Identification of a novel pathogenic variant in PALB2 and BARD1 genes by a multigene sequencing panel in triple negative breast cancer in Morocco

Pathogenic variants (PVs) in BRCA genes have been mainly associated with an increasing risk of triple negative breast cancer (TNBC). The contribution of PVs in non-BRCA genes to TNBC seems likely since the processing of homologous recombination repair of double-strand DNA breaks involves several genes. Here, we investigate the susceptibility of genetic variation of the BRCA and non-BRCA genes in 30 early-onset Moroccan women with TNBC. Methods: Targeted capture-based next generation sequencing (NGS) method was performed with a multigene panel testing (MGPT) for variant screening. Panel sequencing was performed with genes involved in hereditary predisposition to cancer and candidate genes whose involvement remains unclear using Illumina MiSeq platform. Interpretation was conducted by following the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) criteria. Results: PVs were identified in 20% (6/30) of patients with TNBC. Of these, 16.7% (5/30) carried a BRCA PV [10% (3/30) in BRCA1, 6.7% (2/30) in BRCA2] and 6.6% (2/30) carried a non-BRCA PV. The identified PVs in BRCA genes (BRCA1 c.798_799delTT, BRCA1 c.3279delC, BRCA2 c.1310_1313del, and BRCA2 c.1658T>G) have been reported before and were classified as pathogenic. The identified founder PVs BRCA1 c.798_799del and BRCA2 c.1310_1313delAAGA represented 10% (3/30). Our MGPT allowed identification of several sequence variations in most investigated genes, among which we found novel truncating variations in PALB2 and BARD1 genes. The PALB2 c.3290dup and BARD1 c.1333G>T variants are classified as pathogenic. We also identified 42 variants of unknown/uncertain significance (VUS) in 70% (21/30) of patients with TNBC, including 50% (21/42) missense variants. The highest VUS rate was observed in ATM (13%, 4/30). Additionally, 35.7% (15/42) variants initially well-known as benign, likely benign or conflicting interpretations of pathogenicity have been reclassified as VUS according to ACMG-AMP. Conclusions: PALB2 and BARD1 along with BRCA genetic screening could be helpful for a larger proportion of early-onset TNBC in Morocco.


