Download An Interstitial Tandem Duplication of 9p23-24

[CANCER RESEARCH 58. 86.1-866. March I. IW8]
Advances in Brief
An Interstitial Tandem Duplication of 9p23-24 Coexists with a Mutation in the
BRCA2 Gene in the Germ Line of Three Brothers with Breast Cancer1
Larissa Savelyeva, Andreas Claas, Simone Gier, Peter Schlag, Lothar Finke, Jonathan Mangion, Michael R. Stratton,
and Manfred Schwab2
Division of Cytogenelics, German Cancer Research Center. D-69120 Heidelberg ¡L.S.. A. C.. S. G., M. S.]; Division for Surgery and Surgical Oncology. Rohen-Rossle-Klinik.
Max-DeIbruck~Centrum Berlin Buch, D-13122 Berlin ¡P.S., L. F.}; and Section of Molecular Carcinogenesis, The Institute of Cancer Research, Sullon. Surrey SM2 5NG, United
Kingdom ¡J.M., M. R. S.]
Abstract
Germ-line mutations
of the BRCA2 gene account for the majority of
families with both male and female breast cancer. However, among inde
pendently ascertained families with the same mutation, cases of male
breast cancer often appear to cluster in a single family or in a particular
branch of one family. This suggests that the risk of male breast cancer
conferred by BRCA2 mutations may be modified by other genetic or
environmental factors. We report a family in which three brothers with
breast cancer carry in their germ line two genetic abnormalities: an
insertion A at nucleotide 2041 in exon 10 of BRCA2, which leads to
premature termination of the encoded protein at codon 615, and a tandem
interstitial duplication involving chromosome bands 9p23-24. We propose
1.1
urn
2.1
2.3
2.2
3.1
Introduction
2.4
BHCA 59y
BRCA 60y
that the coexistence of this rare chromosomal abnormality with BRCA2
mutation may be augmenting the risk of male breast cancer conferred by
the BRCA2 mutation.
3.2
3.3
3.4
3.6
BRCA55y
insilu
BRCASey
BRCASly
Fig. 1. Breast cancer family with four affected male members. Strfid pedigree symbols,
affected individuals; slash through symbol, deceased individual; O and •¿.
females; D and
•¿
males.
Breast cancer affects 1 in 1000 men and is therefore approximately
100 times less common than in women (1-3). Among the established
genetic risk factors are Klinefelter's syndrome (4); mutation in the
androgen receptor gene (5, 6); and, particularly,
BRCA2 gene on chromosome 13ql2-13 (7-19).
1.2
mutations in the
Materials and Methods
Recent analyses indicate that mutations in BRCA2 account for 75%
of families in which there is at least one case of male breast cancer and
at least three more cases of breast cancer (either in males or in females
diagnosed before age 60).3 However, the pattern of male breast
Cells, Chromosome Preparations. Cytogenetic analysis of short-term
blood cultures as well as of EBV-transformed lymphocytes from patients was
cancers in such families is unusual. Most families have a single case,
yet a remarkable number of the remainder have three, four, or five
cases. Moreover, among breast cancer families with an identical
BRCA2 mutation, male breast cancers often cluster in one family or in
one branch. A recent formal analysis of the large Icelandic breast
cancer kindred caused by the BRCA2 999del5 mutation (13) showed
that the clustering of male breast cancer cases is unlikely to be due to
chance, indicating that there exists a genetic modifying factor aug
menting the risk conferred by the BRCA2 mutation in some branches.4
our laboratory. FISH, YACs, and cosmids were labeled by nick translation
with biotin-14-dCTP (Life Technologies, Inc.) or DIG-11-dUTP (Boehringer
done according to routine procedures.
DNA Probes, Labeling, and FISH Analysis.
