Characterization of BRCA1 and BRCA2 splicing variants:

Breast Cancer Res Treat
DOI 10.1007/s10549-011-1674-0
PRECLINICAL STUDY
Characterization of BRCA1 and BRCA2 splicing variants:
a collaborative report by ENIGMA consortium members
Mads Thomassen • Ana Blanco • Marco Montagna • Thomas V. O. Hansen •
Inge S. Pedersen • Sara Gutiérrez-Enrı́quez • Mireia Menéndez • Laura Fachal
Marta Santamariña • Ane Y. Steffensen • Lars Jønson • Simona Agata •
Phillip Whiley • Silvia Tognazzo • Eva Tornero • Uffe B. Jensen •
Judith Balmaña • Torben A. Kruse • David E. Goldgar • Conxi Lázaro •
Orland Diez • Amanda B. Spurdle • Ana Vega
•
Received: 19 May 2011 / Accepted: 5 July 2011
Ó Springer Science+Business Media, LLC. 2011
Abstract Mutations in BRCA1 and BRCA2 predispose
carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of
variants with unknown clinical significance. The Evidence-
Amanda B. Spurdle and Ana Vega contributing equally to this study.
This study is conducted on behalf of the ENIGMA Consortium
Splicing Working Group.
based Network for the Interpretation of Germline Mutant
Alleles (ENIGMA) consortium was initiated to evaluate
and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial
project of the ENIGMA Splicing Working Group, we
report splicing and multifactorial likelihood analysis of 25
BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to
identify aberrant transcripts. The findings were compared
Electronic supplementary material The online version of this
article (doi:10.1007/s10549-011-1674-0) contains supplementary
material, which is available to authorized users.
M. Thomassen (&) T. A. Kruse
Department of Clinical Genetics, Odense University Hospital,
Soenderboulevard 29, 5000 Odense C, Denmark
e-mail: [email protected]
A. Blanco L. Fachal A. Vega
Fundación Pública Galega de Medicina Xenómica-SERGAS,
Grupo de Medicina Xenómica-USC, CIBERER, IDIS, Santiago
de Compostela, Spain
M. Montagna S. Agata S. Tognazzo
Immunology and Molecular Oncology Unit, Istituto Oncologico
Veneto IOV-IRCCS, Via Gattamelata 64, 35128 Padua, Italy
T. V. O. Hansen A. Y. Steffensen L. Jønson
Genomic Medicine, Department of Clinical Biochemistry,
Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9,
2100 Copenhagen, Denmark
I. S. Pedersen
Department of Clinical Biochemistry, Section of Molecular
Diagnostics, Aalborg University Hospital, Reberbansgade 15,
9000 Aalborg, Denmark
S. Gutiérrez-Enrı́quez O. Diez
Oncogenetics Laboratory, Vall d’Hebron Institute of Oncology
(VHIO), University Hospital Vall d’Hebron, Barcelona, Spain
M. Menéndez E. Tornero C. Lázaro
Genetic Diagnosis Unit, Hereditary Cancer Program, Institut
Català d’Oncologia, Hospital Duran i Reynals-Bellvitge
Biomedical Research Institute (IDIBELL), L’Hospitalet de
Llobregat, Barcelona, Spain
M. Santamariña
Grupo de Medicina Xenómica-USC, University of Santiago de
Compostela, CIBERER, IDIS, Santiago de Compostela, Spain
P. Whiley A. B. Spurdle
Genetics and Population Health Division, Queensland Institute
of Medical Research, 300 Herston Rd, Herston, Brisbane, QLD
4006, Australia
U. B. Jensen
Department of Clinical Genetics, Aarhus University Hospital,
Skejby, Brendstrupgaardsvej 21C, 8200 Aarhus N, Denmark
J. Balmaña
Medical Oncology Department, University Hospital Vall
d’Hebron, Barcelona, Spain
D. E. Goldgar
Department of Dermatology, University of Utah, Salt Lake City,
UT, USA
123
Breast Cancer Res Treat
to bioinformatic predictions using four programs. The
posterior probability of pathogenicity was estimated using
multifactorial likelihood analysis, including co-occurrence
with a deleterious mutation, segregation and/or report of
family history. Abnormal splicing patterns expected to lead
to a non-functional protein were observed for 7 variants
(BRCA1 c.441?2T[A, c.4184_4185?2del, c.4357?1G
[A, c.4987-2A[G, c.5074G[C, BRCA2 c.316?5G[A,
and c.8754?3G[C). Combined interpretation of splicing
and multifactorial analysis classified an initiation codon
variant (BRCA2 c.3G[A) as likely pathogenic, uncertain
clinical significance for 7 variants, and indicated low
clinical significance or unlikely pathogenicity for another
10 variants. Bioinformatic tools predicted disruption of
consensus donor or acceptor sites with high sensitivity, but
cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also
provide further evidence that clinical RNA-based assays
should be extended from analysis of invariant dinucleotides
to routinely include all variants located within the donor
and acceptor consensus splicing sites. Importantly, this
study demonstrates the added value of collaboration
between laboratories, and across disciplines, to collate and
interpret information from clinical testing laboratories to
consolidate patient management.
Keywords BRCA1 BRCA2 Splicing ENIGMA Splicing sites Splice mutations Multifactorial analysis
Introduction
Germline mutations in the BRCA1 (MIM 113705) and
BRCA2 (MIM 600185) genes predispose carriers to breast
and ovarian cancer [1–3]. Unfortunately, a considerable
proportion of sequence variants identified during routine
clinical testing are missense mutations, silent variations,
in-frame deletions and insertions, and intronic variants of
uncertain clinical significance, creating difficulties for
patient and family management. The Breast Cancer Information Core database (BIC, http://research.nhgri.nih.
