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Oncogene (1999) 18, 4160 ± 4165
1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00
http://www.stockton-press.co.uk/onc
SHORT REPORT
Identi®cation of a 3 kb Alu-mediated BRCA1 gene rearrangement in two
breast/ovarian cancer families
Marco Montagna*,1, Maria Santacatterina1, Arianna Torri1, Chiara Menin1,2, Daniela Zullato1,
Luigi Chieco-Bianchi1 and Emma D'Andrea1
1
Department of Oncology and Surgical Sciences, Oncology Section, Interuniversity Center for Research on Cancer, University of
Padova, via Gattamelata, 64; I-35128 Padova, Italy; 2IST Biotechnology Section, Interuniversity Center for Research on Cancer,
University of Padova, via Gattamelata, 64; I-35128 Padova, Italy
Most of the hereditary breast cancers are attributed to
constitutive alterations of either BRCA1 or BRCA2
genes; nonetheless, germline mutations of these genes in
`high risk' families are found less frequently than
expected from linkage data. Recent ®ndings suggest that
major genomic rearrangements of the BRCA1 gene
might account for at least some of the apparently
mutation negative cases. We studied 60 a€ected
probands belonging to families with a strong history of
breast and/or ovarian cancer who scored negative for
BRCA1 gene mutations by PTT and SSCP analysis.
DNA was analysed by the Southern blotting procedure
using three di€erent restriction enzymes, and two probes
obtained by RT ± PCR of the 5' and 3' BRCA1 coding
sequence. A 3 kb deletion encompassing exon 17 and
causing a frameshift mutation was identi®ed in two
independently ascertained families. RT ± PCR and longrange DNA PCR were employed to characterize the
rearrangement that was ®nally shown to be the result of
a recombination between two very similar Alu repeats.
This type of mutation is not identi®ed by the
conventional methods of mutation detection which are
based on PCR ampli®cation of single exons. Therefore,
further search for gene rearrangements is needed to
better de®ne the proportion of `high risk' families that
might be explained by gross genomic alterations of the
BRCA1 gene.
Keywords: BRCA1; genomic rearrangement; breast
cancer; ovarian cancer; Alu repeats
About 5 ± 10% of all breast cancers are due to a strong
genetic predisposition segregating as a highly penetrant
autosomal dominant trait (Claus et al., 1991). Among
the genes associated with the disease, BRCA1 and
BRCA2 are thought to account for most of the families
with multiple breast and/or ovarian cancers (Ford et
al., 1998). Germline point mutations in these genes are
scattered throughout their entire coding sequence, and
mostly recur only in families sharing the same ancestry
(BIC: The Breast Cancer Information Core); they
*Correspondence: M Montagna
Received 23 October 1998; revised 14 January 1999; accepted 16
February 1999
include nonsense mutations, small deletions and
insertions causing frameshift mutations, missense
mutations occurring at crucial aminoacid positions
within well conserved domains, and mutations a€ecting
the splice sites with loss of one or more exons in the
transcript. The frequency of these types of mutations
varies greatly depending on the racial or ethnic group,
and, in general, is lower than expected from data based
on linkage analysis (Szabo and King, 1997). In
particular, in a recent report from the Breast Cancer
Linkage Consortium it was estimated that only 63% of
families linked to BRCA1 have a mutation detectable
by the standard screening methods (Ford et al., 1998),
thus suggesting that other types of gene inactivation
might account for some of the predisposed kindreds.
Based on the diminished or absent expression of one
BRCA1 allele, an inhibition of BRCA1 transcription by
`regulatory mutations' was advanced in some cases; to
date, however, this kind of gene alteration has been
rarely reported (Miki et al., 1994; Gayther et al., 1995;
Serova et al., 1996; Xu et al, 1997).
Major genomic rearrangements of the BRCA1 gene,
which escape most of the current PCR-based mutation
detection methods, are likely to account for at least
some of the cases without apparent BRCA1 mutations
(Petrij-Bosch et al., 1997; Swensen et al., 1997; Puget et
al., 1997). This possibility is also supported by the high
density of Alu repeats dispersed in the BRCA1 genomic
sequence (Smith et al., 1996); indeed, Alu sequences
can mediate major genomic rearrangements by
promoting unequal crossing-over or other types of
recombination.
