Functional Analysis of BARD1 and BRCA1 Variants of Uncertain Significance In

Functional Analysis of BARD1 and BRCA1 Variants of Uncertain Significance in

Homology-Directed Repair

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

in the Graduate School of The Ohio State University

Aleksandra Igorevna Adamovich, B.S.

Biomedical Sciences Graduate Program

The Ohio State University

2019

Dissertation Committee

Jeffrey Parvin, M.D., Ph.D., Advisor

Michael Freitas, Ph.D.

Paul Goodfellow, Ph.D.

Amanda Toland, Ph.D.

Copyrighted by

Aleksandra Igorevna Adamovich

2019

Abstract

BRCA1 and BARD1 encode proteins that form a heterodimer critical for mediating tumor suppression. Individuals with deleterious germline mutations in BRCA1 and BARD1 are at an increased risk for breast and ovarian cancers associated with poor disease outcomes.

Although increased cancer screening has helped identify genetic variants, there is not enough information regarding many of them to ascertain clinical risk. These variants are known as variants of uncertain significance (VUS). Functional assays can be used to predict the clinical predisposition of gene variants. The homology-directed repair (HDR) assay is one such assay that has been developed to examine the function of variants in

DNA repair. While BRCA1 has been extensively examined in HDR, with loss of function correlating with pathogenicity, its binding partner BARD1 has not been. In this dissertation, 76 potentially cancer-causing BARD1 truncation and missense variants were identified using whole exome sequencing and examined in the singleton HDR assay. Two were known to be benign variants and found to be functional in HDR, and three were known pathogenic variants found to have loss of function, supporting the notion that the

HDR assay can be used to predict the clinical risk of BARD1 variants. Four variants in the ankyrin repeat domain were found to be non-functional in HDR, indicating that there are DNA repair functions associated with this domain that have not been closely examined. To examine whether BARD1-associated loss of HDR function results in DNA

ii damage sensitivity, cells expressing non-functional BARD1 variants were treated with ionizing radiation or cisplatin. These cells were found to be more sensitive to DNA damage, and variations in the residual HDR function of non-functional variants did not correlate with variations in sensitivity.

While over 130 BRCA1 variants have been examined in the HDR assay, more comprehensive analysis of several BRCA1 functional domains, including the repair- mediating BRCT domain, has not been conducted. In this dissertation, all potential missense and truncation variants in amino acids 1577 to 1863 were generated and subjected to multiplex HDR assays. More than 500 variants between amino acids 1577 and 1768 were characterized from an integrated library of over 2000 variants in the multiplex assay, including thirty variants currently classified as VUS. All of the non- functional missense mutants identified were located in the BRCT domain. Results from the multiplex HDR assay also correlated with results from singleton HDR assays.

Variants classified as functional by the multiplex HDR assay were also functional in assays examining BRCA1 transcriptional activation (TA) and cell proliferation via saturation genome editing (SGE). However, several known pathogenic variants and variants classified as non-functional in the TA and SGE assays were functional in the multiplex HDR assay. Despite discrepancies between functional assays, the majority of result comparisons were consistent. In addition, variants that were non-functional in the multiplex HDR assay were always non-functional in the TA and SGE assays when comparisons were available. These findings help improve understanding of the BARD1

iii and BRCA1 functional domains in DNA repair and identify regions and variants of importance for cancer predisposition.

Dedication

Dedicated to my family.

Acknowledgments

I would like to thank my family, my advisor Jeffrey Parvin, my committee, and all past and present members of the Parvin lab for their support and feedback over the years.

Vita

2005-2009…………………………………Dublin Scioto High School

2009-2013…………………………………B.S. Microbiology, The Ohio State University

2013-2014, 2018-2019……………………Distinguished University Fellowship, The

Ohio State University

2014-2015…………………………………HHMI Med into Grad Fellowship

2015 to present……………………………Graduate Research Associate, Department of

Biomedical Informatics, The Ohio State

University

2019………………………………………The Ohio State University College of Medicine

Trainee Research Day Travel Award

Publications

Adamovich, AI and Parvin, JD. (2019). Characterizing BRCA1 Variants Using

Multiplex Functional Assays. Arch. Appl. Med. 1, 1-8.

Adamovich AI, Banerjee T, Wingo M, et al. (2019). Functional Analysis of BARD1

Missense Variants in Homology-Directed Repair and Damage Sensitivity. PLoS Genet.

15, e1008049.

vii

Adamovich AI, Toland AE and Parvin JD. (2019). F-Box Protein-Mediated Resistance to PARP Inhibitor Therapy. Mol Cell. 73, 195-196.

Starita LM, Islam MM, Banerjee T, Adamovich AI et al. (2018). A Multiplex

Homology-Directed DNA Repair Assay Reveals the Impact of More Than 1,000 BRCA1

Missense Substitution Variants on Protein Function. Am J Hum Genet. 103, 498-508.

Fields of Study

Major Field: Biomedical Science

viii

Table of Contents

Abstract ...... ii Dedication ...... v Acknowledgments...... vi Vita ...... vii List of Tables ...... x List of Figures ...... xi Chapter 1. Characterizing BRCA1 and BARD1 Variants Using Functional Assays ...... 1 Chapter 2. Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair and Damage Sensitivity ...... 25 Chapter 3. Multiplex Homology-Directed Repair Analysis of BRCA1 C-terminus Missense Variants ...... 69 Chapter 4. Discussion ...... 102 Bibliography ...... 112 Appendix A. F-box Protein Mediated Resistance to PARP Inhibitor Therapy ...... 136 Appendix B. Variants that Passed Read-Count Thresholding in Multiplex Homology- Directed Repair Analysis of BRCA1 1577-1768...... 142 Appendix C. Primers Used in BRCA1 C-terminus Mutagenesis ...... 156

List of Tables

Table 2-1. GFP expression percentages for BARD1 variant HDR assays...... 32 Table 3-1. Percentages of GFP-positive cells in each HDR assay replicate...... 77 Table 3-2. The number of GFP-positive and GFP-negative cells collected for each HDR sorting replicate...... 78 Table 3-3. The number of variants identified from filtered and unfiltered BRCA1 amino acid 1577-1768 sequence-function data and their depletion scores...... 81 Table 3-4. Functional results for variants tested in the TA, SGE and multiplex HDR assays...... 88

List of Figures

Figure 1-1. BRCA1 and BARD1 protein structure...... 3 Figure 1-2. The homology-directed repair (HDR) assay...... 7 Figure 1-3. BRCA1 variants assessed in homology-directed repair (HDR)...... 9 Figure 1-4. In multiplex assays, BRCA1 variant libraries are generated and inserted into reporter cells or organisms...... 19 Figure 2-1. Selection of BARD1 missense variants for functional analysis using sequencing data of cancer patient samples...... 29 Figure 2-2. Functional analysis of 76 BARD1 variants...... 36 Figure 2-3. Functional analysis of 105 BARD1 variants...... 39 Figure 2-4. BARD1 protein sequence differences between mammalian species...... 42 Figure 2-5. Immunoblots of truncated BARD1 variants...... 46 Figure 2-6. HBT-tagged BARD1 variants expressed from a single FRT site function similarly to transiently transfected variants in HDR...... 48 Figure 2-7. Sensitivity of BARD1 variants to ionizing radiation...... 51 Figure 2-8. Sensitivity of BARD1 variants to cisplatin...... 53 Figure 3-1. BRCA1 variant library diversity maps...... 75 Figure 3-2. Sequence-function map of variants in BRCA1 amino acids 1577-1768...... 80 Figure 3-3. Structure of the BRCA1 BRCT domain with labeled potentially intolerant regions...... 83 Figure 3-4. Comparison of multiplex HDR assay results to clinical data and other functional assays...... 85 Figure A-1. Observed mechanisms of PARPi resistance in cancer cells...... 139

Chapter 1. Characterizing BRCA1 and BARD1 Variants Using Functional Assays

A majority of the text from this chapter has been published as a review (Adamovich and Parvin, 2019).

Hereditary breast and ovarian cancers (HBOCs) are caused by germline mutations in tumor suppressor genes, and account for 5-10% of breast cancer cases (Larsen et al.,

2014) and 23% of ovarian cancer cases (King et al., 2011). A plurality of HBOCs are caused by mutations in BRCA1 and BRCA2, which account for 25% of hereditary breast cancers (Melchor and Benítez, 2013) and 34-84% of hereditary ovarian cancers (Ramus and Gayther, 2009). Several other genes, including BARD1, are also associated with

HBOC (Couch et al., 2017; Norquist et al., 2016). While a minority of individuals with breast and ovarian cancers have hereditary disease, individuals with pathogenic BRCA1 variants have a 65% lifetime risk for breast cancer and a 39% risk for ovarian cancer

(Eyfjord et al., 2003), as compared to the 12.4% breast cancer risk and 1.3% ovarian cancer risk present in the general population according to the National Cancer Institute.

Individuals with pathogenic BRCA1 and BARD1 variants are also at increased risk for triple negative breast cancers, which have low survival rates (Metzger-Filho et al., 2012;

Shimelis et al., 2018).

BRCA1

The BRCA1 protein has many functions associated with tumor suppression. BRCA1 encodes a 1863 amino acid protein that features an amino-terminal RING domain, a DNA binding domain (DBD), coiled coil (CC) domain, and two tandem carboxy-terminal

BRCT domains (Roy et al., 2012) (Figure 1-1).

Figure 1-1. BRCA1 and BARD1 protein structure. The BRCA1 gene encodes an 1863 amino acid protein consisting of an amino-terminal RING domain, DNA binding domain (DBD), coiled coil (CC) domain, and two tandem carboxy-terminal BRCT domains. The BARD1 gene encodes a 777 amino acid protein consisting of an amino-terminal RING domain, a tandem ankyrin (ANK) repeat domain, and two tandem carboxy-terminal BRCT domains. BRCA1 and BARD1 form an obligate heterodimer via their RING and BRCT domains, as indicated by the arrows.

The BRCA1 protein forms an obligate heterodimer with another tumor suppressor protein, BARD1, via their RING (Wu et al., 1996) and BRCT (Lee et al., 2015; Simons et al., 2006) domains. This interaction mediates tumor suppressor activity through direct mediation of DNA repair via the recruitment of RAD51 to sites of DNA double-strand breaks (Moynahan et al., 1999; Scully et al., 1996; Snouwaert et al., 1999). In addition, the BRCA1 and BARD1 RING domains function as an E3 ubiquitin ligase (Hashizume et al., 2001; Lorick et al., 1999; Ruffner et al., 2001). The BRCA1 BRCT domains also bind to several phosphoproteins (Rodriguez et al., 2003; Yu et al., 2003). Binding to

Abraxas mediates BRCA1 recruitment to sites of DNA damage (Wang et al., 2007),

BRIP1 is a DNA helicase involved in DNA repair (Cantor et al., 2001; Clapperton et al.,

2004) and CtIP mediates DNA end resection following DNA damage (Yun and Hiom,

2009). BRCA1 has also been shown to activate transcription via its C-terminus

(Chapman and Verma, 1996), mediate checkpoint activation (Xu et al., 2002a), and ubiquitinate γ-tubulin to regulate the centrosome number in the cell (Starita et al., 2004).

BARD1

BARD1 encodes a 777 amino acid protein that consists of an amino-terminal RING domain, an ankyrin repeat domain, and two tandem BRCT domains (Irminger-Finger et al., 2016) (Figure 1-1). In addition to the various tumor suppressor functions mediated by the BRCA1-BARD1 heterodimer, BARD1 regulates several BRCA1-independent processes. BARD1 has been shown to mediate apoptosis through the binding of p53 to the ANK domain (Berardi et al., 2005). The oncoprotein BCL3, which interacts with the 4 transcription factor NF-κB, binds to BARD1 via their ankyrin domains (Dechend et al.,

1999). BARD1 also regulates mRNA processing of 3′ ends by binding and degrading polyadenylation factor CstF-50 (Kleiman et al., 2005). It has also been shown that the

BARD1 BRCT domain interacts with poly(ADP-ribose) (PAR) to recruit the BRCA1-

BARD1 complex to areas of DNA damage (Li and Yu, 2013). BARD1 interaction with

HP1 retains this complex and allows for the accumulation of DNA helicase FANCJ at these sites (Miyoshi et al., 2015; Wu et al., 2016).

Clinical Significance

Thanks to the increased accessibility and affordability of clinical sequencing and cancer screening, a large number of BRCA1 and BARD1 missense variants have been identified.

However, the sequencing data are insufficient to differentiate benign and pathogenic missense variants, and many variants do not occur frequently enough in the population to assign a clinical risk. These are known as variants of unknown clinical significance

(VUS). According to the ClinVar database (Landrum et al., 2018), which records the clinical significance of gene variants, about 50% of known BRCA1 variants, and about

60% of known BARD1 variants, are considered VUS or have conflicting reports of pathogenicity. VUS in BRCA1 and BRCA2 are found in 10-20% of clinical tests (Eccles et al., 2015), indicating that individuals are often informed of their uncertain cancer risk.

Individuals with pathogenic BRCA1 variants are heterozygous for wild-type BRCA1 and are at risk for loss of heterozygosity (LOH), where expression of the wild-type allele is lost and cells become homozygous for the non-functional BRCA1 variant, which may 5 increase cancer risk (Merajver et al., 1995). There exist preventative measures for breast and ovarian cancers such as mastectomy and salpingo-oophorectomy, but these are invasive procedures that should only be done if necessary (Hartmann and Lindor, 2016).

Based on the potential severity of the disease and the exponentially higher risk associated with HBOCs, there is an essential and pressing need to identify which germline variants are associated with disease. Functional assays may help categorize this large number of

BRCA1 and BARD1 VUS by correlating protein function with cancer predisposition.

BRCA1 and BARD1 Functional Assays

While frame-shift or nonsense variants are likely to cause loss of protein function, it is much more difficult to predict the effect of missense variants that change a single amino acid. Numerous assays have been developed to examine BRCA1 function, with one such assay also used to examine BARD1 function, although assay sensitivity and specificity can vary. Many involve DNA repair. The homology-directed repair (HDR) assay examines the ability of variants to mediate homologous recombination (Lu et al., 2015;

Pierce et al., 2001; Ransburgh et al., 2010) (Figure 1-2).

Figure 1-2. The homology-directed repair (HDR) assay. (A) The HDR assay utilizes the reporter HeLa-DR cell line containing two non-functional GFP alleles, one of which is interrupted by a cut site for the rare restriction endonuclease I-SceI. Endogenous expression of the gene of interest is depleted using siRNA. An siRNA-resistant gene variant and I-SceI restriction enzyme are co-expressed, the latter inducing a double-strand break. If the variant is functional in HDR, the donor GFP will be used to repair the broken GFP, resulting in green fluorescence that can be quantified using flow cytometry. If the variant is not functional in HDR, other repair methods will be utilized and green fluorescence will not be observed. Variant protein expression is observed via immunoblotting. (B) Photographs of HeLa-DR cells in the HDR assay. Cells treated with control siRNA express GFP, while GFP expression is diminished when BRCA1 expression is depleted, as BRCA1 is necessary for HDR. Figures adapted from (Ransburgh et al., 2010).

The HDR assay uses a reporter cell line containing two non-functional GFP coding sequences, one of which is interrupted by a cut site for the rare-cutting restriction endonuclease I-SceI. If variants are functional in HDR, the other GFP allele will be used as a template to repair a double-strand break induced by I-SceI, resulting in functional

GFP expression that can be quantified (Pierce et al., 2001; Ransburgh et al., 2010).

BRCA1 variants have also been tested in a similar assay for single-strand annealing

(SSA), where a double-strand break in a GFP allele is induced in a reporter HeLa-SSA cell line (Towler et al., 2013). DNA resecting reveals homology with an upstream GFP allele, resulting in repair and green fluorescence.

The HDR assay has been used to examine the function of BRCA1 and BARD1 variants, as BRCA1 and BARD1 are found in an obligate heterodimer and both are necessary for mediating DNA repair. Nearly 150 BRCA1 variants have been examined using the HDR assay, and with the exception of splicing variants, the assay has shown 100% specificity and sensitivity (Figure 1-3).

Continued

Figure 1-3. BRCA1 variants assessed in homology-directed repair (HDR). 138 BRCA1 9

Figure 1-3 Continued single missense substitutions were tested for function in the HDR assay (Lu et al., 2015; Ransburgh et al., 2010; Starita et al., 2015; Towler et al., 2013). HeLa-DR cells (Ransburgh et al., 2010) were treated with siRNA specific to the BRCA1 3'-untranslated region (UTR) and empty vector (lane 2) or BRCA1 expression plasmid (lanes 1, 3-140). Cells depleted of endogenous BRCA1 with wild-type BRCA1 rescue (lane 1) were used as a positive control. Cells treated with empty plasmid and BRCA1 3'UTR siRNA were used as a negative control (lane 2). HDR function was characterized by the percentage of GFP-positive cells measured using flow cytometry. Results in each experiment were normalized to the wild-type rescue (lane 1), which was set equal to 1. Missense variants are labeled with colors according to the functional domain they are located in. Variants that are not located in a functional domain are labeled in gray. Truncation variants are labeled as “Trunc.”, and controls as “Cont.” in the legend. Variants that are known to be either benign or pathogenic according to ClinVar are labeled with blue or red dots respectively. Variants with uncertain interpretations are labeled with gray dots.

Results were combined from four papers that examined BRCA1 variants in HDR (Lu et al., 2015; Ransburgh et al., 2010; Starita et al., 2015; Towler et al., 2013) and variants were color-coded by functional domain. HDR activity, as quantified by percent GFP- positive cells, was normalized to the activity of cells complemented with wild-type

BRCA1, which was set to one (lane 1). Co-transfection of a plasmid expressing wild-type

BRCA1 fully rescued loss of endogenous BRCA1 expression. Cells depleted of BRCA1

(lane 2) showed a 10-fold drop in DNA repair activity. Known benign variants were labeled with blue dots, known pathogenic variants were labeled with red dots, and VUS were labeled with gray dots. In one BRCA1 study (Lu et al., 2015), germline and tumor samples were evaluated for LOH, and 17 variants in BRCA1 were found to have significantly elevated LOH in the tumor. Of these only six were non-functional in the

HDR assay, suggesting that LOH does not accurately predict loss of function variants.

Non-functional BRCA1 missense variants were located in the RING and BRCT domains, consistent with the locations of known pathogenic variants and suggesting that future analysis could focus on the N- and C-termini. While BARD1 variants have also been examined in the HDR assay, it is not as extensive as the work focused on BRCA1

(Billing et al., 2018; Laufer et al., 2007; Lee et al., 2015). As a result, the roles of the

BARD1 domains in HDR are not as well-characterized. The function of BARD1 variants in HDR will be further discussed in Chapter 2.

Similarly, the rescue of radiation resistance (RRR) assay uses the BRCA1-/- HCC1937 human breast cancer cell line to examine whether BRCA1 variants repair double-strand breaks caused by ionizing radiation (Abbott et al., 1999; Cortez et al., 1999; Ruffner et

11 al., 2001; Scully et al., 1999). A yeast-associated assay also examines BRCA1 missense variants for their ability to affect homologous recombination and cell growth, as deleterious variants have been shown to cause increased homologous recombination in yeast (Caligo et al., 2009).

Other BRCA1 complementation-based functional assays also exist. In the centrosome duplication assay (Kais et al., 2012), cells expressing BRCA1 variants are examined for centrosome numbers, as loss of BRCA1-mediated centrosome regulation results in chromosomal instability (Starita et al., 2004; Xu et al., 1999). Non-functional and known deleterious variants had increased centrosome numbers and unpaired centrioles (Kais et al., 2012). Another complementation assay examines BRCA1 variant function in Brca1- deficient mouse embryonic stem cells via cell proliferation and sensitivity to DNA damage agents such as ionizing radiation and cisplatin (Bouwman et al., 2013; Chang et al., 2009).

Several assays can only be utilized for specific regions of BRCA1 based on the functions examined. BRCA1 RING domain-mediated E3 ubiquitin ligase activity is assayed by examining loss of binding to the E2 ubiquitin enzyme UbcH5a and BARD1 with yeast two-hybrid screenings and ubiquitin ligase activity assays (Morris et al., 2006). Several functional assays have been also designed for the BRCA1 C-terminus. The transcription activation assay fuses the BRCA1 C-terminus to heterologous DNA binding domains in yeast and mammalian two-hybrid assay systems to examine transcription activation function, and non-functional variants are interpreted as likely pathogenic (Chapman and

Verma, 1996; Monteiro et al., 1997; Vallon-Christersson et al., 2001). Results for this

12 assay are identical for most variants in both yeast and mammalian systems (Carvalho et al., 2007; Phelan et al., 2005; Vallon-Christersson et al., 2001). Another yeast assay utilized for the functional analysis of C-terminal variants is the small colony phenotype

(SCP) assay, where functional BRCA1 has been shown to inhibit growth in yeast and non-functional variants do not (Coyne et al., 2004; Humphrey et al., 1997; Millot et al.,

2011). Variants in the N-terminus did not affect colony growth in the SCP assay

(Humphrey et al., 1997). Variants in the C-terminus have also been examined for sensitivity to proteolytic digestion because pathogenic BRCA1 variants have been shown to be unstable and sensitive to digestion (Lee et al., 2010b; Williams and Glover, 2003;

Williams et al., 2001, 2003). Another assay examines whether BRCA1 variants bind phosphopeptides, based on the several phosphoproteins that bind to the C-terminus (Lee et al., 2010b; Williams et al., 2004).

Several functional assays examine BRCA1 localization. In the yeast localization phenotype (YLP) assay, functional BRCA1 accumulates in a single inclusion body in the yeast nucleus, and this localization is disrupted by non-functional and pathogenic variants

(Millot et al., 2011). Another assay examines BRCA1 localization to the nucleus in human cells and nuclear foci formation following DNA damage (Chen et al., 1996;

Scully et al., 1997a, 1997b). Localization of BRCA1 to the cytoplasm has also been associated with breast and ovarian cancer (Chen et al., 1995), and pathogenic variants in the BRCA1 RING and BRCT domains do not form damage-associated foci (Au and

Henderson, 2005; Rodriguez et al., 2004).

A large number of functional assays utilize cDNA to examine variant function. However, pathogenic variants may also be caused by splicing defects. Mini-gene splicing assays have been used to identify changes in BRCA1 splicing by inserting BRCA1 exons into a vector, expressing them in cells, and harvesting the RNA to make cDNA and examine transcript changes (Ahlborn et al., 2015; Steffensen et al., 2014).

In addition to biological assays, computational methods have been developed to determine whether variants are non-functional and deleterious. Programs such as Sorting

Tolerant from Intolerant (SIFT), A-GVGD, PolyPhen-2 and Combined Annotation-

Dependent Depletion (CADD) can be used to predict whether certain amino acid substitutions, based on properties such as sequence, structure and evolutionary conservation, affect protein function (Adzhubei et al., 2010; Kircher et al., 2014; Ng and

Henikoff, 2003; Tavtigian et al., 2006). Programs like VarScan, Pindel and GATK can also be used to identify potentially pathogenic variants from sequencing data (McKenna et al., 2010; Shen et al., 2012; Ye et al., 2009). Ideally, the information gained from functional assays of BRCA1 and BARD1 can also be extended to similar proteins.

Functional analysis from BRCA1 BRCT domain variants tested in the transcription activation assay has been used to update the VarCall model and identify potentially pathogenic variants in other proteins containing BRCT domains (Woods et al., 2016).

While in silico and bioinformatics analysis can help evaluate a large number of variants, the conclusions are not always accurate and there is not enough preexisting, validated data in these programs. Such programs are best used in conjunction with functional

14 assays to characterize variants and provide more empirical evidence to improve program output.

Multiplexed Assays

While functional assays are very useful for classifying BRCA1 and BARD1 variants, they are limited by the fact that only a single variant can be expressed in a tested cell population and therefore each needs to be tested one at a time. With each additional variant tested, more time and resources are needed. To accurately examine the function of a protein like BRCA1 or BARD1, all potential variants in a region of interest or even the whole gene need to be examined. That would require testing hundreds or thousands of variants, which is not feasible for the experimental procedures discussed earlier.

Multiplexed approaches allow for a greater number of variants to be tested by expressing hundreds to thousands of variants in the same cell population.

Multiplex assays require experimental procedures that allow for the examination of large numbers of variants. Multiplex CRISPR/Cas9 knockout screens have been used to identify essential genes in various cancer cell lines to better target cancer treatments (Hart et al., 2015). A large number of assays utilize deep mutational scanning, where every possible amino acid change at every protein position is generated and functional effects are examined (Fowler and Fields, 2014). Several methods have been developed for generating hundreds or thousands of mutations. Variants can be introduced at random by controlling the PCR mutagenic rate (Wilson and Keefe, 2004). Variants can also be generated through targeted methods such as programmed allelic series (PALS) 15 mutagenesis, where mutagenic primers to the region of interest are synthesized on a microarray and a degradable wild-type template introduces the mutations (Kitzman et al.,

2015). Mutagenic forward and reverse primers for each codon can also be designed for inverse PCR (Jain and Varadarajan, 2014). Mutagenesis by integrate tiles (MITE) uses ligation to introduce a microarray of all potential single nucleotide variants (SNVs) in a region into a full-length construct (Melnikov et al., 2014). SNVs can also be generated using saturation editing by inserting a library of all potential DNA hexamers into a region of interest and inserting the mutagenized region into the genome with CRISPR/Cas9

RNA-guided cleavage (Findlay et al., 2014). It is useful to examine SNVs because they account for the majority of human mutations (Ng et al., 2008), but SNV studies do not provide the same structure-function information available with studies that examine all possible amino acid changes. Functional variants are identified via high-throughput DNA sequencing to examine which variants are depleted by comparing the variant distribution in the input sample versus the experimental sample, and the frequency of a given variant in each pool is used to determine function (Fowler and Fields, 2014; Fowler et al., 2014).

A variety of protein and gene functions have been examined using multiplex assays.

