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Abstract

Background:

Breast cancer remains a major global health challenge, as it is the most commonly diagnosed malignancy worldwide, particularly among women. Germline variants in cancer-predisposing genes play a critical role in breast cancers with familial origin.

Objectives:

To identify genetic variants in cancer-predisposing genes among breast cancer patients and individuals at risk in a selected cohort from two imaging facilities in the Central Province of Sri Lanka.

Design:

A genetic association study involving breast cancer confirmed patients, at-risk individuals, and healthy controls.

Methods:

Blood samples were collected from consenting patients, and genomic DNA was extracted from the samples and subjected to Next Generation Sequencing and Sanger sequencing. The inherited predisposition to breast cancer was evaluated to find genes associated with breast cancer using the Ion Torrent PGM platform followed by bioinformatics analysis.

Results:

Variants were detected in several high- and moderate-penetrance genes, including BRCA1 [c.3113A>G; p.Glu1038Gly], BRCA2 [c.6509A>G; p.Lys2170Arg; c.7879A>T; p.Ile2627Phe; c.5574_5577delAATT; p.Ile1859LysfsTer3], PALB2 [c.1592delT; p.Leu531fs], BRIP1 [c.2400C>T;], and in MRE11A [c.508C>A; p.Gln170Lys] genes. Among these, BRCA2 mutations and the PALB2 frameshift deletion were classified as pathogenic germline variants. The benign BRCA1 variant [c.3113A>G; p.Glu1038Gly] was the most frequent variant observed. Common missense variants included BRCA1 [c.3113A>G; p.Glu1038Gly; c.3548A>G; p.Lys1183Arg; c.2612C>T; p.Pro871Leu], BRCA2 [c.7397T>C; p.Val2466Ala], and ATM [c.5948A>G; p.Asn1983Ser], while frequently detected intronic variants were found in MUTYH [c.1468-40C>G;], PALB2 [c.3114-51T>A;], and NF1 [c.288+41G>A; c.328+37C>G;]. Pathogenic variants occurred in fewer than 10% of individuals in any group, and other variants were identified in different frequencies. Conclusion: Evaluation of germline variants in this cohort revealed the presence of pathogenic mutations and other variants with benign or uncertain significance. Three pathogenic variants, BRCA2 [c.6509A>G; c.7879A>T; c.5574_5577delAATT] and PALB2 [c.1592delT], were identified in high-risk genes important for breast cancer prediction. Identification of population-based variants may improve breast cancer screening and management in Sri Lanka.

Keywords

Breast cancer cancer-predisposing genes germline variants multigene panel testing pathogenic variants

Introduction

Breast cancer (BC) is characterized by the uncontrolled growth of breast tissue and remains the most frequently diagnosed cancer globally with significant implications for women’s health. According to the International Agency for Research on Cancer, there were 2.3 million new cases and 670 000 deaths due to BC in 2022. In Sri Lanka, the National Cancer Control Program reported approximately 3000 new cases per year in 2020 and 2021, accounting for about 27% of all female cancers.^1,2 As a consequence, numerous risk factors have been studied and found to cause this cancer. Some of the BC-associated risk factors are age, body mass index, hormone replacement therapy, exposure to radiation, reproductive factors, prior history of tumor or hyperplasia in breasts, and genetic background.³ Furthermore, many studies have identified that certain risk factors significantly increase relative risk. Particularly, genetic predisposition is identified as one of the most significant risk factors associated with BC. A study conducted to rate the risk factors for BC identified that genetic predisposition significantly affects the risk of BC, ranging from 3 to 200.^4,5

When genetic predisposition is considered, family history and specific germline mutations of cancer susceptibility genes were well explained in several studies. A previous study³ reported that the degree of the risk associated with family history is a function of the type of affected relative, age of cancer development, and the number of relatives affected. Another study revealed that a twofold increased risk is associated with having a first-degree relative compared with the general population.⁵ However, only 10% to 20% of BCs are found to be family clustered, and only 30% to 40% of familial BCs are known to predispose genetically.⁶ Additionally, BCs can be caused by specific germline mutations of cancer-predisposing genes, and hundreds of genes were evaluated for gene variants^7,8

