Analysis of BRCA1/2 variants of unknown significance in patients with breast cancer from a prospective KOHBRA study


 Background Genetic testing for BRCA1 and BRCA2 genes is crucial for diagnosing hereditary breast and ovarian cancer syndromes (HBOC). Testing for such genes has been on the rise due to the development of multigene panel tests. However, results classified as variants of uncertain significance (VUS) present challenges to clinicians trying to interpret their functions and choose appropriate management plans.Methods We reviewed a total of 676 breast cancer patients who had VUS on BRCA mutation tests between November 2007 and April 2013 in the KOHBRA study. These results were compared to the ClinVar database. We calculated the incidence and odds ratios for these variants using the Korean Reference Genome Database (KRGDB).Results A total of 58 and 91 distinct VUS in BRCA1 and 2 were identified in the KOHBRA study (comprising 258 and 453 patients, respectively). A total of 27 variants in the KOHBRA study were not registered in the SNP database. Of the BRCA1 VUS, 20 variants were reclassified as benign or likely benign, 4 variants were reclassified as pathogenic or likely pathogenic, and 8 variants remained as VUS according to the ClinVar database. Of the BRCA2 VUS variants, 25 variants were reclassified as benign or likely benign, two variants were reclassified as pathogenic or likely pathogenic, and 33 variants remained as VUS according to the Clinvar database. There were 12 variants with conflicting interpretations of pathogenicity for BRCA1 and 18 variants for BRCA2. Among them, p.Leu1780Pro showed a particularly high odds ratio.Conclusions Six pathogenic variants and one conflicting variant identified using ClinVar could be reclassified as pathogenic variants in this study. Using updated ClinVar and calculating odds ratios can be helpful when reclassifying VUS in BRCA1/2.


Abstract
Background Genetic testing for BRCA1 and BRCA2 genes is crucial for diagnosing hereditary breast and ovarian cancer syndromes (HBOC). Testing for such genes has been on the rise due to the development of multigene panel tests. However, results classi ed as variants of uncertain signi cance (VUS) present challenges to clinicians trying to interpret their functions and choose appropriate management plans.
Methods We reviewed a total of 676 breast cancer patients who had VUS on BRCA mutation tests between November 2007 and April 2013 in the KOHBRA study. These results were compared to the ClinVar database. We calculated the incidence and odds ratios for these variants using the Korean Reference Genome Database (KRGDB).
Results A total of 58 and 91 distinct VUS in BRCA1 and 2 were identi ed in the KOHBRA study (comprising 258 and 453 patients, respectively). A total of 27 variants in the KOHBRA study were not registered in the SNP database. Of the BRCA1 VUS, 20 variants were reclassi ed as benign or likely benign, 4 variants were reclassi ed as pathogenic or likely pathogenic, and 8 variants remained as VUS according to the ClinVar database. Of the BRCA2 VUS variants, 25 variants were reclassi ed as benign or likely benign, two variants were reclassi ed as pathogenic or likely pathogenic, and 33 variants remained as VUS according to the Clinvar database. There were 12 variants with con icting interpretations of pathogenicity for BRCA1 and 18 variants for BRCA2. Among them, p.Leu1780Pro showed a particularly high odds ratio.
Conclusions Six pathogenic variants and one con icting variant identi ed using ClinVar could be reclassi ed as pathogenic variants in this study. Using updated ClinVar and calculating odds ratios can be helpful when reclassifying VUS in BRCA1/2.