Mannheim). Suppression of repetitive sequences, denaturation, hybridization,
and fluorescence detection were as described (20, 21). FITC-avidin (Vector
Laboratories) and biotinylated anti-avidin D (Vector) were used for detection
of hybridized biotinylated DNA. Anti-DIG-mouse IgGlx (Boehringer Mann
heim) and Cy3-conjugated sheep antimouse IgG (Dianova) were used to detect
DIG-labeled probes. .Slides were counterstained
with 4',6-diamidino-2phenylindole, mounted in antifade solution, and analyzed with a Zeiss Axiophot microscope. Images were taken with a cooled charge-coupled device
camera (KAF 1400; Photometries) and processed using IPLab Spectrum soft-
Results
Received 11/14/97; accepted 1/15/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by Deutsches Krebsforschungzentrum funds through the
We have studied the family represented in Fig. 1. The index patient,
3.3, and his brother, patient 3.6, developed infiltrating ductal breast
cancer at the ages of 55 and 51 years, respectively. Patient 3.5
underwent a prophylactic mastectomy at age 52. Histological exam
ination of the resected breast tissue revealed extensive ductal carci
noma in situ. The father of the three brothers (patient 2.3) died at age
Organ Tumors: Breast Cancer program and by the United Kingdom Cancer Research
Campaign. The study contains parts of the Ph.D. thesis of A. C.
2 To whom requests for reprints should be addressed, at Division of Cytogenetics,
German Cancer, Research Center. Im Neuenheimer Feld 28Ü.D-69120 Heidelberg.
Germany. Phone: 6221-42 32 20; Fax: 6221-42 32 77; E-mail: [email protected].
1 D. Ford, D. F. Easton. and the Breast Cancer Linkage Consortium,
All YACs5 were obtained
from the CEPH Mega-YAC library. Cosmid C6A11 (WI-3357) was isolated in
submitted for
5 The abbreviations used are: YAC, yeast artificial chromosome; FISH, fluorescence in
publication.
4 D. F. Easton et al., manuscript in preparation.
situ hybridization: DIG. digoxigenin.
863
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TANDEM
3.3
DUPLICATION
OF 9p23-24
AND MALE BREAST CANCER
3.5
3.6
Fig. 2. Duplication of 9p23-24 in lymphocytes of male breasl cancer patients 3.3, 3.5, and 3.6. a, cosmid C67 containing a portion of the gene pió (green) and YAC799D2 (red)
encompassing D9SI852, D9S281. and D9SI849 were hybridized to metaphase chromosomes. Arrows, duplication of DNA detected by YAC799D2, but not by C67. on one of the
chromosome 9 homologues, h, cohybridization of the probes C6A11 (green) detecting Wl-3357 and YAC799D2 (red) shows that the duplicated DNA in this region is arranged in
tandem, c. YAC95IG6 (green) encompassing D9S1779 and D9S1858 and YAC799D2 (red) were hybridized to metaphase chromosomes. Arrows, duplication of DNA detected by
YAC799D2. YAC951G6, which is distal to 799D2, is present in a single copy. Cosmid probes C67 and C6A11 and YAC951G6 were labeled with biotin; YAC799D2 was labeled
with DIG. The chromosomes were counterstained wilh 4'.6-diamidino-2-phenylindole.
864
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TANDEM
DUPLICATION
OF 9p23-24
AND MALE BREAST CANCER
59 from breast cancer. One of his sisters (2.1) also died of breast
cancer at age 60; the other sister did not develop breast cancer. No
other cancers or other abnormal phenotypes have been reported in this
family. Samples were only available from patients 3.3, 3.5, and 3.6, as
well as from the unaffected member, patient 3.1.
Cytogenetic analysis of constitutional karyotypes of Giemsa-
showed migration shifts. This revealed the mutation 2041 insA in
exon 10, which is predicted to generate a frameshift and premature
termination of translation at codon 615. The mutation was present in
all three brothers. This mutation has been detected in at least two other
breast cancer families.7 In one family, Montreal 133, there are 11
cases of female breast cancer and 1 case of ovarian cancer." In the
stained chromosomes from metaphases of both primary cultures and
EBV-transformed peripheral blood lymphocytes directed our attention
other, Berkeley 9, there are 13 cases of female breast cancer and none
of ovarian cancer (17). In neither of these families with 2041 insA has
a case of male breast cancer been reported. The unaffected family
member, patient 3.1, did not reveal the 2041 insA mutation.
