combining homologous recombination and phosphopeptide … · 2018-10-31 · dna damage and repair...

DNA Damage and Repair

Combining Homologous Recombination andPhosphopeptide-binding Data to Predict theImpact of BRCA1 BRCT Variants on Cancer RiskAmbre Petitalot1,5, Elodie Dardillac2,3, Eric Jacquet4, Naima Nhiri4,Jos�ee Guirouilh-Barbat2,3, Patrick Julien1, Isslam Bouazzaoui5, Dorine Bonte2,Jean Feunteun2, Jeff A. Schnell4, Philippe Lafitte1, Jean-Christophe Aude5,Catherine Nogu�es1, Etienne Rouleau1, Rosette Lidereau1, Bernard S. Lopez2,3,Sophie Zinn-Justin5, and Sandrine M. Caputo1,6, on behalf of the UNICANCERGenetic Group BRCA network

Abstract

BRCA1mutations have been identified that increase the riskof developing hereditary breast and ovarian cancers. Geneticscreening is now offered to patients with a family history ofcancer, to adapt their treatment and the management of theirrelatives. However, a large number of BRCA1 variants ofuncertain significance (VUS) are detected. To better under-stand the significance of these variants, a high-throughputstructural and functional analysis was performed on a largeset of BRCA1 VUS. Information on both cellular localizationand homology-directed DNA repair (HR) capacity wasobtained for 78 BRCT missense variants in the UMD-BRCA1database and measurement of the structural stability andphosphopeptide-binding capacities was performed for 42mutatedBRCTdomains. This extensive and systematic analysisrevealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1HRactivityin cells. Furthermore, binding to a set of 5 different phospho-peptides was tested: all causal variants showed phosphopep-

tide-binding defects and no neutral variant showed suchdefects. A classification is presented on the basis of mutatedBRCT domain solubility, phosphopeptide-binding proper-ties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCTdomains either insoluble in bacteria or defective for phos-phopeptide binding, lead to an increased cancer risk.Furthermore, the data suggest that variants with a WT HRactivity and whose BRCT domains bind with a WT affinity tothe 5 phosphopeptides are neutral. The case of variants withWT HR activity and defective phosphopeptide bindingshould be further characterized, as this last functional defectmight be sufficient per se to lead to tumorigenesis.

Implications: The analysis of the current study on BRCA1structural and functional defects on cancer risk and classifica-tion presented may improve clinical interpretation and ther-apeutic selection. Mol Cancer Res; 1–16. �2018 AACR.

IntroductionBRCA1 encodes a large, 1863-residue protein that functions as a

hub, coordinating a large range of cellular pathways includingDNA repair, transcriptional regulation, cell-cycle control, centro-some duplication, and apoptosis (1). Mutations in BRCA1 havebeen identified that predispose to breast and/or ovarian cancer.

Further screening of patients revealed mutations throughout thewhole BRCA1 gene sequence. The causal nature of mutationscausing a premature stop is generally accepted. Other variantscorrespond to missense variations, deletions/insertions, or intro-nic variations preserving the reading frame. The causal or neutralnature of these variants is more difficult to establish. Therefore,they are called variants of uncertain significance (VUS). In France,they are detected in about 10% of the tested population in theabsence of a causal mutation. Initial attempts to evaluate theclinical significance of VUS in BRCA1/2 were mainly based onfamily data including family history and cosegregation with thedisease. From these data, VUS were classified as either neutral(class 1), likely neutral (class 2), associated to an unknown cancerrisk (class 3), likely causal (class 4), and causal (class 5) (2).However, the majority of the VUS are rare, and therefore thesefamily-based clinical analyses often lack statistical power. High-throughput experimental assays are then the most reliable way todetermine the functional impact of variants with nontruncatingchanges in protein residue composition.

Our goal is to improve the understanding of cancer suscepti-bility caused by BRCA1 gene variations by developing a high-throughput and robust experimental approach for missense VUS

1Service de G�en�etique, D�epartement de Biologie des Tumeurs, Institut Curie,Paris, France. 2Institut Gustave Roussy, CNRS UMR 8200, Universit�e Paris-Saclay, Villejuif, France. 3Team labeled "Ligue 2014," Villejuif, France. 4Institutde Chimie des Substances Naturelles, CNRS UPR 2301, Universit�e Paris-Saclay,Gif-sur-Yvette, France. 5Institut de Biologie Int�egrative de la Cellule, CEA, CNRS,Universit�e Paris Sud, UMR 9198, Universit�e Paris-Saclay, Gif-sur-Yvette, France.6Paris Sciences Lettres Research University, Paris, France.

Note: Supplementary data for this article are available at Molecular Cancer Research Online(http://mcr.aacrjournals.org/).

Corresponding Authors: Sandrine M. Caputo, Institute Curie, 26 rue d'Ulm, Paris75005, France. Phone: 331-7238-9367; Fax: 331-5310-2665; E-mail:[email protected]; and Sophie Zinn-Justin, Universit�e Paris-Saclay,Gif-sur-Yvette, France. E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0357

�2018 American Association for Cancer Research.

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functional characterization. At the present time and according tomost frequently used BRCA1 genetic testing criteria, a causalmutation used for genetic counselling is found in approximately10%of tested index cases (individuals for whoma complete studyof both genes is performed; ref. 3). The carriers from such variantsof class 5 affected with breast or ovarian cancer may now benefitfrom therapies based on platinum agents or PARP inhibitors thatare specifically efficient against BRCA1/2-related tumors (4, 5).Moreover, geneticists can provide informative tests for relativesand adapt their management (reinsurance or preventive manage-ment) according to the test results (6). If the variant is classified asneutral (class 1), another molecular etiology for the familial riskshould be found. However, today, most variants are assigned toclass 3, which corresponds to a lack of knowledge on the VUSimpact. Study of the impact of these VUS is based on differentapproaches (cosegregation, cooccurrence with a causal mutation,family history, RNA stability and quality, amino acid conserva-tion, structural impact of the mutation, functional studies, loss ofheterozygosity in tumors and tumor phenotype, and the use ofmultifactor risk models; refs. 7–11). A combination of structuraland functional information is particularly well adapted to thecharacterization of rare missense variants, with the aim of eval-uating the risk of cancer and taking clinical decisions.

Several groups have undertaken the description of the structureand function of a set of VUS, to predict whether the correspondingmutations or deletions/insertions are likely to affect biologicalpathways involvingBRCA1and lead to tumorigenesis.Most of thestudies have analyzed the impact of mutations in the N-terminaland C-terminal globular domains, whose functions are largelydescribed: the N-terminal RING domain associated to the proteinBARD1 regulates BRCA1 localization and, through its E3 ubiqui-tin ligase activity, contributes to the G2–M cell-cycle checkpoint,whereas the C-terminal BRCT domains are responsible for tran-scription activation and binding to phosphorylated proteinsinvolved in DNA double-strand break signaling and repair(12–14,53). The C-terminal region of BRCA1 encompasses twoBRCT repeats (aa 1646–1736 and aa 1760–1855) that adoptsimilar structures and are packed together in a head-to-tailarrangement (15). These BRCT domains interact specifically withphosphorylated protein targets containing the sequence pSer-x-x-Phe (16–20, 52). The structural impact of the BRCT missensevariations compiled in the BRCA1 Circos resource from theENIGMA consortium was analyzed using limited proteolysis andphosphorylated BACH1–binding assays, and for a subset of thesemutations, by expression in bacteria and characterization of thethermodynamic stability of the purifiedmutants (21–25). Impactof these mutations in cells was revealed using transcriptionalactivation assays (23), and for a small subset of these mutations,homology-directed DNA repair (or homologous recombination;HR) assays (26). However, a high-throughput approach thatprovides both structural and functional information on the sameVUS and is recognized as sufficient to conclude in most casesabout the impact of the VUS on tumorigenesis is still lacking.

To further understand the link between protein stability, phos-phopeptide recognition, localization in cells, HR, and tumori-genesis, we decided to systematically analyze the 3D structure andfunction of the 78 BRCT missense variants deposited in theBRCAShare (ex-UMD-BRCA1 database; refs. 27, 28), includingall VUS detected in France, except missense mutations with asplice impact (29, 30). Therefore, we developed high-throughputassays to (i) measure the capacity of the VUS to repair double-

strand breaks by HR in cells, (ii) test their capacity to relocalize tothe nucleus after addition ofMitomycin (MMC), (iii)measure thethermostability of the corresponding expressed/purified mutatedBRCT domains, and (iv) investigate their binding to a large panelof phosphorylated peptides from different BRCA1 partners(ACC1, BACH1, CtiP, and Abraxas). Half of the selected VUS arealso listed in other databases such as the BIC, KConfab, andClinVar databases. For these VUS, we were able to compare ourexperimental data with previously published results obtainedon subsets of VUS through either in vitro or in cell approaches,thus validating our results (23, 25, 26). Our large-scale analysisfrom both in vitro and in cell data provides a solid basis fordiscussing the impact of the different structural and functionaldefects on increased cancer risk.

Materials and MethodsLentivirus-inducible BRCA1 shRNA system

An anti-BRCA1 shRNA construct was made targeting the30-untranslated region (30-UTR) of the HsBRCA1 gene (Genbankaccession number NC_000017.10). The target sense sequence is50-TATAAGACCTCTGGCATGAAT-30 with a loop TTCAAGAGA.The sequence was cloned into AgeI and EcoRI of a pLKO-Tet-Onvector purchased fromAddgene (31). The pLKO-Tet-On expressesconstitutively a tetracycline-controlled transcriptional suppressorTet, which in turn controls expression of the anti-BRCA1 shRNAsequence inserted in the shRNA cloning site of the vector to beunder the human H1 promoter. In the absence of doxycycline (atetracycline derivative), expression of the anti-BRCA1 shRNA isblocked. When doxycycline is added to the culture medium,transcription of the anti-BRCA1 shRNA occurs, which results inthe knockdown of theHsBRCA1 gene in a highly dose-dependentmanner.

