novel and recurrent brca1/brca2 mutations in early onset and familial breast and ovarian cancer...
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SHORT COMMUNICATION
Novel and recurrent BRCA1/BRCA2 mutations in early onsetand familial breast and ovarian cancer detected in the Programof Genetic Counseling in Cancer of Valencian Community(eastern Spain). Relationship of family phenotypes with mutationprevalence
Inmaculada de Juan Jimenez • Zaida Garcıa Casado • Sarai Palanca Suela •
Eva Esteban Cardenosa • Jose Antonio Lopez Guerrero • Angel Segura Huerta •
Isabel Chirivella Gonzalez • Ana Beatriz Sanchez Heras • Ma Jose Juan Fita •
Isabel Tena Garcıa • Carmen Guillen Ponce • Eduardo Martınez de Duenas •
Ignacio Romero Noguera • Dolores Salas Trejo • Mercedes Goicoechea Saez •
Pascual Bolufer Gilabert
� Springer Science+Business Media Dordrecht 2013
Abstract During the first 6 years of the Program of
Genetic Counselling in Cancer of Valencia (eastern Spain),
310 mutations (155 in BRCA1 and 155 in BRCA2) in 1,763
hereditary breast (BC) and ovarian cancer (OC) families
were identified. Of the mutations found 105 were distinct (53
in BRCA1 and 52 in BRCA2), eight new and 37 recurrent.
Two of the novel mutations were frame-shift placed in exons
2 and 11 of BRCA1 and the remaining six were placed in
BRCA2; four frame-shift (three in exon 11 and one in exon
23), one deletion of the entire exon 19 and one in the inter-
vening sequence of exon 22. The BRCA1 mutations with
higher recurrence were c.66_68delAG, c.5123C [ A,
c.1961delA, c.3770_3771delAG and c.5152?5G [ A that
covered 45.2 % of mutations of this gene. The age of onset of
BCs of c.68_69delAG mutation carriers occurs later than for
the other recurrent mutations of this gene (45 vs. 37 years;
p = 0.008). The BRCA2 mutations with higher recurrence
were c.9026_9030delATCAT, c.3264insT and c.8978_
8991del14 which represented 43.2 % of all mutations in this
gene, being the most recurrent mutation by far c.9026_
9030delATCAT that represents 21.3 % of BRCA2 mutations
and 10.6 % of all mutations. Probands with family histories
of BC and OC, or OC and/or BC in at least two first degree
relatives, were the more likely to have BRCA1/BRCA2
mutations (35.2 % of the total mutations). And that most
BRCA1mutations (73.19 % mutations) occurred in probands
with early-onset BC or with family history of OC.
This study is conducted on behalf of the Group for Assessment for
Hereditary Cancer of Valencian Community.
I. de Juan Jimenez � S. Palanca Suela � E. Esteban Cardenosa �P. Bolufer Gilabert (&)
Laboratory of Molecular Biology, Service of Clinical Analysis,
Escuela de Enfermerıa 7a, Hospital Universitario La Fe, Avd.
Campanar 21, 46009 Valencia, Spain
e-mail: [email protected]
Z. Garcıa Casado � J. A. Lopez Guerrero
Laboratory of Molecular Biology, Instituto Valenciano
de Oncologia (IVO), Valencia, Spain
A. Segura Huerta
Genetic Counseling Unit, Hospital La Fe, Valencia, Spain
I. Chirivella Gonzalez
Genetic Counseling Unit, Clinic Hospital, Valencia, Spain
A. B. Sanchez Heras � C. Guillen Ponce
Genetic Counseling Unit, Hospital de Elche, Alicante, Spain
M. J. Juan Fita � I. Romero Noguera
Genetic Counseling Unit, Instituto Valenciano de Oncologia
(IVO), Valencia, Spain
I. Tena Garcıa � E. Martınez de Duenas
Genetic Counseling Unit, Hospital General de Castellon,
Castellon, Spain
D. Salas Trejo � M. Goicoechea Saez
Department of Public Health, Cancer Plan Office, Conselleria
de Sanitat, Valencia, Spain
123
Familial Cancer
DOI 10.1007/s10689-013-9622-2
Keywords Hereditary breast and ovarian cancer
(HBOC) � BRCA1 � BRCA2 � Recurrent mutations � Novel
mutations � Relationship genotype-phenotype
Introduction
Germline mutations in BRCA1 and BRCA2 (BRCA1/
BRCA2) genes predispose to breast cancer (BC) and/or
ovarian cancer (OC). Genetic studies on BRCA1/BRCA2 [1,
2] are poorly efficient in detecting mutations since rarely
exceed the 20–25 % of mutations in a population at risk of
BC or OC. In most populations, recurrent BRCA1/BRCA2
mutations are not relevant, except those described in Ash-
kenazi Jewish [3, 4], Iceland [5] or Chinese populations [6,
7]. Moreover, the mutational spectrum varies widely among
ethnic groups and geographic regions [7–9] and often new
mutations are detected. However, knowledge of the muta-
tional spectra of populations together with the identification
of the most relevant recurrent mutations may be of help to
address the genetic studies of BRCA1/BRCA2 mutations,
beginning by the most prevalent recurrent mutations.
The association of personal and family history with a
higher likelihood of having BRCA1/BRCA2 mutations has
been reported [11, 12]. However, by applying such criteria
in only 20–25 % of cases pathogenic mutations are
detected in BRCA1/BRCA2, while the remaining 80 %
are non-informative, in whom no pathogenic mutations are
found. The low efficacy of these criteria demands the
search for new efficient criteria to select the individuals or
families with higher probability of harbouring deleterious
mutations in BRCA1/BRCA2 genes [10].