Background
Hereditary breast and ovarian cancer syndrome (HBOC) is related to germline mutations in BRCA1 and BRCA2 [1].
Demands for genetic testing to identify pathogenic mutations in BRCA1 and BRCA2 have steadily increased since the identi cation of these genes [2]. A BRCA genetic test shows 4 possible results: no mutation detected, pathogenic mutation, benign mutation, or variant of uncertain signi cance (VUS). A VUS is an alteration in the gene sequence that has an unknown effect on the function of the gene product.
Identi cation of a pathogenic BRCA mutation in a patient diagnosed with breast cancer affects treatment and prognosis, but also enables prevention of other cancers [3]. Guidelines for the management of pathogenic variants in BRCA1 and BRCA2 genes recommend consideration of risk-reducing medications or surgeries [4,5]. Therefore, identifying the function of the mutation is important for patients and their families. Furthermore, the cost of genetic testing has rapidly decreased due to recent technological progress and the use of large-scale nextgeneration sequencing (NGS) requiring shorter processing times [2].
A major problem with genetic testing for BRCA is the interpretation and management of VUSs. This leaves patients with uncertainty due to the lack of interpretation in the clinical context, as well as the lack of speci c guidelines regarding genetic counseling or prophylactic management in mutation carriers and their relatives [6].
Overall, a VUS rate of 7-15% in women who received BRCA testing has been reported [7]. The frequency of VUS reports varies worldwide depending on testing prevalence and population ancestry [7,8]. Researchers reported that the frequency of VUSs was 21% in African-Americans, 5-6% in individuals of European ancestry in the USA, and 15% in European laboratories [9,10]. Myriad Genetic Inc. (Salt Lake City, UT, USA) reported that they decreased the proportion of VUS to 2.1% using their accumulated data [11]. However, these databases are not public or accessible.
In this study we aimed to explore the prevalence of VUS and reclassify these variants using the ClinVar database and the Korean Reference Genome Database (KRGDB) in the Korean population.

Subjects
The study population was obtained from the Korean Hereditary Breast Cancer (KOHBRA) study [12]. The KOHBRA study is a multicenter prospective cohort study to identify the prevalence and cause of hereditary breast cancer in the Korean population. Through the study, 3,015 subjects were recruited between May 2007 and December 2013 from 36 institutions [13]. The eligibility criteria were as follows: (1) breast cancer patients with a family history of breast or ovarian cancer; (2) breast cancer patients without a family history of breast or ovarian cancer (nonfamilial) who were 40 or younger at diagnosis, and were diagnosed with bilateral breast cancer or another primary malignancy; (3) male breast cancer patients; and (4) family members of BRCA 1/2 mutation carriers. After excluding several subjects, a total of 2,953 subjects (1,228 familial breast cancer patients, 1,175 non-familial breast cancer patients, and 550 family members of affected carriers) were evaluated. We identi ed 676 breast cancer patients with VUSs on BRCA mutation tests.
These results were reclassi ed using the ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/) based on refSNP (RS) numbers. The odds ratio (OR) for each variant was calculated using Korean population data from the KRGDB, which was established by conducting whole genome sequencing of 1,722 Koreans [14]. Variants that are not registered in the Single Nucleotide Polymorphism (SNP) database are shown in Table 1. In this study, variations without RS numbers were also included in the denominator when checking frequency. BRCA 1/2 mutation analysis BRCA 1/2 genetic testing was performed using genomic DNA from the peripheral blood by uorescence-based con rmation sensitive gel electrophoresis, denaturing high performance liquid chromatography, or direct sequencing, based on the procedure of the DNA testing laboratories linked to each institution. All BRCA 1/2 variants were categorized as pathogenic, variants of unknown signi cance (VUSs), or polymorphic (benign).