Lentivirus productionThe constructed plasmid pLKO-Tet-On/Anti-BRCA1-shRNA,

and the plasmids pMD2.G and pCMV-dR8.74 (kindly providedby Anne Galy, Genethon, France) were transfected into theHEK293T cells using Lipofectamine 2000 (Invitrogen) followingthe manufacturer's protocol. The plasmid pMD2.G encodes theenvelope protein from the vesicular stomatitis virus (VSV-G)and the plasmid pCMV-dR8.74 encodes the proteins from thegenes Gag and Pol from the human immunodeficiency virustype 1 (HIV-1). The media were changed 24 hours posttrans-fection. Then, the supernatants were collected at 48, 72, and 96hours posttransfection. For each time point, after eliminationof the cell debris, the collected supernatant was ultracentrifugedthrough a sucrose cushion (20%) at 20,000 rpm for 2 hours at12�C. The supernatant was removed and the virus pellet wasresuspended in 500 mL of complete media overnight at 4�C. Thefollowing day, the virus was pooled in the first tube. Finally, thevolume was increased to 1 mL, and the solution was filtered,aliquoted, and stored at �80�C.

Design of the RG37 cell line containing the doxycycline-inducible shBRCA1

RG37 cells are SV40-transformedhumanfibroblasts containinga chromosomally integrated DR-GFP substrate that specificallymonitors gene conversion (32). RG37 cells were infected withlentiviral particles containing a shRNA directed against the 30UTRpart of the BRCA1messenger and the puromycin resistance gene.

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Puromycin was added 3 days after infection and cells werereseeded at low density to isolate individual clones. Cloneswere then screened for deficiency in the formation of BRCA1 fociafter ionizing radiation, deficiency in HR (using the DR-GFPsubstrate; Fig. 2) and for the extinction of BRCA1 expressionmonitored by Western blot analysis. The new cell line was calledRG37-shBRCA1.

Plasmid constructions for expression in mammalian cellsCloning of the mutated BRCT1-BRCT2 gene into the pcDNA3

(modified)-full-length Brca1 was performed by nested PCR (33).First insert was generated by PCR from the gene coding for GST-BRCT1-BRCT2 using the primers: 50TCAACAGAAAGGGTCAA-CAAAAGAAT30 and 50GCCGATATCATCGATTCAGTAGTGG-CTGTG30 (1). Second insert was generated by PCR from pcDNA3(modified)-full-length BRCA1 gene using the primers: 50ACA-CCCAGGATCCTTTCTTGATTG30 (2) and 50ATTCTTTTGTTGA-CCCTTTCTGTTGA30. These inserts were associated by PCR usingtheprimers 1 and3. Thenew insertwas cloned into theBamHIandEcoRV sites of the pcDNA3 (modified)-full-length BRCA1. Allconstructs were verified by DNA sequencing.

HR assays in human RG37-shBRCA1 cellsCellswere pretreated (or not)with10mg/mLdoxycycline 3days

before plating. Theywere then seeded at 2� 105 per well in 6-wellplates. Twenty-four hours after plating, expression vectors encod-ing for BRCA1 (or its variant forms) and the HA-tagged mega-nuclease I-SceI were cotransfected using JetPEI reagent (PolyPlus,Ozyme). Forty-eight hours after transfection, cells were trypsi-nized and GFPþ cells were directly measured by flow cytometry.

Western blot analysis of BRCA1 proteinThe expression of BRCA1 and I-SceI wasmonitored byWestern

blot analysis (Supplementary Fig. S1A). Therefore, proteinextracts (25–50 mg) were resolved on 9% SDS-PAGE, then trans-ferred to a nitrocellulose membrane (0.22 mm) and probed withthe following specific antibodies: anti-BRCA1 (mouse, ab16780,Abcam), anti-HA (mouse, sc-7392, Santa Cruz BiotechnologyInc.), anti-aTubulin (mouse, T5168, Sigma-Aldrich). Immuno-reactivity was visualized using an Enhanced ChemiluminescenceDetection Kit (WesternBright ECL, Advansta Inc.).

Statistical analysisEach batch of assays was run with positive and negative controls

and each variant was tested at least in triplicate in at least fourindependent experiments. The statistical analysis was performedusing theR language release3.5.1 (34) runningonaLinuxserver.Wehave compared the variant BRCA1HR to theWTBRCA1HRusing aone-waypairedStudent t test.Toaccount for themultiple testing, theP values were adjusted using the Benjamini–Hochberg method(35); we controlled the false discovery rates at a level a ¼ 0.05.

Localization assaysRG37-shB1 cells were treated with doxycycline two days before

transfection. Cells were seeded at a density of 1 � 105 per well in6-well plates each containing a glass cover slide and transfectedwith various BRCA1 variants using JetPEI reagent. Twenty-fourhours later, cells are treated with 2.5 mmol/L mitomycin for 2hours and then themediumwas changed. After 8 hours, cells werefixed (PBS, 2% paraformaldehyde, 10 minutes) and permeabi-lized (PBS, 0.1% Triton X-100, 5 minutes). Cells were then

incubated with PBS containing 1% BSA, 0.05% Tween, andanti-BRCA1 antibody (ab16780, Abcam) for 45 minutes at 37�C.After washing three times, cells were then incubated with PBScontaining 1% BSA, 0.05% Tween, and Alexa 568–conjugatedsecondary antibodies (Jackson ImmunoResearch). Cells werethen stained with DAPI and examined with fluorescence micro-scope. Statistical significance was calculated using a two-tailedStudent t test with GraphPad.

Plasmid construction for bacterial expressionThe gene coding for the BRCA1 region BRCT1-BRCT2 (aa

1646–1863) was cloned using the LIC technology (LigaseIndependent Cloning) into the pETM-10 and pETM-30 vectors(EMBL) using the following primers: forward 50GGCAG-GAGCAGCCTCGGAGAATCTTTATTTTCAGGGCGTCAACAAAA-GAATGTCCATGGTGGT30; and reverse 50GCAAAGCACCGG-CCTCGttaTCAGTAGTGGCTGTGGGGGATCT30. All mutationswere generated by Site-Directed Mutagenesis Kit (Stratagene) andverified by sequencing.

Peptides for binding studiesThe following peptides were synthetized by GeneCust Europe:

one control peptide CTRL-P (PTRV-pS-SPVFGAT), 4 monopho-sphorylated peptides CTiP-P (PTRVS-pS-PVFGAT), BACH1-P(ISRST-pS-PTFNKQTK), ACC1-P (DSPPQ-pS-PTFPEAGH),Abraxas-1P (or AB-1P) (GFGEYSR-pS-PTF), and 1 biphosphory-lated peptide Abraxas-2P (or AB-2P) (GFGEY-pS-R-pS-PTF).

Protein expression and solubility assayThe BRCT variants cloned into the pETM-30 expression vector

were introduced into the E. coliBL21(DE3) strain and expressed in96-well microplates using a self-inducible medium. After lysis,soluble protein fractions were separated from insoluble fractions.Soluble fractions were transferred to new 96-well microplates forgel analysis and the rest of the soluble fractionwas incubatedwith20 mL Hi-Trap chelating beads, washed, and diluted into SB1x.Bacteria pellets were suspended in 50 mL 2% SDS. Protein expres-sion and solubility were further analyzed by SDS-PAGE.

Purification of BRCA1 BRCT domain variantsThe BRCTs variants cloned into the pETM-30 expression vector

were introduced into the E. coli BL21(DE3) strain andwere grownat 37�C in LB medium containing 75 mg/mL kanamycin to anOD600 nm of 1.0. Protein overproduction was induced at 20�Cwith 1 mmol/L isopropyl b-D-thiogalactoside (IPTG) for 12hours. The bacteria were then harvested by low-speed centrifu-gation at 6,000 rpm for 15 minutes. The bacterial pellet wassuspended in 30 mL lysis buffer [100 mmol/L Tris pH 7.5,150 mmol/L NaCl, 1% (w/v) Triton X-100, 10% glycerol,1 mmol/L EDTA, 10 mmol/L DTT, 1 mmol/L phenylmethylsul-fonyl fluoride (PMSF), 2 mmol/L ATP, 10 mmol/L MgSO4] andincubated with 1% lysozyme overnight at 4�C. Then, 1 mL ben-zonase (Sigma), 2 mmol/L ATP, 10 mmol/L MgSO4 and 10mmol/L MgCl2 and 10 mmol/L DTT were added. After 30 min-utes, the extractwas centrifuged at 20,000 rpm for 30minutes, andthe soluble protein fraction was loaded on a 30 mL Glutathionesepharose 4FF column previously equilibrated in 50mmol/L Tris,150 mmol/L NaCl pH 7.5. The column was washed with a buffercontaining 1 mol/L NaCl, then 150 mmol/L NaCl, and then with50 mmol/L Tris, 150 mmol/L NaCl pH 7.5. The TEV protease wasadded and incubated overnight at 4�C. After TEV cleavage, the

Classification of BRCA1 BRCT Missense VUS

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elution from the GST-trap column was loaded onto a HisTrapcolumnequilibrated in 50mmol/L Tris pH7.5, 150mmol/LNaCl.The protein was eluted with 50 mmol/L Tris pH 7.5, 150 mmol/LNaCl, and 10 mmol/L imidazole. Fractions highly enriched inBRCT variants were diluted 5-fold in 50 mmol/L Tris pH 7.5 andloaded on a 5 mL Q-Sepharose High Performance columnequilibrated in 50 mmol/L Tris pH 7.5. Bound proteins wereeluted with between 200 and 350 mmol/L NaCl. In both cases,the collected fractions were analyzed by 0.1% SDS-15% PAGE,using as a marker the broad range prestained protein marker(Bio-Rad). The purified protein was characterized by electro-spray ionization mass spectroscopy.

Isothermal titration calorimetryITC was performed using a high-precision VP-ITC calorimetry

instrument. To characterize interactions between the BRCT1-BRCT2 domains and peptides, all proteins were dialyzed against50 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 10 mmol/Lb-mercaptoethanol, and protease inhibitors (Roche). BRCT1-BRCT2 domains (10–20 mmol/L) in the calorimetric cell at 30�Cwere titrated with the peptide (at a concentration of 100–200mmol/L in the injection syringe). Analyses of the data were per-formed using the Origin software provided with the instrument.