Another unsolved issue nowadays is the genotype-phe-
notype relationship and also the different expressivity of
BRCA mutations on the age of onset of cancer [13, 14]. In the
present study, we wish to take advantage of the experience
gathered during the first 6 years of the Program of Genetic
Counseling in Cancer of Valencia (eastern Spain) trying to
clarify the aforementioned issues. This study describes the
novel and relevant recurrent mutations detected in BRCA1/
BRCA2 genes in individuals or families with high risk of BC
and OC studied in the Program and investigates the rela-
tionship of the different types of personal or family history
with the likelihood of harboring mutations.
Patients and methods
Patients and subjects
The 1,763 index cases (ICs) of individuals or families at
risk of hereditary BC/OC (HBOC) here included, were
selected by the units of Genetic Counselling in Cancer
according to any of the eight distinct individual or family
phenotypes established in the Program of Genetic Coun-
selling in Cancer of Valencia Community [15] (Table 1).
All tumor diagnoses of ICs were confirmed by medical and
histological reports.
All patients were informed of the purpose of the study,
their implications and genetic testing limitations. All the
patients showed their agreement by signing the informed
consent elaborated by the Health Department in accordance
with the recommendations of the Declaration of Human
Rights, the Conference of Helsinki [16] and institutional
regulations, and approved by the Hospital Ethics Committee.
Methods
BRCA1/BRCA2 mutation analysis
Genomic DNA was extracted from peripheral blood. All
BRCA1/BRCA2 exons and exon–intron flanking boundaries
were amplified by PCR using the primer pairs and PCR
conditions reported in the Breast Cancer Information Core
(BIC) [17]. Mutational screening was carried out by pre-
screening the heteroduplex of PCR amplicons by confor-
mation sensitive gel electrophoresis [18], followed by
direct sequencing of the PCR products where heterodu-
plexes were identified [19]. Large genomic rearrangements
(LGRs) of BRCA1/BRCA2 were performed by multiplex
ligation-dependent probe amplification (MLPA) technique
[20] on DNA using MRC Holland kits (Netherlands),
according to the manufacturer’s instructions.
Mutation nomenclature
Nucleotide numbering has been taken from RefSeqGene
(http://www.ncbi.nlm.nih.gov/refseq/rsg/), NC_000017.10
(41196312–41277500) for BRCA1 and NC_000013.10
(32889617–32973809) for BRCA2. The mutations have
been designed according to the guidelines of the Human
Genome Variation Society (HGVS) [21].
We consider recurrent mutations those that occurred
more than once, although in this study we only pointed out
those with prevalence above 6 %.
Statistics
The estimation of mutation carrier probabilities in BRCA1
or BRCA2 genes for the different familial phenotypes
(inclusion criteria) was computed by conditional logistic
regression with covariates (step by step; backwards Wald).
Familial phenotypes were introduced as independent vari-
ables and mutation types as dependent variables.
To compare two proportions we used the Z-test.
The non-parametric test U-Mann–Whitney (UMW) or
I. de Juan Jimenez et al.
123
Ta
ble
1-
Fam
ily
ph
eno
typ
esan
do
nco
log
ical
feat
ure
so
fth
ed
iffe
ren
tg
rou
ps
esta
bli
shed
Incl
uss
ion
crit
eria
&F
amil
y
ph
eno
typ
es
BR
CA
XIC
ICw
ith
BR
CA
1m
uta
tio
nIC
wit
hB
RC
A2
mu
tati
on
No
(%)
BC
/
bB
C
OC
/
BC
?O
C/
bB
C?
OC
Ag
eat
dia
gn
osi
saN
o(%
)B
C/
bB
C
OC
/
BC
?O
C/
bB
C?
OC
Ag
eat
dia
gn
osi
saN
o(%
)B
C/
bB
C
OC
/
BC
?O
C/
bB
C?
OC
Ag
eat
dia
gn
osi
sa
Fam
ilie
sw
ith
asi
ng
leca
se
wit
hB
Co
rO
C
25
3(1
7.4
)2
15
38
31
(21
–8
0)
32
(20
.6)
17
15
34
.5(2
0–
61
)2
0(1
2.9
)1
55
33
(27
–6
1)
1B
C\
30
yea
rs5
6(3
.8)
56
02
8(2
6–
29
)5
(3.2
)5
02
4.5
(20
–2
9)
6(3
.87
)6
03
0(2
0–
30
)
2Is
ola
ted
or
bil
ater
alB
C
\4
0y
ears
15
5(1
0.7
)1
54
13
2(3
1–
40
)1
2(7
.7)
12
03
3(3
2–
40
)7
(4.5
1)
70
32
(31
–4
0)
3B
Can
dO
Cin
the
sam
e
pat
ien
t
42
(2.9
)5
37
53
.5(2
6–
80
)1
5(9
.7)
01
54
5(3
2–
61
)7
(3.8
7)
25
47
(41
–6
1)
Fam
ilie
sw
ith
two
firs
td
egre
e
rela
tiv
esw
ith
BC
or
OC
78
3(5
3.9
)7
26
57
44
(21
–8
5)
83
(53
.5)
40
43
45
(26
–7
9)
81
(52
.2)
69
12
44
(27
–7
8)
4T
wo
rela
tiv
esw
ith
BC
or
bil
ater
alB
C,
atle
ast
on
of
them
dia
gn
ose
d
bef
ore
50
yea
rs
62
9(4
3.3
)6
23
64
4(2
4–
85
)3
2(2
0.6
)2
93
43
(26
–6
8)
62
(40
.26
)5
93
41
(27
–7
3)
5T
wo
or
mo
reO
Cin
1st
deg
ree
rela
tiv
es
25
(1.7
)7
18
44
(27
–6
8)
13
(8.4
)2
11
46
(34
–6
8)
1(0
.65
)1
04
4(4
4)
6O
ne
BC
and
on
eO
Cin
two
1st
deg
ree
rela
tiv
es
91
(6.3
)5
83
34
8(2
1–
75
)3
8(2
4.5
)9
29
46
(29
–7
9)
12
(8.4
4)
39
54
(36
–7
8)
7O
ne
BC
inw
om
anan
d
on
ein
man
1st
deg
ree
rela
tiv
es.
