ABSTRACT

Objective:

Breast cancer (BC) is the most common cancer type in women and may be inherited, mostly in an autosomal dominant pattern. The clinical diagnosis of BC relies on the published diagnostic criteria, and analysis of two genes, BRCA1 and BRCA2, which are strongly associated with BC, are included in these criteria. The aim of this study was to compare BC index cases with non-BC individuals in terms of genotype and diagnostic features to investigate the genotype/demographic information association.

Materials and Methods:

Mutational analyses for the BRCA1/BRCA2 genes was performed in 2475 individuals between 2013-2022 from collaborative centers across Turkey, of whom 1444 with BC were designated as index cases.

Results:

Overall, mutations were identified in 17% (421/2475), while the percentage of mutation carriers in cases of BC was similar, 16.6% (239/1444). BRCA1/BRCA2 gene mutations were detected in 17.8% (131/737) of familial cases and 12% (78/549) of sporadic cases. Mutations in BRCA1 were found in 4.9%, whereas 12% were in BRCA2 (p<0.05). Meta-analyses were performed to compare these results with other studies of Mediterranean-region populations.

Conclusion:

Patients with BRCA2 mutations were significantly more common than those with BRCA1 mutations. In sporadic cases, there was a lower proportion with BRCA1/BRCA2 variants, as expected, and these results were consistent with the data of Mediterranean-region populations. However, the present study, because of the large sample size, revealed more robust findings than previous studies. These findings may be helpful in facilitating the clinical management of BC for both familial and non-familial cases.

Key Points

• Breast cancer

• BRCA1

• BRCA2

• Genomic profiling

• Population study

Introduction

Breast cancer (BC) is a condition affecting approximately two million people per year, globally. The incidence is estimated as 1:8 in women and 1:833 in men (1). The clinical diagnosis of BC relies on the published diagnostic criteria (2). Two genes have been identified as being strongly associated with BC but not all cases are due to inherited factors. These two genes are breast cancer (BRCA) 1 and BRCA2. The BRCA1 gene, located on chromosome 17, codes for breast cancer type 1 susceptibility protein. This gene has 22 exons distributed over approximately 110 kb of genomic DNA. In contrast with the BRCA1 gene, the BRCA2 gene has 27 exons over approximately 84.2 kb of genomic DNA on chromosome 13 (3). To date, more than 3242 disease-causing mutations have been identified in either BRCA1 or BRCA2 (4). It has been suggested that patients with BC without detected variants in BRCA1 or BRCA2 probably have mutations on other cancer related genes or large gene deletions, somatic mosaic mutations, and mutations in un-analysed gene noncoding regions of BRCA1 and/or BRCA2 (1, 2).

There is clinical interest in whether the phenotypic presentation of BC differs depending on disease-causing variants in BRCA1 or BRCA2. Early studies from Mediterranean countries, even the population-based studies, which have reported genotype/phenotype correlations have not found any evidence for phenotypic differences between patients with BRCA1 mutations vs. patients with no identified mutation or between patients with BRCA1 vs. BRCA2 mutations (4, 5, 6, 7, 8). These studies, however, tend to have relatively small sample sizes. The largest and most recent studies showed BRCA2 was found more frequently in individuals with BC in the region. The main studies included patients without family history but are also limited by the low number of index cases in the study group. On the other hand, BRCA2 positivity reported with relatively higher frequencies in the Mediterranean region of Turkey when compared with other international studies (9, 10).

In this study, mutational analysis for the BRCA1 and BRCA2 genes was performed in 2475 individuals, of whom 1444 had been diagnosed with BC and were considered index cases. Comparisons were then made between BC patients and those without BC and between patients with by BRCA1 or BRCA2 variants in terms of diagnostic and demographic features to describe the genotype/demographic association in BC in this population. Mutation type, either protein truncation or missense, was also compared in terms of phenotypic features, as well as with the probands with positive family histroy. These latter comparisons were made to determine whether there was additional prognostic information that can be provided to families, based on genetic test results or mode of inheritance.

