Breast Cancer Res Treat (2008) 107:431–441
DOI 10.1007/s10549-007-9571-2
EPIDEMIOLOGY
Greek BRCA1 and BRCA2 mutation spectrum: two BRCA1
mutations account for half the carriers found among high-risk
breast/ovarian cancer patients
Irene Konstantopoulou Æ Theodore Rampias Æ Angela Ladopoulou Æ George Koutsodontis Æ
Sophia Armaou Æ Theodore Anagnostopoulos Æ George Nikolopoulos Æ Smaragda Kamakari Æ
George Nounesis Æ Antonis Stylianakis Æ Charisios Karanikiotis Æ Evangelia Razis Æ Helen Gogas Æ
Antonios Keramopoulos Æ Vassiliki Gaki Æ Christos Markopoulos Æ Dimosthenis Skarlos Æ
Nikos Pandis Æ Thalia Bei Æ Iordanis Arzimanoglou Æ George Fountzilas Æ Drakoulis Yannoukakos
Received: 12 March 2007 / Accepted: 16 March 2007 / Published online: 24 April 2007
Springer Science+Business Media B.V. 2007
Abstract 127 Greek breast/ovarian cancer families were
screened for germline BRCA1/2 mutations by dHPLC followed by direct sequencing. Our results indicated 16 and 5
breast/ovarian cancer families bearing deleterious mutations in the BRCA1 and BRCA2 genes, respectively. Two
novel BRCA2 germline mutations (G4X and 3783del10)
are reported here for the first time. Subsequent compilation
of our present findings with previously reported mutation
data reveals that in a total of 287 Greek breast/ovarian
cancer families, 46 and 13 carry a deleterious mutation in
BRCA1 and BRCA2, respectively. It should be noted that
two BRCA1 mutations, 5382insC and G1738R, both located in exon 20, account for 46% of the families found to
carry a mutation. Based on our mutation analysis results,
we propose here a hierarchical, cost-effective BRCA1/2
mutation screening protocol for individuals of Greek ethnic
Irene Konstantopoulou and Theodore Rampias equally contributed to
this work.
I. Konstantopoulou A. Ladopoulou S. Armaou T. Anagnostopoulos G. Nikolopoulos G. Nounesis N. Pandis I. Arzimanoglou G. Fountzilas D. Yannoukakos (&)
Molecular Diagnostics Laboratory, I/R-RP, National Center for
Scientific Research ‘‘Demokritos’’, Athens, Greece
e-mail: [email protected]
I. Konstantopoulou T. Rampias A. Ladopoulou G. Koutsodontis S. Kamakari T. Bei
BioGenomica, Centre for Genetic Research & Analysis,
Athens, Greece
A. Stylianakis
KAT Hospital, Athens, Greece
C. Karanikiotis
Department of Medical Oncology, 424 Army General Hospital,
Thessaloniki, Greece
E. Razis
Diagnostic and Therapeutic Centre of Athens HYGEIA,
Athens, Greece
H. Gogas
First Department of Internal Medicine, University of Athens,
Athens, Greece
A. Keramopoulos V. Gaki
Breast Cancer Unit, Iaso Women’s Hospital, Maroussi, Greece
C. Markopoulos
Hellenic Breast Surgeons Society, Athens, Greece
D. Skarlos
2nd Oncology Department, Henri Dunant Hospital, Athens,
Greece
N. Pandis
Department of Genetics, Saint Savvas Anticancer Hospital,
Athens, Greece
I. Arzimanoglou
Instiute of Human Genetics, University of Aarhus, Aarhus,
Denmark
G. Fountzilas
Department of Medical Oncology, ‘‘Papageorgiou’’ Hospital,
Aristotle University of Thessaloniki School of Medicine,
Thessaloniki, Greece
G. Fountzilas
Hellenic Cooperative Oncology Group,
Athens, Greece
123
432
origin. The suggested protocol can impact on the clinical
management of breast-ovarian cancer families on a national healthcare system level.
Keywords BRCA1 BRCA2 Greece Breast cancer Ovarian cancer Genetic testing Germline mutation dHPLC
Introduction
Genetic linkage studies have led to the identification of
highly penetrant genes, which are associated with the
familial breast and ovarian cancer syndrome. Tumors in
breast and/or ovarian cancer women with parallel family
history of breast/ovarian cancer are characterized by
alterations in certain genes, mainly BRCA1 (MIM#
113705) and BRCA2 (MIM# 600185), but also CHK2,
ATM, PALB2 and others (for a recent review see [1]). The
lifetime risk to develop breast and/or ovarian cancer conferred by a mutated BRCA1 or BRCA2 gene was calculated
to be 60–85% for breast and 15–40% for ovarian cancer
[2]. Therefore, it is widely believed that germline mutations in BRCA1 and BRCA2 predispose to breast and
ovarian cancer, with significantly smaller lifetime risks
noted for other types of cancer [3, 4].
