Are missense variants in
BRCA1 of clinical significance?
Cancer Genetics Laboratory, Dept of
Medical and Molecular Genetics, King’s
College Medical School, Guy’s Hospital
BREAST CANCER GENETICS
• 1 / 10 Lifetime risk
• 90 – 95% sporadic
• 5 – 10% familial
• Large extended families: -
breast and/or ovarian
-
± male breast
-
highly penetrant
-
dominantly acting
• Familial clusters
• BRCA1 and 2
•BRCA ? …
-
Lower penetrance
more common?
Dominant inheritance of Breast/Ovarian Cancer susceptibility
BRCA1/BRCA2
Only 17% of inherited predisposition
is accounted for by BRCA1/BRCA2
high penetrance, single gene
Familial “Clusters”
“Complex” genetic predisposition
Probably multiple genes of low penetrance
•
•
•
•
•
Mutations in BRCA1 account for approx.
35% of dominant familial breast cancer
(approx. 50% breast & ovarian).
Most mutations are small
insertions/deletions leading to in frame stop
codons.
Many missense variants (more later).
Penetrance, known mutations confer a
substantial lifetime risk of breast (80% risk
by age 70) and ovarian cancer(40% risk).
testing for breast and ovarian cancer
susceptibility has become integrated into the
practice of clinical oncology.
Signalling
Centrosome duplication
Transcription
BRCA2
ERa
UbcH5 BAP1
RAD51
ZBRK ?
-Tubulin
E2F
?
P300/CBP
BARD1hMSH2 cMyc
Ubiquitin
ligation
hMSH6
CstF50
ATM
p53
BRCA1
RAD50 BLM
Mre11
NBS1
ATR CdK2
HDACs
Branched
P53
pRB
MEKK3
?
DNA
P
Brg1
RHA CtIPBACH1
Pol II
P300/CBP
DNA processing
RNA processing
DNA repair
Cell cycle regulation
•
Chromatin remodelling
Which is relevant to disease?
CLINICAL DILEMMAS - BRCA1 & BRCA2
Large genes
Mutation screening difficult
Full sequencing
Which families?
Pathogenic mutations?
Missense / polymorphisms
Mutation-specific risks /
counselling
Penetrance
Counselling
- Affected
- Unaffected
The Breast Cancer Information Core (BIC) was established
in 1995 to catalogue the range and frequency of germline
BRCA1 and BRCA2 mutations.
Data largely arisen from sequencing (Myriad).
http://research.nhgri.nih.gov/bic/
BRCA1
Exon 2
No data on segregation with disease.
BIC database
(Population enriched for family history)
9556 entries,.
1539 distinct mutations, polymorphisms and variants
2734/9556 missense.
411 different non-polymorphic missense variants,
16 thought to be pathogenic, rest unclassified.
Missense variant
•
Polymorphism (>1%)
•
Rare non-pathogenic variant
•
Rare pathogenic variant
Missense variants are
difficult to classify
because...
•
•
•
•
•
Segregation with disease analysis
requires sampling of individuals in the
pedigree and has been achieved for few
missense variants.
Absence from controls ?
Present with a known disease causingvariant ?.
LOH- requires tumour samples.
Substitution of a conserved amino acid
-understanding of functional
relevance.
Missense variants
If we assume missense variants occur
across the gene more or less evenly we
might see a peak of variants at sites
important to disease pathogenesis, in a
population selected for family history.
15
51 0
-1
10 00
11
15 5 0
12
20 0 0
12
25 5 0
13
30 0 0
13
35 5 0
14
40 0 0
14
45 5 0
15
50 0 0
15
55 5 0
16
60 0 0
16
65 5 0
17
70 0 0
17
75 5 0
08
80 0 0
18
85 5 0
19
90 0 0
195 95
1 0
10 -10
01 00
10 -1 0
51 50
12 1 2
01 00
12 -1 2
51 50
13 1 3
01 00
13 -1 3
51 50
14 1 4
01 00
14 -1 4
51 50
15 1 5
01 00
15 -1 5
51 50
16 1 6
01 00
16 -1 6
51 50
17 1 7
01 00
17 -1 7
51 50
18 1 8
01 00
-1
86
3
No. different substitutions per bin
Non-polymorphic missense variants from a population enriched
for personal/family history of disease (BIC database) across the
BRCA1 gene.
25
20
**
15
10
5
0
bins of 50 amino acids
RING
BRCT BRCT
•
•
•
44 different missense variants
reported in patients in first 100aa.
The most frequently occurring
known missense mutations occur
within it
BRCA1 RING bearing N-terminus
has E3 ubiquitin ligase activity in
purified in vitro systems.
Ub
E1
Ub
UbcH5a
RING
BRCTBRCT
BARD1
BRCA1
(E2)
Ub
BRCA1
BRCTBRCT
RING
BARD1
Ub
Substrate
Ub
Ub
Ub
Substrate
Ub
Ub
Ub
Ub
BRCA1
BARD1
UbcH5 Ub
(E2)
Ub
E1
Ub
BRCA1 Ubiquitin ligase activity:
How relevant is it to disease
susceptibility?
•
Structurally deleterious known pathogenic
missense mutations in the RING inhibit the
activity...
Random Mutagenesis
•
•
Substrate
Ub
Ub
Ub
Ub
Find out which amino acids
BRCA1
are required for the BRCA1
UbcH5 Ub
(E2)
interaction with ubiquitin BARD1
Ub
ligase components- make an
E1
‘experimental population’.