38
(2.6
)3
80
58
(32
–7
8)
0(0
)0
0–
6(3
.90
)6
05
3(4
0–
77
)
Fam
ilie
sw
ith
thre
eo
rm
or
seco
nd
deg
ree
rela
tiv
esw
ith
BC
,at
leas
ttw
om
ust
be
firs
t
deg
ree
40
8(2
8.1
)4
08
04
7(2
1–
86
)4
0(2
5.8
)4
00
42
(25
–7
3)
52
(33
.55
)5
20
39
(29
–7
7)
8F
amil
ies
wit
ho
ut
OC
and
wit
hth
ree
or
mo
re
case
so
fB
C.T
wo
of
them
hav
eto
be
firs
t-
deg
ree
40
8(2
8.1
)4
08
04
7(2
1–
86
)4
0(2
5.8
)4
00
42
(25
–7
3)
52
(34
.42
)5
20
39
(29
–7
7)
Oth
ercr
iter
iab
9(0
.6)
63
41
.5(2
3–
66
)0
(0)
00
2(1
.30
)2
04
6(3
3–
59
)
To
tal
(%)
14
53
(10
0)
13
55
(93
.3)
98
(6.7
)4
4(2
1–
86
)1
55
(10
0)
97 (6
2.6
)
58
(37
.4)
42
.8(2
0–
79
)1
55
(10
0)
13
8 (89
.6)
17
(10
.4)
41
.2(2
7–
78
)
ICin
dex
case
s,N
on
um
ber
of
IC,
BC
bre
ast
can
cer,
bB
Cb
ilat
eral
bre
ast
can
cer,
OC
ov
aria
nca
nce
ra
Med
ian
(ran
ge)
bR
elat
ives
wit
hco
lore
ctal
can
cer
dia
gn
ose
db
efo
reth
eag
eo
f5
0o
rp
atie
nts
wh
od
idn
ot
fulfi
llth
ein
clu
sio
ncr
iter
iast
rict
ly
Relationship of family phenotypes
123
Kruskal–Wallis (KW) were used for comparison of two
independent or more independent quantitative variables,
respectively. A p value \0.05 was considered statistically
significant. All the computations were performed using the
SPSS v11.0 statistical package (Chicago, IL).
Results
We identified 310 pathogenic mutations among 1,763
HBOC families studied (17.6 %), 155 in BRCA1 and 155 in
BRCA2 (Table 1). From these, 105 were distinct mutations
(53 in BRCA1 and 52 in BRCA2), eight novel and 37
recurrent mutations.
Novel and recurrent mutations of BRCA1
Mutational spectra
In BRCA1 we found 53 distinct mutations in 155 families
(Table 2). The most prevalent mutations were frame-shift
(FS) (52.2 %) and missense (MS) (14.8 %). Nonsense (NS),
mutations in the intervening sequence (IVS) and large rear-
rangements (LGRs) represent *11 % all together.
Novel mutations
In BRCA1, we identified two novel mutations that are not
listed in the conventional databases [17, 22, 23] and, as far
as we know, have not been published (Table 2). The
c.51_63del13 of exon 2 found in a 50-year-old male with
three basal cell carcinomas and the c.3084insT of exon 11
detected in a 40-year-old female with OC.
Recurrent mutations of BRCA1
The mutations with recurrences above 6.0 % were c.66_
68delAG, c.1961delA, c.3770_3771delAG, c.5123C [A and c.5152?5G[ A, with recurrences of 12.9, 9.0, 7.1,
10.3 and 5.8 %, respectively, all together adding 45.1 % of the
total mutations of this gene (Table 2). The predominant
family phenotypes of ICs were the 3, 4 and 6 and the OC was
present in 22.85 % of them (16/70).
Novel and recurrent mutations of BRCA2
Mutational spectra
In BRCA2 we detected 52 distinct mutations in 155 ICs.
The most frequent mutations were, by far, the FS that
represents the 81.29 % of all mutations, followed by NS
with 12.9 % (Table 3). IVS was present in 5.16 % and only
one LGR was found.
Novel mutations
Six novel mutations were found in this gene, four FS (three
in exon 11 and one in exon 23), one deletion of the entire
exon 19 and one mutation in the IVS of exon 22 that were
not included in the conventional databases [17, 22, 23] and,
as far as we know, have not yet been published (Table 3).
Of the novel FS detected, three were located in exon 11 of
BRCA2, the c.3265_3266delAG identified in an IC diag-
nosed of BC at 43 years, the c.3381delT detected in an IC
with early onset BC diagnosed at 31 years, the c.6129delA
was identified in a patient with bilateral BC (bBC) diagnosed
at 50 and the c.9023delT of exon 23 found in a patient with
BC diagnosed at age of 47 (Table 3). The deletion of the
entire exon 19 was found in a patient with bBC diagnosed at
42 years and the c.8954-5A [ G of exon 22 were detected in
two patients with BC diagnosed at a median age of 44.
Recurrent mutations in BRCA2
The mutations of BRCA2 with recurrence above 5 % were
c.9026_9030delATCAT, c.3264insT and c.8978_8991del14
with recurrences of 21.29, 14.19 and 7.74 %, respectively, all
together adding 43.2 % of the mutations in this gene
(Table 3). Here, the predominant family types of ICs were the
4 and 8, and the major tumor was BC with 68.6 % (46/67),
whereas OC was only present in 14.9 % (10/67) of the ICs.
Mutations and tumors of IC
Of the 1,763 HBOC ICs studied, 90.2 % had BC or bBC
and 9.8 % OC, with or without BC (Table 1).