Introduction
Breast cancer (BC) is the most commonly diagnosed malignancy and the leading cause of cancer death among women worldwide. An estimated 19.3 million new cases and 10 million cancer deaths occurred in 2020 [1]. According to the GLOBOCAN Cancer Tomorrow prediction tool, incident cases are expected to increase by more than 46% by 2040 [2].The increasing global BC burden is mainly observed in low and medium human development index (HDI) countries [3], particularly women under the age of 50. Marked changes in lifestyle, socio-cultural contexts, and built environments are having a major impact on the prevalence of risk factors for BC burden in lowand medium HDI countries [4]. In North Africa (i.e., in Morocco, Algeria, Tunisia, Libya and Mauritania) BC has rapidly overtaken cervical cancer as the most commonly diagnosed cancer [5]. The incidence among women aged 15-49 is lower than in Western countries, but the very low incidence among women over 50, combined to the young age pyramid of North-Africa, makes the relative proportions of young patients substantially higher (50-60% versus 20% in France) [5]. The size and grade of breast tumors are increased, while the median age of onset (48) is more than ten years younger than the European/North American median of 61 [6]. Moreover, the relative frequency of triple negative and inflammatory breast cancer is also higher in North Africa [5].
BC is a heterogeneous and polygenic disease that can be divided into different molecular sub-types based on histological and genomic features. Increasing steroid hormone receptors expression (estrogen and/or progesterone receptors) defines different BC subtypes using immunohistochemistry (IHC) markers together with clinic-pathologic indexes. Approximately 70% of BCs are estrogen receptors (ER) positive (ER+) and/or progesterone receptor (PR) positive (PR+) tumors [7]. The human epidermal growth factor receptor type (HER) 2 amplification defines a second type, with an incidence of about 20% of BCs [8]. The remaining belongs to the triple negative breast cancer (TNBC) subtype, i.e. those that do not over-express ER, PR or HER2. TNBC is associated with advanced-stage disease and higher-grade tumors at diagnosis and is associated with an increased recurrence risk and poor five-year survival rates relative to other BCs [9].
Current evidence suggests the association between specific molecular subtypes and BRCA mutational status. BRCA1 PV carriers mainly increase TNBC, whereas BRCA2 carriers are more likely to increase ER+ and/or PR+ tumors [10]. Besides BRCA genes, an increasing number of studies have investigated genetic predisposition to TNBC using gene panel analysis [11,12]. PVs in non-BRCA genes have been observed in women with TNBC, and subsequent studies showed that PVs in BARD1, BRIP1, PALB2, RAD51C, and RAD51D are more common in TNBC compared to other BC subtypes [13]. PVs in established BC genes as well as other cancer susceptibility genes were identified in 14.4% (8.4% BRCA genes and 6.0% non-BRCA) of TNBC patients. PVs in BARD1, BRCA1, BRCA2, PALB2, and RAD51D were associated with high risk (Odds Ratio (OR)> 5.0) of TNBC. PVs in BRIP1, RAD51C, and TP53 were associated with moderate risk (OR > 2) of TNBC [11]. Moreover, PVs in BARD1, RAD51C, and RAD51D placed the patient at moderate risk for ER-BC and TNBC. Conversely, PVs in ATM, CDH1, and CHEK2 were associated with ER+ BC. Additionally, there was a higher prevalence of PVs in BRCA1, BRCA/2 and PALB2 observed with TNBC compared with ER+ BC, 8.13% versus 1.84%, respectively [14].
In North Africa, BRCA mutations frequency varied widely from ≈1% (Morocco) in sporadic BC [15] to 37.5% (Tunisia) in hereditary breast or/and ovarian cancer patients (HBOC) [16]. The pooled prevalence of BRCAPVs among HBOC was 16% [17]. However, the contribution of BRCA and non-BRCA genes to TNBC has not yet been determined. In a recent study from Morocco, it has been reported that 22% of TNBC patients harbor PVs in the BRCA genes using Ion AmpliSeq BRCA1 and BRCA2 Panel [18]. Thus, there is a great need to investigate the frequency and importance of PVs in BRCA and non-BRCA genes among TNBC patients. Herein, we present the first Next Generation Sequencing (NGS)-based Multigene Panel Test (MGPT) study of 30 early onset (≤ 41 years of age) Moroccan women with TNBC. Panel sequencing was performed with genes involved in hereditary predisposition to cancer and candidate genes whose involvement remains unclear. This provides an advantage to map susceptibility genes for TNBC in North Africa.

Study subjects
Newly diagnosed women with TNBC were identified at the department of Obstetrics and Gynecology of Mohammed V Military Teaching Hospital in Rabat. TNBC patients were chosen according to the following criteria: age at diagnosis ≤ 41 years without family history or belonging to a family history. This family history of cancer was defined as diagnosed BC or OC in first-and second-degree relatives. Clinico-pathological data of TNBC, including tumor site, histological type and grade and TNM classification were collected at BC diagnosis. A total of 30 early onset TNBC patients were enrolled between January 2020 and June 2020, including 18 (60%) without a family history and 12 (40%) with a family history. Informed consent was obtained from all participants at the time of peripheral blood draw. Patients consenting to participate in the study completed epidemiology and family history questionnaires and donated 10 ml of blood for genetic analyses.

Immunohistochemical analysis
The status of ER, PR, and HER2 receptors was determined using IHC analysis. Briefly, IHC analysis to determine ER and PR status was performed using standard procedures on 4 μm sections of paraffin embedded tissue specimens stained with the monoclonal antibodies 6F11 and 1A6 for ER and PR, respectively. Nuclear staining 1% was considered a positive result. The Hercept test was carried out in the institute since 2007 and was determined for all patients during the course of this study. Assays are scored with a 4-tiered system (0 -3+). HER2 positivity was defined as strong complete membrane staining in at least 10% of tumor cells. Patients were considered HER2 if they had IHC 3+ by DAKO Hercept test. Tumors exhibiting equivocal HER2 expression, denoted as 2+ membranous staining of tumor cells, are confirmed by fluorescence in situ hybridization (FISH) at an outside laboratory. A signal ratios (HER2: CEP17) of ≥ 2.2 were classified as amplified. In the absence of positive FISH data, tumors scored 2+ by IHC were considered negative for HER2.