In this study we report the identi®cation of a 3 kb
genomic deletion comprising exon 17 that caused a
frameshift mutation leading to a premature stop codon
in two independently ascertained breast/ovarian cancer
families.
Sixty Italian probands with breast and/or ovarian
cancer and belonging to high risk families living in the
Veneto Region were selected from a list of cases that
scored negative for BRCA1 gene mutations as assessed
by PTT of exon 11 and SSCP analysis of the remaining
coding sequence (Montagna et al., 1996, and data not
shown). Of the 60 families, 40 were site-speci®c breast
cancer families, and 20 included both breast and
ovarian cancer patients. Each family satis®ed at least
one of the following selection criteria: (i) at least three
breast and/or ovarian cancer cases with a ®rst-degree
relationship; (ii) two cases of breast and/or ovarian
BRCA1 gene rearrangements in Italian breast/ovarian cancer families
M Montagna et al
cancer with at least one of them a€ected before age 50;
(iii) at least two ovarian cancer patients.
A blood sample was obtained from each proband
after written informed consent, and genomic DNA
was analysed by Southern-blotting; each DNA sample
was digested using HindIII, EcoRI and BamHI
restriction enzymes, blotted and hybridized with
probes pbr5' and pbr3' obtained by RT ± PCR of
exons 2 ± 10 and 12 ± 24, respectively (both probes also
include part of exon 11). The resulting restriction
patterns were con®rmed on a restriction map of the
BRCA1 genomic sequence derived from the L78833
GenBank sequence (Figure 1).
Probe pbr5' was obtained by a nested RT ± PCR
using previously reported primers (Friedman et al.,
1994 and Hogervorst et al., 1995): c1F and 11AR for
the ®rst PCR, BR2F2 and 11AiR for the second PCR.
This probe disclosed an abnormal EcoRI restriction
pattern in probands B96 and B37: however, a normal
band pattern was obtained with both BamHI and
HindIII. Further investigation of these two cases, using
additional restriction enzymes as well as cDNA
analysis, suggested the presence of an EcoRI restriction fragment length polymorphism (data not shown).
On the contrary, probe pbr3' revealed aberrant
bands in patient B74 after both HindIII and EcoRI
DNA digestion (Figure 2); in particular, both
restriction patterns were consistent with the loss of a
*3 kb segment internal to the 19 kb HindIII fragment
and removing an EcoRI site between exons 16 and 17
(Figure 1). The apparently normal restriction fragment
pattern obtained with BamHI was likely due to the low
resolution of the agarose matrix for high molecular
weight fragments (*36 kb versus *33 kb).
To investigate whether this putative rearrangement
could a€ect the BRCA1 gene the BRCA1 transcript
was analysed focusing on the region including exons 16
and 17, which are both located approximately within a
3 kb distance from the EcoRI site removed by the
rearrangement. RT ± PCR of RNA from patient B74
was performed using primers located in exon 15
(forward primer) and exon 20 (reverse primer); in
addition to the expected 737 bp band, a smaller and
fainter band was observed in patient B74 and not in
normal controls (Figure 3a). The size of the abnormal
band was incompatible with the loss of the entire exon
16 which consists of 311 nucleotides, but was
compatible with the loss of either exon 17, 18 or 19.
A second PCR was therefore performed with a forward
primer overlapping the junction between exons 16 and
17; in this case no abnormal bands were revealed, thus
suggesting that the missing exon was number 17
(Figure 3b). The absence of exon 17 was ®nally
demonstrated by sequence analysis of the cloned
PCR-fragment corresponding to the mutant allele.
This rearrangement caused a sequence frameshift in
exon 18 leading to a premature stop codon at
aminoacid position 1672, thus generating a truncated
protein product.