The ICs carrying BRCA1 mutations showed a distinct
spectrum of tumors than the BRCA2 mutation carriers and
non-informative cases. Thus, prevalence of OC, alone or
combined with BC or bBC in BRCA1 carriers is significantly
higher than that in BRCA2 mutation carriers and non-infor-
mative cases [37.4 % in BRCA1 carriers vs. 7.1 % in BRCA2
carriers plus non-informative cases; p = 0.0001; Table 1].
Additionally BRCA1 mutation carriers were particularly
linked with OC [20.6 % in BRCA1 vs. 4.1 % in BRCA2 plus
non-informative cases; p \ 0.001] and with bBC [10.3 % in
BRCA1 vs. 8.3 % in BRCA2 plus non-informative cases;
p = 5.14E-06]. However BRCA2 mutation carriers and
non-informative cases share similar tumor spectra, although
bBC showed a higher prevalence in BRCA2 carriers than in
non-informative cases [12.9 % in BRCA1 mutations vs.
7.8 % bBC in non-informative, p = 0.012)].
Mutations and age of onset of the tumors in ICs
With regard to recurrent mutations, we found that the
median age of onset of BCs of c.68_69 delAG BRCA1
I. de Juan Jimenez et al.
123
Ta
ble
2B
RC
A1
mu
tati
on
spec
tru
man
do
nco
log
ical
and
ph
eno
typ
icch
arac
teri
stic
so
fth
ein
dex
case
s
Exon
/
intr
on
Muta
tion
(HG
VS
Nom
encl
ature
)
Mut
Tip
e
Pro
tein
chan
ge
No
Cas
es
(%)
Med
ian
Age
(Ran
ge)
Fam
ily
phen
oty
pes
aT
um
ors
of
ICR
efer
ence
s
12
34
56
78
BC
bB
CB
C?
OC
bB
C?
OC
OC
Oth
ers
1–2
Del
Ex
1–2
LG
R5
(3.2
2)
44
(25–48)
11
12
21
11
1–24
Del
Ex
1–24
LG
R1
(0.6
4)
29
11
3D
elE
x3
LG
R1
(0.6
4)
32
11
3–5
Del
Ex
3–5
LG
R4
(2.5
8)
32
(30–45)
11
11
31
5–7
Del
Ex
5–7
LG
R1
(0.6
4)
34
11
8D
elE
x8
LG
R1
(0.6
4)
38
11
8–13
Del
Ex
8–13
LG
R3
(1.9
3)
47
(27–79)
11
12
1
20
Del
Ex
20
LG
R1
(0.6
4)
42
11
2c.
51_63del
13
FS
p.A
la17A
lafs
X2
1(0
.64)
46
11
Novel
2c.
68_69d
elA
GF
Sp
.Glu
23V
alf
sX17
20
(12.9
)44
(30–73)
11
24
15
613
12
4[1
7,
19
,22
,24
,
25
,33
]
5c.
211A
[G
MS
p.A
rg71G
ly4
(2.5
8)
43
(30–60)
21
12
11
[17
,19
,22
,25
,
29
,33
,34
]
I–5
c.212
?1G
[A
IVS
4(2
.58)
44
(34–48)
11
11
11
2[1
7,
19
,22
,25
,
29
,32
,33
]
I–5
c.213–12A
[G
IVS
1(0
.64)
33
11
[17
]
7c.
304_305in
sAF
Sp.A
la102A
spfs
X4
1(0
.64)
39
11
[33
]
11
c.958_959in
sAC
FS
p.A
rg320A
snfs
X20
1(0
.64)
49
11
[22
]
11
c.981_982del
AT
FS
p.C
ys3
28X
1(0
.64)
35
11
[17
,22
]
11
c.1121_1123del
insT
FS
p.T
hr3
74Il
efsX
31
(0.6
4)
49
11
[17
,22
,25
]
11
c.1504_1508del
TT
AA
AF
Sp.L
eu502A
lafs
X2
2(1
.28)
42
(38–45)
11
2[1
7,
19
,26
,29
,
31
]
11
c.1570del
GF
Sp.A
la524G
lnfs
91
(0.6
4)
33
11
[17
,32
]
11
c.1674del
AF
Sp.G
ly559V
alfs
X13
1(0
.64)
43
11
[17
,22
]
11
c.1687C
[T
NS
p.G
ln563X
1(0
.64)
29
11
[17
,22
]
11
c.1953_1956del
GA
AA
FS
p.L
ys6
53S
erfs
X47
2(1
.28)
33
(25–40)
22
[17
,19
,22
]
11
c.1961d
elA
FS
p.L
ys6
54S
erfs
X47
14
(9.0
3)
43
(24–68)
11
33
51
61
16
[17
,19
,22
,25
]
11
c.2690_2691in
sAF
Sp.P
ro897P
rofs
X6
1(0
.64)
29
11
[17
]
11
c.3084_3085in
sTF
Sp.A
rg1028S
erfs
X2
1(0
.64)
40
11
Novel
11
c.3257T
[G
NS
p.L
eu1086X
4(2
.58)
44
(41–51)
11
11
11
2[1
7,
22
]
11
c.3331_3334del
CA
AG
FS
p.G
ln1111A
snfs
X5
5(3
.22)
51
(42–59)
11
12
31
1[1
7,
22
]
11
c.3359_3360del
TT
FS
p.V
al1120G
lufs
X12
1(0
.64)
61
11
[17
,22
]
11
c.3481_3491del
11
FS
p.G
lu1161P
hef
sX3
3(2
.0)
58
(38–63)
12
21
[17
,22
,25
]
11
c.3581del
CF
Sp.T
hr1
194T
hrf
sX16
1(0
.64)
53
11
[17
,19
]
11
c.3627_3628in
sAF
Sp.G
lu1210A
rgfs
X9
3(2
.0)
48
(37–51)
11
11
11
[17
,22
]
11
c.3695_3699del
GT
AA
AF
Sp.V
al1234G
lnfs
X8
1(0
.64)
61
11
[22
]
11
c.3759_3760in
sTF
Sp.S
er1253P
hef
sX2
1(0
.64)
43
11
[14
]
11
c.3770_3771d
elA
GF
Sp
.Glu
1257G
lyfs
X9
11
(7.1
)43
(31–59)
11
27
61
21
1[1
7,
19
,22
,29
]
Relationship of family phenotypes
123
Ta
ble
2co
nti
nu
ed
Exon
/
intr
on
Muta
tion
(HG
VS
Nom
encl
ature
)
Mut
Tip
e
Pro
tein
chan
ge
No
Cas
es
(%)
Med
ian
Age
(Ran
ge)
Fam
ily
phen
oty
pes
aT
um
ors
of
ICR
efer
ence
s
12
34
56
78
BC
bB
CB
C?
