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Abstract

Background

Breast cancer remains a leading cause of mortality among Nigerian women, with triple-negative breast cancer (TNBC) being particularly prevalent. Variations in BRCA1 and BRCA2 genes remain key risk factors for this disease. However, there are gaps in the frequency and spectrum of these variants in Nigerian populations, as well as a dearth in the local capacity to characterize these variations.

Objective

This study aimed at identifying and characterizing the germline variations in BRCA1/2 in Nigerian breast cancer patients and healthy age-matched controls to understand the genetic risk profile of breast cancer in this population.

Methods

A prospective case-control study was conducted involving 45 breast cancer patients and 51 controls recruited from four major hospitals. DNA was extracted from blood samples, followed by targeted sequencing of BRCA1/2 exonic and intronic regions using the Ampliseq BRCA panel and Illumina MiSeq platform. Variant calling was performed, clinical significance was evaluated on ClinVar and BRCA Exchange databases, and haplotype analysis was performed using NIH LDlink and Haploview 4.2 software.

Results

Pathogenic BRCA1/2 variants were identified in 6.7% of breast cancer patients, all with TNBC and a family history of cancer. Two pathogenic BRCA1 variants were detected: a frameshift deletion BRCA1 c.133_134delAA (p.Lys45 fs) (rs397508857) and a missense variant BRCA1 c.5324T > A (p.Met1775Arg) (rs41293463). A BRCA2 frameshift deletion BRCA2 c.8817_8820del (p.Lys2939 fs) (rs397508010) was also identified. These variants were absent in controls. Haplotype analysis revealed distinct BRCA1 and BRCA2 haplotypes in the breast cancer group.

Conclusion

This study identifies key BRCA1/2 pathogenic variants and unique haplotypes in Nigerian breast cancer patients, highlighting the need for population-specific genetic screening. Integrating genetic testing into breast cancer management strategies could facilitate early detection, personalized treatment planning, and genetic counseling in Nigeria.

Keywords

breast cancer BRCA1 BRCA2 Nigeria germline variants haplotypes genetic screening

Background

Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer-related deaths among women globally,¹ with an estimated 2.3 million new cases and 666,103 deaths in 2022.¹ Africa reported approximately 198,553 cases, contributing 8.6% to the global burden, with Nigeria alone representing 16.3% of these cases.¹

Despite a lower incidence compared to high-income countries, Africa faces increasing BC mortality rates, primarily attributed to inadequate healthcare infrastructure, low public awareness, and frequent late-stage diagnoses.^2–4 Nigerian patients, in particular, exhibit a younger median age at BC diagnosis and often present with aggressive forms of the disease due to a higher prevalence of the triple-negative BC (TNBC) subtype.^4–6 The TNBC subtype continues to pose a management challenge, and this contributes to elevated mortality rates in the West African region.^6,7 Although the racial disparity in predisposition to and outcomes from BC are well documented, emerging evidence reveals significant population disparities in the African BC landscape, including variations in the frequency of germline predisposition mutations, profiles of somatic mutation landscapes, and distinctive mutational signatures.^8–10

The established risk of developing BC is associated with mutations in the homologous recombination pathway, with BRCA1 and BRCA2 genes, which account for approximately 70% and 23% of the mutational burden, respectively.¹¹ BRCA1 and BRCA2 are tumor suppressor proteins on chromosomes 17q21 and 13q12, respectively.¹² Studies indicate that germline pathogenic variants in BRCA 1/2 significantly contribute to inherited BC occurring in 5–10% of all women with BC, with a greater prevalence in the TNBC subtype.¹³ In West African populations, only one potential founder variant, the BRCA1 943ins10 variant, has been identified to date.¹⁴ However, its presence varies regionally; for example, it is absent in specific Nigerian populations.^15,16 This underscores the importance of exploring non-founder populations, such as those in Nigeria, to understand better the spectrum of recurrent pathogenic variants and their implications for BC risk.¹⁵ Although few studies have reported some unique BRCA1/2 in the Nigerian population,^15–17 it is questionable whether the sequencing and data analysis were performed indigenously, thereby exacerbating the gap in the skill of African indigenous investigators to meet the human resource workforce for cancer control.¹⁸

Early screening of pathogenic or likely pathogenic variants through genetic screening holds promise for improved diagnostic procedures and therapeutic outcomes. Integrating genetic testing into Nigeria's BC control strategy could provide valuable insights into the prevalence and impact of BRCA 1/2 in this population, facilitating targeted interventions and personalized treatment strategies. This study, therefore, aimed at identifying germline BRCA 1/2 variants among Nigerian BC patients and healthy controls.

Methods

Study Design and Population

This prospective case-control study recruited 45 patients with histopathological confirmed BC receiving treatments from the following teaching hospitals in Nigeria: National Hospital Abuja (NHA), Lagos University Teaching Hospital (LUTH), Lagos State University Teaching Hospital (LASUTH), and Federal Medical Centre Abeokuta (FMC), between May 2021-April 2022 and January-May 2024. The participants were diagnosed within the same period. Additionally, 51 controls aged 20–80 years with no previous history of cancer were included in the study. All BC participants were females aged 20–80 years old who had been diagnosed with BC and agreed to provide written consent after understanding the research details. Participants who were frail or diagnosed with other cancers or had other comorbidities that could interfere with result outcomes were excluded from the research.

The reporting of the study conforms to the STROBE guidelines for case-control studies.¹⁹

Blood Sample Collection and DNA Extraction

A total of 5 mL of venous blood was collected from BC patients and healthy controls into EDTA vacutainer tubes. The blood was centrifuged within 2 h of sample collection at 4000 rpm for 15 min to obtain the buffy coat fraction. Genomic DNA (gDNA) was subsequently extracted from either 200ul of peripheral blood or the buffy coat fraction using the DNA extraction kit from Aidlab Biotechnologies (HaiDian District, Beijing, China) following the manufacturer's protocol. Extracted gDNA was quantified with the Denovix broad range assay kit on the DeNovix DS-11 FX Fluorometer. Samples were then diluted to 50 ng/µL with TE buffer (10 mM Tris-HCl (pH 8.0) + 0.1 mM EDTA) for library preparation.