The intensity ratio between the wild type and the
exon 17-deleted band in the RT ± PCR product of
patient B74 clearly showed a lower expression of the
Figure 1 Structure and restriction maps of the BRCA1 gene. Probes pbr5' and pbr3' and the corresponding exons are indicated at
the top of the ®gure; HindIII, BamHI, and EcoRI restriction patterns are shown below the DNA exon-intron organization of the
BRCA1 gene; numbers on the restriction maps refer to the size (in kb) of the restriction fragments involved in the rearrangement;
arrows mark the location of the rearrangement breakpoints
4161
BRCA1 gene rearrangements in Italian breast/ovarian cancer families
M Montagna et al
4162
Figure 2 Southern blot analysis of the DNA of six a€ected
probands. Six mg of DNA derived from lymphoblastoid cells were
digested overnight with EcoRI, BamHI, HindIII restriction
enzymes, electrophoresed on 0.8% agarose gel, transferred onto
Hybond-N+ nylon membranes (Amersham), and hybridized with
the 32P-labeled pbr3' probe (exons 12 ± 24) obtained by a nested
RT ± PCR using previously reported primers (Friedman et al.,
1994; Hogervorst et al., 1995): primers 11PF and 24R for the ®rst
PCR, and primers BR11F5 and BR24R2 for the nested PCR.
Arrows indicate the aberrant bands caused by the rearrangement
in patient B74. M, molecular weight markers
mutant allele (Figure 3a). This allelic-imbalance is
attributable to a mechanism known as nonsensemediated m-RNA decay, which drives the preferential
degradation of transcripts in which premature translation termination has occurred (Jacobson and Pelts,
1996).
To further characterize this rearrangement at the
genomic level, we performed a long-range DNA PCR
and ampli®ed the region from exons 16 ± 18 containing
the breakpoint. A *7 kb fragment was obtained from
both patient B74 and a normal control, while a *4 kb
fragment, corresponding to the mutant allele, was
ampli®ed selectively from patient B74, thus further
con®rming the deletion of a *3 kb fragment (Figure
4a). The preferential ampli®cation of the 4 kb fragment
in patient B74 is likely due to the di€erent size of the
mutant (4 kb) versus the wild type (7 kb) fragment. The
breakpoint-containing region was then narrowed down
by using di€erent restriction enzymes; in this analysis,
HincII was the most informative restriction enzyme, and
mapped the deletion breakpoint to a 1000 bp HincII
fragment (Figure 4b). Thus, a couple of PCR primers
were designed to amplify this HincII fragment, which
was then sequenced. A comparison with the L78833
GenBank sequence con®rmed a deletion of 3.2 kb,
comprising exon 17. Moreover, the sequences adjacent
to the breakpoint were disclosed to be part of two Alu
repeats located in introns 16 (nt. 58 500 ± 58 798) and
17 (nt. 61 616 ± 61 918), and sharing an overall homology of 85% (Figure 5). This ®nding strongly suggested
that the deletion was the result of a homologous
recombination between two Alu sequences.
Subsequent Southern blot analysis disclosed a
restriction pattern in patient B58 that was identical to
Figure 3 RT ± PCR analysis of patient B74 BRCA1 transcript. (a) RNA, isolated from lymphoblastoid cells using RNAzol (TelTest Inc.), was retrotranscribed with the avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) and PCR-ampli®ed
with primers c8F and c10R (Friedman et al., 1994) located in exons 15 and 20, respectively; the arrow indicates the mutant allele in
patient B74; (b), ampli®cation of the same samples using primer c10F, located over the junction between exons 16 and 17, and
primer c10R
BRCA1 gene rearrangements in Italian breast/ovarian cancer families
M Montagna et al
4163
a
b
Figure 4 DNA ampli®cation and mapping of the breakpoint-containing region in patient B74. (a), long-range PCR was performed
by amplifying the genomic sequence between exons 16 and 18 at the following conditions: after an initial denaturation (4 min at
958C) of the template DNA in PCR bu€er with primers 16F and 18R (Friedman et al., 1994), a combination of 5U of Taq DNA
polymerase (Perkin Elmer) and 5U of Taq extender PCR additive (Stratagene) was added to the reaction; cycling conditions were as