Mutation nomenclature
All sequence variations are described according to HUGO-approved systematic nomenclature (http://www.hgvs.org/mutnomen/) using GenBank reference sequences (NM_007294.2 for BRCA 1 and NM_000059.3 for BRCA 2). The breast cancer information core nomenclature is also described for convenience.  (Fig. 1). RS numbers were reviewed for 262 and 440 subjects with BRCA1 and BRCA2 mutations, respectively. Table 1 shows the reclassi cation results for VUS according to the ClinVar database. We classi ed the results into four groups: benign/likely benign, VUS, con icting in interpretations of pathogenicity, and pathogenic/likely pathogenic. "Benign/likely benign" was the most common reclassi cation in both BRCA1 and BRCA2 VUS. Table 2 shows the rank of VUS genes based on the number of patients.
Of the 278 patients with BRCA1 VUS, 58 types of VUS were identi ed and 44 types had RS numbers. 20 of these, found in 193 patients, were classi ed as "benign/likely benign;" the least common VUS was classi ed as "pathogenic/likely pathogenic" and comprised 4 types in 6 patients (Table 1).
Of the 453 patients with BRCA2 VUS, 91 types of VUS were identi ed and 78 types had RS numbers. The most common type of VUS was "benign/likely benign", with 25 types in 328 patients, and "pathogenic/likely pathogenic" was the least common and included 2 types of VUS in 3 patients.
In this study, six gene mutations previously classi ed as VUS were reclassi ed as "likely pathogenic" based on We calculated ORs for each variant using the KRGDB as shown in Fig. 2. The OR of BRCA1 c.5339T > C (p.Leu1780Pro) was signi cantly high, although BRCA1 c.5339T > C had con icting pathogenicity interpretations based on ClinVar.

Discussion
We re-evaluated genetic results in patients with VUS in BRCA1 and BRCA2 using the ClinVar database. Because genetic characteristics can vary by ethnicity [7,9,10]. this study aimed to identify the prevalence of VUS in the Korean population and to re-classify the results of initial genetic testing. The KOHBRA study is a multicenter prospective cohort study that aims to identify the prevalence and cause of hereditary breast cancer in the Korean population [12]. Although the initial genetic testing results showed 676 patients with VUS (278 patients for BRCA1 and 453 patients for BRCA2) among 2,403 breast cancer patients (28.13%, 676/2,403), re-evaluation showed a decreased frequency of VUS (8.03%, 193/2,403). About a third of the variants that were originally classi ed as VUS in the KOHBRA study were reclassi ed as benign/likely benign or pathogenic/likely pathogenic, accounting for two-thirds of all VUS patients (71.45%, 483/676). This result suggests that a re-classi cation approach using the ClinVar database can reduce the frequency of VUS in the Korean population.

Pathogenic/ likely pathogenic mutations
In this study, six mutations in nine patients were reclassi ed from VUS to "pathogenic or likely pathogenic". Because all six mutations have su cient evidence in functional studies (PS3) [18][19][20], have not been reported in the genomic database from the general population (PM2), are classi ed as pathogenic in ClinVar (PP5), and are deleterious mutations (PP3) in an in silico study [21], they should be reclassi ed as likely pathogenic or pathogenic mutations. For patients with these mutations, additional genetic counseling and proper management such as familial genetic testing, risk-reducing medications, or risk-reducing surgery are needed for prevention of cancer.

Con icting interpretations of pathogenicity
Calculating ORs Interestingly, we identi ed several cases with "con icting interpretations of pathogenicity" and reviewed the current status of reported evidence (Table 3). Calculating ORs can help to reclassify VUS in BRCA1/2. The KRGDB is built for precision medicine research and is a large-scale, single-race database collected from 1,722 Koreans. We

