Abstract
Background
The spectrum of BRCA1 and BRCA2 mutations varies among populations; however, some mutations may be frequent in particular ethnic groups due to the “founder” effect. The c.3700_3704del mutation was previously described as a recurrent BRCA1 variant in Eastern European countries. This study aimed to investigate the frequency of c.3700_3704del BRCA1 mutation in Albanian breast and ovarian cancer patients from North Macedonia and Kosovo.
Materials and methods
A total of 327 patients with invasive breast and/or ovarian cancer (111 Albanian women from North Macedonia and 216 from Kosovo) were screened for 13 recurrent BRCA1/2 mutations. Targeted NGS with a panel of 94 cancer-associated genes including BRCA1 and BRCA2 was performed in a selected group of 118 patients.
Results
We have identified 21 BRCA1/2 pathogenic variants, 17 (14 BRCA1 and 3 BRCA2) in patients from Kosovo (7.9%) and 4 (1 BRCA1 and 3 BRCA2) in patients from North Macedonia (3.6%). All BRCA1/2 mutations were found in one patient each, except for c.3700_3704del BRCA1 mutation which was observed in 14 unrelated families, all except one originating from Kosovo. The c.3700_3704del mutation accounts for 93% of BRCA1 mutation positive cases and is present with a frequency of 6% among breast cancer patients from Kosovo.
Conclusions
This is the first report of BRCA1/2 mutations among breast and ovarian cancer patients from Kosovo. The finding that BRCA1 c.3700_3704del represents a founder mutation in Kosovo with the highest worldwide reported frequency supports the implementation of fast and low-cost screening protocol, regardless of the family history and even a pilot population-based screening in at-risk population.
Keywords: BRCA 1, c.3700_3704del, founder mutation, breast cancer, Kosovo
Introduction
Breast cancer is the leading cause of global cancer incidence in 2020, representing 11.7% of all cancer cases worldwide [1]. Mutations in BRCA1 and BRCA2 genes account for at least 20% of hereditary breast cancer cases. In general population, about 5–10% of all breast cancer and 10–15% of ovarian cancer cases can be attributed to these well characterized genetic risk factors [2, 3]. The lifetime risk for breast and ovarian cancer for BRCA1 mutation carriers has been estimated between 65–85% and 39–93%, respectively. For BRCA2 mutation carriers the lifetime risk ranges from 45–85% for breast and 11–27% for ovarian cancer [4].
The incidence of pathogenic BRCA1 and BRCA2 mutations differs between populations worldwide as well as within Europe. Various populations present a wide spectrum of different mutations, although some mutations may be frequent in particular ethnic groups that are geographically and/or reproductively isolated due to the “founder” effect. The highest frequency of BRCA1/2 founder mutations is found in Ashkenazi Jews (2–2.5%) where only three mutations (185delAG and 5382insC in BRCA1 and 6174delT in BRCA2) represent 95% of all BRCA1/2 mutations identified in this population [5]. Founder mutations are present in several European populations, with a strong founder effect observed in individuals of Slavic descent, including the Czech Republic, Poland, Russia and Belarus [6]. There is limited knowledge about the BRCA1/2 mutational profile in breast and ovarian cancer patients in West Balkan countries. In our previous study we reported the presence of c.3700_3704del mutation in BRCA1 only among our cohort of Albanian breast cancer patients [7, 8]. The mutation was previously described as a recurrent BRCA1 variant in Eastern European countries in multiple individuals affected with hereditary breast and ovarian cancer [9–15]. This study aimed to investigate the frequency of c.3700_3704del mutation in larger Albanian breast and ovarian cancer cohort from the Republic of North Macedonia and Republic of Kosovo. The findings of this study may help to optimize the genetic testing strategy for breast and ovarian cancer among individuals with Albanian ethnic origin in the region.
Materials and methods
A total of 327 Albanian patients diagnosed with invasive breast and/or ovarian cancer during the years 2010–2021 were included in this study (111 Albanian women from N. Macedonia and 216 from Kosovo). The patients were recruited from the Histopathology Laboratory of the Clinical Hospital Acibadem Sistina, Skopje, and University Clinic of Radiotherapy and Oncology, Medical Faculty, Skopje. Written informed consent was obtained from each participant. The study was approved by the Ethics Committee of the Macedonian Academy of Sciences and Arts, Skopje, Republic of North Macedonia. Personal data (origin, age at diagnosis and familiar history of the disease), histopathological classification, stage, receptor status (estrogen, progesterone and HER2) are given in Table 1. Median age at diagnosis was 50 years (range 22–81 years) and 19% of patients reported first-degree relative(s) with breast or/and ovarian cancer. Genomic DNA was isolated from peripheral white blood cells using standard phenol-chloroform extraction.
