Simple Summary
Breast cancer is one of the most common cancers among women, but its genetic causes remain poorly characterized in North African populations. In this study, we investigated germline genetic variants in 165 Tunisian breast cancer patients using targeted next-generation sequencing of a multigene cancer panel. We identified pathogenic or likely pathogenic variants (P/LPVs) in BRCA1 and BRCA2 genes in 19 patients (11.5%), with several recurrent variants and additional variants not previously reported in our Tunisian cohort. P/LPV carriers were more frequently younger at diagnosis and showed significant associations with family history and several clinical features. We also identified P/LPVs in other genes. In addition, 56 variants of uncertain significance (VUS) were detected, of which 7 were prioritized through computational analyses. Additional functional or segregation evidence should be collected to establish pathogenicity. Our findings expand the available genetic data on breast cancer in Tunisia and highlight the importance of population-specific genomic studies for improving variant interpretation and genetic counseling.
Keywords: breast cancer, BRCA1, BRCA2, pathogenic/likely pathogenic variant, variant of unknown significance, targeted next-generation sequencing
Abstract
Background/Objectives: This study aims to investigate the mutational spectrum of BRCA1 and BRCA2 genes in a cohort of breast cancer (BC) patients from southern Tunisia, and to evaluate their clinical and prognostic significance. Additionally, this study explores the contribution of other cancer predisposition genes and the prevalence of variants of uncertain significance (VUS). Results: Among the 165 patients included, pathogenic or likely pathogenic variants (P/LPVs) in BRCA1/BRCA2 were identified in 19 cases (11.51%), including 8 in BRCA1 and 11 in BRCA2. The presence of BRCA P/LPVs associated with young patients (p = 0.006) and those with TNBC (p = 0.036). Beyond BRCA1/2, PV/LPVs were detected in other cancer-related genes, including TP53 (n = 3), CHEK2, RAD50 (n = 2 cases each), and MUTYH, BARD1, and BRIP1 (one case each). Furthermore, 56 VUS were identified; among them, 7 were prioritized based on in silico predictive analyses, suggesting a potential deleterious effect. However, these VUS should not be used for clinical decision-making without additional evidence from functional and familial segregation studies. Conclusions: Our findings provide novel insights into the genetic landscape of breast cancer in southern Tunisia, highlighting the clinical relevance of BRCA1/2 mutations and the contribution of other susceptibility genes. These results support the personalized management of breast cancer patients and the implementation of expanded multigene panel testing in routine clinical practice to improve genetic counseling.
1. Introduction
Breast cancer (BC) is one of the leading causes of death worldwide in the female population, with an estimated 2,600,000 cases and 685,000 deaths in 2022 [1,2]. In Tunisia, the incidence of BC is approximately 30/100,000 per year [3,4]. Several risk factors, such as hormonal regulation and lifestyle, have been associated with BC development [5,6]; however, heredity remains one of the most significant risk factors [7]. In fact, 5 to 10% of BCs are attributable to hereditary syndromes with autosomal dominant transmission, such as hereditary breast and ovarian cancer syndrome (HBOC). Two major predisposition genes (BRCA1 and BRCA2) are responsible for 30–50% of HBOC cases [8]. BRCA1 (MIM#113705) and BRCA2 (MIM# 600185) are tumor suppressor genes that play a crucial role in DNA repair through the homologous recombination pathway [8]. Loss of the BRCA function impairs DNA repair, resulting in high mutation rates and thus contributing to tumor development [9,10].
Indeed, women carrying pathogenic germline mutations in BRCA1/BRCA2 genes have an increased risk of developing HBOC compared to those with wild-type BRCA genes [8,11,12].
Advances in genomics through next-generation sequencing (NGS) have revolutionized cancer diagnosis and management, stratifying risk, guiding surveillance, and informing therapeutic decisions. In BC, sequencing is essential for genetic counseling of at-risk relatives, tailored medical follow-up, and eligibility for PARP inhibitor therapies [3,4].
Thousands of pathogenic and likely pathogenic variants (PV/LPVs) have been identified in the BRCA1 and BRCA2 genes since they were first studied [12].
Importantly, the prevalence and spectrum of BRCA1/2 PVs/LPVs vary considerably between different ethnic groups and geographic regions. Because of this variability, many population-based studies have been conducted to map the mutational profiles of these genes in various countries [13,14].
In the Middle East and North Africa (MENA) region, where BC incidence, age at onset, and clinical presentation often differ from those of Western populations, characterizing the BRCA1/2 mutational profile is particularly important. Studies conducted in several MENA countries have reported the prevalence of germline BRCA1/2 PVs/LPVs among BC patients, emphasizing both shared and unique variant patterns compared to other populations [15,16,17,18]. These findings bring attention to the need for region-specific genetic testing panels and personalized clinical management approaches.
Tunisia has a complex population history due to its geographical position at the crossroads of North Africa and the Mediterranean. Its genetic diversity reflects contributions from North African, Mediterranean, Middle Eastern, and sub-Saharan populations. The population also shows varying degrees of endogamy and consanguinity, which may contribute to the persistence of recurrent genetic variants in some communities.
