation heterogeneity within a sample.
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Using this method, several groups have evaluated BRCA1 promoter methylation in breast cancer cell lines and tumors [7,27–30]. The results from these studies indicate that, in breast cancer tumors, aberrant BRCA1 methylation can be extensive, with nearly all CpG sites methylated in multiple clones from the same sample. The pattern of methylation can also be heterogeneous, with methylation patterns varying significantly among multiple clones from the same sample. A consistent observation is that tumors in which the BRCA1 promoter was extensively hypermethylated throughout the sample had low BRCA1 expression. Ovarian cancer has a very high mortality rate, in large part due to the lack of early detection methods. The majority of ovarian cancer cases are identified only after significant disease dissemination. The detection of aberrant promoter methylation in a panel of genes has been proposed as a screening tool for the early detection of cancer cells in readily available body fluids such as blood and urine. In a recently published feasibility study, tumor-specific hypermethylation of a panel of six tumor suppressor promoters, including BRCA1, was detected in the serum of ovarian cancer patients with 100% specificity and 82% sensitivity [31]. The availability of detailed information about promoter methylation patterns and changes in genes that occur with disease progression would permit the design of methylationspecific primers that are both highly specific and highly sensitive. These MS-PCR primers would have immediate application for the early detection of ovarian cancer, which could significantly improve patient survival. Although two groups have analyzed BRCA1 promoter methylation in ovarian tumors using bisulfite genomic sequencing, only a limited portion of the promoter region (326 bases containing 16 CpG sites) was sequenced. In addition, neither group reported details of the methylation patterns [29,32]. The purpose of this study was to carefully define the extent and pattern of methylation of the BRCA1 promoter in ovarian tumor samples in order to determine (a) the frequency of cytosine methylation at each CpG site within 660 bp of the proximal promoter and (b) whether hypermethylation of BRCA1 occurs in early stage cancer and is, therefore, a candidate for early detection screening. Secondary objectives were to determine whether BRCA1 methylation is predictive of decreased BRCA1 expression and whether the primers currently used to detect methylation by MSPCR are at optimal targets. 2. Materials and methods 2.1. Tissue specimens Tumor samples were obtained from Magee-Womens Hospital Tissue Procurement Program (Pittsburgh, PA) and the Gynecologic Oncology Group (GOG) Internal Tissue Bank. The tissue was rinsed of blood, snap-frozen after surgery, and stored at ⫺80⬚C. A hematoxylin and eosin slide was prepared for each sample to ensure that the vast majority
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(⬎85%) of the tissue was tumor. All surgical specimens were reviewed by pathologists to confirm their ovarian histology. This protocol was approved by the MageeWomens Hospital Institutional Review Board. 2.2. Methylation-specific PCR and sequencing Genomic DNA was extracted using the Puregene DNA extraction kit (Gentra, Minneapolis, MN) as per the manufacturer’s instructions. The DNA concentration was determined using a Hoefer (San Francisco, CA) TKO 100 fluorometer. DNA (1.2 µg) from each sample was treated with sodium bisulfite and purified using the CpGenome kit (Chemicon, Temecula, CA) as per the manufacturer’s instructions. Polymerase chain reaction of the β-globin gene was performed before and after bisulfite treatment, to determine the efficiency of bisulfite conversion, using the following primers: 5′-GAAGAGCCAAGGACAGGTAC-3′ and 5′-CAACTTCATCCACGTTCAC-3′. These primers bind positions ⫺195 through ⫺176 and 54 through 73, using the A in the initiation codon as position 1 [33]. The primers amplify native DNA, but do not amplify bisulfite converted DNA. Any sample that had a positive β-globin PCR band after bisulfite treatment was retreated. The MS-PCR primers used in this study were designed so that the reverse primer is the initiating primer, in a region where the native DNA is rich in cytosines, which are converted to uracil by the bisulfite treatment. It thus discriminates between bisulfite-converted and native DNA and serves as an additional control for the fidelity of the bisulfite conversion. The forward primers were designed to discriminate between methylated and unmethylated DNA. Methylated forward primer: TTG GGT GGT TAA TTT AGA GTT TC; methylated reverse primer: CCG TCC AAA AAA TCT CAA CGA A. The PCR conditions consisted of an initial denaturing step of 94⬚C for 6 minutes, followed by 35 cycles of 94⬚C for 45 seconds, 61⬚C for 30 seconds, and 72⬚C for 30 seconds, ending with a 7-minute final extension at 72⬚C. PCR was also performed using primers specific for unmethylated DNA. Unmethylated forward primer: TTG GTT TTT GTG GTA ATG GAA AAG TGT; unmethylated reverse primer: CAA AAA ATC TCA ACA AAC TCA CAC CA. The PCR conditions were the same except that the annealing temperature was 67⬚C. For sequencing, bisulfite-converted DNA was amplified using two rounds of PCR with the following nested primers (5′ to 3′). Reaction 1, forward primer: GTT GGA TGG GAA TTG TAG TTT TT; reverse primer: CAA ATA CCC CAA AAC ATC ACT TAA A. The product from the first round reaction was used at a final dilution of 1:10 in a second PCR reaction, with primers as published by Rice and Futscher [34]. Forward primer: AAC TCT ACT ACC TTT ACC CAA AA; reverse primer: GTT TAT AAT TGT TGA TAA GTA TAA G. The final PCR products were cloned using the TA-Cloning kit (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from Escherichia coli using QIAprep Spin Miniprep kits (Qiagen, Valencia, CA) and the
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inserts were sequenced at the University of Pittsburgh Genetics Core. 2.3. Total RNA isolation and cDNA synthesis Total RNA from frozen tissue sections was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol, followed by treatment with DNaseI (Invitrogen) to digest any endogenous DNA and then heat-inactivated. cDNA was synthesized from 2 µg of total RNA using the Superscript III First Strand synthesis system (Invitrogen) as per the manufacturer’s protocol. 2.4. Real-time quantitative PCR Real-time PCR was performed using the ABI 7700 sequence detector (Applied Biosystems, Foster City, CA). BRCA-1 primers and probe overlap the exon 23/24 splice junction, avoiding amplification of genomic DNA, as previously described [35]. Forward primer: 5′-CAGAGGACAATGGCTTCCATG-3′; reverse primer: 5′-CTACACTGTCCAACACCCACT CTC-3′; Taqman probe: (6-FAM) 5′-AATTGGGCAGATGTGTGAGGCACCTG-3′. The BRCA1 gene and the endogenous reference gene (GAPDH) were amplified in a singleplex 50-µL reaction with cDNA equivalent to 100 ng of total RNA. GAPDH primers and probe (H599999905) were designed by Applied Biosystems as an Assay-on-Demand kit. A typical 50-µL reaction sample also contained 25 µL of Taqman universal PCR master mix (1× Taqman buffer, 200 µmol/L dNTPs, 5 mmol/L MgCl2,
1.25 U AmpliTaq Gold, and 0.5 U of Amperase uracilN-glycosylase (UNG) along with the Taqman primers and minor groove binder probes. Thermal cycling conditions were 2 minutes at 50⬚C and 10 minutes at 95⬚C followed by 40 cycles at 95⬚C for 30 seconds and 60⬚C for 1 minute. Relative quantification of gene expression was performed using the comparative CT (cycle–threshold) method, which consists of the normalization of the number of target gene copies (BRCA1) to an endogenous reference gene (GAPDH). Samples for which the GAPDH CT was greater than 28 were excluded. A calibrator was used to determine relative expression. The reported BRCA1 expression of each sample is the average of two replicates. 2.5. Statistical analyses The Mann–Whitney U-test and Fisher’s exact test were performed using the Simple Interactive Statistical Analysis Web-based program and a P value ⭐ 0.05 was considered significant.