OC
bB
C?
OC
OC
Oth
ers
11
c.3785C
[A
NS
p.S
er1262X
1(0
.64)
53
11
[17
,19
]
11
c.4065_4068del
TC
AA
FS
p.A
sn1355L
ysf
sX10
1(0
.64)
48
11
[17
,22
,24
,30
]
12
c.4161_4162del
TC
FS
p.G
ln1388G
lufs
X2
1(0
.64)
44
11
[17
,19
]
13
c.4287C
[A
NS
p.T
yr1
429X
5(3
.22)
48
(31–57)
12
11
31
1[1
9,
22
]
13
c.4307_4308del
CT
FS
p.S
er1436P
hef
sX4
2(1
.28)
44
(37–50)
11
11
[22
,33
]
13
c.4357del
GF
Sp.A
la1453G
lnfs
X3
1(0
.64)
42
11
[33
]
14
c.4375A
[T
NS
p.L
ys1
459X
1(0
.64)
41
11
[17
]
15
c.4552C
[T
NS
p.G
ln1518X
1(0
.64)
31
11
[17
]
I–15
c.4675
?1G
[A
IVS
1(0
.64)
35
11
[17
]
16
c.4810C
[T
NS
p.G
ln1604X
1(0
.64)
40
11
[17
,22
]
17
c.5030_5033del
CT
AA
FS
p.T
hr1
677Il
efsX
21
(0.6
4)
60
11
[17
,22
]
18
c.5095C
[T
MS
p.A
rg1699T
rp2
(1.2
8)
50
(47–52)
11
11
[17
,22
]
18
c.5117G
[C
MS
p.G
ly1706A
la1
(0.6
4)
41
11
[17
,22
,25
]
18
c.5123C
>A
MS
p.A
la1708G
lu16
(10.3
)36
(26–68)
26
62
10
22
2[1
7,
19
,22
,25
–
29
]
I–18
c.5152
15G
>A
IVS
r.5194_5271d
el9
(5.8
)37
(20–61)
12
22
11
52
2[1
7,
22
,25
]
I–18
c.5153–1G
[A
IVS
1(0
.6)
48
11
[17
,19
,25
,26
,
29
]
19
c.5154G
[A
NS
p.T
rp1718X
3(2
.0)
45
(34–49)
12
3[1
7,
33
]
20
c.5363_5364in
sCF
Sp.S
er1755S
erfs
X74
2(1
.2)
46
(46)
22
[17
,24
,30
]
21
c.5311_5333del
FS
p.P
ro1771S
erfs
X50
1(0
.64)
49
11
[17
,22
,32
,33
]
TO
TA
L53
155
43
(20–73)
614
18
36
18
44
748
88
16
17
132
1
Under
lined
muta
tions:
no
chan
ges
des
crib
edbef
ore
,bold
muta
tions:
Rec
urr
ent
var
iati
ons
ICin
dex
case
s,N
onum
ber
of
IC,
BC
bre
ast
cance
r,bB
Cbil
ater
albre
ast
cance
r,O
Covar
ian
cance
r,L
GR
larg
ere
agem
ent,
FS
fram
e-sh
ift,
IVS
inte
rven
ing
sequen
ce,
NS
nonse
nse
,M
Sm
isse
nse
aS
eeT
able
1
I. de Juan Jimenez et al.
123
Ta
ble
3B
RC
A2
mu
tati
on
spec
tru
man
do
nco
log
ical
and
ph
eno
typ
icch
arac
teri
stic
so
fth
ein
dex
case
s
Ex
on
/
Intr
on
Mu
tati
on
(HG
VS
No
men
clat
ure
)
Mu
t
Tip
e
Pro
tein
Ch
ang
eN8
Cas
es
(%)
Med
ian
Ag
e
(Ran
ge)
Fam
ily
Ph
eno
typ
esa
Tu
mo
rso
fIC
Ref
eren
ces
12
34
56
78
[8
BC
bB
CB
C?
OC
OC
3c.
14
5G
[T
NS
p.G
lu4
9X
2(1
.29
)4
5(3
2–
58
)1
12
[17
,2
2,
33
]
3c.
26
1_
26
2d
elC
TF
Sp
.Leu
88
Ala
fsX
12
3(1
.93
)3
4(3
3–
44
)3
3[1
7,
22,
33
]
4c.