Previous genetic studies have identified both Tunisian-specific and variants shared with other North African and Middle Eastern populations, highlighting the genetic diversity of the region. Therefore, characterizing BRCA1/2 variation specifically in Tunisian patients may help identify recurrent or population-associated variants that could be relevant to genetic testing and clinical management in Tunisia [19].
In Tunisia, previous studies have reported that between 14.1% and 37% of BC patients carry pathogenic BRCA variants [18,19,20,21]. Several recurrent mutations have been identified within this population, suggesting the existence of population-specific mutational patterns [22]. Identifying these recurrent variants is crucial for the development of targeted genetic screening strategies and for improving clinical management tailored to the Tunisian context.
The primary objective of this study is to expand the mutational spectrum of the BRCA1 and BRCA2 genes by identifying germline pathogenic and likely pathogenic variants with or without family history of cancer in patients selected from the south Tunisian region. This effort aims to support therapeutic decision-making and provide more effective genetic counseling for Tunisian BC patients and their families.
2. Materials and Methods
2.1. Study Population
A cohort of 165 unrelated Tunisian breast cancer patients (162 females and 3 males), enrolled from the Habib Bourguiba Hospital of Sfax-Tunisia’s Medical Oncology Department between 2020 and 2024, was included in this study. All patients underwent germline genetic testing based on clinical features, including early onset of breast cancer (<35 years), bilateral disease, triple-negative phenotype, or family history of breast and/or ovarian cancer. Among these patients, 116 (70.3%) have a familial history of cancer according to the NCCN guidelines [11] and 37/165 (22.4%) were diagnosed with triple-negative breast cancer (TNBC).
Written informed consent was obtained from all participants, and the study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Faculty of Medicine of Sfax, Tunisia.
2.2. DNA Extraction and Targeted Next-Generation Sequencing
Genomic DNA was extracted from peripheral blood using QIAamp Blood DNA (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Concentration and purity of the DNA were measured using a NanoDrop spectrophotometer and Qubit fluorometer (dsDNA HS kit, Thermo Fisher Waltham, Masachussetts, MA, USA).
DNA libraries were generated using the OncoRisk Cancer Panel (Celemics, Seoul, Republic of Korea) according to the manufacturers’ protocols. This panel includes 31 genes: APC, ATM, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PRSS1, PTEN, RAD50, RAD51C, RAD51D, SLX4, SMAD4, STK11, TP53, and VHL.
Sequencing was performed on an Illumina MiSeq Instrument (Illumina, San Diego, CA, USA) with read lengths of 2 × 151 bp using V3 flow cell (Illumina). The average sequencing depth was 1405.85, with a minimum of 100× for variant calling. Raw reads were analyzed using the basespace cloud-based tool by Illumina.
2.3. Variant Classification
Variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines into five categories: pathogenic, likely pathogenic, variant of uncertain significance (VUS), likely benign, and benign. Pathogenicity was assessed after comparing the data with sequence databases: ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), accessed on 1 April 2026, Varsome (https://varsome.com) and Franklin (Qiagen). Only pathogenic variants, likely pathogenic variants, and VUS are discussed in this study.
2.4. In Silico Pathogenicity Prediction
All VUS identified in this study underwent extended bioinformatic characterization to assess their potential clinical relevance. Each VUS was investigated using a panel of computational tools covering different mechanisms: Protein function impact: PolyPhen-2 (probably damaging/benign), SIFT (deleterious/tolerated), Mutation Taster (disease-causing to most sensitive), REVEL, Alpha missense, FATHM, and CADD (Combined Annotation Dependent Depletion); Threshold used: CADD ≥ 20 means potentially pathogenic, in addition to Regulome, SpliceAI, and SPIP, for intronic variants. Variants appearing in two or more unrelated patients were flagged as recurrent VUS, potentially indicating founder effects or population enrichment.
2.5. Statistical Analysis
Descriptive statistics were generated using SPSS V.30.0.
The following analyses were performed: frequency of PV/LPV and VUS per gene, distribution by age and clinical parameters, comparison of recurrent vs. non-recurrent VUS. A p-value < 0.05 was considered significant. Kaplan–Meier was used to correlate the overall survival in patients carrying or not germline P/LPVs in BRCA1/2 genes.
3. Results
3.1. Patient Cohort and Clinical Features
A total of 165 BC patients were enrolled in our study. The median age at diagnosis was 43.9 (range, 24–75 years); most were female (98.1%) and all were from the south region of Tunisia. A family history of breast and/or ovarian cancer was reported in 70.31% of patients. Overall, 92 patients (55.8%) had SBR II invasive ductal carcinoma, 21 patients (12.72%) had bilateral BC and 37 (22.4%) had triple-negative BC. Twenty-five patients (15.15%) died of the disease (Table 1).
Table 1.
