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