Up to date, several hundreds of distinct mutations,
polymorphisms and variants have been identified in the
BRCA1 and BRCA2 genes and reported to the Breast
Cancer Information Core Database (BIC, http://www.
nhgri.nih.gov/Intramural_research/Lab_transfer/Bic). Taking into account the length of the two genes, the plethora of
mutations, each presumably conferring a different cancer
risk, and the ambiguous effect of the numerous unclassified
sequence variants, it is understandable why there is a need
to develop a comprehensive and focused mutation analysis
for the purpose of a more efficient clinical management of
at-risk individuals.
Several studies have demonstrated that BRCA1 and
BRCA2 disease-causing mutations are confined to certain
ethnic populations and geographic areas [5, 6]. Some
populations are characterized by a very short number of
BRCA1 and BRCA2 pathological alterations, for instance
three mutations in Ashkenazi Jews [7, 8] and only one in
Icelanders [9] account for almost all alterations detected in
these ethnic groups; in the same line, three BRCA1 mutations account for 50% of all pathological alterations observed in Norwegians [10]. On the other hand, other
countries appear to exhibit a much broader BRCA1/2
mutation spectrum. This seems to be the case of Greece,
where previous studies involving breast-ovarian cancer
families showed that BRCA1 5382insC is in fact the most
prevalent mutation, with a number of other deleterious
123
Breast Cancer Res Treat (2008) 107:431–441
mutations found in parallel once or at a very low frequency
[11–14]. Consequently, there is a need to perform BRCA1/
2 screening on a large number of Greek breast/ovarian
cancer families to narrow down the number of mutations to
those that are biologically significant for this particular
ethnic group. To accomplish this, we screened the entire
BRCA1 and BRCA2 genes (all exons and intron-exon
boundaries) in 127 Greek families for the presence of
molecular alterations. Here, we report the Greek BRCA1/2
mutation spectrum, and propose a time- and cost-effective
protocol for future genetic testing of at-risk individuals of
Greek ethnic origin.
Subjects and methods
Patients and families
Index patients of 127 families with breast and/or ovarian
cancer were screened for BRCA1 and BRCA2 mutations.
Patients were selected ad hoc from several Greek hospitals
in collaboration with the Hellenic Cooperative Oncology
Group (HECOG). The study was approved by the hospitals
ethics and research committees and was in agreement with
the 1975 Helsinki statement, revised in 1983. Informed
consent was obtained from all individuals before genetic
analysis was performed and patients were counselled about
the implications of genetic testing. Index patients were
classified into four categories, according to family or personal disease history: (i) families with more than three
cases of breast and/or ovarian cancer (n = 15), (ii) families
with three cases of breast and/or ovarian cancer (n = 24),
(iii) families with two cases of breast and/or ovarian cancer
(n = 58), (iv) patients with no family history, including
cases of early-onset (<35 years) breast cancer, bilateral
breast cancer, male breast cancer and breast and ovarian
cancer in the same patient (n = 30).
Screening for BRCA1 and BRCA2 germline mutations
Genomic DNA was isolated from peripheral blood using
standard protocols. To amplify exons and exon-intron
boundaries of the above genes, primer pairs were either
obtained from the BIC database (http://www.nhgri.nih.gov/
Intramural_research/Lab_transfer/Bic/Member/
BRCA1_mutation_database.html) or designed using the
‘Primer3’ software at http://www-genome.wi.mit.edu/cgibin/primer/primer3_www.cgi. PCR was performed using a
TM
TaKaRa PCR Dice Thermal Cycler (Takara Bio Inc.,
Shiga, Japan) and a DNA Engine Dyad model (MJ Research-BioRad Laboratories Inc., Hercules, CA, USA).
Mutational screening for BRCA1 and BRCA2 was carried out in two steps: first by pre-screening heteroduplexes
Breast Cancer Res Treat (2008) 107:431–441
using denaturing high-performance liquid chromatography
(dHPLC), and then by direct sequencing. For heteroduplex
formation, all PCR amplicons were subjected to an additional 3-min 95C denaturing step followed by gradual
reannealing from 95 to 65C over a period of 30 min prior
to analysis. Denaturing high-performance liquid chromatography was carried out using the WAVE DNA Fragment
Analysis System equipped with a DNASep column
(Transgenomic Inc., San Jose, CA). Running conditions for
the dHPLC analyses were optimized using WAVETM
Maker software (Transgenomic Inc., San Jose, CA).
