BRCA1 and BRCA2 germline mutations in Chinese Hakka breast cancer patients

Objective To investigate the prevalence of BRCA1/2 gene variants and evaluate the clinical and pathological characteristics associated with these variants in Chinese Hakka breast cancer patients. Methods A total of 409 breast cancer patients were analyzed based on next-generation sequencing results, with 337 categorized as non-carriers and 72 as carriers of BRCA1/2 variants. Data on the patients’ BRCA1/2 gene mutation status, clinical and pathological characteristics, as well as menstrual and reproductive information, were collected, analyzed, compared, and tabulated. Logistic regression analysis was performed to explore the relationship between clinical characteristics and pathogenic variants. Results Among the patients, 72 were identified as carriers of pathogenic or likely pathogenic variants in BRCA1/2, while 337 had likely benign or benign mutations. The BRCA1 c.2635G > T (p. Glu879*) variant was detected at a high frequency, accounting for 12.5% (4/32) of the BRCA1 mutations, while the c.5164_5165del (p.Ser1722Tyrfs*4) variant was common among the BRCA2 mutations, accounting for 17.5% (7/40). It was observed that a higher proportion of BRCA1 carriers had the triple-negative breast cancer subtype, whereas more BRCA2 carriers exhibited estrogen receptor (ER) + and progesterone receptor (PR) + subtypes. Multivariate logistic regression analysis revealed that a family history of cancer (OR = 2.36, 95% CI = 1.00–5.54), bilateral cancer (OR = 4.78, 95% CI 1.61–14.20), human epidermal growth factor receptor 2 (HER2)- (OR = 8.23, 95% CI 3.25–20.84), and Ki67 ≥ 15% (OR = 3.88, 95% CI 1.41–10.65) were associated with BRCA1/2 mutations, with the age at diagnosis, age at menarche, and premenopausal status serving as covariates. Conclusions The most common pathogenic variant of the BRCA1 and BRCA2 in breast cancer patients was c.2635G > T and c.5164_5165del, respectively. Additionally, a family history of cancer, bilateral cancer, HER2-, and Ki67 ≥ 15% were identified as independent predictors of BRCA1/2 pathogenic variants. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-023-01772-9.


Background
Female breast cancer is a widespread and significant health concern, ranking as the fifth leading cause of cancer-related deaths [1].Understanding the various factors contributing to the development of breast cancer in women is crucial, as it involves a complex interplay of genetic, reproductive, lifestyle, and environmental influences [2].One particularly noteworthy factor in hereditary breast cancer is the presence of pathogenic variants in the breast cancer susceptibility genes BRCA1 and BRCA2 [3].These genes play a crucial role in DNA damage repair and act as tumor suppressors, contributing to genome stability [4].Carriers of BRCA1 and BRCA2 mutations face a cumulative breast cancer risk of 72% and 69% respectively by the age of 80 [5].Consequently, germline genetic screening for BRCA1/2 mutations has become an essential tool for cancer prediction and clinical management, enabling carriers to benefit from surveillance, chemoprevention, and preventive surgery to mitigate breast cancer risk [6].Moreover, individuals with metastatic breast cancer and BRCA1/2 mutations can benefit from treatment with poly ADP-ribose polymerase inhibitors [7] or in combination with cisplatin [8].Hence, genetic counseling and testing are imperative in the context of hereditary breast cancer.
A family history of cancer, negative human epidermal growth factor receptor 2 (HER2), high Ki67 index, and lymph node status have been identified as closely associated with BRCA mutations [9].Additionally, Khalis et al. [10] observed a significant association between menstrual history, fertility status, and an increased risk of breast cancer.Indeed, this article explores the potential influence of the BRCA1/2 gene on clinical and pathological features, as well as on ovulation and the menstrual cycle.
Hakka is a Han nationality group with a unique genetic background.Hakka mainly lives in southern China and has a wide distribution in Southeast Asia [11].However, there is limited reporting on the BRCA1/2 mutation sites and their associated clinical and pathological characteristics, as well as menstrual and reproductive status among the Chinese Hakka population.Consequently, this article aims to investigate the clinical and pathological features, menstrual patterns, and reproductive conditions in individuals carrying germline BRCA1/2.