gov/bic) contains almost 1,800 distinct variants that are
reported as having unknown clinical significance, more than
half of the total variants reported in BIC. A possible pathogenic mechanism for a subset of these variants is disruption
of normal mRNA splicing, particularly variants located near
to exon/intron boundaries that could lead to exon skipping,
cryptic splicing, or intron retention. Several bioinformatic
algorithms implemented in web-based programs have been
proposed for prediction of splicing effects of nucleotide
variants [4–9]. While these methods may become stand-
123
alone diagnostic tools in the future, it is important that splice
assays are performed in parallel with the bioinformatic predictions at this point in time. Firstly, it is acknowledged that
their sensitivity and specificity to predict likelihood of
disrupted splicing requires improvement, particularly for
variants lying outside the highly conserved AG and GT
acceptor and donor dinucleotides [10]. Secondly, the algorithms show poor performance for prediction of alternatively
used mechanisms, especially cryptic splice sites [11, 12], for
variants located within or outside the consensus dinucleotides. Thus, RNA analyses are necessary to determine the
alternatively used mechanisms for each variant, including
variants at the invariant dinucleotide positions assumed to
disrupt splicing, to identify transcripts of uncertain clinical
significance such as in-frame deletions and upregulation of
naturally occurring transcripts [13–16] for subsequent classification using statistical methods such as multifactorial
likelihood analysis. This alternative method for evaluating rare
sequence variants integrates data from several different
approaches, targeting independent characteristics associated
with known pathogenic mutations [17]. The model can include
data from co-segregation of variant and disease, evolutionary
conservation and physical/chemical properties of amino acid
changes, tumor pathological parameters, and family history
[10, 17]. Results from splicing and functional analysis may be
integrated in future likelihood models as well [18].
An international consortium entitled the Evidence-based
Network for the Interpretation of Germline Mutant Alleles
(ENIGMA; http://www.enigmaconsortium.org) has recently
been founded to promote large-scale collaborative studies of
BRCA1 and BRCA2 sequence variants that improve and
develop current variant classification methods, and to provide standardized classifications to relevant variant databases. The Splicing Working Group within this researchbased consortium has initiated several projects, including
studies aimed at identifying optimal standardized protocols
and prediction tools for characterizing splicing aberrations,
and assessing the consistency of interpretation of clinical
significance of splicing assay results. In addition, in this
preliminary ENIGMA Splicing Working Group survey, we
collated previously unpublished assay results from 7 different laboratories assessing splicing aberrations associated
with 25 different BRCA1 and BRCA2 variants, and applied 4
different bioinformatic prediction algorithms (SSF, MaxEntScan, NNSPlice, and GeneSplicer) to evaluate their
performance compared to the observed splicing results. In
addition, we evaluated the clinical significance of variants
using multifactorial likelihood modeling. Our results demonstrate the value of collaboration between laboratories and
across disciplines to improve the clinical interpretation of
rare sequence variants for clinical use.
Breast Cancer Res Treat
Materials and methods
Identification of sequence variants and testing
of patients
ENIGMA Splicing Working Group members were invited
to submit unpublished splicing assay results for BRCA1 and
BRCA2 variants for a combined publication. After exclusion of variants already classified as non pathogenic by
IARC (http://brca.iarc.fr/LOVD) or with similar splicing
analyses previously published by other groups, 25 variants
assayed across 7 different laboratories were identified as
suitable for inclusion: three Danish (Aalborg Hospital—
AAS, Odense University Hospital—OUH and Copenhagen
Breast Cancer Study, Rigshospitalet, Copenhagen University Hospital—CBCS), three Spanish (Fundación Pública
Galega de Medicina Xenómica, Santiago de Compostela—
FPGMX, Vall d’Hebron University Hospital—HVH, Catalan Institute of Oncology—ICO) and one Italian (Istituto
Oncologico Veneto, IOV, Padua). All variants were discovered by clinical testing in index cases of high risk
families for hereditary breast and ovarian cancer undergoing genetic counseling according to the respective
national recommendations. Written informed consent was
obtained in all cases. BRCA1/BRCA2 testing was performed using different approaches including sequencing,
denaturing high-performance liquid chromatography
(dHPLC), temperature gradient capillary electrophoresis
(TGCE), protein truncation test (PTT), conformation
sensitive capillary electrophoresis (CSCE), and multiplex
ligation dependent probe amplification assay (MLPA).
Variant nomenclature
The DNA numbering is based on the cDNA sequences,
NM_007294.3 for BRCA1 and NM_000059.3 for BRCA2,
except that exon numbering for BRCA1 was performed
according to U14680. The nomenclature follows the recommendations from the Human Genome Variation Society
(HGVS).
mRNA analysis assays
The 7 participating centers used two main strategies to
characterize the effect of sequence variants: RT-PCR followed by sequencing, and a mini gene assay. The laboratory protocols, PCR conditions, and primers used are
summarized in Supplementary Tables 1 and 2. In brief,
RNA was extracted from fresh whole blood, blood cultured
with phytohaemaglutinin (blood-PHA), or EBV transformed lymphoblastoid cell lines (LCLs). Cultured cells
were treated with puromycin to prevent RNA degradation
by nonsense-mediated decay (NMD) in most instances.
A minigene assay was applied to 3 BRCA2 variants as
recently described [19].
Bioinformatic splice predictions
For bioinformatic prediction of variant induced splice
aberrations, we used Alamut software (http://www.inter
active-biosoftware.com/alamut.html). This software includes
SpliceSiteFinder (SSF), [4], MaxEntScan [5], NNSplice [6],
and GeneSplicer [7] for prediction of donor and acceptor
sites. Biological significance variation (expressed as ‘‘disruption of intron–exon junction’’) was considered when at
least one tool predicted a 100% splice site score reduction.
We also investigated prediction of cryptic sites (a site
defined by the wildtype sequence, but only used when a
variant disrupts the native donor or acceptor site) for variants
that resulted in disruption of the intron–exon junction, and
the prediction of de novo splice sites (a site that is created by
the variant) for variants lying outside of the invariant dinucleotide positions, considering the sequence covering variant
and proximal consensus site plus 100 bp upstream and
downstream.
Multi-factorial likelihood analysis
Likelihood ratios for segregation were derived using
methods described previously by Mohammadi et al [20],
and implemented as a web-based calculator (http://www.
msbi.nl/cosegregation/). Likelihood ratios reported for
family history were based on the statistical model developed by Easton et al. 2007 [21], derived from the Myriad
Genetics Laboratories dataset of 70,000 BRCA1 and
BRCA2 tests. Likelihood scores for co-occurrence with a
pathogenic mutation were derived as previously described
[17], from the same dataset, and also for clinical sample
sets from all the participating laboratories that recorded any
of the 25 variants under study. Probabilities were derived
for each of the components included in the study, under the
assumption that each factor was statistically independent.