37
0d
elA
FS
p.M
et1
24
Trp
fsX
12
1(0
.65
)5
81
1[3
3]
10
c.1
02
3T
[C
NS
p.C
ys3
41
X1
(0.6
5)
27
11
[22
]
10
c.1
12
6d
elT
FS
p.P
he3
76
Leu
fsX
23
1(0
.65
)3
81
1[2
2,
33]
10
c.1
31
0_
13
13
del
AA
GA
FS
p.L
ys4
37
Ilfs
X2
21
(0.6
5)
33
11
[17
,2
2,
25
,3
3]
10
c.1
36
6_
13
67
del
GA
FS
p.G
lu4
56
Glu
fsX
51
(0.6
5)
29
11
[33
]
10
c.1
44
8_
14
49
del
AG
insT
TA
CF
Sp
.Ala
48
3A
lafs
X3
1(0
.65
)3
71
1[3
3]
10
c.1
60
7_
16
07
insA
FS
p.S
er5
36
X4
(2.5
8)
37
(34
–3
9)
22
4[1
7,
25,
32
]
10
c.1
64
6_
16
49
del
AG
GA
FS
p.L
ys5
49
Arg
fsX
71
(0.6
5)
34
11
[33
]
10
c.1
84
2d
up
FS
p.A
sn6
15
X1
(0.6
5)
57
11
[17
,3
3]
11
c.2
70
1d
elC
FS
p.A
la9
02
Leu
fsX
23
(1.9
3)
33
(27
–3
8)
21
3[2
2,
27]
11
c.2
80
8_
28
11
del
AC
AA
FS
p.A
la9
38
Pro
fsX
21
5(3
.22
)3
6(3
0–
44
)1
11
11
41
[17
,1
9,
22
,2
5,
26,
29]
11
c.3
17
0_
31
27
4d
elA
GA
AA
FS
p.L
ys1
05
7T
hrf
sX8
1(0
.65
)3
71
1[1
7,
22]
11
c.3
26
4_
32
65
insT
FS
p.G
ln1
08
9S
erfs
X1
02
2(1
4.2
)4
0(2
9–
69
)2
18
31
71
63
12
[17
,2
2,
25,2
7–
29,
33]
11
c.3
26
5_
32
66
del
AG
FS
p.G
ln1
08
9H
isfs
X9
1(0
.65
)4
31
1N
ov
el
11
c.3
38
1d
elT
FS
p.P
he1
12
7L
eufs
X2
31
(0.6
5)
31
11
No
vel
11
c.3
84
7_
38
48
del
GT
FS
p.V
al1
28
3L
ysf
sX2
1(0
.65
)3
41
1[1
7,
22,
33
]
11
c.3
92
2G
[T
NS
p.G
lu1
30
8X
7(4
.51
)4
5(2
9–
56
)3
13
61
[17
,1
9,
22
,2
5,
29,
33]
11
c.4
79
7d
elT
FS
p.A
sn1
59
9L
ysf
s18
3(1
.93
)3
4(3
3–
39
)1
23
[17
,1
9,
32
,3
3]
11
c.4
93
6_
49
39
del
GA
AA
FS
p.G
lu1
64
6G
lnfs
X2
31
(0.6
5)
38
11
[17
,1
9,
22
,2
9,
33]
11
c.5
11
2_
51
15
del
AA
TA
FS
p.A
sn1
70
6L
eufs
X5
1(0
.65
)2
91
1[2
2,
33]
11
c.5
12
9_
51
32
del
AT
GT
FS
p.T
yr1
71
0fs
X1
(0.6
5)
37
11
[17
,2
2]
11
c.5
14
6_
51
49
del
TA
TG
FS
p.T
yr1
71
6L
ysf
sX8
1(0
.65
)4
11
1[2
6,
27,
31
]
11
c.5
28
6_
52
87
del
TC
FS
p.T
yr1
76
2T
yrf
sX1
51
(0.6
5)
42
11
[17
,3
3]
11
c.5
34
5_
53
46
insA
FS
p.G
ln1
78
2G
lnfs
X5
1(0
.65
)4
01
1[3
0]
11
c.5
57
6_
55
79
del
TT
AA
FS
p.I
le1
85
9L
ysf
sX3
3(1
.93
)5
8(4
6–
60
)1
11
11
1[1
7,
25
–2
7,
33]
11
c.5
72
0_
57
23
del
CT
CT
FS
p.S
er1
90
7X
2(1
.29
)4
0(3
3–
46
)2
2[1
7,
33]
11
c.5
89
0d
elA
FS
p.L
ys1
96
4S
erfs
X4
01
(0.6
5)
46
11
[17
,3
3]
11
c.5
94
6d
elT
FS
p.S
er1
98
2A
rgfs
X2
21
(0.6
5)
47
11
[17
,2
2,
30
,3
3]
11
c.6
07
1d
elA
FS
p.G
ln2
02
4A
rgfs
X1
61
(0.6
5)
58
11
[17
,3
3]
Relationship of family phenotypes
123
Ta
ble
3co
nti
nu
ed
Ex
on
/
Intr
on
Mu
tati
on
(HG
VS
No
men
clat
ure
)
Mu
t
Tip
e
Pro
tein
Ch
ang
eN8
Cas
es
(%)
Med
ian
Ag
e
(Ran
ge)
Fam
ily
Ph
eno
typ
esa
Tu
mo
rso
fIC
Ref
eren
ces
12
34
56
78
[8
BC
bB
CB
C?
OC
OC
11
c.6
12
9d
elA
FS
p.T
hr2
04
2T
hrf
sX9
1(0
.65
)5
01
1N
ov
el
11
c.6
27
5_
62
76
del
TT
FS
p.L
eu2
09
2P
rofs
X7
6(3
.87
)4
3(3
2–
73
)3
12
51
[17
,2
2,
25
,2
6,
29,
30]
11
c.6
48
6_
64
89
del
AC
AA
FS
p.L
ys2
16
2A
snfs
X5
2(1
.29
)4
1(3
2–
49
)2
2[1
7,
22]
11
c.6
49
4d
elT
FS
p.L
eu2
16
5T
rpfs
X3
1(0
.65
)3
61
1[3
2,
33]
11
c.6
62
9_
66
30
del
AA
FS
p.G
lu2
21
0G
lyfs
X1
41
(0.6
5)
76
11
[17
,2
2,
25
,3
3]
14
c.7
17
1_
71
72
insA
FS
p.G
lu2
39
1A
rgfs
X6
01
(0.6
5)
31
11
[33
]
14
c.7
23
4_
72
35
insG
FS
p.T
hr2
41
2A
spfs
X2
1(0
.65
)3
21
1[2
2,
33]
I–1
5c.