Library Preparation

BRCA1 and BRCA2 exonic and intronic regions were targeted using the Ampliseq for BRCA panel (Illumina, San Diego, CA, USA). The Ampliseq for BRCA panel consists of two primer pools spanning 265 amplicons. 50 ng of gDNA was used to prepare libraries using the Ampliseq library PLUS for Illumina kit (Illumina, San Diego, CA, USA) and Ampliseq CD indexes set A for Illumina (IDT, Coralville, IA, USA) following the manufacturer's reference guide (Document # 1000000039405 v06). Quality control of individual libraries was performed using the Agilent 4150 ScreenTape Station System (Agilent Technologies), with the High Sensitivity D5000 Screen Tape and reagents (Agilent Technologies), and the High Sensitivity Denovix kit on the DeNovix DS-11 FX Fluorometer. Libraries were then pooled in equimolar volumes, diluted to 20 pM, and loaded in the Miseq v3 reagent cartridge (Illumina). Sequencing was performed by the Illumina Miseq Sequencer with a 300 cycle (150 bp paired end reads).

Data Analysis

The primary analysis was conducted using the on-instrument software SCS/RTA (Illumina). Subsequently, a secondary analysis was performed using the DNA Amplicon workflow available on the Local Run Manager Version 3.0.1.1 (Illumina, San Diego, CA, USA), with reference to the hg19 genome assembly using the BRCA.dna_manifest.20180509.txt manifest file. Alignment was conducted with the software aligner BWA-MEM Whole-Genome (v0.7.9a-isis-1.0.2), variant calling was performed with Pisces Variant Caller (v5.2.9.23), and metrics accessed using Bam Metrics (v0.0.22) and SAMtools (v0.1.19-isis-1.0.3), all provided by Illumina (San Diego, CA, USA). To ensure the reliability of the identified variants, a quality score filter of Q30 was applied, a minimum read coverage of 10 was required, and only variants that met the quality thresholds (quality score >100 and ‘pass’ filter) were included in the analysis. All variant nomenclature and clinical significance were cross-verified with the dbSNP (https://www.ncbi.nlm.nih.gov/snp/),²⁰ ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/)²¹ and BRCA Exchange databases (https://www.brcaexchange.org).²² Variant tables were then imported into RStudio for further visualization and analysis using the maftools package²³ on R (v4.3.3).

To categorize variants of unknown significance (VUS), the pathogenicity effect was evaluated using three in silico mutation interpretation tools: SIFT (Sorting intolerant form tolerant), PROVEAN (Protein variation effect analyzer), and MutationTaster2.^24–26 The VUS were further characterized using the Genebe.net online platform²⁷ following the guidelines set by the American College of Medical Genetics and Genomics (ACMG) for reporting and characterizing variants.²⁸

For the analysis of linkage disequilibrium (LD) patterns in BRCA1/2 variants, the NIH LDlink (ldlink.nih.gov) was used, using the Yoruba in Ibadan, Nigeria (YRI) and Esan in Nigeria (ESN) populations from the 1000 Genomes Project as reference population.^29,30 Genotypes were phased using the geneHapR package,³¹ and haplotype analysis in both cases and controls was carried out using the Haploview 4.2 software.³² Fifteen BRCA1 and twenty-two BRCA2 haplotype tagging SNPs (htSNPs) were selected to determine the common haplotypes as described by.³³

Results

Clinicopathological Characteristics of BC Patients and Controls

This study recruited 45 breast cancer (BC) patients and 51 control subjects. The mean age of the BC patients was 48.33 ± 10.60 years, ranging from 31 to 76 years, while the mean age of the control subjects was 46.41 ± 11.69 years. Fifteen patients (35.6%) had invasive breast cancer of no special type (IBC-NST), 29 (64.4%) had invasive ductal carcinoma (IDC), and one patient had invasive lobular carcinoma (ILC). Fifteen of these patients were diagnosed with the triple-negative breast cancer subtype (TNBC), seven with the luminal subtype, and two with the human epidermal growth factor receptor 2 positive (HER2+) subtype; immunohistochemistry results were unavailable for 21 patients. Table 1 summarizes the clinical and pathological characteristics of both the BC patients and the control subjects.

Table 1.

Clinical and Pathological Characteristics of BC and Control Subjects.

Variable	Cases (n = 45)	Control (n = 51)
Age(years)
≤ 40	11 (24.4%)	12 (23.53%)
41–50	17 (37.8%)	27 (52.94%)
> 50	15 (33.3%)	12 (23.53%)
Unknown	2 (4.4%)	0 (0%)
BMI	29.27 ± 9.21	27.04 ± 7.81
Clinical Information (Cases)
Tumor Location	Number	Percentage
LB	18	40%
RB	21	46.7%
BB	1	2.2%
Unknown	5	11.1%
Histopathology
IBC(NST)	15	33.3%
IDC	29	64.5%
ILC	1	2.2%
Immunohistochemistry
Luminal	7	15.6%
TNBC	15	33.3%
HER2+	2	4.4%
Unknown	21	46.7%
Risk
Smoking
Stopped smoking	2	4.4%
Never smoked	43	95.6%
Alcohol intake
Never drank	39	86.7%
Current drinker	1	2.2%
Stopped drinking	5	11.1%
Menopausal status
Yes	20	44.4%
No	17	37.8%
Choose not to say	8	17.8%
Family history
Yes	9	20%
No	36	80%
Oral contraceptive use
Yes	10	22.2%
No	29	64.4%
Choose not to say	6	13.3%

Pathogenic BRCA1 and BRCA2 Variants in BC and Controls

In this study, BRCA1 and BRCA2 heterozygous pathogenic variants were identified in 6.7% of cases, with a total of three pathogenic variants observed (3/45). These pathogenic variants were found exclusively in individuals with BC and were absent in the control group. Specifically, the pathogenic variants included two BRCA1 (c.133_134delAA and c.5324T > A) and one BRCA2 (c.8817_8820del). Among the BRCA1 variants, one was a frameshift deletion, and the other a missense variant. The BRCA2 variant was also a frameshift deletion. All individuals carrying a pathogenic variant had a reported family history of cancer and were diagnosed with the TNBC subtype, as detailed in Table 2. The pedigree illustrating the patient with the BRCA2 c.8817_8820del (p.Lys2939 fs) is shown in Figure 1.