Table 1.
Clinical characteristics of 327 breast and/or ovarian cancer patients
| Origin | % (n/total) |
|---|---|
| Albanian patients from North Macedonia | 33.9 (111/327) |
| Albanian patients from Kosovo | 66.1 (216/327) |
| Age of diagnosis | |
| ≤ 50 | 53.2 (174/327) |
| > 50 | 46.8 (153/327) |
| Family history of cancer | |
| Breast cancer/Ovarium FH | 19.7 (62/315) |
| Other cancer FH | 19.7 (62/315) |
| No family history | 60.6 (191/315) |
| ND | 3.6 (12/327) |
| Localization | |
| BC Unilateral | 96.9 (310/320) |
| BC Bilateral/Contralateral | 3.1 (10/320) |
| OC/OC + BC | 1.5 (5/324) |
| ND | 0.9 (3/327) |
| Histopatology data | |
| Ductal | 87.8 (281/320) |
| Lobular | 6.6 (21/320) |
| Mixed (Ductal & Lobular) | 2.2 (7/320) |
| Medullary | 0.9 (3/320) |
| Papillar | 1.2 (4/320) |
| Mucinous | 0.9 (3/320) |
| Paget disease of the breast | 0.3 (1/320) |
| ND | 0.9 (3/323) |
| Stage | |
| I | 16.3 (52/318) |
| II | 46.2 (147/318) |
| III | 37.1 (118/318) |
| IV | 0.3 (1/318) |
| ND | 2.8 (9/327) |
| Tumor ER status | |
| Er+ | 77.5 (244/315) |
| Er− | 22.5 (71/315) |
| ND | 2.5 (8/323) |
| Tumor PR status | |
| Pr+ | 66.7 (210/315) |
| Pr− | 33.3 (105/315) |
| ND | 2.5 (8/323) |
| Tumor HER2 status | |
| Her2+ | 26.3 (83/316) |
| Her2− | 73.7 (233/316) |
| ND | 2.2 (7/323) |
| Tumor TN status | |
| TN | 10.4 (33/316) |
| non-TN | 89.6 (283/316) |
| ND | 2.2 (7/323) |
FH — Family history; BC — breast cancer; OC — ovarian cancer; ND — no data; ER — estrogen receptor; PR — progesterone receptor; TN — triple negative receptor status
All patients were screened for 13 recurrent BRCA1/2 mutations detected among Macedonian population (c.181T>G, c.1102G>T, c.3700_3704del, c.4035del, c.4065_4068del, c.5266dup, c.68_69del in BRCA1 and c.5722_5723del, c.5946del, c.7879A>T, c.8317_8330del, c.8168A>G, c.9098dup in BRCA2) by a single nucleotide primer extension assay utilizing the ABI PRISM™ SNaPshot Multiplex Kit (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions [16]. The analysis of the amplified fragments was performed on the ABI PRISM® 3130 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). Additionally, for 118 patients we performed targeted next generation sequencing (NGS) using TruSight Cancer sequencing panel to analyze 94 cancer-associated genes including BRCA1 and BRCA2 (Illumina, San Diego, CA, USA). The sequencing was done on the NGS MiSeq Illumina Personal Sequencer. The patients for NGS were selected according to their family history of cancer, early age of onset, and bilateral and/or triple negative (TN) breast cancer. The identified BRCA1/2 variants have been evaluated and classified based on the American College of Medical Genetics and Genomics (ACMG) classification guidelines [17].
Results
In our cohort of 327 Albanian women, we identified 21 pathogenic variants in BRCA1 and BRCA2 genes with overall mutation detection rate of 6.4% (21/327). The mean age of diagnosis among 21 BRCA1/2 pathogenic carriers was 39.3 years (range 22–56 years) and 38.1% reported positive family history for breast and/or ovarian cancer. The frequency of BRCA1 and BRCA2 pathogenic variants was 4.6% (15/327) and 1.8% (6/327), respectively. We found only two different deleterious mutations in BRCA1 and six in BRCA2 mutation positive cases (Tab. 2). All BRCA1 and BRCA2 mutations were found in one patient each, except for 3700_3704del which was identified in 14 patients, representing 93.3% (14/15) of all BRCA1 observed mutations and general mutation frequency of 4.3% (14/327). Overall mutation frequency for BRCA1 and BRCA2 genes in Albanians from Kosovo was 7.9% (17/216), of which 6.5% (14/216) were BRCA1 and 1.4% (3/216) were BRCA2 carriers. In Albanians from North Macedonia we observed lower overall mutation frequency compared to Kosovo (3.6%, 4/111) where BRCA1 was mutated in only one patient (0.9%, 1/111) and BRCA2 in three patients (2.7%, 3/111). The 3700_3704del mutation was detected in 76% (13/17) of the BRCA1/2 mutation positive patients and represent 93% (13/14) of BRCA1 mutation positive patients from Kosovo. In comparison, the 3700_3704del mutation was detected in only one Albanian patient from North Macedonia and was the only mutation detected in the BRCA1 gene.
