Allelic imbalance on chromosomes 13 and 17 and mutation analysis of BRCA1 and BRCA2 genes in monozygotic twins concordant for breast cancer
Asta Försti1,
Liping Luo,
Igor Vorechovsky,
Magnus Söderberg2,
Paul Lichtenstein3 and
Kari Hemminki
Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge,
2 Department of Pathology, Huddinge Hospital, 14186 Huddinge and
3 Institute of Environmental Medicine, Karolinska Institute, Box 210, 17177 Stockholm, Sweden
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Abstract
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To study genetic changes associated with the development of breast cancer and the extent of its hereditary predisposition, paraffin-embedded tissue samples were obtained from monozygotic twin pairs concordant for breast cancer through the linked Swedish Twin and Cancer Registries. DNA samples extracted from the matched tumour and normal tissues of nine twin pairs were analysed for allelic imbalance using a series of microsatellite markers on chromosomes 13 and 17, containing loci with known tumour suppressor genes. Multiple losses of constitutional heterozygosity (LOH), consistent with a loss of large genomic region, the whole chromosome or chromosome arm, was found in at least three pairs of twins. One double mitotic crossover was identified in one tumour sample in a pair concordant for LOH at multiple loci on both chromosomes. Recombination breakpoints were mapped to regions delineated by D13S218 and D13S263, and D13S155 and D13S279, respectively. In general, no genetic effect of losing the same allele within a twin pair was found. However, for one marker at chromosome 13 (D13S328, between the BRCA2 and the Rb-1 loci) and two markers on chromosome 17 (D17S786, distal to the p53 locus, and D17S855, an intragenic BRCA1 marker) the proportion of twin pairs with the same LOH was significantly higher than expected. These regions may reflect hereditary genomic changes in our sample set. In addition, tumour DNA samples from a subset of 12 twin pairs were analysed for BRCA1 and BRCA2 mutations using exon-by-exon single-strand conformation polymorphism analysis. Two unclassified BRCA2 variants, with a putative pathogenic effect, were identified, but no pathogenic alterations were found in the BRCA1 gene.
Abbreviations: LOH, loss of constitutional heterozygosity; SSCP, single-strand conformation polymorphism.
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Introduction
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Cytogenetic and molecular genetic analysis of breast cancer samples suggests that tumour development results from the accumulation of genetic alterations, including activation of oncogenes and inactivation of tumour suppressor genes (1). According to the paradigm first observed in retinoblastoma (2), one normal allele of a tumour suppressor gene is inactivated by a mutation or a deletion and, in the process of cancer development, it is converted to a homozygous form by a second independent alteration involving chromosomal nondisjunction and deletion, mitotic recombination or gene conversion (3). The conversion of a cancer cell to homozygosity at or around the critical locus (loss of heterozygosity, LOH) has been common to all the tumour suppressor genes identified. Several loci of frequent LOH have been reported for breast cancer, with most frequent LOHs at chromosomal arms 1p, 1q, 3p, 6q, 11p, 11q, 13q, 16q, 17p, 17q and 22q (1).
Family history is a well established and important risk factor for breast cancer. It has been estimated that 510% of all breast cancers are due to hereditary predisposition (4). Two genes, BRCA1, located on chromosome 17q21 (5,6), and BRCA2, located on 13q12-13 (79), have been shown to predispose to familial breast cancer. Breast cancer has been linked to BRCA1 in an estimated 52% of high-risk breast cancer families collected by the Breast Cancer Linkage Consortium and to BRCA2 in 32% of families (10). Mutations in BRCA1 and BRCA2 are rare in sporadic breast tumours (1114). Yet, allelic imbalance and LOH on several regions of chromosomes 13 and 17, including the BRCA1 and BRCA2 loci, have been observed frequently in sporadic breast tumours (1520). Because a decrease in BRCA1 expression has been observed in sporadic breast cancer (21), other mechanisms, such as methylation of the CpG island within the promoter of BRCA1 and BRCA2, have been discussed (2225). However, methylation alone does not explain the high frequency of LOH at the BRCA1 and BRCA2 regions. This suggests that other tumour suppressor genes on chromosomes 13 and 17 are important for sporadic tumour development.