Enhancer activity has been extensively studied via deep mutational sequencing multiplex reporter assays (Inoue and Ahituv, 2015; Muerdter et al., 2015). All possible SNVs of the liver enhancers ALDOB, ECR11 and LTV1 were generated to examine changes in enhancer function (Patwardhan et al., 2012). Thousands of candidate liver enhancers have also been examined for function via lentivirus-based multiplex reporter assays (Inoue et al., 2017). Protein properties have been studied as well - single amino acid variants of

PTEN and TPMT have been examined to identify low abundance, potentially pathogenic variants by fusing them to EGFP and sorting cells based on fluorescence (Matreyek et al.,

2018). Protein abundance assays could be applicable to many different proteins, as several tested proteins showed protein abundance differences between variants (Matreyek et al., 2018). Protein stability and folding has been examined with deep mutational scanning via the degradation and loss of cellular export of misfolded TEM-1 and LGK variants (Bacik et al., 2017). Multiplex sequencing has also been used to examine different features of mRNA processing, such as alternative splicing (Julien et al., 2016;

Rosenberg et al., 2015) and 3′ untranslated regions (Shalem et al., 2015; Zhao et al.,

2014).

In addition to examining widely applicable functions, specific proteins have been functionally characterized via multiplex assays. APH(3’)II, which provides resistance to aminoglycoside antibiotics, has been mutated to examine regions necessary for that resistance (Melnikov et al., 2014). Levoglucosan kinase variants have been examined for function in the pyrolysis oil catabolic pathway (Klesmith et al., 2015). Variants in the U- box domain of the ubiquitination factor Ube4b have also been examined to identify residues necessary for E3 ubiquitin ligase activity and ubiquitin transfer (Starita et al.,

2013). Some proteins have also been characterized in multiplex assays to identify pathogenic variants. Variants of the tumor suppressor PTEN have been examined for protein function through variant protein abundance (Matreyek et al., 2018) and examination of variant lipid phosphatase activity (Mighell et al., 2018). PPARγ variants have been examined for inducing CD36 expression in macrophages following stimulation

17 with PPARγ agonists to identify variants that predispose to lipodystrophy (Majithia et al.,

2016). Multiplexing has also been used to examine the function of H-Ras, a signaling protein and known oncogene, by coupling the binding of Ras•GTP and its binding partner

C-Raf to expression of a chloramphenicol resistance factor to evaluate variant fitness

(Bandaru et al., 2017).

Multiplexed Assays for Analyzing the Function of BRCA1 Missense Variants

BRCA1 has been extensively examined via deep functional sequencing using several different functional assays (Figure 1-4).

Figure 1-4. In multiplex assays, BRCA1 variant libraries are generated and inserted into reporter cells or organisms. Functional variants are selected for using methods such as ubiquitin activity (Starita et al., 2015), binding to BARD1 (Starita et al., 2015), HDR activity (Starita et al., 2018) or growth fitness (Findlay et al., 2018). After selection, the remaining variants are identified by sequencing the degenerate barcodes attached to them. Depleted variants are identified as depleted based on their presence in reporter cells prior to selection conditions.

Variants in the first 300 amino acids of BRCA1, including those in the RING domain, have been examined for their E3 ubiquitin ligase activity and BARD1 binding activity

(Starita et al., 2015). Variants were expressed on the surface of T7 bacteriophage and those that autoubiquinated (Starita et al., 2013) in the presence of ubiquitin were selected.

Binding between the BRCA1 and BARD1 RING domains was examined using multiplexed yeast two-hybrid assays. Examination of E3 ligase activity showed that the zinc ion residues that maintained RING domain structure and residues that interact with

E2 ubiquitin enzyme UbcH5c (Fox et al., 2003) were most frequently non-functional due to mutations. Results were similar with regards to BARD1 binding – variants that affected the zinc ions, and subsequently RING domain structure, were most intolerant to substitution.

HDR has been directly assessed to examine the function of missense variants in the

BRCA1 N-terminus (Starita et al., 2018). Missense variants in the first 192 amino acids of BRCA1 were generated and subjected to the HDR assay - variants were sorted into

GFP-positive and GFP-negative pools, and then the barcodes attached to each variant were sequenced. All of the non-functional variants identified were nonsense variants or were located in the RING domain, which has extensively studied repair functions. Known benign variants were functional in HDR, while all known pathogenic variants, except for the splicing variant R71G (Vega et al., 2001), were non-functional, resulting in 87.5% sensitivity and 100% specificity for the assay. Examining the relationship between HDR function and protein structure also identified regions where glycine substitutions were not tolerated.

Multiplex analysis of BRCA1 function has also been measured via the growth of human haploid HAP1 cells, where BRCA1 and other HDR-related genes are necessary for cell survival (Findlay et al., 2018). Instead of generating all potential amino acid variants, the authors examined the function of all potential SNVs. Saturation genome editing (SGE) of the BRCA1 gene in situ was used to generate all possible variants in the 13 exons that make up the RING and BRCT domains (Findlay et al., 2014). SNVs that inhibited

BRCA1 function were selected against by increasing HDR rates in HAP1 cells, and the gDNA and RNA of variants present in the input population was sequenced and compared to the variants present in the population after multiple passages. Using SGE to generate all potential SNVs allowed the authors to examine splicing (Findlay et al., 2014), finding that SNVs that disrupted splice sites were non-functional and had depleted mRNA. Most non-functional missense SNVs also did not have reduced RNA levels, indicating that loss of function was mostly due to protein defects. As previously seen in the multiplex assays of E3 ubiquitin ligase function and HDR, variants that affected RING domain folding, which affects binding to BARD1, and buried hydrophobic residues were non-functional.

When the results of all four multiplexed assays for BRCA1 function were compared, nearly all of the results were consistent (Starita et al., 2018). The multiplexed HDR and

SGE assays had results that were consistent for over 100 variants. The L22S missense variant was found to be functional in the ubiquitin ligase and BARD1 binding assays

(Starita et al., 2015) but was non-functional in HDR, and only five variants had discrepant results when comparing the HDR and SGE assays.

The Future of Examining BRCA1 and BARD1 Variant Function

Assays examining BARD1 and BRCA1 functions, such as HDR, transcription activity and nuclear localization, have been used to characterize variants and infer pathogenicity.

In recent years, new developments in multiplexed approaches have been applied to study

BRCA1 variants as well to characterize BRCA1 functional domains more broadly and comprehensively. Multiplexed assays examining ubiquitin ligase activity, BARD1 binding activity, HDR, and cell fitness examine the BRCA1 N- and C-termini, and have had a general consensus of functional results.

With the existence of multiple functional assays for BRCA1 and multiplexing making it more feasible to characterize thousands of variants at once, this begs the question of what is the “best” assay to use for identifying pathogenic variants. BRCA1 has been shown to be involved in functions such as DNA repair, centrosome regulation and transcription, so which of these functions is the best for predicting clinical risk? It appears that no one assay is perfect, and they all serve different purposes because function is context-specific.

Testing the same variants in the HDR and SSA DNA repair processes has shown that all known pathogenic variants were defective in both processes (Towler et al., 2013).

However, the SSA assay also identified several benign variants as defective, indicating that different regions of the BRCA1 protein are involved in both processes. There were also differences in what was considered a non-functional variant between the HDR and centrosome duplication assays (Kais et al., 2012). Similarly, more variants were non- functional in the multiplexed E3 ubiquitin ligase activity assay than in the multiplexed

BRCA1-BARD1 RING binding assay, and evidence suggests that ubiquitin ligase 22 activity is not needed for tumor suppressor function (Laufer et al., 2007; Shakya et al.,

2011; Starita et al., 2015). In addition, multiple BRCA1 BRCT missense variants have been tested via the same assays – sensitivity to proteolysis, binding to BRIP1 phosphopeptides, and transcription activation via fusion to a heterologous binding domain – and complete concordance occurred in assays about 55% of the time (Lee et al.,

2010b). Another factor that many functional assays do not address is splicing variants, as many of the assays discussed utilize cDNA and do not account for them. The variety of responses regarding BRCA1 function that are provided by these assays show that there is no one true functional assay, and that multiple assays are needed to fully characterize a gene like BRCA1.

BARD1 function has been examined using the HDR assay, and loss of variant function consistently correlates with pathogenicity. BARD1 has not been as extensively researched as BRCA1, and the multiplexed HDR assay would be helpful for characterizing BARD1 functional domains. As several BARD1 functions are dependent on interaction with BRCA1, it is likely that functional assays developed for BRCA1 can be used to examine BARD1 function as well. Multiplex assays examining E3 ubiquitin ligase function and BRCA1/BARD1 binding could be conducted using BARD1 variants.

In addition, BARD1 variants could be tested using the multiplex SGE assay, as it uses a cell line where HDR-associated genes are necessary for cell survival. Non-multiplex assays, such as nuclear localization or stem cell complementation, may also be utilized to characterize BARD1 more effectively.

Ultimately, functional assays should augment genetic information and enable a clinician to advise individuals about their risk for breast and ovarian cancer. As more functional assays are conducted, more confidence can be placed on their predictive value. All four published multiplexed BRCA1 assays had results that were overall consistent with each other. We suggest that the confidence in the predictive value is higher for the variants that have common results, as indicated by the more than 100 variants whose function was consistent between the HDR and SGE assays (Starita et al., 2018). When results differ, it could be due to technical error, or possibly due to real differences in the amino acid requirements of the two assays. The multiplexing of additional BRCA1 functional assays, as well as the application of already-validated multiplex assays to BARD1 and other domains of BRCA1, would allow for better understanding of both BRCA1 and BARD1 and provide improved classification of variants.

Chapter 2. Functional Analysis of BARD1 Missense Variants in Homology-Directed Repair and Damage Sensitivity

Aleksandra I. Adamovich1, Tapahsama Banerjee1, Margaret Wingo1, Kathryn Duncan1,

Jie Ning2, Fernanda Martins Rodrigues2, Kuan-lin Huang2, Cindy Lee1, Feng Chen2, Li

Ding2, and Jeffrey D. Parvin1*

1 Department of Biomedical Informatics, Ohio State University Comprehensive Cancer

Center, Ohio State University, Columbus, OH

2 McDonnell Genome Institute, Washington University School of Medicine, Washington

University in St. Louis, St. Louis, MI

Address correspondence to [email protected]

Author contributions: A.A. performed experiments, analysis and wrote the manuscript,

T.B., M.W., K.D. and C.L. helped perform clonogenic and/or functional assays, J.N. cloned mutants, K.H., F.C. and F.M.R. performed data analysis, L.D. and J.P. supervised experiments and edited the manuscript.

Introduction

Variants in BRCA1 and BRCA2 account for a plurality of hereditary breast and ovarian cancer (HBOC) cases, and are associated with risks of 50–85% for breast cancer and 15–

40% for ovarian cancer (Antoniou and Easton, 2003; Godwin et al., 2010; Levy-Lahad and Friedman, 2007; Parmigiani et al., 2007). BARD1 forms an obligate heterodimer with BRCA1, which functions as both an E3 ubiquitin ligase (Hashizume et al., 2001;

Wu et al., 1996) and as a direct mediator of homologous recombination for the recruitment of RAD51 to the sites of DNA double-strand breaks (Moynahan et al., 1999;

Scully et al., 1996; Snouwaert et al., 1999). Truncated BARD1 variants have been identified in breast and ovarian cancers (De Brakeleer et al., 2010; Ghimenti et al., 2002;

Lee et al., 2010a) and germline variants in the BARD1 gene are associated with increased cancer risk (Hart et al., 2017). Still, for both BRCA1 and BARD1, the functional and clinical consequences are often unknown for sequence changes that replace the encoded amino acid residue.

Both BRCA1 and BARD1 are tested on clinical gene panels for breast and ovarian cancer susceptibility. Many BRCA1 variants, as well as a few BARD1 variants, have been determined to be clinically pathogenic. However, many more variants, which are generally missense substitutions, do not occur frequently enough in the population to assign a cancer risk and are classified as variants of uncertain significance (VUS). The

ClinVar database (Malheiro et al., 2017) gathers information on pathogenic and benign variants, but most variants in its database are VUS. A gene panel testing 25 breast cancer- associated genes found 42% of all tests have findings of a VUS in one or more genes, 26 indicating many people have such variants and there is a growing need for their classification (Tung et al., 2015). Datasets such as the Cancer Genome Atlas (TCGA) gather information on missense variants, but are unable to be used for the accurate prediction of the cancer predisposition of a specific VUS. Assays examining homology- directed repair (HDR) function have demonstrated that known pathogenic BRCA1 variants are non-functional in HDR, while benign variants are functional (Lu et al., 2015;

Ransburgh et al., 2010; Starita et al., 2015; Towler et al., 2013). BARD1 consists of an amino-terminal RING domain, three ankyrin repeat domains, and two carboxy-terminal

BRCT domains (Bowcock et al., 2002; Wu et al., 1996). Previous work in our lab has examined the HDR function of 29 BARD1 variants, focusing on the RING and BRCT domains (Lee et al., 2015).

In this study, we identified 76 BARD1 missense and truncation variants that were potentially cancer-associated from a large dataset containing exome-sequencing data on matched germline and tumor samples (Huang et al., 2018; Lu et al., 2015), and tested them for HDR function. Several HDR-deficient variants were identified in both the ankyrin repeat and BRCT domains. To examine the effects caused by loss of HDR function, cells expressing HDR-deficient BARD1 variants were treated with DNA damaging cisplatin or ionizing radiation. Cells expressing HDR-deficient variants were more sensitive to DNA damage and formed significantly fewer colonies than cells expressing wild-type BARD1. Although cells expressing HDR-deficient variants were more sensitive to damage than wild-type cells, quantitative variations in HDR deficiency did not correlate with differences in sensitivity to DNA damage agents. The results of this

27 study reveal functional domains of BARD1 and suggest that the functional analysis of

BARD1 HDR activity is predictive of breast and ovarian cancer risk.

Identification of BARD1 variants as potential cancer-associated loss of function variants.

BARD1 missense variants with potential cancer predisposition were identified in a set of

10,389 TCGA cancer samples from 33 cancer types using whole exome sequencing

(Figure 2-1A) (Huang et al., 2018; Lu et al., 2015). Sixty-two rare germline variants and

14 somatic variants were found with variant calling. The variant allele frequency (VAF) and loss of heterozygosity (LOH) of germline variants were also examined to identify variants that could be functionally important. Six variants—S339T, T343I, V523A,

N450H, G451fs and L239Q—were identified as having significantly higher LOH, indicating they had an increased likelihood of being pathogenic (Figure 2-1B). Variants are listed by amino acid change instead of nucleotide change. At the beginning of this study, variants were selected from a cohort of 4,034 samples (Kandoth et al., 2013; Lu et al., 2015) that later became part of a larger set of 10,389 samples (Huang et al., 2018;

Kumar et al., 2018). Because of changes to selection criteria and data analysis, most, but not all, of the variants analyzed in this study were present in the larger data set, which was used to update the variant calling (Figure 2-1C). While several variants were not present in our newer data set, they are still likely present in the exome sequencing data.

Analyzed variants were present in 24 of the 33 cancer types examined, not just breast or ovarian cancer, as might be predicted for a BRCA1 binding partner (Figure 2-1D). 28

Continued

Figure 2-1. Selection of BARD1 missense variants for functional analysis using sequencing data of cancer patient samples. (A) BARD1 missense variants of interest were identified in a cohort of 4,034 samples from 12 cancer types and a larger set of 10,389 TCGA samples from 33 cancer types with whole exome sequencing (Huang et al., 2018; Lu et al., 2015). (B) Identification of LOH in BARD1 through comparison of VAF in 29

Figure 2-1 Continued tumor and normal samples. Each dot depicts one variant. The diagonal line denotes neutral selection of the germline variant where the normal and tumor variant allele frequencies (VAFs) are identical. LOH was considered significant at False Discovery Rate (FDR) ≤ 0.05. (C) Number of samples containing each of the 76 BARD1 variants in the 10,389 cohort (Huang et al., 2018). (D) Number of samples affected by each BARD1 variant for each of the 33 cancer types.

Analysis of BARD1 variants in Homology-Directed Repair (HDR).

Seventy-six BARD1 missense variants, a majority of which were located in the ankyrin repeat and BRCT domains or between these domains, were tested for function in the homology-directed repair (HDR) assay (Figure 2-2A). For the HDR assay, a cell line that has two non-functional GFP coding sequences integrated into its DNA is used to examine DNA repair function. One of these GFP-encoding genes contains a recognition site for the rare-cutting restriction endonuclease I-SceI. When the I-SceI expression plasmid is transiently transfected into these cells, a double-strand break is made in one of the GFP sequences. If homology-directed repair uses the second GFP coding sequence as a template to repair across the double-strand break, then the encoded GFP is rendered functional (Pierce et al., 2001; Ransburgh et al., 2010). We used a HeLa-derived cell clone called HeLa-DR, which has the GFP-encoding recombination substrate integrated at a single site. After transfection of the I-SceI expression plasmid, 10–20% of the cells were converted to GFP-positive (Ransburgh et al., 2010). Endogenous BARD1 expression was depleted in HeLa-DR cells by two rounds of transfection of a siRNA that targets the 3’-UTR of the BARD1 mRNA. Simultaneously with the silencing of 30 endogenous BARD1, BARD1 variants were expressed from transiently transfected plasmids that were resistant to the siRNA. Two days following the first transfection, the siRNA and plasmid were transfected again into the cells along with the plasmid that expresses the I-SceI endonuclease. Three days after the second transfection, the number of GFP-positive cells was determined using flow cytometry (Table 2-1). Full HDR activity was observed under conditions of mock depletion of BARD1 by transfection with a control siRNA (Figure 2-2A, bar 1) and by depletion of BARD1 using the 3’-UTR targeted siRNA with rescue by transfection of a plasmid that expressed wild-type

BARD1 (Figure 2-2A, bar 3). Cells depleted of BARD1 and transfected with an empty vector had a 25-fold decrease in HDR activity measured as the percentage of GFP- positive cells (Figure 2-2A, bar 2). We set the level of GFP expression following a double-strand break to a value of 1 relative to wild-type rescue (Figure 2-2A, bar 3) to facilitate comparison between experiments.

Experiment 1 Experiment 2 Experiment 3 Sample GFP Sample GFP Sample GFP % % % Control 5.8 Control 8 Control 11.71 Empty 0.1 Empty 0.3 Empty 0.75 WT 6 WT 7.5 WT 9.55 E223G 6.5 S241C 8.5 E67K 10.78 E223G 6.9 S241C 9 E67K 10.6 E223G 6.7 S241C 7.9 E67K 10.59 D230E 6.8 I249V 7.1 M104I 11.37 D230E 6.2 I249V 7 M104I 11.09 D230E 7.1 I249V 8.1 M104I 10.67 L239Q 6.8 I258T 8.8 S109R 10.61 L239Q 7.3 I258T 9.4 S109R 11.63 L239Q 6.8 I258T 9.5 S109R 11.83 R322H 9.9 N118S 12.06 R322H 9.8 N118S 10.96 R322H 10.3 N118S 11.61 N326D 8.2 K140N 10.58 N326D 10.2 K140N 10.97 N326D 8.4 K140N 10.78 S151N 11.22 S151N 12.47 S151N 11.16 V154fs 1.15 V154fs 1.25 V154fs 1.33 D190N 10.96 D190N 11.66 D190N 11.3 D190Y 11.64 D190Y 10.59 D190Y 10.73 R194K 11.07 R194K 11.36 R194K 12.16

Continued

Table 2-1. GFP expression percentages for BARD1 variant HDR assays.

Table 2-1 Continued

Experiment 4 Experiment 5 Experiment 6 Sample GFP Sample GFP Sample GFP % % % Control 15.73 Control 12.62 Control 14.56 Empty 0.39 Empty 0.57 Empty 0.63 WT 13.35 WT 9.71 WT 13.78 N326S 13.61 I434F 8.76 N488S 10.77 N326S 13.24 I434F 8.41 N488S 11.04 N326S 13.25 I434F 8.39 N488S 10.96 S339N 11.64 A435V 10.09 H506R 11.15 S339N 12.76 A435V 10.27 H506R 12.65 S339N 11.5 A435V 10.38 H506R 11.77 S339T 12.16 L447C 10.66 V507A 2.86 S339T 12.23 L447C 10.17 V507A 2.14 S339T 13.24 L447C 10.15 V507A 2.38 S342N 11.32 N450H 9.4 V510A 12.12 S342N 11.88 N450H 9.64 V510A 10.95 S342N 11.23 N450H 10.76 V510A 12.18 T343I 11.39 G451fs 1.68 A518V 8.26 T343I 11.8 G451fs 1.86 A518V 9 T343I 12.41 G451fs 1.73 A518V 8.89 T351M 10.64 A460T 4.32 V523I 12.68 T351M 10.5 A460T 4.24 V523I 11 T351M 11 A460T 4.87 V523I 11.73 L359fs 0.83 L465F 5.11 V523A 11.14 L359fs 0.81 L465F 4.71 V523A 9.81 L359fs 0.59 L465F 5.2 V523A 10.11 E361D 13.56 L480S 2.08 P530L 3.19 E361D 12.4 L480S 2.22 P530L 2.93 E361D 13.29 L480S 2.25 P530L 2.98 S389C 12.9 H483R 9.05 Y533F 12.05 S389C 12.49 H483R 8.94 Y533F 11.52 S389C 12.69 H483R 8.91 Y533F 11.07 H606D 0.77 H606D 0.68 H606D 0.59

Continued

Table 2-1 Continued

Experiment 7 Experiment 8 Experiment 9 Sample GFP Sample GFP Sample GFP % % % Control 15.23 Control 13.22 Control 10.07 Empty 0.75 Empty 0.54 Control 11.25 WT 13.38 WT 14.3 Control 11.41 K540N 11.92 H606D 0.76 Empty 0.46 K540N 10.57 H606D 0.85 Empty 0.55 K540N 11.01 H606D 0.84 Empty 0.5 S551* 1.97 D612V 14.39 WT 12.25 S551* 1.85 D612V 13.57 WT 12.14 S551* 2.29 D612V 13.58 WT 11.23 H556D 11.3 L625I 10.57 C62S 13.24 H556D 11.57 L625I 9.37 C62S 11.75 H556D 11.15 L625I 9.98 C62S 11.42 S558P 11.91 R641Q 13.55 I69M 13.36 S558P 11.15 R641Q 13.04 I69M 13.12 S558P 11.27 R641Q 13.48 I69M 13.34 Q564* 2.1 R642G 13.68 V85L 11.69 Q564* 1.84 R642G 13.01 V85L 13.29 Q564* 1.82 R642G 13.39 V85L 11.01 S660R 0.7 R664T 11.9 N98S 11.03 S660R 0.51 R664T 12 N98S 11.82 S660R 0.49 R664T 11.98 N98S 12.22 R565C 7.47 G698D 0.57 S103N 12.28 R565C 7.59 G698D 0.57 S103N 11.38 R565C 7.34 G698D 0.67 S103N 11.58 R565H 10.13 P707S 5 S575N 11.69 R565H 9.96 P707S 4.57 S575N 10.83 R565H 10.07 P707S 5.06 S575N 11.24 G574D 9.69 G753D 2.77 S616N 11.81 G574D 9.71 G753D 2.76 S616N 12.54 G574D 9.99 G753D 3.09 S616N 11.19 T598I 8.59 V767fs 0.56 G656R 8.87 T598I 7.63 V767fs 0.76 G656R 8.93 T598I 7.28 V767fs 0.73 G656R 7.48

Continued

Table 2-1 Continued

Experiment 7 Experiment 8 Experiment 9 Sample GFP Sample GFP Sample GFP % % % F677L 8.19 F677L 8.33 F677L 9.19 V713M 7.94 V713M 8.72 V713M 8.44 T719A 6.81 T719A 8.04 T719A 7.87 R731H 11.11 R731H 12.27 R731H 10.42 R731C 10.36 R731C 10.65 R731C 9.66

Continued

Figure 2-2. Functional analysis of 76 BARD1 variants. (A) 76 BARD1 single missense 36

Figure 2-2 Continued substitutions were tested for function in the HDR assay. HeLa-DR cells (Ransburgh et al., 2010) were treated with control siRNA (lane 1) or siRNA specific to the BARD1 3'- untranslated region (UTR) (lanes 2-80) and empty vector (lanes 1,2) or BARD1 expression plasmid (lanes 3-80). Two positive controls were used: cells treated with empty vector and control siRNA (lane 1), and cells depleted of endogenous BARD1 with wild-type BARD1 rescue (lane 3). Cells treated with empty plasmid and BARD1 3'UTR siRNA were used as a negative control (lane 2). HDR function was characterized by the percentage of GFP-positive cells measured using flow cytometry. Results in each experiment (±S.E.M.) were normalized to the WT rescue (lane 3), which was set equal to 1. Results represent three independent transfections per BARD1 plasmid. Variants that are benign and pathogenic according to ClinVar are labeled blue and red respectively. Variants with conflicting interpretations are labeled gray. HDR-deficient variants are marked by an asterisk and classified by having HDR function less than 0.6 and p < 0.01 when compared with endogenous BARD1 (control siRNA) using the Student’s t-test. (B) BARD1 variants tested in the HDR assay were examined for their expression relative to endogenous BARD1. Replicates were pooled together to examine BARD1 expression. The BARD1 protein is indicated with an arrow, as the BARD1-specific band migrated more slowly than a cross-contaminating band. The endogenously expressed BARD1 in control transfections (lanes 1, 14, 23, 29, 33, 41, 53, 65, 77, 90, 100) and the depleted BARD1 without rescue (lanes 2, 15, 24, 30, 34, 42, 54, 66, 78, 91, 101) can be compared with the expression of variant BARD1 proteins as indicated. A tagged BARD1 V507A plasmid was used to confirm BARD1 expression (lanes 31 and 32, upper arrow) and migrated more slowly than endogenous BARD1 (lane 29, lower arrow). All missense variants had expression higher than the endogenously expressed BARD1.

The 76 variants tested were from across the full coding sequence of BARD1. Variants whose HDR activity was significantly different from endogenous BARD1 and whose expression was greater than or equal to endogenous BARD1 were considered to be repair-deficient (Figure 2-2A, B). The eight variants located in the RING domain, as well the 22 in the region between the RING and ankyrin repeat domains, all had HDR activity similar to wild-type. Previous work in our lab (Lee et al., 2015) examined the HDR activity of 29 BARD1 missense variants, including additional variants in the RING domain. We combined the current HDR results with the previously published results into a single table containing 105 variants (Figure 2-3). In this previous work, the variants

L44R, C53W, and C71Y in the RING domain were found to be defective in HDR due to defective binding to BRCA1.