The individual likelihood ratios were multiplied to calculate
an overall multifactorial likelihood ratio. For variants outside the donor or acceptor dinucleotide, a prior probability of
pathogenicity of 0.26 was assigned [21]. For variants in the
donor or acceptor dinucleotides, a prior probability of 0.96
was assigned, based on the midpoint of estimate ranges
(CI 91 to 100%) reported for ±1/2 variants highly likely to
disrupt splicing [21]. The prior for silent exonic alterations
was assumed to be that of a C0 missense substitution. Bayes
rule was then used to calculate a posterior probability that the
variant was pathogenic from the multifactorial likelihood
ratio and the prior probability. Variants were classified
according to the 5 class IARC quantitative scheme [22],
based on the posterior probability.
123
Breast Cancer Res Treat
Results
Characteristics of families and variants assayed
The 25 variants were identified in 33 families. The clinical
characteristics of the families are shown in Table 1. The
majority of index cases presented with breast cancer under
age 50 or bilateral breast cancer (20 cases), and/or a family
history of breast cancer in at least one relative (22 cases).
Previous report of the variants in the BIC database and in
HapMap samples are shown in Table 2, together with
results from mRNA analysis for the variants. Four of the
intronic variants were located in the conserved dinucleotide
position, and the remaining 12 intronic variants assayed
were located 3–107 bp from the intron–exon boundary.
The 9 exonic variants assayed included 1 variant predicted
to disrupt the initiation codon, and another 8 variants
located between 1 and 1247 bp from the neighboring
intron. Twelve of the 25 variants have been reported
previously to the BIC database, but only BRCA1
c.4357?1G[A was classified by BIC as clinically important, based on position alone. Thirteen of the variants had
been previously identified in HapMap samples, with frequency data available for only BRCA2 c.1788T[C p.=
(rs11571642) and BRCA2 c.7397C[T p.Ala2466Val
(rs169547). Both of these variants are reported to occur at
polymorphic frequency ([1%) in at least one population,
suggesting a priori that they are unlikely to be associated
with a high risk of disease.
Experimental analysis of splicing aberrations
As summarized in Table 2, a splicing aberration that is
predicted to lead to a truncated protein was observed for all
4 variants in the conserved dinucleotide sites (BRCA1
c.441?2T[A, c.4184_4185?2del, c.4357?1G[A, and
c.4987-2A[G), for an exonic variant located 1 bp from the
intron–exon boundary (BRCA1 c.5074G[A), and for an
intronic variant located 3 bp from the intron–exon boundary (BRCA2 c.8754?3G[C). An in-frame whole exon
deletion was observed for an additional intronic variant
located 5 bp from the intron–exon boundary (BRCA2
c.316?5G[A). In addition, an intronic variant located 6 bp
from the boundary (BRCA1 c.301?6T[C) was associated
with very low levels of aberrant transcript (detectable by
dHPLC analysis but not by gel electrophoresis) that is
predicted to encode an in-frame 3 amino acid deletion at
the exon 6–7 junction. All these aberrant transcripts were
characterized by sequencing. Laboratory results for these
variants are shown in Figs. 1 and 2. No evidence of aberrant transcript leading to a cryptic start site was obtained in
the variant located at the initiation codon (BRCA2 c.3G[A)
and both alleles were equally present in the sequence of the
123
cDNA, justifying the high prior probability of pathogenicity for multifactorial analysis. No variant-specific
aberration was observed for the remaining variants.
As noted in Fig. 1B.1, C.1 and D.1, although PCR
products indicative of aberrant transcripts were detectable in
all assays from whole blood RNA, the detection of aberrant
transcripts was greatly enhanced by the addition of puromycin to cultured cells to prevent NMD. Moreover, the
identification and interpretation of aberrant transcripts was
greatly facilitated when the template used for sequencing
reactions was purified from agarose excised bands (as
opposed to cDNA pool), with sequencing of both aberrant
and full-length transcripts providing information to assess
whether the variant allele produces aberrant transcript only,
or a combination of aberrant and full-length transcripts. For
BRCA1 c.5074G[C, located in the last base of exon 17,
sequencing of the 290 bp PCR product representing the
wildtype transcript identified only the wild type G allele at
position c.5074, indicating complete inactivation of correct
splicing for the variant C allele. This was supported by
sequencing of the re-amplified upper band which identified a
mutant transcript with retention of part of intron 17
(Fig. 1F.2). Similarly, the assay for BRCA2 c.316 ? 5G[A
identified upregulated skipping of exon 3, an in-frame
transcript isoform that is also detected as a minor fraction in
mRNA from healthy controls (Fig. 1G [23, 24]. The extent
of exon 3 loss from the mutated allele was investigated
further by allele specific sequencing of another variant
c.-26G[A carried by the patient (data not shown): this analysis revealed no detectable exon 3 sequence for the A-specific
primer, and a low amount of exon 3 loss for the G-specific
primer comparable to the fraction of mRNA missing exon 3 in
a control sample (Fig. 1G.2). This indicates complete loss of
exon 3 from the allele containing BRCA2 c.316?5G[A.
The splicing aberration observed for BRCA2 variant
c.8754?3G[C, shown to be associated with aberrant
splicing by RT-PCR assays of RNA from whole blood was
investigated further by minigene assay of the exon 21
region, to investigate the extent of consensus site disruption
and cryptic site usage (Fig. 2). This analysis indicated
cryptic splicing of approximately 84 % of mRNA from the
variant C allele (Fig. 2(3)), and sequencing of the minigene assay PCR product identified a cryptic splice site
46 bp after exon 21 (Table 2).