Abnormal dHPLC peak variants were directly sequenced
from PCR amplified products using ABI Prism Dye
Terminator Kit v3.1 (Applied Biosystems, Foster City, CA,
USA) and ABI Prism 3100-Avant automated DNA sequencer. Primer sequences and PCR & dHPLC running
protocols are available upon request.
Mutation results
Our results were evaluated using the BLAST (http://
TM
www.ncbi.nlm.nih.gov/BLAST) and SeqScape
v2.0
(Applied Biosystems, Foster City, CA, USA) software
packages. Any mutation found was confirmed on a second
DNA sample isolated from a duplicate tube of blood
followed by sequencing in both forward and reverse
directions. All nucleotide numbers refer to the wild type
cDNA sequence of BRCA1 (U14860.1) and BRCA2
(NM_000059.2) as reported in GenBank.
In silico evaluation of intronic sequence variants
Intronic variants were evaluated for potential splicing effects with the following online analysis tools: NNSPLICE
(http://www.fruitfly.org/seq_tools/splice.html), SpliceSiteFinder
(http://violin.genet.sickkids.on.ca/~ali/splicesitefinder.html), GenScan (http://genes.mit.edu/GENSCAN.
html), and NetGene2 (http://www.cbs.dtu.dk/services/
NetGene2/).
Results
Complete screening of 127 Greek breast/ovarian families
for all BRCA1 and BRCA2 exons along with flanking intronic sequences was performed. Our results revealed sixteen breast/ovarian cancer families carrying deleterious
BRCA1 mutations (16/127 = 12.5%) and five carrying
BRCA2 mutations (5/127 = 4%), thus 16.5% of the families studied (21/127) carried either a BRCA1 or a BRCA2
mutation. Of the seven different BRCA1 mutations reported
here, three are frameshift (5382insC found x7, 3819del5
found x1, and 3896delT found x1), two nonsense (R1203X
433
found x1, R1751X found x1), and two missense (G1738R
found x4, and C61G found x1). Each of the five BRCA2
mutations is observed only once; three are frameshift
(3783del10, 4637delTA, 5950delCT), one is nonsense
(G4X), and one is a single base substitution that results in
aberrant splicing (7235G > A, skipping of exons 12–13)
[15]. All but two of the above mutations have been previously reported in the BIC database (http://www.nhgri.
nih.gov/ Intramural_research/Lab_transfer/Bic/Member/
BRCA1_mutation_database.html). The two novel BRCA2
mutations described here for the first time are G4X and
3783del10, located in exons 2 and 11, respectively. The
former is a nonsense mutation resulting from a substitution
of a G to T at cDNA position 238, thus generating a stop
codon from glycine (GGA > TGA) at position 4 of the
protein sequence. The latter is a deletion of 10 nucleotides
(GTTGAAATTA) starting at cDNA position 3783 (codon
1185) that results in a frame shift generating a premature
stop codon at protein position 1205.
Table 1 summarizes mutation analysis data of the 21
breast/ovarian cancer families found to carry a BRCA1 or
BRCA2 mutation including mutation carrier status for all
tested family members. A comparative assessment indicated that the mean age of disease onset did not differ
significantly among BRCA1 and BRCA2 carriers, and was
43 years and 44 years for breast cancer patients bearing
BRCA1 and BRCA2 mutations respectively, and 45 years
for ovarian cancer patients with BRCA1 mutations; no
BRCA2 carriers with ovarian cancer were available for
testing.
Apart of the above-noted pathological molecular alterations, we also detected several common polymorphisms
and a number of variants of unknown biological significance (Table 2). Five of those variants were located in the
coding sequence; three of those five have been previously
reported to the BIC database as unclassified variants
(missense mutations A524V in BRCA1, M1915T in
BRCA2, and the in-frame deletion N1355del in BRCA1),
whereas the remaining two are reported here for the first
time (P1812A in BRCA1 and A3102G in BRCA2).