Size-exclusion chromatographySize-exclusion chromatography experiments aiming at identi-

fying interactions between BRCT1-BRCT2 domains and peptidesafter ITC were performed using a Superdex-75 10/300GL column(GE Healthcare) preequilibrated in the ITC buffer (50 mmol/LTris-HCl pH 7.5, 150mmol/L NaCl, 10 mmol/L b-mercaptoetha-nol and protease inhibitors; Roche). Proteins were concentratedafter ITC (calorimetric cell) to obtain a volumeof 500 mL andwereloaded on the column at a flow rate of 0.5 mL/minute at 4�C.

Thermostability measurements by fluorescence-based thermalshift assay

The FBTSA method is used to monitor the BRCT domainthermal denaturation and changes induced by a mutation and/or a binding event. To measure the thermostability of WT andmutated BRCT domains, we mixed 1 mg of purified protein in50 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl, 2 mmol/Lb-mercaptoethanol, and the SYPRO Orange dye (diluted 400-fold from a 5,000-fold stock solution, Invitrogen). The sameexperimental conditions were used to investigate the interactionof the BRCT domains and the phosphorylated peptides CTRL-P,ACC1-P, CTIP-P, BACH1-P, AB-1P (one phosphorylation),and AB-2P (two phosphorylations). The peptides were first dilut-ed in the same buffer as the BRCT domains and then addedto the reaction mixture with increasing concentrations up to256 mmol/L. Reaction mixtures were made in duplicate in aMicroAmp Optical 384-well reaction plate at a final volume of10 mL (4 mmol/L) and each experiment was repeated at least twiceindependently. Experiments were carried out in QuantStudio12KFlex qPCRmachine (Applied Biosystems) with a temperaturegradient in the range of 15–95�C at 3�C/minute.

ResultsSelection of 78 BRCT VUS from the UMD-BRCA1 database

The VUSwere selected from the French UMD-BRCA1 database,which gathered in November 2015, 2,020 distinct variants col-

lected from 8,446 families, and included 1,028 distinct VUS ofclass 3 (http://www.umd.be/BRCA1/; ref. 27). A large majority ofthese VUSof class 3were found in a unique family of patients. Theexon containing the largest number of variants (exon 18) corre-sponded to the first BRCT domain (BRCT1; ref. 27). We focusedour study on variants located in the C-terminal BRCT1 and BRCT2domains of BRCA1 (aa 1646–1859). We selected the 65 VUS ofclass 3 that corresponded to a missense variation in the BRCTdomains of BRCA1 (Fig. 1A; Supplementary Table S1). We alsoselected 6 variants of class 5 (V1665E, R1699W,G1706R, A1708E,S1715N in BRCT1 and M1775R in BRCT2;¼causal variants) and1 variant of class 4 (G1706E in BRCT1; ¼likely causal variant) aswell as 3 variants of class 2 (M1652T in BRCT1, R1751Q in linker,M1783T inBRCT2;¼likely neutral variants) and3 variants of class1 (M1652I, T1720A in BRCT1, V1804D in BRCT2; ¼neutralvariants), to serve as controls for our experiments (Fig. 1A). The78 selected mutations are distributed all along the BRCT domainsequence. They are also located on the whole 3D structure: in theBRCTdomainhydrophobic cores, at the interface between the twoBRCT domains, as well as in solvent-exposed regions and inparticular the phosphopeptide-binding region (Fig. 1B). All 78VUS were evaluated through a dedicated high-throughput work-flow combining tests performed in cells (HR assay, localizationafter DNA damage) and in vitro (protein stability upon expressionin bacteria and binding to a set of 5 phosphopeptides) to providea comprehensive description of their function.

Impact of the missense mutations on HRTo address this question, we designed a novel cell line RG37-

shB1. This cell line was derived from RG37 cells, which are SV40-transformed human fibroblasts containing an integrated DR-GFPsubstrate to specificallymonitor gene conversion upon expressionof the meganuclease I-SceI (Fig. 2A; ref. 32). RG37 cells wereinfected with a retroviral construct containing a doxycycline-induced shRNA raised against the 30-UTR of the endogenousBRCA1 gene (RG37-shB1cells). Therefore, supplying doxycyclinespecifically silenced the endogenous BRCA1without affecting theexpression of the exogenous transfected BRCA1 cDNA. Figure 2Bshows that supplying doxycycline strongly decreased the expres-sion of BRCA1 and reduced the efficiency of I-SceI–induced HR2-fold. Cotransfection of a wild-type BRCA1 cDNA with theI-SceI–coding plasmid stimulated the frequency of HR in cellsunexposed to doxycycline, as already described with RG37 cells,and, importantly, rescued HR frequency in doxycycline-treatedcells (Fig. 2B). Interestingly, all tested BRCA1 variants reportedas causal (V1665E, R1699W, G1706R, A1708E, S1715N,M1775R) or likely causal (G1706E) were unable to complementBRCA1 deficiency for HR (Fig. 2C).

Using the same assay, we measured the capacity of the 78missense variants (including the 7 causal mutations shownabove) to complement BRCA1 silencing for I-SceI–inducedHR. Either WT BRCA1 or the VUS were expressed in thissystem, as checked by Western blot analysis using anti-BRCA1antibodies (Supplementary Fig. S1A). Variation of HR efficien-cy as a function of the position of the variation in the sequencedid not reveal any functionally critical hotspot (Supplemen-tary Fig. S1B). Expression of variants reported as neutral(M1652Ib due to G>A, T1720A, V1804D) or likely neutral(M1652T, R1751Q, M1783T) systematically complementedHR efficiency at least as much as WT-BRCA1 (Fig. 3A; Table1). VUS M1652Ia (due to G>T; class 3) was tested, which also

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affects position 1652: it consistently showed a WT HR effi-ciency. On the basis of this unique assay, we could alreadyconfirm the link between causality and HR defect with asensitivity of 100% (7/7 causal variants show an HR defect)and a specificity of 100% (6/6 neutral variants have no HRdefect). This first analysis confirmed the essential role of HRdefect in tumorigenesis.

Global analysis of the HR assay results performed on the 78variants revealed that 31VUS (40%) failed to complement BRCA1deficiency for HR (Fig. 3A). These VUS include the 7mutations ofclass 4 or 5, 24 VUS of class 3, and no VUS of class 1 or 2. Positionsthat are mutated in defective VUS are distributed all along theBRCA1 BRCT domains, from aa 1655 to aa 1837. However, 19among the 31 defective VUS are mutated within BRCT1. More-over, most mutated residues corresponding to defective VUS areburied in the 3D structure of the BRCT domains (25 within 31are less than 20% solvent accessible), whereas only half of thepositions mutated in variants characterized by a normal HR areburied in the hydrophobic cores of the BRCT domains or at the

BRCT1/BRCT2 interface. This analysis shows that if a position iseither in BRCT1or buried, itsmutation ismore likely to negativelyaffect HR.

At given positions, several mutations were identified, corre-sponding to different VUS. For example, position 1665 ismutatedin 3 VUS that showed either no defect (V1665M, V1665L) or asignificant defect (V1665E, causal) in HR efficiency, suggestingthat any hydrophobic residue is tolerated at this buried position,but the introduction of a charged amino acid causes a HR defect.At position 1699, which is solvent-exposed and is responsible forcontacts with the phosphopeptide main chain (Fig. 5B), variationtoQ had the same functional impact as the causal mutation toW.Impact of these mutations on BRCA1 structure and phosphory-lated BACH1 binding are consistent with the analysis of Coquelleand colleagues (21), who showed that the R1699W mutationdestabilized the BRCA1 protein structure and likely interferedwith the docking of the peptide phenylalanine into the BRCTpeptide–binding groove, whereas the R1699Qmutation caused asteric clash between the glutamine side-chain carbonyl and the

Figure 1.

Distribution of the 78 variationscorresponding to the selected VUS.A, Position of the mutations along theBRCA1 sequence. On the top view, thewhole BRCA1 sequence is displayed,with its 2 globular domains in red(N-terminal RING domain) and orange(C-terminal BRCT region), and itsnuclear export/import signals in black(NES) and green (NLS), respectively.The lower view is a zoom of the BRCA1BRCT region. It contains 2 BRCTdomains indicated in gray. The 78variations corresponding to theselected VUS are indicated on thiszoom. The 7 causal and 6 neutralselected variants are displayed in redand blue, respectively. B, Position ofthe mutated residues in 3D structureof the BRCT domains (in dark gray) incomplex with a phosphorylatedBACH1 peptide (in light gray)(PDBcode 1T15). Mutated positions arefound throughout the whole structure.Those corresponding toVUSof classes1 and 2 are colored in blue, classes 4and 5 in red, and class 3 in yellow. VUSof classes 1, 2, 4, and 5 are labeled.






main-chain carbonyl of the peptide phenylalanine residue. Atposition 1706, which is buried, variation to A did not affect HR,whereas the causal mutations to charged residues (G1706E andG1706R) did. Similarly, at position 1708, which is also buried,variation to V did not affect HR, whereas the causal mutation to Ror E did. Such analysis underlines the consistency of our HRassay results and highlights the variability of the functionalconsequences of variations at a same position as a function ofthe physico-chemical properties of the WT versus variant aminoacid. Clearly, at buried positions, mutation to a hydrophobicresidue is tolerated but mutation to a charged residue impairsHR efficiency. At solvent-exposed positions, if the mutatedresidue is involved in the recognition of a functionally essentialpartner, then any type of replacement might impair recognitionand increase cancer risk.

Impact of the missense mutations on BRCA1 nuclearlocalization after DNA damage in mammalian cells

A first explanation for the deleterious effect of a mutation onHR is the overall low concentration and/or the decreased nuclearlocalization after DNA damage of the corresponding variant. Asno significant difference in protein amounts was observedthroughout the VUS byWestern blot analysis, we further revealedby immunofluorescence BRCA1 localization in RG37-shB1 cellsafter addition of mitomycin C. This molecule is a chemothera-peutic drug that upon reduction is converted into a highly reactiveintermediate alkylatingDNA.Weobserved thatWTBRCA1aswellas a large set of VUS correctly localized at the nucleus afteraddition of this drug. However, 34 variants were not correctlylocalized in response to mitomycin C (Table 1; Fig. 3B and C).These variants include 4 causal variants (all except R1699W and

Figure 2.