follows: 10 cycles at 948C ± 10 s, 648C ± 30 s, 688C ± 5 min followed by 22 additional cycles at 948C ± 10 s, 608C ± 30 s, 688C ± 5 min
with an autoextension of 15 s/cycle, and a 688C ± 20 min ®nal extension. Arrows indicate the wild type (7 kb) and mutant (4 kb)
alleles. C, normal control. M, molecular weight marker. (b) HincII restriction pattern of the mutant (B74) and wild type (C) alleles
as shown in the scheme. The arrow indicates the HincII fragment containing the breakpoint. M, molecular weight marker
BRCA1 gene rearrangements in Italian breast/ovarian cancer families
M Montagna et al
4164
Figure 5 Comparison of the two Alu sequences involved in the
rearrangement of patient B74. The HincII fragment containing
the breakpoint was ampli®ed with primers 1/17F2 (5'-ggagaagccagaattgacag-3') and 1/17R2 (5'-ccagcaggtagataggagtt-3'), cloned
with the `PCR TA cloning kit' (Invitrogene), and sequenced with
the `PCR product sequencing kit' (Amersham). Sequence identity
between the two Alu repeats is indicated by double dots; the
rearranged sequence is shown in bold, the boxed sequence
indicates the region where the breakpoint occurred
a site-speci®c breast cancer family including seven
breast cancers, whereas family B74 also presents two
cases of ovarian cancer. This variability in phenotype
suggests the presence of di€erent modi®er factors in the
two families.
In this study we identi®ed a genomic rearrangement
in two of 60 probands with undetectable BRCA1
mutations; this frequency might actually be higher as
other rearrangements in genomic regions not well
covered by the pbr5' and pbr3' probes and deletions
of the entire BRCA1 locus might have been missed.
The numerous Alu repeats present within the
BRCA1 gene (Smith et al., 1996), and the recent
observation of other genomic deletions (Petrij-Bosch et
al., 1997; Swensen et al., 1997), one of which also
includes exon 17 (Puget et al., 1997), suggest that (a)
Alu-mediated recombination and recombination-like
events constitute a further mechanism for the
pathological inactivation of the BRCA1 gene, and (b)
the genomic region surrounding exon 17 might be a
site more prone than others to Alu-mediated rearrangements. The involvement of these types of mutations
might have been underestimated to date, because most
of the current mutation detection strategies are based
on DNA ampli®cation of single exons. Our approach
and/or long-range DNA PCR of the BRCA1 genomic
sequence are apparently the best methods for
identifying this type of mutation. This and further
studies will help to complete the BRCA1 mutational
spectrum, and explain at least some of the `high risk'
families without apparent BRCA1 mutations.
that obtained in patient B74, thus suggesting the
occurrence of the same DNA rearrangement. PCR
ampli®cation and sequence analysis of the HincII
fragment from patient B58 con®rmed this hypothesis
by showing an identical breakpoint. Furthermore,
probands B74 and B58 were shown to share one
allelic pattern over ®ve microsatellite markers
(D17S1321,
D17S855,
D17S1322,
D17S1323,
D17S1327) spanning the BRCA1 gene (Neuhausen et
al., 1996), thus suggesting a possible common ancestry
for the two families (data not shown).
It is noteworthy that the phenotypes in the two
families are di€erent despite the same mutation; B58 is
Acknowledgements
We thank the family members and the physicians (S
Aversa, A Bononi, C Di Maggio, E Ferrazzi, A
Fornasiero, V Fosser, O Nascimben, O Nicoletto, A
Rosa-Bian, G Zavagno) who contributed to this study; M
Quaggio for her expert technical assistance; P Gallo for
artwork and P Segato for help in preparing the manuscript.
M Montagna and M Santacatterina are fellows of the
Associazione and Fondazione Italiana per la Ricerca sul
Cancro (AIRC/FIRC), respectively. This study was
supported by grants from Ministero della Ricerca Scienti®ca e Tecnologica (MURST), Italian Hereditary Breast/
Ovarian Cancer Consortium/AIRC-Coordinated Project,
Regione Veneto/Ricerca Sanitaria Finalizzata, and Fondazione Cassa di Risparmio di Padova e Rovigo, Italy.
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