Six intronic variants located as far as 26 nucleotides
apart from the intron-exon junctions (IVS6 + 7G > A,
IVS6 + 26AC > CA, IVS13-10C > T in BRCA1, and
IVS2-7A > T, IVS5 + 26T > C, IVS17-12T > C in
BRCA2–see also Table 2) were evaluated for potential
splicing effects with four online analysis tools. Of these
six, only IVS2-7A > T of BRCA2 showed a relatively
significant change in score probability (P > 0.2) for recognition as an acceptor site with the NNSPLICE tool (0.68
compared to 0.94 for the wild-type sequence). In the same
line, NetGene failed to identify BRCA2 exon 3 when the
change was introduced. Unlike the above, the other two
tools SpliceSiteFinder (SA + BPS 169.3 compared to
123
123
p
*
5 (37t+, 43, 56
57, 77)
335
BRCA1/exon11
1 (63)
1 (36p,t+)
2 (49p,t+, xxt+)
1 (40p,t+)
244
347
1 (48)
1 (61)
1 (56)
)
1 (48
1 (66p,t+)
1 (48p,t+)
1 (33p,t+)
1 (28p,t+)
211
290
296
349
135
p,t+
1 (50p,t+)
1 (xx)
1 (37t+)
1 (36p,t+)
344
369
230
BRCA2/exon13
1 (58)
proband,
t+
tested, found to be carrier,
t–
tested, non-carrier; xx, age unknown
F < 30t+
F40t+, F43t+,
F64t+, F40t–
BRCA2/exon11
BRCA1/exon20
BRCA2/exon11
BRCA1/exon20
BRCA1/exon11
BRCA2/exon11
BRCA1/exon20
BRCA1/exon20
BRCA1/exon20
BRCA1/exon20
1 (40p,t+)
BRCA1/exon20
390
F34t–
1 (33p,t+)
M65
2 (46, xx)
1 (33
2 (53, 54 )
BRCA1/exon20
BRCA1/exon11
BRCA2/exon2
BRCA1/exon20
394
t+
Mxxt+, Mxxt–
BRCA1/exon20
365
)
t+
1 (32t+)
2 (46p,t+, > 65)
287
p,t+
2 (45, 55)
1 (42p,t+)
,
1 (xx)
4 (45 p,t+, 53,
60, 80)
393
p,t+
3 (60p,t+, xx, xx)
362
280
4 (25, 46p,t+,
52t+, 65 t+)
285
BRCA1/exon20
BRCA1/exon20
1 (xxt+)
9 (30, 30, 35,
35, 36p,t+, 38t+,
xx, xx, xx)
85
Fxxt–, Fxxt–, Fxxt–,
Mxxt–, Fxxt+
BRCA1/exon5
1 (47)
4 (37p,t+, 40,
35/40, 44)
Gene/exon
324
novel mutations described here for the first time
No family
history
Families with
two cases
Families with
three cases
Families with >
three cases
Sex, age and carrier
status of healthy
relatives tested
No. of OvCa
cases (age of
onset)
Total no. of BrCa
cases (age of onset)
Patient
no.
4637delTA
5382insC
3783del10*
R1751X
3896delT
R1203X
7235G > A
5950delCT
5382insC
5382insC
G1738R
5382insC
5382insC
G1738R
3819del5
G4X*
5382insC
5382insC
G1738R
G1738R
C61G
Mutation
Table 1 Mutation description and disease history of the 21 Greek families found to carry a BRCA1/2 germline mutation
ter 1480
ter 1829
ter 1205
ter 1751
ter 1263
ter 1203
del exon13
ter 1909
ter 1829
ter 1829
1738Gly > Arg
ter 1829
ter 1829
1738Gly > Arg
ter 1242
ter 4
ter 1829
ter 1829
1738Gly > Arg
1738Gly > Arg
61Cys > Gly
Effect
Br and OvCa in the same patient
Br and OvCa in the same patient
Br and OvCa in the same patient,
one case of lung cancer, patient’s
mother of Russian descent
Patient was adopted
proband’s father of Russian descent
One case of endometrial ca, one
case of pancreatic ca
One case of male BrCa, one case of
CRC, one case of unknown
primary ca
One case of prostate cancer, two
cases of gastric cancer
Br and CRC in the same patient,
three cases of lung ca, one case of
unknown primary ca
Br and OvCa in the same patient,
bilateral BrCa, two cases of colon
cancer, one case each of prostate,
larynx, gastric cancer, four cases
of unknown primary ca
Comments/other cancers
434
Breast Cancer Res Treat (2008) 107:431–441
Breast Cancer Res Treat (2008) 107:431–441
435
Table 2 Cumulative BRCA1 and BRCA2 polymorphisms and unclassified variants in Greek breast/ovarian cancer women (underlined variants
are under further investigation for possible association with disease)
Gene/exon
Nucleotide
Codon
Base change
BRCA1/5¢ UTR
1802a
5¢ UTR
C to G