Participants
This retrospective study included 409 breast cancer patients who were admitted to Meizhou People's Hospital (Huangtang Hospital), Meizhou Academy of Medical Sciences, between September 2017 and November 2021.The inclusion criteria were as follows: (1) female patients who had been diagnosed with breast cancer; (2) undergoing BRCA gene testing; and (3) complete clinical records, including clinical characteristics and menstrual and reproductive case data.Exclusion criteria involved cases where the genetic test results were of uncertain significance.This study was approved by the Ethics Committee of Medicine, Meizhou People's Hospital (Huangtang Hospital), Meizhou Academy of Medical Sciences.All participants signed informed consent by the Declaration of Helsinki.

BRCA1/2 testing
Approximately 2 mL of peripheral blood was collected in a tube containing EDTA, and genomic DNA was extracted according to the QIAamp DNA Blood Mini Kit instructions (Qiagen, Germany).The genomic DNA samples were sent to CapitalBio (Beijing, China) and subjected to next-generation sequencing on the Ion Proton instrument (Life Technologies).All procedures were performed according to the standard operating procedures of the Life Technology Company.The sequencing results were compared with the BRCA1 (NM_007294.3) and BRCA2 (NM_000059.3)reference sequences for variant detection.According to the Human Genome Variation Society (HGVS) guidelines, there are five grades of variants: pathogenic variants, likely pathogenic variants, variants of uncertain significance (VUS), likely benign variants, and benign variants [12].
This study divided breast cancer patients who had been tested for BRCA1/2 gene variants into two groups: BRCA1/2 mutation carriers with pathogenic and likely pathogenic variants, and non-BRCA1/2 mutation carriers.The demographic data, clinical and pathologic characteristics, and menstrual and reproductive status of the two groups of patients were tabulated, and the two groups of patients were compared.

Immunohistochemical examination
All of the patients involved in the analysis underwent definitive surgery.The tumor histopathology molecular subtypes were determined by detecting estrogen receptor (ER) and pregnancy receptor (PR) status, HER2 status, and Ki67 marker index.The tumor was defined as ER and/or PR positive if more than 1% of the tumor cells have ER and/or PR positive nuclei.HER2 staining patterns were divided into 4 groups: 3 + (strong and diffuse staining in 10% of cancer cells), 2 + (moderate and diffuse staining), 1 + (local staining), and 0. HER2 positivity was defined as HER2 staining of 3 + and 2 + supplemented with a positive FISH test.HER2-is defined as HER2 staining 0, 1 + , and FISH negative when HER2 staining 2 + .The Ki67 labeling index is expressed as the percentage of positive cells in each case, and the threshold of 15% indicates a high proliferation index.

Guidance for patient treatment
Breast cancer patients with a diagnosis age younger than 40 years, family history of breast, ovarian, or colorectal cancer; premenopausal breast cancer, bilateral breast cancer; triple-negative breast cancer; HER2-, and Ki67 ≥ 15%, should receive BRCA genetic counseling for BRCA testing.
Patients with negative genetic test results are considered non-mutant and are advised to undergo regular follow-ups.However, for patients with likely pathogenic or pathogenic mutations, it is essential to explain the risk of carrying the mutated gene to their family members and the possibility of passing it on to their offspring.Moreover, it is recommended to conduct BRCA1/2 genetic testing for immediate family members of these patients.
For women with a confirmed pathogenic or likely pathogenic variant of BRCA1/2 or those with a high degree of suspicion based on the presence of a known or possibly pathogenic variant in the family, post-test counseling should encompass a discussion of risk-reducing mastectomy.This counseling should include an exploration of the degree of cancer risk reduction/protection, surgeryrelated risks, breast reconstruction options, management of menopausal symptoms, and discussions about reproductive requirements [13].Additionally, Olaparib and other PARP inhibitors can be used as maintenance therapy in breast cancer patients with BRCA1/2 mutations [14].Carboplatin may be recommended in patients with advanced triple-negative breast cancer with BRCA1/2 mutations.