3. Results 3.1. Hypermethylation of BRCA1 promoter detected in early- and late-stage ovarian tumors by MS-PCR The methylation-specific PCR, using primers we designed (Fig. 1), demonstrated BRCA1 promoter methylation in 16% (8 of 50) of ovarian tumors. A sample of MS-PCR
Fig. 1. The proximal 660 bases of the BRCA1 promoter. CpG dinucleotides are underlined and in upper case letters. The boxed a indicates the transcription start site (⫹1). The arrows indicate the positions of primers used in this and other publications: primer [1] was used in the present study; primer [2], used by Strathdee et al. [20]; primer [3], Esteller et al. [22]; primer [4], Baldwin et al. [21]. A CREB site that is important for basal transcription and is regulated by methylation is shown at ⫺173 [29,46,47]. A putative Myb binding site, which is methylation-sensitive, is at ⫺29 [42,48]. The four most frequently methylated CpGs are in gray boxes. The three least frequently methylated CpGs are in gray circles with black outline.
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results is shown in Fig. 2. The methylated samples included 3 of 28 serous, 1 of 9 endometrioid, 1 of 3 mucinous, 1 of 3 clear cell, and 2 of 7 adenocarcinomas of mixed or undifferentiated histology. The methylation frequencies within the different histological subtypes were not statistically significantly different (Table 1). In addition, there was no correlation of BRCA1 promoter methylation with increasing stage or grade (Table 1). Of the 15 stage I tumors analyzed, 3 (20%) were MS-PCR positive. DNA samples from 42 of 50 ovarian tumors showed no methylation at the BRCA1 promoter by MS-PCR.
3.2. BRCA1 promoter methylation pattern is heterogeneous The results from the MS-PCR screening were used to select methylated and unmethylated samples for further analysis by bisulfite genomic sequencing. We sequenced 660 bp of the bisulfite-converted proximal promoter, containing 31 CpG sites, from all 8 MS-PCR-positive (⫹) and 14 MSPCR-negative (⫺) tumor samples. Five to 13 clones were sequenced per sample, with 179 clones sequenced in toto. We sequenced 84 clones from the 8 MS-PCR⫹ tumors. As anticipated, the MS-PCR⫹ samples had a consistently higher level of methylation than the MS-PCR⫺ samples (Figs. 3 and 4). All of the MS-PCR⫹ samples (8 of 8), yielded at least one clone that was extensively methylated;
Fig. 2. Screening of the methylation state of DNA in primary ovarian tumors. The upper and lower panels show ethidium bromide-stained PCR products that were generated using primers specific to methylated bisulfitetreated genomic DNA or unmethylated bisulfite-treated genomic DNA. The sample order is the same in each panel. Upper panel: The presence of a 202-bp band indicates methylated DNA. The forward and reverse primers are specific for methylated cytosines at ⫺134 and ⫹27 to ⫹44, respectively (see Fig. 1). Lower panel: The presence of an 86-bp product indicates unmethylated DNA. The forward and reverse primers are specific for unmethylated CpG sites at ⫺37 to ⫺19 and ⫹16 to ⫹27, respectively. Unmethylated bisulfite-treated (U) DNA from normal placenta, methylated bisulfitetreated (M) DNA (CpGenome Kit control), untreated genomic DNA (No BS), and a no DNA (water) control were included in every MS-PCR assay.
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Table 1 Clinicopathologic comparison of ovarian tumors with or without hypermethylation of the BRCA1 promoter Characteristics Histology Papillary serous Endometrioid Mucinous Clear cell Adenocarcinoma, othera FIGO stage I IA IC II IIA IIB IIC III IV Grade 1 2 3 All tumors
Tumors, no.
Proportion BRCA1 methylated (%)
P
28 9 3 3 7
3/28 1/9 1/3 1/3 2/7
(11) (11) (33) (33) (29)
0.288 0.555 0.937 0.937 0.929 0.824
1 2 2 25 5
3/15 (20) 2 1 1/5 (20) 0 1 0 4/25 (16) 0/5 (0)
13 12 25 50
2/13 1/12 5/25 8/50
12 3
(15) (8) (20) (16)
0.824
0.649 0.401 0.659 0.373 0.351
a Adenocarcinoma, other: four tumors of mixed histology, two undifferentiated and one not otherwise specified (NOS). Differences were considered statistically significant when P ⭐ 0.05 according to Fisher’s exact test.
