The frequency of identified TP53 clinically significant germline variants was 0.4% (13/3455) and in all cases TP53 mutations were identified in females with breast cancer. Therefore, the proportion of TP53-associated breast cancer within all breast cancer cases in our cohort was 0.44% (13/2957), consistent with existing literature [19]. The predominance of this localization of cancer types is attributed to the high prevalence of breast cancer patients in our cohort and the elevated risk of breast cancer in the spectrum of tumors typical for LFS.

Among patients with breast cancer aged up to 31 y.o., the proportion of carriers of pathogenic TP53 variants was 3.0% (3/100), aligning with previously reported rates of 3.8%-6.0% for patients ≤ 31 y.o. [19, 20].

According to the TP53 Database (version R20, ISB-CGC, https://Tp53.isb-cgc.org/), the most frequent germline variants in the TP53 gene are located at positions Arg175, Arg213, Gly245, Arg248, Arg273, Arg282, and Arg337, accounting for 40% (937/2358) of missense mutations in the database. Excluding the position Arg337, characteristic of the southern Brazilian population, these positions account for 32% of mutations. In our study, variants categorized as frequent according to the TP53 Database accounted for 30.8% of the identified TP53 variants: c.524G > A (Arg175; n = 1; 7.7%), c.743G > A (Arg248; n = 2; 15.4%), and c.818G > A (Arg273; n = 1; 7.7%). The majority of TP53 variants in our study, consistent with scientific literature, were represented by missense variants within the DNABD domain.

One TP53 variant identified in our study absent in the GnomAD population frequency database and are not described in literature or databases. In patient P02, harboring the variant c.376-1G > C in intron 4, the family history and clinical features align with Chompret criteria (Fig. 4). Scientific literature describes a different nucleotide substitution in the same DNA position—c.376-1G > A, clinically significant, disrupting the canonical splice acceptor site and detected in patients exhibiting features of LFS [21,22,23,24]. Such genetic alterations predominantly result in protein loss of function (LOF) [25]. Based on this data, the previously undescribed variant is annotated as pathogenic.

In the present study, it was observed that a significant proportion of TP53 pathogenic variants appear to occur outside of LFS families, which is a noteworthy finding. However, a recent report from a larger study indicates a higher proportion of carriers complying with the modified Chompret criteria [26]. This discrepancy may be attributed to the lower sensitivity of the Chompret criteria in our study, which could be due to the small number of patients in the group of TP53 mutation carriers. A larger cohort may provide a more comprehensive understanding of the relationship between TP53 variants and compliance with the Chompret criteria, highlighting the need for further investigation in this area.

Our study revealed two pairs of unrelated patients harboring described pathogenic variants. The first pair (P05 and P06) exhibited variant c.542G > A in exon 5, leading to the p.Arg181His substitution. The minor allele frequency is 0.00001314 (GnomAD Genomes). Patient P05, aged 38, was diagnosed with unilateral breast cancer, whereas patient P06, aged 47, was diagnosed with synchronous bilateral breast cancer. Both reported familial history of breast cancer and lung cancer (Fig. 4).

The second pair (P08 and P09) exhibited variant c.743G > A in exon 7, resulting in the p.Arg248Gln substitution. The substitution at position Arg248 is the most prevalent according to TP53 Database, accounting for 9.1% of all described germline pathogenic TP53 variants. Patient P09, aged 49, had unilateral breast cancer, while patient P08 had Hodgkin's lymphoma at age 20 and breast cancer at age 29. Both reported a familial history of breast cancer (Table 4).

Our study involved 138 patients with synchronous/metachronous bilateral breast cancer, accounting for 4.7% (138/2957) of all breast cancer patients. Among TP53 mutation carriers, 23.1% (3/13) exhibited bilateral breast cancer (OR = 5.9289, 95% CI: 1.6130–21.7933, p-value = 0.0074). Kwong A. et al. [27] results indicated an OR = 7.0011 (95% CI: 2.8449–17.2292, p-value < 0.0001), aligning with our findings. Thus, synchronous/metachronous bilateral breast cancer is significantly more prevalent among breast cancer patients with TP53 gene mutations compared to those without mutations.

In the study by Giacomelli AO et al. [17] an experimental assessment of various TP53 gene mutations impact on p53 protein function was demonstrated. Several thousands malignant tumor cell lines with different TP53 mutations were analyzed, subjected to substances activating p53: nutlin-3 (inhibitor of MDM2 and p53 binding) or etoposide (topoisomerase II inhibitor, causing DNA damage). The presence of Dominant Negative (DN) and Loss of Function (LOF) effects for each mutation was determined through experiments. Analyzing TP53 Database data reveals that these characteristics statistically significantly influence the age of disease manifestation (Fig. 5). For breast cancer patients the average age of manifestation is 38.9 y.o. with LOF + (95% CI: 36.4–41.3) and 53.6 y.o. (95% CI: 47.0–60.1) with LOF— mutations. This pattern holds true for malignant tumors at other locations. For lung cancer, the average age of manifestation is 50.7 (95% CI: 44.3–57.1) and 60.4 (95% CI: 52.2–70.7) y.o., and for brain cancer, it is 22.5 (95% CI: 18.5–26.6) and 34.9 (95% CI: 25.0–44.8) y.o. (LOF + and LOF- respectively).

Age of manifestation of cancer depending on the location and functional type of mutation. LOF ± – presence or absence of the loss of function effect according to Giacomelli et al. [17], NF is a non-functional variant, PF is a partially functional variant according to data from [18]. The majority of mutations in the TP53 gene identified in our study are classified as LOF + /NF. LOF—/PF mutations were detected in 5 patients (P05, P06, P07, P11, P12). Given the wide age range at manifestation for each functional mutation type, this parameter does not allow to perform precise prognosis for a specific carrier. However, it may be valuable for assessing the clinical significance of the variant and evaluating the risk of early manifestation of cancer in a family

In the study by Kato S et al. [18] the assessment of p53 protein function with different mutations was conducted using a different method. For each TP53 mutation in yeast culture the median transcriptional activity of p53 was calculated across 8 specific promoters (activity expressed as a percentage of wild-type protein activity). Missense variants were classified as "non-functional" if the median was ≤ 20%; "partially functional" if the median was > 20% and ≤ 75%; "functional" if the median was > 75% and ≤ 140%; and finally, "super-functional" if the median was > 140. According to scientific literature in TP53 Database most breast cancer patients with breast cancer are carriers of non-functional (107/133) or partially functional (21/133) variants, with an average age of tumor manifestation of 39.2 (95% CI: 36.7–41.8) and 48.7 (95% CI: 42.3–55.1) y.o. respectively. Functional and super-functional variants (5/133) in breast cancer patients are classified as benign or likely benign according to ACMG criteria.

Thus, functional features of the p53 protein determined in cell culture experiments can help determine the clinical significance of different TP53 mutations.