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Technical Briefs |
1
Laboratory of Molecular Oncology and Departments of
2
Immunology and
3
Oncology, San Carlos University Hospital, 28040 Madrid, Spain;
a address correspondence to this author at: Laboratorio de Oncología Molecular, Planta Baja Sur, Hospital Clinico San Carlos, c/Martin Lagos s/n, 28040 Madrid, Spain
BRCA1 is a tumor suppressor gene (1) responsible for one-half of familial breast/ovarian cancer syndromes and 40% of breast-only cancer syndromes (2)(3). BRCA1 codes for a 220-kDa nuclear phosphoprotein that has been suggested to play a role in cellular processes, including DNA repair and recombination (4)(5), transcriptional regulation (6)(7), and appropriate chromosomal segregation (8). It is unclear which of BRCA1 functions are important for decreasing breast/ovarian cancer susceptibility.
BRCA1 testing and genetic counseling services are offered to families with histories of breast and/or ovarian cancer (9). The available screening methods to detect germ-line BRCA1 mutations are expensive and time consuming because the gene is large, prevalent BRCA1 mutations are not found (except in ethnic communities), and mutations are scattered throughout the coding sequence (10).
Gene screening often detects a BRCA1 variant that does not imply a frameshift or a splicing alteration but represents a missense mutation not previously reported or registered in the Breast Cancer Informative Core Database (11). To address the question of whether these mutations represent new cancer-predisposing mutations or rare polymorphisms, one must consider characteristics such as absence of the variant in a control group of sufficient size, cosegregation with cancer in some families, and occurrence in a highly conserved protein sequence or in a putative functional domain. These considerations imply the study of large-pedigree families, which very often are not available and the conclusions of which are not always compatible with genetic counseling practice. As an indication of these limitations, only BRCA1 missense mutations that abolish the BRCA1 C-terminal transcriptional activity in a transfection assay (12) or disturb the RING-finger domain structure (13) have been defined as cancer-predisposing mutations (11). The lack of complete understanding of BRCA1 makes it difficult to design a reliable BRCA1 functional test similar to those for other tumor suppressor genes such as p53 (14).
BRCA1 is a classical tumor suppressor gene that follows Knudson's two-hits hypothesis (10). BRCA1 somatic mutations have not been detected in sporadic breast cancer (15) and are very uncommon in sporadic ovarian cancer (16). These findings indicate that selective retention of one BRCA1 allele in a breast or ovarian tumor is a good predictor of the cancer-predisposing role of this allele.
Very often, index cases from families under study in genetic counseling services are women already diagnosed as having breast or ovarian cancer, and fresh or paraffin-embedded tissue samples from their tumors are readily available. In these cases, a loss of heterozygosity (LOH) study at the BRCA1 locus is possible. Traditionally, tumor LOH has been studied with the help of microsatellite markers. Unfortunately, this method does not permit discrimination of whether the unclassified mutant allele has been the one selectively retained in the tumor DNA. Hence, additional sequencing analysis is needed to address this issue.
We have developed an analysis based on PCR-denaturing gradient gel electrophoresis (DGGE) that permits us to demonstrate the selective retention of deleterious BRCA1 alleles in DNA extracted from either fresh or paraffin-embedded breast/ovarian tumor tissues.
The extraction protocol for paraffin-embedded tissue DNA was modified from Sarkar et al. (17). Basically, two 10-mm sections from a block of paraffin-embedded tissue were placed in a 1.5-mL microcentrifuge tube after excess paraffin was removed with a scalpel. To dissolve the paraffin, the sections were immersed in 1 mL of xylene, gently mixed, and incubated for 15 min at room temperature. The tube was then centrifuged for 15 min at 13 800g. The liquid was then removed, and the entire procedure was repeated once. The tissue was rehydrated by repeating the above procedure, first with 1 mL of ethanol and finally with 1 mL of 700 mL/L ethanol. After complete ethanol evaporation, 500 µL of lysis buffer (10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 2.5 mmol/L MgCl2, 4.5 mL/L Tween® 20, 100 mg of proteinase K) was added, followed by incubation at 55 °C for 2 h and then at 48 °C until the tissue was completely degraded (4 days at 48 °C, with 50 mg of proteinase K added the second day). Samples were boiled for 10 min and centrifuged at 10 000g for 10 min. The liquid was carefully removed, and the proteins were extracted with standard phenol/chloroform procedures. The DNA was then precipitated by the addition of a 1:10 volume of 3 mol/L sodium acetate and 2.5 volumes of ethanol (50 µL of sodium acetate and 1375 µL of ethanol to a 500-µL sample).
Peripheral blood lymphocytes and fresh tissue DNA were extracted according to standard protocols.