BCs may be classified as sporadic, familial, or hereditary. Sporadic BC occurs in individuals with no notable family history and accounts for the majority of cases. Familial cases cluster within families but lack a clearly identifiable inherited mutation, while hereditary BC is caused by germline mutations with high penetrance. Familial cases may arise due to a combination of shared genetic factors with low to moderate penetrance and environmental or lifestyle influences.⁹ Recent reviews, such as “Understanding genetic variations in familial breast cancer” (2024), have further emphasized this classification of BC susceptibility genes based on their penetrance and functional roles in DNA repair and cell cycle control. These studies highlight how the integration of multigene panel testing has improved the identification of high-, moderate-, and low-risk genetic variants, aiding in risk assessment and clinical decision-making.¹⁰

Most of the genes responsible for cancer predisposition play a significant role in cell cycle regulation and DNA damage repair. Among them, BRCA1 and BRCA2 are considered as major susceptibility genes, with BRCA1 being the most significant. Mutations in highly penetrant BRCA1 and BRCA2 confer a high risk of BC. It has been identified that female carriers with mutations in BRCA1 and BRCA2 have a 50% to 85% lifetime risk.⁸ Pathogenic variants in the predisposition genes ATM, BRCA1, BRCA2, CHEK2, and PALB2 are associated with increased risks of BC, and pathogenic variants in BARD1, RAD51 C, and RAD51D are associated with increased risks of ER-negative BC.^11,12 PALB2 has recently been confirmed as a high-risk BC gene.^11,12 Many studies have utilized multigene panel testing for cancer screening and assessing risk management strategies for women with pathogenic variants in cancer-predisposing genes in the general population. These studies found that protein-truncating variants in BRCA1, BRCA2, and PALB2 fall into the high-risk category, while those in ATM, BARD1, CHEK2, RAD51C, and RAD51D are categorized as moderate risk. This highlights their clinical utility for inclusion in BC risk prediction panels.^11,12

It is important to scrutinize the genetic variants in a population since it provides an insight into several aspects regarding BC prediction and patient management. Genetic testing of familial BC patients facilitates the identification of population-based genetic variants, which can enhance our understanding of the disease process and lead to more targeted treatments. Exploring the patterns and role of genetic heterogeneity in sporadic BC patients is also important.¹³ Therefore, genetic testing can be used as a powerful predictive tool. Individuals who have a strong family history of BC are more prone to get the disease, and gene panel testing has a growing clinical importance in treatment options such as prophylactic mastectomy and chemopreventive drugs.⁸ However, as BC risk is influenced by other genetic and lifestyle factors, in addition to family history, it is important to consider the genetic test results in risk assessment. Considering all the above facts, this study aims to reveal genetic polymorphisms in cancer-predisposing genes among the BC confirmed patients and individuals at risk of developing BC in a selected group of patients presented to two BC imaging facilities located in the Central Province of Sri Lanka.

Materials and Methods

This study was approved by the Ethics Review Committee, Faculty of Allied Health Sciences, University of Peradeniya, Sri Lanka (AHS/ERC/2018/001). The guidelines and methodology approved by the Ethics Review Committee were followed to complete the study. This is a descriptive, retrospective study carried out from July 2018 to July 2019. Written informed consent for the study was obtained from the entire study participants after providing pre-test counseling. A detailed questionnaire was given to the study group to obtain data on a voluntary basis. The questionnaire was pilot tested in approximately 12% of participants to ensure clarity and cultural appropriateness. Participants were recruited from two breast imaging units in Kandy, Sri Lanka. Privacy and the confidentiality of all the data collected from the patients are maintained. Altogether, the cohort consisted of 79 subjects. Sample size was determined by the availability of eligible patients within the study period. Guidelines given by the American College of Molecular Genetics and Genomics were adopted when selecting the patients for this study. Study participants were recruited as four groups. (i) familial BC patients with a positive family history (n = 13), (ii) sporadic BC patients without a family history (n = 20), (iii) at risk individuals who had at least one first- or second-degree relative with BC but no personal history of the disease (n = 26), and (iv) healthy controls frequency matched by age and without personal or family history of any cancer (n = 10). The BC patients were selected for the study after reviewing their mammography and histopathology reports. Family history is defined by at least one first- or second-degree female relative reported to have had BC. Those who were in the terminally ill stage and those who had not unconsented were excluded from the study. An additional group of age-matched at-risk individuals (n = 10) was included solely for primer validation. These samples were analyzed using Sanger sequencing to confirm successful amplification of target regions in BRCA2 and PALB2 and to ensure the reliability of the designed PCR assays. Their data were not included in the main cohort analysis.