76
17
?1
G[
AIV
S1
(0.6
5)
37
11
[17
,2
2]
17
c.7
86
3T
[A
NS
p.T
yr2
62
1X
1(0
.65
)3
91
1[1
7,
33]
17
c.7
97
7–
1G
[C
IVS
4(2
.58
)4
6(3
6–
51
)1
21
31
[17
,2
2]
18
c.8
04
2_
80
43
del
CA
FS
p.T
hr2
68
1S
erfs
X1
11
(0.6
5)
38
11
[17
,3
3]
19
Del
Ex
19
LG
R1
(0.6
5)
42
11
No
vel
I–2
0c.
86
32
?2
T[
GIV
S1
(0.6
5)
27
11
[33
]
I–2
1c.
89
54
-5A
[G
IVS
2(1
.29
)4
4(3
9–
49
)2
2N
ov
el
23
c.8
97
8_
89
91
del
14
FS
p.S
er2
99
3P
hef
sX2
01
2(7
.7)
39
(31
–5
5)
11
21
61
10
11
[17
,1
9,
22
,2
5,
28,
33]
23
c.8
98
8_
89
90
del
AT
Ain
sTT
FS
p.L
eu2
99
6P
hef
sX5
1(0
.65
)5
11
1[3
3]
23
c.9
01
8C
[A
NS
p.T
yr3
00
6X
7(4
.51
)4
4(3
4–
61
)1
21
12
7[1
7,
19
,2
2,
25,
33]
23
c.9
02
3d
elT
FS
p.I
le3
00
8Il
efsX
20
1(0
.65
)4
71
1N
ov
el
23
c.9
02
6_
90
30
del
AT
CA
TF
Sp
.Ty
r30
09
Ser
fsX
73
3(2
1.3
)4
4(3
0–
78
)3
21
43
37
12
07
51
[26
,2
8,
31
]
25
c.9
28
6G
[T
NS
p.G
lu3
09
6X
1(0
.65
)5
91
1[1
7,
22,
25
]
25
c.9
46
6C
[T
NS
p.G
ln3
15
6X
1(0
.65
)3
81
1[1
7,
32,
33
]
To
tal
52
15
54
1(2
7–
78
)6
77
62
11
26
52
21
19
20
97
un
der
lin
edm
uta
tio
ns:
no
chan
ges
des
crib
edb
efo
re,
bo
ldm
uta
tio
ns:
Rec
urr
ent
var
iati
on
s
ICin
dex
case
s,N
on
um
ber
of
IC,
BC
bre
ast
can
cer,
bB
Cb
ilat
eral
bre
ast
can
cer,
OC
ov
aria
nca
nce
r,L
GR
larg
ere
agem
ent,
FS
fram
e-sh
ift,
IVS
inte
rven
ing
seq
uen
ce,
NS
no
nse
nse
aS
eeT
able
1
I. de Juan Jimenez et al.
123
mutation carriers occurs later than the BCs of recurrent
mutations placed in other exons of this gene [med-
ian(range) = 45(30–73) vs. 37(20–71) years; KW = 0.008].
However, we did not find differences in the age of presentation
of BC for the recurrent mutations of BRCA2.
Overall, BRCA1/BRCA2 mutation carriers showed earlier
age of onset of tumors than non-informative cases (median
age of 42 years in carriers vs. median of 44 years in non-
informative; UMW, z = -2.29; p = 0.02; Table 1). This is
due to the early age of onset of BC in mutation carriers
[median of 40 years in carriers vs. median of 43 years in non-
carriers; UMW; z = 4.05; p = 0.0001)]. However, the age
of onset of OC between carriers and non-carriers was not
modified [median of 50 years in carriers vs. 48 years in non-
carriers; UMW, z = -1.179; ns].
Family phenotypes and mutation prevalence
When we compare the different types of family phenotypes
of ICs BRCA1/BRCA2 mutation carriers with non-infor-
mative cases applying the logistic regression, the model
selects the phenotypes 1, 2, 3, 5, 6 and 8 as the more prone
to have pathogenic mutations, excluding phenotypes 4 and
7 (Table 4). From these, the ICs of families with pheno-
types 3, 5 and 6 showed the highest likelihood of having
mutations (86 mutations out of 244 ICs; 35.2 %) and the
phenotypes 1, 2 and 8 showed an intermediate chance of
harboring mutations (16.5 %; 122 mutations out of 741
families).
Family phenotypes and genes affected
We analyzed the influence of family phenotypes of ICs
with the likelihood of having a BRCA1 mutation. The
logistic regression selected the familial phenotypes 1, 3, 5,
6 and 8 as significantly linked with BRCA1 mutations. In
fact, these families gathered the 71.6 % of the pathogenic
mutations in this gene (Table 4).
