Introduction

Breast cancer (BC) is the most common female malignancy worldwide. Mutations in BRCA1 and BRCA2 genes are responsible for hereditary BC. Individuals with inherited mutations in BRCA genes should be offered the risk-reduction strategies, such as screening (mammography and breast magnetic resonance imaging), surveillance (clinical breast examination, breast self-examination), chemoprevention, and risk-reduction surgery [1].

BC is also caused by mutations in the TP53, ATM, CDH1, PTEN, and STK11 genes associated with hereditary syndromes. Mutations in the genes mentioned above can inhibit DNA repair pathways [2, 3].

In Russians, mutations of BRCA1/2 genes were found only among Slavic women (newcomers), and were not found in Khakas, Buryats, Tyvans, Yakuts and others women (indigenous) [4, 5]. However, the cancer burden in Khakas, Buryats, Tyvans has risen and the cancer risk assessment has been limited [6,7,8]. The main purpose of the study was to identify molecular changes associated with hereditary BC in Russian Mongoloid BC patients.

Materials and methods

Thirty-nine patients were included in the study. The median age of patients at BC diagnosis was 42 years (range: 26–55). Eighty-one percent of patients were diagnosed with BC before the age of 50. More than one-third of patients under the age of 50 had a family history of BC. Almost all tested women were diagnosed with invasive (ductal) carcinoma of no special type. Information, including age at diagnosis, family history, histological type of cancer, and family origin was obtained. Reported clinical characteristics of the patients are given in Table 1.

Table 1 Clinical characteristics of the study cohort
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Blood samples were collected in ethylenediaminetetra-acetic acid-containing tubes. Genomic DNA from peripheral blood was extracted using the phenol–chloroform method. Purity of the DNA was assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA) and then quantified using the Qubit 2.0 fluorometer and HS dsDNA Assay Kit (Thermo Fisher Scientific, USA). Integrity of the DNA (DIN) was verified on a 2200 TapeStation system (Agilent, USA). The positive control sample with BRCA1 c.3755_3758delTGTC pathogenic mutation was included as an inner control.

DNA library were prepared using the Hereditary Cancer Solution™ kit (Sophia GENETICS, Switzerland) to cover 27 genes: ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PMS2CL, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, and XRCC2. Paired-end sequencing (2 × 150 bp) was conducted using NextSeq 500 system (Illumina, USA). The pathogenic variants were validated using Sanger sequencing (SeqStudio, Thermo Fisher Scientific, USA).

Bioinformatics analysis

Sequencing data was analyzed according to the GATK best practice recommendation for Whole Exome Sequencing using GRCh37 as a reference for Burrows-Wheeler alignment. The obtained variants were annotated with ANNOVAR software and ranged according to population frequency (genomic exome, gnomAD genome, and ExAC), ClinVar, CADD, and literature data [9,10,11]. Detected sequence variants were annotated using PolyPhen2, Mutation Taster, and SIFT [12,13,14].

Results

In our study, 8% (3/39) of patients harbored one pathogenic variant and 15% (6/39) of patients harbored likely pathogenic variant. In addition, 8% of patients had VUS, 15% had conflicting variants and 54% had only benign variants (Fig. 1a).

Fig. 1
figure 1

a Proportion of unrelated BC patients by functional status of the observed gene variants in the coding regions and b distribution after grouping by age of onset

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It should be noted that the pathogenic variants were found in two non-BRCA1/2 susceptibility genes and were diagnosed only in BC patients under 45 years old (Figs. 1b, 2). Table 2 illustrates variants that are described as highly pathogenic by dbPubMed, likely pathogenic (possibly/probably damaging by PolyPhen2 or deleterious by SIFT).

Fig. 2
figure 2

Sequenograms of the RAD51D (rs137886232, NC_000017.10:g.33428366G>A, pathogenic variant) (a) and PTEN (rs786201044, NC_000010.10:g.89692922T>C, pathogenic variant) (b) genes

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Table 2 Functional annotation of the identified genetic variants in the Buryat BC patients
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The RAD51D variant (rs137886232) was observed in two unrelated individuals. One of these patients had a family history of late-onset stomach cancer in second-degree relatives. Another pathogenic variant was observed in the PTEN gene (rs786201044) in a BC patient aged 38 with no family history of BC.