Each study participant was subjected to venipuncture under strict aseptic condition and 2 ml blood sample was collected into an ethylene diaminetetra acetic acid (EDTA) tube and immediately stored at −20°C. Genomic DNA was extracted from peripheral white blood cells using the Promega genomic DNA purification kit ® (Promega, Madison, USA). Then, these DNA samples were subjected to Next-Generation sequencing (NGS), and 10 samples were analyzed by Sanger sequencing. The inherited predisposition to BC was evaluated using 18-gene panel test on the Credence breast platform to analyze genes having BC predisposition (BRCA1, BRCA2, TP53, ATM, BARD1, BRIP1, CDH1, CHEK2, MRE11A, MUTYH, NBN, NF1, PALB2, PEN, RAD50, RAD51C, RAD51D, and STK11). Genomic regions were amplified using oligonucleotides, prepared into single-ended libraries of 200 bp size followed by Next Generation Sequencing on Ion Torrent PGM. The DNA sequences were mapped to the published Human Genome (GRCh37/hg19) using TMAP aligner version 4.0.6, followed by Variant Calling (TVC: Version 4.2.3.) and annotation (VEP: Version 75) using Credence Breast Cancer Pipeline version 1.1.2 (ClinVar:02: Mar:2015 Update). Variants were classified according to the ACMG/AMP 5-tier system (pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign). After analyzing the results, reports were provided to participants along with post-test counseling as a benefit of their participation. The sequences obtained for 10 samples completed by Sanger sequencing were checked for mutations and variants using BioEdit software (version 7.2). Primers were designed to amplify the pathological mutations identified for BRCA2 and PALB2 genes in this cohort to develop a PCR-based assay. Among these 10 samples, six were representative at-risk individuals with a positive family history who had been included in the main cohort, and four were additional age-matched at-risk individuals. All 10 samples were used exclusively for primer validation. PCR products obtained using the designed primers were sequenced by the Sanger method, and the resulting sequences were analyzed through multiple sequence alignments and BLAST searches to verify the presence of single-nucleotide polymorphisms (SNPs) or mutations. To minimize potential sources of bias, standardized data collection methods were used for all participants, and genetic analyses were performed using the same laboratory protocols and blinded sample identifiers. The reporting of this study conforms to the STROBE statement for observational studies;¹⁴ the completed checklist is provided as Supplementary File 4.

Results

The cohort comprised 79 participants: 13 familial BC patients (16.5%), 20 sporadic BC patients (25.3%), 26 at-risk individuals (32.9%), 10 healthy controls (12.7%), and 10 age-matched at-risk individuals were Sanger sequenced for primer validation (12.7%). (Table 1). The mean ages of individuals analyzed using NGS were 57.2, 52.2, 49.3, and 50.5 years for familial, sporadic, at-risk, and healthy control groups, respectively (Table 2). In this study, germline variants of 18 cancer-predisposing genes were studied. Some of these genes were highly penetrance while the others were moderate and low. In the current study, frequencies of different pathogenic variants and other protein-truncating variants such as missense and intronic variants were also studied.

Table 1.

Details of the study population.

Study population	No. of participants	Percentage
Familial breast cancer patients	13	16.5%
Sporadic breast cancer patients	20	25.3%
At risk individuals—without disease, but with a family history of breast cancer	26	32.9%
Additional at-risk individuals	10	12.7%
Controls	10	12.7%
Total	79	100%

Table 2.

History of the participants.

	Familial breast cancer patients (N = 13)	Sporadic breast cancer patients (N = 20)	At risk individuals (N = 26)
Age at diagnosis	57.2 ± 7.1	52.2 ± 13.3	49.3 ± 9.4
Number of family members with breast cancers
N = 0	00	20	00
N = 1	11	00	17
N = 2	02	00	07
N = 3	00	00	02
Degree of the affected relative
First	05	00	16
Second	05	00	03
Both	03	00	07
Ethnicity
Sinhalese	11	17	24
Tamils	02	01	01
Muslims	00	02	00
Others	00	00	01

Data from the 10 additional at-risk individuals used for primer validation are not included in this table, as these samples were analyzed separately for methodological optimization only.