DNA isolation
Genomic DNA was isolated from 200 μl peripheral blood anti-coagulated with Ethylene diamine tetra acetic acid on Blood DNA Maxi kit (Qiagen, Hilden, Germany) following the manufacturer's manual. DNA concentrations were assessed with the dsDNA HS assay kit by the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA).

Gene panel testing
We performed targeted capture sequencing with a gene panel that is associated with high, intermediate and low cancer risk and candidate genes whose involvement remains unclear.

Panel sequencing
Sonic fragmentation of DNA was performed on a Bioruptor instrument (Diagenode). Kapa HTP library preparation and SeqCap EZ Choice probes and reagents (Roche) were used for library preparation and capture. Briefly, 20ng of genomic DNA was fragmented and processed by end-repairing, A-tailing and adapter ligation of paired-end indexed adapters, and a 7-cycle pre-capture PCR amplification. Further, the libraries were enriched through hybrid capture based method using specific probes. This was followed by PCR based enrichment, cleanup, and quantification of double stranded DNA using high sensitivity Qubit (Invitrogen, USA) measurement. Agencourt AMPure XP beads (Beckman Coulter, USA) were widely used for PCR amplicon purification and DNA size selection. The quality of fragmentation, library, and capture were evaluated using the Agilent 2100 Bioanalyzer system. A final library concentration ranging from 8 to 10 pM was used to carry out cluster generation and was sequenced on a Standard Flow Cell using V2 sequencing reagent kit (300 cycles) on MiSeq Instrument (Illumina, San Diego, CA). A typical sequencing run consists of 12 samples. All steps were performed following the providers' guidelines. No analysis of exons 11 to 15 of PMS2 and exons 1, 13, and 14 of SDHA, and no quantitative analysis of WRN exon 10 were performed, due to high identity with paralogs genes.

Bioinformatics Analysis
De-multiplexing was performed using bcl2fastq2 Conversion Software (Illumina). Sequencing reads were aligned to the University of California Santa Cruz hg19 reference genome assembly by Burrows-Wheeler Aligner [19]. Recalibration of base quality scores (BaseRecalibrator) and realignment (Realigner-TargetCreator, IndelRealigner) were carried out using Genome Analysis Toolkit (GATK) and PICARD tools, as recommended by Eurogentest guidelines [20]. A variant calling was made using GATK Haplotype-Caller and annotated using Ensemble Variant Effect Predictor [21].Variants were filtered for quality score ≥ 30, depth ≥ 30x, and presence in ≥ 20% of reads.

Interpretation of variants
An Interpretation of variants was conducted by following the classification system recommended by the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) Standards and Guideline for the Interpretation of Sequence Variants [22]. The process can result in 1 of 5 classifications: benign, likely benign, unknown/uncertain significance (VUS), likely pathogenic, and pathogenic. Likely benign and benign variants were not clinically reported. All classifications were ultimately evaluated by AL, MC and referred to NU. The clinical significance of each sequence variant was also based on a set of criteria such as allele frequency as well as the information from clinical genome databases including ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/).

Complementary analysis
Identified PVs were confirmed on a second patient sample. PVs in BRCAand BRCA2 geneswere examined by NGS with Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies). The Sanger sequencing was performed for the novel PV in PALB2 and BARD1 genes using a 3500xl instrument and Big Dye terminator kit 3.1 (Applied Biosystems).

Statistical analysis
The statistical analysis used the chi-squared test, with p< 0.05 taken as the threshold for a significant difference.

