DNA repair capacity is impaired in healthy BRCA1 heterozygous mutation carriers
Breast Cancer Research and Treatment
Tereza Vaclová1, Gonzalo Gómez-López2, Fernando Setién3, José María García Bueno 4, José Antonio
Macías5, Alicia Barroso1, Miguel Urioste6, Manel Esteller3,7,8, Javier Benítez1,9,10, Ana Osorio1,9,*
1
Human Genetics Group, Human Cancer Genetics Programme, Spanish National Cancer Research Centre
(CNIO), Madrid, 28029, Spain
2
Bioinformatics Unit, Structural Biology and Biocomputing Programme, Spanish National Cancer
Research Centre (CNIO), Madrid, 28029, Spain
3
Cancer Epigenetics Group, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical
Biomedical Research Institute (IDIBELL), Barcelona, 08908, Spain
4
Medical Oncology Section, Complejo Hospitalario Universitario de Albacete, Albacete, 02006, Spain
5
Hereditary Cancer Unit, Medical Oncology Service, Hospital Morales Meseguer, Murcia, 30008, Spain
6
Familial Cancer Unit, Human Cancer Genetics Programme, Spanish National Cancer Research Centre
(CNIO), Madrid, 28029, Spain
7
Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona,
08907, Spain
8
Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, 08010, Spain
9
Spanish Network on Rare Diseases (CIBERER), Madrid, 28029, Spain
10
Genotyping Unit (CEGEN), Human Cancer Genetics Programme, Spanish National Cancer Research
Centre (CNIO), Madrid, 28029, Spain
*corresponding author
aosorio@cnio.es
Online Resource 2. (a) Correlation between mRNA and hypo-P BRCA1 protein levels.
Statistically significant positive Spearman correlation between gene and protein expression in
the panel of lymphoblastoid cell lines. Wild type samples are represented by light grey dots,
lymphoblastoid cell lines with missense mutation are shown as grey squares, and cell lines
carrying a truncating mutation are displayed as black triangles. (b) BRCA1 dephosphorylation
of BRCA1 by Calf Intestinal Alkaline Phosphatase (CIP). Nuclear proteins of sample 11S67-L
were subjected to CIP treatment (1U/μg protein) at 37°C for 60min or non-treated. Samples
were separated by SDS-PAGE to resolve the upper hyper-phosphorylated and lower hypophosphorylated band of BRCA1 (see Materials and Methods section for details). Immunoblots
were probed with mouse monoclonal anti-BRCA1 antibody (Calbiochem, #OP92).
Online Resource 3. Effect of the BRCA1 germline mutation on the level of DNA damage and
repair. (A) High-throughput microscopy quantification of gamma-H2AX signal intensity in the
nuclei of wild type lymphoblastoid cell lines and cells harboring monoallelic missense or
truncating mutations in BRCA1. The dots represent signal intensity detected in 240 individual
nuclei of each lymphoblastoid cell lines in the group and the red line indicates the mean
intensity of the gamma-H2AX signal. The differences between groups were evaluated using the
Mann-Whitney U test (*** denotes P<0.001) (B) Average number of RAD51 foci per nucleus in
control cells and cells with heterozygous missense or truncating mutations in BRCA1. The bars
represent the mean from 240 analyzed nuclei of each lymphoblastoid cell lines in the
particular group +/- SEM. The P-value was calculated using the Mann-Whitney U test (**
denotes P<0.01).
Online Resource 4. BRCA1 is not required to maintain normal RAD51 protein levels in
lymphoblastoid cell lines . (A) Western blot analysis of RAD51 expression in a panel of
lymphoblastoid cell lines
(WT= wild type, MIS = missense, TRUN = truncating). RAD51 was
detected using a rabbit polyclonal anti-RAD51 antibody (Santa Cruz; #sc-8349) and β-actin
served as a loading control. The first sample in both blots is identical (Wild type cell line
06S179-L) and was used to apply between blot-normalization when analyzing protein band
densities by ImageJ. * indicates lymphoblastoid cell lines which could not be included in the
final panel of analyzed cell lines. (B) RAD51 protein level in control lymphoblastoid cell lines
and cells harboring missense (MIS) or truncating (TRUN) heterozygous mutations (two-tailed
Student’s t-test; no significant differences between groups; mean of 1.390 ± 0.24 (SEM) for
WT, 1.408 ± 0.12 for MIS, and 1.509 ± 0.12 for TRUN). The intensity of protein bands was
quantified by Image-J and normalized to WT sample 06S179-L.
Online Resource 5. Unsupervised hierarchical clustering of lymphoblastoid cell lines
harboring wild type BRCA1 or heterozygous missense or truncating mutations. Dendrograms
derived from unsupervised hierarchical clustering based on expression of the 42807 transcripts
that remained after normalization and pre-processing. Color labels define BRCA1 mutation
status/type: wild type in blue, missense in red, and truncating in black.
Online Resource 6. Description of the 20 genes whose expression strongly differentiates
groups of lymphoblastoid cell lines defined by the type of BRCA1 mutation.