Description of the high-throughput cellular assay set up for measurement of VUS HR efficiency. A, Description of the HR substrate (DR-GFP). Two inactiveGFPgenes are organized into direct repeats. The 50 GFPcassette is inactivatedbecause of deletions in both the 50 and the 30 sequences. Expressionof I-SceI generatesa cleavage (DSB) targeted into the substrate. HR between the two GFP genes, with I-SceI, can generate a functional GFP gene through gene conversionwithout crossing over. Recombinant cells are thus GFP-positive (GFPþ) and can be monitored by FACS (54). The DR-GFP substrate is stably integrated into achromosome of SV40-transformed fibroblasts in the RG37 cell line (25). B, Inducible knockdown of endogeneous BRCA1. One cell line containing adoxycycline-inducible shRNA against the endogenous BRCA1 was derived from the RG37 cell lines (which bear the HR substrate): the RG37 cells have beentransduced with a lentivirus coding for a shRNA against the 30UTR of the endogenous BRCA1 mRNA. This allows to specifically targeting the endogenousBRCA1 and not the exogenous BRCA1 that will be then used. In addition, expression of this shRNA is inducible by the doxycycline. Supplying doxycycline (DOX)induces the expression of the shRNA leading to the silencing of the expression of the endogenous BRCA1 (left) and to reduced HR frequencies, monitoredusing the DR-GFP (right). This cell line (named RG37shB1) was then used to test all the BRCA1 variants. Because this shRNA does not affect the expression ofexogenous BRCA1, expression of exogenous wild-type BRCA1 (WT-BRCA1) is able to stimulate HR frequency, as already shown for BRCA1 overexpressionin RG37 cells (55), and to rescue decreased HR frequency in DOX-exposed cells, i.e., silenced for the expression of the endogenous BRCA1. The values correspondto at least 3 independent experiments. C, Impact of BRCA1 bearing causal mutations on the rescue of HR in cells silenced for the endogenous BRCA1. Thevalues are shown normalized to WT-BRCA1 (in black). They correspond to four to eight independent experiments.

Petitalot et al.





A1708E), the likely causal G1706E, and 29 VUS of class 3. Withinthese 29 variants, 18 are defective for HR. The remaining 11 VUSare capable of compensating their localization defect to carry outnormal DNA repair byHR. Theymight still show other functionaldefects. The 6neutral or likely neutral VUS showedno localizationdefect. Finally, 6 VUS of class 3 showed HR defects but nolocalization defects, demonstrating that localization defect is notthe only mechanism leading to impaired HR. In summary, thissecond assay enabled classification of 5 of 7 causal variants(sensitivity ¼ 71.4%) and 6 of 6 neutral variants (specificity ¼100%), but only 18 VUS of class 3 are identified as both HR-defective (within 24 VUS) and localization defective (within 29VUS), suggesting that the two assays provide only partially over-lapping conclusions.

Impact of the missense mutations on solubility in E. coli andthermostability of recombinant BRCT domains

Another explanation for the loss of HR activity of 40% of theVUS is the impact of BRCA1 variations on the BRCT domainthermostability leading to a global loss of function of thesedomains. We measured the structural impact of the 78 missensevariations by expressing the mutated domains, purifying thesoluble domains andmeasuring the thermostability of the result-ing mutated BRCT domains by fluorescence-based thermal shiftassay (FBTSA).

First, 23 mutated proteins were insoluble in E. coli (Table1; Fig. 4A; Supplementary Fig. S2). We classified these proteinsas highly unstable, and no further work was carried out on them.They corresponded to 4 variants of class 5, 1 variant of class 4, 17

Figure 3.

Impact of the BRCA1 missensemutations on HR. A, Plot of the HRefficiencies measured after expressionof either WT BRCA1 or VUS normalizedto the HR efficiency after expression ofWT BRCA1. Statistical significance wascalculated using a one-side pairedStudent t test. To account for themultiple testing, the P values wereadjusted using the Benjamini–Hochbergmethod at a level a ¼ 0.05. HRefficiencies significantly different fromthe WT value are marked by asterisks.They are indicated by � if P < 0.05 and�� if P < 0.01. All 7 (likely) causal (notedLC or C) variants (in red) cause asignificantly decreased HR efficiency,whereas all 6 (likely) neutral (noted LNor N) variants (in blue) cause nosignificant HR difference. Twenty-onevariants could not provide WT HRefficiency. B, Identification of the VUSdefective for nuclear localization afteradditionofMMC,plotted as a functionofincreasing HR efficiencies as in A. Forobserving VUS nuclear localizationdefects, plates were examined afteraddition of mitomycin to RG37 cellstransfected with VUS plasmids. Cellswere immunostained with an anti-BRCA1 antibody followed by an AlexaFluor 488–conjugated secondaryantibody and the nucleus was stainedwith DAPI. BRCA1 localization wasvisualized by fluorescence microscopy.Statistical significance was calculatedusing a two-tailed Student t test withGraphPad. This analysis revealed that34VUSwerenotcorrectly addressed to thenucleus. The bars corresponding tothese VUS are colored in green. A morequantitative report of the percentage ofnuclear fluorescencemeasured for eachVUS can be found in SupplementaryTable S3. As an illustration, in the toppart of B, images from controlexperiments showing that BRCA1 WT iscorrectly localized in the nucleus aftermitomycin treatment (lane 1) as well asimages revealing the localization of aneutral (M1652T; lane 2), and a causal(V1665E; lane 3) VUS are displayed.






VUS of class 3, and 1 variant of class 2. Interestingly, 21 of 23(91%) of these VUS also showed significant HR defects. Only twoof these VUS shows a normal HR function: M1652T that isclassified as likely neutral and A1752E that belongs to class 3.Thus, a very large majority of highly unstable BRCT domainscorrespond to VUSwith a defective HR activity. Reciprocally, only4 VUSwith impairedHR activities (13%of theHR-defective VUS)correspond to domains that could be purified in vitro: R1699W,R1699Q, M1775R, and V1833M. These results revealed a nicecorrelation between the HR activity of the whole BRCA1 VUS andthe solubility of the mutated BRCT fragments in our conditions.

When going further into this in vitro analysis, 13 poorly solublemutated BRCT domains could not be purified because of aggre-gation, 26 could be expressed and purified but with low yields,and only 16 were obtained with a yield close to that of the WT-BRCT domains (Table 1; Fig. 4A). This classification seems tocorrelate with HR results, because 46% of the aggregatingmutants, but only 12% of the poorly soluble mutants and 6%of the WT-like mutants, are HR-defective.

Finally, we measured the thermostability of the 42 purifiedmutated domains using a high-throughput fluorescence assay byFBTSA (Supplementary Fig. S3). The melting temperature mea-

sured for the WT is 52�C � 0.3�C. No clear distinction could beobserved between the results obtained for the two causal variantsand the two likely neutral variants: causal variants M1775R andR1699W showed melting temperatures of 41.9�C and 43.8 �0.1�C, respectively, whereas likely neutral mutants R1751Q andM1783T showed melting temperatures of 42.7�C and 44.9 �0.1�C, respectively. However, more generally, the four HR-defec-tive variants for which a melting temperature could be measuredare distributed within the 45% variants with the lowest meltingtemperatures: their melting temperatures range between 40.4�Cand 48.8�C. Also, the three neutral variants have significantlyhigher melting temperatures comprised between 50.5�C and51.6�C.

In summary, the causality and HR assay results are stronglyrelated to the observation of insolubility versus solubility of theBRCT domains in bacteria. We observed that five of seven causalvariants (that are all HR-defective) exhibit BRCT domains insol-uble in bacteria (sensitivity ¼ 71.4%), whereas five of six neutralvariants (that all show WT HR activity) have BRCT domains thatare soluble in bacteria (specificity ¼ 83%). Moreover, among the24 HR-defective VUS of class 3, 16 correspond to insolubledomains, 6 to domains that aggregate during purification, 1 to

Table 1A. Summary of the experimental results presented in this studyHGVS DNA

nomenclature c.Amino Acid

ChangeUMD-BRCA1

Class HRLocalisation after

MMC

Expression in E. coli & Stability if

Soluble

Loss of phosphopeptide

bindingClassification

HGVS DNAnomenclature c.

Amino AcidChange

UMD-BRCA1 Class HR

Localisation afterMMC

Expression in E. coli & Stability if

Soluble

Loss of phosphopeptide

bindingClassification

c.4949T>C M1650T like wt like wt B -0,8 1P c.5192A>G E1731G 1,12 ns 1,12 ns B -2,7 1P

c.4955T>C M1652T 2 0,88 ns like wt C NA 2P c.5192A>T E1731V 1,2 ns 1,2 ns B -0,9 1P

c.4956G>A M1652I b 1 like wt like wt B -0,3 1P c.5203G>A E1735K 0,97 0,97 B- NA 2P

c.4956G>T M1652I a like wt B -0,4 1P c.5213G>A G1738E 0,828877005 0,828877005 B- NA 3P

c.4963T>G S1655A 0,62974268 like wt C NA 3P c.5213G>T G1738V 0,828877005 0,97 B- NA 3P

c.4991T>C L1664P like wt B -1,3 1P c.5216A>G D1739G 1,27 ns like wt B- NA 2P

c.4993G>A V1665M like wt like wt B -0,9 1P c.5216A>T D1739V 1,25 ns like wt B- NA 2P

c.4993G>C V1665L 1,07 like wt B -0,1 1P c.5236C>A H1746N like wt A 8 2P

c.4994T>A V1665E 5 0,632716049 C NA 3P c.5252G>A R1751Q 2 1,08403589883057 ns like wt B -1,8 1P

c.4999A>G K1667E like wt like wt A -0,01 1P c.5254G>A A1752T 0,828877005 0,828877005 B- NA 3P

c.5005G>T A1669S 1,04 like wt B -0,01 1P c.5254G>C A1752P 0,597 0,828877005 B- NA 3P

c.5008A>G R1670G 0,98 like wt B like wt 1P c.5255C>A A1752E 0,95 0,828877005 C NA 2P

c.5053A>G T1685A 0,660550459 C NA 3P c.5282T>C F1761S 0,619369369 0,828877005 C NA 3P

c.5057A>G H1686R 0,622568093 C NA 3P c.5291T>C L1764P 0,828877005 0,828877005 C NA 3P

c.5060T>A V1687D 0,776176929 problem of re localisation C NA 3P c.5309G>T G1770V 0,95 0,828877005 B- NA 2P