Figure 1.

Informative pedigree for the BC participant with the BRCA2 c.8817_8820del (p.Lys2939 fs) variant.

Table 2.

Characteristics of Pathogenic BRCA1 and BRCA2 Variants.

Age	Subtype	Family History	Tribe	Gene	HGVS	Variant Classification	MAF (NIG)	Rs_ID (dsSNP)
31	TNBC	Yes	Yoruba	BRCA1	BRCA1 c.133_134delAA (p.Lys45 fs)	FrameShift	0.230⁽¹⁵⁾	rs397508857
37	TNBC	Yes	Ora	BRCA1	BRCA1 c.5324T > A (p.Met1775Arg)	Missense	0.691⁽¹⁵⁾, 0.843⁽¹⁶⁾	rs41293463
55	TNBC	Yes	Yoruba	BRCA2	BRCA2 c.8817_8820del (p.Lys2939 fs)	FrameShift	0.461⁽¹⁵⁾	rs397508010

HGVS = Human Genome Variation Society, MAF = Minor allele frequency, NIG = Nigeria.

Benign/Likely Benign Variants

A total of ninety-seven heterozygous benign or likely benign variants in BRCA1 and BRCA2 were identified in both BC and control groups, comprising 36 BRCA1 variants and 61 BRCA2 variants. All identified variants were heterozygous.

For BRCA1, the variants were classified as follows: 12 missense, 14 intronic, 8 synonymous, 1 splice site, and 1 3’ UTR variant. Of these, 23 variants of benign or likely benign significance were found in both the BC and control groups. Additionally, 8 variants were exclusive to the control group, while 5 variants were identified only in the BC group.

For BRCA2, the variants were categorized as follows: 26 missense, 16 intronic, 16 synonymous, 1 splice site, and 2 5’ UTR variants (see Table 3). Among these, 38 variants were common to both BC and control groups, 18 variants were exclusive to the control group, and 5 variants were identified in the BC group only (see Table 3).

Table 3.

Benign/Likely Benign BRCA1/2 Germline Variants in BC and Controls.