Clinical characteristics of BC patients. Correlation of clinical features with mutational status. TNBC: triple-negative breast cancer.
| Characteristics | N (%) |
|---|---|
| Age (y) | |
| ≤35 | 39 (23.6) |
| 36–55 | 107(64.84) |
| >55 | 19 (11.51) |
| Hormonal status | |
| Luminal A | 92 (55.8) |
| Luminal B | 28 (17) |
| TNBC | 37 (22.4) |
| Her2+ | 8 (4.8) |
| Family history | |
| Negative | 49 (29.69) |
| Positive | 116 (70.31) |
| Tumor Site | |
| Unilateral | 144 (87.28) |
| Bilateral | 21 (12.72) |
| SBR Grade | |
| I | 10 (6.1) |
| II | 92 (55.8) |
| III | 63 (38.2) |
| Metastasis | |
| Absence | 116 (70.3) |
| Presence | 49 (29.7) |
| Ki67 index | |
| >20% | 92 (55.8) |
| ≤20% | 73 (44.2) |
| Prognosis | |
| Dead | 25 (15.2) |
| Alive | 140 (84.8) |
3.2. Pathogenic/Likely Pathogenic Variants
Among 165 BC patients, 19 (11.51%) carried germline BRCA1 (n = 8) and BRCA2 (n = 11) P/LPVs, while 10 patients (6.06%) had P/LPVs in other cancer-related genes such as RAD50, CHEK2, TP53, etc. (Figure 1). Most of these variants had been previously reported in the ClinVar and dbSNP databases, whereas three were novel (Table 2). Recurrent PVs in BRCA2 (c.8486A>G and c.3248delA) and BRCA1 (c.1129del) were each identified in two unrelated patients (Table 2). Most P/LPVs were frameshift variants (six in BRCA1 and five in BRCA2), while four were missense, two of which were not previously reported in the ClinVar database (Table 2). Furthermore, one patient carried both a frameshift and splice P/LPV in BRCA2 (c.1310_1313del./c.632-1G>A). Besides BRCA1/2, we identified P/LPVs in MUTYH, RAD50, Chek2, TP53, and BRIP1 genes in 10 patients (Table 3). It is important to mention that BRCA1, BRCA2, TP53, and CHEK2 were considered in the context of breast cancer susceptibility, whereas variants identified in BRIP1, MUTYH, RAD50, and APC were interpreted according to their established or uncertain gene–disease associations and were not considered evidence of breast cancer predisposition in this cohort.
Figure 1.
Spectrum of the germline genomic profile in Tunisian BC patients: distribution of all germline variants identified in 165 Tunisian BC patients, classified according to ACMG guidelines.
Table 2.
BRCA1/2 PVs/LPVs in 165 BC patients. List of pathogenic/likely pathogenic variants detected in BRCA1/2 genes.
| Patient ID | Variant Location | HGVS Nomenclature | Age | Family History of Cancer | dbSNP rs | Reported |
|---|---|---|---|---|---|---|
| BRCA1 (NM_007294.4) | ||||||
| 204 | Exon10 | c.1129del. p.(Ser377Ala fsTer17) |
32 | Yes Yes |
rs2053988613 | Yes |
| 316 | 33 | |||||
| 208 | Exon10 | c.3968-3971del. p.(Gln1323Arg fsTer12) | 41 | Yes | rs886040181 | Yes |
| 259 | Exon 13 | c.4484G>A, p.(Arg1495Lys) |
47 | Yes | rs80357389 | Yes |
| 336 | Exon 16 | c.5030_5033del. p.(Thr1677IlefsTer2) | 31 | Yes | rs80357580 | Yes |
| 350 | Exon 5 | c.248_249insGAGGA, p.(Glu84fs) | 52 | Yes | No | |
| 367 | Exon 19 | c.5266dupC, p.Gln1756fs |
31 | Yes | rs80357906 | Yes |
| 330 | Exon 10 | c.4067_4071del p.(Gln1356ArgfsTer10) |
45 | Yes | rs2154266021 | Yes |
| BRCA2 (NM_000059.4) | ||||||
| 289 | Exon 19 | c.8486A>G, p.(Gln2829 Arg) |
51 | Yes | rs80359100 | Yes |
| 224 | 40 | |||||
| 255 196 |
Exon 11 | c.3248del. p.(Asn1083IlefsTer4) |
46 | Yes | rs886040466 | Yes |
| 341 | Exon 3 | c.289G>T, p. (Glu97Ter) | 41 | Yes | rs397507646 | Yes |
| 363 | Exon 3 | c.315-316del. p.(Gly106fs) | 39 | No | ||
| 327 | Exon 11 | c.5073dup. p.(Trp1692MetfsTer3) |
31 | Yes | rs80359479 | Yes |
| 328 | Exon 11 | c.2064T>A, p.(Tyr688 Ter) |
48 | Yes | - | No |
| 334 | Exon 10 | c.1813del. p.(Ile605TyrfsTer9) |
45 | Yes | rs80359306 | Yes |
| 384 | Exon 10 | c.1310_1313del./c.632-1G>A p.Lys437IlefsTer22 |
67 | Yes | rs80359277 |
Yes |
| 399 | Exon 15 | c.7533T>G, p.(Tyr2511 Ter) | 41 | Yes | - | Yes |
Table 3.