c.5062G>T V1688F 0,598949212 C NA 3P c.5321A>G N1774S 0,89ns like wt A -1,2 1P

c.5068A>G K1690Q 0,638795987 like wt C NA 3P c.5324T>G M1775R 5 1,02 0,828877005 B 6,8 3P

c.5071A>G T1691A 1,05 like wt B like wt 1P c.5344T>C W1782R 1,11442786069652 ns problem of re localisation B -0,8 1P

c.5072C>A T1691K 0,711409396 C NA 3P c.5348T>C M1783T 2 1,11442786069652 ns like wt B -1,2 1P

c.5072C>G T1691R 0,658291457 C NA 3P c.5355G>T Q1785H 0,94 like wt A -0,8 1P

c.5072C>T T1691I 0,59 C NA 3P c.5359T>G C1787G 0,97 like wt A -0,4 1P

c.5085T>A F1695L like wt like wt like wt like wt 1P c.5365G>T A1789S 1,02 0,828877005 B -1,6 2P*

c.5089T>C C1697R 0,821672355 C NA 3P c.5371G>C V1791L 0,98 like wt A -0,4 1P

c.5095C>T R1699W 5 0,663316583 like wt B 4,5 3P c.5411T>A V1804D 1 1,11324376199616 ns like wt B -0,4 1P

c.5096G>A R1699Q 0,713567839 like wt A 5,9 3P c.5419A>G I1807V 1,02 like wt A -0,01 1P

c.5099C>T T1700I 0,649425287 like wt C NA 3P c.5426T>G V1809G 0,596928983 0,828877005 C NA 3P

c.5116G>A G1706R 5 0,659090909 C NA 3P c.5429T>G V1810G 0,86 ns like wt A -0,01 1P

c.5117G>A G1706E 4 0,56 C NA 3P c.5432A>G Q1811R 0,614203455 problem of re localisation C NA 3P

c.5117G>C G1706A 0,890909090909091ns like wt B -0,8 1P c.5458G>A G1820S 1,05 problem of re localisation A -0,2 1P

c.5123C>A A1708E 5 0,585014409 like wt C NA 3P c.5461T>C F1821L 1,1 like wt A -0,01 1P

c.5123C>T A1708V 1,10384 ns like wt B -1,7 2P* c.5474G>A G1825E 1,12ns like wt A -1,5 1P

c.5129G>A G1710E 0,97 like wt B -2,5 1P c.5497G>A V1833M 0,828877005 like wt B -3,3 2P

c.5138T>C V1713A 0,678341272 0,678341272 C NA 3P c.5498T>G V1833G 0,739626556 problem of re localisation B- NA 3P

c.5144G>A S1715N 5 0,703305352 0,678341272 C NA 3P c.5506G>A E1836K 0,91 like wt A 3,7 2P

c.5150T>A F1717Y 1,22 ns like wt B 8,2 2P c.5509T>C W1837R 0,828877005 problem of re localisation B- NA 3P

c.5155G>T V1719L 1,1 problem of re localisation B -0,01 1P c.5509T>G W1837G 0,836785162287481 ns problem of re localisation B- NA 2P

c.5158A>G T1720A 1 1,03 like wt B -0,3 1P c.5522G>A S1841N 0,893877551020408ns problem of re localisation B- NA 2P

c.5165C>A S1722Y 0,94 B- NA 2P c.5531T>C L1844P 0,931313131 like wt A -0,01 1P

c.5177G>T R1726I 1,22 ns problem of re localisation B -1,7 1P c.5566C>T P1856S 1,03 like wt A -0,4 1P

NOTE: In column "HR," variants with defective HR are indicated in red and variants withWT HR in blue. In column "localization after MMC," variants with defective localization are again in red.In column "expression in E. coli and stability during purification if soluble," VUS with BRCT domains that were mostly insoluble in E. coli appear in red, poorly soluble mutated domainsthat could not be purified because of aggregation in light red, domains that could be expressed and purified but with low yields in light blue and mutated domains that were obtainedwith a yield close to that of the WT BRCT domains in blue. In column "loss of phosphopeptide-binding," VUS with domains binding as the WT BRCT domains are indicated in blue and VUSwith domains that do not bind in red (light red: defect in BACH1-P binding; bright red: binding defect observed with the 5 phosphopeptides). Boxed VUS are mutated at positions buried inthe 3D structure of the BRCA1 BRCT domains. In column "classification," we have distributed the 78 variants in 3 distinct classes (1P no impact, 2P intermediate impact, 3P severe impact) as afunction of our results, as explained in Table 1B. Red stars associated to the class type indicate that inconsistent binding datawere previously published. Therefore, the corresponding VUSwereclassified as 2P.

Petitalot et al.





a poorly soluble domain, and 1 to a WT-like domain and 1 to aWT-like domain. We have represented on the 3D structure of theBRCA1 BRCT domains the localization of the mutated positionscorresponding to both HR-deficient VUS and insoluble (brown)or aggregating (yellow) BRCT domains (Fig. 4B). Clearly, thesepositions aremainly found in BRCT1 and in particular close to thephosphorylated serine of the BRCA1 partner, underlying thenecessity for BRCA1 to interact with phosphorylated partners topromote HR. Fragment from residue 1685 to residue 1708,comprising a b-strand, a loop, and a a-helix interacting withthe consensus motif Ser-X-X-Phe of BRCA1-phosphorylatedpartners (further named the phosphopeptide binding loop),concentrates 13 within the 27 identified positions colored inbrown in Fig. 4B.

Impact of the missense variants on the binding of the BRCTdomains to phosphorylated peptides

The BRCA1 BRCT domains bind to phosphoproteins such asAbraxas, ACC1, BACH1, and CtiP, which share a common Ser-X-X-Phe motif phosphorylated on the serine residue (17–19).Because several mutations leading to an increased cancer riskdisrupt the binding surface of the BRCT domains to phosphor-ylated peptides, it was suggested that BRCA1 phosphopeptidebinding is essential for BRCA1's tumor-suppressing function (26,

36).Moreover, a previous studyofmice carrying aBRCTmutant ofBRCA1 that is defective in recognition of phosphorylated proteinsalso suggested that BRCT phosphoprotein recognition is requiredfor BRCA1 tumor suppression (14). Using the same high-throughput fluorescence assay as for the thermostability mea-surements, revealing thermostability shifts due to peptidebinding here, we tested binding of the 42 purified mutatedBRCT domains to five different phosphopeptides. These arefragments of the DNA repair protein Abraxas (belonging to theso-called BRCA1-A complex), the acetyl-CoA carboxylase 1(ACC1), the DNA helicase BACH1 (belonging to the so-calledBRCA1-B complex), and the transcriptional corepressor CtiP(belonging to the so-called BRCA1-C complex). ACC1 is anenzyme essential for cancer cell survival that catalyzes fatty acidbiosynthesis; phosphopeptide (ACC1-P) recognition is impor-tant for the regulation of fatty acid biosynthesis (37). BACH1phosphopeptide (BACH1-P) recognition is involved in G2–Mcheckpoint control and DSB repair and CtiP phosphopeptide(CTIP-P) recognition is essential for DNA end resection of DSBsduring HR. Abraxas and the BRCA1-A complex recruit BRCA1 toDNA double-strand break sites (DSB) through an ATM-depen-dent ubiquitin-mediated signaling pathway. The interaction ofphosphorylated Abraxas (mono-phosphorylated peptide AB-1P and di-phosphorylated peptide AB-2P) with BRCA1 is

Table 1B. Classification of the variants

Func�on in cells

BACH1-P ACC1-P/CTIP-P AB-1P/AB-2P HR

2 1 1 1 3

Phosphopep�de binding in vitroSolubility in bacteria

and duringpurifica�on

3P

0-1 2 2-3 3 5-6

Binds to the 5phosphopep�des in

this study

Binds tophosphopep�des as

reported in theliterature

Contradictory bindingdata

Binds tophosphopep�des as


Do not bind tophosphopep�des as


Do not bind to the 5phosphopep�des in

this studyAll cases

Undetermined Undetermined Undetermined Likely Causal Causal

0

No impact

P2P1

NOTE: The scoring scheme is a result of our analysis. First, because of the strong relationship observed between both BRCA1 HR defect and BRCA1 phosphopeptide-binding defect andtumorigenesis, our score largely depends on these BRCA1 properties (þ3 for an HR defect and þ3 if binding to all 5 phosphopeptides is impaired). Also, as impaired solubility in bacteria isgenerally associatedwith causality,we scoredþ2 in caseof insolubility or aggregationduring the purification. VUSwith noobserveddefect (score¼0)were classified as 1P,with an intermediateimpact (scores between 1 and 3) as 2P, and with defects observed both in vitro and in cells (scores higher than 4) as 3P. Moreover, VUS with a score of 0 for which we measured WT bindingproperties that are not consistent with the literature were classified as 2P.






critical for the function of Abraxas in DNA repair of DSBs andmaintenance of genomic stability (23).

First, to confirm the binding capacity of the purified WT BRCTdomains, we measured their affinity for a set of phosphorylatedpeptides using isothermal titration calorimetry (ITC). We mea-sured the affinities of the interactions between the BRCT domainsand either ACC1-P or BACH1-P. We obtained affinities of 2.1 �0.2 mmol/L and 0.19 � 0.01 mmol/L, respectively, consistentlywith the literature (Supplementary Fig. S4A and S4B; refs. 37, 38).We also measured the affinities of the interactions betweenthe BRCT domains and either AB-1P or AB-2P. We obtainedaffinities of 25 � 2 nmol/L and 6.2 � 1.2 nmol/L, respectively(Supplementary Fig. S4C and S4D). These last interactions arecharacterized by a large affinity increase compared with theinteractions with ACC1-P and BACH1-P. Finally, we ran size-exclusion chromatography experiments on these complexes. TheBRCA1andBRCA1þBACH1-P elution volumes correspond to themolecular mass of a monomer, whereas that of BRCA1þAB-1P isindicative of a monomer–dimer equilibrium and that ofBRCA1þAB-2P reveals a dimer, which is also consistent with theliterature (Supplementary Fig. S5A and S5B; ref. 23).