that is, methylated at 25 or more of the 31 CpG sites. Complete methylation at all 31 CpG sites was observed in 18 of 84 (21%) clones analyzed. These clones were from five of the eight MS-PCR⫹ tumor samples. In nearly half of the clones (40 of 84; 48%), 24 or more CpG sites were methylated, while 23 of 84 (27%) clones from these samples were completely unmethylated. Four examples of MS-PCR⫹ tumor samples are shown in Fig. 4. One of these, sample 480, was noteworthy in the scarcity of methylation among clones analyzed; it was heavily methylated in only 1 of 10 (10%) clones. To verify that we were not missing methylated samples in our MS-PCR screen, we sequenced 95 clones from 14 MS-PCR⫺ tumors. As expected, those clones had a very low level of methylation in the promoter region. The number of CpG sites that were methylated in a single clone from an unmethylated tumor sample ranged from 0 to 3 of the 31 CpG sites. The majority of clones (59 of 95; 62%) were completely unmethylated in the region sequenced. Two typical examples of MS-PCR⫺ tumor samples nonetheless showing very low levels of methylation are shown in Fig. 4 on the left (no. 18 and no. 203). 3.3. CpG sites at ⫺37 and ⫺29 are most frequently methylated in CpG island Evaluation of the sequence data derived from both MSPCR⫹ and MS-PCR⫺ tumors suggest that the two CpG sites most distal to the transcription start site (⫺567, ⫺565) are
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the most frequently methylated (bottom of Fig. 3). These sites are methylated in 69% and 80% of clones sequenced that contained any methylation, including many MS-PCR⫺ (i.e., screened as unmethylated) clones, and most likely marks the boundary of the BRCA1 CpG island (see Discussion). The next most frequently methylated CpG sites (⫺37, ⫺29) are located adjacent to and within a putative v-Myb binding site (Fig. 1). The CpG at ⫺37 is also notable because it is the only site where the two cytosines preceding the guanine (CCG, ⫺38 and ⫺37) were methylated in many clones (Figs. 3 and 4). Eight other guanines within the CpG island are preceded by multiple cytosines, but only at ⫺38 and ⫺37 are multiple sequential cytosines methylated. This implies
that methylation of two cytosines preceding the guanine at position ⫺36 is a nonrandom event. 3.4. BRCA1 expression correlates with methylation status The relative expression of BRCA1 mRNA was determined for 37 of 50 samples using real-time reverse transcriptase PCR. The expression of BRCA1 was evaluated according to histological type, grade, and FIGO stage. There were no significant differences using the Mann–Whitney U-test. BRCA1 expression was also compared between MS-PCR⫹ and MS-PCR⫺ samples. Expression ranged from 0.2 to 57.1 relative expression units (REUs) in the MS-PCR⫺ samples. By contrast, the relative expression in MS-PCR⫹ samples
Fig. 3. High-resolution analysis of the methylation state of DNA in primary ovarian tumors. Bisulfite genomic sequencing detected the methylation status of 31 CpG sites within the proximal promoter. CpG sites are indicated across the top of the figure. The ⫹1 position is the transcription start site. The arrows at the top right indicate the primers used to detect methylation during the MS-PCR screening. Each row of circles represents a summary of the percent methylation at each CpG site for a tumor sample. Percent methylation was determined by adding up the number of times the cytosine at a specific CpG site was methylated in all of the clones from an individual tumor and dividing by the total number of clones analyzed for that tumor, which is indicated in the column at the right. Results from the MS-PCR screen are indicated in the column at the far right. The stage of the tumor sample and sample number are in the first two columns. At the bottom of the figure, are listed the frequency of methylation (%) at each CpG site for all clones (Total), MSPCR⫹ and MS-PCR⫺. Clones containing no methylation were excluded from these frequency calculations. The four most frequently and three least frequently methylated CpGs are indicated by shaded boxes and ovals, respectively, in the Total frequency row. The numbering of bases in Fig. 1 is retained for comparison.
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Fig. 4. Methylation patterns of individual clones of six representative tumor samples. Each row of circles represents the methylation pattern for an individual clone of 660 bp of the promoter from that tumor. The samples are arranged in pairs from Stage I to III. Those with lower levels of methylation are shown on the left. The individual clones from each ovarian tumor sample were used to calculate the percent methylation at each site. Those summaries are shown in Fig. 3.
ranged from 0.7 to 4.5 REUs. The average relative BRCA1 expression for the MS-PCR⫹ samples was 2.45 ⫾ 1.32. This was statistically significantly lower than the average of 18.15 ⫾ 14.24 for the MS-PCR⫺ samples (P ⬍ 0.0005).
4. Discussion This is the first extensive analysis of the methylation state of the BRCA1 promoter in sporadic ovarian cancer. Promoter methylation is an important mechanism of gene silencing in cancer cells. Since most aberrant hypermethylation occurs at CpG islands in promoter regions, laboratory analysis of these regions offers a means to ascertain quickly any epigenetic modifications. DNA from tumors is often found in patients’ sera, and aberrantly methylated DNA has been detected in the sera of ovarian, breast, cervical, prostate, and numerous other cancer patients [31,36–41]. Previous studies have reported BRCA1 methylation frequencies of between 10 and 31% using MS-PCR [20–24]. In this study, we found that 16% of all sporadic ovarian
tumors were positive by MS-PCR. Verification of this percentage was achieved by sequencing the promoter region following bisulfite conversion. The assay was sensitive in that we could detect as little as 10% methylation of the target DNA within a tumor sample with the MS-PCR assay. Our primary objective was to determine the frequency of cytosine methylation at each CpG site within 660 bp of the proximal promoter. Such information is critical in designing high-fidelity MS-PCR assays. Uniform methylation of all 31 CpG sites in the proximal promoter appears to be uncommon, as it occurred in only 21% (18 of 84) of the hypermethylated clones analyzed. Even when aberrant methylation was detected, certain CpG sites commonly remained unmethylated; these should therefore be avoided when designing MS-PCR primers. In every MS-PCR⫹ ovarian tumor sample, methylation of 24 CpG sites (24 of 31) was consistently detected. Three of these 24 sites, at positions ⫺565, ⫺37, and ⫺29, were methylated in a respective 84, 85, and 84% of methylated clones from MS-PCR⫹ samples. The cytosines at ⫺37 and ⫺29 were also methylated, at a low level, in several