In silico prediction of splicing aberrations
All variants were tested bioinformatically using Alamut
software that includes SSF, MaxEntScan, NNSPlice, and
GeneSplicer for splice signal prediction (Table 3). An
initial observation is that one or more of the algorithms did
not identify the conserved donor and acceptor sites, preventing the assessment of the effect of variation at or near the
Breast Cancer Res Treat
Table 1 Clinical characteristics of included families
Gene
Variant
Laboratory
Diagnosis
index case
Age
diagnosis
index case
No breast cancer in
the family (age at
diagnosis)a
Number of ovarian
cancer in the family (age
at diagnosis)1
Other cancers in the
family (age at
diagnosis)b
BRCA1
c.301?6T[C
IOV
BC
51
3 (52,60,50)
1 (61)
LU, LA, MYL
(NA)
BRCA1
c.441?2T[A
FPGMX
BC
25
2 (47c,56)
1 (55c)
0
BRCA1
c.548-8delT
OUH
BC
42
2 (50, 69)
0
LU (NA)
BRCA1
c.4097-15T[C
FPGMX
BC
45
1 (54)
0
0
BRCA1
c.4184_4185?2del
FPGMX
BC
27
2 (30,40)
0
ORL (42), LEU
(20), GYN (40),
ESO (54)
BRCA1
BRCA1
c.4357?1G[A
c.4358-4delA
FPGMX
OUH
BC
BC
26
48
0
1 (68)
0
0
0
0
BRCA1
c.4987-2A[G
FPGMX
OC
61
0
0
Unknown
BRCA1
c.5074G[C
p.Asp1692His
IOV
OC
33
3 (60,60,42)
1 (NA)
CRC (69)
BRCA1
c.5075-107A[G
ICO
Bilat BC
46, 46
1 (62)
0
GAS (82), cancer of
unknown origin
(NA)
BRCA1
c.5075-107A[G
ICO
BC
32
2 (39, 70)
0
0
BRCA1
c.5333A[G
p.Asp1778Gly
ICO
BC
42
3 (58,59,74)
0
1 CRC (66)
BRCA2
c.3G[A p.Met1?
HVH
BC
45
2 (37, 45)
0
0
BRCA2
c.316?5G[A
AAS
BC
30
2 (38)
0
PC (62), BCC (52),
AB (56)
BRCA2
c.425?33A[G
ICO
Bilat BC
37, 42
0
0
LU (NA)
BRCA2
BRCA2
c.425?33A[G
c.425?33A[G
ICO
HVH
Bilat BC
BC
53, 53
47
1 (50, 51)
1 (69)
0
0
HN (60), LYM (59)
2 HN (55, 85); 2
LU (55, 55);
LYM (70)
BRCA2
c.426-22G[T
CBCS
Bilat BC
60, 65
1 (42)
0
Unknown
BRCA2
c.516?18T[C
CBCS
BC
54
2 (49, 42)
0
Unknown
BRCA2
c.632-16A[C
HVH
Bilat BC
49, 53
0
0
PC (61)
BRCA2
c.992A[T
p.Lys331lle
ICO
BC
27
1 (45)
0
0
BRCA2
c.1096T[G
p.Leu366Val
FPGMX
OC
47
0
0
CRC (70); LEU
(NA)
BRCA2
c.1788T[C p.=
FPGMX
BC
40
1 (48)
0
0
BRCA2
c.3156A[C p.=
FPGMX
BC
52
2 (58,NA)
0
0
BRCA2
c.7397C[T
p.Ala2466Val
HVH
BC
26
5 (28, 52, 38, 52, 70)
0
PA (49), TES (60)
BRCA2
BRCA2
c.7435?6G[A
c.8421G[T p.=
HVH
FPGMX
BC
BC
33
50
0
1 (35)
0
0
0
0
BRCA2
c.8754?3G[C
CBCS
None
(male)
NA
3 (43, 36, 43)
0
2 PC (73, 62), CRC
(74), REC (73)
NA not available
a
Excluding index case
b
BC breast cancer, OC ovarian cancer, PC prostate cancer, LU lung cancer, TES testicular cancer, GAS gastric cancer, LA larynx, AB abdominal
cancer, MYL myeloma, CRC colorectal cancer, BCC basal cell carcinoma, LYM lymphoma, HN head and neck cancer, LEU leukemia, GYN
gynecological, ORL oral, REC rectal
c
Same patient
123
123
E12 I12
I13
c.441?2T[A
c.548-8delT
c.4097-15T[C
c.4184_4185?2del
c.4357?1G[A
c.4358-4delA
c.4987-2A[G
c.5074G[C
p.Asp1692His
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
E17
I16
I13
I11
I8
I7
I6
c.301?6T[C
BRCA1
Exon
or
intron
position
Variant
Gene
Donor
consensus site
(1 bp)
Invariant
dinucleotide
Acceptor
consensus site
Invariant
dinucleotide
Invariant
dinucleotide
Intronic
Polypyrimidine
tract
Invariant
dinucleotide
Donor
consensus site
Location
(distance from
intron)
Table 2 Analysis of BRCA1 and BRCA2 variants
IOV
FPGMX
OUH
FPGMX
FPGMX
FPGMX
OUH
FPGMX
IOV
Laboratory
3
0
0
21
0
2
0
0
0
No.
in
BIC
rs80187739
-
-
rs80358027
-
-
rs1799736
(reported
as c.5486delT)
-
-
Variation
viewera
rsID for
the variant
NFD
-
-
NFD
-
-
NFD
-
-
MAF
(population)a
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR,
DHPLC
Method
Cryptic splice site 153
nucleotides in intron 17.
(frame-shift;
p.Asp1692ArgfsX15)
Skipping of exon 17
(frame-shift;
p.Asp1692MetfsX10)
None
Skipping of exon 13
(frame-shift;
p.Arg1397TyrfsX2)
Skipping of exon 12
(frame-shift;
p.Gly1366AlafsX8)
None
None
Donor cryptic splice site in
exon 7 and acceptor in
exon 8 (frame-shift;
p.Ser127ArgfsX10)
Low amount of transcript
lacking 9 nucleotides at
exon 6–7 junction (inframe change of 4 amino
acids;
Gly98_Tyr101delinsAsp)
Observed splicing
aberration (predicted
effect on encoded
protein)
Disruption of
intron–
exon
junction
Disruption of
intron–
exon
junction
No changes
predicted
Disruption of
intron–
exon
junction
Disruption of
intron–
exon
junction
No changes
predicted
No changes
predicted
Interruption
of intron–
exon
junction
Disruption of
intron–
exon
junction
Interpretation
based on
splicing
prediction
toolsb
Class 3
uncertain
Class 4 likely
pathogenic
Class 3
Uncertain
Class 5
pathogenic
Class 4
Likely
Pathogenic
Class 3
uncertain
Class 3
uncertain
Class 4 likely
pathogenic
Class 1 not
pathogenic/
low clinical
significance
Multifactorial
likelihood
classification
according to
the IARC 5
class systemc
Class 5 pathogenic
(observed
splicing
aberration)
Class 5
pathogenic
(observed
splicing
aberration)
Class 3 Uncertain
Class 5
pathogenic
(multifactorial,
observed
splicing
aberration)
Class 5
Pathogenic
(observed
splicing
aberration)
Class 3 uncertain
Class 3 uncertain
Class 5
pathogenic
(observed
splicing
aberration)
Class 1 not
pathogenic/low
clinical
significance
Combined
interpretation
of frequency
information,
multifactorial
analysis, and
splicing results
Breast Cancer Res Treat
I6
I7
c.5333A[G
p.Asp1778Gly
c.3G[A p.Met1?
