Affiliation of authors: Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Holden Comprehensive Cancer Center, University of Iowa, Iowa City.
Correspondence to: Richard E. Buller, M.D., Ph.D., Department of Obstetrics and Gynecology, University of Iowa Hospitals and Clinics, 200 Hawkins Dr., Iowa City, IA 52242 (e-mail: richard-buller{at}uiowa.edu).
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ABSTRACT |
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INTRODUCTION |
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Testing for BRCA1 mutations in a large cohort is both cumbersome and impractical because of the large size of this gene, which is encoded by nearly 5500 bases located in an approximately 117-kilobase-pair segment of chromosome 17q. Our group (6) demonstrated that the protein truncation test was superior to single-strand conformational polymorphism (SSCP) analysis in screening ovarian cancers for BRCA1 null mutations. The protein truncation test was not only more sensitive than SSCP but also more efficient, using less than one eighth of the number of reactions required for SSCP. Unfortunately, the protein truncation test fails to detect missense mutations, which account for an estimated 15% of the BRCA1 mutations in tumors. However, the functional importance of such mutations is questioned (7).
Denaturing gradient gel electrophoresis and its offshoot, two-dimensional gene scanning, have also been proposed as screening tools (8). This technique is equivalent to DNA-based protein truncation testing of exon 11 but has not been compared with complementary DNA (cDNA)-based protein truncation testing for the other portions of the open reading frame. Both protein truncation testing, as described by our laboratory, and two-dimensional gene scanning take only five polymerase chain reactions (PCRs) per tumor. However, two-dimensional gene scanning, which also detects missense mutation and polymorphisms, generates at least 37 PCR fragments from its use of five multiplex PCRs, which increases the difficulty of analysis (6,8). Two-dimensional gene scanning detects a number of missense mutations of unknown functional importance. Still another investigator (9) has recommended a combined approach, including DNA-based protein truncation testing for exon 11, allele-specific oligonucleotide assay for the eight most common mutations, and DNA-based sequencing of the rest of the gene. This combined approach is still less efficient than a protein truncation test-based approach.
Although not as cumbersome as SSCP or two-dimensional gene scanning with denaturing gradient gel electrophoresis, performing protein truncation tests on all newly diagnosed epithelial ovarian, peritoneal, or fallopian tube cancers is clearly still time consuming. Early in the 1990s, Amos and Struewing (10) demonstrated the importance of a family history of ovarian cancer as a risk factor for ovarian cancer in general. More recently, Moslehi et al. (1) found that family history of ovarian cancer was an important risk factor for BRCA1 mutations in ovarian cancer. Still others (11) have found a high rate of BRCA1 mutations associated with ovarian cancer, in certain populations, regardless of a family history of ovarian cancer. Loss of heterozygosity (LOH), which occurred in all BRCA1 mutations, whether germline or somatic, was a key finding in the BRCA1 sequencing study of ovarian cancers by Berchuck et al. (12) that was also confirmed in our laboratory (6). A family history of ovarian cancer was compared with LOH as the first step in determining on whom further testing should be performed. The purpose of this study was to develop an efficient screening strategy to detect various types of BRCA1 dysfunction that would have a high yield without compromising the efficiency of the process and to determine the frequency of BRCA1 dysfunction in ovarian cancer.
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MATERIALS AND METHODS |
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Fig. 1 shows a schematic of our proposed screening strategy. LOH analysis is the first step narrowing the number of patients on which the protein truncation test must be performed. The protein truncation test then identifies important candidate truncating mutations and candidate tumors that failed to express BRCA1 protein for methylation-specific PCR (MS-PCR) analysis. This strategy will not detect missense mutations.
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BRCA1 intragenic polymorphic loci (D17S855, D17S1322, and D17S1323) were amplified by PCR with radiolabeled primers by use of blood and snap-frozen tumor samples. PCR fragments were analyzed on 8% polyacrylamide sequencing gels that were fixed, dried, and exposed to Scientific Imaging FilmTM (Kodak, Rochester, NY) for 30 minutes to 3 hours to show distinct bands for analysis. Mutations identified by direct sequencing from protein truncation test candidates provided additional LOH information when the polymorphic markers were not informative. To be informative, two alleles had to be visualized in the DNA PCR product from peripheral blood. LOH was recorded if the difference in intensity between two bands in the tumor DNA PCR product was visually greater than or equal to 2 : 1. The concepts of LOH and noninformative alleles are demonstrated in Fig. 2.
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The open reading frame of BRCA1 was divided into five overlapping fragments as described previously (6). Both DNA and cDNA templates (generated from random hexamers) were used. DNA templates were used to generate fragments that encompass only exon 11. DNA cannot be used for exons 210 or 1224 inclusive because of the size of the intervening introns. (In these regions, truncating mutations were detected by appropriately designed in-frame primers for PCR amplification of cDNA.) The technique for RNA isolation and cDNA synthesis for reverse transcriptionPCR starting from snap-frozen tumor samples stored at 140 °C was described previously (14). Failure to amplify a product in the cDNA reactions also provided candidate tumors where epigenetic phenomenon, including promoter silencing, may occur (i.e., if the products were identified for genomic DNA [exon 11] but no products from cDNA [exons 211 and 1124], despite appropriate amplification of a housekeeping gene sequence, such as glycerol 3-phosphate dehydrogenase [G3PD]).