P/LPVs in other cancer-related genes in BC patients. List of pathogenic/likely pathogenic variants detected in other cancer-related genes.
| Patient ID | Gene | Variant | Age | dbSNP |
|---|---|---|---|---|
| 292 | MUTYH NM_001128425.1 |
c.1227_1228dup p.Glu410GlyfsTer43 | 45 | rs587780078 |
| 334 | RAD50 NM_005732.4 | c.2801del p.Asn934IlefsTerTer6 | 45 | rs748536322 |
| 325 |
RAD50 NM_005732.4 |
c.2801dup p.Asn934Lysfs10 | 43 | rs748536322 |
| 289 | BARD1, NM_000465.4 | c.16C>T p.Gln6Ter |
51 | - |
| 421 | CHEK2, NM_007194.4 | c.1427C>T p.Thr476Met | 43 | rs142763740 |
| 196 |
CHEK2 NM_007194.3 |
c.1260C>A p.Cys420Ter |
32 | rs762205611 |
| 290 | TP53, NM_000546.6 | c.792_794del p.Leu265del |
29 | |
| 285 |
TP53 NM_000546.6 |
c.497C>G p.Ser166Ter | 55 | |
| 341 |
TP53 NM_000546.6 |
c.613T>G p.Tyr205Asp | 68 | rs1057520008 |
| 328 |
BRIP1 NM_032043.3 |
c.585del p.Asn196ThrfsTer7 | 48 |
Interestingly, five patients carried concomitant pathogenic variants, including a pathogenic BRCA2 variant together with an additional pathogenic variant in another cancer-susceptibility gene. Notably, all five patients had a strong family history of breast and/or ovarian cancer and were diagnosed at a relatively young age (35–51 years) (Table 2 and Table 3).
Furthermore, we reviewed previously published Tunisian studies, which revealed a heterogeneous BRCA1/2 mutational spectrum, with several recurrent P/LPVs reported across different cohorts. Notably, BRCA1 c.211dupA and c.5266dupC, BRCA1 c.5030_5033delCTAA, and BRCA2 c.1310_1313delAAGA have been repeatedly identified in Tunisian families or patients (Table 4).
Table 4.
Reported Tunisian P/LP variants in the literature.
| Gene | HGVS c. Variant | Cancer Phenotype | Reported in Tunisian Studies/Population | References |
|---|---|---|---|---|
| BRCA1 | c.798_799delTT | BC | High-risk families from central Tunisia | [3] |
| BRCA1 | c.212 + 2insG | BC | High-risk families from central Tunisia | [3] |
| BRCA1 | c.211dupA | BC | Recurrent; reported in several Tunisian cohorts | [21,23] |
| BRCA1 | c.3331_3334delCAAG | BC | High-risk families from central Tunisia | [3] |
| BRCA1 | c.1504_1508delTTAAA | BOC families | Familial/early-onset Tunisian cases | [21] |
| BRCA1 | c.1612C>T | BOC | Reported in Tunisian carriers | [10] |
| BRCA1 | c.2418dupA | BC | Tunisian patients | [10] |
| BRCA1 | c.2433delC | BC | Tunisian patients | [10] |
| BRCA1 | c.3751dup | BOC | Tunisian hereditary cancer families | [23] |
| BRCA1 | c.4041_4042del | BOC | Tunisian hereditary cancer families | [23] |
| BRCA1 | c.4067_4071delAAGAA | BOC | Southern Tunisia | [4] |
| BRCA1 | c.5266dupC | BOC | Recurrent, reported across multiple Tunisian regions | [3,21,23] |
| BRCA1 | c.5030_5033delCTAA | BOC | Highly recurrent in southern Tunisia; detected in both BC and OC patients | [4] |
| BRCA2 | c.1310_1313delAAGA | BOC | Recurrent Tunisian mutation, particularly reported in northern and southern cohorts | [4,23] |
| BRCA2 | c.1313dupT | Familial/early-onset BOC | Novel mutation initially reported in Tunisian families | [21] |
| BRCA2 | c.7654dupT | Familial/early-onset BOC | Novel mutation initially reported in Tunisian families | [21] |
| BRCA2 | c.17_20delAAGA | BOC | Southern Tunisia | [4] |
| BRCA2 | c.1796_1800delCTTAT | BOC | Southern Tunisia | [4] |
3.3. Association of BRCA P/LPV and Clinico-Pathological Features
BRCA status was significantly associated with age at diagnosis, with a higher prevalence of BRCA mutations among younger patients (p = 0.006, Table 5). In addition, BRCA P/LPVs were significantly associated with a positive family history (p = 0.042), hormonal status (p = 0.036), and presence of metastasis (p = 0.048) (Table 5). Survival analysis revealed that carriers of BRCA1/2 pathogenic or likely pathogenic variants (P/LPVs) had significantly shorter overall survival than non-carriers (log-rank p = 0.002; Figure 2A). In addition, Cox proportional hazards regression analysis demonstrated that BRCA1/2 mutation status was significantly associated with overall survival (HR = 6.336, 95% CI: 1.745–23.007; p = 0.005; Figure 2B).