Materials and Methods

Results

Discussion and Conclusion

Mutations were sought in all coding exons and exon-intron junctions of the BRCA1 and BRCA2 genes in DNA from 2475 diagnosed and screening patients from Turkey and 221 (51.3%) previously reported pathogenic mutations, 142 (33%) VUS and 15 (3.7%) novel mutations were found, while the overall BRCA1 and BRCA2 mutation detection rate was 9.9%.

No mutation in BRCA1/2 could be identified in 82.9% of all patients. Despite being one of the largest cohorts of BRCA1/2 screening in the literature, as a limitation of our study, we were not able to examine gross deletion and duplication status of BRCA1 and BRCA2 genes in all mutation negative patients due to different infrastructures of collaborative centers. As noted in previous studies, the mutation detection rate varies from 2.7% to 19% for patients with positive family history but without clinical information in different populations (8, 9, 13).

One of the main focuses of this study was to pool a nationwide Mediterranean country dataset that will increase the power of further analysis for clinical interpretations, both in familial and non-familial cases and the cases with BRCA1 and BRCA2 mutations.

In multifactorial disorders such as cancers, correlation between genotype variation and demographic information is not as well understood as it is in Mendelian disorders. Analysis and interpretation of genetic test results should be considered with the patient’s clinical and family history. This study also showed that a significant percentage of BRCA1 and BRCA2 variations are still classified as VUS. Thus, improvement of genetic variation databases is crucial for correct diagnosis. In the light of the fact that the genotype and phenotype correlation for BC is still controversial, these results can enhance our knowledge on this complicated, common and severe condition.

It was also observed that the most common mutations in the BRCA1 and BRCA2 genes in a representative Turkish population were not among the 10 most common mutations that were reported in a study that included all continents. BRCA1 c.1444_1447delATTA p.I482* and BRCA2 c.7689delC p.H2563Qfs*85 mutations can be considered to be founder mutations for Turkish population and a screening program can be planned for early diagnosis of BC (14).

We also demonstrated the importance of looking at the frequency of each variant per specific ethnic groups as opposed to the overall gnomAD frequency. Our analysis highlighted 56 pathogenic variants that had MAF ≤0.001 (Minor Allele Frequency) in the aggregated gnomAD population but were common in our population. Furthermore, when a more stringent MAF cut-off value (≤0.0001) was used, 123 pathogenic variants should be re-classified as more frequent and might be suggested as founder mutations for our population. In brief, these data also suggest that a number of variants still classified as pathogenic are not truly disease causing or the variants with the higher observed frequency are not truly benign.

The overall BRCA1/2 mutation detection rate for patients with BC in Turkey was 9.9% in this study. The proportion of BRCA1 to BRCA2 mutations was approximately 2 to 2.5 for BC cases. Moreover, in patients with no family history of BC, BRCA1 mutations accounted for 34.6% and BRCA2 mutations accounted for 65.4% among mutation positive cases. Our study summarizes the interpretation process using the most important criteria as per ACMG guidelines, gene specific databases for analysis of the variant frequencies in the largest available population, together with local datasets and results of the computational predictions for a broadly representative but heterogeneous Turkish population.

Patient Characteristics

Patients with a diagnosis of BC and healthy individuals with family history of BC were enrolled between 2013 and 2020 with informed written consents. The study was approved by the institutional review boards of all participating universities and the ethics board at Cukurova University. All the cases were diagnosed with invasive ductal BC with no other types of cancers or any other precancerous conditions. Similarly, individuals that were studied for screening were not affected with any other malignancies. For the familial studies, individuals who had family history of invasive ductal BC were included. Patient selection was made according to the American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines (11, 12). Enrolled patients were evaluated by all our collaborators from Turkey, including from the Mediterranean, Aegean, Black Sea, Central Anatolia, Marmara, Eastern Anatolia, and Southeastern Anatolia Regions, and also from Northern Cyprus. As this study was conducted retrospectively, patient selection criteria were re-evaluated according to the up-to-date ASCO and NCCN guidelines prior to genetic analyses. The goal was to identify if variants were present in the BRCA1 and BRCA2 genes in these patients with invasive ductal BC. For familial BC cases that were enrolled, we included only the index patients for phenotypic analyses.