c.316?5G[A
c.425?33A[G
c.426-22G[T
c.516?18T[C
c.632-16A[C
c.992A[T
p.Lys331lle
c.1096T[G
p.Leu366Val
c.1788T[C p.=
BRCA1
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
E10
E10
E10
I4
I4
I3
E2
E22
I17
c.5075-107A[G
BRCA1
Exon
or
intron
position
Variant
Gene
Table 2 continued
Exonic
(122 bp)
Exonic
(303 bp)
Exonic
(199 bp)
Intronic
Intronic
Intronic
Intronic
Donor
consensus site
Kozak
consensus
start site
(41 bp)
Acceptor
consensus site
(1 bp)
Intronic
Location
(distance from
intron)
FPGMX
FPGMX
ICO
HVH
CBCS
CBCS
ICO and
HVH
AAS
HVH
FPGMX
and ICO
ICO
Laboratory
1
rs11571642
-
rs80359253
3d
0
rs81002905
rs81002834
-
-
rs81002840
rs80358650
rs80357041
-
Variation
viewera
rsID for
the variant
3
1
0
0
1
5
1
0
No.
in
BIC
0.025
(HapMapYRI)
0 (HapMapJPT)
0 (HapMapHCB)
0 (HapMapCEU)
-
NFD
NFD
NFD
-
-
NFD
NFD
NFD
-
MAF
(population)a
RT-PCR
RT-PCR
RT-PCR
RT-PCR
Minigene
Minigene
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
Method
None
None
None
None
None
None
None
Skipping of exon 3.
(in-frame deletion;
p.(Asp23_Leu105del)
None
None
None
Observed splicing
aberration (predicted
effect on encoded
protein)
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
Disruption of
intron–
exon
junction
No changes
predicted
No changes
predicted
No changes
predicted
Interpretation
based on
splicing
prediction
toolsb
Class 2 likely
not
pathogenic
Class 2 likely
not
pathogenic
Class 2 likely
not
pathogenic
Class 3
uncertain
Class 3
uncertain
Class 3
uncertain
Class 2 likely
not
pathogenic
Class 3
uncertain
Class 4 likely
pathogenic
Class 2 likely
not
pathogenic
Class 3
uncertain
Multifactorial
likelihood
classification
according to
the IARC 5
class systemc
Class 1 not
pathogenic/low
clinical
significance
(frequency)
Class 2 likely not
pathogenic
Class 2 likely not
pathogenic
Class 3 uncertain
Class 3 uncertain
Class 3 uncertain
Class 2 likely not
pathogenic
Class 4 likely
pathogenic
Class 4 likely
pathogenic
Class 2 likely not
pathogenic
Class 3 uncertain
Combined
interpretation
of frequency
information,
multifactorial
analysis, and
splicing results
Breast Cancer Res Treat
123
123
c.3156A[C p.=
c.7397C[T
p.Ala2466Val
c.7435?6G[A
c.8421G[T p.=
c.8754?3G[C
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
I21
E19
I14
E14
E11
Exon
or
intron
position
Donor
consensus site
Exonic (66 bp)
Donor
consensus site
Exonic (38 bp)
Exonic
(1247 bp)
Location
(distance from
intron)
CBCS
FPGMX
HVH
HVH
FPGMX
Laboratory
0
0
14
49
0
No.
in
BIC
-
-
rs81002852
rs169547
-
Variation
viewera
rsID for
the variant
-
-
NFD
0.095
(HapMapYRI)
0 (HapMapJPT)
0.037
(HapMapHCB)
0.014
(HapMapCEU)
-
MAF
(population)a
RT_PCR,
Minigene
RT-PCR
RT-PCR
RT-PCR
RT-PCR
Method
Cryptic splicing 46
nucleotides downstream
(frame-shift;
p.Gly2919ValfsX4)
None
None
None
None
Observed splicing
aberration (predicted
effect on encoded
protein)
Disruption of
intron–
exon
junction
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
Interpretation
based on
splicing
prediction
toolsb
Class 3
uncertain
Class 2 likely
not
pathogenic
Class 2 likely
not
pathogenic
Class 2 likely
not
pathogenic
Class 1 not
pathogenic/
low clinical
significance
Multifactorial
likelihood
classification
according to
the IARC 5
class systemc
Class 4 likely
pathogenic
Class 2 likely not
pathogenic
Class 2 likely not
pathogenic
Class 1 not
pathogenic/low
clinical
significance
(frequency)
Class 1 not
pathogenic /
low clinical
significance
Combined
interpretation
of frequency
information,
multifactorial
analysis, and
splicing results
d
c
b
One of these families is included in this study
Extracted from Supplementary Table 4
Interpretation based on information in Table 3
BRCA1: http://www.ncbi.nlm.nih.gov/sites/varvu?gene=672; BRCA2: http://www.ncbi.nlm.nih.gov/sites/varvu?gene=675. Note that none of the variants studied were reported at frequency[0.01 in Caucasians in the HapMap phase 1 dataset explored at the time this study was initiated. NFD no frequency data
a
Variant
Gene
Table 2 continued
Breast Cancer Res Treat
Breast Cancer Res Treat
Fig. 1 RT-PCR analysis of 6 BRCA1 variants and 1 BRCA2 variant
disrupting the consensus donor or acceptor sites. 1 Gel images of RTPCR products from agarose (a, f, g) or Agilent Bioanalyzer (b, c, d,
e). LCL lymphoblastoid cell lines, Blood-PHA blood treated with
phytohaemaglutinin, P puromycin, 29PCR product of re-amplification, Ctr control, Ca carrier of the variant; Sequencing of 949 bp
band from carrier blood-PHA treated with puromycin shows both fulllength and D62 bp-exon7&3 bp-exon8 transcripts; this 949 bp band
also appears at minimal concentrations in blood and blood-PHA
without puromycin carrier samples, suggesting the formation of a
heteroduplex between the full length and the mutant transcript;
à
1210 bp band appears also in control samples; §An additional 875 bp
band is shown in Agilent electrophoresis, it does not appears in
agarose gel from which only the full-length transcript was obtained in
the sequencing of purified band; *An additional 537 band is merged
with the 504 band in agarose gel, this extracted band shows both fulllength and Dexon17 transcripts. 2 a Electropherogram of cDNA pool
from LCLs without puromycin. b–d Agarose gel and electropherograms of purified agarose bands from blood-PHA grown with
puromycin. e Agarose gel and electropherograms of purified agarose
bands from blood. f Agarose gel and electropherograms of purified
agarose bands from LCLs without puromycin. G: Electropherogram
of cDNA pool from blood. The exon structure and reference sequence
is indicated above the electropherograms. 3 High resolution DHPLC
Chromatogram from cDNA products
relevant consensus site. Nevertheless, for all 4 variants in the
invariant dinucleotide donor/acceptor positions, and the
exonic variant located 1 bp from the intron–exon boundary,
all available predictions (ranging from one to four) indicated
complete inactivation of the site. Prediction of disrupted
splicing was not as clear-cut for the two intronic variants
located 3 and 5 bp from the intron–exon boundary shown to
be associated with obvious splicing aberrations (BRCA2
c.8754?3G[C and c.316?5G[A, respectively), with complete inactivation of the native consensus site indicated by
Genesplicer only. The BRCA1 c.301?6T[C variant associated with very minor splicing aberration demonstrated
complete inactivation of the site using NNSplice, but less
marked loss in score for SSF (–7.4%) and MaxEntScan
(–37.1%).