Functional study
The interpretation of VUS is still complex. Functional studies for reclassifying VUS could be a promising approach.
Traditionally, VUS interpretation depended on inductive conclusions based on information from individual patients [22]. However, many potential variants in BRCA have a low frequency and the phenotype is incompletely penetrant.
Findlay et al. reported the application of saturation genome editing (SGE) to measure the functional outcomes of all possible single nucleotide variants (SNVs) in key areas of BRCA1 [18]. Functional effects were almost concordant with established assessments of pathogenicity. Function scores using SGE could help interpret the signi cance of VUS by providing functional classi cation and assessment of ambiguous or newly-discovered variants.
The four BRCA1 mutations (c.5089T>C (p.Cys1697Arg), c.5509T>C (p. Trp1837Arg), c.5516T>C (p.Leu1839Ser), and c.81-9C>G) identi ed as "likely pathogenic" using the ClinVar database were identi ed as non-functional in Findlay's study [18]. The c.5339T> C (p.Leu1780Pro) variant identi ed as "con icting interpretations of pathogenicity" in the ClinVar database was also identi ed as "non-functional" in the functional study results, which suggests that this variant is pathogenic [18]. On the other hand, other variants with con icting interpretations of pathogenicity in the ClinVar database (including c.154C>T (p.Leu52Phe), c.5068A>C (p.Lys1690Gln), and c.5332G>A (p.Asp1778Asn)) were categorized as functional or intermediate [18]. The other BRCA1 variants with con icting interpretations of pathogenicity in the ClinVar database could not be evaluated according to the Findlay's study because it analyzed only RING and BRCT domains as targets [18].
Patients whose results indicate "con icting interpretations of pathogenicity," should be managed with a multidisciplinary team approach.

L1780P interpretation
With regard to c.5339T>C (p.Leu1780Pro), four reports interpret this as "pathogenic or likely pathogenic" and one report stated that it was of "uncertain signi cance".
When ORs were calculated using the KRGDB for all KOHBRA data, the OR of BRCA1 c.5339T>C (p.Leu1780Pro) was found to be signi cantly elevated (Fig. 2). This variant was also identi ed as non-functional in Findlay's study.
In addition, several studies have suggested that this mutation is pathogenic based on other evidence including strong family history of breast and ovarian cancer, absence in general population data, impaired function demonstrated by in silico studies, and triple negativity in clinicopathologic features [16,17,23]. It is advisable to make management decisions based on 'likely pathogenic' variants. Previous studies have used a similar approach to reclassify some variants [24, 25].

Limitation
One limitation of the study is that we reviewed VUSs by assigning them to a database based on a mostly Caucasian population. Researchers contributing to the SNP database or ClinVar tend to be concentrated in Western countries, leading to a lack of registration of major variants in the Asian population or a lack of interpretation of variants such as L1780P. Nevertheless, it is meaningful that we con rmed VUS status in Koreans using a prospective study and laid the groundwork for broadening our understanding of VUS and conducting further research. Another limitation of the study is that there is missing information in the KOHBRA data that is necessary to reclassify VUS (e.g. 27 variants not submitted to the SNP database). However, lacking data comprised only 4.29% of the cohort and is unlikely to weaken the power of the current study.

Conclusions
Taken together, most of the mutations that were classi ed as VUS in the KOHBRA study were reclassi ed as benign. Six patients with BRCA1 VUS (4 mutations) and three patients with BRCA2 VUS (2 mutations) were reclassi ed as "pathogenic or likely pathogenic". When ORs were calculated using the KRGDB for all KOHBRA data, the OR of BRCA1 c.5339T > C (p.Leu1780Pro) was signi cantly high, although ClinVar considered BRCA1 c.5339T > C to have con icting interpretations of pathogenicity. These seven mutations could be reclassi ed as likely pathogenic or pathogenic mutations according to the American College of Medical Genetics (ACMG) guidelines. Mutations classi ed as benign in ClinVar have a high normal frequency, so it is desirable to judge them as benign.
However, still some VUSs have been reported as having con icting interpretations of pathogenicity rather than being assessed as benign or pathogenic. Their character is expected to be more clearly distinguished when more information is gathered. When VUSs are reclassi ed as pathogenic/likely pathogenic, appropriate management including risk-reducing medication and surgery should be discussed with patients and their families. In addition to collecting individual data, functional studies using genetic techniques such as SGE will also contribute to the functional classi cation and assessment of VUS.  Figure 1 Schematic diagram of patient selection: BRCA1 and 2 (n=55) Figure 2 Odd ratios (ORs) using Korean population data from KRGDB.