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clones from MS-PCR samples (Figs. 1 and 4; no. 18, 203, 222). This region appears to mark a cluster of methylation, perhaps a region of methylation initiation. We hypothesize that the MS-PCR⫺ samples that had these sites methylated would, over time, show methylation throughout their promoters. This concept is supported by the observation that none of the clones from our control normal placental DNA ever showed methylation in the BRCA1 promoter. These sites, at ⫺37 and ⫺29, are also of particular interest in regard to their positions, which are within and adjacent to a methylation-sensitive v-Myb consensus binding site [42]. Thus, the v-Myb transcription factor may be a methylationsensitive regulator of BRCA1 expression in ovarian surface epithelial cells. Unique methylation of these same two CpG sites (⫺37 and ⫺29) was strongly correlated with very low BRCA1 expression in a breast cancer sample as well [27]. Our data support the hypothesis that the region from ⫺38 to ⫺29 may be a nucleation site for methylation with consequent elimination of v-Myb binding and a reduction in BRCA1 expression. The other commonly methylated site in MS-PCR⫺ samples was at ⫺565. This site was methylated in 80% of clones containing any methylation from MS-PCR⫹ and MS-PCR⫺ tumors. Previous studies have shown that it is also highly methylated in both normal cells (peripheral blood lymphocytes) and cancer cells (breast cancer cell lines), indicating that these CpGs are usually methylated and may, therefore, reside outside the BRCA1 CpG island [34]. Another question we addressed was the timing of detectable methylation in the BRCA1 promoter. We were able to detect extensive hypermethylation of the BRCA1 promoter in both early and late stage tumors, including stage IA tumors, indicating that, in fact, BRCA1 promoter methylation does occur early in ovarian carcinogenesis. This observation confirms a recent report [24] and strengthens the case for developing assays for the detection of aberrant tumor suppressor promoter hypermethylation of genes such as BRCA1, as a tool for early diagnosis of ovarian cancer [24,43,44]. We also postulate that early BRCA1 gene silencing through methylation could be a mechanism whereby sporadic ovarian tumors develop a BRCA1-associated gene expression profile in the absence of BRCA1 mutations [45]. Although these tumors do not harbor BRCA1 mutations, hypermethylation of the BRCA1 promoter throughout these tumors would lead to decreased BRCA1 expression and subsequent dysregulation of downstream effectors, according to current models [18]. In support of this notion is our finding that all tumors with BRCA1 promoter methylation had very low BRCA1 gene expression. Sequencing data from 179 clones from both MS-PCR⫹ and MS-PCR⫺ tumors was evaluated to determine the fidelity of primers currently used to detect methylation by MS-PCR and to locate putative optimal targets for primer binding. For our initial screen, we detected methylation using MS-PCR primers that bind the CpG sites at positions ⫺134 (forward) and ⫹27 and ⫹44 (reverse). We sequenced nearly half of
the samples screened by MS-PCR (44%, 22 of 50). None of the MS-PCR⫺ samples were methylated at ⫺134, ⫹27 or ⫹44. Furthermore, 100% of the eight MS-PCR⫹ tumor samples contained DNA methylated at two of those CpG sites and 75% (6 of 8) contained DNA methylated at all three sites. For MS-PCR⫹ samples 2 and 3, which were otherwise heavily methylated, we detected no methylation at position ⫺134 in any of the 23 clones sequenced. The reason that these samples were MS-PCR⫹ is not clear; either the primers were not entirely specific or else the assay was able to pick up a minor percentage of DNA that did have methylation at this site, but was not among the clones sequenced. The forward primer most frequently used in other studies binds the CpG at position ⫺19 [22]. This site was methylated in 75% of the MS-PCR⫹ samples analyzed in this current study. Together, these data point to the utility of a deeper analysis of promoter sites for methylation-specific PCR studies. In conclusion, our sequence analysis reveals that BRCA1 promoter methylation can be extensive and can occur as an early event. We were able to detect methylation by MS-PCR when ⬍10% of the target DNA was methylated. Although the precise mechanism of DNA methylation initiation and spread during tumorigenesis is unknown, our findings suggest that certain CpGs may be preferentially methylated in the BRCA1 promoter in ovarian tumors. Thus, these CpGs may define attractive targets to design rapid and highly sensitive MS-PCR assays for early detection of ovarian cancer via analysis of the aberrant methylation of BRCA1 as part of an assay of multiple tumor suppressor promoters.
Acknowledgments We would like to thank Jacqueline Jones for technical support and Brian Wilcox for careful reading of the manuscript and helpful discussion. Financial support for this work was provided by the Scaife Family Foundation.
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[23] Rathi A, Virmani AK, Schorge JO, Elias KJ, Maruyama R, Minna JD, Mok SC, Girard L, Fishman DA, Gazdar AF. Methylation profiles of sporadic ovarian tumors and nonmalignant ovaries from high-risk women. Clin Cancer Res 2002;8:3324–31. [24] Wang C, Horiuchi A, Imai T, Ohira S, Itoh K, Nikaido T, Katsuyama Y, Konishi I. Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene. J Pathol 2004;202:215–23. [25] Dobrovic A, Simpfendorfer D. Methylation of the BRCA1 gene in sporadic breast cancer. Cancer Res 1997;57:3347–50. [26] Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996;93:9821–6. [27] Rice JC, Ozcelik H, Maxeiner P, Andrulis I, Futscher BW. Methylation of the BRCA1 promoter is associated with decreased BRCA1 mRNA levels in clinical breast cancer specimens. Carcinogenesis 2000; 21:1761–5. [28] Rice JC, Massey-Brown KS, Futscher BW. Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells. Oncogene 1998;17:1807–12. [29] Mancini DN, Rodenhiser DI, Ainsworth PJ, O’Malley FP, Singh SM, Xing W, Archer TK. CpG methylation within the 5′ regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site. Oncogene 1998;16:1161–9. [30] Miyamoto K, Fukutomi T, Asada K, Wakazono K, Tsuda H, Asahara T, Sugimura T, Ushijima T. Promoter hypermethylation and posttranscriptional mechanisms for reduced BRCA1 immunoreactivity in sporadic human breast cancers. Jpn J Clin Oncol 2002;32:79–84. [31] Ibanez de Caceres I, Battagli C, Esteller M, Herman JG, Dulaimi E, Edelson MI, Bergman C, Ehya H, Eisenberg BL, Cairns P. Tumor cell-specific BRCA1 and RASSF1A hypermethylation in serum, plasma, and peritoneal fluid from ovarian cancer patients. Cancer Res 2004;64:6476–81. [32] Chan KY, Ozcelik H, Cheung AN, Ngan HY, Khoo US. Epigenetic factors controlling the BRCA1 and BRCA2 genes in sporadic ovarian cancer. Cancer Res 2002;62:4151–6. [33] Bauer HM, Ting Y, Greer CE, Chambers JC, Tashiro CJ, Chimera J, Reingold A, Manos MM. Genital human papillomavirus infection in female university students as determined by a PCR-based method. JAMA 1991;265:472–7. [34] Rice JC, Futscher BW. Transcriptional repression of BRCA1 by aberrant cytosine methylation, histone hypoacetylation and chromatin condensation of the BRCA1 promoter. Nucleic Acids Res 2000;28:3233–9. [35] Favy DA, Lafarge S, Rio P, Vissac C, Bignon YJ, Bernard-Gallon D. Real-time PCR quantification of full-length and exon 11 spliced BRCA1 transcripts in human breast cancer cell lines. Biochem Biophys Res Commun 2000;274:73–8. [36] Ziegler A, Zangemeister-Wittke U, Stahel RA. Circulating DNA: a new diagnostic gold mine? Cancer Treat Rev 2002;28:255–71. [37] Muller HM, Widschwendter A, Fiegl H, Ivarsson L, Goebel G, Perkmann E, Marth C, Widschwendter M. DNA methylation in serum of breast cancer patients: an independent prognostic marker. Cancer Res 2003;63:7641–5. [38] Hu XC, Wong IH, Chow LW. Tumor-derived aberrant methylation in plasma of invasive ductal breast cancer patients: clinical implications. Oncol Rep 2003;10:1811–5. [39] Widschwendter A, Muller HM, Fiegl H, Ivarsson L, Wiedemair A, Muller-Holzner E, Goebel G, Marth C, Widschwendter M. DNA methylation in serum and tumors of cervical cancer patients. Clin Cancer Res 2004;10:565–71. [40] Kang GH, Lee S, Lee HJ, Hwang KS. Aberrant CpG island hypermethylation of multiple genes in prostate cancer and prostatic intraepithelial neoplasia. J Pathol 2004;202:233–40. [41] Goessl C, Krause H, Muller M, Heicappell R, Schrader M, Sachsinger J, Miller K. Fluorescent methylation-specific polymerase chain reaction for DNA-based detection of prostate cancer in bodily fluids. Cancer Res 2000;60:5941–5.