With a few exceptions, the bioinformatic predictions
for variants without observable splice aberrations did not
suggest significant disruption of consensus sites, or creation of de novo sites. The maximum reduction in score for
a consensus site was 39.8% for BRCA1 c.548-8delT,
predicted by MaxEntScan only and comparable to a
similar MaxEntScan score for BRCA1 c.301?6T[C that
was observed to be associated with only very minor levels
of aberrant splicing. For the variants that did not disrupt a
donor or acceptor site, 3 were predicted to create a de
novo site: BRCA2 c.632-16A[C at c.636 (MaxEntScan);
BRCA2 c.1096T[G at c.1097 (SSF, MaxEntScan) and
BRCA2 c.3156A[C at c.3161 (SSF). However, the prediction scores were always weaker than the closest donor/
acceptor, consistent with experimental results that failed
123
Breast Cancer Res Treat
Fig. 2 Minigene splicing and RT-PCR analyses of BRCA2
c.8754?3G[C. (1) RT-PCR analysis of c.8754?3G[C using RNA
from fresh blood as template. (2) Schematic figure showing the
plasmid construct containing exon 21 and flanking sequences. (3) RTPCR analysis of mini gene product using pSPL3 specific primers.
Wild type band of 299 and a larger cryptic splicing product are
indicated
to identify aberrant transcripts associated with these
variants.
For the 8 variants resulting in disruption of acceptor or
donor sites, the location of cryptic splice sites was predicted
using bioinformatic algorithms, and compared to results
from RNA assays (Supplementary Table 3). Cryptic sites
were predicted by at least one program for each variant.
Results from splicing assays indicated that 4 variants
resulted in exon skipping aberrations only, with no cryptic
site usage. The other 4 variants resulted in transcript aberrations that did involve the use of cryptic splice sites
(Table 2; Figs. 1, 2). All 5 cryptic sites identified by splicing
assays were recognized by at least one of the bioinformatic
programs when the input sequence was increased to cover
the relevant region: the low level aberration associated with
BRCA1 c.301?6T[C resulted from use of a cryptic site at
c.292 (SSF,MaxEntScan, NNSplice); the aberrant transcript
for BRCA1 c. 441?2T[A was generated by use of a cryptic
donor at c.379 (SSF, MaxEntScan, NNSplice) and the
cryptic acceptor at c.445 (MaxEntScan, NNSplice, GeneSplicer); BRCA1 c.5074G[C was associated with cryptic
site use at c.5074?153 (MaxEntScan, GeneSplicer); and
BRCA2 8754?3G[C was associated with cryptic site use at
c.8754?46 (all four algorithms). The validated cryptic sites
did not necessarily have markedly higher prediction scores,
or a greater number of predictions, compared to sites that
were not validated by experimental splicing assays.
Multifactorial likelihood analysis
Multifactorial likelihood analysis of the 25 variants (Supplementary Table 4) included prediction of prior
123
probability of pathogenicity (based on sequence conservation, position and missense alteration if appropriate), and
at least one other data point for each variant. As summarized in Table 2, 2 variants were classified as Class 1 not
pathogenic/low clinical significance (including BRCA1
c.301?6T[C with a minor level splicing aberration), eight
were Class 2 likely not pathogenic (none with splicing
aberrations, with the 2 variants BRCA2 c.1788T[C p.=
(rs11571642) and BRCA2 c.7397C[T p.Ala2466Val
(rs169547) reported to occur at clearly polymorphic frequency ([2%) in at least one non-Caucasian sample population). Another 4 variants were Class 4 likely pathogenic
(3 located in a consensus site and shown to result in
splicing aberrations considered to be consistent with
pathogenicity [10], and the 4th at the initiation codon), and
one consensus site variant was classified as Class 5 pathogenic (also shown to alter splicing consistent with
pathogenicity).
There was insufficient information to provide robust
multifactorial classification for the remaining 10 variants
(Class 3 uncertain), however 3 of these (BRCA1 c.5074G
[C, BRCA2 c.316?5G[A, BRCA2 c.8754?3G[C) were
associated with splicing aberrations that could be considered
consistent with a pathogenic classification (BRCA1
c.5074G[C, major aberration arising from the variant allele)
or likely pathogenic classification (BRCA2 c.316?5G[A
resulting in in-frame transcript and BRCA2 c.8754?3G[C,
splicing aberration that would benefit from further quantitative studies). After combined interpretation of the frequency
information, multifactorial analysis results and splicing data
(Table 2), 5 variants were considered class 5 pathogenic, 3
variants were class 4 likely pathogenic, 6 variants were likely
not pathogenic (class 2), another 4 variants were class 1 not
pathogenic/low clinical significance and the remaining 7
variants remain uncertain (class 3).
