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 . 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 classified as VUS in the KOHBRA study were reclassified 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-classification approach using the ClinVar database can reduce the frequency of VUS in the Korean population.
About two thirds of the VUS in the KOHBRA study were reclassified as benign or likely benign (193/278 (69.42%) of BRCA1 patients, 328/453 (72.41%) of BRCA 2 patients, and 471/676 (69.67%) of all patients). A third of the mutation types classified as VUS in the KOHBRA study were downgraded to “benign or likely benign” (20/58 mutations in BRCA1 and 25/91 in BRCA2, 30.2%). These results were consistent with previous studies [1, 15, 16]. So et al. reported that 30/75 (40%) VUS patients were reclassified to “benign or likely benign” . In our study, six patients with BRCA1 VUS (4 mutations) and three patients with BRCA2 VUS (2 mutations) were reclassified as “pathogenic or likely pathogenic”.
Pathogenic/ likely pathogenic mutations
In this study, six mutations in nine patients were reclassified from VUS to “pathogenic or likely pathogenic”. BRCA1 c.5089T>C (p.Cys1697Arg), BRCA1 c.5509T>C (p. Trp1837Arg), BRCA1 c.5516T>C (p.Leu1839Ser), and BRCA1 c.81-9C>G were interpreted as likely pathogenic in BRCA1. BRCA2 c.8023A>G (p.Ile2675Val) and BRCA2 c.9004G>A (p.Glu3002Lys) were interpreted as pathogenic/ likely pathogenic in BRCA2 based on the ClinVar database.
Because all six mutations have sufficient evidence in functional studies (PS3) [18-20], have not been reported in the genomic database from the general population (PM2), are classified as pathogenic in ClinVar (PP5), and are deleterious mutations (PP3) in an in silico study , they should be reclassified 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.
Conflicting interpretations of pathogenicity
Interestingly, we identified several cases with “conflicting 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 calculated ORs using the KRGDB (Figure 2). Several mutations (7 BRCA1 and 9 BRCA2 mutations, orange colored dots) were evaluated. Most of them showed no significance. However, BRCA1 c.5339T>C (p.Leu1780Pro) showed possible pathogenicity, while BRCA1 c.154C>T (p.Leu52Phe) and BRCA2 c.964A>C (p.Lys322Gln) were potentially benign. BRCA 1 c.5014_5016delCAC, BRCA 1 c.5332G>A, BRCA2 c.182T>C, BRCA 2 c.1909+22delT, BRCA 2 c.8486A>G, and BRCA2 c.8954-5A>G have no reports in the general population; they should remain classified as VUS because there are no reports on their deleterious function.
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 . 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 . Functional effects were almost concordant with established assessments of pathogenicity. Function scores using SGE could help interpret the significance of VUS by providing functional classification 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) identified as “likely pathogenic” using the ClinVar database were identified as non-functional in Findlay's study . The c.5339T> C (p.Leu1780Pro) variant identified as “conflicting interpretations of pathogenicity” in the ClinVar database was also identified as “non-functional” in the functional study results, which suggests that this variant is pathogenic . On the other hand, other variants with conflicting 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 . The other BRCA1 variants with conflicting 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 .
Patients whose results indicate "conflicting interpretations of pathogenicity," should be managed with a multi-disciplinary team approach.
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 significance”.
When ORs were calculated using the KRGDB for all KOHBRA data, the OR of BRCA1 c.5339T>C (p.Leu1780Pro) was found to be significantly elevated (Fig. 2). This variant was also identified 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].
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 confirmed 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.