Continued

Figure 2-3. Functional analysis of 105 BARD1 variants. 105 BARD1 single missense 39

Figure 2-3 Continued substitutions were tested for function in the HDR assay. Results from Lee at al. 2015 are included with variants tested in this chapter. HeLa-DR cells (Ransburgh et al., 2010) were treated with control siRNA (lane 1) or siRNA specific to the BARD1 3'-untranslated region (UTR) (lanes 2-109) and empty vector (lanes 1, 2) or BARD1 expression plasmid (lanes 3-109). Two positive controls were used: cells treated with empty vector and control siRNA (lane 1), and cells depleted of endogenous BARD1 with wild-type BARD1 rescue (lane 3). Cells treated with empty plasmid and BARD1 3'UTR siRNA were used as a negative control (lane 2). HDR function was characterized by the percentage of GFP-positive cells measured using flow cytometry. Results in each experiment (±S.E.M.) were normalized to the WT rescue (lane 3), which was set equal to 1. Results represent three independent transfections per BARD1 expression plasmid. Variants that are benign and pathogenic according to ClinVar are labeled blue and red respectively. Variants with conflicting interpretations are labeled gray. HDR-deficient variants are marked by an asterisk and classified by having HDR function less than 0.6 and p < 0.01 when compared to endogenous BARD1 (control siRNA) using the Student’s t-test.

Surprisingly, in the current study, four of the 17 variants in the ankyrin domain, which has no known DNA repair function, were found to express full-length BARD1 and be defective in HDR. Variants A460T, L465F, L480S, and P530L had HDR activity lower than 0.6, which was significantly lower than cells expressing endogenous BARD1. The five variants located between the ankyrin repeat and BRCT domains were proficient in

DNA repair with the exception of R565C, whose HDR activity was just below the cutoff of 0.6. Of the 19 missense variants tested in the BRCT domain, which is known to be involved in recruiting and retaining the BRCA1-BARD1 heterodimer to areas of DNA damage, five were found to be defective in HDR (Li and Yu, 2013; Miyoshi et al., 2015).

The variants S660R and G698D had HDR function comparable to cells transfected with empty vector. The variants T598I, P707S, and G753D had activity higher than empty vector but still significantly lower than endogenous BARD1. A larger fraction of residues conserved across several mammalian species were mutated in repair-deficient variants

(9/10) than in functional ones (38/55) (Figure 2-4). Five truncation variants were also tested, and all were about as equally defective as the empty vector in the HDR assay.

Previous work has suggested that filtering using high LOH could be used to identify

BRCA1 variants defective in HDR (Lu et al., 2015). However, BARD1 variants that were found to have high LOH (Figure 2-1B) were all functional, with the exception of truncation variant G451fs.

Continued

Figure 2-4. BARD1 protein sequence differences between mammalian species. 42

Figure 2-4 Continued

Human (Homo_sapiens), domestic cat (Felis_catus), domestic dog (Canis_lupis_familiaris), mouse (Mus_musculus), gray short-tailed opossum (Monodelphis_domestica) and domestic sheep (Ovis_aries) BARD1 protein sequences were aligned using Clustal Omega to examine conserved residues. Asterisks (*) indicate positions with a single, fully conserved residue. Colons (:) indicate residue conservation between groups of strongly similar properties. Periods (.) indicate conservation between groups of weakly similar properties. Residues that were mutated in HDR-deficient, non- truncating variants are highlighted in yellow. Residues mutated in HDR-functional variants are highlighted in green.

Testing the HDR function of BRCA1 variants has shown that, with the exception of variants that impact mRNA splicing, known pathogenic variants of BRCA1 are HDR- defective, while known benign variants are not (Lu et al., 2015; Ransburgh et al., 2010;

Towler et al., 2013). Similarly, the BARD1 variants S241C and E361D, which have been found in patients with breast cancer and are benign according to ClinVar, are functional in HDR (Figure 2-2A, blue dots). Truncation variants V154fs, S551*, and Q564*, where the asterisk indicates a stop codon, are listed as pathogenic according to ClinVar and were non-functional in the HDR assay (Figure 2-2A, red dots). Several other variants that were tested have been identified in breast cancer patients and have conflicting interpretations of pathogenicity (Figure 2-2A, gray dots).

We tested whether the level of expression of any BARD1 variants could have affected their HDR activity. Expression of BARD1 variants was examined via immunoblot

(Figure 2-2B). The relative expression of the endogenous BARD1 and siRNA-depleted

BARD1 were shown (Figure 2-2B lanes 1, 2, 14, 15, 23, 24, 29, 30, 33, 34, 41, 42, 53,

54, 65, 66, 77, 78, 90, 91, 100,101). Though the expression levels of missense variants differed, they all expressed at higher levels than the endogenous BARD1. As an example,

BARD1 L480S (lane 63) had lower expression than the plasmid encoded wild-type (lane

55), but both had more intensely labeled bands than the endogenously expressed BARD1

(lane 53). The variant BARD1 H606D is present in the immunoblots in Figure 2-2B (lane

103), but is not listed in Figure 2-2A because full length protein was not detected, and it was found to contain a nonsense mutation at codon 125. Similarly, BARD1 L359fs is present in Figure 2-2B (lane 50) and Figure 2-5 (lanes 5, 14) but is not listed in Figure 2-

2A because it was a miss-call during variant selection. Frameshift and nonsense codon variants (Figure 2-2B lanes 10, 60, 81, 84, 112) lacked full length BARD1. Truncation variants G451fs, S551*, Q564* and V767fs expressed truncated BARD1, while variant protein V154fs was not detected (Figure 2-5). We infer that repair defects observed in truncation variants with poor protein expression were due to the absence of protein instead of expressed, non-functional variant protein. We conclude that for the missense variants, a low level of HDR activity was not due to low expression of the BARD1 protein.

Figure 2-5. Immunoblots of truncated BARD1 variants. Truncated BARD1 variants tested in the HDR assay were examined for their expression relative to endogenous BARD1. Replicates were pooled together to examine BARD1 expression. The BARD1 protein is indicated with an arrow. The endogenously expressed BARD1 in control transfections (lanes 1, 10) and BARD1 WT rescue (lanes 3, 12) were compared with the expression of truncated BARD1 variant proteins. Variants were run on 4-12% Bis-Tris (top) and 6% acrylamide (bottom) gels due to a contaminating band that co-migrated with BARD1 WT on 4-12% Bis-Tris gels. Variants G451fs (lanes 6, 15), S551* (lanes 7, 16), Q564* (lanes 8, 17) and V767fs (9, 18) expressed truncated protein. Variant V154fs (lanes 4, 13) does not contain the residue recognized by the BARD1 antibody used, and may express truncated protein. 46

Comparison of BARD1 HDR activity with sensitivity to DNA damage by ionizing radiation and cisplatin.

BARD1 variants A460T, P707S, G753D, and V767fs were selected for further analysis, as they covered a range of HDR activities below 0.6 when transiently expressed. The selected variants and wild-type BARD1 were tagged with the His-Biotin-Tobacco Etch

Virus (HBT) tag (Tagwerker et al., 2006) and integrated into the FRT site of a HeLa-DR derivative cell line called HeLa-DR-FRT (Starita et al., 2018). The advantage of these

FRT site-containing cells was that the BARD1 gene was stably expressed from a single site and should have consistent levels of expression. We tested the stably expressed variants in the HDR assay to confirm repair proficiency was the same as the transiently expressed variants. The HDR activities of these variants from Figure 2-2 are shown in isolation (Figure 2-6A). The same repair trends were observed in both transiently expressed and stably expressed BARD1 variants (Figure 2-6A, B). BARD1 A460T- integrated cells had the most residual repair activity among these variants, and cells integrated with BARD1 P707S, G753D and V767fs had decreasing levels of repair proficiency respectively. Expression of the integrated BARD1 variants was also greater than or equal to that of endogenous BARD1 (Figure 2-6C). BRCA1 expression in variant-integrated cell lines was similar in cells expressing endogenous BARD1 and the various defective BARD1 variants (Figure 2-6C). Thus, none of the changes in HDR observed with these BARD1 variants were attributable to changes in BRCA1 expression.

Continued Figure 2-6. HBT-tagged BARD1 variants expressed from a single FRT site function similarly to transiently transfected variants in HDR. (A) Of the 76 BARD1 variants examined for HDR functionality (Figure 2-2A), BARD1 A460T, P707S, G753D, and V767fs were selected for further study based on their range of HDR activity. (B) BARD1 variants integrated into a single FRT site in HeLa-DR-FRT cells (Starita et al., 2018) functioned similarly to the same variants expressed in HeLa-DR cells by transient 48

Figure 2-6 Continued transfection. HBT-tagged BARD1 WT and variants were integrated into HeLa-DR-FRT cells via the FRT site for consistent single-site expression. Cells were treated with control or BARD1 3'UTR siRNA and examined via the HDR assay. Unintegrated HeLa-DR-FRT cells were used as a control. For comparison, results from transiently transfected cells were selected from Figure 2-2 (Figure 2-6A), and the results from expressing the integrated BARD1 variants are shown. While HDR activity was lower in cells containing integrated BARD1 as compared with transiently expressed BARD1, the integrated variants exhibited the same trend as the transiently expressed variants—BARD1 A460T was the most proficient, followed by BARD1 P707S, G753D, and V767fs. (C) Endogenous BARD1 was knocked down in treated stable cell lines, and BRCA1 and BARD1 expression were examined. HBT-tagged BARD1 variants migrated more slowly on electrophoresis gels than endogenous BARD1, as indicated by the upper (HBT- tagged) and lower (endogenous) arrows. HBT BARD1 was unaffected by the 3'UTR- targeted siRNA, while endogenous BARD1 was depleted. All of the BARD1 variants expressed at higher levels than the endogenously expressed BARD1 (middle). BRCA1 protein levels were not affected in variant-integrated cells (top).

We examined whether the quantitative loss of HDR proficiency correlated with the sensitivity of cells to extrinsic DNA damage. Clonogenic cell sensitivity assays were performed on HeLa-DR-FRT cells expressing integrated BARD1 wild-type and variants, as well as endogenous-only unintegrated cells. Cells were depleted of endogenous

BARD1 or BRCA1 and subjected to ionizing radiation (IR) (Figure 2-7) or cisplatin

(Figure 2-8). Depletion of BARD1 or BRCA1 from the endogenous-only cells

(E/siBARD and E/siBRCA) was used to determine the effect of a non-rescued HDR defect on sensitivity to IR and provided a baseline for DNA damage sensitivity (Figure 2-

7A, bottom). E/siBARD and E/siBRCA cells formed significantly fewer colonies after IR than control cells (E/siCON). BARD1 variant-integrated cells depleted of endogenous

BARD1 (Variant/siBARD) all formed significantly fewer colonies following IR than the same cells treated with control siRNA (Variant/siCON) (Figure 2-7A, top). For ease of comparison, we included results from Variant/siBARD, E/siBARD, E/siBRCA and

WT/siBARD cells on one graph (Figure 2-7B). Immunoblots were done to confirm knockdown of endogenous BARD1 (Figure 2-7C). While expression of each BARD1 variant differed, it was still greater than or equal to that of endogenous BARD1.

Continued

Figure 2-7. Sensitivity of BARD1 variants to ionizing radiation. (A) Endogenous-only cells and cells expressing BARD1 WT, A460T, P707S, G753D, and V767fs were treated with control, BARD1 3'UTR or BRCA1 3’UTR siRNA and X-ray irradiated at doses of 1, 2, 4 and 6 Gy. Colonies were counted after 12 days of growth followed by staining with crystal violet. Results in each experiment (±S.E.M.) were carried out in triplicate and 51

Figure 2-7 Continued converted to logarithmic scale. In each plot, the dashed line indicates control siRNA (siCON), and the solid line indicates depletion with the BARD1 (siBARD) or BRCA1 (siBRCA) 3'UTR-targeted siRNA. The Student’s t-test was done to examine the growth of variant and endogenous-only cell lines treated with BARD1 or BRCA1 siRNA relative to variant and endogenous-only cells treated with control siRNA (indicated by asterisks; * = p < 0.05, ** = p < 0.01). Cells expressing the four BARD1 variants, as well as endogenous-only cells depleted of BARD1 and BRCA1, were significantly different from variant and endogenous-only cells treated with control siRNA across most irradiation doses. (B) Results from panel A are shown for the BARD1 WT and variant cell lines treated with BARD1 3'UTR siRNA and endogenous-only cells treated with BARD1 or BRCA1 3’UTR siRNA. A Student’s t-test was done to compare the colony formation of BARD1 variant and BRCA1 or BARD1-depleted cell lines to BARD1 WT cells (indicated by asterisks). Colony counts of variants and BRCA1 or BARD1-depleted cell lines were significantly different (p < 0.01) from WT at all concentrations except siBARD 6 Gy (p < 0.05) and P707S 1 Gy, G753D 1 Gy, and G753D 2 Gy (p > 0.05). (C) An examination of the expression of endogenous BARD1 and BRCA1 in treated BARD1 variant-expressing and endogenous-only cell lines. Cells treated with BARD1 3'UTR siRNA had depleted endogenous BARD1 expression, while HBT BARD1 was unaffected. Cells treated with BRCA1 3’UTR siRNA showed depletion of endogenous BRCA1 and BARD1 expression. In all cases, the band representing the expression of the variant HBT BARD1 was denser than the faster migrating band from the endogenous BARD1 protein.

Continued

Figure 2-8. Sensitivity of BARD1 variants to cisplatin. (A) Cells expressing BARD1 WT, A460T, P707S, G753D, and V767fs cells, as well as endogenous-only cells, were treated with control, BARD1 3'UTR or BRCA1 3’UTR siRNA and treated with cisplatin at concentrations of 1.875, 3.75, 7.5, and 15 μM. Colonies were counted after 12 days of

Figure 2-8 Continued growth followed by staining with crystal violet. Results in each experiment were done in triplicate and converted to logarithmic scale (±S.E.M.). In each plot, the dashed line indicates control siRNA, and the solid line indicates depletion with the BARD1 or BRCA1 3'UTR-targeted siRNA. The Student’s t-test was done to examine the growth of variant and endogenous-only cell lines treated with BARD1 or BRCA1 siRNA relative to variant and endogenous-only cells treated with control siRNA (indicated by asterisks; * = p < 0.05, ** = p < 0.01). Cells expressing the four BARD1 variants, as well as endogenous- only cells depleted of BRCA1 and BARD1, were significantly different from variant and endogenous-only cells treated with control siRNA across most cisplatin concentrations. (B) Results from panel A are shown for BARD1 WT, variant and endogenous-only cell lines treated with BARD1 or BRCA1 3'UTR siRNA. A Student’s t-test was done to compare the colony formation of BARD1 variant cell lines, as well as endogenous-only cells depleted of BRCA1 or BARD1, to BARD1 WT cells. Colony counts from variants and endogenous-only cells were significantly different (p < 0.01) from BARD1 WT at most concentrations, excepting A460T 3.75 μM, siBRCA1 3.75 μM and A460T 15 μM (p < 0.05) and A460T 1.875 μM and siBRCA 15 μM (p > 0.05). (C) An examination of endogenous BARD1 and BRCA1 expression in treated BARD1-expressing and control cell lines. Cells treated with BARD1 3'UTR siRNA had depleted endogenous BARD1 expression, while HBT BARD1 was unaffected. Cells treated with BRCA1 3’UTR siRNA showed depletion of endogenous BRCA1 and BARD1 expression. The band representing the expression of variant HBT BARD1 was denser than the endogenous BARD1 protein band in all cases.

E/siBARD, E/siBRCA and Variant/siBARD cells formed significantly fewer colonies than WT/siBARD cells at most irradiation concentrations (Figure 2-7B). It was expected that increased HDR deficiency would result in increased sensitivity to DNA damage agents. For example, we expected that cells expressing BARD1 V767fs, the most HDR- deficient variant, would form the least number of colonies. Cells expressing BARD1

A460T, the variant with the most residual HDR activity, were expected to have the largest number of colonies compared with the other variants. Interestingly, this was not the trend observed in the results. Instead, HDR-defective BARD1 variants were all equally sensitive to IR, and as sensitive as non-rescued cells depleted of BARD1 or

BRCA1. Similarly, all four BARD1 variants were more sensitive than wild-type to treatment with cisplatin (Figure 2-8A). Variant/siBARD, E/siBARD and E/siBRCA cells formed significantly fewer colonies than Variant/siCON and E/siCON cells.

Variant/siBARD, E/siBARD and E/siBRCA cells also formed significantly fewer colonies than WT/siBARD cells (Figure 2-8B). As seen with IR, all BARD1 variants and non-rescued cells depleted of BARD1 or BRCA1 were equally sensitive to cisplatin. In addition, BARD1 variant expression remained consistently equal to or greater than that of endogenous BARD1 in cells, indicating that decreased colony formation was not associated with decreased variant expression (Figure 2-8C). While decreased HDR function resulted in decreased colony formation, quantitative differences in the HDR activity did not correlate to quantifiable changes in sensitivity to cisplatin or IR.

Discussion

In this study, we found: 1) from 10,389 cancer samples across 33 cancer types, 76

BARD1 missense variants were identified as potentially pathogenic and were selected for functional analysis. 2) Fifteen of the 76 tested variants were defective for HDR, suggesting that these were potentially pathogenic variants. 3) Four of the 17 variants tested in the ankyrin repeat domain, for which there was no previously known DNA repair function, were deficient in homologous recombination. 4) Five of the 19 variants tested in the BRCT domain, which does have known DNA repair functions, were deficient in homologous recombination. 5) Variants that were deficient in HDR rendered the cells sensitive to treatment with DNA-damaging cisplatin or IR. 6) Quantitative differences in HDR deficiency among defective variants did not translate to quantitatively different sensitivity to DNA damage.

The BRCA1-BARD1 heterodimer is necessary for tumor suppressor function (Nandula et al., 2008; Wu et al., 1996). Variants that affect binding between BRCA1 and BARD1 have been linked to familial breast cancer or are non-functional in the HDR assay (Couch and Weber, 1996; Lee et al., 2015; Shattuck Eidens et al., 1995). Loss of BARD1 has been linked to increased susceptibility to hereditary breast and ovarian cancer (HBOC) and is associated with loss of tumor suppressor activity (Baer and Ludwig, 2002; Eyfjord et al., 2003; Hart et al., 2017; Levy-Lahad and Friedman, 2007; McCarthy et al., 2003;

Thai et al., 1998). The importance of BARD1 in cancer development indicates how significant it is to determine whether BARD1 VUS are benign or pathogenic.

A rise in the quantity of genomic data has led to an increasing number of VUS, uncertain 56 due to their low frequency and conflicting reports of pathogenicity. In this chapter, potentially pathogenic BARD1 variants were identified in a dataset of 10,389 cancer samples from 33 different cancers (Huang et al., 2018). From germline and somatic samples, 76 variants from across the entire BARD1 gene were identified as suggestive for being pathogenic. Analysis from the tumor sequencing data indicated BARD1 S339T,

T343I, V523A, N450H, G451fs, and L239Q had significantly increased LOH, suggesting that these variants were more likely to be pathogenic. However, with the exception of the

G451fs truncation variant, the other five variants were found to be functional in HDR.

Previous work (Lu et al., 2015) has proposed that HDR-deficient BRCA1 variants could be identified using filtering based on increased LOH. In contrast, we have found that increased LOH does not correlate with HDR deficiency for BARD1 variants. As we are unaware of other studies regarding the relationship between LOH and HDR-deficient

BRCA1 and BARD1 variants, these contrary results suggest that increased LOH is not a reliable indicator of non-functional variants. However, studies have shown that 60-90% of tumors with pathogenic germline BRCA1 or BRCA2 mutations have LOH (Johnson et al., 2002; Osorio et al., 2002). It is possible that the sequence analysis used to predict pathogenic BARD1 variants does not differentiate between driver and passenger mutations, making it difficult to filter for pathogenic variants using LOH. Data suggest that the HDR assay is a more effective method for identifying deficient variants. As the variant expression plasmids used only contain the mRNA coding sequence and we do not have access to patient samples, we cannot accurately examine the mRNA expression levels. However, if the mRNA expression levels of LOH mutants are lower than wild-

57 type BARD1, this could indicate an HDR deficiency that we do not observe in our protein expression experiments.

In addition, the variant A460T, which was non-functional in HDR, was identified as potentially pathogenic during the original analysis of exome sequencing data (Lu et al.,

2015) but was not considered pathogenic after updated analysis. These differences demonstrate the importance of empirical results, such as the HDR assay, for evaluating whether a given variant is predictive of cancer. Conversely, the results from functional assays may provide feedback for the improvement of the bioinformatic interpretation of genomic data. While large scale genomic analyses should be used to identify functionally significant variants, functional assays must still be utilized for more comprehensive characterization of these variants.

The 76 BARD1 missense variants were tested in the HDR assay to examine DNA repair function. Variants were considered non-functional if their HDR activity was significantly different from endogenous BARD1. Previous work in our lab (Lee et al., 2015) characterized fully non-functional variants as those with HDR activity significantly different from endogenous BARD1 but not empty vector, and intermediate variants as those significantly different from both endogenous BARD1 and empty vector. We have changed our classification standards based on the strength of the reproducibility in this study and for ease of analysis. We now interpret variants as functional or nonfunctional and do not include the intermediate phenotype. This interpretation is supported by the new data that the BARD1 A460T variant would have been ranked as intermediate using the previous interpretation, but assays measuring DNA damage sensitivity using IR and

58 cisplatin indicated that BARD1 A460T was just as sensitive as the V767fs variant, which scored similar to the empty vector in the HDR assay.

Based on BRCA1 variant function in HDR, it was anticipated that BARD1 variants with impact on HDR function would map to the RING and BRCT domains. In this study, we tested eight variants in the BARD1 RING domain and none of them were defective for

HDR. In a prior study (Lee et al., 2015), we had analyzed another nine variants in the

BARD1 RING domain; three of these were defective for DNA repair activity. In the current study, we tested 19 variants in the BARD1 BRCT domain, and five of these were defective. BARD1 T598I, S660R, G698D, P707S, and G753D were found to be non- functional in the BRCT domain. The BRCT domain has been shown to interact with the

HP1 protein in order to retain both the BRCA1-BARD1 complex and CtIP, which is involved in DNA end resection, at the damage site (Miyoshi et al., 2015). The BARD1-

HP1 interaction is also necessary for the accumulation of DNA helicase FANCJ at sites of DNA damage (Wu et al., 2016). BARD1 L570E/V571E and L570A/V571A variants have been shown to inhibit the interaction between BARD1 and HP1 (Miyoshi et al.,

2015). The BARD1 BRCT domain is also necessary for binding poly(ADP-ribose)

(PAR), allowing for rapid recruitment of the BRCA1-BARD1 complex to areas of DNA damage (Li and Yu, 2013). The variants K619A, C645R and V695L have been shown to disrupt BARD1-PAR interaction (Li and Yu, 2013). The T598 residue tested in this study

(T598I) is located on the surface of the protein next to K619, and could possibly affect

PAR binding. However, the relationship between BARD1-PAR binding and HDR is unclear since the BARD1 K619A and V695L variants, as well as several others that

59 disrupt PAR binding, have been shown to be functional in HDR (Billing et al., 2018;

Laufer et al., 2007). BRCA1 also binds to the BARD1 BRCT domain (Simons et al.,

2006), and previous work in our lab has identified that this binding is affected by the

BARD1 G623E variant (Lee et al., 2015). Most of the non-functional variants we identified in the BRCT domain, with the exception of T598I, are not located near known binding sites for proteins associated with DNA repair.

To our surprise, we found four variants, BARD1 A460T, L465F, L480S, and P530L, were identified as non-functional in the ankyrin repeat domain. Prior to this study, the ankyrin repeat domain had no known reported function in DNA repair. Previous work has shown that a large deletion of the ankyrin repeat domain results in chromosome instability and loss of HDR function (Laufer et al., 2007). In addition, the BARD1 ankyrin domain has been shown to interact with p53 to mediate apoptosis (Berardi et al.,

2005). The oncoprotein Bcl-3, which interacts with BARD1, is also involved in the regulation of NF-κB transcription via ankyrin repeat domain-associated protein interactions (Dechend et al., 1999). Our results indicate that the ankyrin repeat domain may have functions that are necessary for DNA repair. For both the ankyrin and BRCT domains, the non-functional variants identified may affect BARD1 folding and structure, which could also affect binding to proteins such as BRCA1 or HP1. For example, the

BARD1 P707 and G753 residues are located near one another on the surface of the protein. As coding substitutions at these amino acids result in loss of HDR they may be part of a binding pocket. The identified variants may also indicate binding sites for proteins whose interaction with BARD1 has not yet been discovered. The

60 characterization of non-functional BARD1 variants in areas that are not well-studied helps to further understand the roles of the ankyrin repeat and BRCT domains in homology- directed repair.

Many of the BARD1 variants tested have been recorded on ClinVar as having been isolated from patients with breast cancer or hereditary cancer-predisposing syndromes.

Variants S241C and E361D, which were functional in HDR, have been identified has likely benign, and truncation variants V154fs, S551*, and Q564* are likely pathogenic in

ClinVar. The variants V85L, R194K, I258T, N326S, R565H, and R641Q, which were functional in HDR, have conflicting reports of pathogenicity, with reports indicating they were VUS or likely benign. Since these variants were functional in the HDR assay, we would interpret such variants as likely benign. The non-functional truncating variant

V767fs also had conflicting reports of pathogenicity, with reports indicating that it was a

VUS or likely pathogenic. The trend observed in this chapter is supplemented by the 29 variants that were previously studied (Lee et al., 2015) —variants V507M and R658C were functional in the HDR assay and are considered benign in ClinVar, and several other functional variants are listed as having conflicting reports of pathogenicity because reports indicate they are VUS or likely benign. Previous work has shown that pathogenic

BRCA1 variants are non-functional in the HDR assay, and benign variants are functional

(Lu et al., 2015; Ransburgh et al., 2010; Towler et al., 2013). Based on the data from

ClinVar, this trend appears to be true for BARD1 variants as well, suggesting that the non-functional variants identified in this chapter would be likely pathogenic.

We also asked whether BARD1 HDR function affected cell sensitivity to DNA damage

61 agents. Cells expressing HDR-deficient variants A460T, P707S, G753D and V767fs, as well as endogenous-only, non-rescued cells depleted of BRCA1 or BARD1, were more sensitive to treatment with IR or cisplatin than cells expressing wild-type BARD1.