Primers containing both a eukaryotic translation initiation site and a T7 promoter were used to generate PCR products that were amenable to protein truncation testing (6). The individual PCRs were performed with complete translation of the product in the TNT® Quick Coupled Transcription/Translation System (Promega Corp., Madison, WI) essentially as in the manufacturer's protocol. After the addition of sodium dodecyl sulfate (SDS) buffer, the samples were heated to 85 °C for 2 minutes. An aliquot of the sample was then subjected to SDSpolyacrylamide gel electrophoresis until the leading dye completely crossed the gel (45 W for 15 minutes). Gels were then fixed, dried, and exposed to Scientific Imaging Film overnight.
Sequencing
Direct PCR-based sequencing was performed on any sample in which a shift in band pattern was observed on the protein truncation test. DNA products were purified with the Wizard PCR DNA Purification System (Promega Corp.) or the QIAquickTM PCR Purification Kit (Qiagen Inc., Valencia, CA). The PCR sequencing reaction was completed by using the DNA Cyclic Sequencing System (Promega Corp.). Sequencing of the candidate mutations (with the use of Licor IR2; LI-COR, Lincoln, NE) based on protein truncation testing was carried out by selecting an appropriate region to sequence on a 41-cm, 7% Long RangerTM polyacrylamide gel (FMC Bioproducts, Rockland, ME) (6). Specific M13-tagged primers were chosen on the basis of the size of the truncated protein product. Electrophoresis was performed at 50 °C, 31.5 W, and 35 mA for approximately 6 hours. Gels were evaluated by use of LI-COR ImagIR 4.2 data-collection software and image manipulator software (LI-COR). Mutations were confirmed with bidirectional sequencing of products from a second independent PCR. A germline mutation was determined by the presence of the same mutation in peripheral blood DNA that was identified in tumor DNA. In contrast, a somatic mutation was defined by a normal BRCA1 sequence from blood DNA in the region where a mutation was sequenced from tumor.
Methylation-Specific Polymerase Chain Reaction
MS-PCR was performed on NaHSO3-converted DNA from tumors that failed to express BRCA1 messenger RNA (mRNA). The NaHSO3 reaction has been described previously (1517). From 0.5 to 5 µg of DNA was incubated first with 0.3 M NaOH at 37 °C. The alkalinized mixture was exposed to 3.6 M NaHSO3 and 1 mM hydroquinone at 55 °C for 14 hours before recovering and desalting the products with PromegaTM Wizard Prep (Promega Corp.). The desalting was performed as recommended by the manufacturer, except for the last elution in which double-distilled H2O was incubated on the column at room temperature for 5 minutes before the final centrifugation (18 620g for 2 minutes at 4 °C). The eluate was then incubated with 0.3 M NaOH at 37 °C again before the addition of 3 M ammonium acetate and 95% ethanol. The mixture was next incubated at 20 °C for 20 minutes and then centrifuged at 18 620g at 4 °C for 30 minutes. The supernatant was removed, and the DNA was lyophilized and then resuspended in 100 µL of double-distilled H2O.
MS-PCR was then performed on the converted DNA with the primers and conditions described previously (18). The primers covered a portion of the CpG island in the BRCA1 promoter (GenBank accession number U37574) that flanks the transcription start site. The methylated product was 75 base pairs (bp), and the unmethylated product was 86 bp (18). CpGenomeTM Universal Methylated DNA (Intergen Company, Gaithersburg, MD) was used as the methylated control after NaHSO3 conversion. DNA from non-neoplastic ovarian epithelium and human placental tissue after NaHSO3 conversion were used as unmethylated controls. An additional control included all reagents except DNA template.
Pedigree Analysis
Three-generation pedigrees are routinely obtained on patients seen at the Gynecologic Oncology Clinic of the Holden Comprehensive Cancer Center, Iowa City, by individuals with special genetics training. A positive family history of ovarian cancer was arbitrarily designated on the basis of a three-generation maternal/paternal pedigree, with at least one family member with ovarian carcinoma or two family members with breast carcinoma (13).
Statistical Analyses
Statistical analyses, including Fisher's exact test, were performed by use of SPSS for Windows version 10.0 (Statistical Package for Social Sciences, Chicago, IL). All statistical tests were two-sided.