AA change
Designation
1802C > Gb
Mutation type
Mutation effect
Times in BIC
P
P
–
b
BRCA1/5
IVS4
–
C to A
IVS4-19C > A
IVS
UV
1b
BRCA1/6
IVS6
–
G to A
IVS6 + 7G > A
IVS
UV
12
BRCA1/6
IVS6
–
AC to CA
IVS6 + 26AC > CA*
IVS
UV
–
BRCA1/8
IVS7
–
C to T
IVS7-34 C > Tb
IVS
P
9
BRCA1/8
IVS8
–
A to T
IVS8 + 140A > T*
IVS
P
–
BRCA1/9
IVS8
–
delT
BRCA1/9
686
189
T to C
BRCA1/9
710
197
C to T
BRCA1/9
IVS9
–
A to T
BRCA1/11
BRCA1/11
1186
1690
356
524
A to G
C to T
BRCA1/11
2196
693
BRCA1/11
2201
BRCA1/11
IVS8-57delT
b
IVS
P
15
Asp to Asp
D189Db
Syn
P
1b
Cys to Cys
b
C197C
Syn
P
28
IVS9 + 146A > T*
IVS
P
–
Gln to Arg
Ala to Val
Q356Rb
A524V
M
M
P
UV
82
1
G to A
Asp to Asn
D693Nb
M
P
16
694
C to T
Ser to Ser
S694Sb
Syn
P
13
2430
771
T to C
Leu to Leu
L771Lb
Syn
P
25
BRCA1/11
2731
871
C to T
Pro to Leu
P871Lb
M
P
26
BRCA1/11
3232
1038
C to T
Glu to Gly
E1038Gb
M
P
37
BRCA1/11
3238
1040
G to A
Ser to Asn
S1040N
M
UV
45
BRCA1/11
3537
1140
A to G
Ser to Gly
S1140G
M
UV
28
BRCA1/11
3544
1145
T to C
Val to Val
V1145Vb
Syn
P
–
BRCA1/11
3567
1150
C to T
Pro to Ser
P1150Sb
M
UV
5
BRCA1/11
3667
1183
A to G
Lys to Arg
K1183Rb
M
P
33
BRCA1/11
3878
1253
T to G
Ser to Ser
S1253Sb
Syn
P
1b
BRCA1/11
4182
1355
del AAT
del Asn
N1355del
IFD
UV
2
BRCA1/13
4427
1436
T to C
Ser to Ser
S1436Sb
Syn
P
35
BRCA1/14
BRCA1/16
IVS13
4956
–
1613
C to T
A to G
Ser to Gly
IVS13-10C > T
S1613Gb
IVS
M
UV
P
25
36
BRCA1/16
4962
1615
G to A
Ala to Thr
A1615Tb
M
UV
2
b
b
b
BRCA1/16
5012
1631
T to C
Ser to Ser
S1631S
Syn
P
BRCA1/16
5075
1652
G to A
Met to Ile
M1652Ib
M
UV
39
BRCA1/17
IVS17
–
G to A
IVS16-68G > Ab
IVS
P
6
BRCA1/17
IVS17
–
G to A
IVS16-92G > Ab
IVS
P
6
C to T
b
IVS
UV
2
BRCA1/18
IVS17
–
IVS17-53C > T
b
b
b
BRCA1/18
IVS18
BRCA1/20
5375
1752
A to C
BRCA1/20
IVS20
–
ins12
IVS20 + 48ins12
IVS
P
37
BRCA1/21
IVS21
–
G to A
IVS21 + 78G > A
IVS
UV
1
BRCA1/23
5553
1812
C to G
P1812A*
M
UV
–
M
UV
4b
M
UV
2b
BRCA1/24
BRCA1/24
5616
5685
G to A
1
1833
1856
G to A
C to T
Ala to Ala
Pro to Ala
Val to Met
Pro to Ser
IVS18 + 65G > A
IVS
P
5
A1752Ab
Syn
P
1b
b
V1833M
P1856S
b
b
BRCA2/5¢ UTR
203
5¢ UTR
G to A
203G > A
5¢ UTR
P
12
BRCA2/2
BRCA2/3
282
IVS2
18
–
C to T
T to A
Arg to Arg
R18Rb
IVS2-7T > A
Syn
IVS
P
UV
–
2
BRCA2/3
426
66
A to G
Gln to Gln
Q66Q*
Syn
P
–
BRCA2/4
IVS4
–
A to C
IVS4 + 67A > C*
IVS
UV
–
BRCA2/5
IVS5
–
T to C
IVS5 + 26T > Cb
IVS
UV
–
BRCA2/8
IVS8
–
C to T
IVS8 + 56C > Tb
IVS
UV
3
123
436
Breast Cancer Res Treat (2008) 107:431–441
Table 2 continued
Gene/exon
Nucleotide
Codon
Base change
AA change
Designation
Mutation type
Mutation effect
Times in BIC
BRCA2/10
1093
289
A to C
Asn to His
N289Hb
M
P
13
BRCA2/10
1342
372
C to A
His to Asn
H372Nb
M
P
9
BRCA2/10
1593
455
A to G
Ser to Ser
BRCA2/11
IVS11
–
C to T
BRCA2/11
2457
743
T to C
BRCA2/11
3147
973
G to A
BRCA2/11
3192
988
BRCA2/11
3199
BRCA2/11
S455S
Syn
P
7
IVS10-73C > T*
IVS
UV
–
His to His
H743H
Syn
P
7
Ser to Ser
S973S
Syn
P
1
T to C
Asp to Asp
D988D*
Syn
P
–
991
A to G
Asn to Asp
N991Db
M
P
6
3624
1132
A to G
Lys to Lys
K1132K
Syn
P
8
BRCA2/11
3744
1172
G to A
Ser to Ser
S1172S
Syn
P
4
BRCA2/11
3843
1205
T to A
Ser to Ser
S1205S*
Syn
P
–
BRCA2/11
4035
1269
T to C
Val to Val
V1269Vb
Syn
P
3
BRCA2/11
4486
1420
G to T
Asp to Tyr
D1420Y
M
P
191
BRCA2/11
5972
1915
T to C
Met to Thr
M1915T
M
UV
7
BRCA2/14
7470
2414
A to G
Ser to Ser
S2414S
Syn
P
10
BRCA2/15
BRCA2/15
7821
7836
2531
2536
T to A
T to C
Val to Val
Ser to Ser
V2531V*
S2536S*
Syn
Syn
P
P
–
–
BRCA2/17
IVS16
–
T to C
IVS
P
15
–
BRCA2/18
IVS17
–
BRCA2/22
IVS21
–
BRCA2/22
9079
2951
G to A
BRCA2/25
9533
3102
C to G
BRCA2/27
10590