Testing non-rescued cells allowed us to set a standard for the effect of non-functional

DNA repair on damaged cells. We had hypothesized that the more HDR-deficient a variant was, the fewer colonies cells expressing that variant would form after damage, indicating a quantitative sensitivity to the DNA damage. The results did not support that expectation. Following treatment with IR or cisplatin, HDR-defective variants were in fact more sensitive to DNA damage, but they were as sensitive to DNA damage as cells depleted of BRCA1 or BARD1, suggesting that residual repair did not affect sensitivity.

The HDR assay indicates which variants are functional and non-functional, but it does not provide information on how this affects cell growth. This chapter has shown that loss of homologous recombination results in increased sensitivity to DNA damage, as indicated by decreased colony formation. The HDR results also reveal a correlation between BARD1 variants that cause a loss of DNA repair function with those that are likely cancer predisposing. While we examined in this study a large number of BARD1 variants across the length of the protein, including all three functional domains, many more BARD1 VUS exist. In future work, we hope to mutagenize the BARD1 functional domains on a larger scale, as we have previously done with the BRCA1 N-terminus

(Starita et al., 2018). Creating a library of all potential BARD1 variants in these functional domains and testing the HDR function of these variants would allow us to identify additional pathogenic variants and regions of interest. The work done in this study helps

62 better understand the role of BARD1 in DNA repair, and how loss of homology-directed repair affects cell growth and sensitivity.

Materials and Methods

Selection of BARD1 Variants: BARD1 missense variants conferring potential cancer predisposition were identified in a cohort of 4,034 samples from 12 cancer types (Lu et al., 2015) that was part of larger set of 10,389 TCGA samples from 33 cancer types

(Huang et al., 2018). Germline single nucleotide variants (SNVs) were identified with variant calling on whole exome sequencing data using GATK (McKenna et al., 2010)

(version 3.5, using its haplotype caller in single-sample mode with duplicate and unmapped reads removed and retaining calls with a minimum quality threshold of 10) and VarScan (Shen et al., 2012) (version 2.3.8 with default parameters, except where – min-var-freq 0.10,–p value 0.10,–min-coverage 3,–strand-filter 1) operating on a mpileup stream produced by SAMtools (version 1.2 with default parameters, except where -q 1 -Q

13). Germline indels were identified using VarScan and GATK (same parameters and version as above) in single-sample mode. Pindel (Ye et al., 2009) (version 0.2.5b8 with default parameters, except where -x 4, -I, -B 0, and -M 3 and excluded centromere regions (genome.ucsc.edu)) was also applied for indel prediction. For all analyses, the

GRCh37-lite reference was used and an insertion size of 500 was specified whenever this information was not provided in the BAM header.

All variants were limited to limited to coding regions of full-length transcripts obtained from Ensembl release 70 plus the additional two base pairs flanking each exon that cover 63 splice donor/acceptor sites. SNVs were based on the union of raw GATK and VarScan calls, while indels were required to be called by at least two out of the three variant callers (GATK, VarScan, Pindel). High-confidence, Pindel-unique calls (at least 30x coverage and 20% VAF) were also included. Further, variants were required to have an

Allelic Depth (AD) ≥ 5 for the alternative allele. Readcount analyses for variants passing these filters were performed in both normal and tumor samples using bam-readcount

(version 0.8.0 commit 1b9c52c, with parameters -q 10, -b 15) in order to quantify the number of both reference and alternative alleles. Variants were required to have at least 5 counts of the alternative allele and an alternative allele frequency of at least 20%. Of these, rare variants were filtered for, with ≤ 0.05 % minor allele frequency in 1000

Genomes and ExAC (release r0.3.1).

Variants passing manual review, with low allele frequencies (MAF < 0.05%), and significant LOH were prioritized for characterization. These variants, their cancer type distributions and frequencies are shown in the latest data release of the 10,389 samples

(Huang et al., 2018) to the research community on NCI Genome Data Commons

(https://gdc.cancer.gov/about-data/publications/PanCanAtlas-Germline-AWG).

Cloning of BARD1 Variants: For transient expression, BARD1 (NCBI Reference

Sequence: NM 000465.3) wild-type and missense variants were cloned into a pcDNA3 vector backbone containing a rabbit β-globin intron upstream of the BARD1 translation initiation site to drive expression of the 777 amino acid human BARD1 transgene.

Variants were cloned using the New England BioLabs Q5 Site-Directed Mutagenesis kit.

For stable integrations, BARD1 wild-type and missense variants were cloned into a

64 pcDNA5/FRT/TO vector backbone containing the His-Biotin-Tobacco Etch Virus (TEV)

(HBT) tag (Tagwerker et al., 2006). PCR reactions were done using PfuUltra II Fusion

HS DNA Polymerase. Vectors and inserts were ligated together using Gibson assembly

(Smith et al., 2009). Colonies with successful ligations were fully sequenced to confirm expected variants. All variants were verified using Sanger sequencing services provided

OSU Comprehensive Cancer Center (OSUCCC) Genomics Shared Resource.

Homology-Directed Repair (HDR) Assay: For examining the HDR function of transiently expressed of BARD1 variants, HeLa-DR-13-9 (HeLa-DR) cells were utilized.

HeLa-DR cells contain two non-functional GFP coding sequences, one of which is interrupted by an I-SceI restriction endonuclease site. Cells were cultured in DMEM media containing 1% penicillin/streptomycin, 1% GlutaMAX, 10% bovine serum, and

1.5 μg/ml puromycin. Cells were seeded in a 24-well plate and transfected with siRNA to the BARD1 3’-UTR (5’-AGCUGAAUAUUAUACCAGAdTdT-3’) or control siRNA (5 pmol), and BARD1 wild-type, variant, or pcDNA3 empty vector (300 ng). All transfections were carried out using Lipofectamine 2000 per the manufacturer’s recommendations. Cells were moved to 6-well plates 24 hours later. 48 hours after the first transfection, cells were transfected with 25 pmol siRNA, 750 ng DNA, and 750 ng of expression plasmid containing the restriction endonuclease I-SceI to induce a double- strand break. If HDR is functional, the break is repaired by gene conversion with the second GFP allele, and cells become GFP-positive (Pierce et al., 2001; Ransburgh et al.,

2010). 72 hours after the second transfection, cells were collected and GFP-positive cells were counted using the FACSCalibur in the OSUCCC Analytical Cytometry Shared

Resource. 10,000 cells were counted, and the remaining cells were used for immunoblotting. Cells transfected with BARD1 siRNA and BARD1 wild-type plasmid

(wild-type rescue), and cells treated with control siRNA and empty vector, served as positive controls. Cells treated with BARD1 siRNA and empty vector were used as a negative control. HDR activity, as defined by the percentage of GFP-positive cells, was normalized to wild-type rescue control and set to 1.

For examining the HDR function of stably integrated BARD1 variants, HeLaDR-FRT cells (Starita et al., 2018) were used. Cells integrated with pcDNA5-FRT/TO-HBT- tagged BARD1 wild-type or variants (A460T, P707S, G753D and V767fs) were seeded in

24-well plates. Cells not integrated with BARD1 variants were used as a negative control.

Cells were transfected with siRNA to the BARD1 3’-UTR or control siRNA. All transfections were carried out using Oligofectamine according to the manufacturer’s recommendations. Transfections were carried out on the same time pattern as detailed for transiently expressed BARD1. For 24-well transfections, 30 pmol siRNA was used, and for 6-well transfections, 50 pmol siRNA and 3 μg of I-SceI expression plasmid were used. Cells were collected and GFP-positive cells counted as detailed for transiently expressed BARD1. HDR activity, as defined by the percentage of GFP-positive cells, was normalized to cells treated with control siRNA for each individual cell line and set at 1.

Alignment of BARD1 Protein Sequences: Homo sapiens, Felis catus, Canis lupis familiaris, Mus musculus, Monodelphis domestica, and Ovis aries BARD1 protein sequences were aligned using Clustal Omega (Remmert et al., 2011) to examine conserved residues.

Integration of Variants into HeLaDR-FRT Cells: pcDNA5-FRT/TO-HBT-tagged

BARD1 wild-type and variants were co-transfected with plasmid expressing the flippase recombinase in a 1:2 ratio into HeLa-DR-FRT cells to induce integration at the flippase recognition target site (Craig, 1988; Sauer, 1994). Transfections were done according to

Lipofectamine 2000 manufacturer’s recommendations. 24 hours after transfection, cells were incubated at 30˚C for 24 hours and then moved back to 37˚C. Integrated cells were selected for with 550 μg/ml Hygromycin-B.

Immunoblotting: For BARD1 variants, replicates were combined, spun down at 1200 rpm for 5 minutes and resuspended in 150 μl of 1X LDS-PAGE dye. Samples were sonicated at 45% for 15 seconds three times. Sample was resolved on 6 or 8% SDS-

PAGE gels and transferred to PVDF membrane. Samples were probed with BARD1

(Bethyl, 1:1000) and BRCA1 (1:500) (Schlegel et al., 2003) antibodies. Antibodies to

RHA1 (1:20000) (Schlegel et al., 2003) and α-tubulin (Sigma, 1:20000) were used as loading controls. Membranes were incubated with fluorescent (LI-COR, 1:20000) or chemiluminescent (GE, 1:5000) rabbit and mouse secondary antibodies.

Clonogenic Assays: HeLaDR-FRT cells stably expressing pcDNA5-HBT-BARD1 WT,

A460T, P707S, G753D and V767fs, as well as control cells expressing only endogenous

BARD1, were seeded in 24-well dishes and transfected with 30 pmol of siRNA to the

BARD1 3’-UTR, BRCA1 3’-UTR or control siRNA. Transfections were done using

Oligofectamine as per the manufacturer’s protocol. Cells were transferred to 6-well dishes after 24 hours, and were treated with 50 pmol siRNA 48 hours after the first transfection. 48 hours after the second transfection, 1000 cells of each treatment

67 condition were plated in 10-cm dishes. Remaining cells were saved for immunoblotting to confirm knockdown. After 24 hours, cells were treated with either ionizing radiation

(IR) or cisplatin. Cells that were treated with IR were subjected to 0, 1, 2, 4, or 6 Gy using the RS 2000 X-Ray Irradiator. For cisplatin treatment, cells were treated with 0,

1,875, 3.75, 7.5, and 15 μM of cisplatin for 2 hours, after which cells were washed twice with 1X PBS and fresh media was added. Untreated cells were used as a control. After two weeks of growth at 37˚C, cells were fixed with cold methanol and stained with crystal violet. Dishes were coded to blind their treatment and cells were counted using

OpenCFU (Geissmann, 2013). The log value of the count was used for comparison.

Statistical Analysis: All BARD1 variants in the HDR and clonogenic assays were tested in triplicate. For HDR assays using transiently expressed BARD1 variants, HDR activity was normalized to wild-type rescue, which was set to 1. The Student’s t-test was applied to determine whether BARD1 variant HDR activity significantly differed (p < 0.01) from endogenous BARD1. Variants that were significantly different and below the cutoff of

0.6 were considered non-functional. For clonogenic assays, the Student’s t-test was carried out to examine whether BARD1 variant-expressing and endogenous-only cells treated with BARD1 or BRCA1 3’-UTR siRNA formed a significantly different number of colonies than variants treated with control siRNA and cells expressing BARD1 wild-type

(p < 0.05).

Chapter 3. Multiplex Homology-Directed Repair Analysis of BRCA1 C-terminus Missense Variants

Aleksandra I. Adamovich1, Mariame Diabate1, Tapahsama Banerjee1, Lea Starita2, and

Jeffrey D. Parvin1*

1 Department of Biomedical Informatics, Ohio State University Comprehensive Cancer

Center, Ohio State University, Columbus, OH

2 Department of Genome Sciences, University of Washington, Seattle, WA

Address correspondence to [email protected]

Author contributions: A.A. performed experiments and wrote the manuscript, A.A. and

M.D. performed the analysis, T.B. helped perform functional assays, L.S. and J.P. supervised experiments and J.P. edited the manuscript.

Introduction

Breast and ovarian cancers are two of the most prevalent cancers among women, with breast cancer posing a 13% lifetime risk and ovarian cancer a 1.3% lifetime risk according to the National Cancer Institute. Approximately 5-10% of breast cancer cases

(Larsen et al., 2014) and 23% of ovarian cancer cases (King et al., 2011) are hereditary breast and ovarian cancers (HBOCs) due to germline mutations in tumor suppressor genes. A plurality of HBOCs are caused by mutations in BRCA1 and BRCA2 (Jack et al.,

2002; Melchor and Benítez, 2013). Individuals with pathogenic BRCA1 variants have, on average, a 39% lifetime risk for ovarian cancer and 65% lifetime risk for breast cancer

(Eyfjord et al., 2003), which is much higher than the risk for the general population.

The 1863 amino acid BRCA1 protein comprises an amino-terminal RING domain, a

DNA binding domain (DBD), coiled coil (CC) domain, and two carboxy-terminal BRCT domains (Roy et al., 2012). BRCA1 forms an obligate heterodimer with BARD1, another cancer susceptibility gene (Couch et al., 2017), via their RING and BRCT domains to mediate homologous recombination (Moynahan et al., 1999; Shakya et al., 2011; Simons et al., 2006; Wu et al., 1996). Several phosphoproteins involved in DNA repair also interact with the BRCA1 BRCT domain. Binding to CtIP mediates DNA end resection

(Yun and Hiom, 2009), binding to BRIP1 mediates helicase activity in DNA repair

(Cantor et al., 2001; Clapperton et al., 2004), and Abraxas binding is associated with recruitment to DNA damage sites (Wang et al., 2007).

Although the genome aggregation database gnomAD (Lek et al., 2016) contains over four million missense variants, only 2% are assigned a clinical significance classification in 70 the ClinVar database (Malheiro et al., 2017). In addition, many ClinVar variants are considered variants of clinical significance (VUS) because there is not enough data to assign a clinical risk. Approximately 50% of known BRCA1 variants have conflicting reports of pathogenicity or are labeled as VUS on ClinVar (Landrum et al., 2018).

BRCA1 and BRCA2 VUS are also identified in 10-20% of cancer screenings (Eccles et al., 2015), signifying that many people remain uncertain of their cancer risk. As people with pathogenic BRCA1 mutations have a much higher chance of developing HBOC, individuals need to be accurately informed of their risk. This information would indicate whether women need to be more vigilant in their cancer screenings or perform preemptive and potentially invasive procedures such as mastectomies and salpingo- oophorectomies (Hartmann and Lindor, 2016) to reduce their risk.

One way to predict BRCA1-associated cancer risk is with functional assays, where loss of function is associated with pathogenicity. One such assay is the homology-directed repair (HDR) assay (Pierce et al., 2001; Ransburgh et al., 2010). The HDR assay utilizes a reporter cell line that contains non-functional GFP coding sequences, one of which is interrupted by a rare I-SceI restriction endonuclease site. Expression of the I-SceI expression plasmid results in a double-strand break in one of these GFP sequences. If the

BRCA1 variant expressed in the reporter cell is functional in homology-directed repair, the second GFP coding sequence will be used as a template to repair the double-strand break and the first GFP is rendered functional. After transfection of the I-SceI expression plasmid, 10–20% of the cells, quantified using flow cytometry, are converted to GFP- positive (Ransburgh et al., 2010). Previous work in our lab has examined over 130

BRCA1 variants in the HDR assay using a HeLa-derived cell clone known as HeLaDR

(Lu et al., 2015; Starita et al., 2015; Towler et al., 2013). With the exception of splicing variants, known pathogenic variants were non-functional in HDR while known benign variants were functional. Non-functional missense variants were located in the RING and

BRCT domains, suggesting that these two domains are the primary regions of BRCA1- mediated tumor suppression. Based on this information, we have concluded that BRCA1 pathogenicity correlates with loss of HDR function.

As BRCA1 mutations can cause cancer, it is very important to comprehensively identify which variants are cancer-associated. This would require testing all potential missense variants in the protein or the functional domains, which consists of hundreds or thousands of variants. The time and resources necessary to examine all these variants would be impossible in the singleton HDR assay, where all the cells in a population express one variant. We have been able to establish a multiplex version of the HDR assay in order to examine thousands of variants at once, and have identified the HDR function of more than 1000 variants in the 192 amino acids covering the amino-terminus of BRCA1, including the RING domain (Starita et al., 2018).

In this chapter, we generated high-coverage libraries of potential amino acid substitutions in the region encompassing BRCA1 amino acids 1577-1863, including the BRCT domain. We examined the HDR function of variants from residues 1577-1768 using the multiplex assay. We were able to identify with confidence the HDR function of over 500 variants from a population of cells integrated with over 2000 variants. Multiplex HDR assay variants that have also been tested in the singleton HDR assay had identical

72 functional classifications. Variant classifications from the multiplex assay were also compared to those from functional assays examining transcription activation (TA) and proliferation via saturation genome editing (SGE). The same variants were functional in all three assays, but several variants that were functional in the multiplex HDR assay were non-functional in the TA assay, SGE assay or both. Variants identified in the multiplex HDR assay were present in the ClinVar database as well. In addition to thirty

VUS, one benign variant and eleven pathogenic or likely pathogenic variants were identified. However, three known pathogenic variants – one splicing variant and two truncation variants – were functional in the HDR assay. Despite these inconsistencies, a majority of multiplex HDR variant classifications correlated with TA and SGE assay classifications and clinical data.

Generation of BRCA1 C-terminus Variant Libraries

In order to perform multiplex HDR on all potential amino acid substitutions in the

BRCA1 BRCT domain, variant library pools covering the last 287 codons of BRCA1

(amino acids 1577-1863) were generated. The BRCA1 C-terminus was cloned into the pUC19 vector and mutagenic inverse PCR was performed for each codon to generate all potential missense and nonsense variants for the region of interest (Jain and Varadarajan,

2014). The first 3 nucleotides of each codon-specific forward primer consisted of a hand- mixed NNN (amino acids 1577-1768) or NNK (amino acids 1769-1863) (N = A, T, C or

G; K = G or T) sequence to introduce all potential nucleotide variations at the codon site.

PCR products were pooled together and ligated back to themselves to produce a fragment 73 variant library. The mutagenized fragment was ligated back into full-length BRCA1 expression plasmid along with a degenerate 16-base barcode to create the final BRCA1 variant library pools. Libraries were sequenced in order to link variant sequences to barcodes and determine variant library diversity. Libraries had uniform variant abundance and had a minimum of 85% variant coverage and 11,000 barcodes (Figure 3-

1).

Figure 3-1. BRCA1 variant library diversity maps. The PacBio Sequel System was used to perform long read sequencing. Variants with zero or one amino acid changes were mapped to determine the diversity of the library. All potential amino acid changes, as well as nonsense mutations (as indicated by an asterisk at the top), are labeled on the y- axis. Variant libraries covered amino acids 1577-1672 (top), 1673-1768 (middle) and 1769-1863 (bottom). Variant abundance is indicated by color, with lighter colors representing less abundant variants and darker colors representing more abundant variants. Wild type amino acids are labeled in dark purple. Variants that were not present are shown in gray. Wild type sequence and position are indicated on the x-axis. The pool covering amino acids 1577-1672 contained 1769 unique variants (88% coverage) and 11,000 barcodes, the pool covering amino acids 1673-1768 contained 1712 unique variants (85% coverage) and 12,000 barcodes, and the pool covering amino acids 1769- 1863 had 1830 unique variants (92% coverage) and 18,000 barcodes.

Function of BRCA1 Variant Libraries in HDR

Variant libraries were integrated into HeLaDR-FRT cells (Starita et al., 2018) via FRT recombination (Craig, 1988) to create cell libraries with consistent variant expression at a single site in the genome. Variant cell libraries were treated with two rounds of transfection 48 hours apart with control siRNA or siRNA targeting the BRCA1 3′UTR to deplete endogenous BRCA1. Cells were also treated with siRNA targeting the BRCA1 coding sequence to deplete total BRCA1 expression and identify baseline GFP expression. The I-SceI restriction enzyme was also expressed during the second round of transfection to induce a double-strand break (Table 3-1). Four days after the first transfection, cells were sorted into GFP-positive and GFP-negative populations (Table 3-

2). Four HDR sorting replicates were performed for each variant cell library. The barcodes in the sorted cell populations were PCR-amplified and sequenced to count the number of times a barcode, and therefore a variant, occurred in the population. The variant frequency in a GFP-positive or GFP-negative sample was calculated using the

equation 푉푎푟푖푎푛푡 푆푎푚푝푙푒 퐹푟푒푞푢푒푛푐푦 = . Variant HDR activity was determined using the equation

( ) 푉푎푟푖푎푛푡 퐻퐷푅 퐴푐푡푖푣푖푡푦 = . Variants were considered ( ) non-functional if variant HDR activity was significantly depleted compared to wild type activity.

% GFP+ Cells Control siRNA BRCA1 3′UTR BRCA1 Coding siRNA Sequence siRNA Pool 4, Replicate 1 12.1 8.1 1.7 Pool 4, Replicate 2 12.1 8.1 1.7 Pool 4, Replicate 3 9.7 5.9 2.0 Pool 4, Replicate 4 9.7 5.9 2.0 Pool 5, Replicate 1 11.3 6.1 1.5 Pool 5, Replicate 2 8.2 4.3 1.2 Pool 5, Replicate 3 7.2 3.8 0.7 Pool 5, Replicate 4 7.2 3.8 0.7 Pool 6, Replicate 1 9.7 6.5 1.5 Pool 6, Replicate 2 9.7 6.5 1.5 Pool 6, Replicate 3 8.3 5.2 1.2 Pool 6, Replicate 4 8.3 5.2 1.2 Table 3-1. Percentages of GFP-positive cells in each HDR assay replicate. Sorted cells were treated with control siRNA, siRNA to endogenous BRCA1 (BRCA1 3′UTR siRNA), or siRNA to the BRCA1 coding sequence. Pool 4 = amino acids 1769-1863, pool 5 = amino acids 1673-1768, pool 6 = amino acids 1577-1672.

Control GFP+ Control GFP- BRCA1 GFP+ BRCA1 GFP- Pool 4, 713,000 2,000,000 913,000 2,000,000 Replicate 1 Pool 4, 915,000 2,000,000 811,000 2,000,000 Replicate 2 Pool 4, 401,000 2,000,000 667,000 2,000,000 Replicate 3 Pool 4, 435,000 2,000,000 636,000 2,000,000 Replicate 4 Pool 5, 544,000 2,000,000 484,000 2,000,000 Replicate 1 Pool 5, 570,000 2,000,000 515,000 2,000,000 Replicate 2 Pool 5, 368,000 2,000,000 379,000 2,000,000 Replicate 3 Pool 5, 428,000 2,000,000 372,000 2,000,000 Replicate 4 Pool 6, 547,000 2,000,000 543,000 2,000,000 Replicate 1 Pool 6, 403,000 2,000,000 530,000 2,000,000 Replicate 2 Pool 6, 403,000 2,000,000 425,000 2,000,000 Replicate 3 Pool 6, 264,000 2,000,000 431,000 2,000,000 Replicate 4 Table 3-2. The number of GFP-positive and GFP-negative cells collected for each HDR sorting replicate. Variant pools were treated with control siRNA or siRNA targeting endogenous BRCA1. Pool 4 = amino acids 1769-1863, pool 5 = amino acids 1673-1768, pool 6 = amino acids 1577-1672.

A sequence-function map covering amino acids 1577 to 1768 was generated to examine variant function in HDR (Figure 3-2). The structure-function map for amino acids 1769 to1863 is pending. Variants were visualized in shades of red based on how many replicates in which they were found to be depleted from the GFP-positive population – variants depleted in no replicates were white while variants depleted in three or four replicates were colored in dark red or maroon. Wild type amino acids were marked with a black dot, and variants with no data were labeled in gray. As in previous multiplex HDR assay work focusing on the BRCA1 amino-terminus (Starita et al., 2018), variants depleted in the GFP-positive population in three or four replicates were considered non- functional. Over 2000 variants were identified from the sequencing of HDR-sorted library pools (Figure 3-2A, Table 3-3). However, many variants were not counted frequently enough in the control population to provide statistical confidence in the results. After read-count filtering for variants that passed statistical confidence, over 500 variants were classified (Figure 3-2B). The list of filtered variants is located in Appendix

B. Non-functional variants, with the exception of truncation variants, were located in the

BRCT domain. Surprisingly, several truncation variants were categorized as non- functional via the assay. A majority of variants were not depleted in any replicates and therefore considered functional (Table 3-3).

Figure 3-2. Sequence-function map of variants in BRCA1 amino acids 1577-1768. (A) The unfiltered sequence-function map of the effect of amino acid substitutions in BRCA1 amino acids 1577-1768. The depletion score for each variant is the number experimental replicates in which it was depleted. Depletion scores are indicated by color and range from never depleted and likely functional (white) to likely non-functional (dark red). Variants depleted in 3 or 4 replicates were considered non-functional. Black dots indicate wild-type residues, and gray boxes indicate missing data. Each position in BRCA1 is arranged along the x-axis, with the position of the BRCT domain indicated on top. All potential amino acid substitutions, as well as nonsense mutations (indicated with an asterisk on the bottom), are labeled on the y-axis. (B) The sequence-function map in (A) after read-count threshold filtering for statistical confidence.

Score # Variants Unfiltered 0 1116 1 188 2 86 3 67 4 573

Filtered 0 434 1 21 2 14 3 18 4 23 Table 3-3. The number of variants identified from filter and unfiltered BRCA1 amino acid 1577-1768 sequence-function data and their depletion scores.

The least tolerated amino acid changes were to proline and arginine, which resulted in seven non-functional variants each, and to tyrosine, which resulted in four non-functional variants. Five residues – T1691, A1708, V1714, F1734 and A1752 – had two or more non-functional amino acid changes, indicating that substitutions at these regions may not be tolerated (Figure 3-3A). While all five residues are visible from the protein surface, the V1714 residue is the least accessible (Figure 3-3B).

Figure 3-3. Structure of the BRCA1 BRCT domain with labeled potentially intolerant regions. (A) Amino acid residues that had multiple non-functional missense variants identified in the multiplex HDR assay (Figure 3-2) were mapped to the BRCA1 BRCT domain and labeled in red. The non-functional amino acid changes to each region are listed. (B) A spacefill model of the BRCA1 BRCT domain with the same amino acid residues labeled.