In this paper we present a new approach to study genetic changes associated with the development of breast cancer and the extent of its hereditary predisposition: analysis of LOH in monozygotic twins with breast cancer. Our previous analysis of the Swedish Twin Register showed clear evidence for a heritable component in the breast cancer (26). For monozygotic twins, the relative risk of developing breast cancer was fourfold in the old cohort (born in 18861925) and ninefold in the young cohort (born in 19261958), about twice as high as the risks in dizygotic twins. If LOH occurs in a genomic region containing a tumour suppressor gene that confers a strong predisposition to breast cancer, then LOH would be expected to be replicated in the concordant co-twin, and the same allele should be predominantly lost at the heterozygous loci in tumour DNA of both twins because monozygotic twins have identical haplotypes. These conditions are rarely met, even if LOH is a common event. The monozygotic twin model thus has a high statistical power to suggest candidate regions of potential tumour suppressor genes, even with a limited number of twin pairs. Although bilateral breast cancers could be used in a similar way as monozygotic twins, they do not signal heritable risks to the same extent as monozygotic twins (27).
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Materials and methods
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Sample collection
Monozygotic twins concordant for breast cancer were identified from the Swedish Twin Registry linked to the Swedish Cancer Registry (26). The cases were diagnosed between 1959 and 1992 in twins born in the period 18861958. Twenty-two monozygotic twin pairs were concordant for breast cancer. Paraffin-embedded tissue samples were collected from the hospitals of diagnosis. One randomly chosen sample of tumour DNA from 12 concordant twin pairs was analysed for BRCA1 and BRCA2 mutations. Tumour tissue samples and at least one normal tissue sample per twin pair were available from nine monozygotic twin pairs. These pairs were used for the LOH analysis. Age at diagnosis of these patients ranged from 32 to 76 years (mean 58 years). Six patients were diagnosed when aged <50 years. The mean age of diagnosis of breast cancer in Sweden is close to 62 years. Both the LOH and the mutational analysis could be carried out from five twin pairs.
DNA isolation
DNA was isolated from the paraffin-embedded tissue samples as described earlier (28). Paraffin material was cut in 10 µm sections, mounted on a glass slide and inspected by a pathologist. Areas of slides containing tumour tissue and those containing normal tissue were microdissected. DNA was isolated by suspending the paraffin chips in 100 µl of digestion buffer (50 mM KCl, 1.5 mM MgCl2, 10 mM TrisHCl pH 9.0, 0.5% Tween 20 and 200 mg/ml proteinase K). The mixture was incubated at 55°C for 648 h and proteinase K was inactivated by heating at 99°C for 10 min. Debris was sedimented at 12 000 r.p.m. for 5 min. For mutation analysis, DNA was purified by phenol extraction and ethanol precipitation. Aliquots (0.5 µl) of the supernatant were used for PCR amplification.
LOH analysis
LOH was analysed in the breast cancer samples using 16 microsatellite markers in the long arm of chromosome 13 and 14 markers distributed over the whole chromosome 17 (Figure 1
). The primer sequences, order of the markers and genetic distances (in cM) between the microsatellite loci were obtained from the Genome Database (http://www.gdb.org, at Johns Hopkins University) and the Généthon human genetic linkage map (29). Primers containing a fluorescent Cy5 dye were obtained from Amersham Pharmacia Biotech. Unlabelled primers were from Ransom Hill Bioscience, Inc. PCR was performed using a PerkinElmer thermal cycler, model 480. The reaction volume was 5 µl, including 1x PCR buffer (MBI Fermentas, Lithuania), 12 mM MgCl2 (MBI Fermentas, Lithuania), 0.110.25 mM dNTPs, 0.21.0 µM primers and, with some primers, 510% dimethylsulfoxide (DMSO) or glycerol. A `hot start' procedure was used in which 0.25 U recombinant Taq polymerase (MBI Fermentas, Lithuania) was added at 80°C after an initial denaturation step at 96°C for 5 min. PCR was carried out for 2739 cycles, typically at 94°C for 45 s, at 55°C for 45 s with a final elongation step at 72°C for 10 min. Amplified products were denatured at 95°C for 3 min in loading dye (95% formamide containing blue dextran and EDTA; Amersham Pharmacia Biotech). The resulting products were separated on a 6% polyacrylamide7.2 M urea gel and visualized and analysed with an automated fluorescent ALF Express sequencer (Amersham Pharmacia Biotech). The results were analysed with Pharmacia DNA Fragment Manager 1.2 (Amersham Pharmacia Biotech). For an informative marker, LOH was defined by a decrease in signal intensity of either allele of
50%.

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Fig. 1. Diagram of chromosomes 13 (a) and 17 (b) showing the approximate positions of the markers used in this study based on the genetic order. Cytogenetic locations of the markers are indicated.