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High-resolution methylation analysis of the BRCA1 promoter in ovarian tumors Cathy B. Wilcox, Bora E. Baysal, Holly H. Gallion1, Mary A. Strange, Julie A. DeLoia* Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, Lab 420, 204 Craft Avenue, Pittsburgh, PA 15213 Received 27 August 2004; received in revised form 9 December 2004; accepted 13 December 2004
Abstract
Both hereditary and sporadic ovarian tumors frequently have decreased BRCA1 expression. One mechanism of downregulating BRCA1 expression is hypermethylation of the BRCA1 promoter. Studies have shown that the BRCA1 promoter is aberrantly hypermethylated in a subset of ovarian tumors, although the proportion varies widely between reports. High-resolution analysis of the BRCA1 promoter in ovarian cancer may provide information regarding the extent and heterogeneity of methylation and guide future studies using methylation-specific polymerase chain reaction (MSPCR). We screened 50 primary epithelial ovarian tumors for BRCA1 promoter hypermethylation using MS-PCR. The BRCA1 promoter was hypermethylated in 16% (8 of 50) of the tumors, including two stage IA tumors. Sequence analysis of the promoter revealed that methylation of the CpG island is both extensive and mosaic in the methylated samples. Two CpG dinucleotides in the BRCA1 promoter, within and adjacent to a Myb consensus binding site, were most frequently methylated in ovarian tumors. BRCA1 expression was significantly lower in methylated than in unmethylated samples. Our analysis of the BRCA1 promoter revealed preferential methylation of specific CpG sites in ovarian tumors. This finding could be exploited in the design of highly sensitive MS-PCR assays for direct assessment of tumor DNA and potentially for early detection of ovarian cancer in body fluids. 쑖 2005 Elsevier Inc. All rights reserved.
1. Introduction Ovarian cancer is the leading cause of death from gynecologic malignancies, with a lifetime risk of diagnosis of 1.7% and a mortality rate of 1.0% [1]. Approximately 5% of patients with ovarian cancer have a constitutional mutation in BRCA1, the breast cancer susceptibility gene [2–5], located at Homo sapiens (HSA) 17q21. The vast majority (90%) of ovarian cancer cases are sporadic, with no family history of disease. Somatic mutations in BRCA1 are rarely identified in these sporadic tumors [6–10]; however, decreased expression of BRCA1 in sporadic ovarian and breast tumors has been observed and may contribute to the pathoetiology of disease [11–14]. Aberrant promoter hypermethylation and transcriptional repression of tumor suppressor genes, including those encoding the proteins Rb [15], p16 [16], and E-cadherin [17], 1 Present address: Precision Therapeutics, Inc., 2516 Jane Street, Pittsburgh, PA 15203. * Corresponding author. Tel.: (412) 641-6070; fax: (412) 641-6156. E-mail address: [email protected] (J.A. DeLoia).
0165-4608/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2004.12.017
have been identified in many types of cancer [18,19]. The BRCA1 promoter contains a very large CpG island, a region with a high concentration of CpG dinucleotides, that is normally unmethylated but is hypermethylated in 10–31% of ovarian tumors [20–24]. Various methods have been used to analyze promoter methylation, including methylation-specific restriction digestion and methylation-specific polymerase chain reaction (MS-PCR) [25,26]. Both of these methods, although rapid, are prone to false positive results due to incomplete digestion and nonspecific primer binding, respectively. In addition, these methods provide information for only a small fraction of the potential methylation sites. A more detailed and reliable analysis of methylation is achieved by sequencing multiple clones of a promoter region following bisulfite conversion. Bisulfite conversion is a chemical reaction that converts unmethylated cytosine to uracil, while leaving methylated cytosine unaltered. Sequencing a promoter region after bisulfite treatment thus permits unambiguous detection of methylation status of every CpG site in the cloned region. Analyzing multiple clones provides a good estimation of the amount of methylation heterogeneity within a sample.
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Using this method, several groups have evaluated BRCA1 promoter methylation in breast cancer cell lines and tumors [7,27–30]. The results from these studies indicate that, in breast cancer tumors, aberrant BRCA1 methylation can be extensive, with nearly all CpG sites methylated in multiple clones from the same sample. The pattern of methylation can also be heterogeneous, with methylation patterns varying significantly among multiple clones from the same sample. A consistent observation is that tumors in which the BRCA1 promoter was extensively hypermethylated throughout the sample had low BRCA1 expression. Ovarian cancer has a very high mortality rate, in large part due to the lack of early detection methods. The majority of ovarian cancer cases are identified only after significant disease dissemination. The detection of aberrant promoter methylation in a panel of genes has been proposed as a screening tool for the early detection of cancer cells in readily available body fluids such as blood and urine. In a recently published feasibility study, tumor-specific hypermethylation of a panel of six tumor suppressor promoters, including BRCA1, was detected in the serum of ovarian cancer patients with 100% specificity and 82% sensitivity [31]. The availability of detailed information about promoter methylation patterns and changes in genes that occur with disease progression would permit the design of methylationspecific primers that are both highly specific and highly sensitive. These MS-PCR primers would have immediate application for the early detection of ovarian cancer, which could significantly improve patient survival. Although two groups have analyzed BRCA1 promoter methylation in ovarian tumors using bisulfite genomic sequencing, only a limited portion of the promoter region (326 bases containing 16 CpG sites) was sequenced. In addition, neither group reported details of the methylation patterns [29,32]. The purpose of this study was to carefully define the extent and pattern of methylation of the BRCA1 promoter in ovarian tumor samples in order to determine (a) the frequency of cytosine methylation at each CpG site within 660 bp of the proximal promoter and (b) whether hypermethylation of BRCA1 occurs in early stage cancer and is, therefore, a candidate for early detection screening. Secondary objectives were to determine whether BRCA1 methylation is predictive of decreased BRCA1 expression and whether the primers currently used to detect methylation by MSPCR are at optimal targets. 2. Materials and methods 2.1. Tissue specimens Tumor samples were obtained from Magee-Womens Hospital Tissue Procurement Program (Pittsburgh, PA) and the Gynecologic Oncology Group (GOG) Internal Tissue Bank. The tissue was rinsed of blood, snap-frozen after surgery, and stored at ⫺80⬚C. A hematoxylin and eosin slide was prepared for each sample to ensure that the vast majority