Comparison to Other Functional Assays and Known Clinical Data

Nine of the variants identified in the multiplex HDR assay have also been examined in the singleton HDR assay (Anantha et al., 2017; Lu et al., 2015; Petitalot et al., 2019)

(Figure 3-4A). Results were consistent for all nine variants.

As BRCA1 is a widely researched protein and many variants are associated with cancer, several functional assays have been developed in order to predict pathogenicity. In the transcription activation (TA) assay, the BRCA1 C-terminus is fused to the heterologous

DNA binding domain in two-hybrid assay systems and transcription activation is examined (Chapman and Verma, 1996; Monteiro et al., 1997; Vallon-Christersson et al.,

2001). If transcription occurs, the variant is considered functional. 27 variants were examined in both the TA assay and multiplex HDR assay (Fernandes et al., 2019) (Figure

3-4B). 15 variants were functional and five variants were non-functional in both assays.

However, seven variants (S1651P, M1652K, D1692Y, R1699L, G1738V, P1749L and

G1763V) that were non-functional in the TA assay were considered functional in the multiplex HDR assay.

Continued

Figure 3-4. Comparison of multiplex HDR assay results to clinical data and other 85

Figure 3-4 Continued functional assays. (A) A comparison of the HDR activity of variants examined in singleton assays versus the multiplex assay. (B) A comparison of the functional categorization of variants tested in both the TA (Fernandes et al., 2019) and multiplex HDR assays. (C) A comparison of the functional scores of variants tested in both the SGE (Findlay et al., 2018) and multiplex HDR assays. Variants with an SGE score > -0.742 were classified as functional (labeled blue) and variants with an SGE score < -1.32 were classified as loss of function (labeled green). Variants were classified as intermediate (labeled orange) if the score fell in-between those two values. (D) A comparison of multiplex HDR classifications to known clinical significance as recorded in ClinVar.

Another multiplexed functional assay that has been used to examine BRCA1 variants is the saturation genome editing (SGE) assay (Findlay et al., 2018). In the SGE assay, the

BRCA1 gene in HAP1 cells was edited in situ to generate all potential SNVs in the RING and BRCT domains, and variants that inhibited BRCA1 cell growth-supporting function were selected against. The cell population after several passages was compared to the input population to determine which variants were depleted following selection. Of the

116 variants tested in both the multiplex HDR and SGE assays, 74 variants were functional and 13 variants were non-functional in both assays (Figure 3-4C).

As with the TA assay, several variants considered non-functional the SGE assay were functional in the multiplex HDR assay. All eight variants categorized as intermediate in the SGE assay (S1631T, T1677I, E1683G, D1692Y, L1705R, G1743A, C1767R,

C1768G) were considered functional in the HDR assay. Twenty-one variants were considered non-functional in the SGE assay and functional in the multiplex HDR assay.

Seven variants were non-functional or had intermediate function in both the TA and SGE assays but were considered functional in the multiplex HDR assay (Table 3-4).

Variants HDR TA SGE Replicates Function Function Depleted K1648Q 0 FUNC FUNC R1649G 0 FUNC FUNC M1650R 0 FUNC FUNC S1651P 0 LOF LOF M1652K 1 LOF LOF D1692Y 0 LOF INT R1699L 0 LOF LOF G1706A 0 FUNC FUNC V1713A 3 LOF LOF S1715R 4 LOF LOF R1737G 0 FUNC FUNC G1738V 0 LOF LOF V1740L 0 FUNC FUNC R1741L 0 FUNC FUNC Q1747L 0 FUNC FUNC G1748A 0 FUNC FUNC P1749L 2 LOF LOF R1751P 4 LOF LOF R1751I 0 FUNC FUNC A1752P 4 LOF LOF K1759M 0 FUNC FUNC G1763V 1 LOF LOF L1764P 4 LOF LOF Table 3-4. Functional results for variants tested in the TA, SGE and multiplex HDR assays. Variants with conflicting function scores between the three assays are bolded.

Nearly fifty of the variants classified in the multiplex HDR assay are also located in the

ClinVar database (Figure 3-4D). Of those 43 variants, 30 variants are classified as VUS by ClinVar, with two of those (G1706R and V1713A) being non-functional in HDR. The known benign variant G1706A was classified as functional by the multiplex HDR assay.

Of the eleven variants categorized as pathogenic or likely pathogenic by ClinVar, eight of them were non-functional in the HDR assay. The splicing variant D1692Y (Ahlborn et al., 2015) and the nonsense variants E1683* and S1587* are non-functional according to

ClinVar, but were categorized as functional in the multiplex HDR assay. However, despite some inconsistencies in the sensitivity of the multiplex HDR assay classification compared to TA, SGE and clinical data, a majority of variant assessments are the same.

All of the variants labeled as non-functional in the multiplex HDR assay were also non- functional in the SGE and/or TA assays.

Discussion

In conclusion: 1) more than 500 BRCA1 variants between amino acids 1577 and 1768 were characterized in the multiplex HDR assay, many of which have no known clinical significance. 2) Non-functional missense variants were located in the BRCT domain. 3)

Most multiplex HDR results correlated with results from singleton HDR assays. 4)

Results from the TA and SGE assays correlated for functional variants but not non- functional variants. 5) Classifications of multiplex HDR variants found in ClinVar correlated with clinical predisposition with the exception of splicing and some truncation variants. 89

Over 2000 variants were identified on the unfiltered multiplex HDR sequence-function map of amino acids 1577 to 1768, covering ~50% of potential variants in the region.

However, after read-count filtering for statistical confidence, only about 500 variants

(~13% coverage) remained. In addition to many variants being filtered out of the sequence-function map, fewer variants were present on the unfiltered map than on the variant diversity maps for the unintegrated plasmid pools, which had ~86% coverage.

Previous multiplex HDR work focusing on BRCA1 amino acids 2-192 had 28% sequence-function variant coverage (Starita et al., 2018), and we would ideally like to improve on it. The presence of many more variants on the unfiltered sequence-function map indicates that coverage could be increased on the filtered map.

There are several potential factors that could address the low coverage observed. A minimum of 100,000 cells were integrated for each library, but the coverage differences seen between the variant diversity and sequence-function maps may indicate more variants need to be integrated for better coverage. Sequencing additional barcodes may also increase the confidence in the variants that were filtered out. We also hope to classify the entire BRCT domain by adding results from the last 96 amino acids of

BRCA1 to the sequence-function map.

All of the non-functional variants, aside from truncation variants, were located within the bounds of the BRCT domain. This correlates with known information on the tumor suppressor properties of the BRCT domain (Shakya et al., 2011). Amino acid substitutions to proline and arginine caused the greatest number of non-functional mutations, with seven non-functional variants each. Prolines substitutions are known to

90 destabilize alpha helices (Richardson and Richardson, 1988) and five of the seven non- functional proline variants (A1708P, W1718P, F1734P, R1751P, A1752P) were located in alpha helices. Arginine mutations are likely to disrupt protein structure due to the large size and charge. In addition, tyrosine variants accounted for four non-functional variants.

Tyrosine may also disrupt the protein structure because it is a large, aromatic amino acid.

Five residues – T1691, A1708, V1714, F1734 and A1752 – had two or three non- functional variants. Although there was not extensive coverage on the filtered sequence- function map, it is possible that these residues are more intolerant to amino acid substitutions. The A1708 residue is present in a protein pocket where several other non- functional variants have been identified, suggesting that this pocket is a protein interaction region (Lu et al., 2015). Variant A1708E is a known pathogenic variant, and

A1708V has diminished function in the singleton HDR assay (Lu et al., 2015). Three of these residues are located in alpha helices (A1708, F1734 and A1752) and one is located in a beta sheet (V1714), which could render these residues more sensitive to amino acid changes that affect structure. The V1714 residue is also closer to the interior of the protein, where mutations may affect protein structure more. None of these residues are located in the known phosphoprotein interaction region (Williams et al., 2004).

Results for variants that had been examined in both the singleton and multiplex HDR assays correlated. Variants that were found to be functional in the TA and SGE assays were functional in the multiplex HDR assay as well. However, a subset of variants that were non-functional in the TA assay, SGE assay or both were considered functional in the multiplex HDR assay. While it is possible that different amino acid residues are

91 necessary for the functions tested in the TA and SGE assays, this could also be due to complications with the multiplex HDR assay. In the HDR assay, a maximum of 10-20% of cells are converted to GFP-positive (Ransburgh et al., 2010). The low percentage of

GFP-positive cells could affect the results of the multiplex HDR assay. In the HDR assays performed for variants in amino acids 1577-1768, the percentage of GFP-positive cells was ~4-7% in cells depleted of endogenous BRCA1. In addition, cells depleted of

BRCA1 still had a baseline GFP expression of ~1-2%. The dynamic range of these experiments is likely lower because of the low GFP expression, which could affect results. The low dynamic range could make it more difficult to determine whether variants were significantly depleted in the GFP-positive population if the number of GFP- positive wild type cells was also low. Subsequently, non-functional variants may be more likely to be scored as not depleted in replicates. This is supported by the fact that several truncation variants, including those that are known to be pathogenic or which eliminate the BRCT domain entirely, were scored functional in the multiplex assay.

In previous work focusing on the BRCA1 amino-terminus, the sequence-function map covering amino acids 97-192 had much better coverage of variants than the portion covering amino acids 1-96. The amino acid 97-192 library also had a higher GFP-positive percentage in cells depleted of endogenous BRCA1 than the amino acid 1-96 library:

~10% GFP-positive versus ~7% GFP-positive. Additional HDR sorting replicates with higher overall GFP percentages could help increase the dynamic range of variants in the

BRCT domain and therefore better classify them. However, it was hypothesized that the pool encompassing variants in amino acids 1-96 had a lower GFP-positive percentage

92 because it covered the RING domain (Starita et al., 2018) where more variants are likely to be non-functional than in a pool encompassing a region that is dispensable to HDR.

This will also be a factor to consider for libraries covering amino acids in the BRCT domain.

Observations similar to the TA and SGE assays were seen with clinical significance data from ClinVar. Three known pathogenic variants – E1587*, E1683* and D1692Y – scored as functional in the multiplex HDR assay. D1692 is a splicing variant (Ahlborn et al.,

2015), and the HDR assay does not account for splicing variants because it utilizes variant expression plasmids containing mRNA coding sequence. Both truncation variants, however, should be non-functional. Variants that were depleted in even one or two HDR sorting replicates were scored only as non-functional or pathogenic in the TA or SGE functional assays or on ClinVar. This differed from the amino-terminus multiplex

HDR results, where variants that were pathogenic or scored as non-functional in other assays were consistently depleted in three or four replicates. This could be due to the low dynamic range of the carboxy-terminus HDR replicates, which was discussed earlier.

Variants that were functional in other assays were not depleted in any HDR sorting replicates.

There is no one true functional assay – different BRCA1 functional assays can report contrary results regarding variant function (Lee et al., 2010b), as the regions and residues regulating protein function can vary. Multiple functional assays should be performed on a region and calibrated with clinical data in order to accurately ascertain its function and gain confidence in the potential clinical risk of a variant. The HeLaDR variant cell

93 libraries established in this chapter could also be treated with DNA damage agents that lead to the accumulation of double-strand breaks such as cisplatin (Adamovich et al.,

2019) or PARP inhibitors (Farmer et al., 2005) in order to observe which variants are not functional in homologous recombination. Similarly, the fitness of BRCA1 variants integrated into a BRCA1-null cell line could be examined after treatment with the same

DNA damage agents. Examining the fitness of these variant libraries in non-HeLa derived cell lines could indicate whether variant function changes depending on cellular properties. For example, HeLa cells do not express the DNA damage-associated protein

Wwox (Qu et al., 2013), which binds to BRCA1 and regulates DNA repair (Schrock et al., 2017). Wwox has also been shown to interact with ATM (Abu-Odeh et al., 2014), which phosphorylates serines in and around the BRCA1 coiled coil domain following

DNA damage and mediates G2/M checkpoint activation (Cortez et al., 1999; Xu et al.,

2002b). Variants that affect this interaction are functional in HDR using the HeLaDR reporter cell line (unpublished data), but may not be in a Wwox-proficient cell line. Such results would increase the confidence in whether variants are benign or pathogenic in addition to data from the multiplex HDR assay.

Comprehensive characterization of the BRCA1 carboxy-terminus and BRCT domain in

HDR provides more information regarding the clinical risk of both known VUS and novel variants. Multiplex HDR analysis also elucidates what protein regions are unlikely to tolerate mutations and therefore critical for HDR. Hopefully, doctors will be able to use this information to advise people of their cancer predisposition, which would increase

94 patient certainty and allow for more vigilant screening or preventative measures for at- risk individuals.

Materials and Methods

Site-Saturation Mutagenesis Libraries and Barcoding: pcDNA5-FRT/TO-HA-BRCA1 was digested with NheI-HF and SbfI-HF (New England Biosciences), and the 1700 bp fragment containing amino acids 1293-1863 was cloned into the pUC19 plasmid. For experiments mutagenizing amino acids 1557-1768, the pcDNA5-FRT/TO-HA-BRCA1 plasmid was modified to include regions of homology for Illumina sequencing. Three site-saturation mutagenesis libraries – pool 4 (amino acids 1769-1863), pool 5 (amino acids 1673-1768) and pool 6 (amino acids 1577-1672) – were constructed using inverse

PCR mutagenesis (Jain and Varadarajan, 2014). 30 base mutagenic primers were designed for each codon, with hand-mixed NNN (pools 5 and 6) and NNK (pool 4) (N =

ACTG, K = GT) bases at the 5′ end of the sense oligonucleotide. Ligations were pooled together, phosphorylated and ligated back to themselves as previously described (Jain and

Varadarajan, 2014). Cells were transformed into MAX Efficiency DH5α competent cells

(Thermo Fisher), and 10 μl was plated on an agar plate with ampicillin (0.1 mg/mL) while the remaining 990 μl were added to 250 ml LB media with ampicillin (0.1 mg/mL).

Plates were counted after 24 hr incubation and libraries with at least 10,000 variants

(approximately five-fold library coverage) were purified using the Qiagen Maxiprep kit.

Pool 4 had approximately 86,000 variants, pool 5 had 85,000 variants, and pool 6 had

104,200 variants. PCR was used to isolate the mutagenized BRCA1 fragment and add a 95

16-base degenerate barcode to variants (primers NheI-Fwd-Long and BRCA1-AscI-

Barcode-XhoI-Gib-Rev for pool 4, BRCA1-NheI-Fwd-Long and BRCA1-SbfI-Barcode-

Rev for pools 5 and 6). PCR primer sequences are located in Appendix C. The BRCA1 fragment was ligated back into pcDNA5-FRT/TO-HA-BRCA1 using Gibson assembly

(Smith et al., 2009) and transformed into MAX Efficiency DH5α competent cells. Pool 4 had 46,300 variants, pool 5 had 10,400 variants, and pool 6 had 11,900 variants.

Assigning Barcodes to Variants: Barcoded fragments were isolated from pools via digestion with restriction enzymes SbfI-HF and NheI-HF. Fragments were purified using the Qiagen Gel Extraction kit and AmpurePB (Pacific Biosciences) beads according to manufacturer instructions. To generate SMRTBell templates, PacBio adapters were added to the purified fragment using the SMRTBell Template Prep Kit 1.0 (Pacific

Biosciences) and eluted in 10 μl of 10 mM Tris (pH 8).

The BRCA1 pool 4 library was sequenced on four SMRT cells on a Pacific Biosciences

RS II sequencer. Pool 5 and 6 BRCA1 libraries were sequenced on one sequel cell each on the Pacific Biosciences Sequel sequencer. Base call files were converted to the bam format from the bax format using bax2bam (version 0.0.2), and bam files for each library from separate lanes were concatenated. The Circular Consensus Sequencing (CCS) algorithm (version 2.0.0) was used to determine consensus sequences for each sequenced molecule with default conditions (ccs and bax2bam can be found in the PacBio GitHub repository). Each consensus sequence was aligned to the BRCA1 wild-type reference sequence with the Burrows Wheeler Aligner (Li and Durbin, 2010). Barcodes and insert sequences were extracted with custom scripts that parsed CIGAR and MD strings. If

96 barcodes were sequenced more than once and barcode-variant sequences differed, the barcode was assigned to the variant that represented more than 50% of the sequences.

Barcodes without a majority variant sequence were assigned the variant with the highest average quality score, as determined by the CCS algorithm. Scripts for barcode-variant extraction and barcode unification can be found at the Shendure lab’s GitHub repository.

Pool 4 had about 18,000 barcodes assigned to variants with zero or one amino acid substitution, encoding 1830 unique variants. Pool 5 had about 12,000 barcodes assigned to variants, encoding 1712 variants. Pool 6 had about 11,000 barcodes assigned to variants, encoding 1769 variants. For each library, a barcode-variant map file was created to contain the nucleotide sequence and its barcode.

Integration of Libraries into HeLaDR-FRT Cells: Ten 10 cm tissue-culture plates of

HeLa-DR-FRT cells at 90% confluency were transfected with 100 μg of the pcDNA5/FRT BRCA1 variant library and 200 μg of pOG44 to express the flippase enzyme. Transfections were done with 300 μl Lipofectamine (Thermo Fisher) and 10 ml of Opti-MEM according to manufacturer’s instructions. Transfected cells were kept in

DMEM media containing 1% GlutaMAX, 1% Sodium Pyruvate and 10% Fetal Bovine

Serum. Cells were incubated at 37˚C for 24 hr, moved to 30˚C for 24 hr, and then back to

37˚C. 72 hr after the transfection, cells were transferred to 20 15 cm plates containing selection media (50% fresh DMEM, 50% filter-sterilized conditioned media, 10% fetal bovine serum, 1% GlutaMax, 1% Sodium Pyruvate and 550 μg/mL Hygromycin-B).

After 24 hrs, the hygromycin-resistant cells were washed with sterile phosphate-buffered saline (PBS), and selection media was replaced. After approximately 1 week, cell

97 colonies were counted. Pool 4 had 102,000 colonies, pool 5 had 108,000 colonies, and pool 6 had 113,000 colonies. After another week, cells were resuspended in 20 ml of culture media and mixed thoroughly. 15 ml of resuspended cells were frozen into 1 mL aliquots. The remaining cells were plated on 15 cm plates and passaged for another week before performing HDR reporter experiments to ensure loss of unintegrated BRCA1 expression plasmid.

HDR Reporter Assays: HDR assays for each pool were performed in quadruplicate.

Cells integrated with variant libraries were seeded at 70% confluency (65 μl from a 90% confluent plate) in 48 wells across two 24-well plates. The next day, each well was transfected with 30 pmol of siRNA, 1.5 μl of Oligofectamine (Thermo Fisher), and 31 μl of Opti-MEM per manufacturer’s recommendations. For each experiment, two wells were treated with siRNA targeting the BRCA1 coding region, 16 wells were treated with control siRNA, and 30 wells were treated with siRNA targeting the BRCA1 3′ UTR, which integrated variants were resistant to. Cells were transferred to four 6-well tissue culture plates 24 hr after transfection. 48 hr after the Oligofectamine transfection, wells were transfected with 5 pmol siRNA, 3 μl of Lipofectamine 2000 (Thermo Fisher), 3 μg pCBASceI and 250 μl Opti-MEM as per manufacturer’s instructions. After 24 hr, two wells for each siRNA condition were analyzed for GFP expression by observing 10,000 cells using the FACSCalibur machine at the OSUCCC Analytical Cytometry Shared

Resource. Experiments proceeded if cells treated with control siRNA and BRCA1 3′UTR siRNA were between 7-9% and 4-7% GFP respectively, and if cells treated with BRCA1 coding sequence siRNA were between 1-2% GFP. GFP percentages are provided in

Table 3-1. 72 hr after Lipofectamine transfection, cells were pooled according to treatment and sorted by FACS using the Aria III in the OSUCCC Analytical Cytometry

Shared Resource. Cells were resuspended and pooled in filter-sterilized sorting buffer containing 1X PBS (Ca2+- and Mg2+-free), 5 mM EDTA, 25 mM HEPES (pH 7.0), and

1% heat-inactivated fetal bovine serum dialyzed against Ca2+ and Mg2+ PBS. A minimum of 264,000 GFP-positive cells and a maximum of two million GFP- cells were collected per pool. The number of cells collected in each experiment is provided in Table 3-2.

Genomic DNA (gDNA) was extracted from GFP-positive and GFP-negative cells with a

DNeasy Blood & Tissue Kit (QIAGEN) according to manufacturer’s instructions. DNA was eluted into 200 μl of Buffer EB (Qiagen).

Barcode Amplification and Sequencing: Barcodes were amplified from gDNA over 16 reactions, each containing 250 ng of gDNA. PCR reactions were done using Kapa2G

Robust Polymerase for 22-23 cycles. For pool 4, the primers Pool4-Nest1-Fwd and FRT-

LacZ-Rev were used and for pools 5 and 6 the primers CTerm56-Nest1-Fwd and FRT-

LacZ-Rev were used. The resultant ~750 bp fragment was purified with 0.8X AmpurePB beads and eluted in 40 μl 10 mM Tris (pH 8). 125 ng of PCR product was re-amplified with primers containing sample indexes and Illumina cluster-generating sequences using

Kapa2G Robust Polymerase. The resultant 150 bp (pool 4) or 170 bp (pools 5 and 6) amplicon was purified using double Ampure purification (0.6X and 0.8X). The samples were multiplexed and barcodes and sample indexes (Seq-i70x and Seq-i50x primers) sequenced on the HiSeq 4000 at the Nationwide Children’s Hospital Genomic Services

Laboratory.

Variant Scoring, Classifications, and Depletion Score: FASTQ files containing sample barcodes and the barcode map for each library was input into the software package

Enrich2 (Rubin et al., 2017). Enrich2 counted barcodes, associated each barcode with a nucleotide variant, and translated unique barcode and amino acid variants. Barcodes assigned to variants containing insertions, deletions and multiple amino acid substitutions were removed from analysis. Counts for each variant were converted to frequencies by dividing variant counts by the total number of variant counts for each sample. The ratio of the frequency of each variant in the GFP-positive population versus its frequency in the GFP-negative population was calculated and normalized to the GFP+/GFP- ratio for wild type BRCA1.

To construct the functional/nonfunctional classifier for variants, the standard deviation of the score decays according to read count was determined in a way that could be modeled by a log10-log10 curve (Starita et al., 2018). The decay of the standard deviation of scores from the control siRNA experiment was then modeled. The model was applied to the standard deviation of scores from the BRCA1 siRNA experiment and used to calculate a p value and a false-discovery-rate-adjusted q value for each variant to determine whether it was similar to or significantly different from the control siRNA experiment (q < 0.055). It was determined where the false positives of the control experiments occurred along read count continuum to assign a read-count threshold for the

BRCA1 siRNA condition. Variants with a read count below the threshold were removed from further analyses. Depletion scores represent the number of replicates in which a variant was found to be depleted from the BRCA1 siRNA GFP-positive population. Only

100 variants that were present above the read-count threshold in at least three replicates were included. Analyses were performed in R Studio.

Protein Modeling: Regions containing multiple non-functional variants were mapped to protein structures using PDB files in Jmol. The PDB ID for the BRCA1 BRCT domain structure used is 1T15 (Clapperton et al., 2004).

101

Chapter 4. Discussion

102

In this dissertation, we have examined VUS of two cancer susceptibility genes associated with HBOC, BRCA1 and BARD1, using a functional assay for HDR. While BRCA1 has been extensively examined in the HDR assay, and loss of function is associated with pathogenicity, BARD1 has not been thoroughly studied previously.

BARD1

From whole exome sequencing analysis to identify potential cancer-causing variants, 76

BARD1 variants from across all three functional domains of the protein were identified and tested in the singleton HDR assay. Two variants that have been identified as clinically benign were functional in the HDR assay, while three variants classified as clinically pathogenic were non-functional. This supports the notion that the HDR assay can be used to predict the pathogenicity of BARD1 variants. Five non-functional variants were identified in the BRCT domain, which mediates the recruitment and retaining of the

BRCA1-BARD1 heterodimer at sites of DNA damage (Li and Yu, 2013; Miyoshi et al.,

2015). However, most of the non-functional variants in the BRCT domain were not located near DNA damage-associated residues. Four non-functional variants were identified in the ankyrin repeat domain, which has no known DNA repair function. Non- functional variants were also more sensitive to DNA damage mediated by cisplatin and

X-ray irradiation, with residual HDR activity not protecting against damage.

103

BARD1 Future Directions

While these data provide more information on how BARD1 functional domain activity mediates HDR, questions remain unanswered. To better examine the function of the ankyrin repeat and BRCT domains, it would be useful to conduct multiplex assays in future experiments. As the BRCA1-BARD1 heterodimer is necessary for DNA repair

(Shakya et al., 2011), the same multiplex assays used to examine HDR in BRCA1 can be used for BARD1. Non-functional variants are selected against in the SGE assay by increasing the HDR rate in cells (Findlay et al., 2018), indicating it may be possible to identify non-functional BARD1 variants in this assay as well. Multiplex BARD1 assays could allow for thorough functional domain analysis, as only testing several variants in each region does not provide a complete range of information. This may identify regions intolerant to mutations within the domains and help better understand how HDR function is mediated by BARD1.

It would also be interesting to examine the mechanism behind why these BARD1 variants were non-functional in HDR. The mutated residue in several of these variants is on the interior of the protein, where proper folding and structure are more likely to be disrupted. The variants that were further examined in chapter 2 – A460T, P707S, G753D and V767fs – were all present on the surface of the protein, where variant changes could affect protein binding. The A460T variant may be more susceptible to phosphorylation due to the threonine mutation, which might affect function. The variants examined were not located at the known binding sites of BARD1 protein interactors, which could indicate there are unknown binding sites for these proteins or novel protein interactions. 104

Interacting proteins could be identified by screening non-functional variants against an array of proteins using a method such as the mammalian two-hybrid system (Fiebitz et al., 2008).