Second, we verified using our FBTSA assay that binding ofthe WT BRCA1 BRCT domains to ACC1-P, BACH1-P, CtiP-P,

AB-1P, and AB-2P induced a measurable increase in theBRCT thermostability that rose with peptide concentration(Supplementary Fig. S6A). Indeed, addition of ACC1-P andCtiP-P caused a maximal stability increase of 5.9�C and 6.2 �0.1�C at 256 mmol/L, respectively, whereas addition of the sameamount of BACH1-P, AB-1P, and AB-2P increased the WT BRCTthermostability by 8.2�C, 14.1�C, and 15.1 � 0.1�C, respec-tively. Control experiments showed that addition of a peptidewith the same sequence as CtiP, but phosphorylated on theother serine residue (CTRL-P; see Fig. 5A) increased the ther-mostability by only 0.1�C.

From this description of the WT BRCT domain–bindingproperties, it was possible to design a large high-throughputexperiment aiming at identifying the VUS defective in phos-phopeptides binding. Figure 5A shows the thermostabilityshifts caused by binding of the peptides CTRL-P, ACC1-P,BACH1-P, CtiP-P, AB-1P, and AB-2P to the BRCT domains ofWT BRCA1 and the 42 mutants that could be expressed andpurified. Clearly, addition of either ACC1-P, CtiP-P, BACH1-P,AB-1P, or AB-2P generally have the same impact on the ther-mostability of the mutated BRCT domains. This confirms thatthe five peptides bind to the same BRCA1 site. Five variantssignificantly lost affinity for the peptides: R1699W andM1775R

Figure 4.

Representation of the solubility inbacteria of the recombinant mutatedBRCT domains. A, Bars of the HR plotof Fig. 3Aare coloredas a functionof thesolubility in E. coli of the correspondingmutated BRCT domains. Brown barsmark mutants that are completelyinsoluble as GST fusion proteins in E.coli. Yellow bars indicate mutants thatare partially soluble in bacteria butaggregate during removal of the GSTtag and purification. Only mutantscorresponding to gray and black barscould be further characterized in vitroby fluorescence. The differencebetween these two last classes is basedon their expression and purificationyields, gray meaning low productionyield and black WT-like productionyield. B, 3D representation of theposition of the residues mutated in VUScharacterized by both (i) defective HRcapacities and (ii) recombinant BRCTdomains that could not be producedand purified from bacteria (in brownand yellow/green as in A).

Petitalot et al.





(Supplementary Fig. S6B) are the two only causal variants thatcould be purified; mutants R1699Q, F1717Y and H1746Ncorrespond to VUS of class 3. In the case of mutants F1717Yand H1746N, no binding of the peptides to the BRCT domainscould be detected (Fig. 5A). Representation of the 4 positionsmutated in these 5 variants on the 3D structure of the BRCTdomains revealed that Arg1699 and Met1775 are directlyinvolved in peptide binding, whereas Phe1717 and His1746are further from the binding site and probably indirectlycontribute to peptide recognition (Fig. 5B; ref. 20). Theseresidues might be critical for correctly positioning the phos-phopeptide binding loop (amino acids 1685 to 1708), as theyinteract with Lys1690 and Tyr1703, respectively. Altogether, thevariants of classes 4 and 5 are all HR-deficient and when testedare phosphopeptide-binding deficient, whereas the variants ofclasses 1 and 2 have a WT HR activity and when tested show aWT phosphopeptide-binding capacity. By taking into accountthe results of the cellular HR assay and the in vitro binding assay,

it is possible to classify 7 of 7 causal variants (sensibility ¼100%) and 6 of 6 neutral variants (specificity ¼ 100%).

DiscussionIn this study, we tested 78 BRCA1 variants using 4 assays that

focus on different properties previously shown as related toBRCA1 oncogenic function. Our challenge was to set up ahigh-throughput protocol to (i) measure BRCA1 HR capacity in6-well plates through a fluorescence reading, (ii) test bacterialexpression of the BRCT domains in 96-well plates and read thestability and phosphopeptides binding of the domains usingfluorescence-based thermal shift assays. Thus, it was possible toprovide results for the 4 assays on a very large set of VUS from theUMD-BRCA1database, corresponding to all VUS that aremutatedin the BRCT domains (Table 1). We will now compare theseresults to the published data available on other VUS databases(Table 2). We will discuss the relationship existing between the

Figure 5.

Evaluation of the impact of missensemutations on the BRCTphosphopeptide-binding capacities. A,Thermal shifts due to the addition ofphosphopeptides at 256 mmol/L ontothe WT and mutated BRCT domains.Mutations are ordered as a function ofthe mutated residue number. Likelyneutral and neutral mutations aremarked by blue LN and N letters,respectively, whereas causal mutationsare marked by a red C. Black, brown,gray and yellow, light orange and darkorange bars correspond to the additionof a control peptide (CTRL-P), ACC1-P,CtiP-P, BACH1-P, AB-1P, and AB-2P,respectively. Bars boxed in greencorrespond to a reductionof the thermalshifts by at least a factor 2 (dark andlight green when residual binding or nobinding was observed, respectively; seeB). The bars boxed in yellow/greenmark a mutation that enhanced bindingto CtiP-P, ACC1-P, AB-1P, and AB-2P butdecreased binding to BACH1-P. B, 3Drepresentation of the positionscorresponding to the mutationsidentified by boxes in A. The BACH1-Ppeptide is displayed in red. Positions indark and light green are in contact or farfrom the phosphopeptide-binding site,respectively. Position in yellow (1836)interacts with Lys995 from BACH1-P. Asthe corresponding residue is specific toBACH1 (see A), mutation of Glu1836 hasdifferent impacts on the binding ofBRCA1 to BACH1-P compared withACC1-P, CtiP-P, AB-1P, and AB-2P.






Table 2. Summary of the structural and functional data available on the 78 VUS, based on this study as well as on previously published studies

DNAnomenclature

Amino AcidChange

ClassProteasesensi�vity

Expression inE. Coli &Stability ifSoluble

Expression inE. Coli &Stability ifSoluble

BACH1-Ppep�de bdg

Loss of affinityfor BACH1-P

Loss of affinityfor Opt-P

Loss ofphosphopep�de binding

Transcrip�onassay

Cispla�nresistance &

HR

Forma�on offoci a�er IR &

HR

Transcrip�onassay

Localisa�ona�er MMC

HR

N,M,F,FF=increasingsensi�vity

S, S-,S--,I =decreasingsolubility

S, S-,S--,I =decreasingsolubility

N, M,F,FF =decreasingaffinity

N = na�ve-likeN,F=

decreasingaffinity

N,M,F =decreasingac�vity

Resist.:N,FHR: +,-

Foci:N,FHR: +,-,--

N,M,F =decreasingac�vity

N,F = na�ve,non na�velocaliza�on

N+,N,F =enhanced,normal,

defec�ve HR

Lee et al. 2010Rowling et al.

2010our study Lee et al. 2010

Coquelle et al.2011

Rowling et al.2010

our study Lee et al. 2010Bouwman etal., 2013

Gaboriau et al.2015

Woods et al.,2016

our study our study

c.5095C>T R1699W 3P N S-- S- FF NO BDG 300 F F F / HR- F N Fc.5324T>G M1775R 3P N S- S- FFFFFFF

FFIFFP3E6071GA>G7115.c - M F / HR- F F Fc.5123C>A A1708E 3P FF I I F - F F F / HR-- F F Fc.5144G>A S1715N 3P F I I M - FF F Fc.4994T>A V1665E 3P I - F F

c.5116G>A G1706R 3P I - F F

c.5096G>A R1699Q 3P N S+ S FF 28,7μM/F 100 F F F / FNF+RH

FFIFP3A5861TG>A3505.c - FFFFFFIFFP3K1961TA>C2705.c - FFFFMIFFP3R7961CC>T9805.c - FFFFFINP3A3171VC>T8315.c - FFFF

SFFP3P2571AC>G4525.c -- FF - FFFFFFIFP3S1671FC>T2825.c - FFFF

c.5291T>C L1764P 3P F I I F - F F / HR- F F F

MIFP3R1181QG>A2345.c - FFFF

c.5509T>C W1837R 3P FF I S-- FF - FFFFF

SFFP3E8371GA>G3125.c -- Loss of specificity - FFFF

IP3A5561SG>T3694.c - N F

IP3R6861HG>A7505.c - FFFIP3D7861VA>T0605.c - F FIP3F8861VT>G2605.c - F FIP3Q0961KG>A8605.c - N FIP3R1961TG>C2705.c - F FIP3I1961TT>C2705.c - F F F

IP3I0071TT>C9905.c - N F

SP3V8371GT>G3125.c --- N F

SP3T2571AA>G4525.c --- F F FIP3G9081VG>T6245.c - F F

SP3G3381VG>T8945.c --- F F

NNMMFFFSFP2N6471HA>C6325.c

SP2Y7171FA>T0515.c - F N N

SFFP2G9371DG>A6125.c -- FF - NNFFFSFFP2V9371DT>A6125.c -- FF - NNFFF

c.5509T>G W1837G 2P FF I S-- F - NFFF

c.5522G>A S1841N 2P FF I S-- F - NF / HR+ F F N

SP2V0771GT>G9035.c --- F / HR- F N

SP2Y2271SA>C5615.c --- N

SP2K5371EA>G3025.c --- Not NNraelC

IP2E2571AA>C5525.c - F N

c.5497G>A V1833M 2P F I S- FNFFNNWT BINDING AND HR

DEFECT

SFP2V8071AT>C3215.c - NNMFNF

FM/Mμ2FFSNP2K6381EA>G6055.c / NNFMN

SNP2S9871AT>G5635.c - NFNNNM

UNCONSISTENTBINDING DATA

Impact

INTERMEDIATE IMPACTAND NO BINDING DATA

INSOLUBLE or BINDINGDEFECT AND HR

DEFECTIVE

STRONG BINDINGDEFECT

Control group ofCAUSAL variants

INTERMEDIATE IMPACTAND DEFECTIVE

BINDING

Loss of binding (Botuyan et al, 2004)