Subjects were enrolled in our research protocol through six different centers across Turkey and Cyprus. Clinical information was not available for every feature of BC on every participant. Some patients were referred and enrolled in the mutation screening process without sending sufficient clinical information to determine diagnostic status. Some of other demographic data, such as ethnicity, were not included due to the heterogeneity of the Turkish population, and lack of the consent of the majority of patient cohort further information. Patients who had no information, such as family history, were not included in the demographic analysis. We have, however, included them in the description of the mutations. Patients who were under 18 years of age, who were all index cases, were included. Moreover, patients who were under 30 years of age and carried TP53 mutations were excluded, due to the purpose of our study.

Screening and Classification of Genetic Variations

DNA was extracted from peripheral blood lymphocytes of both healthy individuals and BC cases. Next generation sequencing was performed for all coding exons and exon-intron junctions of the BRCA1 and BRCA2 genes. In addition, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed for 591 AGENTEM’s primary index patients, as this is the national reference center for BRCA1/BRCA2. MLPA assay was not performed in the other collaborative centers. Nucleotide change was considered as pathogenic, a polymorphism or a variant of unknown significance (or unclassifiable variant) when it was novel and parents were unavailable for study. American College of Medical Genetics and Genomics (ACMG) criteria were followed for variant classification. The variations that were not identified in the Human Gene Mutation Database (HGMD) and The Single Nucleotide Polymorphism Database (dbSNP) or any other clinical databases (ClinVar and VarSome) were assessed as novel changes. Novel variants were then investigated through in silico analysis for variant classification. In silico analysis tools, including PolyPhen, Mutation Taster, CADD, SIFT, BLOSUM, PhyloP, GeneSplicer, B-SIFT, MaxEntScan, QCI Inferred Activation, BayesDel, DANN, SpliceAI, GenoCanyon, fitCons, MUT Assesor, Varity, FATHMM-XF, FATHMM-MKL, EIGEN PC, LRT were used, based on the genomic location, population frequencies, type and possible impacts on protein of the variations.

Statistical Analysis

The BC disease features for the following groups were compared using student’s t test: (1) gene loci mutated BRCA1 versus BRCA2 and (2) familial versus sporadic using Graph Pad Prism (8.0.1.) Patient clinical findings were analyzed after grouping by gender, familial or sporadic, and location of mutation in BRCA1 or BRCA2. As patients came from different sources and may not have all demographic criteria assessed, the numbers for each analysis varied. Only information from patients with a definite diagnosis was used for statistical analyses.

Population Comparison

GnomAD v2.1.1 data set (GRCh37/hg19) was used for the population comparison, which spans 125,748 exome sequences and 15,708 whole-genome sequences from unrelated individuals. The highest frequency of specific genetic alteration in gnomAD data set from various populations was used in order to compare our results with global data.

GnomAD v2.1.1 data set is the largest publicly available population data to date, and categorizes the populations as follows; African/African-American, Amish, Latino/Admixed American, Ashkenazi Jewish, East Asian, South Asian, Middle Eastern, European (Finnish), European (non-Finnish) and other. However, the proportion of the gnomAD population did not cluster with any of the Mediterranean populations. Therefore, it is more likely that Mediterranean populations were classified as “other”, which includes individuals of mixed background, as in Turkey.

The MAF cut-off of 0.001 that is recommended for variant discovery in dominant inherited Mendelian diseases was used to classify variants as rare frequency (MAF ≤0.001) supporting variants’ pathogenic effect, and common frequency (MAF ≥0.001) which are unlikely to be causative.

Patient Characteristics

BRCA1 and BRCA2 mutational analyses were performed in 2475 subjects. However, we were unable to curate all data for phenotypic features and not all subjects were interviewed for family history. Therefore we include 1444 (58.3%) cases contributing to results. Among 1444 BC patients, 737 (51%) of them had positive family history while 549 (49%) cases had no invasive ductal BC in their family. In the remaining patients (n = 158), family history of BC was unknown. Among BRCA1/BRCA2 positive patients with a definite diagnosis, identification of a genetic alteration for familial patients was higher (54.8%; 131/239) than for patients with sporadic BC (32.6%; 78/239), and this was significant. The remaining variant positive patients (n = 30) were the individuals with unknown familial history of BC.