BRCA1

With the increased availability of high-throughput sequencing, many multiplex assays are being developed to examine protein function comprehensively. We have previously utilized a multiplex HDR approach to characterize all potential missense and truncation variants the BRCA1 amino terminus and RING domain (Starita et al., 2018). This dissertation expands on that work with the examination of variants in the carboxy- terminus and BRCT domain. High-coverage (>85%) variant libraries covering BRCA1 amino acids 1577-1863 were generated and integrated into the HeLaDR-FRT reporter cell line. More than 500 variants from amino acids 1577-1768, many of which have no known clinical significance, were classified from an integrated library of over 2000 variants. Over one hundred variants examined in the multiplex HDR assay had also been tested in the BRCA1 TA assay (Fernandes et al., 2019), multiplex SGE assay (Findlay et al., 2018) or both. Multiplex HDR results correlated for a majority of variants also tested in the SGE and/or TA assays and with known clinical significance data. However, a subset of variants that were functional in the multiplex HDR assay were classified as clinically pathogenic or non-functional in the TA or SGE assays. Despite this, variants identified as non-functional in the multiplex HDR assay were consistently non-functional

105 in the TA and SGE assays, suggesting that other variants categorized as non-functional in the multiplex HDR assay were also pathogenic.

BRCA1 Future Directions

While over 500 variants in BRCA1 1577-1768 and 1000 variants in BRCA1 2-192

(Starita et al., 2018) have been classified in HDR, this work has shown that examining the HDR activity of variants through multiplex methods can be improved upon. The sequence-function map covering BRCA1 2-192 had ~28% coverage, and the map covering BRCA1 1577-1768 had ~13% coverage. The multiplex SGE assay was able to examine the function of ~96% of SNVs in the region of interest (Findlay et al., 2018).

This indicates that there are improvements to be made to multiplex examination of HDR function. One of the primary issues with the HDR assay is GFP expression in reporter cells, which is maximally between 10 and 20% (Pierce et al., 2001; Ransburgh et al.,

2010). While this is acceptable in the singleton assay because results are normalized to wild type and compared to endogenous expression, there appear to be limits when multiplexing. Pools covering regions that mediate HDR, like the BRCT and RING domains, are more likely to have more defective variants, which would lower the overall

GFP expression of the library. Fewer variants were classified in the variant library containing the RING domain than in the library covering a region inessential to HDR, and this could also be a problem in the BRCT domain. Integrated carboxy-terminus variant libraries had even lower percentages overall than the amino-terminus RING library (4-7% vs 7%), which results in a lower dynamic range and may cause the false 106 negative classifications observed. The low dynamic range of the carboxy-terminus likely resulted in low numbers of wild type BRCA1 in the GFP-positive population, so it would be difficult for the HDR activity of non-functional variants to be significantly different from that of functional variants. This would also explain why none of the intermediate variants in the multiplex SGE assay were depleted in the multiplex HDR assay, because it would have been even more difficult for those variants to be significantly depleted. We also observed that it was difficult to obtain at least 500,000 GFP-positive HeLa cells in

FACS analysis, and it would be ideal to obtain more cells to sequence more variant barcodes and increase result confidence.

The multiplex HDR results from the BRCA1 carboxy-terminus were classified using the same rubric as those from the amino terminus – variants depleted in three or four replicates were considered non-functional. This resulted in a very high margin of error when results from the multiplex HDR assay were compared to results from the SGE and

TA assays. For example, using these standards, 21 out of 116 variants (18%) tested in both the SGE and HDR multiplex assays were non-functional in the SGE assay and functional in the HDR assay. While this is a small percentage overall, we want these results to be used to predict cancer risk for women in the clinic. A nearly 20% rate of uncertainty in whether a predicted benign variant is actually pathogenic is very high and not useful for this purpose. However, it was observed that in comparisons with the TA and SGE assays, replicates were only depleted in the multiplex HDR assay for variants that were non-functional in the other assays and never for variants that were considered functional by these other assays (Figure 3-4). This is probably a factor of the lowered

107 dynamic range – it is much more difficult for a variant to be significantly depleted compared to wild type, so non-functional variants may be depleted in very few replicates or none at all. By loosening the replicate cutoff to categorize variants as non-functional if they are depleted in any HDR sorting replicates, more pathogenic variants are likely to be correctly classified as non-functional. The 21 out of 116 variants considered non- functional in the SGE assay but functional in the multiplex HDR assay would decrease to

10 variants (~9%), which is better than the previous margin of error. Relaxing the number of depleted replicates needed to classify a variant as non-functional is unlikely to result in functional variants being labeled as non-functional. As discussed earlier, assay comparisons showed that variants scored as functional in the SGE or TA assays (78 total) never had any replicates depleted in the multiplex HDR assay. Splicing variants would be the exception to these guidelines, as the multiplex HDR assay variant libraries consist of expression plasmids containing mRNA coding sequence and splicing variants are not accurately assessed.

Based on the limitations observed in the multiplex HDR assay, we have considered how to improve the examination of HDR variant function. Considering the low GFP expression observed in cells, it may be better to develop an assay that does not rely on

GFP sorting. DNA damage agents such as cisplatin (Sorenson and Eastman, 1988) and

PARP inhibitors (Farmer et al., 2005) result in the accumulation of double-strand breaks in the cell, which can only be fixed using HDR. Cells expressing variants that are not functional in HDR would therefore be rendered non-viable. Adapting this idea to a multiplex assay could be a more effective method for examining variant HDR activity.

108

These experiments could easily be performed in the HeLaDR-FRT variant libraries that have already been created, and results can be compared to those that have already been obtained from the multiplex HDR assay. The overall experimental workflow would be fairly similar as well – libraries would be transfected with control siRNA, siRNA to endogenous BRCA1, or siRNA to the BRCA1 coding sequence in two rounds 48 hours apart. Two days after the second transfection, cells would be treated with a DNA damage agent at a concentration that would allow functional variants to survive (see Chapter 2).

Cells from the control population and the cells depleted of endogenous BRCA1 would be collected when the latter population reaches confluency. Cells fully depleted of BRCA1 would be used to estimate how many non-functional variants survived and determine the amount of experimental background noise. Afterwards, variant barcodes could be sequenced to identify which variants were depleted in the population of cells expressing only integrated variants. Those variants would be classified as non-functional in HDR and therefore potentially pathogenic. Four replicates could be performed for each library and variants classified based on how many replicates they were depleted in.

The work discussed in Chapter 2 included testing the DNA damage sensitivity of BARD1 variants non-functional in HDR using X-ray irradiation and cisplatin (Adamovich 2019).

In the clonogenic assays performed, HeLaDR-FRT cells depleted of BRCA1 had a tenfold drop in colony formation and survival when treated with 7.5 μM of cisplatin for two hours as compared to wild type cells (Figure 2-8A). Although not all of the non- functional cells may die, the number that remains may result in a reasonably high signal to noise ratio. Our work has also shown that cells expressing endogenous BARD1 have

109 similar cisplatin and X-ray sensitivities with or without non-functional BARD1 variants integrated (Figures 2-7, 2-8).

This multiplex proliferation assay we have described may be more straightforward than the current multiplex HDR assay we have utilized. It would not require FACS sorting, and fewer samples in each replicate would need to be collected and sequenced. The multiplex HDR assay requires four samples to be collected: control GFP-positive, control

GFP-negative, endogenous BRCA1-depleted GFP-positive, and endogenous BRCA1- depleted GFP-negative. The multiplex proliferation assay would only require two samples to be collected: control treated and endogenous BRCA1-depleted treated. As there are millions of cells in a confluent 10 cm dish, many more variants and replicates could be amassed in a multiplex proliferation assay than the multiplex HDR assay, where only about 500,000 GFP-positive cells were obtained in many HDR sorts. Additionally, as fewer samples are being pooled together for sequencing, more of each sample, and therefore more variant barcodes, can be added to the multiplexed sample.

The proliferation assay could be adapted to other cell lines if an FRT site is present for integration of the variant library. The phenotypes of different cell lines could affect repair function. As previously discussed in Chapter 3, cell lines that express the DNA damage- associated protein Wwox, unlike the HeLa cell line, could worth testing. Wwox interacts with ATM (Abu-Odeh et al., 2014), which phosphorylates BRCA1 after DNA damage

(Cortez et al., 1999), and while phosphorylation variants are HDR-functional in HeLa cells they may not be in a Wwox-proficient cell line.

110

Overall Conclusions

Multiplex assays are likely the future of functional testing as high-throughput sequencing becomes easier and cheaper to perform. These assays identify a much larger amount of variants more efficiently than singleton assays, even if regional coverage may not be extensive. With improvements in such areas, they are likely to become even more effective over time. The multiplexing of multiple functional assays would also allow for thorough examination of a protein with multiple functions, such as BRCA1, to accurately determine which variants truly affect pathogenicity. The overall objective of the research performed in this dissertation is to better inform women of their BRCA1 or BARD1- associated cancer risk. The work conducted has identified the HDR function and predicted the pathogenicity of hundreds of BRCA1 and BARD1 variants, many of which had no previously known clinical significance. Hopefully, these results will diminish uncertainty as they were intended to and future work will advance this intent even further.

111

Bibliography

Abbott, D.W., Thompson, M.E., Robinson-Benion, C., Tomlinson, G., Jensen, R.A., and

Holt, J.T. (1999). BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J. Biol. Chem.

274, 18808–18812.

Abu-Odeh, M., Salah, Z., Herbel, C., Hofmann, T.G., and Aqeilan, R.I. (2014). WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response. Proc. Natl. Acad. Sci. 111, E4716-25.

Adamovich, A.I., and Parvin, J.D. (2019). Characterizing BRCA1 Variants Using

Multiplex Functional Assays. Arch. Appl. Med. 1, 1–18.

Adamovich, A.I., Banerjee, T., Wingo, M., Duncan, K., Ning, J., Rodrigues, F.M.,

Huang, K., Lee, C., Chen, F., Ding, L., et al. (2019). Functional Analysis of BARD1

Missense Variants in Homology-Directed Repair and Damage Sensitivity. PLoS Genet.

15, e1008049.

Adzhubei, I.A., Schmidt, S., Peshkin, L., Ramensky, V.E., Gerasimova, A., Bork, P.,

Kondrashov, A.S., and Sunyaev, S.R. (2010). A method and server for predicting damaging missense mutations. Nat. Methods 248–249.

Ahlborn, L.B., Dandanell, M., Steffensen, A.Y., Jønson, L., Nielsen, F.C., and Hansen,

T.V.O. (2015). Splicing analysis of 14 BRCA1 missense variants classifies nine variants 112 as pathogenic. Breast Cancer Res. Treat. 150, 289–298.

Anantha, R.W., Simhadri, S., Foo, T.K., Miao, S., Liu, J., Shen, Z., Ganesan, S., and Xia,

B. (2017). Functional and mutational landscapes of BRCA1 for homology-directed repair and therapy resistance. Elife 6, e21350.

Antoniou, A.C., and Easton, D.F. (2003). Polygenic Inheritance of Breast Cancer:

Implications for Design of Association Studies. Genet. Epidemiol. 25, 190–202.

Au, W.W.Y., and Henderson, B.R. (2005). The BRCA1 RING and BRCT domains cooperate in targeting BRCA1 to ionizing radiation-induced nuclear foci. J. Biol. Chem.

280, 6993–7001.

Bacik, J.-P., Whitehead, T.A., Wrenbeck, E.E., Klesmith, J.R., and Michalczyk, R.

(2017). Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning. Proc. Natl. Acad. Sci. 114, 2265–2270.

Baer, R., and Ludwig, T. (2002). The BRCA1 / BARD1 heterodimer , a tumor suppressor complex with ubiquitin E3 ligase activity. Curr. Opin. Genet. Dev. 12, 86–91.

Bandaru, P., Shah, N.H., Bhattacharyya, M., Barton, J.P., Kondo, Y., Cofsky, J.C., Gee,

C.L., Chakraborty, A.K., Kortemme, T., Ranganathan, R., et al. (2017). Deconstruction of the ras switching cycle through saturation mutagenesis. Elife 6, e27810.

Berardi, P., Cartier, L., Irminger-Finger, I., Jefford, C.E., Feki, A., Krause, K.-H., and

Wu, J.-Y. (2005). BARD1 induces apoptosis by catalysing phosphorylation of p53 by

DNA-damage response kinase. Oncogene 24, 3726–3736.

Billing, D., Leuzzi, G., Nanez, S.A., Lin, C.-S., Szabolcs, M., Zha, S., Baer, R., Jiang,

W., Ciccia, A., Taglialatela, A., et al. (2018). The BRCT Domains of the BRCA1 and

113

BARD1 Tumor Suppressors Differentially Regulate Homology-Directed Repair and

Stalled Fork Protection. Mol. Cell 72, 127-139.e8.

Bitler, B.G., Watson, Z.L., Wheeler, L.J., and Behbakht, K. (2017). PARP inhibitors:

Clinical utility and possibilities of overcoming resistance. Gynecol. Oncol. 695–704.

Bouwman, P., Aly, A., Escandell, J.M., Pieterse, M., Bartkova, J., Van Der Gulden, H.,

Hiddingh, S., Thanasoula, M., Kulkarni, A., Yang, Q., et al. (2010). 53BP1 loss rescues

BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat. Struct. Mol. Biol. 17, 688–695.

Bouwman, P., van der Gulden, H., van der Heijden, I., Drost, R., Klijn, C.N., Prasetyanti,

P., Pieterse, M., Wientjens, E., Seibler, J., Hogervorst, F.B.L., et al. (2013). A high- throughput functional complementation assay for classification of BRCA1 missense variants. Cancer Discov. 3, 1142–1155.

Bowcock, A.M., Baer, R., Ayi, T.-C., Hwang, L.-Y., and Tsan, J.T. (2002). Conservation of function and primary structure in the BRCA1-associated RING domain (BARD1) protein. Oncogene 17, 2143–2148.

De Brakeleer, S., De Grève, J., Loris, R., Janin, N., Lissens, W., Sermijn, E., and

Teugels, E. (2010). Cancer predisposing missense and protein truncating BARD1 mutations in non-BRCA1 or BRCA2 breast cancer families. Hum. Mutat. 31, E1175-85.

Bryant, H.E., Schultz, N., Thomas, H.D., Parker, K.M., Flower, D., Lopez, E., Kyle, S.,

Meuth, M., Curtin, N.J., and Helleday, T. (2005). Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434, 913–917.

Caligo, M.A., Bonatti, F., Guidugli, L., Aretini, P., and Galli, A. (2009). A yeast

114 recombination assay to characterize human BRCA1 missense variants of unknown pathological significance. Hum. Mutat. 30, 123–133.

Cantor, S.B., Bell, D.W., Ganesan, S., Kass, E.M., Drapkin, R., Grossman, S., Wahrer,

D.C.R., Sgroi, D.C., Lane, W.S., Haber, D.A., et al. (2001). BACH1, a novel helicase- like protein, interacts directly with BRCA1 and contributes to its DNA repair function.

Cell 105, 149–160.

Carvalho, M.A., Marsillac, S.M., Karchin, R., Manoukian, S., Grist, S., Swaby, R.F.,

Urmenyi, T.P., Rondinelli, E., Silva, R., Gayol, L., et al. (2007). Determination of cancer risk associated with germ line BRCA1 missense variants by functional analysis. Cancer

Res. 67, 1494–1501.

Chang, S., Biswas, K., Martin, B.K., Stauffer, S., and Sharan, S.K. (2009). Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations. J. Clin. Invest. 119, 3160–3171.

Chapman, M.S., and Verma, I.M. (1996). Transcriptional activation by BRCA1. Nature

678–679.

Chaudhuri, A.R., Callen, E., Ding, X., Gogola, E., Duarte, A.A., Lee, J.E., Wong, N.,

Lafarga, V., Calvo, J.A., Panzarino, N.J., et al. (2016). Replication fork stability confers chemoresistance in BRCA-deficient cells. Nature 535, 382–387.

Chen, Y., Chen, C.F., Riley, D.J., Allred, D.C., Chen, P.L., Von Hoff, D., Osborne, C.K., and Lee, W.H. (1995). Aberrant subcellular localization of BRCA1 in breast cancer.

Science (80-. ). 270, 789–791.

Chen, Y., Farmer, A.A., Chen, C.F., Jones, D.C., Chen, P.L., and Lee, W.H. (1996).

115

BRCA1 is a 220-kDa nuclear phosphoprotein that is expressed and phosphorylated in a cell cycle-dependent manner. Cancer Res. 56, 3168–3172.

Clapperton, J.A., Manke, I.A., Lowery, D.M., Ho, T., Haire, L.F., Yaffe, M.B., and

Smerdon, S.J. (2004). Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer. Nat. Struct. Mol. Biol. 11, 512–

518.

Cortez, D., Wang, Y., Qin, J., and Elledge, S.J. (1999). Requirement of ATM-dependent phosphorylation of Brca1 in the DNA damage response to double-strand breaks. Science

(80-. ). 286, 1162–1166.

Couch, F.J., and Weber, B.L. (1996). Mutations and polymorphisms in the familial early- onset breast cancer (BRCA1) gene. Hum. Mutat. 8, 8–18.

Couch, F.J., Shimelis, H., Hu, C., Hart, S.N., Polley, E.C., Na, J., Hallberg, E., Moore,

R., Thomas, A., Lilyquist, J., et al. (2017). Associations between cancer predisposition testing panel genes and breast cancer. JAMA Oncol. 3, 1190–1196.

Coyne, R.S., McDonald, H.B., Edgemon, K., and Brody, L.C. (2004). Functional characterization of BRCA1 sequence variants using a yeast small colony phenotype assay. Cancer Biol. Ther. 3, 453–457.

Craig, N. (1988). The Mechanism Of Conservative Site-Specific Recombination. Annu.

Rev. Genet. 22, 77–105.

Dechend, R., Hirano, F., Lehmann, K., Heissmeyer, V., Ansicau, S., Wulczyn, F.G.,

Scheidereit, C., and Leutz, A. (1999). The Bcl-3 oncoprotein acts as a bridging factor between NF-κB/Rel and nuclear co-regulators. Oncogene 18, 3316–3323.

116

Do, K., Wilsker, D., Ji, J., Zlott, J., Freshwater, T., Kinders, R.J., Collins, J., Chen, A.P.,

Doroshow, J.H., and Kummar, S. (2015). Phase I study of single-agent AZD1775 (MK-

1775), a wee1 kinase inhibitor, in patients with refractory solid tumors. J. Clin. Oncol.

33, 3409–3415.

Eccles, E.B., Mitchell, G., Monteiro, A.N.A., Schmutzler, R., Couch, F.J., Spurdle, A.B.,

Gómez-García, E.B., Driessen, R., Lindor, N.M., Blok, M.J., et al. (2015). BRCA1 and

BRCA2 genetic testing-pitfalls and recommendations for managing variants of uncertain clinical significance. Ann. Oncol. 26, 2057–2065.

Eyfjord, J.E., Risch, H.A., Evans, C., Manoukian, S., Peto, J., Radice, P., Olsson, H.,

Olah, E., Easton, D.F., Hopper, J.L., et al. (2003). Average Risks of Breast and Ovarian

Cancer Associated with BRCA1 or BRCA2 Mutations Detected in Case Series

Unselected for Family History: A Combined Analysis of 22 Studies. Am. J. Hum. Genet.

72, 1117–1130.

Farmer, H., McCabe, H., Lord, C.J., Tutt, A.H.J., Johnson, D.A., Richardson, T.B.,

Santarosa, M., Dillon, K.J., Hickson, I., Knights, C., et al. (2005). Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434, 917–921.

Fernandes, V.C., Golubeva, V.A., Pietro, G. Di, Shields, C., Amankwah, K.,

Nepomuceno, T.C., de Gregoriis, G., Abreu, R.B.V., Harro, C., Gomes, T.T., et al.

(2019). Impact of amino acid substitutions at secondary structures in the BRCT domains of the tumor suppressor BRCA1: Implications for clinical annotation. J. Biol. Chem. 294,

5980–5992.

Fiebitz, A., Nyarsik, L., Haendler, B., Hu, Y.H., Wagner, F., Thamm, S., Lehrach, H.,

117

Janitz, M., and Vanhecke, D. (2008). High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays. BMC Genomics 9, 68.

Findlay, G.M., Boyle, E.A., Hause, R.J., Klein, J.C., and Shendure, J. (2014). Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123.

Findlay, G.M., Daza, R.M., Martin, B., Zhang, M.D., Leith, A.P., Gasperini, M., Janizek,

J.D., Huang, X., Starita, L.M., and Shendure, J. (2018). Accurate classification of

BRCA1 variants with saturation genome editing. Nature 562, 217–222.

Fowler, D.M., and Fields, S. (2014). Deep mutational scanning: A new style of protein science. Nat. Methods 801–807.

Fowler, D.M., Stephany, J.J., and Fields, S. (2014). Measuring the activity of protein variants on a large scale using deep mutational scanning. Nat. Protoc. 2267–2284.

Fox, D., Brzovic, P.S., Nishikawa, H., Keeffe, J.R., Klevit, R., Ohta, T., Miyamoto, K., and Fukuda, M. (2003). Binding and recognition in the assembly of an active

BRCA1/BARD1 ubiquitin-ligase complex. Proc. Natl. Acad. Sci. 100, 5646–5651.

Geissmann, Q. (2013). OpenCFU, a New Free and Open-Source Software to Count Cell

Colonies and Other Circular Objects. PLoS One 8, e54072.

Ghimenti, C., Sensi, E., Presciuttini, S., Brunetti, I.M., Conte, P., Bevilacqua, G., and

Caligo, M.A. (2002). Germline mutations of the BRCA1-associated ring domain

(BARD1) gene in breast and breast/ovarian families negative for BRCA1 and BRCA2 alterations. Genes Chromosom. Cancer 33, 235–242.

Godwin, A.K., Miron, A., Blank, S. V., Huzarski, T., Kriege, M., Borg, A., Hamann, U.,

Couch, F.J., Paterson, J., Milgrom, R., et al. (2010). Common Breast Cancer

118

Susceptibility Alleles and the Risk of Breast Cancer for BRCA1 and BRCA2 Mutation

Carriers: Implications for Risk Prediction. Cancer Res. 70, 9742–9754.

Hart, S.N., McFarland, R., Polley, E.C., Lilyquist, J., Feng, B., Thomas, A., Hu, C.,

Dolinsky, J.S., Couch, F.J., Shimelis, H., et al. (2017). Associations Between Cancer

Predisposition Testing Panel Genes and Breast Cancer. JAMA Oncol. 3, 1190–1196.

Hart, T., Chandrashekhar, M., Aregger, M., Steinhart, Z., Brown, K.R., MacLeod, G.,

Mis, M., Zimmermann, M., Fradet-Turcotte, A., Sun, S., et al. (2015). High-Resolution

CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. Cell

163, 1515–1526.

Hartmann, L.C., and Lindor, N.M. (2016). The Role of Risk-Reducing Surgery in

Hereditary Breast and Ovarian Cancer. Obstet. Gynecol. Surv. 454–468.

Hashizume, R., Fukuda, M., Maeda, I., Nishikawa, H., Oyake, D., Yabuki, Y., Ogata, H., and Ohta, T. (2001). The RING Heterodimer BRCA1-BARD1 Is a Ubiquitin Ligase

Inactivated by a Breast Cancer-derived Mutation. J. Biol. Chem. 276, 14537–14540.

Huang, K., Mashl, R., Wu, Y., Ritter, D., Wang, J., Oh, C., Paczkowska, M., Reynolds,

S., Wyczalkowski, M., Oak, N., et al. (2018). Pathogenic Germline Variants in 10,389

Adult Cancers. Cell 173, 355-370.e14.

Humphrey, J.S., Salim, A., Erdos, M.R., Collins, F.S., Brody, L.C., and Klausner, R.D.

(1997). Human BRCA1 inhibits growth in yeast: potential use in diagnostic testing. Proc.

Natl. Acad. Sci. U. S. A. 94, 5820–5825.

Inoue, F., and Ahituv, N. (2015). Decoding enhancers using massively parallel reporter assays. Genomics 159–164.

119

Inoue, F., Kircher, M., Martin, B., Cooper, G.M., Witten, D.M., McManus, M.T., Ahituv,

N., and Shendure, J. (2017). A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity. Genome Res. 27, 38–52.

Irminger-Finger, I., Ratajska, M., and Pilyugin, M. (2016). New concepts on BARD1:

Regulator of BRCA pathways and beyond. Int. J. Biochem. Cell Biol. 72, 1–17.

Jack, E., Bradley, L., Kwan, E., Narod, S.A., Rosen, B., Abrahamson, J.L.A.,

McLaughlin, J.R., Fan, I., Cole, D.E.C., Vesprini, D.J., et al. (2002). Prevalence and

Penetrance of Germline BRCA1 and BRCA2 Mutations in a Population Series of 649

Women with Ovarian Cancer. Am. J. Hum. Genet. 68, 700–710.

Jain, P.C., and Varadarajan, R. (2014). A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library.

Anal. Biochem. 449, 90–98.

Johnson, S.M., Shaw, J.A., and Walker, R.A. (2002). Sporadic breast cancer in young women: Prevalence of loss of heterozygosity at p53, BRCA1 and BRCA2. Int. J. Cancer.

Julien, P., Miñana, B., Baeza-Centurion, P., Valcárcel, J., and Lehner, B. (2016). The complete local genotype-phenotype landscape for the alternative splicing of a human exon. Nat. Commun. 7, 11558.

Kais, Z., Chiba, N., Ishioka, C., and Parvin, J.D. (2012). Functional differences among

BRCA1 missense mutations in the control of centrosome duplication. Oncogene 31, 799–

804.

Kandoth, C., McLellan, M.D., Vandin, F., Ye, K., Niu, B., Lu, C., Xie, M., Zhang, Q.,

McMichael, J.F., Wyczalkowski, M.A., et al. (2013). Mutational landscape and

120 significance across 12 major cancer types. Nature 502, 333–339.

King, M.-C., Norquist, B., Roeb, W., Wickramanayake, A., Swisher, E.M., Pennington,

K.P., Walsh, T., Lee, M.K., Garcia, R.L., Stray, S.M., et al. (2011). Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc. Natl. Acad. Sci. 108, 18032–18037.

Kircher, M., Witten, D.M., Jain, P., O’roak, B.J., Cooper, G.M., and Shendure, J. (2014).

A general framework for estimating the relative pathogenicity of human genetic variants.

Nat. Genet. 46, 310–315.

Kitzman, J.O., Starita, L.M., Lo, R.S., Fields, S., and Shendure, J. (2015). Massively parallel single-amino-acid mutagenesis. Nat. Methods 12, 203–206.