Comparison
Gene symbola
Gene name
Chromosome
logFC
Downregulated in
adenylate cyclase 1 (brain)
plastin 3
interferon, gamma
tetraspanin 5
LINE-1 type transposase domain containing 1
interferon regulatory factor 5
inducible T-cell co-stimulator
peroxisome proliferator-activated receptor gamma
integrin, beta 5
plexin domain containing 2
wingless-type MMTV integration site family, member 11
DEAD (Asp-Glu-Ala-Asp) box polypeptide 43
chronic lymphocytic leukemia up-regulated 1 opposite strand
7
WT vs MIS
ADCY1
PLS3
IFNG#
TSPAN5
L1TD1
IRF5#
ICOS#
PPARG
ITGB5#
PLXDC2
WNT11
DDX43
CLLU1OS
12
4
1
7
2
5
3
10
11
6
12
2.07
2.32
-2.00
-2.02
-2.05
-2.13
-2.13
-2.15
-2.17
-2.26
-2.51
-2.55
-2.81
WT
WT
MIS
MIS
MIS
MIS
MIS
MIS
MIS
MIS
MIS
MIS
MIS
WT vs TRUN
LY6D
MMP7
lymphocyte antigen 6 complex, locus D
matrix metallopeptidase 7 (matrilysin, uterine)
8
11
-2.04
-2.14
TRUN
TRUN
SEPT10
RNF130
TNK1
RAMP1
septin 10
ring finger protein 130
tyrosine kinase, non-receptor, 1
receptor (G protein-coupled) activity modifying protein 1
UDP-N-acetyl-alpha-D-galactosamine:polypeptide
acetylgalactosaminyltransferase 14 (GalNAc-T14)
2
5
17
2
2.01
2.22
-2.09
-2.14
TRUN
TRUN
MIS
MIS
2
-2.98
MIS
MIS vs TRUN
GALNT14
N-
X
NOTE: logFC, logarithmic fold change. Only differentially expressed genes with |logFC| > 2 are shown.
a
genes involved in immune response (according to the Reactome_Immune_system gene set) are
marked with the # symbol
WT= wild type; MIS= Missense; TRUN=Truncating
Online Resource 7. Molecular and cellular functions related to genes differentially expressed
between wild type cells and cells with a missense mutation in BRCA1.
Molecular and cell functions
Range of p-value(s)
Molecules involved (from our dataset)
Cell Death and Survival
1,58E-05-4,48E-02
WNT11,IFNG,ICOS,PPARG,IRF5
Cellular Development
1,95E-05-4,48E-02
ITGB5,IFNG,ICOS,PPARG
Cell-To-Cell Signaling and
Interaction
1,97E-05-3,61E-02
ITGB5,IFNG,ICOS,PPARG
Cellular Movement
5,12E-05-4,94E-02
WNT11,ITGB5,ICOS,IFNG,PPARG
Cellular Growth and Proliferation
2,3E-04-4,48E-02
ADCY1,PLS3,ITGB5,IFNG,ICOS,PPARG
Amino Acid Metabolism
6,93E-04-6,93E-04
IFNG
Cell Cycle
6,93E-04-1,99E-02
IFNG,PPARG
Cell Morphology
6,93E-04-4,41E-02
PLS3,ICOS,IFNG,PPARG
Cell Signaling
6,93E-04-3,07E-02
ADCY1,IFNG,PPARG
Cellular Compromise
6,93E-04-1,17E-02
IFNG,ICOS
Cellular Function and Maintenance
6,93E-04-4,35E-02
PLS3,IFNG,ICOS,PPARG
DNA Replication, Recombination,
and Repair
6,93E-04-2,13E-02
IFNG
Gene Expression
6,93E-04-3,85E-02
IFNG,PPARG
Lipid Metabolism
6,93E-04-4,74E-02
IFNG,PPARG
Molecular Transport
6,93E-04-4,74E-02
IFNG,ICOS,PPARG
Nucleic Acid Metabolism
6,93E-04-2,15E-02
DDX43,ADCY1,IFNG
Protein Trafficking
6,93E-04-6,93E-04
ICOS
Small Molecule Biochemistry
6,93E-04-4,74E-02
DDX43,ADCY1,IFNG,PPARG
Drug Metabolism
1,38E-03-1,1E-02
IFNG,PPARG
RNA Post-Transcriptional
Modification
1,39E-03-3,46E-03
IFNG
Cellular Assembly and
Organization
2,77E-03-4,41E-02
PLS3,IFNG,ICOS
Cellular Response to Therapeutics
2,77E-03-2,77E-03
IFNG
Vitamin and Mineral Metabolism
2,77E-03-2,77E-03
IFNG
Carbohydrate Metabolism
3,46E-03-2,33E-02
IFNG,PPARG
Free Radical Scavenging
1,1E-02-4,08E-02
IFNG
Energy Production
3,48E-02-3,48E-02
PPARG
Online Resource 8. Involvement of the differentially expressed genes in cells with missense
mutations in cell death and inflammatory response pathways. The network shows interaction
of 27 cell death pathway genes and 16 inflammatory response pathway genes. Underlined
genes belong to the set that were differentially expressed between wild types and cells with
missense mutations in BRCA1. The legend specifies the molecule type and type of interaction
between molecules.
Online Resource 9. Molecular and cellular functions related to genes differentially expressed
between wild type cells and cells with truncating mutations in BRCA1.
Molecular and cell functions
Range of pvalue(s)
Molecules involved (from our dataset)
Cellular Movement
2.31E-04-1.11E-02
LY6D,MMP7
Cellular Development
3.46E-04-1.2E-02
MMP7
Cellular Growth and Proliferation
3.46E-04-1.2E-02
MMP7
Cell-To-Cell Signaling and
Interaction
4.62E-04-4.62E-04
MMP7
Cell Death and Survival
1.31E-02-2.84E-02
MMP7
Post-Translational Modification
1.56E-02-1.56E-02
MMP7
Protein Degradation
1.56E-02-1.56E-02
MMP7
Protein Synthesis
1.56E-02-1.56E-02
MMP7
Online Resource 10. Downstream effect analysis of genes differentially expressed between
wild type cells and cells with truncating mutations in BRCA1. The top 5 molecular and cell
functions significantly altered in cells carrying heterozygous truncating mutations in BRCA1 are
shown. The grey line represents the significance threshold of 0.05.