Results
The mean age at diagnosis of TNBC was 38 (± 2.8) years. Most TNBC patients were diagnosed with infiltrating ductal carcinoma (IDC). The Scarff-Bloom-Richardson grades II and III were predominant (33.3% and 36.7% of cases, respectively). Axillary lymph nodes contained metastasis (N+) in 5 (16.7%) cases. Unilateral BC was diagnosed in all patients, and one (3.3%) had both BC and OC. The characteristics of the early onset TNBC patients are summarized in Table 2.
PVs were identified in 20% (6/30) of patients with TNBC. Of these, 16 PVs. The BRCA1 c.798_799del (p.Ser267LysfsX19) variant was found in two unrelated TNBC patients. Both carriers were diagnosed less than 38 years of age and showed a strong family history of BC. The BRCA1 c.798_799delTT variant, located in exon 11, is a frame-shift variant including two small deletions, two bases (TT) deletion. The deletion causes a frame-shift which changes a Serine to a Lysine at codon 267, and creates a premature stop codon at position 19 of the new reading frame. The BRCA1 c.798_799delTT variant, previously reported as BRCA1 917_918delTT using alternate nomenclature, has been reported in association with familial and early-onset BC and OC and has been described as a North African identified founder variant [15,[23][24][25][26].  [27], was detected in early onset women diagnosed with both TNBC (at the age of 35) and OC (at the age of 38). The BRCA2 c.1310_1313delAAGA variant, located in exon 10, causes a frame-shift which changes a Lysine to an Isoleucine at codon 437 and creates apremature stop codon at position 22 of the new reading frame. It has previously reported as BRCA2 1310del4, BRCA21537del4, and BRCA21538del4 using alternate nomenclature.
The BRCA2 c.1658T>G (p.Leu553Ter) variant, located in exon 11, causes a non-sense substitution. It was observed in one patient without family history, diagnosed with TNBC at the age of 36. In databases, theBRCA2c.1658T>G variant was associated with breast-ovarian cancer, hereditary cancer-predisposing syndrome, or hereditary breast or/and ovarian cancer syndrome.
Our panel sequencing allowed identification of several sequence variations in most investigated genes, among which we found novel variation in PALB2 and BARD1 genes. The PALB2 c.3290dup (p.Lys1098) variant has not been reported before in the BIC database. The PALB2 c.3290dup variant was detected in a young patient diagnosed with TNBC at an age ≤ 36 years. Family history was negative in the PV carrier. Thus, we concluded that this PV is not linked to family history of BC and/or OC. The PALB2 c.3290dup variant is a frame-shift variation due to the insertion of C nucleotide at acid 3290 of codon 1097 in exon 12, which is predicted to lead to a premature stop codon 1098 and a truncated protein. The BARD1 c.1333G>T (p.Glu445), that has not been reported previously, is classified as pathogenic (Class 5). The BARD1 c.1333G>T variant occurred with BRCA2 c.1658T>G (p.Leu553Ter) in early onset TNBC women diagnosed with pancreatic cancer at the age of 32. The early disease onset and having two cancers in this case may be the result of harboring two PVs in BRCA2 and BARD1 genes. Details of PVs detected by NGS-based MGPT in our study are reported in Table 3.
We also identified 42 VUS in 70% (21/30) of TNBC patients, including 50% (21/42) missense variants. The other VUSs were characterized as intronic or synonymous variants. A high rate was expected given the number of genes included in our panel gene. The BC susceptibility gene carrying the highest number of VUS was ATM (13%,4/30). Additionally, 35.7% (15/42) variants initially wellknown as benign, likely benign or conflicting interpretations of pathogenicity have been reclassified as VUS according to the ACMG-AMP classification. Details of the identified VUS are shown in Table 4.

Discussion
The absence of specific molecular markers for TNBC has made the targeted treatments extremely challenging and the death rates very high compared to the other BC subtypes. The NGS offers several clinical applications in cancer and precision oncology that are significant for risk predictors, early detection of disease, diagnosis by sequencing and medical imaging, accurate prognosis, biomarker identification and identification of therapeutic targets for novel drug discovery [28]. To identify TNBC patients who might benefit from treatment strategies, BRCA and non-BRCA genes testing through NGS could lead to a more accurate prediction of the responsiveness to platinum and poly (ADP-ribose) polymerase (PARP) inhibitors. Thus, this strategy should be considered in management and precision medicine [29]. Recent data have hypothesized that patients with advanced-stage TNBC associated with PVs in BRCA genes might be specifically sensitive to PARP inhibition; both Olaparib and Talazoparib are currently approved for such situation [30,31]. Moreover, there is some evidence that adding platinum-agents in the neoadjuvant setting improves the pathologic complete response [32,33]. The role of PARP inhibitors in the setting of non-BRCA associated cancers has been limited. Recently, Lapatinib plus Veliparib therapy have a manageable safety profile and promising antitumor activity in advanced TNBC [34].

