Spectrum of BRCA1/BRCA2 variants in 1419 Turkish breast and ovarian cancer patients: a single center study

Introduction

According to the latest cancer statistics reports published in Turkey and all around the world, the most common cancer type in females and the second most common cancer type in both genders, is breast cancer [1]. Ovarian cancer is the second most common cancer among gynecological cancers. Because most of the patients are diagnosed in advanced stages, ovarian cancer is known as the most mortal gynecological cancer [2].

Hereditary breast and ovarian cancer syndrome (HBOC) is a hereditary cancer predisposition syndrome, characterized by increased incidence of breast cancer, ovarian cancer and the other solid tissue tumors in the family, respectively [3]. Other solid tumor cancers commonly seen in BRCA1/2 carriers are pancreas and prostate tumors [4]. It is considered that, 5–10% of all breast and ovarian cancer cases may be hereditary. Approximately 20–25% of HBOC are associated with two susceptibility genes, BRCA1 and BRCA2 [5]. Both BRCA genes are tumor suppressor genes that encode proteins that function in the DNA repairing process [6], [7]. HBOC patients inherit one mutated allele in BRCA1 or BRCA2 from one of their parents, but they have a second, wild type allele. If the second allele becomes non-functional as a result of a somatic mutation, cancer could develop. This is defined as “two-hit hypothesis” [8].

Although the BRCA1 and BRCA2 genes are known as the most competent genes, there are also genes such as TP53, PTEN, CDH1, ATM, CHEK2 and PALB2 which are involved in tumor suppressor mechanisms and cause familial cancer as a result of germline mutations [9]. Women with pathogenic mutations in BRCA1 or BRCA2 genes are at risk of developing breast and ovarian cancer up to 87% and 54%, respectively, until they are 70 years old [10], [11]. The overall population risk in pancreatic cancer is 0.5%. This ratio increases to 1–3% in BRCA1 mutations and 2–7% in BRCA2 mutations. In terms of prostate cancer; the overall population risk is 6% (up to age of 69). In BRCA1 and BRCA2 mutations this ratio increases to 8.6% and 20%, respectively [4]. The exact cancer risk also differs slightly depending on whether the type and the localization of BRCA1 or BRCA2 pathogenic variants. Effected individuals can benefit from prophylactic mastectomy and prophylactic salpingo-oforectomy, chemoprevention and improved surveillance strategies; therefore, identification of mutation carriers is crucial for cancer prevention and control [12], [13].

Especially before the era of next generation sequencing technology, identification of the BRCA status was not easy to access. Up to date, a total of 2987 germline pathogenic or likely pathogenic variants in BRCA1 and 3407 germline pathogenic or likely pathogenic variants in BRCA2 genes have been reported in the NCBI’s ClinVar database. In addition to the recognized pathogenic or likely pathogenic variants, a large number of variants classified as unknown significance (VUS). The rate of VUS variants is influenced by regional differences and the knowledge on common polymorphisms in a national population database. In this sense, a Turkish population genome database that we can use in our study has not been completed yet.

The prevalence of BRCA mutations varies with ethnicity and region [14]. There may be contradictory views about the pathogenic potential of the variants. In order to make a clear interpretation, it is important to provide genetic counseling in the light of detailed clinical information of the patient. Although, data from different populations presented with databases and articles continues to increase day by day, the majority of studies include the prevalence and spectrum of BRCA1 and BRCA2 mutations in European, North American, African, and African-American populations. Hereby, there is a need for better understanding of the mutation spectrum of BRCA1 and BRCA2 genes and cancer risk prediction in Turkish people. For developing national health strategies for genetic screening, it is crucial to determine the spectrum of damaging alterations in causative genes of HBOC and to describe frequent founder mutations.

Materials and methods

Results

Of 1419 patients, 134 (9.4%) were found to carry a pathogenic and 5 (0.3%) were found to carry a likely pathogenic mutation. Of those, 58 patients were found to carry a mutation in BRCA1 and 64 in BRCA2. Altogether, 57 different pathogenic and likely pathogenic mutations were detected. These pathogenic and possible pathogenic variants were found to be equally distributed between the BRCA1 and BRCA2 genes (Tables 1 and 2).

VUS alteration was detected in 91 patients (6.4%) with 64 different variants. The spectrum of pathogenic and likely pathogenic mutations comprises 71 (51.07%) frame-shift variants, 16 (11.5%) large deletions, 1 (0.7%) large duplication, 31 (22.3%) nonsense variants, 10 (7.1%) splice site defects, and 10 (7.1%) missense variants. The spectrum of VUS variants comprises 71 (78%) missense variants, 15 (16.4%) nonsense variants, 4 (4.3%) small in frame deletions/insertions, 1 (1%) splice site alteration (Tables 3 and 4). All the pathogenic, likely pathogenic and VUS variants except for large deletions and duplications are listed in Tables 1–4.

Discussion

Sequence analysis for BRCA1 and BRCA2 genes for detecting the tendency of breast and ovarian cancer is the first cancer genetic test in clinical routine [15]. Dr. Abdurrahman Yurtaslan Ankara Oncology Training and Research Hospital is the only third step, national oncology hospital, which is affiliated with Turkish Ministry of Health. Since 2017, BRCA1 and BRCA2 genetic tests have been studied in our molecular genetics laboratory within the scope of public health services. So far, many centers in different countries have published their BRCA1 and BRCA2 gene mutation spectrum from their in-house data [16], [17], [18]. There are also reports from Turkey, based on Turkish population, but they are so limited in terms of the number of study group (less than 200 individuals) and most of the samples were performed by conventional sequencing method, instead of next generation sequencing systems [19], [20], [21]. This study has the highest number of Turkish patients ever published.