Table 5.
Association of BRCA status with clinicopathological parameters.
| Patient Characteristics | BRCA Wild-Type |
BRCA1 Mutant |
BRCA2 Mutant | p-Value |
|---|---|---|---|---|
| Age (years) | ||||
| ≤35 (39) | 28 (71.8) | 5 (12.8) | 6 (15.4) | 0.006 |
| 36–55 (107) | 100 (93.5) | 3 (2.8) | 4 (3.7) | |
| >55 (19) | 18 (94.7) | 0 (0) | 1 (5.3) | |
| Hormonal status | ||||
| Luminal A | 85 (58.2) | 3 (37.5) | 4 (6.5) | 0.036 |
| Luminal B | 27 (18.5) | 0 (0) | 1 (9.1) | |
| TNBC | 27 (18.5) | 5 (62.5) | 5 (45.5) | |
| Her2+ | 7 (4.8) | 0 (0) | 1 (9.1) | |
| Family history | ||||
| Negative (49) | 48 (98) | 0 (0) | 1 (2) | 0.042 |
| Positive (116) | 98 (84.5) | 8 (6.9) | 10 (8.6) | |
| Tumor Site | ||||
| Unilateral (144) | 129(89.6) | 7 (4.9) | 8 (5.6) | 0.325 |
| Bilateral (21) | 17 (81) | 1 (4.8) | 3 (14.3) | |
| Grade | ||||
| I (10) | 10 (100) | 0 (0) | 0 (0) | 0.062 |
| II (92) | 86 (93.5%) | 2 (2.2) | 4 (4.3) | |
| III (63) | 50 (79.4%) | 6 (9.5) | 7 (11.1) | |
| Metastasis | ||||
| Absence | 107 (92.2) | 3 (2.6) | 6 (5.2) | 0.048 |
| Presence | 39 (79.6) | 5 (10.2) | 5 (10.2) | |
| Ki67 index | ||||
| ≥20% (92) | 88 (95.7) | 2 (2.2) | 2 (2.2) | 0.005 |
| <20% (73) | 58 (79.5) | 6 (8.2) | 9 (12.3) | |
Figure 2.
Prognostic impact of BRCA mutation status on overall survivaland Cox regression analysis. (A) Kaplan–Meier overall survival curves comparing patients harboring BRCA pathogenic variants (BRCA Mut) with those carrying wild-type BRCA (BRCA WT). (B) Forest plot of the univariable Cox proportional hazards regression analysis evaluating the association between clinicopathological variables and overall survival.
3.4. Variants of Uncertain Significance (VUS)
Several variants of uncertain significance (VUS) were identified across the following genes: ATM (n = 11), SLX4 (n = 7), BRCA2 and BRCA1 (n = 6 each), APC, BARD1, MLH1 (n = 3 each), CDH1, MRE11 (n = 2 each), BLM, BRIP1, CDNK2A, CHEK2, ERCC1, FANCM, MSH3, MSH6, MUTYH, PMS2, NBN, RAD50, RAD51D, NSD1, and WT1 (n = 1 each) (Supplementary Table S1). Overall, 64 patients (38.7%) harbored VUS distributed across 25 genes. ATM and SLX4 accounted for the largest number of VUS, with 11 and 7 variants, respectively (Figure 3).
Figure 3.
Distribution of variants of uncertain significance (VUS) identified in the study cohort. The pie chart shows the distribution of VUS across the different genes, with the highest frequencies observed in ATM, BRCA2, and BRCA1.
Three recurrent VUS in ATM (p.Phe1463Cys, p.Leu1498Ile, and p.Arg2105Gly) were each identified in two unrelated patients. In addition, other recurrent VUS in BLM (c.-78del), MUTYH (c.583A>G), and SLX4 (c.-416G>A) were identified in two and four unrelated patients, respectively (Supplementary Table S1).
Co-occurrence of a VUS with a P/LPV in another gene was documented in nine patients. Notably, one patient (334) harbored two PVs in BRCA2 and RAD50, in addition to a VUS in BLM (Table 2 and Table 3). This patient has a strong family history of BC and is aged 45 years old.
Among the identified VUS, seven variants were considered of particular interest, being predicted as deleterious by in silico prediction tools and/or expected to affect protein function (Table 6). They include five missense variants localized in ATM and CHEK2 genes, one novel splicing variant in BRCA2, and one in-frame delins variant in the BRCA1 gene (Table 6).
Table 6.