TNBC and BRCA genes
Genetic susceptibility to TNBC has been associated with rare germline variants occurring in BRCA genes, and BRCA2 PVs were less common than BRCA1 PVs [10,35]. In our study, the BRCA PV prevalence was 16.7% among TNBC women aged <41. Among BRCA carriers, BRCA1 gene was found to be mutated in 10% (3/30), while BRCA2 gene was mutated in 6.7% (2/30). Our findings highlight that the TNBC phenotype at young age at onset can provide a valuable tool for identifying individuals with high likelihood of being BRCAPV carriers. This information emphasizes the recommendation for genetic testing in women diagnosed with TNBC at a young age because they have an increased risk of carrying BRCA PVs particularly in BRCA1. Data from a previous Tunisian study showed that diagnosis before the age of 40 could be the effective BRCA testing selection criterion among women with triple-negative tumors [36]. Recent findings highlight that receptor triple negative could be an effective selection of patients for BRCA1 analysis and should therefore be considered in genetic screening guidelines in Tunisia [37]. In Algeria, the BRCA1 PVs have been detected in Algerian patients with TNBC diagnosed at age ≤45 [38]. Thus, an earlier age should be considered as a guideline for BRCA genetic testing in women with TNBC in North Africa.
By analyzing the incidence rates of BRCA1 PVs in both TNBC and non-TNBC, Tun et al. found that women with high-risk TNBC are much more likely to have PVs in BRCA1 gene compared with women with non-TNBC and provides a relative risk of 5.65 (95% CI, 4.15-7.69). Furthermore, two out of nine (≈22%) TNBC patients harbor a PV in BRCA1 gene [39].
Armstrong et al. reported a concordant finding, although the estimates of BRCA1 PV prevalence were mostly lower than the estimate by Tun et al. [40]. In four studies of TNBC patients that reported on BRCA PVs prevalence, values ranged from 9.3% in an Australian study (n=439) [35] to 15.4% in a US study (n=207) [41]. Both of these studies were done in early and advanced tumor stages BC populations. In UK, Robertson et al. showed that diagnosis of TNBC below 50 years would be a suitable age threshold for BRCA testing and may be a cost effectiveness strategy [42]. Lu et al. recommended genetic testing for TNBC patients diagnosed before the age of 50, a population with BRCA PV frequency of 17.5%. The authors also highlighted data showing that the estimated BRCA PV frequency of individuals with any type of BC diagnosed before the age of 40 is 11% [43]. Overall, testing in the UK has an unusually high threshold in comparison with the other European countries, where in all cases <41 years would be eligible for screening [44].
When stratified by family history, our study showed that 13% (4/30) early onset TNBC patients with a family history were identified with PVs in BRCA genes, compared to 3.3% (1/30) without a family history. Couch et al. revealed that 12.2% (66/539) TNBC patients with a family history carry BRCA PVs, compared to 8.6% (83/969) patients without a family history [12]. Hartman et al. identified 21 BRCA PVs (13 in BRCA1 and 8 in BRCA2) in a cohort of 199 unselected women with TNBC and providing an overall prevalence rate of 10.6%. Additionally, 5.2% (8/153) PVs were found in 153/199 patients without significant family history [45]. In an unselected cohort study in 77 TNBC patients, it was found that 19.5% (15/77) had BRCA PVs including 15.6% (12/77) in BRCA1 and 3.9% (3/77) in BRCA [46]. In a Canadian TNBC cohort (n=54) with no familial BC aggregation, 9% (5/54) with PVs in BRCA1 and 2% (1/54) of patients with PVS in BRCA2 were detected [47]. Lu et al. stated the importance of BRCA testing regardless of family history as few female family members or small families can mask genetics relative to BC and OC [43]. Muendlein et al. conclude that the prevalence of BRCA PVs is high in TNBC patients and are not restricted to young women or patients with a positive family history [48]. Although family history is commonly used to identify individuals with a possible predisposition to BC, Zang et al. showed that family history could not predict an underlying predisposition cancer syndrome in most patients. Furthermore, some individuals with cancer have de novo mutations, whereas others inherit them with incomplete penetrance; where, the family history is likely to be negative [49].