3¢ UTR
A to C
a
IVS16-14T > C
b
T to C
IVS17-12T > C
IVS
UV
IVS21-66T > C
IVS
P
4
Ala to Thr
A2951T
M
P
40
Ala to Gly
A3102G*
M
UV
–
10590A > C*
3¢ UTR
P
–
Polymorphism within the beta-promoter sequence of BRCA1 (accession number U37574)
b
Previously published mutations by us are also included in order to give a complete figure of the observed mutations in Greek population for
future use as markers
* novel alterations first described in this study
AA, amino acid; BIC, Breast Cancer Information Core; IFD, in-frame deletion, IVS, intervening sequence, M, mutation; P, polymorphism; Syn,
synonymous; UTR, untranslated region; UV, unclassified variant
174.4) and GenScan (exon 3 P value: 0.965 compared
to 0.978) did not confirm any significant change in p value.
BRCA1 variant IVS13-10C > T was found in a homozygotic state in one of our patients, clearly indicating that it is
not associated with the disease, since there is strong
evidence that biallelic BRCA1 deleterious mutations result
in embryonic lethality [16]; furthermore, the same variant
has been reported to occur in trans with a deleterious
mutation [17].
Discussion
In the present study we identified 21 disease-causing
BRCA1/BRCA2 mutations among 127 Greek breast/ovarian
cancer families subjected to genetic testing. This work
concludes our effort to obtain a more complete picture of
the BRCA1/BRCA2 mutation spectrum in the Greek population. Our present data were combined with previously
123
published results involving 160 families [11–14, 18], which
were recruited in the same manner and selected using the
same criteria as here.
As a result, a cumulative mutation analysis from 287
Greek breast/ovarian cancer women +/– family history
demonstrated deleterious BRCA1 or BRCA2 mutations in
59 cases (20.5%). The prevalence of BRCA1 mutations was
approximately 3.5 times the prevalence of BRCA2 mutations: 46 families (78%) carried a BRCA1 mutation, while
only 13 families (22%) carried a BRCA2 mutation. It is
noteworthy that 56% of all mutations found are located in
exon 20 of BRCA1; moreover, 64% of all mutations are
located at the 3¢ end of this gene (exons 20–24). On the
other hand, almost one third (31%) of the mutations
identified are located in the large exons of the two genes
(11 of BRCA1, 10 and 11 of BRCA2), which represent
60% of their coding sequence. A graphic representation of
the Greek population mutation spectrum is given in
Figure 1.
Breast Cancer Res Treat (2008) 107:431–441
437
Fig. 1 Mutational spectrum of
BRCA1and BRCA2 genes found
in the total of 287 Greek
families screened with history
of breast and/or ovarian cancer.
Every cycle/triangle represents
one family; black: families with
breast cancer cases only; grey:
breast and ovarian cancer cases;
white: ovarian cancer cases
only. Triangles depict families
with large genomic
rearrangements. * described in
(18). ** described in (14) and
(18)
A mutation profile consisting of 26 different mutations,
with 15 in BRCA1 and 11 in BRCA2, was identified in our
series of Greek breast/ovarian cancer families (Table 3). Of
those, eight were observed in at least two unrelated families and thus characterized as recurrent, with the remaining
18 to be rather unique in our cohort of Greek breast/ovarian
cancer women. The broader and more variable BRCA1/
BRCA2 mutation spectrum presented here, as opposed to
that observed in other genetically homogeneous populations such as Iceland (21), Norway (22) and Poland (23), is
consistent with the already described genetic heterogeneity
of the Greek population (24).