List of relevant VUS.
| Patient | Gene | NM | rs | cDNA Change | Exon | Protein Change | Variant Type | ClinVar | gnomAD Exome Frequency | SIFT | PolyPhen-2 | Mutation Taster | CADD Score | Alpha Missense | Revel | FATHMM | Regulome db | Splice AI Lookup | SPIP | DynaMut ∆∆G |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 341 & 268 | ATM | NM_000051.3 | rs138327406 | c.4388T>G | exon 29 | p.Phe1463Cys | Missense | Conflicting | 0.0016 | D | PD | D | 26.8 | LP (0.83) |
D (0.758) |
D (0,88) |
- | - | - | −1.62 Destab |
| 327 & 267 | ATM | NM_000051.4 | rs879253983 | c.6313A>G | exon 43 | p.Arg2105Gly | Missense | VUS | 0.00000397 | D | PD | D | 24.8 | LP (0.68) |
D (0779) | D (0.759) |
- | - | - | −1.02 Destab |
| 285 | ATM | NM_000051.3 | rs201314561 | c.7381C>T | exon 50 | p.Arg2461Cys | Missense | Conflicting | 0.0000835 | D | PD | D | 29.4 | LB (0.72) |
D (0.707) |
D (0.142) |
- | - | - | 0.536 Stab |
| 331 | ATM | NM_000051.3 | rs876658907 | c.5531A>G | exon 37 | p.Tyr1844Cys | Missense | VUS | - | D | PD | B | 24.3 | LB (0.08) |
D (0.67) |
D (0.523) |
- | - | - | −1.73 Destab |
| 300 | CHEK2 | NM_007194.3 | rs74751600 | c.1117A>G | exon 11 | p.Lys373Glu | Missense | VUS | 0.00040 | D | PD | D | 27.7 | LP (0.96) |
D (0.369) |
D (0.727) |
- | - | - | 0.722 Stab |
| 364 | BRCA1 | NM_007294.4 | _ | c.2077-2082 delins | exon 10 | p.Ser694_Asp695del | In-frame deletion | VUS | - | - | - | - | - | - | - | - | - | - | - | - |
| 318 | BRCA2 | NM_000059.3 | rs876659568 | c.1909+3A>T | intronic | _ | splicing | VUS | - | - | - | - | 11.2 | - | - | - | 5 | Donor Loss | Alteration of the consensus splice site | - |
Two ATM variants (c.4388T>G and c.6313 A>G) were each found in two unrelated patients with a strong family history of BC. Both variants sit within the regulatory FAT domain of the ATM protein and are likely to destabilize the protein according to their scores in DynaMutΔΔG of −1.62 and −1.02, respectively. Additionally, among the other investigated ATM variants, and based on DynaMut predictions, we found that the Tyr1844Cys may lead to protein destabilization while the Arg2461Cys may have a stabilizing effect (Figure 4).
Figure 4.
Prediction of the 3D structure of wild-type and mutant proteins of selected VUS. (A): Phe1463Cys, (B): Tyr1844Cys, (C): Arg2105Gly in ATM, and (D): Lys373Glu in CHEK2 using DynaMut2. Protein structures are displayed as ribbon representations, while the mutated residues and neighboring amino acids are shown as sticks.
Furthermore, a novel BRCA2 VUS was identified in a young patient diagnosed at 28 years with a triple-negative breast cancer (TNBC). Splice-site prediction analysis suggested that this variant may substantially impair the donor splice site (reference score: 82.26; mutated score: 71.06; Δ = −13.62%), indicating a potential disruption of the normal mRNA processing and thus the BRCA2 protein function.
Another novel in-frame deletion–insertion (c.2077_2082delGACAGCinsAACAGT) in exon 10 of the BRCA1 gene was detected in one patient diagnosed with breast and ovarian cancer at the age of 40. This alteration is predicted to remove two amino acids (Ser694 and Asp695) within the DNA-binding domain of BRCA1 protein, which may have strong implications for the structural stability and the function of the protein.
Importantly, these in silico predictions were used solely to prioritize VUS for further investigation and do not establish their pathogenicity or clinical significance. Accordingly, the identified VUS should not be used for clinical decision-making in the absence of additional evidence from functional and familial segregation studies.
4. Discussion
In this study, we report the molecular profiling results of 165 Tunisian BC patients using targeted next-generation sequencing (NGS). In total, we identified 29 pathogenic or likely pathogenic variants (P/LPVs) distributed across eight cancer-predisposition genes. As expected, BRCA1 and BRCA2 constituted the major altered genes, accounting for 19 out of 29 (65.51%) P/LPVs. The remaining variants were distributed amongst TP53, CHEK2, RAD50, MUTYH, BARD1, and BRIP1 genes, reflecting the genetic heterogeneity of BC susceptibility in Tunisia.
It is well documented that the prevalence of BRCA1/BRCA2 P/LPVs in BC varies considerably among populations due to differences in ethnic background, founder effects, selection criteria, and testing strategies [13,14,18]. In North African countries, reported frequencies range between 11% to 37%, highlighting both regional heterogeneity and the under-representation of this population in global genetic studies [15,16,17,18]. In our cohort, 16 BRCA1/BRCA2 PVs were detected in 19 out of 165 patients (11.5%), which aligns with the range of previously reported frequencies for the region [4,22].