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(⬎85%) of the tissue was tumor. All surgical specimens were reviewed by pathologists to confirm their ovarian histology. This protocol was approved by the MageeWomens Hospital Institutional Review Board. 2.2. Methylation-specific PCR and sequencing Genomic DNA was extracted using the Puregene DNA extraction kit (Gentra, Minneapolis, MN) as per the manufacturer’s instructions. The DNA concentration was determined using a Hoefer (San Francisco, CA) TKO 100 fluorometer. DNA (1.2 µg) from each sample was treated with sodium bisulfite and purified using the CpGenome kit (Chemicon, Temecula, CA) as per the manufacturer’s instructions. Polymerase chain reaction of the β-globin gene was performed before and after bisulfite treatment, to determine the efficiency of bisulfite conversion, using the following primers: 5′-GAAGAGCCAAGGACAGGTAC-3′ and 5′-CAACTTCATCCACGTTCAC-3′. These primers bind positions ⫺195 through ⫺176 and 54 through 73, using the A in the initiation codon as position 1 [33]. The primers amplify native DNA, but do not amplify bisulfite converted DNA. Any sample that had a positive β-globin PCR band after bisulfite treatment was retreated. The MS-PCR primers used in this study were designed so that the reverse primer is the initiating primer, in a region where the native DNA is rich in cytosines, which are converted to uracil by the bisulfite treatment. It thus discriminates between bisulfite-converted and native DNA and serves as an additional control for the fidelity of the bisulfite conversion. The forward primers were designed to discriminate between methylated and unmethylated DNA. Methylated forward primer: TTG GGT GGT TAA TTT AGA GTT TC; methylated reverse primer: CCG TCC AAA AAA TCT CAA CGA A. The PCR conditions consisted of an initial denaturing step of 94⬚C for 6 minutes, followed by 35 cycles of 94⬚C for 45 seconds, 61⬚C for 30 seconds, and 72⬚C for 30 seconds, ending with a 7-minute final extension at 72⬚C. PCR was also performed using primers specific for unmethylated DNA. Unmethylated forward primer: TTG GTT TTT GTG GTA ATG GAA AAG TGT; unmethylated reverse primer: CAA AAA ATC TCA ACA AAC TCA CAC CA. The PCR conditions were the same except that the annealing temperature was 67⬚C. For sequencing, bisulfite-converted DNA was amplified using two rounds of PCR with the following nested primers (5′ to 3′). Reaction 1, forward primer: GTT GGA TGG GAA TTG TAG TTT TT; reverse primer: CAA ATA CCC CAA AAC ATC ACT TAA A. The product from the first round reaction was used at a final dilution of 1:10 in a second PCR reaction, with primers as published by Rice and Futscher [34]. Forward primer: AAC TCT ACT ACC TTT ACC CAA AA; reverse primer: GTT TAT AAT TGT TGA TAA GTA TAA G. The final PCR products were cloned using the TA-Cloning kit (Invitrogen, Carlsbad, CA). Plasmid DNA was purified from Escherichia coli using QIAprep Spin Miniprep kits (Qiagen, Valencia, CA) and the
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inserts were sequenced at the University of Pittsburgh Genetics Core. 2.3. Total RNA isolation and cDNA synthesis Total RNA from frozen tissue sections was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol, followed by treatment with DNaseI (Invitrogen) to digest any endogenous DNA and then heat-inactivated. cDNA was synthesized from 2 µg of total RNA using the Superscript III First Strand synthesis system (Invitrogen) as per the manufacturer’s protocol. 2.4. Real-time quantitative PCR Real-time PCR was performed using the ABI 7700 sequence detector (Applied Biosystems, Foster City, CA). BRCA-1 primers and probe overlap the exon 23/24 splice junction, avoiding amplification of genomic DNA, as previously described [35]. Forward primer: 5′-CAGAGGACAATGGCTTCCATG-3′; reverse primer: 5′-CTACACTGTCCAACACCCACT CTC-3′; Taqman probe: (6-FAM) 5′-AATTGGGCAGATGTGTGAGGCACCTG-3′. The BRCA1 gene and the endogenous reference gene (GAPDH) were amplified in a singleplex 50-µL reaction with cDNA equivalent to 100 ng of total RNA. GAPDH primers and probe (H599999905) were designed by Applied Biosystems as an Assay-on-Demand kit. A typical 50-µL reaction sample also contained 25 µL of Taqman universal PCR master mix (1× Taqman buffer, 200 µmol/L dNTPs, 5 mmol/L MgCl2,
1.25 U AmpliTaq Gold, and 0.5 U of Amperase uracilN-glycosylase (UNG) along with the Taqman primers and minor groove binder probes. Thermal cycling conditions were 2 minutes at 50⬚C and 10 minutes at 95⬚C followed by 40 cycles at 95⬚C for 30 seconds and 60⬚C for 1 minute. Relative quantification of gene expression was performed using the comparative CT (cycle–threshold) method, which consists of the normalization of the number of target gene copies (BRCA1) to an endogenous reference gene (GAPDH). Samples for which the GAPDH CT was greater than 28 were excluded. A calibrator was used to determine relative expression. The reported BRCA1 expression of each sample is the average of two replicates. 2.5. Statistical analyses The Mann–Whitney U-test and Fisher’s exact test were performed using the Simple Interactive Statistical Analysis Web-based program and a P value ⭐ 0.05 was considered significant.
3. Results 3.1. Hypermethylation of BRCA1 promoter detected in early- and late-stage ovarian tumors by MS-PCR The methylation-specific PCR, using primers we designed (Fig. 1), demonstrated BRCA1 promoter methylation in 16% (8 of 50) of ovarian tumors. A sample of MS-PCR
Fig. 1. The proximal 660 bases of the BRCA1 promoter. CpG dinucleotides are underlined and in upper case letters. The boxed a indicates the transcription start site (⫹1). The arrows indicate the positions of primers used in this and other publications: primer [1] was used in the present study; primer [2], used by Strathdee et al. [20]; primer [3], Esteller et al. [22]; primer [4], Baldwin et al. [21]. A CREB site that is important for basal transcription and is regulated by methylation is shown at ⫺173 [29,46,47]. A putative Myb binding site, which is methylation-sensitive, is at ⫺29 [42,48]. The four most frequently methylated CpGs are in gray boxes. The three least frequently methylated CpGs are in gray circles with black outline.
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results is shown in Fig. 2. The methylated samples included 3 of 28 serous, 1 of 9 endometrioid, 1 of 3 mucinous, 1 of 3 clear cell, and 2 of 7 adenocarcinomas of mixed or undifferentiated histology. The methylation frequencies within the different histological subtypes were not statistically significantly different (Table 1). In addition, there was no correlation of BRCA1 promoter methylation with increasing stage or grade (Table 1). Of the 15 stage I tumors analyzed, 3 (20%) were MS-PCR positive. DNA samples from 42 of 50 ovarian tumors showed no methylation at the BRCA1 promoter by MS-PCR.