When we analyzed the distribution of family phenotypes
between BRCA1 or BRCA2 mutations, the logistic regression
selected the phenotypes 1, 3, 5 and 6 as strongly linked with
BRCA1 mutations. In fact, these phenotypes constituted the
Table 4 Mutation prevalence and family genotypes
Mutations Phenotypes selecteda N.Families Number positives % § OR 95 % CI
BRCA1/2 versus non-informative 1 67 11b 16.4 1.8 0.98–3.30
N = 1753 2 174 19b 10.9 0.48 0.25–0.91
3 64 22b 34.4 2.72 1.49–4.96
5 39 14b 35.9 4.03 2.05–7.91
6 141 50b 35.5 3.7 2.47–5.52
8 500 92b 18.4 1.53 1.13–2.08
Total 985 208 21.11
BRCA1 versus non-informative 1 67 5c 7.5 4.05 1.19–8.58
N = 1608 3 64 15c 23.4 5.95 2.83–12.49
5 39 13c 33.3 12.73 6.12–26.48
6 141 38c 26.9 9.24 5.58–15.30
8 500 40c 8.0 2.23 1.43–3.54
Total 811 111 13.7
BRCA2 versus non-informative 2 174 7d 4.0 0.34 0.14–0.79
N = 1608
Total 174 7 4.0
BRCA1 versus BRCA2 1 11 5c 45.5e 0.3 0.01–0.91
N = 310 3 22 15c 88.2e 0.33 0.12–0.92
5 14 13c 92.8e 0.043 0.005–0.331
6 50 38c 76.0e 0.21 0.11–0.419
Total 97 71 73.2
Logistic regression with covariate
N: Cases in analysisa See Table 1, § = No positives/No families with this phenotypeb BRCA1/2 positives, c BRCA1 positives, d BRCA2 positivese BRCA1 positive/total positive for this family phenotype
Relationship of family phenotypes
123
73.19 % of the mutations of BRCA1, whereas only 26.8 % of
the mutations were in BRCA2 (Table 4).
Discussion
In this study, we identified eight novel mutations, two in
BRCA1 and six in BRCA2, which together with the nine we
already reported [33] make a total of 17 novel mutations
detected in the population/inhabitants of eastern Spain.
This relatively high incidence of novel mutations confers a
singularity to the population studied with regard to other
regions of Spain.
The c.66_68delAG recurrent mutation showed the
highest prevalence of BRCA1 of all the mutations in this
gene. This mutation of Ashkenazim ancestry was origi-
nated 46 generations ago [24]. The mutation is widespread
throughout many regions of Spain [19, 25, 27, 28, 31], with
the exception of Castilla-Leon [29] (the central area of the
Iberian Peninsula) where it is surprisingly absent.
The recurrent mutation c.1961delA with a prevalence of
9 % of BRCA1 mutations is related in ICs with OC. The
remainder recurrent mutations of BRCA1, c.3770_3771de-
lAG, c.5123C [ A and c.5152?5G [ A, showed a range of
prevalence between 5.8 % and 10.3 %. All these mutations
detected in Valencia Community were also spread through-
out different areas of Spain [19, 25–27, 31] with similar
prevalence [28]. However, no association was found
between particular family phenotypes and any of the recur-
rent mutations in BRCA1.
The recurrent mutation c.9026_9030delATCAT of
BRCA2 showed the highest prevalence in this gene and
among all mutations. This mutation was reported for the
first time by Neuhausen et al. [30] in French families and
later in families of Almeria [31] and the Basque Country
[26] in Spain. The prevalence of this mutation, of 21.3 % is
significantly higher than the 12.4 % (p = 0.043) reported
by Diez et al. [28] in other regions of Spain. The recurrent
mutation c.3264insT, apart from the Valencia Community
[33] it is also found in different areas of Spain [27–29].
However, the prevalence of 14.19 % here found for this
mutation is significantly higher (p = 0.017) than the 3.8 %
reported by Diez et al. [28] for the other Spanish areas. For
the recurrent mutation c.8978_8991del14 the prevalence of
7.7 % here found is similar to that detected in other areas
of Spain [25, 28]. As in BRCA1, no association was found
between some particular phenotype and any of the recur-
rent mutations for BRCA2.
The high prevalence of the recurrent mutations here
found enables to screen 41.3 % of our mutational spectra
(128/310) by testing only the seven recurrent, four in
BRCA1 and three in BRCA2.
The results here found show that the c.68_69delAG
BRCA1 recurrent mutation behaves as a low penetrant
mutation in comparison with the other recurrent mutations
of this gene, since the carriers of this mutations show a
later age of onset of BCs as it has been already reported
[35]. But in addition, the age of diagnosis of BCs in
mutation carriers occurs earlier than in non-informative
cases, in concordance with the increased risk conferred by
these mutations although it has not been confirmed com-
paring hereditary and sporadic breast cancer [36]. How-
ever, the presence of mutations in BRCA1 or BRCA2 did
not affect the age of onset of OC.
Our results support, as it has been reported, [11] that ICs
with family histories with large burden of OC, isolated or
in combination with BC, showed the highest prevalence of
pathogenic mutations (86 mutations in 244 ICs; 35.2 %).
Furthermore, these families together with BC of early onset
represent the 73.19 % (71 out of 97 mutations of these
family groups) of the mutations detected in BRCA1.
However, for BRCA2 mutations we did not find differences
in the prevalence among the different family types.
Conclusions
We should remark the relatively high incidence of novel
mutations what seems to be a singularity of our study
population.
Our results emphasize the importance of recurrent
mutations in our population, since with seven highest
recurrent mutations, 4 in BRCA1 and 3 in BRCA2, we cover
the 41.3 % (128 out 310) of the mutational spectra. At this
regard it is worth mentioning that the recurrent mutations
of the study population of BRCA2, particularly the
c.9026_9030delATCAT, showed a clearly higher preva-
lence than the found in other regions of Spain.
We have shown that the BRCA mutation carriers have
an early age of onset as compared with non-informative
cases what has not been shown for OC. In addition, the
c.68_69delAG BRCA1 recurrent mutation behaves as a low
penetrance mutation in comparison with the other recurrent
mutations of this gene.
The probands with family histories of BC and OC, or
OC and/or BC in at least two first degree relatives, were the
more likely to have BRCA1/BRCA2 mutations (35.2 % of
total mutations). And that most of mutations of BRCA1
(73.19 % of mutations) occurred in probands with early
onset BC or with family history of OC.
Acknowledgments We should express here our gratitude for the
good work and dedication to the laboratory technicians Virginia
Gonzalez, Teresa Barrachina and Gemma Perez, the last granted by
the Instituto de Investigacion Sanitaria del H.U. La Fe.
I. de Juan Jimenez et al.
123
Conflict of interest None to be declared.
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