Table 2.
Pathogenic variants in BRCA1 and BRCA2 genes identified in 327 Albanian patients from North Macedonia (NMK) and Kosovo (KS).
| Exon no. | HGVS nomenclature | AA change | Variant ID | Times observed in patients | Times observed in family members |
|---|---|---|---|---|---|
| BRCA1 (NM_007300.3) | |||||
| 11 | c.2933dup | p.Tyr978Ter | rs878853292 | 1 (KS) | 0 |
| 11 | c.3700_3704del | p.Val1234GlnfsTer8 | rs80357609 | 14 (13 KS + 1 NMK) | 1 (KS) |
| BRCA2 (NM_000059.3) | |||||
| 9 | c.775A>T | p.Arg259Ter | rs397507937 | 1 (KS) | 0 |
| 11 | c.5722_5723del | p.Leu1908ArgfsTer2 | rs80359530 | 1 (NMK) | 0 |
| 11 | c.6468_6469del | p.Gln2157IlefsTer18 | rs80359596 | 1 (NMK) | 0 |
| 17 | c.7895del | p.Ala2632GlufsTer16 | – | 1 (KS) | 0 |
| 25 | c.9304del | p.Ala3102GlnfsTer2 | – | 1 (NMK) | 0 |
| 25 | c.9352_9353del | p.Met3118ValfsTer31 | rs786203318 | 1 (KS) | 0 |
Patient and tumour characteristics of the c.3700_3704del carriers are summarized in Table 3. All c.3700_3704del carriers originated from Kosovo, except for one and the mean age of onset was 39.8 years (range 22–56 years). Nine patients (64.3%) reported family history for cancer and seven of them had relatives affected with breast and/or ovarian cancer. Thirteen carriers were diagnosed with invasive ductal carcinoma; one had both breast and ovarian cancer, while another had only ovarian cancer. Triple negative receptor status was found in 71.4% (10/14) of the c.3700_3704del tumours.
Table 3.
Clinical characteristics of 14 breast and/or ovarian cancer patients with 3700_3704del BRCA1 mutation
| N | Origin | Age at diagnosis (years) | Histopatology data | Stage | ER/PR pos. status | TN pos. status | Family history of cancer |
|---|---|---|---|---|---|---|---|
| 1 | N.Macedonia | 48 | Ductal | IIA | Yes | No | Yes (BC) |
| 2 | Kosovo | 41 | Ductal | IIB | Yes | No | Yes (OC) |
| 3 | Kosovo | 30 | Ductal | IA | No | Yes | Yes (BC) |
| 4 | Kosovo | 34 | Ductal | IIIA | No | Yes | No |
| 5 | Kosovo | 50 | Ductal | IIA | No | Yes | Yes (BC) |
| 6 | Kosovo | 41 | Ductal | IIA | No | Yes | No |
| 7 | Kosovo | 56 | Ductal bc and oc | IIA | No | Yes | No |
| 8 | Kosovo | 33 | Ductal | IA | Yes | No | No |
| 9 | Kosovo | 50 | Oc | IIIC | – | – | Yes (other Ca) |
| 10 | Kosovo | 39 | Ductal with medull. feat. | IIA | No | Yes | Yes (BC) |
| 11 | Kosovo | 22 | Ductal with medull. feat. | IA | No | Yes | Yes (BC) |
| 12 | Kosovo | 30 | Ductal with medull. feat. | IIIC | No | Yes | ND |
| 13 | Kosovo | 35 | Ductal with medull. feat. | IIA | No | Yes | Yes (other Ca) |
| 14 | Kosovo | 49 | Ductal | IIA | No | Yes | No |
ER — estrogen receptor; PR — progesterone receptor; TN — triple negative receptor status; pos. — positive; BC — breast cancer; OC — ovarian cancer; ND — no data; Ca — cancer; medull. feat. — medullary features
Discussion
The spectrum of BRCA1 and BRCA2 mutations varies extensively among populations. In some populations, a wide spectrum of different mutations exists, however, in others only specific BRCA1/2 mutations are present with high frequency as a consequence of a founder effect. Founder effects are most prominent in geographically, culturally or religiously isolated populations that undergo rapid expansion from a limited number of ancestors. Identification of founder mutations in certain ethnic groups is a very important step towards the development of screening protocols and improvement of genetic counselling.