Pathogenic variants

This study revealed the presence of some pathogenic variants. However, less frequent pathogenic variants were also found in both high- and low-penetrance genes. In high-penetrance gene BRCA2 (NM_000059.4) [c.6509A>G; p.Lys2170Arg; c.7879A>T; p.Ile2627Phe] missense variants and [c.5574_5577delAATT; p.Ile1859LysfsTer3] frameshift deletion were observed which can be pathogenic. In the high-penetrance gene PALB2 (NM_024675.4) [c.1592delT; p. Leu531fs], frameshift deletion was observed. All these variants were found in less than 10% of individuals in each category. PALB2 has been identified as a high-penetrance gene causing BC, whereas BRIP1 & MRE11A are not considered as BC-causing genes currently.¹¹

Harmless missense variants

BRCA1 (NM_007294.4) [c.3113A>G; p.Glu1038Gly=; c.3548A>G; p.Lys1183Arg=; c.2612C>T; p.Pro871Leu], BRCA2 (NM_000059.4) [c.7397T>C; p.Val2466Ala], ATM (NM_000051.4) [c.5948A>G; p.Ser1983Asn] were the five commonly identified missense variants. BRCA1 [c.3113A>G; p.Glu1038Gly] was shown among 69% of familial BC patients, 80% of sporadic BC patients, and 69% of those at risk. However, this variant was observed in all the healthy controls as well while the clinical significance being benign. Sixty-nine percent of familial BC patients were found to have BRCA1 [c.3548A>G; p.Lys1183Arg and c.2612C>T; p.Pro871Leu] variants, whereas it was 60% in sporadic BC patients and 62% in at-risk individuals. Almost all the controls were found to have these variants. BRCA2 [c.7397T>C; p.Val2466Ala] variant was found among almost all the family history positive BC patients, at-risk individuals, and the controls. In addition, 75% of sporadic BC patients were also positive for the particular variant. About 85% study participants in familial BC patients, 75% of sporadic BC patients, and all the members in the at-risk group and control group showed the ATM [c.5948A>G; p.Asn1983Ser] variant.

The TP53 (NM_000546.6) c.215C>G (p.Pro72Arg) variant has been associated with differences in how certain drugs affect cancer cells, particularly in TP53-related cancers. It is important to note that TP53 mutations, especially pathogenic ones, can be associated with Li-Fraumeni syndrome, a rare cancer predisposition syndrome according to MedlinePlus (.gov) and the Dana-Farber Cancer Institute.^15,16 This TP53 variant was found in 31% familial cases, 35% sporadic cases, and 42% at-risk individuals. The presence of this variant in patients may help predict variability in treatment outcomes rather than disease susceptibility. In our cohort, 2 out of 13 familial BC patients, 1 out of 20 sporadic BC patients, and 2 out of 26 at-risk individuals carried this pharmacogenetic variant that could potentially affect drug response in chemotherapy. Including these data helps to highlight the relevance of TP53 polymorphisms not only in cancer development but also in personalizing chemotherapy strategies for better clinical management.

Table 3 presents the number and percentage of missense variants analyzed by NGS in this study. Missense variants with uncertain significance were also found in this study (refer Supplementary File 1: Table 1).

Table 3.

Missense Variants Confirmed by NGS.

		Familial breast cancer patients (N = 13)		Sporadic breast cancer patients (N = 20)		At risk individuals(N = 26)
Gene	Mutation	No.	%	No.	%	No.	%
BRCA1	c.3113A>G	09	69%	16	80%	18	69%
	c.3548A>G	09	69%	12	60%	16	62%
	c.2612C>T	09	69%	12	60%	16	62%
BRCA2	c.7397T>C	13	100%	15	75%	26	100%
ATM	c.5948A>G	11	85%	15	75%	26	100%
TP53	c.215C>Gdrug response	0402	31%15%	0701	35%5%	1102	42%8%

All variants reported above were also observed in healthy control individuals. Data from the 10 additional at-risk individuals used for primer validation are not included in this table, as these samples were analyzed separately for methodological optimization only.

Intronic variants

When considering the intronic variants, few common variants were identified in which the biotype was protein-coding. The most common variants identified were MUTYH (NM_012222.3 [c.1468-40C>G;] and PALB2 (NM_024675.3) [c.3114-51T>A;], having more than half of each population positive for the variants. Additionally, 54% of the familial BC patients were found to have NF1 (NM_001042492.3) [c.288+41G>A; c.328+37C>G;]. NF1 [c.288+41G>A;] was found in 40% of sporadic BC patients and in 62% of the at-risk individuals, whereas the other was positive among 65% and 77%, respectively. One-fourth of the controls showed above intronic variants. Table 4 presents the intronic variants found in this study.

Table 4.

Intronic variants.