Frequencies of the deleterious variations of BRCA1 and BRCA2 are known to vary between populations. For example, the prevalence of BRCA1/2 in Japan is reported to be 2.6%, while in the US it is as high as 11.1% [22]. Prevalence of BRCA1 and BRCA2 germline mutations is 9.4% in this study. Although the sample size was very low compared to this study, the prevalence of BRCA1/2 in Turkish breast and ovarian cancer patients was between 8 and 19% in previous studies [19], [20], [21]. Moreover, considering that the variant assessment method is standardized with ACMG 2015 criteria, it is understood that some patient results evaluated before 2015 are open to discussion. Deleterious variations in BRCA2 are more than in BRCA1 in this study. This situation can be change in favor of BRCA1 within different populations [23]. There is no definite information that anyone is seen more often.

There may be founder mutations in each population detection of these mutations allow for population-based genetic screening. However, no population has showed founder mutations as striking as those determined for Icelanders and the Ashkenazi Jewry. Interestingly, the founder mutations in Ashkenazi Jewish population, BRCA1:c.185delAG and BRCA2:c.6174delT; and the Icelandic founder mutation BRCA2:c.999del5, were not detected in this study. The most frequent mutations of the study are BRCA1:c.5266dupC and BRCA2:c.1773_1776delTTAT (Tables 1 and 2). These two mutations have the same number of patients, and their total proportion in pathogenic and likely pathogenic mutations is 12.9%. BRCA1:c.5266dupC is also described as one of the most frequent mutation in Bulgaria, Croatia, Egypt, Greece, Iran, Israel, Italy, Lebanon, Macedonia, Romania, Serbia, Slovenia, Tunisia, Uzbekistan, Yugoslavia [24], but there is no evidence about the founder effect of BRCA2:c.1773_1776delTTAT among different populations. The second most frequent mutations are BRCA1:c.2800C>T and BRCA2:c.5969delA, with totally 8.6% of all pathogenic and likely pathogenic mutations of the study. The high frequency of these frequent mutations also appears to be specific to the Turkish population. According to these results, due to the wide spectrum of mutations in Turkish population, it is recommended to perform all exons and introns of BRCA1/2 genes. Although Turkey is at a location adjacent to Europe and the Middle East, Central Asia is based on the origins of Turkish society. In this manner, BRCA mutation spectrum studies of Middle East populations have important differences with this study. None of the most frequent variants in Arab/Middle East populations were found [25], [26].

Exon 10 for BRCA1 and Exon 11 for BRCA2 are the hot-spot regions, concordant with ClinVar and HGMD. No novel mutations were reported in this study. Describing novel variants in BRCA1 and BRCA2 genes is very difficult, because currently 2319 and 2205 variants have been identified in HGMD database for BRCA1 and BRCA2 genes, respectively.

The frequency of large deletions/duplications of BRCA1/2 varies considerably among populations. The highest proportion of BRCA1 large deletions/duplications has been reported from northern Italy, where the large genomic deletions/duplications are 23% of all pathogenic mutations [27]. In contrast, a study from western Denmark, prevalence of BRCA1 deletions/duplications is 3.8% in HBOC cases [28]. This study also is the largest study in terms of the number of patients who underwent MLPA in BRCA1/2 negative patients in the next generation sequencing in Turkish population. In this study, the rate of MLPA positive patients among HBOC patients was 12.2%. This ratio shows that MLPA method should be used as second step diagnostic method in sequence analysis negative patients in order not to skip BRCA positive patients in Turkish population.

Seventy-five percent of the VUS variants detected in our study were on the BRCA2 gene (Table 4). The most common VUS variant, BRCA2:c.9976A>T, causes a nonsense alteration at the penultimate exon. The last 93 amino acids are truncated, as a result of this substitution. This variant was reported in 2327/296226 healthy individuals (including eight homozygotes) at a frequency of 0.0078555. Moreover, some submitters of reputable clinical databases (ClinVar, HGMD) have classified this variant as benign or likely benign. However, the high prevalence of this variant in HBOC cases suggests that it may be a risk factor [29].

BRCA1 and BRCA2 de novo mutation rate was estimated to be 0.3% (0.1%; 0.7%) [30]. In this context, family screening with Sanger sequencing was performed for all of the BRCA1/2 positive patients, and it was detected that all the mutations is segregated from the parents of the patients.

In conclusion, the spectrum of BRCA1/2 mutations in the Turkish population has been shown in the largest patient group to date. The thesis that founder mutations show diversity in different populations has been confirmed in our study and the mutations that are common in Turkish population have been presented in this study. In addition, the proportion of large BRCA rearrangements in Turkish HBOC patients was found to have an average value (12.2%) among different reported populations (3–23%). The controversial VUS variants were compared with the Turkish patient data, and this contributed to the understanding of the risk of these conflicting variants. It is thought that the studies to be performed in larger patient groups will be complementary in understanding BRCA mutations in Turkish population in the future studies.