TNBC and non BRCA-genes
In recent years it has become clear that truncating PALB2 variants have been shown to be associated with a high risk for breast, ovarian and pancreatic cancers [50][51][52]. In our investigation, the truncating variant PALB2 c.3290dup was found in 3.3% (1/30) of TNBC cases. According to the ACMG-AMP guidelines, the PALB2 c.3290dup variant was characterized as pathogenic (class 5) and considered predisposing for TNBC. Family history was negative in a PALB2 c.3290dup carrier. Genetic testing for PALB2 would provide another key genetic marker to identify women at elevated risk of TNBC regardless of their family history of BC. Our results suggest that the PALB2 gene contributes to the risk of TNBC in North Africans and highlights the need to identify PVs in the PALB2gene which has a significant impact on an individual's risk of TNBC in younger women.
Similar observations are made when comparing the frequency of PVs reported here to that reported in the study of Zanati et al. in which 4.3% of TNBC patients carried PVs in PALB2 [52]. The observed frequency of PALB2 PVs in our study (3.3%) and the last one appears to be higher compared to other populations (1.3-1.4%) [11,13]. Low prevalence of PALB2 germline mutation (~1%) was observed in 347 Australian TNBC women, similar to the prevalence of PALB2 germline mutation of 1% in familial non-BRCA genes BC cohorts [11]. PALB2 PVs might be overrepresented in patients with TNBC in earlier studies performed in European cohorts [54,55]. It is estimated that 30% [56] and 34% [57] of BC patients with a germline PALB2 PV have a TNBC subtype. Shimelis et al. found that PVs in PALB2 were found to be associated with a high-risk of TNBC with an OR of 14.41 (95%CI; 9.27-22.60) and were enriched in patients with TNBC compared to non-TNBC tumors with an OR of 2.12 (95%CI; 1.63-2.74) [11]. The relatively poor survival from BC in patients with a PALB2 PV was not attributable to the high prevalence of triple-negative phenotype [57]. Due to the lower PV carrier frequency in the PALB2 gene in the population, broad-based studies are needed to refine the genetic testing criteria and the management of the patients and their family members.
Similar observations are made when comparing thefrequency of BRAD1PVs reported here to that reported in previous studies. De Brakeleer et al. suggest that TNBC patients are enriched for PVs in BRAD1 ascompared to control samples and high BCrisk families when they identified four harbored variants in BARD1, of which two protein-truncating variants (c.1347A>G and c.1972C>T) have been confirmed as pathogenic [58]. In a study of 105 women with TNBC from a trial exploring the antitumor activity of neoadjuvant carboplatin/docetaxel chemotherapy, BARD1 PVs were detected in two TNBC patients (1.9%) [59]. The Analysis of 1824 TNBC patients unselected for age or family history of BC led to the identification of 0.5% (9/1824) cases with a BARD1-truncating variant [12]. In Buys's study, the prevalence of BARD1 PVs was higher among women with TNBC (3.3%) than among women with non-TNBC (1.7%) [13]. Shimelis et al. identified 25 individuals harboring BARD1 PVs (0.61%) and reported an OR of 5.92 for TNBC cases of African American and Caucasian populations [11]. Rofes et al. identified ten BARD1 PV carriers from 680 TNBC patients (carrier frequency = 0.9%), resulting in an OR = 5.40 [60]. Similar observations were reported in other studies [61,62], indicating that BARD1 is a risk gene for TNBC. Although the BARD1gene offers a new hope for improving the TNBC therapy, the low number of BARD1 PV carriers, the non-identification of a recurrent hotspot variant, a study with an sufficient sample size, a lack of geographically matched population controls have complicated the interpretation of the results and could hamper the strength of the association of BARD1 PVs with TNBC risks [56]. Further studies in larger cohorts will be necessary to more precisely assess the BARD1associated risk with this tumor phenotype.