Gene	Mutation	Familial breast cancer patients (N = 13)		Sporadic breast cancer patients (N = 20)		At-risk individuals (N = 26)
Gene	Mutation	No.	%	No.	%	No.	%
MUTYH	c.1468-40C>G	7	54%	13	65%	17	65%
PALB2	c.3114-51T>A	8	62%	11	55%	15	58%
NF1	c.288+41G>A	7	54%	8	40%	16	62%
	c.328+37C>G	7	54%	13	65%	20	77%

Approximately one-fourth of the controls carried each of the above variants. Data from the 10 additional at-risk individuals used for primer validation are not included in this table, as these samples were analyzed separately for methodological optimization only.

Some of the intronic variants with uncertain significance found in this study are presented in Table 5.

Table 5.

Intronic variants with uncertain significance.

Gene	Chromosome	Sequence variant	Predicted effect at protein level
ATM	chr11	c.980G>A	.
ATM	chr11	c.7397T>C	.
BRCA1	chr17	c.1873+30T>C	.
		c.548-58delT	.
		c.441+36_441+38delCTT	.
BRCA2	chr13	c.681+56C>T	.
BRCA2	chr13	c.7008-62A>G	.
NF1	chr17	c.1873+30T>C	.

PCR-based test to identify common mutations in BRCA2 and PALB2

Based on the results obtained from this study it was found that pathogenic mutations are present in BRCA2 and PALB2 genes in patients with positive family history in this cohort. Few patient’s samples confirmed by NGS for these pathological mutations and samples obtained from additional at-risk individuals were amplified using these primers. PCR products were sequenced using Sanger sequencing method. Analysis of the sequences revealed that these pathological mutations could be repeatedly amplified using the primers designed. The PALB2 c.1592delT frameshift mutation identified in this cohort disrupts the protein-coding sequence and likely contributes to disease risk. The altered sequence region would appear as a continuous variant downstream of the deletion, consistent with the predicted pathogenic effect.

Discussion and Conclusion

The Scope of this study was to identify genetic variants among familial BC patients, sporadic BC patients, and individuals who are at risk of developing BC in a patient cohort of Sri Lanka. Sixty-nine individuals were included in the study for Next Generation Sequencing, and pathogenic variants, frequent missense, and intronic variants were identified. In contrast to earlier reports suggesting that familial BCs present at younger ages, our cohort demonstrated a higher mean age at diagnosis in familial patients (57.2 ± 7.1 years) compared with sporadic patients (52.2 ± 13.3 years). This difference could be influenced by our limited sample size, so broader studies will be important to clarify this pattern.¹²

Four pathogenic variants in highly penetrant genes, BRCA2 and PALB2, were identified in our study. These genes play key roles in DNA repair and maintaining genomic integrity.¹³ All identified variants have been previously reported and are listed in the Breast Cancer Information Core (BIC) database.¹⁵ Consistent with our findings, several studies have shown that a greater number of pathogenic variants are associated with BRCA2 than with BRCA1.^16
-19 However, the relative risk of developing BC remains higher for individuals with BRCA1 mutations.¹⁸

Several studies have highlighted the unique spectrum of germline variants in Sri Lankan BC patients. Initial work conducted by De Silva et al identified two novel pathogenic frameshift mutations, c.3086delT and c.5404delG along with several novel intronic and missense variants. This highlighted the genetic heterogeneity within this population.²⁰ A follow-up study on BRCA2 in a similar cohort revealed a higher frequency of potentially pathogenic variants, including c.2403insA and c.2667insT, suggesting that BRCA2 may play a more significant role than BRCA1 in hereditary BC predisposition in Sri Lanka.²¹ In young familial BC patients, 23% carried pathogenic BRCA2 variants, emphasizing the need for early, targeted testing.²² More recent multigene panel testing revealed additional mutations in moderate-risk genes such as CHEK2, ATM, PALB2, and CDKN2A, supporting the value of comprehensive genetic screening beyond BRCA1/2 in the Sri Lankan population.²³

BRCA1 [c.3113A>G; p.Glu1038Gly] variant was the most common variant in our cohort and has also been reported at high frequencies in Algerian and Indian populations.^24,25 In both studies, the variant was found to exert a benign effect. It was found in both BC confirmed patients (familial and sporadic) and at-risk individuals in the present study. BRCA2 deleterious mutation [c.5574_5577delAATT; p.Ile1859LysfsTer3] had originally been reported in a study intended to identify the prevalence and spectrum of BRCA1/2 germline mutations in women with BC in China²⁶ and BRCA2 [c.6509A>G; p.Lys2170Arg] was barely found in general.²⁷ On the contrary, BRCA2 [c.7879A>T; p.Ile2627Phe] was most commonly found in Macedonian and Lithuanian populations.^19,28 However, none of these variants were reported in Sri Lankan studies conducted earlier.^15,20
-29 PALB2 [c.1592delT; p.Leu531fs], a high-risk deleterious mutation, was also identified in our study.