to an abnormal banding pattern in 9p in the index patient, 3.3 (not
shown). This abnormal banding pattern involved the distal portion of
one copy of 9p within bands p23-24 and was detected also in the two
affected brothers, patients 3.5 and 3.6. Both the type of banding and
the distal 9p location exclude the possibility that this alteration simply
represents a chromosome 9 variant characterized by a pericentromeric
C-band heteromorphism, which has been seen in constitutional karyo
types of patients with various types of cancer (22-25). We did not
observe this abnormal banding pattern in distal 9p when we inspected
constitutional karyotypes from more than 50 independent tumor pa
tients (not shown). Furthermore, a literature search did not produce
any previous report indicating that 9p might be a normally occurring
polymorphism.6
To characterize the distal 9p alteration in lymphocytes of the three
brothers, patients 3.3, 3.5, and 3.6, in greater detail, we used chro
mosomal FISH. We used two-color FISH with cosmid probe C67,
representative of the MTS1 (pl6INK4. CDKN2) gene (4) at 9p21 and
distal YAC or cosmid probes. All probes were mapped, by FISH, on
Giemsa-stained chromosomes of metaphases of normal peripheral
lymphocytes and were found to be nonchimeric (data not shown).
Fluorescence microscopy clearly revealed a single copy signal for
MTSÃŒprobe C67 on both copies of chromosome 9 in lymphocytes of
the three brothers, patients 3.3, 3.5, and 3.6 (Fig. la). A distal probe
YAC799D2 (defined by D9SI852, D9S28Ì,and D9S1841) produced
two clearly distinct hybridization signals on the abnormal chromo
some 9 of all three brothers, but not on the other normal chromosome
9 copy (Fig. la). The unaffected male family member, patient 3.1 (age
60 years) is negative for the 9p duplication (not shown). Also, the
duplication was not seen when we tested, by FISH, 25 unrelated tumor
patients (not shown).
To find out whether the duplication was tandem or inverted, we
used two-color FISH with probes of two duplicated loci, YAC799D2
and cosmid C6A11 (the latter defined by WI-3357). On the normal
chromosome 9, these two probes produced clearly distinguishable
single-copy signals, with C6A11 more proximal (green) and
YAC799D2 more distal (red; see Fig. 2h). On the abnormal chromo
some 9, the signals were duplicated in the same order (green-red,
green-red). This clearly shows a tandem arrangement of the duplicated
Discussion
This study has established the coexistence of a 2041 insA mutation in
BRCA2 and a duplication of 9p23-24 sequences in three brothers with
breast cancer, whose father also developed the disease. It is highly likely
that the mutation in BRCA2 is contributing to breast cancer susceptibility
in this family. The 9p abnormality, to our knowledge, has not been
previously documented. Moreover, familial clusters of four male breast
cancer cases are extremely unusual, and the abnormality on 9p is present
in all three brothers. Finally, there is prior evidence that modifying genes
influence the risk of male breast cancer conferred by BRCA2. The BRCA2
mutation found in this family has been detected in two other families with
cases of female breast cancer and ovarian cancer. In neither of these was
a case of male breast cancer reported, suggesting that in the family with
the 9p duplication, a modifying element is likely to be present. We
therefore propose that, in this family, altered function of a gene(s)
associated with the duplication on 9p is acting to augment the risk of male
breast cancer associated with the BRCA2 mutation. Future studies will be
directed at clarifying the genetic abnormality on 9p and investigating
whether this locus (altered by duplication or some other mechanism) is
responsible for modifying the male breast cancer risk of BRCA2 in other
families.
Acknowledgments
Cosmid probe C67 was made available by J. Weaver-Feldhaus.
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866
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An Interstitial Tandem Duplication of 9p23−24 Coexists with a
Mutation in the BRCA2 Gene in the Germ Line of Three
Brothers with Breast Cancer
Larissa Savelyeva, Andreas Claas, Simone Gier, et al.
Cancer Res 1998;58:863-866.
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