SNP1A0271TG>A8515.c - NNNNNNNc.5252G>A R1751Q 1P M S- S- NNNNNNN

SNP1D4081VA>T1145.c - NNNNNNNc.4956G>A M1652Ib 1P N S- S- NNNNNN

c.5348T>C M1783T 1P N S-- S- NMNNM / HR+ M N N

c.4991T>C L1664P 1P N S- S- NFNNNNNc.4993G>A V1665M 1P N S- S- NNMNNNNc.5005G>T A1669S 1P N S S- NNMNNNN

NNMNNNSNP1L5961FA>T5805.cNNMNNNSNP1H5871QT>G5535.cNNNNNNSNP1S6581PT>C6655.c

c.5117G>C G1706A 1P N S- S- NNNNN / NNN+RHSP1T0561MC>T9494.c - NNNSP1aI2561MT>G6594.c - NFNNNSP1L5661VC>G3994.c - NNN

NNNSP1E7661KG>A9994.cSP1G0761RG>A8005.c - NNNSP1A1961TG>A1705.c - NNNSP1E0171GA>G9215.c - NNNSP1L9171VG>A8515.c - N F NSP1I6271RT>G7715.c - N F NSP1G1371EG>A2915.c - NNNSP1V1371ET>A2915.c - NNN

NNNSP1S4771NG>A1235.cNNNSP1G7871CG>T9535.cNNNNSP1L1971VC>G1735.cNNNSP1V7081IG>A9145.cNNNSP1S0281GA>G8545.cNNNSP1L1281FC>T1645.cNNNSP1E5281GA>G4745.cNNNNSP1P4481LC>T1355.c

SP1R2871WC>T4435.c - N F N

NNFFNNSNP1G0181VG>T9245.c

NINP2T2561MC>T5594.c - NNNNINTERMEDIATE IMPACT

AND WILD-TYPEBINDING

Control group ofNEUTRAL variants

NO IMPACT

NOTE: Experimental results essential for classification are boxed in red and blue. The 4 columns with a yellow background correspond to the data obtained in this study. Nineadditional columns present the results of other studies on subset of variants. The 78 studiedVUS are presented as a function of their impact. All causal control variants are identifiedas 3P. A group of 12 VUS of class 3, which exhibit defective HR and either a strong phosphopeptide-binding defect in our study or insoluble/aggregatingBRCT domains in our studyand a binding defect in the literature, are also classified as 3P. They show,whenmeasured, defective transcription activation and cisplatin resistance (17–19). For an additional groupof 11 VUS, data are only available from this study; however, these VUS have a defective HR activity and their BRCT domains are poorly soluble in bacteria; therefore, they are alsoidentified as 3P. The 15 VUS identified as 2P (intermediate impact) are presented as a function of their phosphopeptide-binding properties. One of these VUS is M1652T of class 2.Within the 14 VUS of class 3, 9 VUS exhibit defective binding, either in this study or in previously published assays. One VUS (V1833M) binds with a WT affinity to the 5 testedphosphopeptide, and for the 5 remaining VUS no binding data are available. Several VUS from this group present transcription activation defects and, when tested, cisplatinresistance defects (17–19). All control variants but one (M1652T, see above) are identified as 1P. A group of 28 VUS showing wild-type binding to the 5 tested phosphopeptides andnormal HR efficiency in cells are classified as 1P. Five of them still show a localization defect in our assays.

Petitalot et al.





measured properties of our VUS and predisposition to cancer andsuggest related mechanisms of tumorigenesis.

Comparison with the results obtained on the VUS from BIC,KConfab, and ClinVar databases

Over the past few years, several functional assays for classifi-cation of BRCA1 VUS have been developed. In particular, a fastcDNA-based functional assay was reported, based on the VUSability to functionally complement BRCA1-deficient mouseembryonic stem cells, that is, restore the proliferation defect dueto the absence of BRCA1 expression and survive to addition ofcisplatin (39). Seventy-four VUS distributed all along the BRCA1gene were tested using this assay. This analysis strongly suggestedthat causal missense variants are confined to BRCA1 RING andBRCT domains. Several other groups restricted their analyses toBRCA1 proteinsmutated in the evolutionarily conserved RING orBRCT domains. They often aimed at understanding the impact ofmutations on protein stability, phosphopeptide binding, andtranscriptional activation (21, 23–25, 40–43). They suggestedthat phosphopeptide binding and transcription assays gaveresults that were correlated to cancer risk, with specificity andsensitivity higher than 80%. Additional studies reported theactivity of a subset of VUS inHR,whichwas also closely associatedwith cancer risk (39, 44, 45). Finally, characterization of 12 BRCTdomain variants performed both in vitro and in cells showed thatall of the variants, regardless of how profound their destabilizingeffects are in vitro, retained some DNA repair activity and therebypartially rescued the chicken BRCA1 knockout (26). In contrast,the variant R1699L, which disrupts the binding to phosphory-lated proteins (but which is not destabilizing), was completelyinactive. These studies raised the question of the link betweenprotein stability, phosphopeptide binding, HR activity, and can-cer risk.

We have compared our results to the data available from thesestudies (Supplementary Table SI). Our VUS list contains 42 VUSalso present in the BIC, KConFab, or ClinVar databases, whichwere characterized by the teams of L.S. Itzhaki, J.N.M. Glover, J.Jonkers, and A.N. Monteiro (Table 2). In particular, the thermo-dynamic stability of 17mutated BRCT domains and the effects offour of the corresponding variants on BRCA1-mediated DNArepair by HR were measured by Itzhaki's team and our team(25, 26). The resulting data are independent measurementsobtained on a subset of VUS that can be used to evaluate therobustness of the approaches. The seven mutants described asinsoluble by Itzhaki's team are mostly insoluble or aggregatingduring purification in our experimental set up. The 10 remain-ing mutants are always soluble and could be purified in bothteams. Within the four VUS studied in cells by both teams, oneis defective for HR in both assays (A1708E, causal), 2 VUS havea WT DNA repair activity in both assays (M1783T, likelyneutral, and S1841N), and 1 VUS shows a moderate DNArepair activity in Itzhaki's assay and a WT in our assay(M1652Ib). This last variant, M1652Ib, is already classified asneutral. We can conclude that the results obtained by the twoteams are in general consistent.

A large-scale analysis of the sensitivity to proteases, phosphor-ylated peptide binding, and in cell transcriptional activity of 117VUS corresponding to mutations in the BRCT domains of BRCA1was performed by Glover's team (21, 23). Most of the in vitroanalysis was performed on BRCT domains produced by in vitrotranscription/translation, which is an approach complementary

to our study. Comparison of all these results provides essentialarguments to guide further interpretation of our results (Table 2).Thirty-nineVUSwere studiedbybothGlover's teamandour team.Within these 39 mutants, 20 could be purified by us. Eighteenwere resistant to degradation by proteases in Glover's team. TheVUS H1746N and V1833Mwere sensitive to proteases in Lee andcolleagues (23), and are significantly less stable (by more than5�C) than theWTBRCTdomains in our study. In the case of the 19mutants that we could not be obtained using a bacterial expres-sion system, 17 were sensitive to proteases, which confirm thatthese mutants were unstable. Only VUS V1713A and M1652Twere not sensitive to proteases in Lee and colleagues (23). Insummary, our results on solubility in bacteria and results of Leeand colleagues on protease sensitivity are mostly consistent;however, because of the different experimental set up used toproduce the proteins, some differences are observed for a few VUS(23). Furthermore, Glover's teamcouldmeasure thebindingof 19variants insoluble in bacteria to a phosphorylated BACH1 pep-tide. They revealed that 17 of these variants have a defectivephosphorylated peptide–binding capacity (only mutantsM1652T and V1833M bind to phospho-BACH1). This analysisshows that mutated BRCT domains insoluble in bacteria, whenproduced in other systems, are in general also defective in phos-phopeptide recognition.

Relationship between phosphopeptide-binding defects andhigh cancer risk

From our data, it is particularly clear that the results from thephosphopeptide-binding assays are highly correlated with can-cer risk. These assays generally gave similar results using any ofthe 5 tested peptides CTiP-P, BACH1-P, ACC1-P, AB-1P, andAB-2P and provided information on 42 mutants including fiveneutral variants that showed WT BRCT–binding properties andtwo causal variants that exhibited defective BRCT phosphopep-tide-binding capacities. One VUS had a different behavior whentested for binding against the five phosphopeptides: E1836Kshowed improved binding to CtiP-P, ACC1-P, AB-1P, and AB-2P but reduced binding to BACH1-P (very close to that of thecausal R1699W; Supplementary Fig. S6C; ref. 46). It had anormal HR activity and was correctly localized after addition ofMMC. From our data, it is still unclear whether reduced bindingto only BACH1-P is sufficient to significantly increase the cancerrisk.

We could not test the binding properties of 36 mutatedBRCT domains because there were insoluble in bacteria.Comparison of Glover's team results with our results suggeststhat most (89%) mutated BRCT domains insoluble in bacteriain our study are also defective in phosphopeptide binding. Inparticular, by producing proteins using in vitro transcription/translation, Glover's team was able to characterize the phos-phopeptide-binding capacities of 5 of our 7 causal mutants.R1699W and M1775R (consistently with our study) as well asG1706E show large binding defects. A1708E and S1715Nshow smaller but still significant binding defects. Glover'steam also tested the binding properties of our insoluble BRCTmutant corresponding to a likely neutral VUS (M1652T) andshowed that this mutant binds phosphorylated BACH1 as theWT BRCT domains. This analysis strongly suggests that allcausal mutations lead to binding defects and all neutralmutations do not impact BRCT phosphopeptide–bindingproperties. It was recently published from the study of mice






carrying a BRCT mutant of BRCA1 that is defective inrecognition of phosphorylated proteins that BRCT phospho-protein recognition is required for BRCA1 tumor suppression(14).

Itwas alsoproposed that a significant defect in phosphopeptidebinding could completely abolish HR (26). From Glover's teamresults and our data, we do not observe a complete correlationbetween the results of the binding and HR assays. R1699W(causal), R1699Q, andM1775R (causal) both show clear bindingandHRdefects (see consistently forM1775R reports of teams of S.Pellegrini and J.D. Parvin, and inconsistently report of the teamofM.A. Caligo; refs. 23, 44, 47). However, E1836K is only defectivefor BACH1-P binding and exhibits a WT HR efficiency. Morestrikingly, F1717Y andH1746N show strongbinding defects to all5 phosphopeptides togetherwith aWTHRactivity. The functionalconsequences of these binding defects and their putative contri-bution to tumorigenesis are unclear.On thebasis of the systematicbinding defects measured for causal variants, impaired phospho-peptide binding might be sufficient per se to be associated withincreased cancer risk.