Several studies have shown that it plays an important role in BC, particularly in cases with a familial origin.^23,30 However, none of the previous studies involving Asians or Sri Lankan populations have reported this mutation. In addition to pathogenic variants in high-risk genes such as BRCA2 and PALB2, variants have also been identified in BRIP1 and MRE11A. However, pathogenic variants in these genes are generally not considered to confer a significant risk for BC development.³¹

Other than the pathogenic polymorphisms, variants with uncertain significance (VUS) exert an important function in BC. A study stated that most of the variants of uncertain significance are basically missense, silent, and intronic variants or in-frame deletions and insertions.³² Consistent with this, Arachchige et al conducted a comprehensive bioinformatics analysis of selected germline VUS identified in a Sri Lankan hereditary BC cohort, revealing that several variants in BRCA1, BRIP1, and MET may have deleterious structural and functional consequences. These findings highlight the need for follow-up functional work in the local context.³³ Therefore, it was essential to study the pathogenicity and the role of these variants in BC. In this study, frequent missense and intronic variants were observed. Missense variant is a change in the type of amino acid without altering the protein. Though the protein is not truncated, it is hypothesized that this may initiate a pathogenic mechanism which leads to BC.³⁴ Also, most of these missense polymorphisms are SNPs. The current study revealed more than 100 missense variants, and some were found in higher frequencies. Those were reported in BRCA1 [c.3113A > G; p.Glu1038Gly; c.3548A>G; p.Lys1183Arg; c.2612C>T; p.Pro871Leu], BRCA2 [c.7397T>C; p.Val2466Ala], and ATM [c.5948A>G; p.Asn1983Ser] like high-penetrance genes. Similarly, these polymorphisms shared with Algeria,²⁶ India,²⁷ Colombia,³⁵ and Bahrain³⁶ studies have a considerably higher frequency of [c.3113A>G, c.3548A>G, c.2612C>T, c.7397T>C] variants. As reported earlier,³⁶ this study also confirmed that the BRCA1:rs799917 (c.2612C>T; p.Pro871Leu) variant was not associated with BC risk. However, ATM [c.5948A>G; p.Asn1983Ser] was also reported in this cohort. In Sri Lanka, two similar experiments have been conducted on multigene panel testing of BC,^23,30 and frequent missense polymorphisms in the present study were not reported in those experiments except for c.823G>A; p.Gly275Ser in BRCA1 gene. Several other missense polymorphisms found in this study are given in Table 5.

In addition to the effect of pathogenic and other missense variants, it is evident that the intronic variants also play a particular significance in terms of BC risk where the exact mechanism is yet to be explored. It is stated that the intronic variants in intronic and exonic boundaries can disturb the splicing capacity.³⁴ Therefore, it is important to have data on common intronic polymorphisms in a community. A present study revealed a high number of intronic variants while three of them being more frequent. MUTYH is a DNA repairing gene which shows a notable association with colorectal cancer patients. However, it is found that mono-allelic and bi-allelic variants of this gene were also noted in different BC cohorts.^36
-38 Based on the National Center for Biotechnology Information (NCBI) database, MUTYH c.1468-40C>G was previously reported as a benign single-nucleotide variant with an unknown disease specificity which was found in this study as well.³⁹ PALB2 c.3114-51T>A was another variant frequently reported in the current study. Similarly, this was identified in almost all individuals in an Australian research with 347 study participants and also in a South African study.^40,41 NF1 acts as a gene producing tumor suppressor protein neurofibromin.⁴² NF1 c.288+41G>A has previously been reported in the NCBI database as a benign single-nucleotide variant.⁴³ However, this variant has not shown a clinical effect in the current study. Similarly, there was no effect in NF1 c.328+37C>G variant that was not reported in previous literature.