Statistical analysis
SPSS statistical software version 22.0 was used for data analysis.Means ± SDs were used to evaluate differences in quantitative data and N (%) described qualitative data.Student's t-test was used to compare continuous variables, and the χ 2 test was used to compare categorical variables.Log-rank test was used to compare the age of breast cancer onset in different mutation states.Odds ratios (ORs) and 95% confidence intervals were calculated by logistic regression.All p values were two-sided, and p < 0.05 was considered statistically significant.

Clinical pathologic characteristics
The clinical characteristics of the study patients are presented in Table 1.The mean age at diagnosis for BRCA1/2 mutation carriers was 43.78 ± 9.06 years, ranging from 28 to 67 years, which was 4.66 years earlier than non-carriers (48.44 ± 9.20, ranging from 26 to 76 years) (P < 0.001).Notably, there was a significant difference in the age of breast cancer onset under different mutation states (P < 0.001) (Fig. 2A).BRCA1 mutation carriers were diagnosed at a younger age, with 53% (17/32) of BRCA1 carriers diagnosed before the age of 40 years.Among 409 breast patients, 35 had a family history of cancer, and the proportion of BRCA1/2 mutation carriers (13/72, 18.1%) was higher than that of non-carriers (22/337, 6.5%), representing a significant difference (P = 0.002).BRCA1/2 mutation carriers had a higher rate of bilateral breast cancer (9/72, 12.5%) compared to noncarriers (11/337, 3.3%).This difference was statistically significant (P = 0.008).Over 95% of the patients had invasive breast cancer, with no significant difference between BRCA1/2 mutation carriers and non-carriers in the rate of invasive breast cancer (Table 1).

Menstrual and reproductive status of breast cancer patients
The average age at menarche in individuals without the BRCA1/2 mutation was 15.61 ± 1.84 years, whereas in patients with the BRCA1/2 mutation, it was slightly younger at 15.14 ± 1.80 years.The difference in age at menarche between the two groups was statistically significant (P = 0.047).Additionally, 18.1% of breast cancer patients with BRCA1/2 variants had menarche ages younger than 13 years, compared to only 12.2% of breast cancer patients with non-BRCA variants.Among BRCA1/2 mutation carriers, 55 (76.4%) of breast cancer patients experienced the onset of breast cancer before menopause, while 17 (23.6%)experienced it after menopause.This was significantly different from non-carriers, with more individuals developing breast cancer before menopause among BRCA -mutated patients (P = 0.026).The mean age of first breastfeeding and natural menopause was 23.88 ± 3.77, and 50.52 ± 3.83 in non-carriers, respectively, compared with 23.38 ± 2.87 years, and 49.60 ± 3.44 years in BRCA1/2-mutated patients.The mean age at first lactation and natural menopause did not show significant differences between BRCA1/2-mutated patients and non-carriers.Furthermore, the number of BRCA1/2-mutated patients with menstrual periods duration less than 3, 4 to 7, or more than 8 days was 5 (6.9%), 65(90.3%), 2 (2.8%) respectively, and the number with menstrual cycles less than 25, 25 to 35, more than 35 days were 3 (4.2%),66 (91.7%), 3 (4.2%),respectively.χ 2 statistics showed that menstrual period duration and menstrual cycle were similar to those of non-carriers.
The average number of children born to BRCA1/2mutated patients was 2.15 ± 1.13 babies, which was similar to non-BRCA mutation patients (2.20 ± 1.05 babies).The duration of breastfeeding was 21.07 ± 13.47 months, and the average breastfeeding duration per child was 9.34 ± 4.69 months in non-carriers, while the duration of breastfeeding was 17.07 ± 9.84 months, and the average child breastfeeding duration was 8.10 ± 2.31 months  in BRCA1/2-mutated patients.Breastfeeding time in BRCA1/2 mutation carriers was shorter than that of non-carriers, but there was no significant difference.The average number of aborted fetuses was 1.53 ± 0.51 in BRCA1/2-mutated patients, and the average number of aborted fetuses was 1.72 ± 1.04 in non-carriers.There were no significant differences in abortion history, the number of abortions, and the rate of recurrent (two or more) abortions between BRCA1/2-mutated patients and non-carriers (Table 2).