S/N	Gene	Variant Classification	ClinVar	Exon	HGVS	BC_Count	Control _Count	SIFT_SCORE (Missense)	Rs_ID (dbSNP)
1	BRCA1	3'UTR	Benign		BRCA1 c.*36C > G	4	7		rs3092995
2	BRCA1	Intron	Benign		BRCA1 c.212 + 23T > A	3	2		rs8176128
3	BRCA1	Intron	Benign		BRCA1 c.4358–2885G > A	11	13		rs8176193
4	BRCA1	Intron	Benign		BRCA1 c.441 + 18CTT	8	13		rs147856441
5	BRCA1	Intron	Likely benign		BRCA1 c.441 + 51T > C	1	0		rs578250989
6	BRCA1	Intron	Benign		BRCA1 c.441 + 52_441 + 63del	2	7		rs536390258
7	BRCA1	Intron	Benign		BRCA1 c.441 + 64del	1	7		rs72434991
8	BRCA1	Intron	Benign		BRCA1 c.4987–20A > C	1	1		rs80358035
9	BRCA1	Intron	Benign		BRCA1 c.4987–68A > G	16	14		rs8176234
10	BRCA1	Intron	Benign		BRCA1 c.4987–92A > G	16	14		rs8176233
11	BRCA1	Intron	Benign		BRCA1 c.5152 + 66G > A	7	13		rs3092994
12	BRCA1	Intron	Benign		BRCA1 c.5152 + 85del	3	5		rs8176259
13	BRCA1	Intron	Benign		BRCA1 c.5406 + 68T > C	3	1		rs8176307
14	BRCA1	Splice Site	Benign		BRCA1 c.5406 + 8T > G	1	5		rs55946644
15	BRCA1	Intron	Likely benign		BRCA1 c.548–155G > A	0	1		rs139893476
16	BRCA1	Intron	Likely benign		BRCA1 c.81–13C > T	0	2		rs56328013
17	BRCA1	Missense	Benign	11	BRCA1 c.2458A > G (p.Lys820Glu)	3	2	0.173	rs56082113
18	BRCA1	Missense	Likely benign	11	BRCA1 c.37T > G (p.Ile379Met)	2	0	0.001	rs56128296
19	BRCA1	Synonymous	Likely benign	11	BRCA1 c.1971A > G (p.Gln657=)	2	0		rs28897679
20	BRCA1	Synonymous	Benign	11	BRCA1 c.2082C > T (p.Ser694=)	18	20		rs1799949
21	BRCA1	Synonymous	Benign	11	BRCA1 c.2109A > C (p.Thr703=)	0	1		rs4986844
22	BRCA1	Missense	Benign	11	BRCA1 c.2167A > G (p.Asn723Asp)	0	2	0.181	rs4986845
23	BRCA1	Synonymous	Benign	11	BRCA1 c.23T > C (p.Leu771=)	9	12		rs16940
24	BRCA1	Missense	Benign	11	BRCA1 c.2612C > G (p.Pro871Arg)	44	46	1	rs799917
25	BRCA1	Synonymous	Benign	11	BRCA1 c.2814A > G (p.Pro938=)	0	2		rs80356851
26	BRCA1	Missense	Likely benign	11	BRCA1 c.33A > C (p.Glu1038Ala)	14	15	0.029	rs16941
27	BRCA1	Missense	Benign	11	BRCA1 c.3418A > C (p.Ser40Arg)	3	1	0.189	rs2227945
28	BRCA1	Missense	Likely benign	11	BRCA1 c.3600G > T (p.Gln1200His)	0	1	0.034	rs56214134
29	BRCA1	Synonymous	Likely benign	11	BRCA1 c.3739G > A (p.Val1247Ile)	1	0	0.478	rs80357191
30	BRCA1	Missense	Benign	11	BRCA1 c.765G > C (p.Glu255Asp)	0	1	1	rs62625299
31	BRCA1	Synonymous	Likely benign	13	BRCA1 c.4308T > A (p.Ser1436=)	12	12		rs1060915
32	BRCA1	Missense	Benign	15	BRCA1 c.4600G > A (p.Val1534Met)	1	1	0.246	rs55815649
33	BRCA1	Missense	Likely benign	16	BRCA1 c.4682C > A (p.Thr1561Asn)	0	1	0.001	rs56158747
34	BRCA1	Missense	Benign	16	BRCA1 c.4691T > C (p.Leu1564Pro)	1	0	0.241	rs56119278
35	BRCA1	Missense	Likely benign	16	BRCA1 c.4837A > G (p.Ser1613Gly)	18	22	0.027	rs1799966
36	BRCA1	Synonymous	Likely benign	17	BRCA1 c.4992C > A (p.Leu1664=)	1	2		rs142459158
37	BRCA2	5'UTR	Benign		BRCA2 c.-11C > T	1	2		rs76874770
38	BRCA2	Splice_Site	Likely benign		BRCA2 c.1909 + 9_1909 + 10del	1	1		rs527732001
39	BRCA2	5'UTR	Benign		BRCA2 c.-26G > A	6	5		rs1799943
40	BRCA2	Intron	Benign		BRCA2 c.425 + 67A > C	1	4		rs11571610
41	BRCA2	Intron	Benign		BRCA2 c.516 + 21A > T	2	3		rs11571622
42	BRCA2	Intron	Likely benign		BRCA2 c.517–19C > T	0	2		rs11571623
43	BRCA2	Intron	Benign		BRCA2 c.681 + 56C > T	21	21		rs2126042
44	BRCA2	Intron	Benign		BRCA2 c.682–30A > C	0	1		rs11571636
45	BRCA2	Intron	Benign		BRCA2 11 c.1909 + 21_1909 + 22del	1	1		rs276174816
46	BRCA2	Intron	Benign		BRCA2 c.7435 + 53C > T	1	3		rs11147489
47	BRCA2	Intron	Benign		BRCA2 c.7617 + 39G > A	0	1		rs190660894
48	BRCA2	Intron	Benign		BRCA2 c.7805 + 13A > G	1	0		rs149769332
49	BRCA2	Intron	Benign		BRCA2 c.7805 + 6C > G	0	1		rs81002819
50	BRCA2	Intron	Benign		BRCA2 c.7806–14T > C	29	41		rs9534262
51	BRCA2	Intron	Likely benign		BRCA2 c.7806–40A > G	1	6		rs9590939
52	BRCA2	Intron	Benign		BRCA2 c.8487 + 19A > C	3	7		rs11571743
53	BRCA2	Intron	Benign		BRCA2 c.8487 + 47C > T	5	2		rs11571744
54	BRCA2	Intron	Benign		BRCA2 c.8755–66T > C	31	33		rs4942486
55	BRCA2	Intron	Benign		BRCA2 c.9648 + 54G > A	1	1		rs11571823
56	BRCA2	Missense	Likely benign	3	BRCA2 c.175C > G (p.Pro59Ala)	0	1	0.228	rs56091799
57	BRCA2	Synonymous	Benign	3	BRCA2 c.231T > G (p.Thr77=)	3	0		rs114446594
58	BRCA2	Missense	Likely benign	10	BRCA2 c.1040A > G (p.Gln347Arg)	1	0	0.606	rs55800493
59	BRCA2	Synonymous	Benign	10	BRCA1 c.1275A > G (p.Glu425=)	0	2		rs34355306
60	BRCA2	Synonymous	Benign	10	BRCA1 c.1365A > G (p.Ser455=)	1	4		rs1801439
61	BRCA2	Synonymous	Benign	10	BRCA1 c.1788T > C (p.Asp596=)	0	1		rs11571642
62	BRCA2	Missense	Benign	10	BRCA1 c.1798T > C (p.Tyr600His)	0	1	0.252	rs75419644
63	BRCA2	Missense	Benign	10	BRCA1 c.865A > C (p.Asn289His)	1	4	0.046	rs766173
64	BRCA2	Synonymous	Likely benign	11	BRCA1 c.1911T > C (p.Gly637=)	1	0		rs11571652
65	BRCA2	Missense	Benign	11	BRCA1 c.2138A > T (p.Gln713Leu)	0	2	0.059	rs55816687
66	BRCA2	Synonymous	Benign	11	BRCA1 c.2229T > C (p.His743=)	1	4		rs1801499
67	BRCA2	Missense	Benign	11	BRCA1 c.2786T > C (p.Leu929Ser)	0	1	0.033	rs2227943
68	BRCA2	Missense	Benign	11	BRCA1 c.2960A > T (p.Asn987Ile)	0	1	0.056	rs2227944
69	BRCA2	Missense	Benign	11	BRCA1 c.2971A > G (p.Asn991Asp)	2	5	1	rs1799944
70	BRCA2	Synonymous	Benign	11	BRCA1 c.3264T > C (p.Pro1088=)	1	3		rs36060526
71	BRCA2	Synonymous	Benign	11	BRCA1 c.3396A > G (p.Lys1132=)	17	20		rs1801406
72	BRCA2	Synonymous	Benign	11	BRCA1 c.3807T > C (p.Val1269=)	15	14		rs543304
73	BRCA2	Missense	Benign	11	BRCA1 c.3869G > A (p.Cys1290Tyr)	3	1	1	rs41293485
74	BRCA2	Missense	Likely benign	11	BRCA1 c.4090A > C (p.Ile1364Leu)	1	2	0.709	rs56248502
75	BRCA2	Missense	Benign	11	BRCA1 c.4241C > T (p.Thr1414Met)	0	1	0.348	rs70953664
76	BRCA2	Synonymous	Benign	11	BRCA1 c.4563A > G (p.Leu1521=)	42	43		rs206075
77	BRCA2	Missense	Benign	11	BRCA1 c.4681C > A (p.His1561Asn)	0	1	1	rs2219594
78	BRCA2	Missense	Likely benign	11	BRCA1 c.5170A > G (p.Ile1724Val)	0	1	0.948	rs35335654
79	BRCA2	Synonymous	Likely benign	11	BRCA1 c.5418A > G (p.Glu1806=)	2	3		rs34351119
80	BRCA2	Missense	Benign	11	BRCA1 c.5704G > T (p.Asp1902Tyr)	0	1	0.477	rs4987048
81	BRCA2	Synonymous	Likely benign	11	BRCA1 c.6057C > T (p.Asn2019=)	0	2		rs147961615
82	BRCA2	Missense	Likely benign	11	BRCA1 c.6220C > A (p.His2074Asn)	1	2	0.223	rs34309943
83	BRCA2	Missense	Benign	11	BRCA1 c.6290C > T (p.Thr2097Met)	1	0	0.096	rs80358866
84	BRCA2	Missense	Likely benign	11	BRCA1 c.6347A > G (p.His2116Arg)	1	1	0.318	rs55953736
85	BRCA2	Synonymous	Benign	11	BRCA1 c.6513G > C (p.Val2171=)	45	46		rs206076
86	BRCA2	Missense	Likely benign	14	BRCA1 c.7017G > C (p.Lys2339Asn)	2	3	0.032	rs45574331
87	BRCA2	Missense	Benign	14	BRCA1 c.7150C > A (p.Gln2384Lys)	0	1	1	rs55977008
88	BRCA2	Synonymous	Benign	14	BRCA1 c.7242A > G (p.Ser2414=)	16	19		rs1799955
89	BRCA2	Missense	Likely benign	14	BRCA1 c.7319A > G (p.His2440Arg)	1	2	0.494	rs4986860
90	BRCA2	Missense	Benign	14	BRCA1 c.7397T > C (p.Val2466Ala)	42	43	0.697	rs169547
91	BRCA2	Missense	Likely benign	15	BRCA1 c.7504C > T (p.Arg2502Cys)	1	1	0.118	rs55716624
92	BRCA2	Synonymous	Benign	19	BRCA1 c.8460A > C (p.Val2820=)	4	8		rs9590940
93	BRCA2	Missense	Benign	20	BRCA1 c.8503T > C (p.Ser2835Pro)	0	1	0.33	rs11571746
94	BRCA2	Missense	Benign	22	BRCA1 c.8830A > T (p.Ile2944Phe)	8	2	0.008	rs4987047
95	BRCA2	Missense	Benign	27	BRCA1 c.10234A > G (p.Ile3412Val)	8	14	0.35	rs1801426
96	BRCA2	Synonymous	Likely benign	27	BRCA1 c.9720T > C (p.Val3240=)	0	1		rs80359810
97	BRCA2	Missense	Benign	27	BRCA1 c.9730G > A (p.Val3244Ile)	1	3	0.713	rs11571831