Classification of variantsMost of themutations responsible for familial breast or ovarian

cancers affect genes that control HR and/or the replication/HRinterface directly or indirectly (48, 49). The two most oftenmutated genes, BRCA1 and BRCA2, are major players in HR(50, 51). This overrepresentation of genes involved in theresponse to DNA damage and the communication betweenreplication and recombination highlights the importance of thesespecific pathways in the etiology of breast cancer. We here presentone of thefirst high-throughput structural and functional analysesof a large set of BRCA1 BRCTs VUS, which provides informationon the cellular localization andDNA repair capacity of all variants,as well as the thermal stability and binding properties of a subsetof variants. Analysis of these results together with the data pub-lished in the literature on overlapping subsets of variants enabledto correlate for the first time on a large-scale protein stability,phosphopeptide-binding, HR activity and cancer risk. From thisanalysis, threemeasurements seem essential to predict cancer risk:(i) BRCT phosphopeptide-binding affinities; (ii) BRCT capacity tobe produced and purified frombacteria; and (iii) VUSHR activity.Combining these three measurements as well as results from theliterature, we sorted the 65 BRCA1 VUS of class 3 into 3 groups(Table 2).

Thirty-three VUS show no HR and phosphopeptide-bindingdefect in our assays; they are proposed to have no impact oncancer risk (1P). They include all variants of classes 1–2 but one(M1652T, previously classified as likely neutral) and 28 VUS ofclass 3 (43% of this class). We cannot further classify the likelyneutral VUSM1652T because its BRCT domains were insoluble inE. coli in our assays. Glover and colleagues reported that thisvariant binds to BACH1-P (23); however, its binding to otherphosphopeptides has not been tested. Supporting our classifi-cation of 28 VUS of class 3 as 1P, it was recently shown fromgenetic data that G1706A and L1844P can be assigned toneutral (personal communication).

Thirty VUS exhibit several defects including systematic HRdefect and either BRCT insolubility in bacteria or phosphopep-tide-binding defect, they are proposed to have a severe impact oncancer risk (3P). They include all variants of classes 4–5 as well as23 VUS of class 3 (35% of this class). In one case (S1655A),

the VUS also showed a decreased binding to a library of phos-phopeptides containing the Ser-X-X-Phe motif (46). Moreover,the corresponding mouse mutation caused a hypersensitivity togenotoxic stress and a defective HR DNA repair (14). Furthersupporting our classification, L1764P, which is HR-deficient andshows insoluble BRCT domains in bacteria, was classified ascausal based on genetic data during our study (11). C1697Ris also HR-deficient and exhibits insoluble BRCT domains inbacteria. We could recently calculate a causality score forC1697R from the French and literature data as described byD. Goldgar, and we classified this variant as causal (Supple-mentary Table SII; refs. 11, 53).

Only 15 VUS are identified as having an intermediate impact(2P). One of them (M1652T) was already classified as likelyneutral and cannot be yet further classified, as explained above.For 3 other VUS of class 3, no HR defect was observed, andeither we did not find the same binding defect as a function ofthe tested phosphopeptide (E1836K), or we measured a WTbinding for the 5 phosphopeptides but a BACH1-P bindingdefect was reported by others (A1708V, A1789S; ref. 23). Hereagain, complementary experiments are needed to confirm thatthese VUS are neutral. In the case of the 11 other VUS, strongdefects were observed by us, which suggests that these VUScould be causal. V1833M shows an HR defect, even if nobinding defect could be measured by us and others (23). Themechanism leading to V1833M HR defect is yet unknown.G1770V, S1722Y, E1735K, and A1752E have insoluble BRCTdomains but show no HR defect. G1770V was recently classi-fied as causal based on genetic data (personal communication).D1739G, D1739V, W1837G, and S1841N have insoluble BRCTdomains that were reported by others as defective in bindingBACH1-P (23) but also show no HR defect. Finally, H1746Nand F1717Y have soluble BRCT domains that poorly bind tothe 5 tested phosphopeptides but show no HR defect. Wepropose that such VUS with a strong binding defect are likelycausal and should systematically be monitored because of theirassociation with high cancer risk.

ConclusionOur analysis strongly suggests that VUS exhibiting WT HR

efficiency as well as WT phosphopeptide-binding properties areneutral. Also, HR-defective VUS are generally causal. Associationof HR deficiency with a binding defect is strongly associated tocausality. Moreover, our study reveals that VUS with BRCTdomains that are insoluble in bacteria are often causal. Theirphosphopeptide-binding properties should systematically becharacterized, and if a defect is identified, they should similarlybe considered as increasing cancer risk. In particular, we identifiedtwo new variants that show a strong binding defect without beingHR deficient and our analysis stresses that these variants are likelycausal. From these new guidelines, we believe that it is possible toimprove the clinical interpretation of BRCA1 variants, and select alarger group of patients for treatment with therapies based onplatinum agents or PARP inhibitors that are specifically efficientagainst BRCA1/2-related tumors.

Disclosure of Potential Conflicts of InterestE. Rouleau has received speakers bureau honoraria from AstraZeneca

and BMS and is a consultant/advisory board member for AstraZeneca, BMS,and Roche. No potential conflicts of interest were disclosed by the otherauthors.

Petitalot et al.





Authors' ContributionsConception and design: E. Rouleau, B.S. Lopez, S. Zinn-Justin, S.M. CaputoDevelopment of methodology: A. Petitalot, E. Dardillac, E. Jacquet, N. Nhiri,J. Guirouilh-Barbat, I. Bouazzaoui, D. Bonte, B.S. Lopez, S. Zinn-Justin, S.M.CaputoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Petitalot, E. Dardillac, E. Jacquet, P. Julien,I. Bouazzaoui, J.A. Schnell, P. Lafitte, C. Nogues, R. Lidereau, B.S. Lopez,S. Zinn-Justin, S.M. CaputoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Petitalot, E. Dardillac, E. Jacquet, N. Nhiri,J.A. Schnell, J.-C. Aude, B.S. Lopez, S. Zinn-Justin, S.M. CaputoWriting, review, and/or revision of the manuscript: A. Petitalot, D. Bonte,C. Nogues, E. Rouleau, B.S. Lopez, S. Zinn-Justin, S.M. CaputoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): E. Dardillac, E. Rouleau, S. Zinn-Justin,S.M. CaputoStudy supervision: S. Zinn-Justin, S.M. CaputoOther (provided reagents): J. Feunteun

AcknowledgmentsThe authors thank the French oncogeneticists, the UNICANCER Genetic

Group BRCA network led by Dr. Catherine Nogu�es and probands for theircooperation. We gratefully acknowledge Sylvie Mazoyer for the BRCA1 full-length plasmid; Carine Tellier and Sylvaine Gasparini for their help duringthe recombinant plasmid construction, Damarys Loew and Vanessa Massonfor their help with mass spectroscopy, and Sophie Demontety for her helpwith causality scores. This work was supported by a joint translationalresearch grant of the French National Cancer Institute (2011-1-PL BIO-09-IC-1; to A.Petitalot) and the `Direction G�en�erale de l'Offre des Soins'(INCa-DGOS: PRTK2011-046 to A. Petitalot, E. Dardillac, P. Julien, I.Bouazzaoui, and P. Lafitte; and PRT-K 14 134, to P. Lafitte) and by the

Association d'Aide �a la Recherche Canc�erologique de Saint-Cloud (ARCS). B.S. Lopez's team is labeled "Ligue 2014."

UNICANCER Genetic Group BRCA network: Francoise Bonnet, NatalieJones, Virginie Bubien, Michel Longy, Nicolas S�evenet: Institut Bergoni�e -Bordeaux; Sophie Krieger, Laurent Castera, Dominique Vaur: Centre FrancoisBaclesse - Caen; Nancy Uhrhammer, Yves Jean Bignon: Centre Jean Perrin -Clermont-Ferrand; Sarab Lizard: CHU de Dijon, Hopital d'Enfants, Servicede G�en�etique M�edicale - Dijon; Aur�elie Dumont, Francoise Revillion: CentreOscar Lambret - Lille; M�elanie L�eone, Nadia Boutry-Kryza, Olga Sinilnikova(deceased): Hospices Civils de Lyon and Centre L�eon B�erard - Lyon; AudreyRemenieras, Violaine Bourdon, Tetsuro Noguchi, Hagay Sobol: Institut Paoli-Calmettes – Marseille, France; Pierre-Olivier Harmand, Paul Vilquin, PascalPujol: Laboratoire de Biologie Cellulaire et Hormonale (CHU Arnaud deVilleneuve) - Montpellier; Philippe Jonveaux, Myriam Bronner, JoannaSokolowska: CHU de Nancy-Brabois - Vandoeuvre-l�es-Nancy; CapucineDelnatte, Virginie Guibert, C�eline Garrec, St�ephane B�ezieau: CHU - Institut deBiologie - Hotel Dieu - Nantes; Florent Soubrier, Erell Guillerm, FlorenceCoulet: Groupe hospitalier Piti�e-Salpetri�ere, Assistance Publique-Hopitaux deParis, Universit�e Pierre et Marie Curie, Laboratoire d'Oncog�en�etique etAngiog�en�etique mol�eculaire - Paris; C�edrick Lefol, Virginie Caux-Moncoutier,Lisa Golmard, Claude Houdayer, Dominique Stoppa-Lyonnet: Institut Curie -Paris; Chantal Delvincourt, Olivia Beaudoux: Institut Jean Godinot - Reims;Dani�ele Muller: Centre Paul Strauss—Strasbourg; Christine Toulas: InstitutClaudius R�egaud - Toulouse; Marine Guillaud-Bataille, Brigitte Bressac-DePaillerets: Institut Gustave Roussy - Villejuif.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 7, 2017; revised February 7, 2018; accepted September 11, 2018;published first September 26, 2018.

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Petitalot et al.




Published OnlineFirst September 26, 2018.Mol Cancer Res Ambre Petitalot, Elodie Dardillac, Eric Jacquet, et al.

BRCT Variants on Cancer RiskBRCA1Phosphopeptide-binding Data to Predict the Impact of Combining Homologous Recombination and

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combining homologous recombination and phosphopeptide … · 2018-10-31 · dna damage and repair...

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