Discussion
Breast cancer is the most common cancer and the leading cause of cancer death in women.There are multiple nongenetic and genetic factors for breast cancer.Age, race, early menarche/late menopause, breast characteristics, etc., are the nongenetic factors.Approximately 5% of breast cancers occur in women under 40 years of age, with the majority being diagnosed in women aged over 50 years [15].The data from this study reveals that individuals carrying BRCA1/2 mutations tend to be diagnosed with breast cancer at a younger age compared to those without the mutations.This trend is particularly noticeable among BRCA1 mutation carriers, which aligns with findings from previous studies [16,17].Early menarche and late menopause have been associated with an increased risk of breast cancer [15].The pregnancy cycle also influences breast cancer risk due to its direct effects on the metabolism, gene expression profiles, and proliferation dynamics of mammary epithelial cells in response to hormones [18].Breast characteristics, including proliferative lesions with atypia and dense breast tissue, are associated with increased risk [19,20].Family history, BRCA mutations, etc., are the genetic factors.BRCA1 and BRCA2 play an important role in maintaining genome stability by promoting efficient and accurate repair of double-strand breaks [21].It has been reported that 5-10% of breast cancer cases result from germline mutations of BRCA genes [22].For breast cancer, initially, the primary tumor, lymph node, and metastasis (TNM) are a standardized classification system for making integrated judgments and precise decisions [23,24].With the rapid development of knowledge of cancer biology and the discovery and validation of biological factors, ER, PR, HER2, histological grade, and multigene prognostic assays, into the staging system [25].Ki67 is a well-known proliferative marker for evaluating cell proliferation.It is highly expressed in malignant cells but almost undetectable in normal cells, and the Ki67 index independently predicts cancer progression [26].Van der Groep et al. reported that in the age group under 54 years, the likelihood of BRCA1-related disease was only 9% when Ki67 was low, but as high as 75% when Ki67 was high [27].
The BRCA gene mutation sites are associated with different populations.In the Chinese Hakka population, the most frequent mutation in the BRCA1 gene was c.2635G > T (p.Glu879*), with a frequency of 4/32 (12.5%), which is consistent with previous reports [28].This variation involves a single nucleotide alteration in exon 10 of BRCA1, resulting in an early translation stop signal that leads to nonfunctional protein products.The mutation has been reported in other populations, including Hong Kong [29,30], Singapore [31], and Malaysia [32].Additionally, this mutation is also common in ovarian cancer [28].In the current study, the 4 carriers with the c.2635G > T mutation had HER2-invasive breast cancer, with ages of breast cancer diagnosis recorded at 32, 38, 48, and 53.Three carriers had premenopausal breast cancer, and one had postmenopausal breast cancer.The stage at diagnosis was 1-2, with a high proliferation rate (Ki67 ≥ 30%).Two of the patients had bilateral breast cancer, and one patient had breast cancer with fallopian tube carcinoma, indicating an increased risk of contralateral breast cancer for this mutation.The other two sites with a high frequency of BRCA1 mutations in this population were c.3756_3759del (p.Ser1253Argfs*10) and c.5072C > A (p. Thr1691Lys).The first variant results in a shift in the reading frame, leading to the loss of BRCA1 protein function.This variant has been detected in various populations, including South Koreans, Australians, and Poles [33][34][35].In the Chinese Hakka population, carriers of this variant presented with invasive breast cancer, HER2-, and higher proliferation (Ki67 ≥ 70%).The age of diagnosis with breast cancer was 28, 48, and 51, with two carriers having premenopausal breast cancer and one having postmenopausal breast cancer.The other variant is the missense variant replaces threonine with lysine at codon 1691 in the BRCT domain of the BRCA1 protein.Functional studies have reported that this variant impacts BRCA1 function in transcription activation, protease sensitivity, and peptide binding [36].This mutation is more common in Chinese individuals [37].In the Chinese Hakka population, carriers of this variant also presented with invasive breast cancer, HER2-, Ki67 ≥ 60%.The age of breast cancer diagnosis in carriers were 34, 36, and 38, with three carriers developing breast cancer before menopause in this study.