5’UTR = 3 prime untranslated region, HGVS = Human Genome Variation Society.

BRCA1/ 2 Variants Associated with Protein Change

Twenty BRCA1 variants associated with a protein change were plotted on a lollipop plot. Among these, BRCA1 variants c.2612C > G (rs799917), c.4837A > G (rs1799966), and c.2082C > T (rs179949) had the highest counts in both the breast cancer (BC) and control groups (Figure 2(a-b)).

Figure 2.

BRCA variants associated with protein change, showing variation in (a) BRCA1 in BC, (b) BRCA1 in control, (c) BRCA2 in BC, and (d) BRCA2 in controls.

Similarly, forty-two BRCA2 variants associated with a protein change were plotted on a separate lollipop plot. The BRCA2 variants c.6513G > C (rs06076), c.7397T > C (rs169547), and c.4563A > G (rs206075) exhibited the highest counts in both the BC and control groups (Figure 2 (c-d)).

Variants of Unknown Significance (VUS)

Eighteen heterozygous VUS were identified in both BC and control groups. This includes 7 BRCA1 and 11 BRCA2 variants. The VUS were subjected to three online prediction tools: SIFT, PROVEAN, and MutationTaster. The three software applications predicted 14 VUS as tolerant, while 4 variants were predicted to be deleterious to protein function (Table 4).

Table 4.

Variants of Unknown Significance (VUS) in BRCA1/2 for Breast Cancer (BC) and Control Groups, with Predictive Analyses.

S/N	Gene	Variant Classification	ClinVar	Exon	HGVS	BC Count	Control Count	SIFT_SCORE (MISSENSE)	AMCG Classification	Rs_ID (dbSNP)
1	BRCA1	Intron	VUS	NA	BRCA1 c.5152 + 80del	0	2		Benign	rs902919427
2	BRCA1	Intron	VUS	NA	BRCA1 c.441 + 83G > T	1	2		Likely benign	rs986645078
3	BRCA1	Missense	VUS	9	BRCA1 c.557C > A (p.Ser186Tyr)	1	3	0.002	Benign	rs55688530
4	BRCA1	Missense	VUS	11	BRCA1 c.1807T > G (p.Ser603Ala)	1	0	1	Likely benign	rs1555591208
5	BRCA1	Missense	VUS	11	BRCA1 c.3190A > G (p.Ser1064Gly)	0	1	0.001	Uncertain significance	rs273899702
6	BRCA1	Missense	VUS	11	BRCA1 c.3548A > T (p.Lys1183Ile)	17	20	1	Benign	rs16942
7	BRCA1	Synonymous	VUS	16	BRCA1 c.4813T > C (p.Leu1605=)	0	1		Benign	rs80356833
8	BRCA1	Missense	VUS	22	BRCA1 c.5348T > C (p.Met1783Thr)	0	2	0.001	Uncertain significance	rs55808233
9	BRCA2	Missense	VUS	4	BRCA2 c.322A > C (p.Asn108His)	1	0	1	Benign	rs80358567
10	BRCA2	Synonymous	VUS	10	BRCA2 c.1263A > G (p.Gln421=)	1	0		Likely benign	rs2072407964
11	BRCA2	Missense	VUS	10	BRCA2 c.1114A > C (p.Asn372His)	12	4	1	Benign	rs144848
12	BRCA2	Missense	VUS	11	BRCA2 c.5756A > G (p.Lys1919Arg)	1	0	1	Likely benign	rs1593906437
13	BRCA2	Synonymous	VUS	11	BRCA2 c.3102T > C (p.Ile1034=)	1	0		Likely benign	rs2072472508
14	BRCA2	Missense	VUS	11	BRCA2 c.3176T > G (p.Leu1059Arg)	0	1	1	Likely benign	rs1064793927
15	BRCA2	Missense	VUS	14	BRCA2 c.7184A > C (p.His2395Pro)	1	0	1	Uncertain significance	rs1593917980
16	BRCA2	Missense	VUS	26	BRCA2 c.9634G > C (p.Gly3212Arg)	0	2	1	Benign	rs55775473
17	BRCA2	Missense	VUS	27	BRCA2 c.10121C > T (p.Thr3374Ile)	1	1	0.06	Benign	rs56309455
18	BRCA2	Missense	VUS	27	BRCA2 c.9925G > A (p.Glu3309Lys)	0	1	0.748	Likely benign	rs80359251