Compare that to the missense
Ub
variants from patients from
BIC database.
Split Hybrid
VP16
BRCA1
E2
LexA
TetR
TetR
HIS3
Library
VP16
BRCA1
LacZ
•
experimental selection against the BARD1 and the E2
conjugating enzyme identified variants that correlated with
patient variants.
•
test directly the relative sensitivities of each protein
interaction to non-polymorphic missense substitutions in
BRCA1 encoded by patient DNA.
VP16
B
A
LexA
His3
BRCA1:BARD1 inhibitory BARD1
BRCA1
substitutions?
1
M
2
D
3
L
4
S
5
A
6
L
7
R
8
V
9
E
10
E
11
V
12
Q
13
N
14
V
15
I
16
N
17
A
18
M
19
Q
20
K
21
I
22
L
23
E
24
C
25
P
26
I
27
C
28
L
29
E
30
L
31
I
32
K
33
E
BARD1 split (high threshold)
+
suspected
+
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
P V S T K C D H I
F C K F C M L K L L N E K K G P S E C P L C K N
+
+
+
+
+
+
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
D I
T K R S L Q E S T R F S Q L V E E L L K I
I C A F Q L D T G L E
p=0.0003
BRCA1:UbcH5a inhibitory
BARD1
substitutions?
BRCA1
1
M
2
D
3
L
4
S
5
A
6
L
7
R
8
V
9
E
10
E
11
V
12
Q
13
N
14
V
15
I
16
N
17
A
18
M
19
Q
20
K
21
I
22
L
23
E
24
C
25
P
26
I
27
C
28
L
29
E
30
L
31
I
32
K
UbcH5
(E2)
33
E
UbcH5a Split (low threshold)
+
suspected
+
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
P V S T K C D H I
F C K F C M L K L L N E K K G P S E C P L C K N
+
+
+
+
+
+
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100
D I
T K R S L Q E S T R F S Q L V E E L L K I
I C A F Q L D T G L E
p=0.018
All selected residues are conserved (p=0.039)
Yeast Two
Hybrid
3AT
BARD1
BRCA1
Yeast Two
Hybrid
UbcH5
BRCA1
(E2)
Substrate
Ub Ub
UbUb
BRCA1
Ub
BARD1 UbcH5
Ub
E1
Ub
Abkevich, et al., (2004)
41;492-507 J. Med. Genet.
•
All known deleterious missense variants inhibit E2 binding
and ubiquitin ligase activity.
•
Variants that inhibit BARD1 binding also inhibit E2 binding
(we were unable to find a variant that inhibited only BARD1
interaction).
•
The majority of unclassified N-terminal missense variants
from patients inhibit E2 binding and Ub-ligase activity (none
inhibit BARD1 binding).
•
All those predicted to be of little clinical significance (by
conservation and substitution analysis) do not inhibit E2
binding or ligase activity. All but one predicted to be
deleterious inhibit E2 binding and activity.
E2:BRCA1:BARD1 complex
Based on the c-Cbl:UbcH7 RING E3:E2 crystal structure by superimposistion of BRCA1 onto the RING of Cbl, and UbcH5b onto
UbcH7.
Many missense variants that inhibit the E2 interaction have
structurally subtle consequences - Loss of E2 binding and Ubligase activity may be sufficient to predispose to disease.
Zinc ligating mutations also inhibit BARD1 interaction - this
class is much more frequent in the BIC - perhaps more likely to
lead to cancer progression.
•
Substrate
Ub
Ub
Ub
Ub
•
BRCA1
BARD1
UbcH5 Ub
(E2)
Ub
E1
Ub
Can the E3 ligase
activity of BRCA1 be
confirmed in vivo?
If so, what is its role?
Endogenous BRCA1 Protein
BRCA1
Conj Ub
Merge
Substrate
Ub
Ub
Ub
Ub
•
Ub
 Conj ubiquitin antibody: Recognises conjugated
but not free ubiquitin.
88.6(16)% BRCA1 foci accompanied by Ubiquitin
90.6 (9.4)% Ubiquitin foci accompanied by BRCA1.
DNA repair of Double stranded breaks
96.4(5.4)% BRCA1 foci colocalised with Ubiquitin,
93.8 (6.6)% Ubiquitin foci colocalised with BRCA1
10Gy IR
84 (9.6)% BRCA1 foci co-localise with Ubiquitin
80.9 (12.6) Ubiqutin foci co-localise with BRCA1.
•Evidence for BRCA1 E3 ligase activity in
vivo.
•BRCA1 is required for endogenous ubiquitin
conjugation.
•Over-expression of BRCA1 with BARD1
causes increased ubiquitin conjugation, this
requires an intact BRCA1 E2 binding face.
•
BRCA1 dependent, K6-ubiquitin dependent
conjugates are formed at sites of DNA repair and
replication.
•
E2-binding and the ability to form ubiquitin
conjugates is inhibited by known pathogenic missense
variants in the N-terminus and the majority of
unclassified variants from individuals with
personal/family history of familial breast and ovarian
cancer.
•
E3 ubiquitin ligase activity of BRCA1 plays a role
linked to DNA repair/replication.
Cancer Genetics Laboratory
 Laurent Pangon
 Toyomasa Katigiri
 Jo Morris
 Ellen Solomon
Collaborators
•Chris Boutell: MRC Virology Unit, Glasgow,
•Nick Keep: School of Crystallography, Birkbeck
College, University of London.
.