Evaluation of germ line variants using 18 gene BC panel testing in this cohort revealed that the pathogenic mutations and other variants with benign or uncertain significance in both familial and sporadic BC patients in many genes of this cohort. Altogether, three pathogenic variants were found, and none of those have been reported from previous studies conducted in Sri Lanka. The benign BRCA1 [c.3113A>G; p.Glu1038Gly] variant identified in this cohort has been found in Algerian and Indian populations as well. Deleterious BRCA2 [c.5574_5577delAATT; p.Ile1859LysfsTer3] found in this study has been previously reported among Chinese. BRCA2 [c.509A>G; p.Lys2170Arg] identified in this study was found in Poland and BRCA2 [c.7879A>T; p.Ile2627Phe] was most commonly found among Macedonians and Lithuanians. The Finnish population was reported to have PALB2 [c.1592delT; p.Leu531fs] deletion. Recent studies in diverse populations support testing beyond BRCA1/2. In Colombia, multi-gene analysis of unselected BC patients identified pathogenic variants in ATM, PALB2, CHEK2, BARD1, and RAD51D in addition to BRCA1/2.⁴⁴ Similarly, in Turkey, BRCA1/2 mutations were linked to earlier onset even in patients without a family history.⁴⁵ These findings highlight the value of broader gene-panel testing for comprehensive risk assessment in genetically diverse populations.

Additionally, in the present study, a diverse array of protein-truncating missense variants and intronic variants were observed in large numbers while a few were more frequent. These variants were mostly SNPs, and the frequency of variants was different within each study group. These results would have been missed if the testing were strictly limited to single-gene or syndrome testing guidelines.

In the Sri Lankan setting, using PCR-based assays to detect the main pathogenic mutations found in BRCA2 and PALB2 offers a practical and affordable alternative to costly NGS-based BC panels. Many people who need genetic risk screening are reluctant to undergo multigene panel testing because of the high expense. As a solution, targeted PCR followed by Sanger sequencing of commonly mutated regions can be used as an initial screening approach. Individuals who test positive can then be referred for full NGS or exome sequencing, helping to use resources more effectively while improving early detection and risk management. In this study, a group of age-matched at-risk individuals (n = 10) was included for primer validation to confirm the reliability of the method. Among these, six were from the 26 at-risk individuals included in the main cohort analysis, and four were additional age-matched individuals. Sanger sequencing of these samples showed clear amplification of the target regions in BRCA2 and PALB2, confirming that the designed primers were specific and efficient. Sequence analysis using BioEdit and BLAST further verified the presence of previously identified mutations and single-nucleotide variants. Overall, the PCR assay successfully amplified the same pathogenic regions detected by NGS in the main cohort, showing its potential as a reliable and low-cost screening tool for BRCA2 and PALB2 mutations in familial BC.

Although this study primarily analyzed BC patients, a group of healthy control individuals was also subjected to NGS analysis, allowing for comparative assessment of variant occurrence. Variants that were shared between patients and healthy controls are likely germline polymorphisms, whereas those uniquely detected in patients may represent disease-associated changes. However, since paired tumor and normal DNA were not analyzed from the same individuals, it is not possible to definitively classify these as somatic or germline mutations. Future studies incorporating matched tumor-normal sequencing would provide greater resolution in determining variant origin.

Limitations and Recommendations

The relatively small cohort size (N = 79) may limit the generalizability of our findings. However, this study is one of the few attempts to genetically characterize BC-associated variants in a Sri Lankan population, which remains significantly underrepresented in global genomic datasets. It is well established that BRCA1 and BRCA2 mutations in Sri Lankan individuals tend to be private mutations rather than founder mutations. Thus, the identification of even a few rare or previously unreported variants is not unexpected but is still clinically relevant. These findings contribute toward building a population-specific mutation database and may eventually support the design of targeted diagnostic tools for hereditary BC risk prediction in Sri Lanka. Our sample size was limited primarily due to funding constraints and the high cost of NGS. Despite this, the study has laid a valuable foundation for understanding the local mutational landscape and highlights the need for broader, multi-center studies with larger cohorts to validate and expand on these preliminary observations. To improve accessibility and cost-effectiveness in low-resource settings like Sri Lanka, we recommend the development of simplified screening approaches. PCR-based simple tests can be used to amplify common mutated regions of at-risk individuals with positive family history. It revealed that mutations are reported in BRCA1, BRCA2, and PALPB2 genes commonly in the Sri Lankan community based on the results obtained from the previous studies and current study. PCR-based tests with Sanger sequencing can be used to check the common mutated regions, and it may be cost-effective to use specific primers to amplify these regions in high-risk genes using multiplex PCR and to analyze the sequences rather than using single-gene tests. Multigene BC panels available in Sri Lanka are expensive because of the use of NGS platforms. PCR & Sanger sequencing-based screening tests would be economically reasonable for the Sri Lankan community who need to screen for hereditary BC risk prediction.