Kleiman, F.E., Wu-Baer, F., Fonseca, D., Kaneko, S., Baer, R., and Manley, J.L. (2005).

BRCA1/BARD1 inhibition of mRNA 3′ processing involves targeted degradation of

RNA polymerase II. Genes Dev. 19, 1227–1237.

Klesmith, J.R., Bacik, J.P., Michalczyk, R., and Whitehead, T.A. (2015). Comprehensive

Sequence-Flux Mapping of a Levoglucosan Utilization Pathway in E. coli. ACS Synth.

Biol. 4, 1235–1243.

Kumar, B., Chan, T.A., Kucherlapati, R.S., Auman, J.T., Stewart, C., Becker, K.-F.,

Frazer, S., Meier, S., Xi, L., Landen, C.N., et al. (2018). Scalable Open Science

Approach for Mutation Calling of Tumor Exomes Using Multiple Genomic Pipelines.

Cell Syst. 6, 271-281.e7.

Landrum, M.J., Lee, J.M., Benson, M., Brown, G.R., Chao, C., Chitipiralla, S., Gu, B.,

Hart, J., Hoffman, D., Jang, W., et al. (2018). ClinVar: Improving access to variant

121 interpretations and supporting evidence. Nucleic Acids Res.

Larsen, M.J., Thomassen, M., Gerdes, A.M., and Kruse, T.A. (2014). Hereditary breast cancer: Clinical, Pathological and molecular characteristics. Breast Cancer Basic Clin.

Res. 8, 145–155.

Laufer, M., Nandula, S. V., Modi, A.P., Wang, S., Jasin, M., Murty, V.V.V.S., Ludwig,

T., and Baer, R. (2007). Structural requirements for the BARD1 tumor suppressor in chromosomal stability and homology-directed DNA repair. J. Biol. Chem. 282, 34325–

34333.

Lee, C., Banerjee, T., Gillespie, J., Ceravolo, A., Parvinsmith, M.R., Starita, L.M., Fields,

S., Toland, A.E., and Parvin, J.D. (2015). Functional Analysis of BARD1 Missense

Variants in Homology-Directed Repair of DNA Double Strand Breaks. Hum. Mutat. 36,

1205–1214.

Lee, M.K., Pennil, C., Walsh, T., Thornton, A.M., Swisher, E.M., King, M.-C., Nord,

A.S., Stray, S.M., Mandell, J.B., and Casadei, S. (2010a). Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. Proc. Natl. Acad. Sci. 107, 12629–12633.

Lee, M.S., Green, R., Marsillac, S.M., Coquelle, N., Williams, R.S., Yeung, T., Foo, D.,

Hau, D.D., Hui, B., Monteiro, A.N.A., et al. (2010b). Comprehensive analysis of missense variations in the BRCT domain of BRCA1 by structural and functional assays.

Cancer Res. 70, 4880–4890.

Lek, M., Karczewski, K.J., Minikel, E. V, Samocha, K.E., Banks, E., Fennell, T.,

O’Donnell-Luria, A.H., Ware, J.S., Hill, A.J., Cummings, B.B., et al. (2016). Analysis of

122 protein-coding genetic variation in 60,706 humans. Nature 536, 285–291.

Levy-Lahad, E., and Friedman, E. (2007). Cancer risks among BRCA1 and BRCA2 mutation carriers. Br. J. Cancer 11–15.

Li, H., and Durbin, R. (2010). Fast and accurate long-read alignment with Burrows-

Wheeler transform. Bioinformatics 26, 589–595.

Li, M., and Yu, X. (2013). Function of BRCA1 in the DNA Damage Response Is

Mediated by ADP-Ribosylation. Cancer Cell 23, 693–704.

Lorick, K.L., Jensen, J.P., Fang, S., Ong, A.M., Hatakeyama, S., and Weissman, A.M.

(1999). RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. Proc. Natl. Acad. Sci. 96, 11364–11369.

Lu, C., Xie, M., Wendl, M.C., Wang, J., McLellan, M.D., Leiserson, M.D.M., Huang,

K.L., Wyczalkowski, M.A., Jayasinghe, R., Banerjee, T., et al. (2015). Patterns and functional implications of rare germline variants across 12 cancer types. Nat. Commun. 6,

1–13.

Majithia, A.R., Tsuda, B., Agostini, M., Gnanapradeepan, K., Rice, R., Peloso, G., Patel,

K.A., Zhang, X., Broekema, M.F., Patterson, N., et al. (2016). Prospective functional classification of all possible missense variants in PPARG. Nat. Genet. 48, 1570–1575.

Malheiro, A., Riley, G., Hart, J., Jang, W., Ovetsky, M., Hoffman, D., Gu, B., Kattman,

B.L., Chao, C., Brown, G.R., et al. (2017). ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 46, D1062–D1067.

Matreyek, K.A., Starita, L.M., Stephany, J.J., Martin, B., Chiasson, M.A., Gray, V.E.,

Kircher, M., Khechaduri, A., Dines, J.N., Hause, R.J., et al. (2018). Multiplex assessment

123 of protein variant abundance by massively parallel sequencing. Nat. Genet. 50, 874–882.

McCarthy, E.E., Celebi, J.T., Baer, R., and Ludwig, T. (2003). Loss of Bard1, the heterodimeric partner of the Brca1 tumor suppressor, results in early embryonic lethality and chromosomal instability. Mol. Cell. Biol. 23, 5056–5063.

McKenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A.,

Garimella, K., Altshuler, D., Gabriel, S., Daly, M., et al. (2010). The genome analysis toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data.

Genome Res. 20, 1297–1303.

Melchor, L., and Benítez, J. (2013). The complex genetic landscape of familial breast cancer. Hum. Genet. 845–863.

Melnikov, A., Rogov, P., Wang, L., Gnirke, A., and Mikkelsen, T.S. (2014).

Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes. Nucleic Acids Res. 42, 1–8.

Merajver, S., Frank, T., Xu, J., Pham, T., Calzone, K., Bennett-Baker, P., Chamberlain,

J., Boyd, J., Garber, J., and Collins, F. (1995). Germline BRCA1 mutations and loss of the wild-type allele in tumors from families with early onset breast and ovarian cancer.

Clin. Cancer Res. 1, 539–544.

Metzger-Filho, O., Tutt, A., De Azambuja, E., Saini, K.S., Viale, G., Loi, S., Bradbury,

I., Bliss, J.M., Azim, H.A., Ellis, P., et al. (2012). Dissecting the heterogeneity of triple- negative breast cancer. J. Clin. Oncol. 1879–1887.

Mighell, T.L., Evans-Dutson, S., and O’Roak, B.J. (2018). A Saturation Mutagenesis

Approach to Understanding PTEN Lipid Phosphatase Activity and Genotype-Phenotype

124

Relationships. Am. J. Hum. Genet. 102, 943–955.

Millot, G.A., Berger, A., Lejour, V., Boulé, J.B., Bobo, C., Cullin, C., Lopes, J., Stoppa-

Lyonnet, D., and Nicolas, A. (2011). Assessment of human nter and cter BRCA1 mutations using growth and localization assays in yeast. Hum. Mutat. 32, 1470–1480.

Miyoshi, Y., Asano, M., Ohta, T., Klevit, R.E., Vittal, V., Wu, W., Fukuda, T., and

Nishikawa, H. (2015). Interaction of BARD1 and HP1 Is Required for BRCA1 Retention at Sites of DNA Damage. Cancer Res. 75, 1311–1321.

Monteiro, A.N.., August, A., and Hanafusa, H. (1997). Common BRCA1 variants and transcriptional activation. Am J Hum Genet 61, 761–762.

Morris, J.R., Pangon, L., Boutell, C., Katagiri, T., Keep, N.H., and Solomon, E. (2006).

Genetic analysis of BRCA1 ubiquitin ligase activity and its relationship to breast cancer susceptibility. Hum. Mol. Genet. 15, 599–606.

Moynahan, M.E., Chiu, J.W., Koller, B.H., and Jasint, M. (1999). Brca1 controls homology-directed DNA repair. Mol. Cell 4, 511–518.

Muerdter, F., Boryń, Ł.M., and Arnold, C.D. (2015). STARR-seq - Principles and applications. Genomics 145–150.

Nandula, S., Ludwig, T., Murty, V., Baer, R., McCarthy, E., Basso, K., Ospina, E.,

Szabolcs, M., and Shakya, R. (2008). The basal-like mammary carcinomas induced by

Brca1 or Bard1 inactivation implicate the BRCA1/BARD1 heterodimer in tumor suppression. Proc. Natl. Acad. Sci. 105, 7040–7045.

Ng, P.C., and Henikoff, S. (2003). SIFT: Predicting amino acid changes that affect protein function. Nucleic Acids Res. 31, 3812–3814.

125

Ng, P.C., Levy, S., Huang, J., Stockwell, T.B., Walenz, B.P., Li, K., Axelrod, N., Busam,

D.A., Strausberg, R.L., and Venter, J.C. (2008). Genetic variation in an individual human exome. PLoS Genet. 4, e1000160.

Norquist, B., Wurz, K.A., Pennil, C.C., Garcia, R., Gross, J., Sakai, W., Karlan, B.Y.,

Taniguchi, T., and Swisher, E.M. (2011). Secondary somatic mutations restoring

BRCA1/2 predict chemotherapy resistance in hereditary ovarian carcinomas. J. Clin.

Oncol. 29, 3008–3015.

Norquist, B.M., Harrell, M.I., Brady, M.F., Walsh, T., Lee, M.K., Gulsuner, S., Bernards,

S.S., Casadei, S., Yi, Q., Burger, R.A., et al. (2016). Inherited mutations in women with ovarian carcinoma. JAMA Oncol. 2, 482–490.

Osorio, A., De La Hoya, M., Rodríguez-López, R., Martínez-Ramírez, A., Cazorla, A.,

Granizo, J.J., Esteller, M., Rivas, C., Caldés, T., and Benítez, J. (2002). Loss of heterozygosity analysis at the BRCA loci in tumor samples from patients with familial breast cancer. Int. J. Cancer.

Parmigiani, G., Chen, S., Iversen, E.S., Friebel, T.M., Finkelstein, D.M., Anton-Culver,

H., Ziogas, A., Weber, B.L., Eisen, A., Malone, K.E., et al. (2007). Validity of models for predicting BRCA1 and BRCA2 mutations. Ann. Intern. Med. 147, 441–450.

Patwardhan, R.P., Hiatt, J.B., Witten, D.M., Kim, M.J., Smith, R.P., May, D., Lee, C.,

Andrie, J.M., Lee, S.I., Cooper, G.M., et al. (2012). Massively parallel functional dissection of mammalian enhancers in vivo. Nat. Biotechnol. 30, 265–270.

Petitalot, A., Dardillac, E., Jacquet, E., Nhiri, N., Guirouilh-Barbat, J., Julien, P.,

Bouazzaoui, I., Bonte, D., Feunteun, J., Schnell, J.A., et al. (2019). Combining

126

Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of

BRCA1 BRCT Variants on Cancer Risk. Mol. Cancer Res. 17, 54–69.

Phelan, C.M., Dapic, V., Tice, B., Favis, R., Kwan, E., Barany, F., Manoukian, S.,

Radice, P., Van Der Luijt, R.B., Van Nesselrooij, B.P.M., et al. (2005). Classification of

BRCA1 missense variants of unknown clinical significance. J. Med. Genet. 42, 138–146.

Pierce, A.J., Hu, P., Han, M., Ellis, N., and Jasin, M. (2001). Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes

Dev. 15, 3237–3242.

Qu, J., Lu, W., Li, B., Lu, C., and Wan, X. (2013). WWOX induces apoptosis and inhibits proliferation in cervical cancer and cell lines. Int. J. Mol. Med. 31, 1139–1147.

Ramus, S.J., and Gayther, S.A. (2009). The Contribution of BRCA1 and BRCA2 to

Ovarian Cancer. Mol. Oncol.

Ransburgh, D.J.R., Chiba, N., Ishioka, C., Toland, A.E., and Parvin, J.D. (2010).

Identification of breast tumor mutations in BRCA1 that abolish its function in homologous DNA recombination. Cancer Res. 70, 988–995.

Remmert, M., Lopez, R., Li, W., Dineen, D., Sievers, F., Gibson, T.J., Soding, J.,

Thompson, J.D., Wilm, A., McWilliam, H., et al. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol.

7, 539.

Richardson, J.S., and Richardson, D.C. (1988). Amino acid preferences for specific locations at the ends of α helices. Science (80-. ). 240, 1648–1652.

Rodriguez, J.A., Au, W.W.Y., and Henderson, B.R. (2004). Cytoplasmic mislocalization

127 of BRCA1 caused by cancer-associated mutations in the BRCT domain. Exp. Cell Res.

293, 14–21.

Rodriguez, M., Yu, X., Chen, J., and Songyang, Z. (2003). Phosphopeptide Binding

Specificities of BRCA1 COOH-terminal (BRCT) Domains. J. Biol. Chem. 278, 52914–

52918.

Rosenberg, A.B., Patwardhan, R.P., Shendure, J., and Seelig, G. (2015). Learning the

Sequence Determinants of Alternative Splicing from Millions of Random Sequences.

Cell 163, 698–711.

Rottenberg, S., Jaspers, J.E., Kersbergen, A., van der Burg, E., Nygren, A.O.H., Zander,

S.A.L., Derksen, P.W.B., de Bruin, M., Zevenhoven, J., Lau, A., et al. (2008). High sensitivity of BRCA1-deficient mammary tumors to the PARP inhibitor AZD2281 alone and in combination with platinum drugs. Proc. Natl. Acad. Sci. 105, 17079–17084.

Roy, R., Chun, J., and Powell, S.N. (2012). BRCA1 and BRCA2: Different roles in a common pathway of genome protection. Nat. Rev. Cancer 12, 68–78.

Rubin, A.F., Gelman, H., Lucas, N., Bajjalieh, S.M., Papenfuss, A.T., Speed, T.P., and

Fowler, D.M. (2017). A statistical framework for analyzing deep mutational scanning data. Genome Biol. 18, 150.

Ruffner, H., Joazeiro, C.A.P., Hemmati, D., Hunter, T., and Verma, I.M. (2001). Cancer- predisposing mutations within the RING domain of BRCA1: Loss of ubiquitin protein ligase activity and protection from radiation hypersensitivity. Proc. Natl. Acad. Sci. 98,

5134–5139.

Sauer, B. (1994). Site-specific recombination: developments and applications. Curr.

128

Opin. Biotechnol. 5, 521–527.

Schlegel, B.P., Starita, L.M., and Parvin, J.D. (2003). Overexpression of a protein fragment of rna helicase a causes inhibition of endogenous brca1 function and defects in ploidy and cytokinesis in mammary epithelial cells. Oncogene 22, 983–991.

Schrock, M.S., Batar, B., Lee, J., Druck, T., Ferguson, B., Cho, J.H., Akakpo, K.,

Hagrass, H., Heerema, N.A., Xia, F., et al. (2017). Wwox-Brca1 interaction: Role in

DNA repair pathway choice. Oncogene 36, 2215–2227.

Scully, R., Ganesan, S., Brown, M., De Caprio, J.A., Cannistra, S.A., Feunteun, J.,

Schnitt, S., and Livingston, D.M. (1996). Location of BRCA1 in human breast and ovarian cancer cells. Science 272, 123–126.

Scully, R., Chen, J., Plug, A., Xiao, Y., Weaver, D., Feunteun, J., Ashley, T., and

Livingston, D.M. (1997a). Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 88, 265–275.

Scully, R., Chen, J., Ochs, R.L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston,

D.M. (1997b). Dynamic changes of BRCA1 subnuclear location and phosphorylation state are initiated by DNA damage. Cell 90, 425–435.

Scully, R., Ganesan, S., Vlasakova, K., Chen, J., Socolovsky, M., and Livingston, D.M.

(1999). Genetic analysis of BRCA1 function in a defined tumor cell line. Mol. Cell 4,

1093–1099.

Shakya, R., Reid, L.J., Reczek, C.R., Cole, F., Egli, D., Lin, C.S., DeRooij, D.G., Hirsch,

S., Ravi, K., Hicks, J.B., et al. (2011). BRCA1 tumor suppression depends on BRCT phosphoprotein binding, but not its E3 ligase activity. Science (80-. ). 334, 525–528.

129

Shalem, O., Sharon, E., Lubliner, S., Regev, I., Lotan-Pompan, M., Yakhini, Z., and

Segal, E. (2015). Systematic Dissection of the Sequence Determinants of Gene 3’ End

Mediated Expression Control. PLoS Genet. 11, e1005147.

Shattuck Eidens, D., Mcclure, M., Simard, J., Labrie, F., Narod, S., Couch, F., Hoskins,

K., Weber, B., Castilla, L., Erdos, M., et al. (1995). A Collaborative Survey of 80

Mutations in the BRCA1 Breast and Ovarian Cancer Susceptibility Gene: Implications for Presymptomatic Testing and Screening. JAMA J. Am. Med. Assoc. 273, 535–541.

Shen, D., Larson, D.E., Mardis, E.R., McLellan, M.D., Wilson, R.K., Ding, L., Miller,

C.A., Lin, L., Koboldt, D.C., and Zhang, Q. (2012). VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 22, 568–

576.

Shimelis, H., LaDuca, H., Hu, C., Hart, S.N., Na, J., Thomas, A., Akinhanmi, M., Moore,

R.M., Brauch, H., Cox, A., et al. (2018). Triple-negative breast cancer risk genes identified by multigene hereditary cancer panel testing. J. Natl. Cancer Inst. 110, 855–

862.

Simons, A.M., Horwitz, A.A., Starita, L.M., Griffin, K., Williams, R.S., Glover, J.N.M., and Parvin, J.D. (2006). BRCA1 DNA-binding activity is stimulated by BARD1. Cancer

Res. 66, 2012–2018.

Smith, H.O., Chuang, R.-Y., Gibson, D.G., Young, L., Hutchison, C.A., and Venter, J.C.

(2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat.

Methods 6, 343–345.

Snouwaert, J.N., Gowen, L.C., Latour, A.M., Mohn, A.R., Xiao, A., Dibiase, L., and

130

Koller, B.H. (1999). BRCA1 deficient embryonic stem cells display a decreased homologous recombination frequency and an increased frequency of non-homologous recombination that is corrected by expression of a Brca1 transgene. Oncogene 18, 7900–

7907.

Sorenson, C.M., and Eastman, A. (1988). Mechanism of Cis-

Diamminedichloroplatinum(H)-Induced Cytotoxicity: Role of G2 Arrest and DNA

Double-Strand Breaks. Cancer Res. 48, 4484–4488.

Starita, L., Pruneda, J., Lo, R., Fowler, D., Kim, H., Hiatt, J., Shendure, J., Klevit, R.,

Brzovic, P., and Fields, S. (2013). Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis. Proc. Natl. Acad. Sci. 110, E1263-72.

Starita, L.M., Machida, Y., Sankaran, S., Elias, J.E., Griffin, K., Schlegel, B.P., Gygi,

S.P., and Parvin, J.D. (2004). BRCA1-Dependent Ubiquitination of γ-Tubulin Regulates

Centrosome Number. Mol. Cell. Biol. 24, 8457–8466.

Starita, L.M., Young, D.L., Islam, M., Kitzman, J.O., Gullingsrud, J., Hause, R.J.,

Fowler, D.M., Parvin, J.D., Shendure, J., and Fields, S. (2015). Massively parallel functional analysis of BRCA1 RING domain variants. Genetics 200, 413–422.

Starita, L.M., Islam, M.M., Banerjee, T., Adamovich, A.I., Gullingsrud, J., Fields, S.,

Shendure, J., and Parvin, J.D. (2018). A Multiplex Homology-Directed DNA Repair

Assay Reveals the Impact of More Than 1,000 BRCA1 Missense Substitution Variants on Protein Function. Am. J. Hum. Genet. 103, 498–508.

Steffensen, A.Y., Dandanell, M., Jønson, L., Ejlertsen, B., Gerdes, A.M., Nielsen, F.C., and Hansen, T. v. O. (2014). Functional characterization of BRCA1 gene variants by

131 mini-gene splicing assay. Eur. J. Hum. Genet. 22, 1362–1368.

Tagwerker, C., Flick, K., Cui, M., Guerrero, C., Dou, Y., Auer, B., Baldi, P., Huang, L., and Kaiser, P. (2006). A Tandem Affinity Tag for Two-step Purification under Fully

Denaturing Conditions. Mol. Cell. Proteomics 5, 737–748.

Tavtigian, S. V., Deffenbaugh, A.M., Yin, L., Judkins, T., Scholl, T., Samollow, P.B., De

Silva, D., Zharkikh, A., and Thomas, A. (2006). Comprehensive statistical study of 452

BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J. Med. Genet. 43, 295–305.

Thai, T.H., Du, F., Tsan, J.T., Jin, Y., Phung, A., Spillman, M.A., Massa, H.F., Muller,

C.Y., Ashfaq, R., Mathis, J.M., et al. (1998). Mutations in the BRCA1-associated RING domain (BARD1) gene in primary breast, ovarian and uterine cancers. Hum. Mol. Genet.

7, 195–202.

Towler, W.I., Zhang, J., Ransburgh, D.J.R., Toland, A.E., Ishioka, C., Chiba, N., and

Parvin, J.D. (2013). Analysis of BRCA1 Variants in Double-Strand Break Repair by

Homologous Recombination and Single-Strand Annealing. Hum. Mutat. 34, 439–445.

Tung, N., Battelli, C., Allen, B., Kaldate, R., Bhatnagar, S., Bowles, K., Timms, K.,

Garber, J.E., Herold, C., Ellisen, L., et al. (2015). Frequency of mutations in individuals with breast cancer referred for BRCA1 and BRCA2 testing using next-generation sequencing with a 25-gene panel. Cancer 121, 25–33.

Vallon-Christersson, J., Cayanan, C., Haraldsson, K., Loman, N., Bergthorsson, J.,

Brondum-Nielsen, K., Gerdes, A., Moller, P., Kristoffersson, U., Olsson, H., et al.

(2001). Functional analysis of BRCA1 C-terminal missense mutations identified in breast

132 and ovarian cancer families. Hum. Mol. Genet. 10, 353–360.

Vega, A., Campos, B., Bressac-de-Paillerets, B., Bond, P.M., Janin, N., Douglas, F.S.,

Domènech, M., Baena, M., Pericay, C., Alonso, C., et al. (2001). The R71G BRCA1 is a founder Spanish mutation and leads to aberrant splicing of the transcript. Hum. Mutat.

17, 520–521.

Wang, B., Matsuoka, S., Ballif, B.A., Zhang, D., Smogorzewska, A., Gygi, S.P., and

Elledge, S.J. (2007). Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316, 1194–1198.

Wang, Y., Bernhardy, A.J., Cruz, C., Krais, J.J., Nacson, J., Nicolas, E., Peri, S., Van Der

Gulden, H., Van Der Heijden, I., O’Brien, S.W., et al. (2016). The BRCA1-Δ11q alternative splice isoform bypasses germline mutations and promotes therapeutic resistance to PARP inhibition and cisplatin. Cancer Res. 76, 2778–2790.

Williams, R.S., and Glover, J.N.M. (2003). Structural consequences of a cancer-causing

BRCA1-BRCT missense mutation. J. Biol. Chem. 278, 2630–2635.

Williams, R.S., Green, R., and Glover, J.N.M. (2001). Crystal structure of the BRCT repeat region from the breast cancer-associated protein BRCA1. Nat. Struct. Biol. 8, 838–

842.

Williams, R.S., Chasman, D.I., Hau, D.D., Hui, B., Lau, A.Y., and Glover, J.N.M.

(2003). Detection of Protein Folding Defects Caused by BRCA1-BRCT Truncation and

Missense Mutations. J. Biol. Chem. 278, 53007–53016.

Williams, R.S., Lee, M.S., Hau, D.D., and Glover, J.N.M. (2004). Structural basis of phosphopeptide recognition by the BRCT domain of BRCA1. Nat. Struct. Mol. Biol. 11,

133

519–525.

Wilson, D.S., and Keefe, A.D. (2004). Random Mutagenesis by PCR. In Current

Protocols in Molecular Biology, p. Unit 8.3.

Woods, N.T., Baskin, R., Golubeva, V., Jhuraney, A., De-Gregoriis, G., Vaclova, T.,

Goldgar, D.E., Couch, F.J., Carvalho, M.A., Iversen, E.S., et al. (2016). Functional assays provide a robust tool for the clinical annotation of genetic variants of uncertain significance. Npj Genomic Med. 163, 1515–1526.

Wu, L.C., Wang, Z.W., Tsan, J.T., Spillman, M.A., Phung, A., Xu, X.L., Yang, M.C.W.,

Hwang, L.Y., Bowcock, A.M., and Baer, R. (1996). Identification of a RING protein that can interact in vivo with the BRCA1 gene product. Nat. Genet. 14, 430–440.

Wu, W., Togashi, Y., Johmura, Y., Miyoshi, Y., Nobuoka, S., Nakanishi, M., and Ohta,

T. (2016). HP1 regulates the localization of FANCJ at sites of DNA double-strand breaks. Cancer Sci. 107, 1406–1415.

Xu, B., Kim, S. -t., and Kastan, M.B. (2002a). Involvement of Brca1 in S-Phase and G2-

Phase Checkpoints after Ionizing Irradiation. Mol. Cell. Biol. 21, 3445–3450.

Xu, B., O’Donnell, A.H., Kim, S.T., and Kastan, M.B. (2002b). Phosphorylation of serine 1387 in Brca1 is specifically required for the Atm-mediated S-phase checkpoint after ionizing irradiation. Cancer Res. 62, 4588–4591.

Xu, X., Weaver, Z., Linke, S.P., Li, C., Gotay, J., Wang, X.W., Harris, C.C., Ried, T., and Deng, C.X. (1999). Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol.

Cell 3, 389–395.

134

Yamano, H. (2013). EMI1, a three-in-one ubiquitylation inhibitor. Nat. Struct. Mol. Biol.

Ye, K., Schulz, M.H., Long, Q., Apweiler, R., and Ning, Z. (2009). Pindel: A pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25, 2865–2871.

Yu, X., Chini, C.C.S., He, M., Mer, G., and Chen, J. (2003). The BRCT Domain Is a

Phospho-Protein Binding Domain. Science (80-. ). 302, 639–642.