PCR and DGGE conditions were as follow for all tested BRCA1 exons. A 100-ng sample of control or tumor DNA was amplified in the presence of 0.4 µmol/L each oligonucleotide primer and 1 U of Taq polymerase (Cetus-Perkin-Elmer) in a final volume of 25 µL of the following solution: 200 µmol/L each dNTP (Promega), 1.5 mmol/L MgCl2, 50 mL/L deionized formamide (Sigma), 0.166 mmol/L (NH4)SO4, 67 mmol/L Tris-HCl, pH 8.8, 0.1 mL/L Tween-20. PCR reactions were carried out in a DNA Thermal Cycler PTC 100 (MJ Research). After denaturation at 95 °C for 5 min, 10 cycles at 94 °C for 40 s, 43 °C for 60 s (-0.5 °C per cycle), and 72 °C for 90 s plus 30 cycles at 94 °C for 40 s, 40 °C for 60 s, and 72 °C for 90 s (1 s added per cycle) were performed, followed by a final extension step of 10 min at 72 °C. Each PCR amplification was terminated with a round of heteroduplexing: 98 °C for 10 min, 58 °C for 30 min, and finally 37 °C for 30 min. DGGE analysis was performed in a DGGE System (DGGE-2000; C.B.S. Scientific). A 6-µL aliquot of control or tumor PCR product was mixed with 2 µL of standard dye loading buffer and electrophoresed through a 20-cm 10% acrylamide/bis-acrylamide (37.5:1) gel (20–80% urea-formamide chemical gradient) in 1x Tris-acetate-EDTA (40 mmol/L Tris, 20 mmol/L sodium acetate, 1 mmol/L EDTA, pH 8) for 12 h at 100V and 58 °C. The gel was stained in a solution of ethidium bromide, and the DNA was photographed under ultraviolet light. The DGGE BRCA1 oligonucleotide primer sequences are available upon request.
A typical DGGE analysis of different BRCA1 exons amplified
from both tumor DNA (lane T) and peripheral blood lymphocytes DNA (lane
N) is shown in Fig 1
. Examples of cancer-predisposing BRCA1 germ-line
mutations (11) identified in three families with
breast/ovarian cancer syndrome studied in our hospital are shown in
Fig. 1A
. Four bands corresponding to wild-type and mutant alleles plus
two heteroduplex hybrids are apparent for the 589delCT and 5242 C
A
cases. Two bands corresponding to wild-type and mutant alleles are also
apparent for the 1370insATCT case, whereas heteroduplex hybrids are not
so obvious, probably because of the instability of a 4-bp mismatch. All
three mutant alleles—589delCT, 1370insATCT, and 5242A—focus at a
lower denaturing reagent concentration than their wild-type
counterparts because their melting temperatures have been reduced.
Signals from both alleles are of identical strength in normal tissue
(lane N). In contrast, the mutant allele has been selectively retained
in the tumor DNA (lane T), confirming the tumor suppressor role of
BRCA1 in those tumors and indicating that any other true
deleterious BRCA1 variant will be easily detected with this
procedure.
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Examples of direct applications of this analytical procedure are shown
in Fig. 1B
. The 3238GA mutation is a BRCA1 mutation
recorded in the Breast Cancer Informative Core Database as an
unclassified variant (11). The experiment clearly shows that
both BRCA1 alleles are equally retained in the tumor,
indicating that 3238 GA (which produces a nonconservative S1040N
amino acid change in the BRCA1 protein) is not a deleterious
BRCA1 mutation and must be considered a nonpathogenic rare
polymorphism. This is invaluable information for genetic counseling.
IVS7-34CT is a common BRCA1 polymorphism (11).
When analyzed in a woman harboring a deleterious BRCA1
mutation (the same woman who demonstrates the 5242CA mutation in
Fig. 1A
), it is obvious that allele IVS7-34C has been selectively
retained in the tumor DNA, indicating that both IVS7-34C and 5242A lie
in the same allele. Making the same analysis with other common
BRCA1 polymorphisms, we can easily determine
BRCA1 mutation-associated haplotypes. Tumor DNA was
amplified from paraffin-embedded breast tumor tissues except for the
3238GA variant, which was amplified from fresh breast tumor tissue.
As far as we know, we report here for the first time the use of DGGE to detect selective retention of deleterious BRCA1 alleles in tumor DNA extracted from either paraffin-embedded or fresh tissues. DNA extracted from paraffin-embedded tissue can be a poor PCR template because it very often is severely damaged, and DGGE primers add difficulties to the PCR reaction because of the long 40- to 50-bp GC-clamps used to improve melting profiles (18). Nonetheless, the method reported here has been applied successfully to the analysis of different BRCA1 exons amplified from several distinct paraffin-embedded tissues.
The present method takes advantage of the fact that BRCA1 is a highly polymorphic gene and that nearly all screened individuals are heterozygous for one of the well-known more common polymorphic sequences (19). The method can be an advantageous alternative to microsatellite studies when analyzing BRCA1 LOH, especially for laboratories that use a DGGE method for screening BRCA1 mutations. However, a comparative study with the standard microsatellite-based LOH assays should be performed.
Analysis of selective allele tumor retention performed in common polymorphic BRCA1 sequences and mutant exons can be a powerful method of defining BRCA1 mutation-associated haplotypes. This method can also be applied to the identification of sporadic breast/ovarian tumor development in women already identified as harboring a germ-line BRCA1 mutation. We also believe that this approach can be applied to the detection of tumor-selective retention of deleterious mutant alleles of tumor suppressor genes other than BRCA1.
Acknowledgments
This work was supported by grants from Rhone-Pôulenc-Rorer and Comunidad de Madrid (08.1/0015/98). We thank our patients for participating in this study. We thank Dr. P. Perez Segura, Department of Oncology, San Carlos University Hospital for clinical assistance and T. Monreal, R. Vivancos, J. Godino, and I. García Carbonero, Molecular Oncology Laboratory, San Carlos University Hospital, for technical assistance.
Footnotes
fax 34-1-3303544, e-mail uinvest2{at}hcsc.es
References
The following articles in journals at HighWire Press have cited this article:
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  T Caldes, M de la Hoya, A Tosar, S Sulleiro, J Godino, D Ibanez, M Martin, P Perez-Segura, and E Diaz-Rubio A breast cancer family from Spain with germline mutations in both the BRCA1 and BRCA2 genes J. Med. Genet., August 1, 2002; 39(8): e44 - 44. [Full Text] [PDF]
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