TNBC and VUS
VUS represent a significant proportion of variants identified in clinical genetic testing, which account for about 40% of the total variants [63]. In our study, 42 VUS were identified in 70% (21/30) of TNBC patients including 50% (21/42) distinct missense variants. The gene with the highest VUS frequency was the ATM (13%, 4/30). The ATM is a very large gene and is one of the genes with more identified VUS (40%) including missense, in-frame, or synonymous mutations [63]. In our MGPT, the genes that contain more VUS are among the top ten genes cited in the literature with the highest number of variants submitted. A previous study has shown that the rate of VUSs was proportional to the number of genes analyzed in the MGPT in TNBC [64]. Identification of the VUS has become a daily fact of life when tailoring genetic counseling, but little guidance is available for how best to approach them, and limited data are available on how they are affecting the medical practice and the well-being of cancer patients [65]. In the lack of reliable clinical information or functional evidence the VUS remains non-informative in risk management and decision making.
Management recommendations for diagnostic and treatment decision-making for the carriers of PVs in BRCA and non BRCA-genes have been established. Recommendations are focused on a combination of annual magnetic resonance imaging (MRI) and mammography for women with familial risk or BRCA PV and a history of BC [66][67][68]. Guidelines also made recommendations for the treatment of individuals with a BRCA carrier or those with a strong familial risk of developing BC. More recent European and US NCCN guidelines have updated recommendations regarding BRCA-targeted PARP inhibitor therapy in BC [69]. Current evidence suggests that the BC risk for PALB2 mutation carriers may overlap with that for BRCA2 mutation carriers, particularly in the context of a significant family history [55]. Accordingly, recently introduced NCCN practice guidelines suggest that a PALB2 carrier should undergo a MRI or a mammography screening [70,71]. Several studies have shown that the BARD1 can potentially become a new target for BC treatment. Li et al. have reported that the BARD1 BRCT domain interacts with PAR, which results in a subsequent recruitment of the BARD1-BRCA1 complex to the damaged DNA [72]. A higher BARD1 and BRCA1 expression is associated with a worse prognosis of early BC patients, especially the ones that received a radiotherapy, indicating the potential use of PI3K inhibitors to reverse chemoresistance and radioresistance in ER + BC patients [73]. Mammography and MRI remain the fundamental imaging modalities for the high andvery high-risk BC patients. An interesting approach might be radiogenomics, which brings together the clinical assessment, imaging results, and the genetic background [74]. This approach would be of interest in relation to the immunohistochemical staining of the BARD1 gene, which in turn can be imaged inmagnetic resonance scans [75].

Limitations
Our study has some limitations that should be acknowledged. The sample size was relatively small. Thus, our data underscore the need for larger series to better understand the frequency and the contribution of PVs in BRCA, PALB2 and BARD1 genes in patients with early onset TNBC. Despite the reduced sample size in our cohort, our findings supports those deleterious PVs in PALB2 and BARD1were enriched in TNBC patients. Our analysis does not include copy number variants. Although having many advantages, large sequencing panels still have limitations compared to the traditional Sanger sequencing test or smaller NGS panels in cancer precision. In our MGPT, Some genes or exons are not well captured and therefore are not covered, resulting in some variants within these regions going undetected and hence being refractory to analysis. Despite these limitations, our findings may help in implementing effective strategies for BRCA, PALB2 and BARD1 PVs testing in TNBC patients.

Conclusion
Defects in homologous recombination DNA repair genes that may be targeted with PARP inhibitors occur in 60 to 69% of TNBC. PARP repairs the damaged DNA and renders the tumor highly sensitive to platinum-based chemotherapy. With the rise of NGS, it is possible to target multiple genes rapidly and simultaneously in a cost-effective manner. Defining groups of TNBC patients with BRCA, PALB2 and BARD1 PVs is important for the clinical management of patients because several new treatment strategies are being evaluated for related tumors. Overall, an improvement in the detection rate by using an extensive panel analysis determines the hereditary cancer to which the patients and families are exposed based on their history and genetics as part of a targeted therapy [76].