Yun, M.H., and Hiom, K. (2009). CtIP-BRCA1 modulates the choice of DNA double- strand-break repair pathway throughout the cell cycle. Nature 459, 460–463.

Zhao, W., Pollack, J.L., Blagev, D.P., Zaitlen, N., McManus, M.T., and Erle, D.J. (2014).

Massively parallel functional annotation of 3’ untranslated regions. Nat. Biotechnol. 32,

387–391.

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Appendix A. F-box Protein Mediated Resistance to PARP Inhibitor Therapy

Aleksandra I. Adamovich1, Amanda Ewart Toland2, and Jeffrey D. Parvin1*

1 Department of Biomedical Informatics, Ohio State University Comprehensive Cancer

Center, Ohio State University, Columbus, OH

2 Department of Cancer Biology and Genetics, Ohio State University Comprehensive

Cancer Center, Ohio State University, Columbus, OH

Address correspondence to [email protected]

Author contributions: A.A. wrote the manuscript, and A.T. and J.P. edited the manuscript.

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Triple negative breast cancers (TNBCs) are more aggressive than other breast cancers, and as they lack the estrogen receptor, progesterone receptor, and the HER2 receptor

(ER-, PR-, and HER-), these tumors lack targets for therapies. Thus, research into new pathways of therapy for these tumors is critical. Many TNBCs contain pathogenic mutations in BRCA1 or BRCA2, tumor suppressor genes that activate DNA repair via homologous recombination (HR). BRCA1 and BRCA2 mediate the recruitment of the recombinase RAD51 to DNA double-strand breaks to catalyze the repair via HR. In cells lacking functional BRCA, poly(ADP-ribose) polymerases (PARPs) become vital for

DNA repair. PARPs catalyze the transfer of ADP-riboses to target proteins, and several

PARPs have been shown to facilitate repair of single-strand breaks. PARPi target the molecular defect in TNBCs containing BRCA mutations. Inhibition of PARP leads to replication fork stalling due to the accumulation of unrepaired single-strand breaks, and replication fork collapse results in double-strand breaks. These double-strand breaks are normally fixed via HR, and therefore cell death occurs in cells with non-functional

BRCA1 or BRCA2.

PARP inhibitors (PARPi) show efficacy in multiple clinical trials for tumors with HR deficiency. Multiple PARPi have been approved by the FDA or are undergoing clinical trials (reviewed in Bitler et al., 2017). The chemical structures of PARPi vary, as do the type of tumors they treat. Olaparib and talazoparib have been approved for the treatment of breast cancers containing genomic BRCA mutations, and olaparib is also used to treat ovarian cancers with these mutations. Rucaparib and niraparib are approved for treatment of ovarian cancers, with rucaparib focusing on tumors with BRCA mutations and

137 niraparib on platinum-sensitive tumors. The compound veliparib is undergoing clinical trials in combination with irradiation on platinum-sensitive ovarian cancers.

Although loss of BRCA function in TNBCs should render PARPi very effective against the tumor cells (Bryant et al., 2005; Farmer et al., 2005), acquired resistance is a common problem. Several hallmarks of PARPi resistance have been observed (Figure A-1). In cells that contain frame-shift mutations in BRCA1 or BRCA2 the mutant gene can acquire a second mutation that restores the frame of the protein sequence, thereby restoring HR function (Norquist et al., 2011). Cells also use alternative splicing to remove nonessential exons and generate isoforms that are PARPi resistant (Wang et al., 2016). Mutations in

53BP1, which drives a competing pathway for the repair of double-strand breaks via non- homologous end joining (NHEJ), allows cells to survive in the absence of functional

BRCA1 (Bouwman et al., 2010). Upregulation of WEE1 also results in PARPi resistance through WEE1-mediated cell cycle arrest, allowing DNA repair to occur (Do et al.,

2015). Increased expression of p-glycoprotein drug efflux pumps decreases the effectiveness of PARPi (Rottenberg et al., 2008). In addition, replication forks can be stabilized through the loss of PTIP, which recruits MRE11 nuclease to the forks, to prevent the accumulation of double-strand breaks that make HR necessary (Chaudhuri et al., 2016).

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Figure A-1. Observed mechanisms of PARPi resistance in cancer cells. In cancer cells containing BRCA mutations or low expression, homologous recombination is normally unable to occur, and PARPi are able to induce DNA damage resulting in cell death. However, these cells acquire resistance to PARPi through a variety of mechanisms, several of which are targetable with other therapies.

139

In the new study by Marzio et al., the authors hypothesized that an F-box protein mediates a novel mechanism of PARPi resistance because F-box proteins regulate the

DNA damage response and cell cycle checkpoints. F-box proteins act as substrate receptors in SCF ubiquitin ligase complexes, which consist of SKP1, CUL1, an F-box protein, and RBX1. The authors performed an siRNA screen of 69 F-box proteins in a

TNBC cell line sensitive to the PARPi olaparib and screened for proliferation in the presence of olaparib. Depletion of one F-box protein – EMI1 – resulted in significant resistance to olaparib via a novel mechanism.

EMI1 is an F-box protein without previously known activity in SCF E3 ubiquitin ligases, although it has been shown to regulate the activity of APC/C during mitosis independent of other components of the SCF complex (Yamano, 2013). Marzio et al. found that EMI1 does indeed associate with the SCF components in an active complex. A screen of proteins bound to EMI1 revealed an interaction with RAD51. EMI1 was found to regulate RAD51 stability, and depleting EMI1 increased RAD51 protein levels.

Overexpression of RAD51, or depletion of EMI1, bypassed the need for BRCA1 and

BRCA2 to direct RAD51 to DNA double-strand breaks, thereby making HR functional and decreasing PARPi sensitivity in cells. These in vitro results were supported in human breast cancer samples, where EMI1 levels were lower in advanced stage TNBC, and

EMI1 and RAD51 protein levels were inversely correlated. This supports the concept that at least in some TNBC tumors, decreases in EMI1 protein could lead to relaxation of the requirement for BRCA proteins in DNA repair. In addition, mouse xenograft experiments indicated that decreased EMI1 levels correlated with increased olaparib resistance.

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Due to the aggressive nature of TNBC and the lack of available treatments compared to other breast cancers, PARPi are valuable compounds. Identifying modes of resistance and solutions for overcoming resistance is important for improving the survival rates of individuals with TNBC. As PARPi chemical structures differ, the specificity of resistance mechanisms for the various PARPi compounds should be tested. Low EMI1 levels may be a contraindication for olaparib, but perhaps another PARPi would inhibit tumor cell growth in this setting. Marzio et al. suggest that ATR and CHK1 inhibitors sensitize cells to treatment with PARPi, as these inhibitors prevent accumulation of RAD51 in cells.

Combining other therapies with PARPi, including platinum-based drugs, topoisomerase to destabilize replication forks, or CDK12 to downregulate repair proteins, is becoming increasingly prevalent clinically as well as in clinical trials (reviewed in Bitler et al.,

2017). Results from this study have revealed potential new combination therapies that can be tested to reduce the possibility of olaparib resistance and provide new treatment possibilities for individuals with TNBC.

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Appendix B. Variants that Passed Read-Count Thresholding in Multiplex Homology- Directed Repair Analysis of BRCA1 1577-1768

142

protVar score1 score2 score3 score4 qval1 qval2 qval3 qval4 S1577A pass drop pass pass notDepleted notDepleted notDepleted notDepleted S1577R pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1577N pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1577F pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1577V pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1578A pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1578L pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1579A pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1579I pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1579S pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1579T pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1579V pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1580Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1580E pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1580L pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1580F pass pass drop pass notDepleted notDepleted notDepleted notDepleted S1580V pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1581N pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1581L drop pass pass pass notDepleted notDepleted notDepleted notDepleted E1581F pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1581P pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1581T pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1582G pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1582L pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1582K pass pass drop pass notDepleted notDepleted notDepleted notDepleted D1582T pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1582V pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1583Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1583L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1583P pass pass drop pass notDepleted notDepleted notDepleted notDepleted A1584N pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1584G pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1584L pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1584M pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1584F pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1585R pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1585H pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1585L pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1587D pass drop pass pass notDepleted notDepleted notDepleted notDepleted 143

S1587L pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1587* pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1587T pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1588H drop pass pass pass notDepleted notDepleted notDepleted notDepleted A1588L pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1588S pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1589L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1589T pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1590C pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1590Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1590I pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1590L pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1590P drop pass pass pass notDepleted notDepleted notDepleted notDepleted V1590T pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1591I pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1591S pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1591* pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1591V pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1592R pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1592L pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1592K pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1592* pass pass pass pass notDepleted notDepleted notDepleted depleted N1592V pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1593Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1594C pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1594L pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1594Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1594V pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1595H drop pass pass pass notDepleted notDepleted notDepleted notDepleted S1596T pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1597R pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1597L pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1597W pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1597Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1598C drop pass pass pass notDepleted notDepleted notDepleted notDepleted S1598G pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1598L pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1598P pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1599P pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1599S pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1600S pass pass drop pass notDepleted notDepleted notDepleted notDepleted 144

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A1615G pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1615K pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1616L pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1616F pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1616P pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1616* pass pass pass pass depleted notDepleted notDepleted notDepleted A1617S pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1617V pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1618G pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1618L pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1618K pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1618Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1618V pass pass drop pass notDepleted notDepleted notDepleted notDepleted T1619G pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1619L pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1620L pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1620V pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1621P pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1623L pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625R pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625E pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625H pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625L pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625K pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625S pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1625V pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1626A pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1626R pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1626Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1626L pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1626S pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1627R pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1627S pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1628A pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1628D pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1629G drop pass pass pass notDepleted notDepleted notDepleted notDepleted E1629S pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1630G pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1630K pass drop pass pass notDepleted notDepleted notDepleted notDepleted E1630F pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1630P pass pass drop pass notDepleted notDepleted notDepleted notDepleted 146

E1630T pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1630Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1631G drop pass pass pass notDepleted notDepleted notDepleted notDepleted S1631T pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1632R pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1632Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1632L drop pass pass pass notDepleted notDepleted notDepleted notDepleted V1632F pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1633R pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1633D pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1633Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1634A pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1634G pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1634I drop pass pass pass notDepleted notDepleted notDepleted notDepleted R1634K pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1634V pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1636R pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1636T pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1637N pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1637V pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1638M pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1638T pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1639T pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1639V pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1640A pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1640N pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1640G pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1640H pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1641G pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1641H pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1641L pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1642A pass pass drop pass notDepleted notDepleted notDepleted notDepleted S1642L pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1642P pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1642T pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1643P pass drop pass pass notDepleted notDepleted notDepleted notDepleted E1644A pass pass drop pass notDepleted notDepleted notDepleted notDepleted R1645P pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1646L pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1646P drop pass pass pass notDepleted notDepleted notDepleted notDepleted V1646S pass pass pass pass notDepleted notDepleted notDepleted notDepleted 147

K1648Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1648G pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1648S pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1648* pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1649G pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1650R pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1651P pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1652R pass pass pass pass notDepleted notDepleted notDepleted depleted M1652G pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1652K pass pass pass pass notDepleted depleted notDepleted notDepleted V1653L pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1653S pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1654D pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1654G pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1654T pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1655N pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1656L drop pass pass pass notDepleted notDepleted notDepleted notDepleted G1656K pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1656M pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1657R pass pass pass pass depleted depleted notDepleted depleted L1657F drop pass pass pass notDepleted notDepleted notDepleted notDepleted L1657S pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1657* pass pass pass pass depleted notDepleted notDepleted depleted T1658A pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1658G pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1658I pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1658L pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1659A pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1659R pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1659L pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1659F pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1659S pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1659T pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1660R pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1660Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1662A pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1662N pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1662E pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1662I pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1662T drop pass pass pass notDepleted notDepleted notDepleted notDepleted F1662V drop pass pass pass notDepleted notDepleted notDepleted notDepleted 148

M1663N drop pass pass pass notDepleted notDepleted notDepleted notDepleted V1665K pass pass pass pass notDepleted depleted notDepleted notDepleted V1665P drop pass pass pass notDepleted notDepleted notDepleted notDepleted Y1666R pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1666L drop pass pass pass notDepleted notDepleted notDepleted notDepleted Y1666F pass pass pass pass notDepleted notDepleted notDepleted notDepleted Y1666* pass pass pass pass depleted depleted depleted depleted K1667C pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1667G pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1667H pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1667I pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1667P pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1667S pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1667V pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1668P pass pass pass pass notDepleted depleted notDepleted notDepleted A1669R pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1669N pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1669F pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1669V pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1670I drop pass pass pass notDepleted notDepleted notDepleted notDepleted H1673L pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1673F pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1673V pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674R pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674N pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674D pass pass pass pass notDepleted notDepleted depleted notDepleted I1674Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674G pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674L pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674F pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674P pass pass pass pass depleted depleted notDepleted notDepleted I1674S pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674* pass pass pass pass depleted depleted depleted notDepleted I1674T pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1674V pass drop pass pass notDepleted notDepleted notDepleted notDepleted T1675C pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1675Q drop pass pass pass notDepleted notDepleted notDepleted notDepleted T1675L pass pass pass drop notDepleted notDepleted notDepleted notDepleted T1675P drop pass pass pass notDepleted notDepleted notDepleted notDepleted T1675S pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1675V pass pass pass pass depleted depleted depleted notDepleted 149

L1676T pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1677H pass pass drop pass notDepleted notDepleted notDepleted notDepleted T1677I pass drop pass pass notDepleted notDepleted notDepleted notDepleted T1677L pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1677S pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1678R pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1678L pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1678P pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1678S pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1678W pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1679N pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1679D pass pass drop pass notDepleted notDepleted notDepleted notDepleted L1679S pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1679W pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1680R pass pass pass pass depleted depleted depleted depleted I1680P pass pass pass drop notDepleted notDepleted notDepleted notDepleted I1680S pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1681L pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1682P pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1683G pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1683S pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1683* pass pass pass pass depleted notDepleted depleted notDepleted E1683V pass pass pass pass notDepleted notDepleted notDepleted depleted T1684R pass pass pass pass depleted depleted depleted depleted T1684C pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1684H pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1685G drop pass pass pass notDepleted depleted notDepleted notDepleted T1685L pass pass pass pass depleted notDepleted notDepleted notDepleted T1685S pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1687G pass pass pass pass notDepleted depleted depleted notDepleted V1688L pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1689L pass pass drop pass notDepleted notDepleted notDepleted notDepleted M1689T pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1690L drop pass pass pass notDepleted notDepleted notDepleted notDepleted K1690F pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1691G pass pass drop pass depleted depleted notDepleted depleted T1691H pass pass pass pass depleted depleted depleted depleted T1691* pass pass pass pass notDepleted notDepleted notDepleted notDepleted T1691Y pass pass pass pass notDepleted notDepleted depleted depleted D1692L pass pass pass pass notDepleted depleted depleted depleted D1692K pass drop pass pass notDepleted notDepleted notDepleted notDepleted 150

D1692T pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1692Y drop pass pass pass notDepleted notDepleted notDepleted notDepleted A1693L pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1693K pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1693F pass pass drop pass notDepleted notDepleted notDepleted notDepleted E1694K pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1694A pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1694K pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1695A pass drop pass pass notDepleted notDepleted notDepleted notDepleted F1695C pass drop pass pass notDepleted notDepleted notDepleted notDepleted F1695K pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1695S pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1695V pass pass pass pass notDepleted notDepleted notDepleted notDepleted C1697N pass drop pass pass notDepleted notDepleted notDepleted notDepleted E1698S pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1699L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1699P pass pass pass pass depleted notDepleted notDepleted notDepleted L1701A pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1701I drop pass pass pass notDepleted notDepleted notDepleted notDepleted F1704S pass pass pass pass depleted depleted depleted depleted F1704* pass pass pass pass depleted depleted depleted depleted F1704T drop pass pass pass notDepleted notDepleted notDepleted notDepleted L1705R pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1705E pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1705H pass drop pass pass notDepleted notDepleted notDepleted notDepleted L1705K drop pass pass pass notDepleted notDepleted notDepleted notDepleted G1706A pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1706R pass pass pass pass depleted depleted depleted notDepleted G1706D pass drop pass pass depleted notDepleted depleted notDepleted G1706S pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1706V pass drop pass pass notDepleted notDepleted notDepleted depleted I1707R pass pass pass pass depleted notDepleted depleted notDepleted I1707L pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1708P pass pass pass pass depleted depleted depleted depleted A1708Y pass pass pass pass depleted depleted depleted depleted G1709S pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1710L pass pass drop pass notDepleted notDepleted notDepleted notDepleted G1710K pass pass drop pass notDepleted notDepleted notDepleted notDepleted G1710S pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1710T pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1711F pass pass pass pass notDepleted notDepleted notDepleted notDepleted 151

K1711T pass pass pass pass notDepleted notDepleted notDepleted notDepleted W1712G pass drop pass pass depleted notDepleted depleted depleted V1713A pass pass pass pass notDepleted depleted depleted depleted V1713D pass pass pass pass depleted notDepleted notDepleted depleted V1714C pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1714S pass drop pass pass depleted notDepleted notDepleted notDepleted V1714T pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1714W pass pass pass pass depleted depleted notDepleted depleted V1714Y pass pass pass pass depleted depleted depleted depleted S1715R pass pass pass pass depleted depleted depleted depleted S1715I pass pass pass pass depleted depleted notDepleted notDepleted S1715L pass pass drop pass depleted depleted notDepleted notDepleted Y1716P pass pass pass pass depleted depleted depleted depleted Y1716V pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1717K pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1717Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted W1718L pass pass pass pass notDepleted notDepleted notDepleted notDepleted W1718P pass pass pass pass depleted notDepleted depleted depleted V1719H pass pass pass pass depleted notDepleted depleted depleted T1720R pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1721N pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1721K pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1721S pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1723C pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1723L pass drop pass pass notDepleted notDepleted notDepleted notDepleted I1723* pass pass pass pass depleted depleted depleted depleted I1723T pass pass pass drop notDepleted notDepleted notDepleted notDepleted K1724L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1726L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1726T pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1727A pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1727L pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1727S pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1728C pass pass pass pass notDepleted notDepleted notDepleted notDepleted M1728P drop pass pass pass notDepleted notDepleted notDepleted notDepleted M1728S pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1729T pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1729Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1730V pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1731G pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1731L pass drop pass pass notDepleted notDepleted notDepleted notDepleted 152

E1731P pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1731Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1733M pass drop pass pass notDepleted notDepleted notDepleted notDepleted F1734R pass pass pass pass notDepleted depleted depleted depleted F1734M pass pass pass pass notDepleted notDepleted notDepleted notDepleted F1734P pass pass pass pass depleted depleted depleted depleted F1734T pass pass pass pass depleted notDepleted notDepleted depleted E1735R pass pass pass pass notDepleted depleted notDepleted notDepleted R1737G drop pass pass pass notDepleted notDepleted notDepleted notDepleted R1737D pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1738V pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1738L pass drop pass pass notDepleted notDepleted depleted notDepleted G1738Y pass pass pass pass depleted depleted depleted depleted V1740L pass drop pass pass notDepleted notDepleted notDepleted notDepleted V1740P pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1741N pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1741E pass pass pass pass notDepleted notDepleted notDepleted notDepleted V1741P pass pass pass pass notDepleted depleted notDepleted notDepleted V1741T pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1742A drop pass pass pass notDepleted notDepleted notDepleted notDepleted N1742R pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1742L pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1742S pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1743A pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1743R pass pass pass pass depleted depleted notDepleted notDepleted G1743C pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1743F pass pass pass pass depleted notDepleted notDepleted notDepleted R1744A pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1744C pass pass pass pass notDepleted notDepleted notDepleted notDepleted N1745G pass pass pass pass notDepleted notDepleted notDepleted notDepleted H1746T pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1747A pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1747D pass pass drop pass notDepleted notDepleted notDepleted notDepleted Q1747C pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1747I pass drop pass pass notDepleted notDepleted notDepleted notDepleted Q1747L pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1748A pass pass drop pass notDepleted notDepleted notDepleted notDepleted G1748T pass pass pass pass notDepleted notDepleted notDepleted notDepleted P1749E pass pass pass pass depleted depleted depleted depleted P1749L pass pass pass pass notDepleted depleted notDepleted depleted P1749* pass pass pass drop notDepleted notDepleted depleted notDepleted 153

K1750L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1751A pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1751L pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1751P pass pass pass pass depleted depleted depleted depleted R1751* pass pass pass pass depleted depleted depleted depleted R1751T pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1751V pass pass pass pass notDepleted notDepleted notDepleted notDepleted A1752H pass pass drop pass notDepleted notDepleted notDepleted notDepleted A1752I pass pass drop pass notDepleted depleted depleted depleted A1752L pass pass pass pass depleted depleted depleted depleted A1752P pass pass pass pass depleted depleted depleted depleted A1752* pass pass pass pass depleted depleted depleted depleted E1754L pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1754F pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1754S pass pass pass pass notDepleted notDepleted notDepleted notDepleted S1755A pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1756A pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1756R pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1756N pass pass pass pass notDepleted notDepleted notDepleted notDepleted Q1756I pass pass pass pass notDepleted notDepleted notDepleted notDepleted D1757A pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1758C pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1758Q pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1758I pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1758T pass pass pass pass notDepleted notDepleted notDepleted notDepleted R1758Y pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1759I pass pass pass pass notDepleted notDepleted notDepleted notDepleted K1759M pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1760A pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1760E pass pass pass pass depleted notDepleted notDepleted depleted I1760L pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1760T pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1760Y pass pass pass pass depleted depleted notDepleted depleted F1761T pass pass pass pass depleted depleted depleted depleted R1762C pass pass drop pass notDepleted notDepleted notDepleted notDepleted R1762G pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1763A pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1763K pass pass pass pass notDepleted notDepleted notDepleted notDepleted G1763* pass pass pass pass depleted depleted notDepleted depleted G1763V pass pass pass pass notDepleted notDepleted notDepleted depleted L1764M pass pass pass pass notDepleted notDepleted notDepleted notDepleted 154

L1764P pass pass pass pass depleted depleted depleted depleted L1764S pass pass pass pass notDepleted notDepleted notDepleted notDepleted L1764* pass pass pass pass depleted depleted depleted depleted L1764W pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1765C pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1765Q drop pass pass pass notDepleted notDepleted notDepleted notDepleted E1765L pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1765P pass pass pass pass notDepleted notDepleted notDepleted notDepleted E1765V pass pass pass pass notDepleted notDepleted notDepleted notDepleted I1766R pass pass pass pass depleted notDepleted depleted depleted I1766L pass drop pass pass notDepleted notDepleted notDepleted notDepleted I1766T pass pass pass drop notDepleted notDepleted notDepleted notDepleted C1767R pass pass pass pass notDepleted notDepleted notDepleted notDepleted C1767N pass pass pass pass notDepleted notDepleted notDepleted notDepleted C1767L pass pass pass pass notDepleted notDepleted notDepleted notDepleted C1767* pass pass pass pass depleted depleted depleted notDepleted C1767T pass pass pass pass notDepleted notDepleted notDepleted notDepleted C1767V drop pass pass pass notDepleted notDepleted notDepleted notDepleted C1768G pass pass pass pass notDepleted notDepleted notDepleted notDepleted C1768L pass pass pass pass notDepleted notDepleted notDepleted notDepleted

155

Appendix C. Primers Used in BRCA1 C-terminus Mutagenesis

156

Name Sequence BRCA1-NheI-Fwd-Long 5′-GAA ACA AAA TGT TCT GCT AGC TTG TTT TCT TCA CAG-3′ BRCA1-AscI-Barcode-XhoI-Gib- 5′-AAC GGG CCC TCT AGA CTC GAG NNN Rev NNN NNN NNN NNN TGG CGC GCC TCA GTA GTG GCT GTG-3′ BRCA1-SbfI-Barcode-Rev 5′-GTG TGC TCT TCC GAT CCC TGC AGG NNN NNN NNN NNN NNN NAG ATC GGA AGA GCG TCG TGT AGG GAA AGA GTG TCT GCA GGT CGA GCC GAT ATC TC-3′ Pool4-Nest1-Fwd 5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT CAC TAC TGA GGC GCG CCA-3′ Cterm56-Nest1-Fwd 5′-TGA GAT ATC GGC TCG ACC TGC AGA CAC-3′ FRT-LacZ-Rev 5′-GGG TAA CGC CAG GGT TTT CCC AGT CAC GAC G-3′ Pool4-Seq-i701 5′-CAA GCA GAA GAC GGC ATA CGA GAT CGA GTA ATG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCC GGG CCC TCT AGA CTC GAG-3′ Pool4-Seq-i702 5′-CAA GCA GAA GAC GGC ATA CGA GAT TCT CCG GAG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCC GGG CCC TCT AGA CTC GAG-3′ Pool4-Seq-i703 5′-CAA GCA GAA GAC GGC ATA CGA GAT AAT GAG CGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCC GGG CCC TCT AGA CTC GAG-3′ Pool4-Seq-i704 5′-CAA GCA GAA GAC GGC ATA CGA GAT GGA ATC TCG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCC GGG CCC TCT AGA CTC GAG-3′ Pool4-Seq-i705 5′-CAA GCA GAA GAC GGC ATA CGA GAT TTC TGA ATG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCC GGG CCC TCT AGA CTC GAG-3′ Seq-i701 5′-CAA GCA GAA GAC GGC ATA CGA GAT CGA GTA ATG TGA CTG GAG TTC AGA CG- 3′ Seq-i702 5′-CAA GCA GAA GAC GGC ATA CGA GAT TCT CCG GAG TGA CTG GAG TTC AGA CG- 3′

157

Seq-i703 5′-CAA GCA GAA GAC GGC ATA CGA GAT AAT GAG CGG TGA CTG GAG TTC AGA CG-3′ Seq-i704 5′-CAA GCA GAA GAC GGC ATA CGA GAT GGA ATC TCG TGA CTG GAG TTC AGA CG- 3′ Seq-i705 5′-CAA GCA GAA GAC GGC ATA CGA GAT TTC TGA ATG TGA CTG GAG TTC AGA CG- 3′ Seq-i501 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT ATA GCC TAC ACT CTT TCC CTA CAC G-3′ Seq-i502 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACA TAG AGG CAC ACT CTT TCC CTA CAC G-3′ Seq-i503 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACC CTA TCC TAC ACT CTT TCC CTA CAC G-3′ Seq-i504 5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG GCT CTG AAC ACT CTT TCC CTA CAC G-3′

158