Interestingly, three recurrent variants, one in BRCA1 (c.1129del) and two in BRCA2 (c.8486A>G and c.3248delA), were each found in two unrelated patients. Other recurrent BRCA PVs were previously reported in Tunisian BC patients such as c.5030_5033delCTAA in BRCA1 and c.1310-1313 delAAGA in BRCA2 [4]. Their recurrence may suggest local founder effects or population-specific mutational hotspots, consistent with prior reports of geographically restricted BRCA variants in North Africa [18]. Interestingly, none of the newly BRCA1/2 P/LPVs identified in the present cohort overlapped with those reported in this previous cohort. This finding indicates that the present study expands, rather than duplicates, the previously reported BRCA1/2 mutational spectrum in southern Tunisia. The identification of additional variants in independent cohorts highlights the genetic heterogeneity of BRCA1/2-associated BC in the Tunisian population and underscores the value of continued accumulation and harmonization of national genomic data. Furthermore, by reviewing the Tunisian literature, we observe a heterogeneous BRCA1/2 mutational spectrum, with several recurrent variants that may be particularly relevant for developing a national genetic testing strategy. Notably, BRCA1 c.211dupA and c.5266dupC, BRCA1 c.5030_5033delCTAA, and BRCA2 c.1310_1313delAAGA have been repeatedly identified in Tunisian families or patients, although their relative frequencies may vary by geographical region and patient-selection criteria.
Moreover, three BRCA1 P/LPVs and two BRCA2 P/LPVs identified in this study have not been previously reported, thereby expanding the mutational landscape of hereditary BC in Tunisian patients and underlining the value of including under-represented populations in global genetic research.
Furthermore, it is important to mention that a comprehensive Tunisian BRCA1/2 variant resource integrating data from different cohorts and institutions would be particularly valuable for establishing variant frequencies, identifying recurrent or population-enriched variants, and supporting evidence-based genetic counseling and testing strategies.
To further contextualize the BRCA1/2 mutational spectrum observed in our Tunisian cohort, we compared our findings with data reported from neighboring North African populations, particularly Morocco. The Moroccan study by Elalaoui et al. [15] analyzed 163 unrelated patients with hereditary breast and/or ovarian cancer and identified a distinct spectrum of recurrent BRCA1/2 variants, with BRCA2 c.1310_1313delAAGA representing the most frequent variant in their cohort. In our cohort, the same BRCA2 c.1310_1313delAAGA variant was identified in one patient, indicating that some PVs are shared across North African populations. In addition, BRCA1 c.5266dupC, identified in our cohort, has previously been reported among Moroccan patients. Conversely, several P/LPVs identified in our cohort were not among the recurrent variants highlighted in the Moroccan cohort, supporting the presence of substantial heterogeneity in the BRCA1/2 mutational spectrum across North African populations.
In our cohort, family history proved to be a strong predictor of BRCA1/BRCA2 PV status. Patients carrying BRCA1/2 P/LPVs were significantly more likely to report a family history of breast or ovarian cancer, a finding that is consistent with previously studies [24,25]. Moreover, 27% of TNBC patients are BRCA positive, which is in line with reported data [4,17,26].
Beyond BRCA1/2, P/LPVs were identified in several additional cancer-related genes, including TP53, CHEK2, RAD50, MUTYH, APC, and BRIP1. However, these findings should not be interpreted uniformly as breast cancer-susceptibility variants. TP53 and CHEK2 have established relevance to hereditary breast cancer risk, whereas the clinical relevance of heterozygous variants in BRIP1, MUTYH, and RAD50 to breast cancer predisposition is not established. In particular, BRIP1 is an established ovarian cancer-susceptibility gene, while heterozygous MUTYH variants are primarily associated with carrier status for MUTYH-associated polyposis rather than autosomal-dominant hereditary BC. Accordingly, these findings were considered as additional germline variants identified through multigene testing rather than as evidence of non-BRCA hereditary BC predisposition [25,27].
In our cohort, four patients presented the co-occurrence of multiple P/LPVs, and one of them harbored two PVs located in BRCA2 and RAD50, together with a VUS located in the BLM gene. This patient had a strong family history and developed BC at an early age. Such findings highlight the complexity of hereditary BC genetics and emphasize the need for careful clinical interpretation when multiple variants are identified.
A substantial number of variants of uncertain significance (VUS) were detected (n = 56), particularly in ATM, SLX4, BRCA1, BRCA2, and APC genes. Based on prediction tools, seven relevant VUS in ATM, CHEK2, BRCA1 and BRCA2 were retained. Interestingly, two ATM VUS were each detected in two unrelated patients. These variants are located within the FAT domain and are predicted to affect protein stability and potentially impair ATM protein function. Nevertheless, additional investigations such as familial segregation studies are required to clarify their pathogenicity and to determine whether they are associated with BC risk. We also identified two novel VUS: a frameshift deletion in the BRCA1 gene and a splice-site variant in the BRCA2 gene, both of which were predicted to affect the function of the corresponding proteins. The BRCA1 VUS is characterized by the deletion of two amino acids (Ser694 and Asp695) within the DNA-binding domain, potentially altering the structural integrity of the protein. The BRCA2 variant is predicted to disrupt the donor splice site, and further RNA-based analyses are needed to confirm whether it induces aberrant splicing, including potential exon skipping. Numerous studies have concentrated on the reclassification of VUS, especially those occurring in BRCA and ATM genes, which are among the most frequently implicated in hereditary BC [28,29].