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Mutation analysis
Mutation analysis was carried out using PCR single-strand conformation polymorphism (SSCP) as previously described (30,31). The oligonucleotide primer pairs for exon-by-exon amplification of the coding region of the BRCA2 gene were obtained from Dr M.Stratton (Institute for Cancer Research, Surrey, UK). The oligonucleotide primer pairs for PCR SSCP analysis of BRCA1 were as previously described (32).
Statistical analysis
The probability of hereditary effect in LOHs between twin pairs was tested by assuming binomial distribution of allelic loss. The expected probability of the twin pairs losing the same allele by chance was calculated assuming as the null hypothesis H0: P(A
B
C) = P(A) x P(B) x 0.5, where A is the situation where one of the twins has LOH, B that the other twin has LOH, C that they have lost the same allele and P is probability. For each chromosome, chromosomal arm and microsatellite locus, the number of twin pairs with LOH in the same allele was divided by the total number of informative pairs. The expected numbers were derived from LOH analysis of all the microsatellite markers tested in chromosomes 13, 17p and 17q. This is a conservative comparison because each marker was included among the expected numbers. The test was considered significant when the observed probability at a locus exceeded the upper 95% confidence interval (95% CI) of the expected probability.
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Results
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LOH analysis
LOH was analysed on chromosomes 13 and 17 in nine monozygotic twin pairs with breast cancer. Examples of the electropherograms of the samples showing LOH are shown in Figure 2
. Altogether, 64% and 52% of the informative samples showed LOH on chromosomes 13 and 17, respectively. Multiple losses of constitutional heterozygosity, consistent with loss of a large genomic region, the whole chromosome or chromosome arm, were found in at least three pairs (Figures 3 and 4
); in two of them (12453/8184 and 552/2317) the pairs were discordant for LOH at most analysed marker loci on both chromosomes. The remaining pair (300/7083) was concordant for LOH on chromosome 13 and at most analysed marker loci on chromosome 17.

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Fig. 2. Examples of the analysis of LOH using the automated fluorescent ALF Express sequencer. Comparison of tumour (T) and normal (N) tissues within a monozygotic twin pair concordant for breast cancer. The arrows point to the lost allele.
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Fig. 3. Schematic representation of LOH on chromosome 13 in nine twin pairs with breast cancer. Patient identification numbers are indicated above each column. Twin pairs are separated from each other by a black line. The markers analysed are shown on the left. Approximate locations of the known tumour suppressor genes are also indicated. Samples that were not amplified are shown by white boxes.
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Fig. 4. Schematic representation of LOH on chromosome 17 in nine twin pairs with breast cancer. Patient identification numbers are indicated above each column. Twin pairs are separated from each other by a black line. The markers analysed are shown on the left. Approximate locations of the known tumour suppressor genes are also indicated. Samples that were not amplified are shown by white boxes.
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In chromosome 13, none of the twin pairs had lost the same allele in the BRCA2 or Rb-1 region (Figure 3
). We identified one twin pair (300/7083) in which the twins had lost a different allele at five markers around the BRCA2 locus. The same pair had lost the same allele at three markers centromeric to the Rb-1 locus, suggesting that LOH in either pair was due to a mitotic recombination event in two loci. The crossovers were mapped between D13S218 and D13S263 (~5 cM), and D13S155 and D13S279 (~7 cM). Since the parental samples were not available, haplotypes could not be constructed. Twin 300 had infiltrative breast cancer, whereas the other twin, 7083, was diagnosed as medium/highly differentiated lobular breast cancer. Telomeric to the Rb-1 locus, the same pair had lost a different allele at two markers (D13S279 and D13S159) located 28 cM from each other. Frequent LOHs in these regions were observed in the other twin pairs as well.
In chromosome 17p, none of the twin pairs showed LOH at the p53 locus, and only two out of 11 informative samples showed LOH. A region more frequently involved in LOH was located distal to the p53 locus, where three pairs had lost the same allele at marker D17S786 and one pair at marker D17S926 (Figure 4
). In three of the pairs LOH was restricted to the telomeric region distal to the p53 locus. In two pairs (3599/1930 and 3379/1766) the p53 marker TP53 had retained heterozygosity and in one pair (300/7083) no p53 mutations were found when tested by SSCP (P.Berggren, personal communication). The fourth pair (106/7910) was homozygous for the p53 marker and we cannot exclude LOH at the p53 gene.
In chromosome 17q, two twin pairs had lost the same allele at the BRCA1 locus (Figure 4
). Two additional regions of LOH on chromosome 17q were also found. Centromeric to the BRCA1 locus, at marker D17S250, two twin pairs had lost a different allele. Distal to the BRCA1 locus, at marker D17S254, one twin pair had lost the same allele and another pair had lost a different allele.