We have previously reported that different BRCA1/2 mutations were present among breast and ovarian cancer patients from R.N. Macedonia with Macedonian and Albanian ethnic origin. In the current study of 327 breast and ovarian cancer patients with Albanian origin, an overall mutation rate of 6.4% was observed and eight different BRCA1/2 mutations were identified. All BRCA1/2 mutations were found in one patient each, except for c.3700_3704del BRCA1 mutation that was observed in 14 unrelated families, all except one originating from Kosovo. The c.3700_3704del mutation represents 93.3% of all BRCA1 observed mutations, thus showing a very strong founder effect among patients from Kosovo. We have not performed haplotype analysis, but the absence of the common BRCA1 coding sequence variants in all c.3700_3704del carriers suggests a common chromosomal background and represents an indirect evidence of the mutation founder effect. The c.3700_3704del carriers originated from different regions/cities in Kosovo. The BRCA1 c.3700_3704del mutation was present in only one of the 111 studied Albanian breast cancer patients from North Macedonia. Furthermore, this mutation was not present among 1023 breast and/or ovarian cancer patients of Macedonian origin screened in our laboratory (unpublished data). Although the majority of Albanians living in North Macedonia and those from Kosovo originate from the same ethnic Albanian sub-group of Ghegs, the frequency of c.3700_3704del mutation was much higher among Albanians from Kosovo. This may suggest the occurrence of the c.3700_3704del mutation after the settlement of the Albanian sub-group of Ghegs in the territory of Kosovo.
The c.3700_3704del BRCA1 mutation was first reported in 1995, in a small study from Pennsylvania (US) with frequency of 0.09% observed in 115 epithelial ovarian cancer patients [15]. The mutation has been found in several European countries with different frequencies, as well as in Australia and the United States. The highest c.3700_3704del mutation frequency was found in ovarian cancer patients from the Republic of Mordovia (Russia) accounting for 40% of all BRCA1 mutation positive cases, where haplotype analysis of 4 microsatellite markers confirmed its founder effect [18]. The mutational spectrum in this Russian region was different from BRCA1 mutation spectrum in St. Petersburg region, where the mutation was observed in 2.3% of the analysed breast cancer patients [19]. In the Czech Republic the mutation was identified in 13–15% BRCA1-carriers and has overall mutation frequency between 2.6%–3.2% in breast and ovarian cancer patients [10, 13, 20]. Furthermore, the mutation was reported several times as one of the recurrent mutations in breast and ovarian cancer patients from Poland with a frequency between 1%–3.3%, with the highest frequency observed in Northern Poland [11, 21–24] and a general frequency in Polish population of 0.1% [9]. The c.3700_3704del mutation was reported in Greece with a frequency between 0.3%–1.5% in breast and ovarian cancer cases [12, 25]. Additionally, the mutation was observed in breast and ovarian cancer patients from Turkey, Germany, Denmark, Romania and Slovakia with a frequency between 0.2–0.9% [26–30]. The observation of the BRCA1 c.3700_3704del mutation in many different populations raise the question of whether it is an ancient mutation with single origin, or whether it has arisen several times in human history. Haplotype analysis of BRCA1 c.3700_3704del carriers from different populations should be performed (are warranted) to resolve this question.
Conclusions
In conclusion, here we report the spectrum of BRCA1/2 mutations in Albanian patients with breast and/or ovarian cancer from Kosovo and North Macedonia. This is the first report of the spectrum of BRCA1/2 mutations in Kosovo. The finding that BRCA1 c.3700_3704del represents a founder mutation in Kosovo, with the highest worldwide reported frequency (6.0%, 13/216), supports the implementation of fast and low-cost screening protocol for this mutation in all breast cancer patients, regardless of the family history and even a pilot population-based screening in at-risk population.
Acknowledgements
We thank all the patients who took part in this study and all the clinicians, nurses and administrative staff for their contributions and commitment to this study.
Footnotes
Conflict of interest
None declared.
Funding
This study was funded by the Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Macedonian Academy of Sciences and Arts.
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