The BRCA1 mutation 5382insC, though often referred as
a Jewish founder, is also the most frequent alteration in
Caucasians worldwide (10% of detected BRCA1 mutations), with varying frequency among different ethnic
populations. In Europe, 5382insC frequencies are markedly
high in Eastern European countries such as Poland, Russia,
Latvia, Hungary and others, and a gradient in prevalence is
observed from the eastern to the western regions of Europe.
Haplotype analysis has placed the origin of the mutation
approximately 36 generations ago, in the Middle Ages,
somewhere in the Baltic area [5, 7, 25]. More recent data
support origin from a single common ancestor [26]. Our
cumulative results clearly demonstrate that, in consistency
with previous data [13], 5382insC is also the most frequent
mutation observed in the Greek population; having being
detected in 18 unrelated families of Greek origin with an
identified mutation, it has a noted prevalence of 31% (18/
59) in the mutation spectrum of both genes (Fig. 2).
The second most frequent mutation found in our large
cohort of Greek breast/ovarian cancer families is the missense BRCA1 variant G1738R, with a frequency of 15% (9/
59 families, Fig. 1 and 2). There is strong evidence, based
on a combination of genetic, biochemical, in silico, histopathological and segregation analysis, that it is a causative
mutation [16, 27–34]. Recent haplotype analysis proves
G1738R to be a Greek founder mutation and provides
additional segregation data towards the pathogenicity
of this mutation (Anagnostopoulos et al. 2007, in
preparation).
Both 5382insC and G1738R were observed in breast/
ovarian cancer families originating from different parts of
the country, including Greek families living outside Greece
(Anagnostopoulos et al. 2007, in preparation; [26, 35]),
suggesting that these two mutations are common and of old
origin in the Greek population. Our ongoing work aims to
determine mutation frequencies in unselected breast cancer
cases and in the general Greek population, as well as to
specify mutation age via haplotype analysis.
Other recurrent mutations in Greek breast/ovarian cancer families include BRCA1 R1751X, also in exon 20,
5586G > A, that results in exon 23 skipping [19], and
BRCA2 2024del5 and 4147delG (Fig. 1).
Three novel large genomic deletions have been found in
five Greek families, del4174Ex20, del4429ins5Ex24 [18]
and del3.2kbEx20 [14]. Our cumulative data indicate that
large genomic rearrangements constitute a significant portion of deleterious alterations of the BRCA1 gene in the
Greek population (11%, 5/46).
123
123
1623
3099
3277
3726
3741
3819
3896
–
–
5331
5370
5382
5586
–
238
2024
3034
3058
3782
4147
4637
5950
6024
6631
7235
BRCA1/exon11
BRCA1/exon11
BRCA1/exon11
BRCA1/exon11
BRCA1/exon11
BRCA1/exon11
BRCA1/exon11
BRCA1/exon20
BRCA1/exon20
BRCA1/exon20
BRCA1/exon20
BRCA1/exon20
BRCA1/exon23
BRCA1/exon24
BRCA2/exon2
BRCA2/exon10
BRCA2/exon11
BRCA2/exon11
BRCA2/exon11
BRCA2/exon11
BRCA2/exon11
BRCA2/exon11
BRCA2/exon11
BRCA2/exon11
BRCA2/exon13
G to A
del AACTT
delTA
delCT
delTA
delG
del GTTGAAATTA
delA
del AAAC
del TTTAT
F
6631del5b
7235G > A
ter 2137
D exon13
M
F
F
5950delCT
numbering refers to genomic BRCA1 sequence (accession number L78833)
novel mutations identified in this study
F
4147delGc
F
F
3783del10*
4637delTA
F
F
3058delAc
3034del4
b
6024delTAc
ter 1943
ter 1909
ter 1480
Stop 1334
ter 1205
Stop 959
Stop 958
Stop 599
F
ter 4
G to T
2024del5b,c
D exon24
D 82651-87079#
N
S
5586G > Ab,d,f
D exon23-24
G to A
R
F
5382insCa,b,e,f
ter 1829
insC
G4X*
M
N
del4429ins5Ex24g
R
G1738Rb,h
R1751Xa
G to A
C to T
Gly to Arg
ter 1751
del3.2kbEx20f,g
D exon20
–
R
del4174Ex20g
D exon20
F
D 71146-75319#
3896delT
F
F
3741insAa
3819del5
N
R1203X
F
3277insGa
F
M
F
a
Mutation type
3099delTa
1623del5
C61G
Designation
ter 1263
ter 1242
ter 1218
ter 1203
ter 1059
ter 999
ter 505
Cys to Gly
AA change
delT
del GTAAA
insA
C to T
insG
delT
del TTAAA
T to G
Base change
S
F
F
F
F
F
F
F
F
F
N
R
S
F
UV
N
R
R
F
F
F
N
F
F
F
M
Mutation effect
19
13
9
42
1
1
–
–
128
12
–
–
3b,d
1063
10b,h
29a
–
–
3
60
2a
28
1a
1a