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RESULTS |
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All tumors (n = 121) determined to have LOH or that were noninformative at the BRCA1 locus were analyzed by the protein truncation test. Thirty-three tumors (27.3%) had a truncated BRCA1 protein. All 33 abnormalities identified by the protein truncation test were confirmed by direct sequencing to be secondary to BRCA1 null mutations (18 germline and 15 somatic mutations; Table 1). A single tumor originally classified as of unknown germline status may be a somatic mutation as shown by a 68-year-old woman with no female relatives in a three-generation pedigree who developed breast or ovarian cancer. However, because no germline cDNA was available, her true germline status is not known. Among 66 of 100 tumors diploid at the BRCA1 locus studied by the protein truncation test, no null mutations were found. Therefore, LOH/noninformative was shown to be a strong predictor of mutation status (Fisher's exact test, P<.001).
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DISCUSSION |
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Application of novel molecular therapeutic strategies requires an efficient screening system to detect and characterize BRCA1 mutations. Our group (6) has demonstrated previously that the protein truncation test is a more efficient screening technique. However, even with the protein truncation test, much work and expense are required to identify the cancers containing BRCA1 mutations. Several investigators (1,12) have demonstrated the importance of both a family history of ovarian cancer and BRCA1 LOH to the risk of ovarian cancer. Using a large unselected cohort, we have shown that BRCA1 LOH is a better predictor of BRCA1 mutation than family history of ovarian cancer because of its higher sensitivity and negative predictive value (100% versus 40.6% and 100% versus 87.5%, respectively). Using LOH as the first step to identify which cancers should be analyzed results in a larger sample size (121 tumors with LOH or noninformative tumors versus 53 tumors from patients with a family history of breast/ovarian cancer) to be tested, but fewer mutations are missed (none of 33 mutations missed by LOH versus 20 of 33 mutations missed by a family history of breast/ovarian cancer). Just over one half of the original population would need to have a protein truncation test instead of the whole cancer cohort.
The use of protein truncation as the second step in this screening strategy (Fig. 1) not only eliminates the need for evaluating genomic DNA of all patients but also may identify types of mutations and gene dysfunction that are missed by genomic DNA-based screening. Unusual splicing mutations and promoter-silencing DNA methylation are two examples not detected by SSCP (6).
These important mechanisms of BRCA1 dysfunction demonstrate the limitations of screening a population with no predisposition to a specific disease. Several authors (1,8,9,22) have tried to use Ashkenazi or other ovarian cancer-founder mutations to screen the population in question. Garvin et al. (23) used only a protein truncation test to screen exon 11 in a series of patients with breast/ovarian cancer. In our population, this type of screening does not appear to be adequate because 50% of the mutations detected occurred in the 40% of the open reading frame not encoded by exon 11 and the paucity of Ashkenazi-founder mutations (three of 33) in our heterogeneous population with European roots.
The rate of truncating BRCA1 mutations in the entire cohort of patients with ovarian cancer (33 [14.9%] of 221) is higher than the rate from other nonselected cohorts (3%8%) (2427) but is similar to the rate in a consecutive series of genomically sequenced tumors (12.6%) reported by Berchuck et al. (12). The differences stem from the number of somatic mutations reported. Our number is consistent with the BRCA1 somatic mutation frequency reported by Berchuck et al. (12). Thus, the effectiveness of this protein truncation test screening strategy approaches that of genomic sequencing for the detection of both germline and somatic BRCA1 mutations.
Strong protein truncation test bands resulted from the exon 211 and exon 1124 PCR fragments amplified from cDNA and detected on agarose gels before carrying out protein truncation test-coupled translation to protein products (6). However, in 16 (8.6%) of the 187 tumors screened, no BRCA1 transcription product was obtained. In each case, G3PD mRNA and other mRNAs were readily detectable. By extrapolation, we would predict that BRCA1 mRNA would be absent in 18 (8.1%) of the entire 221 tumor cohort, an observation that likely reflects BRCA1 promoter hypermethylation (18,28). To confirm this observation, we carried out MS-PCR to analyze the BRCA1 promoter for CpG island methylation near the transcription start site. Indeed, hypermethylation was found, which may have resulted in the absence of BRCA1 in all of the 16 cancers with absent BRCA1 mRNA. In contrast to the report by Esteller et al. (18), we have found that BRCA1 promoter hypermethylation may occur in BRCA1 diploid tumors. The extrapolated 8.1% (18 of 221) incidence of promoter hypermethylation in our entire cohort is slightly lower than that reported by Baldwin et al. (28) (13.3%) who also used MS-PCR.
All germline BRCA1 null mutations should be detected with protein truncation tests. The additional dysfunction caused by promoter biallelic methylation could be detected with a single cDNA PCR amplifying the fragment from exons 11 to 24. On the basis of this frequency of BRCA1 dysfunction in unselected cancers, we urge that the development of BRCA1-targeted therapeutics continue.
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NOTES |
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We thank Dr. Frederick E. Domann for his general laboratory assistance in the development of the MS-PCR techniques and Amara Lucke, Marisa A. Dolan, Matthew Buller, and Lisa M. Blake for their technical assistance, Holden Comprehensive Cancer Center, University of Iowa, Iowa City.
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Manuscript received June 6, 2001; revised October 15, 2001; accepted November 8, 2001.
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