Discussion
Our study of 25 sequence variants has consolidated information from a variety of sources across multiple clinical
testing laboratories to provide evidence of relevance for
genetic counseling of patients carrying these variants.
According to guidelines proposed for the interpretation of
variant pathogenicity based on splicing results [10], 7
variants are associated with aberrations expected to encode
a non-functional protein, and may be considered to be
clinically significant or likely clinically significant. When
sufficient information was available, multifactorial likelihood classification supported this conclusion. In addition,
the combined interpretation of splicing and multifactorial
analysis was able to classify a variant located in the initiation codon as likely pathogenic. The posterior probability
c.441?2T[A
c.548-8delT
c.4097-15T[C
c.4184_4185?2del
c.4357?1G[A
c.4358-4delA
c.4987-2A[G
c.5074G[C
c.5075-107A[G
c.5333A[G
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
BRCA1
c.3G[A
c.301?6T[C
BRCA1
BRCA2
Variant
Gene
A
A
A
D
A
A
D
D
A
A
D
D
Site
91.50
73.01
92.62
71.90
84.55
74.82
85.16
82.52
89.19
–
–
78.12
(-100%)
(-100%)
(-100%)
(-100%)
91.50
76.92
(?5.4%)
92.62
–
–
75.79
(?1.3%)
–
–
89.19
–
–
72.34
(-7.4%)
Variant scorea
–
–
–
–
–
–
–
–
–
–
–
–
9.79
8.67
8.96
7.48
6.69
7.21
6.64
8.59
8.15
2.82
3.23
8.46
Wild
type
score
9.79
9.73
(?12.3%)
8.96
– (-100%)
– (-100%)
5.92
(-17.9%)
– (-100%)
– (-100%)
8.43
(?3.4%)
1.70
(-39.8%)
– (-100%)
5.32
(-37.1%)
Variant
scorea
Consensus splice
site
Consensus splice site
Wild
type
score
[0–12]
[0–100]
De novo
splice site
location
and scoreb
MaxEntScan
SSF
Table 3 Prediction of aberrant splicing using prediction algorithms in Alamut software
–
–
–
–
–
–
–
–
–
–
–
–
De novo
splice site
location
and scoreb
0.92
0.67
–
0.92
0.61
–
0.99
0.95
0.89
–
–
0.97
Wild
type
score
0.92
0.89
(?32.5%)
–
– (-100%)
– (-100%)
–
– (-100%)
– (-100%)
0.90
(?1.1%)
–
–
–(-100%)
Variant
scorea
Consensus splice
site
[0–1]
NNSPLICE
–
–
–
–
–
–
–
–
–
–
–
–
De novo
splice site
location
and scoreb
6.62
6.38
8.21
–
1.36
–
5.43
5.17
10.70
–
–
–
Wild
type
score
6.35
(-4.0%)
7.51
(?17.7%)
8.21
–
– (-100%)
–
– (-100%)
– (-100%)
9.45
(-11.7%)
–
–
–
Variant
scorea
Consensus splice
site
[0–15]
GeneSplicer
–
–
–
–
–
–
–
c.4185?57
0.49
–
–
–
–
De novo
splice site
location
and scoreb
No changes
predicted
No changes
predicted
No changes
predicted
Disruption of
intron–
exon
junction
Disruption of
intron–
exon
junction
No changes
predicted
Disruption of
intron–
exon
junction
Disruption of
intron–
exon
junction
No changes
predicted
No changes
predicted
Interruption
of intron–
exon
junction
Disruption of
intron–
exon
junction
Interpretation
based on
splicing
prediction
tools
Breast Cancer Res Treat
123
123
c.316?5G[A
c.425?33A[G
c.426-22G[T
c.516?18T[C
c.632-16A[C
c.992A[T
c.1096T[G
c.1788T[C
c.3156A[C
c.7397C[T
c.7435?6G[A
c.8421G[T
c.8754?3G[C
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
BRCA2
D
D
D
D
A
D
A
A
A
D
A
D
D
Site
87.25
84.50
–
–
87.93
76.52
88.81
88.81
93.33
87.54
86.73
84.19
95.87
81.73
(-6.3%)
84.50
–
–
87.93
76.52
88.81
88.81
93.33
87.54
86.73
84.19
83.72
(-12.7%)
Variant scorea
–
–
–
–
c.3161
71.09
–
c.1097
72.41
–
–
–
–
–
–
7.66
9.46
5.64
5.64
10.77
8.16
9.62
9.62
8.03
8.88
8.99
9.11
9.66
Wild
type
score
b
a
–
–
–
–
–
–
c.1097
0.66
–
c.636 0.14
–
–
–
–
De novo
splice site
location
and scoreb
When the algorithm predicts the creation of a de novo site, its location and the predicted score are indicated
Consensus splice site prediction score for the variant sequence, with the percentage of variation in parenthesis
D donor site, A acceptor site, – no predicted donor or acceptor site
5.24
(-31.5%)
9.46
4.71
(-16.6%)
5.64
10.77
8.16
9.62
9.62
9.07
(?13.0%)
8.88
8.99
9.11
6.62
(-31.5%)
Variant
scorea
Consensus splice
site
Consensus splice site
Wild
type
score
[0–12]
[0–100]
De novo
splice site
location
and scoreb
MaxEntScan
SSF
Scores consistent with a prediction of a splicing aberration are shown in bold font
Variant
Gene
Table 3 continued
0.98
0.95
–
–
0.98
0.95
0.90
0.90
0.96
0.98
0.99
0.94
1.00
Wild
type
score
0.63
(-35.5%)
0.95
–
–
0.98
0.95
0.90
0.90
0.98
(?2.1%)
0.98
0.99
0.94
0.99
(-0.5%)
Variant
scorea
Consensus splice
site
[0–1]
NNSPLICE
c.8754?8
0.57
–
–
–
–
–
–
–
–
–
–
–
–
De novo
splice site
location
and scoreb
3.23
–
–
–
6.50
–
4.92
4.92
5.78
–
–
0.88
2.89
Wild
type
score
– (-100%)
–
–
–
6.50
–
4.92
4.92
6.58
(?13.8%)
–
–
1.02
(?15.7%)
– (-100%)
Variant
scorea
Consensus splice
site
[0–15]
GeneSplicer
–
–
–
–
–
–
–
–
–
–
–
–
–
De novo
splice site
location
and scoreb
Disruption of
intron–
exon
junction
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
No changes
predicted
Disruption of
intron–
exon
junction
Interpretation
based on
splicing
prediction
tools
Breast Cancer Res Treat
Breast Cancer Res Treat
was indicative of low clinical significance or unlikely
pathogenicity for another 10 variants, including 2 variants
identified to be clearly polymorphic in non-Caucasian
samples, and one variant with an observable but apparently
minor splicing aberration. Altogether, these findings will
influence patient management for 18 of the 25 variants
assessed, according to generic clinical guidelines linked to
the IARC 5 class variant classification system [22]. Our
results also show the relevance of assessing apparent
missense substitutions for possible splicing aberrations.