The median (range) age for all index patients (n = 1444) was 51.5 (15-88) years, and the average age was 48.6 years. Figure 1 details the demographic characteristics of our study population.

Mutation Analysis

Pathogenic mutations were identified in 218 individuals and variants of unknown significance for 139; in affected BC cases 114 of them had pathogenic variants and 85 cases had VUSs. Total variants, their pathogenicity, and internal frequencies are given in supplementary data (Supplementary Table 1). No genetic change could be identified for 2054 patients (82.9%) in total, and for 1205 (83.5%) of the BC cases. Among 737 BC cases with positive family history, 36 cases (4.9%) had variations in BRCA1 and 95 cases (12.89 %) had variations in BRCA2, while 6 (4.6%) patients had genetic alterations in both genes resulting in a BRCA1:BRCA2 ratio of 1/2.6. Twenty-seven of 549 patients (4.9%) without family history had variants in BRCA1 and 51 patients (9.2%) had variants in BRCA2, resulting in a BRCA1:BRCA2 ratio of approximately 1/2.

The mutations identified in BRCA1 and BRCA2 genes in all 2475 individuals were distributed as follows: 51.3% pathogenic, 15.5% likely pathogenic and 33% VUS (Table 1). Variant classifications for affected BC cases are also shown separately in Table 1.

The most frequent variants that were detected in both BRCA1 and BRCA2 are listed in Table 2. The most frequent variants were distributed equally across both genes. From the perspective of pathogenicity, pathogenic variants were present relatively more frequently, with nine variants. Novel genetic variations in both BRCAs are listed in Table 3. In contrast with the frequent variant list, BRCA2 was more commonly found to be the site of novel variants with 14 versus one novel variant in BRCA1.

Clinical Features and Demographic Comparisons

The distribution of family history and the gender of cases for BC patients in this study are listed in Table 4. Phenotypes of these patients were compared by gender and mutation. Observed frequencies of clinical features listed in Table 5 for BC patients in this study. A proportion of cases were male, 20 of 1444 (1.39%) and pathogenic variations in BRCA2 were present in two of the male BC patients. In this multicenter study, other demographic data, such as ethnicity, were not included due to the heterogeneity of the Turkish population and other legal issues in terms of the law on protection of personal data.

Subjects With A Positive Family History Versus Sporadic BC Cases

The phenotypic effects of mutation between the BRCA1 gene and the BRCA2 gene and BC features were investigated in 737 familial index patients and 549 sporadic BC patients. The median age was 52 years for familial index patients and 48.5 years for sporadic BC patients with average ages of 43.3 and 43.5 years, respectively. Comparison of the disease features of these two groups did not show any significant difference. However, patients with a positive family history were more likely to harbor BRCA1/2 gene mutations than sporadic BC patients.

Impact of Mutation Types

The type of mutations in many genetic related disorders affects disease severity. To evaluate the effect of the type of mutations on the presence of BC features, we compared features of patients.

The proportions of mutations types detected are listed in Table 6.

Allele Frequency Comparison

Among a total of 220 different types of detected variations, 190 (86.4%) of them had higher allele frequencies than their aggregated gnomAD allele frequency. With a 0.001 MAF cut-off, 134 (60.9%) of the 220 variants were evaluated as rare and as all of them showed higher frequency in our study, they were considered as more likely to be pathogenic. In addition, 73.7% (56/76) of the globally common variants (MAF ≥0.001) were more frequent in our study while 20 (26.3%) showed lower frequencies than aggregated gnomAD. Distribution of common (MAF ≥0.001) and rare BRCA1/2 variants (MAF ≤0.001) by gnomAD population and the aggregated gnomAD are given as supplementary data (Supplementary Table 2).

The frequencies of pathogenic variants and VUSs were compared across several ethnic groups and the local whole exome sequencing databases. The analysis showed that out of 28 pathogenic variants located in BRCA1, 31 occurred as a higher frequency than aggregated gnomAD data and distinctive populational gnomAD data. Details are given in supplementary data (Supplementary Table 2).