Statistical analysis
Statistical analysis of the probability for the same allelic loss within a twin pair was carried out by assuming binomial distribution (Table I
). On chromosomes 13 and 17, no genetic effect of losing the same allele within a twin pair was found. In fact the observed probability of 0.12 on chromosome 13 was significantly less than expected (0.20). Only at 17p was the proportion of twin pairs with the same LOH higher than expected (observed, 0.23; expected, 0.12). When the probabilities of losing the same allele within a twin pair at different markers were calculated, only one marker on chromosome 13 (D13S328, between the BRCA2 and Rb-1 loci) showed a significantly higher proportion of twin pairs with the same LOH than expected (Table I
). On chromosome 17, two markers showed a significantly higher proportion of twin pairs with the same LOH than expected (Table I
). The first one was D17S786, distal to the p53 locus. This explains the higher proportion of twin pairs than expected had the same LOH in the 17p arm. The other marker was, D17S855, which is an intragenic BRCA1 marker. The power of the model is seen in Table I
: even one pair of twins rendered the results formally significant at many markers. We, however, applied a more conservative criterion of at least two twin pairs with the same LOH before calling the findings significant.
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Table I. Observed probability (Pobs) that the same allele is lost within a twin pair as compared with the expected probability (Pexp).
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Mutation analysis
No BRCA1 mutations were found in the subset of samples from a representative concordant twin pair, although LOH on 17q was frequently observed in our sample set, including two pairs who had lost the same allele at the BRCA1 marker. Apart from two polymorphisms detected in BRCA2, two missense changes were found with a putative pathogenic effect (Table II
; Figure 5
). In the LOH analysis one of the samples showing a missense change (11031) was not informative at marker D13S260, and had retained heterozygosity at marker D13S171, flanking the BRCA2 gene. In the other case (12850), samples were not available for the LOH analysis.

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Fig. 5. BRCA2 mutation in patient 12850. (a) SSCP analysis; the arrow indicates mobility shift in tumour DNA. (b) ABI electropherogram of tumour DNA sequence. (c) ABI electropherogram of control DNA sequence. The arrows indicate the G A mutation.
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Discussion
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We have analysed LOH on chromosomes 13 and 17 in monozygotic twins with breast cancer, which can give information on hereditary genetic changes in tumour suppressor genes predisposing to the development of breast tumours. We did not find any general genetic effect of losing the same allele within a twin pair. Thus, hereditary effects in the development of breast cancer can be suspected when the twins have lost the same allele at a specific marker. However, as comparisons were done in a conservative way within the same chromosome, the high overall rate of LOH on chromosomes 13 and 17 may mask moderate increases in LOH rates.
Chromosomes 13 and 17 contain the two known tumour suppressor genes, BRCA2 at 13q12-13 and BRCA1 at 17q21, that predispose to breast cancer (59). In planning this study, we had no exact way of estimating the expected numbers of BRCA1 and BRCA2 mutations because nothing was known about these mutations in concordant twins and because the population frequency of the mutations in Sweden was not known. However, in families prone to breast cancer or to breast and ovarian cancer, where the risk of breast cancer may be less than in concordant monozygotic twins, 11% and 14% of families had mutation in BRCA2 and BRCA1, respectively (33,34). According to such crude estimates, our 12 twin pairs could have included one or two mutations of each of these genes, well within the observed two BRCA2 mutations.
No BRCA1 mutations were found in the 12 twin pairs that we were able to analyse. However, since the number of samples included in the study was small and the sensitivity of the method is <100%, the presence of a cancer-predisposing mutation cannot be excluded. Apart from two polymorphisms detected in BRCA2 (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/), two missense changes were found with a putative pathogenic effect. One of them has been reported on a number of occasions (http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/Bic/), probably representing a rare polymorphism. The other missense change in BRCA2 in our sample set has not been reported previously. It was not found in any of 186 chromosomes analysed using PCR SSCP.
On chromosome 13, the region of another tumour suppressor gene, Rb-1 [located at 13q14 (35)], is often lost together with the BRCA2 locus (15,16), but the normal expression of Rb protein excludes it as a candidate breast tumour suppressor (36). Another tumour suppressor gene between the BRCA2 and the Rb-1 loci seems to be a likely explanation for the observed LOH on chromosome 13 in sporadic breast cancer. In our study we did not find any twin pairs who had lost the same allele at the BRCA2 or the Rb-1 region. Instead, we were able to separate the region between the BRCA2 and Rb-1 loci into two smaller segments. The first one was located around the BRCA2 locus between markers D13S260 and D13S218. The second one was located between markers D13S263 and D13S155, including marker D13S328, which was the only marker on chromosome 13 for which a significantly higher proportion of twin pairs than expected had the same LOH.