While our study shows splicing aberration for c.5074G[C,
previous functional assay erroneously attributed impaired
repair of double strand breaks in a heterozygous lymphoblastoid cell line to the amino acid substitution [25].
Another exonic variant BRCA1 c.5333A[G with class 2
classification from multifactorial analysis was not associated with splicing aberrations, despite its close proximity to
the intron–exon boundary. We also provide support that
BRCA2 c.3G[A located close to the start of exon 2 does
not lead to a new aberrant transcript-driven initiation site,
and is thus likely to interfere with initiation of translation.
These findings support the growing argument that there is
clinical benefit gained from undertaking RNA assays of
variants located in any position of the consensus splicing
sequences. As detailed above, aberrant splicing was
observed for the exonic variant BRCA1 c.5074G[C located
1 bp from the intron–exon boundary. In addition, our analysis of BRCA2 c.8754?3G[C showed that this variant
induced retention of 46 nucleotides in the transcript. This is
in agreement with a very recent publication reporting
splicing results for this variant [26] and is an effect similar to
that previously reported for c.8754?1G[A [27]. Similarly,
the detection of complete in-frame skipping of exon 3 from
the mutant allele for BRCA2 c.316?5G[A is comparable to
findings from Bonnet et al [16] for c.316?5G[C, and our
study demonstrates the importance of quantifying the contribution of the variant allele to aberrant and variant transcripts when a variant is associated with upregulation of the
naturally occurring delta exon 3 isoform identified in healthy
controls. Further studies may help clarify the debate
regarding the clinical significance of variants reported to
result in exon 3 skipping [23, 24, 28–31].
More generically, our combined analysis of RNA assays
for splicing aberrations and bioinformatic prediction of
splicing provides further information to assess the value
and limitations of bioinformatic predictions, and to highlight the importance of RNA assays to characterize aberrations associated with rare sequence variants. As
expected, the 4 variants located in invariant positions
within the acceptor/donor site disrupted normal splicing,
but only one algorithm predicted the donor site for BRCA1
c.441?2T[A. The results also confirm that the consequences of constitutive site inactivation are not predicted
with great specificity using current bioinformatic tools, and
may include exon skipping or intron retention. Bioinformatic algorithms were able to detect several cryptic sites
along the analyzed sequence, but criteria to identify the
specific site used (as identified experimentally) were not
obvious. Moreover, even confirmed aberrations may all
lead to in-frame and/or low-level transcripts which would
not necessarily be considered clinically significant without
additional supportive evidence [13, 29]. In this study we
demonstrate the importance of further study of variants
associated with minor splicing aberrations, using alternative approaches to confirm their role in disease
predisposition.
From a technical perspective, our comparison of results
across different laboratories using a variety of tissue types
supports the routine use of NMD inhibitors to enhance the
detection of aberrant transcripts, and sequencing of cDNA
from purified bands to facilitate the identification of transcripts. We also show that interpretation of findings is
improved when it is possible to estimate the fraction of the
mutated allele. While this is much simpler for a variant in the
transcribed sequence, it is also achievable for intronic
variants using alternative approaches such as mini gene
assay. These observations will be used to inform large-scale
multi-site projects recently initiated by the ENIGMA
Splicing Working Group to optimize protocols and prediction tools for characterizing splicing aberrations, towards
standardizing splicing analysis of unclassified variants.
In conclusion, we have examined 25 BRCA1/BRCA2
variants identified in breast/ovary cancer patients from 7
different laboratories contributing to the ENIGMA consortium, and provide findings of clinical utility for a large
subset of them. Importantly, this study also demonstrates
the value of collaboration between laboratories, and across
disciplines, to collate and interpret information from clinical testing laboratories that often remains unpublished due
to time and resource constraints. The results arising from
this comprehensive approach will hopefully provide
impetus for future larger collaborative studies that draw on
information from clinical testing laboratories.
Acknowledgments We thank the families participating in this
research, the clinical personnel involved in aspects of recruitment and
clinical data collection, and the clinical and research institutions
supporting the combined research efforts. Melissa Brown is thanked
for collecting the protocols used by the different laboratories.
FPGMX: This work was partially supported by grants from the Xunta
de Galicia (10PXIB 9101297PR) and FMM Foundation given to A.V.
L.F is supported by Isabel Barreto program from Xunta de Galicia and
Fondo Social Europeo. ICO: Contract grant sponsor: Spanish Health
Research Fund; Carlos III Health Institute; Catalan Health Institute
and Autonomous Government of Catalonia. Contract grant numbers:
ISCIIIRETIC RD06/0020/1051, PI10/01422 and 2009SGR290. HVH:
This work was partially funded by two grants (OD, 2008; SGE, 2008)
from Fundación de Investigación Médica Mutua Madrileña. CBCS:
We would like to thank the NEYE foundation and Familien Hede
123
Breast Cancer Res Treat
Nielsens fond for financial support. ABS is supported by an NHMRC
Senior Research Fellowship, and her research on BRCA1/2 variants is
funded by an NHMRC project grant. IOV: This study was supported
by ‘‘Ministero della Salute’’ (grant numbers RFPS 2006-5-341353,
ACC2/R6.9 and ‘‘Progetto Tumori Femminili’’)
Conflicts of interest
14.
All authors declared no conflicts of interest.
15.
References
16.
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Pedersen IS, Bisgaard ML, Nielsen FC, Kruse TA, Gerdes AM
(2008) BRCA1 and BRCA2 mutations in Danish families with
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