3.2. BRCA1 promoter methylation pattern is heterogeneous The results from the MS-PCR screening were used to select methylated and unmethylated samples for further analysis by bisulfite genomic sequencing. We sequenced 660 bp of the bisulfite-converted proximal promoter, containing 31 CpG sites, from all 8 MS-PCR-positive (⫹) and 14 MSPCR-negative (⫺) tumor samples. Five to 13 clones were sequenced per sample, with 179 clones sequenced in toto. We sequenced 84 clones from the 8 MS-PCR⫹ tumors. As anticipated, the MS-PCR⫹ samples had a consistently higher level of methylation than the MS-PCR⫺ samples (Figs. 3 and 4). All of the MS-PCR⫹ samples (8 of 8), yielded at least one clone that was extensively methylated;
Fig. 2. Screening of the methylation state of DNA in primary ovarian tumors. The upper and lower panels show ethidium bromide-stained PCR products that were generated using primers specific to methylated bisulfitetreated genomic DNA or unmethylated bisulfite-treated genomic DNA. The sample order is the same in each panel. Upper panel: The presence of a 202-bp band indicates methylated DNA. The forward and reverse primers are specific for methylated cytosines at ⫺134 and ⫹27 to ⫹44, respectively (see Fig. 1). Lower panel: The presence of an 86-bp product indicates unmethylated DNA. The forward and reverse primers are specific for unmethylated CpG sites at ⫺37 to ⫺19 and ⫹16 to ⫹27, respectively. Unmethylated bisulfite-treated (U) DNA from normal placenta, methylated bisulfitetreated (M) DNA (CpGenome Kit control), untreated genomic DNA (No BS), and a no DNA (water) control were included in every MS-PCR assay.
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Table 1 Clinicopathologic comparison of ovarian tumors with or without hypermethylation of the BRCA1 promoter Characteristics Histology Papillary serous Endometrioid Mucinous Clear cell Adenocarcinoma, othera FIGO stage I IA IC II IIA IIB IIC III IV Grade 1 2 3 All tumors
Tumors, no.
Proportion BRCA1 methylated (%)
P
28 9 3 3 7
3/28 1/9 1/3 1/3 2/7
(11) (11) (33) (33) (29)
0.288 0.555 0.937 0.937 0.929 0.824
1 2 2 25 5
3/15 (20) 2 1 1/5 (20) 0 1 0 4/25 (16) 0/5 (0)
13 12 25 50
2/13 1/12 5/25 8/50
12 3
(15) (8) (20) (16)
0.824
0.649 0.401 0.659 0.373 0.351
a Adenocarcinoma, other: four tumors of mixed histology, two undifferentiated and one not otherwise specified (NOS). Differences were considered statistically significant when P ⭐ 0.05 according to Fisher’s exact test.
that is, methylated at 25 or more of the 31 CpG sites. Complete methylation at all 31 CpG sites was observed in 18 of 84 (21%) clones analyzed. These clones were from five of the eight MS-PCR⫹ tumor samples. In nearly half of the clones (40 of 84; 48%), 24 or more CpG sites were methylated, while 23 of 84 (27%) clones from these samples were completely unmethylated. Four examples of MS-PCR⫹ tumor samples are shown in Fig. 4. One of these, sample 480, was noteworthy in the scarcity of methylation among clones analyzed; it was heavily methylated in only 1 of 10 (10%) clones. To verify that we were not missing methylated samples in our MS-PCR screen, we sequenced 95 clones from 14 MS-PCR⫺ tumors. As expected, those clones had a very low level of methylation in the promoter region. The number of CpG sites that were methylated in a single clone from an unmethylated tumor sample ranged from 0 to 3 of the 31 CpG sites. The majority of clones (59 of 95; 62%) were completely unmethylated in the region sequenced. Two typical examples of MS-PCR⫺ tumor samples nonetheless showing very low levels of methylation are shown in Fig. 4 on the left (no. 18 and no. 203). 3.3. CpG sites at ⫺37 and ⫺29 are most frequently methylated in CpG island Evaluation of the sequence data derived from both MSPCR⫹ and MS-PCR⫺ tumors suggest that the two CpG sites most distal to the transcription start site (⫺567, ⫺565) are
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the most frequently methylated (bottom of Fig. 3). These sites are methylated in 69% and 80% of clones sequenced that contained any methylation, including many MS-PCR⫺ (i.e., screened as unmethylated) clones, and most likely marks the boundary of the BRCA1 CpG island (see Discussion). The next most frequently methylated CpG sites (⫺37, ⫺29) are located adjacent to and within a putative v-Myb binding site (Fig. 1). The CpG at ⫺37 is also notable because it is the only site where the two cytosines preceding the guanine (CCG, ⫺38 and ⫺37) were methylated in many clones (Figs. 3 and 4). Eight other guanines within the CpG island are preceded by multiple cytosines, but only at ⫺38 and ⫺37 are multiple sequential cytosines methylated. This implies
that methylation of two cytosines preceding the guanine at position ⫺36 is a nonrandom event. 3.4. BRCA1 expression correlates with methylation status The relative expression of BRCA1 mRNA was determined for 37 of 50 samples using real-time reverse transcriptase PCR. The expression of BRCA1 was evaluated according to histological type, grade, and FIGO stage. There were no significant differences using the Mann–Whitney U-test. BRCA1 expression was also compared between MS-PCR⫹ and MS-PCR⫺ samples. Expression ranged from 0.2 to 57.1 relative expression units (REUs) in the MS-PCR⫺ samples. By contrast, the relative expression in MS-PCR⫹ samples
Fig. 3. High-resolution analysis of the methylation state of DNA in primary ovarian tumors. Bisulfite genomic sequencing detected the methylation status of 31 CpG sites within the proximal promoter. CpG sites are indicated across the top of the figure. The ⫹1 position is the transcription start site. The arrows at the top right indicate the primers used to detect methylation during the MS-PCR screening. Each row of circles represents a summary of the percent methylation at each CpG site for a tumor sample. Percent methylation was determined by adding up the number of times the cytosine at a specific CpG site was methylated in all of the clones from an individual tumor and dividing by the total number of clones analyzed for that tumor, which is indicated in the column at the right. Results from the MS-PCR screen are indicated in the column at the far right. The stage of the tumor sample and sample number are in the first two columns. At the bottom of the figure, are listed the frequency of methylation (%) at each CpG site for all clones (Total), MSPCR⫹ and MS-PCR⫺. Clones containing no methylation were excluded from these frequency calculations. The four most frequently and three least frequently methylated CpGs are indicated by shaded boxes and ovals, respectively, in the Total frequency row. The numbering of bases in Fig. 1 is retained for comparison.
