massively parallel functional analysis of missense
TRANSCRIPT
Massively parallel functional analysis of missense variants in the breast/ovarian cancer gene RAD51C
1CHU de Québec-Université Laval, Québec City, QC, G1V 4G2, Canada*Co-corresponding authors
Montalban G1, Milano L1, Rodrigue A1, Coulombe Y1, Desjardins S1, Dumont M1, Joly-Beauparlant C1, Soucy P1, Masson J-Y1* and Simard J1*
24.5%
BRCA2
14.1%
BRCA1
11.4%
ATM
9.9%NF1
6.4% BRIP1
6.9% PALB2
5.1%CDH14.9%
CHEK2
3.4%
TP53
3.1%
PTEN
5%
BARD1
2.8%STK11
2.4%RAD51C
20,627VUS
1990 1995 2000 2005 2010 2015 20190
100
200
300
400
500
# re
sear
ch a
rticl
es
Functional studies
Genetic studies
Genetic studies
Functional studies
• A proportion of hereditary breast/ovarian cancer (BC/OC)cases are due to pathogenic mutations in genes involvedin DNA repair (BRCA1, BRCA2, PALB2, ATM, CHEK2,RAD51C and RAD51D, among others).
• The application of next-generation sequencing has led toan increased detection of variants of unknown clinicalsignificance (VUS).
• The association of protein truncating variants in RAD51Cwith overall breast cancer risk has been confirmedrecently in a large case-control study (Odds Ratio (95%CI) = 1.93 (1.20-3.11) (Breast Cancer AssociationConsortium, N Engl J Med 2021).
• OBJECTIVE: To implement a massively parallel functionalapproach to measure the impact of all possible RAD51Cmissense substitutions.
INTRODUCTION
Data extracted from ClinVar and Pubmed databases
Number of sites: 376 amino acid positionsRequested # of variants: 7516 (20 aa/position)Sites passed: 369Sites failed: 7Variants passed QC: 98.04%
RAD51C LIBRARY - QC
HeLa “landing pad” systemDeep mutational scanning pipeline
METHODS
Mutagenesis library
N-terminal domain
C-terminal domain
WalkerA
Walker B NLS
RAD51C 376aa
.. ... ..... ..
..... ......
... . .... . . .. ..
. .. ........
Library
integration
PARPitreatment
Illumina seq Enriched = neutral
Depleted = deleterious
Variant %
RAD51C-/- cells
attPTet BFP iCasp9 Blast
Tet RAD51C Variant iCasp9 BlastIRES mCherry
RAD51C library plasmids
“landing pad” genomic site
attB
RAD51C
mCherryIRES
barcode
attL attR
Recombination
Genomic integration (1 variant/cell)
BFP
DAPIGFP
mCherry
double0
20
40
60
80
100
% re
com
bina
tion
46%
18%
0.4%
Validation of single landing pad insertion per cell
BFP
RESULTS I – Optimization assays
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 50
20
40
60
80
100
120
Olaparib (uM)
% S
urvi
val
siRNA UTRs
siCTRLsiIDT1
siIDT2
siIDT3siIDT2+siIDT3
siCTRLsiUTR1siUTR2siUTR3siUTR2+3
siRAD51C (UTR)1 2 2d 2+33 3d
d=double
RAD51C
vinculin
0 2 4 620
40
60
80
100
Olaparib (uM)
% S
urvi
ving
cel
ls
HeLa LP clones
siRAD51C+WT+L138F
siCTRL
0 2 4 620
40
60
80
100
Olaparib (uM)
% S
urvi
ving
cel
ls
HeLa LP clones
siRAD51C+WT+L138F
siCTRL
A.
B. C.
1 2 3 4 5
20
40
60
80
100
Olaparib (uM)
% S
urvi
val
SURVIVAL 22 OLAP
U2OS C+
U2OS KO
A. Optimization of siRNA silencing of endogenous RAD51C.B. Complementation assay of HeLa landing pad cells with WT and L138F (pathogenic missense). WT complemented cells rescue drug
sensitivity, whereas L138F expressing cells are sensitive to olaparib treatment.C. Complementation assay of U2OS RAD51C KO model generated for validation of candidate missense variants.
RESULTS II – Pilot screen (integration of 160 missense variants – RAD51C residues 137-144)
siCTRL-N
T
siCTRL-O
LAP
siRAD-N
T
siRAD-O
LAP0.0
0.2
0.4
0.6
0.8
1.0
1.2
Norm
aliz
ed c
ount
s
Positive controls
L138F
WT
Q143R
-DOX
+DOX
51C-mCherryHoechstA.
B.
C.137 138 139 140 141 142 143 144
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
stop -0.4
-0.2
0
0.2
0.4
0.6
0.8
x
⦁
⦁
⦁
⦁
⦁
⦁
⦁
⦁⦁ = WTX = L138F
WT sequenceQ L A V D V Q I
Fold
cha
nge
aa position137 138 139 140 141 142 143 144
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
stop -0.4
-0.2
0
0.2
0.4
0.6
0.8
A. Microscopy images confirming the expression of RAD51C-mCherry integrated in the HeLa landing pad cell system after 24h of doxycycline induction.B. Normalized variant read counts obtained for 11 variants that were used as controls (8 stop codons, the pathogenic missense L138F, the VUS Q143R
and the WT) in siRNA control and siRNA RAD51C non-treated and olaparib treated conditions, respectively. A reduction of read counts is observedfor the Olaparib treated cells when RAD51C is not functional.
C. Functional scores obtained for 160 missense variants. Negative scores (red) indicate potential loss of functionality.
• We have developed a large-scale functional approach to measure the impact of all missense variants in theRAD51C gene using PARP inhibitors sensitivity as a readout.
• Experimental replicates and calculation of loss-of-function scores (LOF) using other DNA damaging agents isongoing.
• Future work will focus on validating our data with published works, clinical databases and complementaryassays (RAD51 foci scoring).
• The final goal is to generate a functional atlas for the RAD51C gene in order to improve the interpretation ofmissense VUS and accelerate their clinical translation.
CONCLUSIONS
Thank you!Merci!
@gemma_montalban