Patterns of Linkage Disequilibrium (LD) and Haplotype Analysis

To identify haplotype tagging SNPs (htSNPs), we analyzed 46 BRCA1 and 72 BRCA2 variants using the LDlink and LDmatrix tools from the NIH, utilizing data from the YRI and ESN populations in the 1000 Genomes database. The analysis revealed strong linkage disequilibrium (LD) among 13 BRCA1 and 15 BRCA2 variants, with r² and D' values ranging from 0.92 to 1 (Figures S1 and S2).

These findings were further validated using Haploview 4.2 with the four-gamete rule. For BRCA1, in the BC group, five haplotype blocks were identified, with Block 2 being the largest at 30 kb and Block 3 at 0 kb (Figure S3). In the control group, four haplotype blocks were observed, with Block 4 being the largest at 33 kb and Block 2 being the smallest at 0 kb (Figure S4). For BRCA2, the BC group showed five haplotype blocks, with Block 5 being the largest at 28 kb (Figure S5). Similarly, in the control group, six haplotype blocks were identified, with Block 6 being the largest at 28 kb (Figure S6).

To represent the BC group for BRCA1, nine htSNPs were selected based on LD patterns from the NIH LD matrix and Haploview results, revealing seven haplotypes (Figure 3). In the control group, twelve htSNPs were selected, resulting in eleven haplotypes (Figure 4). For BRCA2, twelve htSNPs were selected in the BC group based on LD patterns, revealing six haplotypes (Figure 5). In the control group, seventeen htSNPs were selected, resulting in eleven haplotypes (Figure 6).

Figure 3.

Linkage disequilibrium patterns and haplotype distribution of BRCA1 htSNPs in BC.

Figure 4.

Linkage disequilibrium patterns and haplotype distribution of BRCA1 htSNPs in controls.

Figure 5.

Linkage disequilibrium patterns and haplotype distribution of BRCA2 htSNPs in BC.

Figure 6.

Linkage disequilibrium patterns and haplotype distribution of BRCA2 htSNPs in controls.

Discussion

Breast cancer remains a significant health challenge for women in Nigeria, characterized by its aggressive nature and high mortality rates.⁴ This study explored the spectrum of BRCA1/2 variants among Nigerian BC patients and healthy controls, shedding light on the genetic predisposition to BC in the Nigerian population.

Our findings revealed two pathogenic BRCA1 variants (c.133_134delAA and c.5324T > A), found exclusively in BC cases and absent in the control group. The BRCA1 c.133_134delAA variant was detected in a 31-year-old woman with IDC of the TNBC subtype of BC, who also reported a family history of BC. This frameshift variant at codon 45 results in a change from the amino acid lysine to isoleucine, leading to the premature termination of protein synthesis, either through mRNA decay or protein truncation.³⁴ A previous study associated this variant with individuals from families affected by breast and ovarian cancers.³⁵ Notably, it was previously reported in a 43-year-old Nigerian BC patient of Yoruba origin.¹⁵ Our finding, along with these previous reports, suggests that this variant could potentially be associated with early onset BC.

Additionally, the pathogenic BRCA1 c.5324T > A variant was detected in a 37-year-old with IDC of the TNBC subtype who had two relatives with a history of BC. This missense variant affects the BRCA1 BRCT domain, disrupting its interactions with the BRCA1 interacting protein (BRIP1) and carboxy-terminal binding protein (CtIP).³⁶ This variant was the first BRCA1 variant identified in an African American family with hereditary breast and ovarian cancer syndrome³⁷ and has been observed in multiple families with a familial history of breast and ovarian cancer, including Nigerians.^{15,16,38–40}

In BRCA2, we identified one pathogenic variant (BRCA2 c.8817_8820del) in a 55-year-old IDC-TNBC patient of Yoruba origin. This variant is a deletion of four nucleotides in exon 22 of the BRCA2 gene, creating a frameshift in the reading frame, with a concomitant introduction of premature stop translation signal. The BRCA2 c.8817_8820del variant has been previously reported in two BC patients of the Yoruba tribe in Nigeria but has not been identified in the general population, and this underscores its potential ethnic specificity.¹⁶

A possible explanation for the low frequency of germline pathogenic BRCA1/2 variants (6.7%, 3/45) observed in this study could be the limited number of patients with a family history of BC (9/45), the inclusion of older patients, and the limited number of TNBC subtype cases in our selection criteria. Studies have shown that a strong family history of BC and younger age have been established as key factors to consider for genetic testing for HBOC syndrome, as highlighted in various guidelines for different populations.⁴¹ Furthermore, previous studies in other populations have reported BRCA1/2 prevalence rates between 11% and 20% in TNBC patients.^42,43 However, this selection criterion has been challenged by several bodies; for example, Manahan⁴¹ in an updated consensus guideline by the American Society of Breast Surgeons, argued that genetic testing be made available to all patients diagnosed with BC, noting that previous guidelines focusing on patients with personal history were more exclusionary than inclusive. Additionally, studies on Ashkenazi Jews have shown that a population-based screening approach yielded a higher detection of BRCA 1/2 variants compared to screening based solely on a strong family history.⁴⁴