28
222
Times in BIC
AA, amino acid; BIC, Breast Cancer Information Core; F, frameshift; M, mutation; N, nonsense; R, large genomic rearrangement; S, splice; UV, unclassified variant
#
*
2336
2135
1932
1908
1470
1307
1185
944
936
599
4
–
1823
1756
1738
1751
–
–
1259
1234
1208
1203
1053
994
502
61
Codon
[11]; b[13]; c[12]; d[19]; e[20]; f[14]; g[18]; hAnagnostopoulos et al. in preparation
300
BRCA1/exon5
a
Nucleotide
Gene/exon
Table 3 Cumulative BRCA1 / BRCA2 deleterious mutations identified in Greek breast/ovarian cancer patients
1
1
1
1
1
2
1
1
1
2
1
2
3
18
9
3
2
1
1
1
1
1
1
1
1
1
Times observed in
Greek families
438
Breast Cancer Res Treat (2008) 107:431–441
Breast Cancer Res Treat (2008) 107:431–441
439
Fig. 2 Prevalence of
deleterious BRCA1 and BRCA2
mutations among the 59 Greek
families found to be carriers.
5382insC and G1738R, both in
BRCA1 exon 20 are the most
frequent mutations observed
(see text)
Apart from the two novel mutations reported here for the
first time (G4X and 3783del10 in BRCA2), other novel
mutations confined to the Greek population are 3099delT
and 3277insG in BRCA1 [11], and 3058delA in BRCA2 [12].
The finding that about 11% of Greek breast/ovarian
cancer patients belonging to high-risk groups carry a
mutation in exon 20 of BRCA1, easily detectable in a single
reaction with routine methods, has important diagnostic
value for the population. Given our mutation results on the
one hand, and our intention to reduce the high cost of a
complete gene mutation analysis on the other hand, we
propose the following hierarchical BRCA1/2 mutation
screening protocol for people of Greek ethnic origin: Stage
1––sequencing of BRCA1 exon 20 (1% of total cost); Stage
2––sequencing of BRCA1 exons 21 and 23 (2% of total
cost); Stage 3––sequencing of BRCA1 exon 11 (10% of
total cost); Stage 4––sequencing of BRCA2 exons 10 and
11 (20% of total cost); Stage 5––screening for large
genomic rearrangements in BRCA1 (25% of total cost);
Stage 6––sequencing of the rest BRCA1 and BRCA2 exons
(42% of total cost). In the case of male breast cancer patients we suggest that stage 4 should precede stage 3.
Following the optimized time- and cost- effective BRCA1/2
clinical genetic protocol, we believe that we contribute to
sooner see the test become widely accessible to the public,
provided that the right welfare mechanisms exist to direct
to the test only the patients fulfilling the appropriate
criteria.
In conclusion, we have shown that inherited breast/
ovarian cancer in Greece is characterized by a wide and
variable spectrum of mainly BRCA1 but also BRCA2
mutations, with two founder BRCA1 exon 20 mutations
playing a prominent role, as they account for almost half
(46%) of the families found to carry a mutation. Our
cumulative data allow optimization of genetic testing and
counseling procedures via the utilization of the proposed
stepwise protocol, specifically tailored for people of Greek
ethnic origin. It is expected that likewise populationspecific protocols can help genetic testing for BRCA1 and
BRCA2 mutations become part of routine practice in many
clinical centers around the world. In addition, work has
begun on the characterization of genetic variants associated
with a lower risk of cancer than that conferred by germline
BRCA1/BRCA2 mutations, which may contribute to an
individual’s overall risk for cancer. Understandably, the
long-term goal is to more accurately assess the risk for
breast and ovarian cancer, which in turn would enable us to
achieve a more efficient clinical management strategy per
individual woman at risk (personalized medicine).
Acknowledgements We are indebted to the patients for their willingness to collaborate for the purpose of this study. This work was
partly supported by the Hellenic Cooperative Oncology Group (HeCOG), the Hellenic Institute for Occupational Health and Safety, the
Greek Ministry of Health & Welfare (MOHAW) and the Greek
General Secretary for Research & Technology (GSRT) Program
‘Research in Excellence II’ funded by 75% from the European Union.
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