Variants in the ATM, MSH6 and MLH1 genes (likely pathogenic) were previously described as VUS by dbPubMed, but were predicted as probably damaging by PolyPhen2 and/or deleterious by SIFT. The probably damaging variants (PolyPhen2) in the ATM (rs150757822) and MSH6 (rs142254875) genes were observed in a 49-year-old patient with a burdened family history and in a 48-year-old patient with an unknown family history, respectively. One MLH1 variant (rs4986984) was classified as probably damaging (PolyPhen2) and deleterious (SIFT), and was found in two patients diagnosed with BC prior to 46 years and in a 52-year-old patient with a burdened family history. Another probably damaging/deleterious variant in the MLH1 gene (rs367654552) was found in a 55-year-old patient with a burdened family history.

Rare genetic variants classified by dbPubMed as VUS are given in Table 3. All variants presented in Table 3 were considered benign by PolyPhen2 or tolerated by SIFT. One missense VUS (rs80359254) was identified in the BRCA2 gene. VUS were most commonly encountered in the ATM (n = 3), MSH6 (n = 2) and MLH1 (n = 3) genes.

Table 3 Variants of uncertain clinical significance (VUS)
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The rs367654552 of the MLH1 gene was previously described only in the East Asia, whereas the rs80359254 variant of the BRCA2 gene was observed exclusively in Europeans. The rs150757822 and rs1800058 variants of the ATM gene and the rs104894994 variant of the MLH1 gene were found in South Asian populations but not in the East Asian populations. In general, 77% (10 out of 13) of the identified variants were previously found among Europeans (Table 4).

Table 4 Frequencies of non-synonymous pathogenic variants and VUS in different populations
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No mutations were found in the MRE11A, PIK3CA, RAD51C or XRCC2 gene.

4. Discussion

Mongoloid population is the most prevalent among all human populations [15]. In accordance with the Asian BRCA Consortium data, there is a significant difference in incidence rate of BC depending on age, as well as spectrum and prevalence of BRCA1/2 mutations and clinical significance of rare VUS between Mongoloid (East Asian) and Caucasoid (European) people [16,17,18,19,20]. However, European strategies to identify familial BC are still applied to the Asian population, including Mongoloids.

There are more than 200 different ethnic groups in Russia. Most of the population in Russia includes Russians (81%), the largest ethnicities are Tatars, Belarusians, Ukrainians, Bashkirs, Chuvash, Chechens, and Armenians (up to 10%), and smallest nationalities include Kazakhs, Yakuts, Buryats, Ingush, Udmurts, Ossetians and others (up to 0.5% of each) [5]. In our study, we continued to search for mutations in BRCA-negative BC women living in Russia (Buryat). Overall, two pathogenic germline variants in RAD51D and PTEN were found in 8% (3/39) of patients under 40. In addition, 8% of patients had VUS, 15% had conflicting variants and 54% had only benign variants.

The pathogenic variant of RAD51D gene (rs137886232) with low minor allele frequency was observed in two young unrelated Buryat patients. The germline mutation of RAD51D gene (rs137886232) was suggested to have a founder effect in Chinese population [21]. This variant was also described in BC families of European ancestry. It was also reported that RAD51D-deficient tumor cells were sensitive to poly-(ADP) ribose polymerase inhibitors [22].

The pathogenic variant of the PTEN gene (rs786201044), which was predicted to be damaging by in silico analysis, was observed in BC patient aged 38 with no family history of BC. Different studies also reported on this variant (rs786201044) of the PTEN gene in families with Cowden syndrome (an autosomal dominant inherited disorder) [23,24,25,26]. Previous studies have also found that this variant (rs786201044) of the PTEN gene may impact on protein stability and lead to increased proteasome activity [27,28,29,30,31].

Interesting, germline variants of RAD51D and PTEN genes were also described in the COSMIC database as somatic mutations, COSM4721157 and COSM5096, respectively.

In 54% of the patients, no clinically significant variants were identified, probably due to some limitations to our study. In particular, we used a panel of only 27 genes that did not include other BC-predisposing genes, such as BLM, ESR1, FANCA and NQO2.

In this study, over 20% of the Buryat BC patients were found to carry a rare VUS. Buryats are characterized by molecular diversity due to the long generation time or the mixed nature of origin compared with other ethnic groups living in Siberia [32,33,34,35]. It is obvious that a more detailed genetic analysis of the Buryats is required.

Conclusion

In this study, we provide the first description of two pathogenic germline variants in the RAD51D (rs137886232) and PTEN (rs786201044) genes in BRCA1/2-negative Mongoloid (Buryat) women with BC.