Distal to the Rb-1 locus, we found two markers for which the twin pairs had lost either the same or a different allele. They are located at 13q14.3q21.1 (D13S279) and 13q32 (D13S159), 28 cM apart. Most studies concerning breast cancer have concentrated on the region between BRCA2 and Rb-1. There is only one study where the region telomeric to the Rb-1 locus has been evaluated (17). In that study, 13q31q34 was shown to be a target for LOH. This region has been suggested as a preferential site of chromosomal loss in head and neck squamous cell carcinoma (37). In each of these studies the lost region is large,
20 cM; more markers are needed to evaluate the importance of this region.
On chromosome 17 several studies have shown that at least two regions of LOH on the p arm, one at the p53 locus and the other distal to it, and at least three regions of LOH on the q arm, both proximal and distal to the BRCA1 locus, are involved in the development of breast cancer (1820,3840). In our study the most important region of LOH on 17p was located distal to the p53 locus. Three twin pairs had lost the same allele at marker D17S786, 14 cM distal to the p53 locus, which was significantly more than expected. One pair had lost the same allele at marker D17S926, 17 cM distal to marker D17S786. This may mean that there are two different targets for LOH distal to the p53 locus, as has been suggested earlier (41). However, because the twin pairs showing the same LOH at markers D17S786 or D17S926 showed either LOH, were uninformative or could not be analysed at the other marker, it is possible that both of these markers are within the same LOH. The telomeric part of 17p contains a tumour suppressor gene, HIC-1 (42), which has been shown to be hypermethylated in breast tumours (43). Our results suggest, that HIC-1 may be more important for breast tumour development than p53.
On chromosome 17q we found three regions of LOH. The first was located at marker D17S250, centromeric to the BRCA1 locus, at 17q11.212. Data from invasive breast cancer suggest that a tumour suppressor gene may be located in this region (19). The ERBB2 oncogene is located 2 cM distal to D17S250 (44). It is amplified in ~20% of invasive breast tumours (1). Amplification of ERBB2 could be seen as an allelic imbalance similar to LOH. We cannot exclude the possibility that amplification of ERBB2 caused the allelic imbalance seen at marker D17S250 in our study. The second region on chromosome 17q was located around the BRCA1 locus, where two twin pairs had lost the same allele at the intragenic BRCA1 marker D17S855. However, BRCA1 mutations could not be analysed from one of them and no BRCA1 mutations were found from the other twin pair. Our results support the data that another tumour suppressor gene may be located near the BRCA1 locus and causes LOH at the BRCA1 locus in sporadic breast cancer (1820,40).
The region distal to the BRCA1 locus has been poorly investigated, even though several studies suggest at least two regions of LOH in the telomeric part of 17q (1820). Only one study has used dense markers in this region (40). In that study, three common regions of LOH distal to BRCA1 were found, one at 17q22, the second at 17q23q24 and the third at 17q25. We found one twin pair who had lost the same allele at our most telomeric marker, D17S254. Another pair had lost a different allele at this marker. D17S254 is located at 17q22q24 and could thus represent any of the regions of LOH reported earlier (1820,40).
In conclusion, no BRCA1 mutations were found in 12 Swedish monozygotic twin pairs with breast cancer, even though frequent LOH was observed at 17q. Two polymorphisms and two missense changes were found in BRCA2. The overall frequency of LOH on chromosomes 13 and 17 was not genetically determined according to the twin model. Our results support the data that another tumour suppressor gene is located between the BRCA2 and the Rb-1 regions at chromosome 13q. On chromosome 17 the region telomeric to the p53 locus seems to be more important than p53 in the development of breast cancer. Even though BRCA1 seems not to be involved in the development of breast cancer in the twins studied, another tumour suppressor gene near BRCA1 may be involved.
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Notes
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1 To whom correspondence should be addressed Email: asta.forsti{at}cnt.ki.se 
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Acknowledgments
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We thank J.Nejaty and R.Partanen for technical assistance. The study was supported by the Swedish Cancer Fund and the Swedish Society for Medical Research.
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Received May 11, 2000;
revised September 8, 2000;
accepted September 13, 2000.