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Fig. 4. Methylation patterns of individual clones of six representative tumor samples. Each row of circles represents the methylation pattern for an individual clone of 660 bp of the promoter from that tumor. The samples are arranged in pairs from Stage I to III. Those with lower levels of methylation are shown on the left. The individual clones from each ovarian tumor sample were used to calculate the percent methylation at each site. Those summaries are shown in Fig. 3.
ranged from 0.7 to 4.5 REUs. The average relative BRCA1 expression for the MS-PCR⫹ samples was 2.45 ⫾ 1.32. This was statistically significantly lower than the average of 18.15 ⫾ 14.24 for the MS-PCR⫺ samples (P ⬍ 0.0005).
4. Discussion This is the first extensive analysis of the methylation state of the BRCA1 promoter in sporadic ovarian cancer. Promoter methylation is an important mechanism of gene silencing in cancer cells. Since most aberrant hypermethylation occurs at CpG islands in promoter regions, laboratory analysis of these regions offers a means to ascertain quickly any epigenetic modifications. DNA from tumors is often found in patients’ sera, and aberrantly methylated DNA has been detected in the sera of ovarian, breast, cervical, prostate, and numerous other cancer patients [31,36–41]. Previous studies have reported BRCA1 methylation frequencies of between 10 and 31% using MS-PCR [20–24]. In this study, we found that 16% of all sporadic ovarian
tumors were positive by MS-PCR. Verification of this percentage was achieved by sequencing the promoter region following bisulfite conversion. The assay was sensitive in that we could detect as little as 10% methylation of the target DNA within a tumor sample with the MS-PCR assay. Our primary objective was to determine the frequency of cytosine methylation at each CpG site within 660 bp of the proximal promoter. Such information is critical in designing high-fidelity MS-PCR assays. Uniform methylation of all 31 CpG sites in the proximal promoter appears to be uncommon, as it occurred in only 21% (18 of 84) of the hypermethylated clones analyzed. Even when aberrant methylation was detected, certain CpG sites commonly remained unmethylated; these should therefore be avoided when designing MS-PCR primers. In every MS-PCR⫹ ovarian tumor sample, methylation of 24 CpG sites (24 of 31) was consistently detected. Three of these 24 sites, at positions ⫺565, ⫺37, and ⫺29, were methylated in a respective 84, 85, and 84% of methylated clones from MS-PCR⫹ samples. The cytosines at ⫺37 and ⫺29 were also methylated, at a low level, in several
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clones from MS-PCR samples (Figs. 1 and 4; no. 18, 203, 222). This region appears to mark a cluster of methylation, perhaps a region of methylation initiation. We hypothesize that the MS-PCR⫺ samples that had these sites methylated would, over time, show methylation throughout their promoters. This concept is supported by the observation that none of the clones from our control normal placental DNA ever showed methylation in the BRCA1 promoter. These sites, at ⫺37 and ⫺29, are also of particular interest in regard to their positions, which are within and adjacent to a methylation-sensitive v-Myb consensus binding site [42]. Thus, the v-Myb transcription factor may be a methylationsensitive regulator of BRCA1 expression in ovarian surface epithelial cells. Unique methylation of these same two CpG sites (⫺37 and ⫺29) was strongly correlated with very low BRCA1 expression in a breast cancer sample as well [27]. Our data support the hypothesis that the region from ⫺38 to ⫺29 may be a nucleation site for methylation with consequent elimination of v-Myb binding and a reduction in BRCA1 expression. The other commonly methylated site in MS-PCR⫺ samples was at ⫺565. This site was methylated in 80% of clones containing any methylation from MS-PCR⫹ and MS-PCR⫺ tumors. Previous studies have shown that it is also highly methylated in both normal cells (peripheral blood lymphocytes) and cancer cells (breast cancer cell lines), indicating that these CpGs are usually methylated and may, therefore, reside outside the BRCA1 CpG island [34]. Another question we addressed was the timing of detectable methylation in the BRCA1 promoter. We were able to detect extensive hypermethylation of the BRCA1 promoter in both early and late stage tumors, including stage IA tumors, indicating that, in fact, BRCA1 promoter methylation does occur early in ovarian carcinogenesis. This observation confirms a recent report [24] and strengthens the case for developing assays for the detection of aberrant tumor suppressor promoter hypermethylation of genes such as BRCA1, as a tool for early diagnosis of ovarian cancer [24,43,44]. We also postulate that early BRCA1 gene silencing through methylation could be a mechanism whereby sporadic ovarian tumors develop a BRCA1-associated gene expression profile in the absence of BRCA1 mutations [45]. Although these tumors do not harbor BRCA1 mutations, hypermethylation of the BRCA1 promoter throughout these tumors would lead to decreased BRCA1 expression and subsequent dysregulation of downstream effectors, according to current models [18]. In support of this notion is our finding that all tumors with BRCA1 promoter methylation had very low BRCA1 gene expression. Sequencing data from 179 clones from both MS-PCR⫹ and MS-PCR⫺ tumors was evaluated to determine the fidelity of primers currently used to detect methylation by MS-PCR and to locate putative optimal targets for primer binding. For our initial screen, we detected methylation using MS-PCR primers that bind the CpG sites at positions ⫺134 (forward) and ⫹27 and ⫹44 (reverse). We sequenced nearly half of
the samples screened by MS-PCR (44%, 22 of 50). None of the MS-PCR⫺ samples were methylated at ⫺134, ⫹27 or ⫹44. Furthermore, 100% of the eight MS-PCR⫹ tumor samples contained DNA methylated at two of those CpG sites and 75% (6 of 8) contained DNA methylated at all three sites. For MS-PCR⫹ samples 2 and 3, which were otherwise heavily methylated, we detected no methylation at position ⫺134 in any of the 23 clones sequenced. The reason that these samples were MS-PCR⫹ is not clear; either the primers were not entirely specific or else the assay was able to pick up a minor percentage of DNA that did have methylation at this site, but was not among the clones sequenced. The forward primer most frequently used in other studies binds the CpG at position ⫺19 [22]. This site was methylated in 75% of the MS-PCR⫹ samples analyzed in this current study. Together, these data point to the utility of a deeper analysis of promoter sites for methylation-specific PCR studies. In conclusion, our sequence analysis reveals that BRCA1 promoter methylation can be extensive and can occur as an early event. We were able to detect methylation by MS-PCR when ⬍10% of the target DNA was methylated. Although the precise mechanism of DNA methylation initiation and spread during tumorigenesis is unknown, our findings suggest that certain CpGs may be preferentially methylated in the BRCA1 promoter in ovarian tumors. Thus, these CpGs may define attractive targets to design rapid and highly sensitive MS-PCR assays for early detection of ovarian cancer via analysis of the aberrant methylation of BRCA1 as part of an assay of multiple tumor suppressor promoters.
Acknowledgments We would like to thank Jacqueline Jones for technical support and Brian Wilcox for careful reading of the manuscript and helpful discussion. Financial support for this work was provided by the Scaife Family Foundation.
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