We also identified ninety-seven BRCA1/2 variants of benign or likely benign clinical significance in both BC and controls. Among the BRCA1 variants, BRCA1 c.2612C > G (p.Pro871Arg) (rs799917), BRCA1 c.2082C > T (p.Ser694=) (rs1799949), and BRCA1 c.4837A > G (p.Ser1613Gly) (rs1799966) had the highest minor allele frequencies in both groups. Specifically, the BRCA1 c.2612C > G variant was present in 97.7% of BC cases and 90.2% of controls. This variant is known to downregulate BRCA1 expression by disrupting the interaction between BRCA1 mRNA and miR-638.^45,46 It has been associated with increased risks of gastric⁴⁵ and lung cancers.⁴⁷ However, its association with BC remains controversial, with some studies reporting an association⁴⁸ and others reporting a lack of association.^49,50 Higher frequencies of the BRCA1 c.2612C > G variant were also reported in Nigerian men with prostate cancer⁴⁶ and Tanzanian BC patients,⁵¹ and this suggests that this variant may be ancestry-driven. Globally, the minor allele of the BRCA1 c.2612C > G variant has a frequency of 0.54 in the 1000 Genomes project, but it is notably higher at 0.89 among African populations.⁵² Consequently, the prevalence of this SNP in Indigenous Africans suggests that this SNP may be a surrogate indication of ancestry rather than a risk marker and may have a yet-to-be-determined adaptational advantage in this population.

Among the BRCA1 variants of benign/likely benign significance, five variants were exclusive to the BC group only (Table 2). These variants had been previously reported in Nigerians and African Americans with BC but were established to be of mild clinical significance.^15,53 For BRCA2, sixty-one variants of benign significance were identified in both BC patients and controls. The minor allele of BRCA2 c.6513G > C (p.Val2171=) (rs206076), BRCA2 c.7397T > C (p.Val2466Ala) (rs169547), and BRCA2 c.4563A > G (p.Leu1521=) (rs206075) had the highest frequencies in BC cases (100%, 93.3%, and 93%) and controls (90.2%, 84.3%, and 84.3%). These frequencies are consistent with global data from the 1000 Genomes Project, which reported prevalence rates of 97.4%, 93.4%, and 92.5%, respectively.⁵² Additionally, five BRCA2 variants of benign/likely benign significance were found exclusively in BC patients and absent from the controls. These variants have been reported at very low frequencies in various populations, including Nigeria.¹⁵ Recent studies suggest that these so-called “harmless” benign variants may aggregate to influence somatic evolution.⁵⁴

Our study also identified 7 BRCA1 (21.3%) and 11 BRCA2 (18.0%) variants of uncertain significance (VUS). Four of these VUS (BRCA1 c.5348T > C, BRCA1 c.3190A > G, BRCA2 c.10121C > T, BRCA1 c.1807T > G) were predicted to be damaging to protein function by three online prediction software tools. High frequencies of BRCA1/2 VUS in African women pose a significant challenge for genetic testing.⁵⁵ This highlights the need for improved strategies to characterize VUS in African populations and the inclusion of African biospecimens in international initiatives like the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium (http://www.enigmaconsortium.org/).

We utilized the LDmatrix tool and Haploview 4.2 to identify haplotype-tagging SNPs (htSNPs) for BRCA1 and BRCA2.^29,32,33 In the BC group for BRCA1, nine htSNPs revealed seven haplotypes. Haplotype analysis of BRCA genes provides valuable insights into genetic predispositions, founder variants, and treatment responses across different populations.^56,57 Several studies have detected certain BRCA1 haplotypes associated with increased BC risk, although these studies have primarily focused on a limited number of tagging SNPs selected for polymerase chain reaction genotyping.^58–61 For instance, Pelletier⁵⁸ identified five distinct BRCA1 haplotypes using three htSNPs (rs12516, rs8176318, and rs3092995) in the 3′ UTR region, which were strongly associated with BC risk in African Americans. However, in our sequencing data, we identified only the rs3092995 variant which exhibited low LD with other variants. Similarly, Freedman⁶⁰ found no association between five htSNPs (rs1799950, rs799917, rs2227945, rs16942, and rs1799966) and BC risk in African Americans; four of these variants were also present in both BC and control samples in our study. Another study utilized six htSNPs (rs1799966, rs1060915, rs16942, rs16941, rs16940, and rs179949) to predict haplotypes in early-onset Malaysian BC patients. Five of these variants, excluding rs179949, were also detected in our study, and they suggested that the identified rare haplotypes may be associated with early-onset BC in the Malaysian population.⁶¹

We compared the haplotypes detected in our BC group with the haplotypes in the 1000 Genomes Project data available through the LDlink tool.²⁹ Our results indicate that six of our haplotypes align with haplotypes from the YRI and ESN populations in the 1000 Genomes Project.³⁰ However, Hap 7, occurring in about 1% of the BC group, was absent in the YRI and ESN populations data, although it was observed in 3% of the Asian population.³⁰ This suggests potential ethnic and geographic variation in BRCA1 haplotypes.⁶² Additionally, two haplotypes from the 1000 genome data (A_A_C_C_A_T_A_T_G, and A_A_C_C_G_C_A_T_G)³⁰ were undetected in our study, and this may be due to our limited sample size.