Recently, a study of Tunisian BC patients reported the reclassification of two VUS located in ATM (c.6115G>A), and CHEK2 (c.592+3A>T) genes, into likely pathogenic variants using a combination of familial segregation analysis and comprehensive in silico prediction tools [30].
While expected in multigene panel studies, the high VUS rate reflects both the genetic heterogeneity of the Tunisian population and the current limitations of international databases, which under-represent North African genomic information, and thus highlights the urgent need for population-specific functional and segregation analyses to clarify its clinical significance, especially in genetically understudied populations such as Tunisia.
This study has some limitations. The sample size may not fully represent the genetic diversity of the Tunisian population. The gene panel used was limited and might have missed some relevant pathogenic variants. Additionally, the identified VUS, and especially the relevant VUS, need further functional validation to clarify their clinical significance. Future studies with larger cohorts and expanded panels are needed.
5. Conclusions
This study provides a comprehensive molecular characterization of Tunisian BC patients using NGS multigene panel testing. By identifying pathogenic and likely pathogenic variants in individuals at high hereditary risk, and by thoroughly annotating and prioritizing variants of uncertain significance (VUS), we reveal both recurrent and novel mutations that point to population-specific genomic patterns. The integration of NGS with multiple in silico prediction tools enabled the prioritization of VUS for further investigation; however, these computational predictions do not establish pathogenicity or clinical significance.
Through the generation of such locally relevant genomic data, this work moves Tunisia closer to the implementation of effective precision medicine strategies, where cancer management is adapted to the genetic landscape of the Tunisian population.
Acknowledgments
The authors thank the CIC (Centre d’Investigation Clinique CHU Habib Bourguiba de Sfax-Tunisie) for his support, and Nesrine Kechaou, for her valuable support in launching the NGS run.
Abbreviations
| ACS | American Cancer Society |
| BC | Breast cancer |
| NCCN | National Comprehensive Cancer Network |
| AJCC | American Joint Committee on Cancer |
| ACMG | American College of Medical Genetics |
| TNBC | Triple-negative breast cancer |
| PVs/LPVs | Pathogenic variants/likely pathogenic variants |
| HGVS | Human Genome Variation Society |
| MENA | Middle East and North Africa |
| HER2 | Human epidermal growth factor receptor 2 |
| HBC | Hereditary breast cancer |
| NGS | Next-generation sequencing |
| PARP | poly ADP-ribose polymerase |
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cancers18172810/s1, Supplementary Table S1: In silico parameters of variants of uncertain significance identified in the study cohort. List of VUS with in silico predictive scores: comparison of deleteriousness predictions from multiple in silico tools (PolyPhen-2, SIFT, CADD, Mutation Taster, REVEL, Alpha missense, FATHM, SpliceAI, SPIP, Regulome) for all the identified VUS. D: Damaging, PD: Probably Damaging, LB: Likely Benign, D*: Deleterious, N: Neutral, P: Pathogenic, LP: Likely Pathogenic.
Author Contributions
N.A.-B. performed library preparation and sequencing, analyzed and interpreted the patient data and wrote the manuscript. R.A.-D. collected patient data and contributed to the sequencing and data analysis. D.B.-A.-G. contributed to the sequencing and data analysis. W.B.K., S.G., A.F. and H.K. contributed to collecting patients‘ data and genetic counseling. A.K. and J.D. were responsible for the patients’ clinical care. A.G. contributed to the discussion of the results and critical reading of the manuscript. G.-H.L. contributed to the critical reading and revision of the manuscript. R.M.-G. contributed to the genetic data analysis and revision, and manuscript writing and revision. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
This study was conducted in accordance with the Declaration of Helsinki. Ethical approval was obtained from The Faculty of Medicine of Sfax, Tunisia (Ethics Committee Decision N°42/26, Sfax on 12 March 2019).
Informed Consent Statement
Written informed consent was obtained from all participants prior to their inclusion in the study.
Data Availability Statement
VCF files are available from the corresponding author upon reasonable request. The data of the current study are involved in the article. The patients’ clinicopathological data are presented in Table 1; the detected SNPs and their accession numbers (rs) are illustrated in Table 2 and Table 3 in the manuscript.
Conflicts of Interest
The authors declare that they have no competing interests.
Funding Statement
This research was supported by the grant of Tunisian ministry of higher education and scientific research (Grant: LR19CBS02).
Footnotes
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
VCF files are available from the corresponding author upon reasonable request. The data of the current study are involved in the article. The patients’ clinicopathological data are presented in Table 1; the detected SNPs and their accession numbers (rs) are illustrated in Table 2 and Table 3 in the manuscript.