Conclusions

This study identified three pathogenic germline variants in the high-penetrance genes BRCA2 and PALB2, present at low frequencies (<10%) among a Sri Lankan cohort of BC patients and at-risk individuals. Multiple benign and uncertain variants, including common missense and intronic variants, were also detected. PCR and Sanger sequencing confirmed the reproducibility of key pathogenic mutations, demonstrating the reliability of the designed primers. Our findings emphasize the genetic heterogeneity of BC in Sri Lanka and support the integration of population-specific genetic information into risk assessment and management strategies. Larger studies are needed to confirm these results and to investigate the functional impact of frequent variants.

Supplemental Material

sj-docx-1-bcb-10.1177_11782234261420605 – Supplemental material for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka

Supplemental material, sj-docx-1-bcb-10.1177_11782234261420605 for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka by Lalani Yatawara, Badra Hewavithana, Ashansa P Ramanayake, Susiji Wickramasinghe and Malithi Amarasiri in Breast Cancer: Basic and Clinical Research

Supplemental Material

sj-docx-2-bcb-10.1177_11782234261420605 – Supplemental material for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka

Supplemental material, sj-docx-2-bcb-10.1177_11782234261420605 for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka by Lalani Yatawara, Badra Hewavithana, Ashansa P Ramanayake, Susiji Wickramasinghe and Malithi Amarasiri in Breast Cancer: Basic and Clinical Research

Supplemental Material

sj-docx-3-bcb-10.1177_11782234261420605 – Supplemental material for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka

Supplemental material, sj-docx-3-bcb-10.1177_11782234261420605 for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka by Lalani Yatawara, Badra Hewavithana, Ashansa P Ramanayake, Susiji Wickramasinghe and Malithi Amarasiri in Breast Cancer: Basic and Clinical Research

Supplemental Material

sj-docx-4-bcb-10.1177_11782234261420605 – Supplemental material for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka

Supplemental material, sj-docx-4-bcb-10.1177_11782234261420605 for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka by Lalani Yatawara, Badra Hewavithana, Ashansa P Ramanayake, Susiji Wickramasinghe and Malithi Amarasiri in Breast Cancer: Basic and Clinical Research

Supplemental Material

sj-docx-5-bcb-10.1177_11782234261420605 – Supplemental material for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka

Supplemental material, sj-docx-5-bcb-10.1177_11782234261420605 for Identification of Genetic Variants Among Breast Cancer Patients and At-Risk Individuals: A Cohort Study in Sri Lanka by Lalani Yatawara, Badra Hewavithana, Ashansa P Ramanayake, Susiji Wickramasinghe and Malithi Amarasiri in Breast Cancer: Basic and Clinical Research

Footnotes

Acknowledgements

We would like to acknowledge the financial assistance given by the University of Peradeniya, Sri Lanka. Furthermore, we would like to acknowledge all the participants of this study.

ORCID iD

Lalani Yatawara

Ethical Considerations

This research was approved by Ethics Review Committee, Faculty of Allied Health Sciences, University of Peradeniya, Sri Lanka (AHS/ERC/2018/001).

Consent to Participate

Written informed consent was obtained from all the study participants.

Consent for Publication

Not applicable

Author Contributions

Lalani Yatawara: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—original draft, Writing—review & editing

Badra Hewavithana: Data curation, Methodology, Project administration, Resources

Ashansa Ramanayake: Formal analysis

Susiji Wickramasinghe: Formal analysis, Software, Validation

Malithi Amarasiri: Writing—review & editing

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by Peradeniya University research grant [URG/2016/14/AHS] awarded to Lalani Yatawara. The funding bodies did not play any role in the study design, collection, analysis, and interpretation of data and in writing the manuscript.

Declaration of Conflicting Interests

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

Data Availability Statement

All data generated or analyzed during this study are included in this published article [and its supplementary information files]. The genomic variants with clinical assertions identified in the current study are available in the ClinVar repository () and can be searched using the HGVS notation or the accession numbers assigned for variants (SCV001477297—SCV001477300).

Use of AI Tools

No scientific data were generated or modified using artificial intelligence. A language editing tool (Grammarly) was used solely to refine the clarity and grammar of the manuscript. Turnitin was used for plagiarism checking.

Supplemental material

Supplemental material for this article is available online.

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