In the control group, twelve htSNPs revealed eleven haplotypes, with Hap 1 being the most common, accounting for 57.4% of the control group. This frequency is lower than the YRI and ESN populations in the 1000 Genomes Project.³⁰ For instance, Hap 2, which accounts for 17.0% of the control group, was found at 6.52% in the 1000 Genomes data, ranking third in that dataset.³⁰ Additionally, Haps 6 and 8–11 were absent in the YRI and ESN populations data but occurred in 1% of our control group population. The 1000 genome data remain the largest open-source reference for global genetic variation, providing a catalog of gene variations across populations, including Nigerians.^30,63

For BRCA2, using twelve haplotype-tagging SNPs (htSNPs) in the BC group, our results revealed six haplotypes. A previous study using fifty genotyped variants revealed an association between some detected haplotypes and BC risk⁶⁴; however, when compared to our detected BRCA2 variants, only nine of the fifty genotyped variants (rs206076, rs169547, rs2126042, rs1801406, rs1799955, rs144848, rs1801426, rs1799944, and rs766173) were found in our study. Similarly, in an investigation with early-onset BC patients in India using twenty BRCA2 htSNPs, of the 20 htSNPs, only three of the variants were detected in our study.⁶⁵ Additionally, BRCA2 haplotype analysis in South African BC patients identified the rs80358810 variant as a founder variant in African populations; this variant was undetected in our study.⁶⁶ This underscores the necessity for population-specific genetic research to capture the unique genetic diversities within each population.^67–69

The detected BRCA2 haplotypes in the BC and control groups were compared with the 1000 Genomics database, and no unique haplotypes were detected in both the BC and control groups compared to those reported for the YRI and ESN populations.³⁰ However, Hap 3 in the BC group, which was present at a frequency of 3.1% in the BC group, had a lower frequency of 0.24% in the YRI and ESN populations.³⁰

These findings highlight the unique genetic diversity of the Nigerian population and emphasize the need for broader regional representation in genetic studies across Nigeria.^69–71 Genomic analysis of already established recurrent variants such as the BRCA1 Y101X potential Yoruba founder variant, is essential for accurately reconstructing haplotypes within the Nigerian population.⁷² Haplotype analysis of recurrent variants is a powerful tool for understanding how genetic variants are inherited, and it could serve as the foundation for developing cost-effective specific genetic testing panels for Nigerian BC patients.⁷³ Similar genomic analysis in other populations, such as Ashkenazi Jews and South African populations, have led to the identification of founder variants, enabling the development of point-of-care (POC) technologies that reduce costs and improve accessibility.⁵⁶^,⁶⁶ Adopting a similar strategy in Nigeria could facilitate early detection, particularly for high-risk individuals, make genetic testing affordable, and ultimately enhance healthcare outcomes for BC patients in Nigeria.

A limitation of this study, however, was the absence of immunohistochemistry data for a substantial number of recruited subjects. This gap made it impossible to determine the prevalence of the TNBC subtype within our cohort. Similar studies have also encountered such limitations, emphasizing that immunohistochemistry is not yet a routine part of point-of-care management in some Nigerian hospitals.^6,17 Additionally, future studies with larger sample sizes could provide deeper insights into the genetic diversity of the Nigerian population and help identify pathogenic variants that may have been missed in this study.

Conclusion

These findings suggest that Nigerian BC patients may exhibit higher frequencies of BRCA1/2 variants, with significant clinical implications for personalized treatment. This population could potentially have a higher degree of “BRCAness,” which would make them suitable candidates for targeted therapies such as poly (ADP-ribose) polymerase (PARP) inhibitors.^8,15 However, the relatively low number of patients with the TNBC subtype in our study likely contributed to the lower detection of BRCA1/2 pathogenic variants. A larger representation of TNBC patients in future studies could increase the detection of BRCA1/2 variants and, consequently, enhance therapeutic outcomes for a broader group of patients in the Nigerian population.

Supplemental Material

sj-docx-1-tct-10.1177_15330338251333012 - Supplemental material for Screening of Germline BRCA1 and BRCA2 Variants in Nigerian Breast Cancer Patients

Supplemental material, sj-docx-1-tct-10.1177_15330338251333012 for Screening of Germline BRCA1 and BRCA2 Variants in Nigerian Breast Cancer Patients by Abimbola F. Onyia, Paul Jibrin, Temitope Olatunji-Agunbiade, Ademola Oyekan, AbdulRazzaq Lawal, Adewumi Alabi, Anthonia C. Sowunmi, Eben A. Aje, Oluwabusayo B. Ogunniyi, Ebenezer S. Nkom, Opeyemi C. De Campos, Oluwakemi A. Rotimi, Jelili O. Oyelade and Solomon O. Rotimi in Technology in Cancer Research & Treatment

Footnotes

Author Contributors

SOR,AFO,and JOO were in conceptualization and study design. PJ,OA,TO,AO,AL,AA,ACS,EAJ,and ESN were involved in data collection. AFO,OBO,OCD,and SOR were involved in writing the original draft,and data analysis. SOR,AFO,OAR,and JOO were involved in reviewing and editing the manuscript. All authors read and approved the final version for publication.

Data Availability

All data utilized in the analysis is available on request. All relevant data to the study have been included in the article or uploaded as supplementary information.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Ethics Statement

Before conducting the study,ethical approval was obtained from the Covenant University Human Research Ethics Committee,Covenant University,Ota,Ogun State,Nigeria (approval number: CU/HREC/SOR/072/21),on 20th May 2021,and The Nigerian National Ethics Committee (approval number: NHREC/01/01/2007-19/01/2024) on 19th January 2024. Additionally,all participants recruited for the study provided written informed consent before and after the details of the study were fully explained and understood. To ensure confidentiality and protect participants’ privacy,their identities were de-identified on the questionnaires and throughout the research process.

Funding

The author(s) disclosed receipt of the following financial support for the research,authorship,and/or publication of this article: SOR is supported by funding from the Office of the Assistant Secretary of Defense for Health Affairs through the Congressionally Directed Medical Research Programs (CDMRP),Prostate Cancer Research Program,Health Equity Research,and Outcomes Improvement Consortium,under Award Number (W81XWH2210972) and by the National Cancer Institute of the National Institutes of Health under Award Number P50CA116201 through the sub-award COV-182363-02,a part of the Mayo Clinic Breast Cancer SPORE – Project 1. JOO is supported by the National Human Genome Research Institute of the National Institutes of Health under Award Number 5U01HG013442. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. AFO received a scholarship from The African Centre for Translational Genomics to support her PhD studentship.

ORCID iDs

Abimbola F. Onyia

Ademola Oyekan

Adewumi Alabi

Oluwakemi A. Rotimi

Supplemental Material

Supplemental material for this article is available online.

LIST OF ABBREVIATIONS

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