The most frequent mutation in the BRCA2 gene observed in the Hakka population was c.5164_5165del (p.Ser1722Tyrfs*4), which is consistent with previous reports [28].This variation results in a shift in the reading frame and loss of function of BRCA2.There were seven cases of this mutation in breast cancer patients in the Hakka population.The seven carriers showed high Ki67 expression, with six carriers having HER2-status, and six cases being classified as luminal B breast cancer.The ages at diagnosis with breast cancer for carriers of this mutation were 35,39,43,47,53,54, and 58 years, respectively.Four carriers had premenopausal breast cancer, and three carriers had postmenopausal breast cancer.Two carriers had a family history of cancer: one had a family member with rectal cancer, and the other had a family member with ovarian cancer.This variant has been detected in the Chinese Han population [38,39] and the Macau population [40].Another high frequency of BRCA2 mutation in the Hakka population was c.2806_2809del (p.Ala938Profs*21).The ages at diagnosis with breast cancer for carriers of this mutation were 29, 48, and 53, and breast cancer occurred before menopause.The variant has been detected in various populations, including Asians, Europeans, and Americans [41,42].
The findings from this study revealed that breast cancer patients were diagnosed at a younger age compared to the statistical results of the larger sample of the Hakka population [43].However, there were no differences observed in the clinical classification, stage, reproductive history, or menstrual status at diagnosis.Compared to non-carriers, the average age at diagnosis of BRCA1/2 mutation carriers was younger, and more individuals under 40 were diagnosed with breast cancer, which is consistent with the results of previous reports [37,44,45].Multivariate analysis revealed that family members with cancer (OR = 2.36, 95% CI 1.00-5.54,p = 0.049) as one of the independent predictors for BRCA1/2 mutation.Among the thirteen BRCA1/2 mutation-carrying families, there were nine breast cancer families, two digestive tract cancer families, and two ovarian cancer families.Mutations in the BRCA gene might also increase the risk of breast cancer [44,45], ovarian cancer [46,47], and colorectal cancer [48,49].Bilateral breast cancer has been identified as an independent predictor for BRCA1/2 mutation.Various studies have shown that the detection rate of BRCA1/2 gene mutations can be high in patients with bilateral breast cancer [50,51].Yuntao Xie et al. recently developed a nomogram, BRCA -CRisk, to accurately predict the risk of contralateral breast cancer in patients with BRCA1/2 mutations [52].BRCA1/2 mutation carriers exhibited a higher rate of bilateral breast cancer compared to non-carriers, which is consistent with previous reports [53,54].More BRCA mutation carriers were HER2-and had no distant metastases (M0 = 88.9%).For individuals carrying BRCA1 or BRCA2 mutations and diagnosed with HER2-breast cancer, treatment with PARP inhibitors such as olaparib and talazoparib may be considered [55,56].Olaparib has been shown to improve the survival time and quality of life of breast cancer patients, as it is generally well-tolerated with no evidence of cumulative toxicity during extended exposure [7,57].Talazoparib is used in patients with advanced breast cancer and a germline BRCA1/2 mutation [58].Additionally, Ki67 ≥ 15% was one of the independent predictors for a BRCA1/2 mutation, as most BRCA1/2 mutation carriers have breast tumors with vigorous mitosis [27].
The impact of BRCA1 and BRCA2 on natural menopausal age remains uncertain and controversial.While some studies have suggested that women with BRCA1/2 mutations experience an earlier average menopause than women without BRCA mutations [60], meta-analyses have not supported this hypothesis [61].In the study, the average age at natural menopause was 49.60 ± 3.44 years for BRCA1/2 mutation carriers and 50.52 ± 3.83 years for control subjects, with no significant difference.Breast cancer patients with BRCA variants are more likely to occur before menopause.Additionally, there were no significant differences in menstrual duration, menstrual cycle, or fertility between BRCA1/2 mutation carriers and non-carriers.The analysis of 2295 matched pairs of women with a BRCA1/2 found that they had similar ages of menarche [62].Univariate logistic regression showed that age at menarche ≥ 18 years (OR = 1.42, 95% CI = 0.72-2.84,p = 0.315) and age at menarche ≤ 13 years (OR = 0.37, 95% CI = 0.13-1.06,p = 0.064) were not an independent predictor of BRCA mutations.There was no difference in menstrual duration, menstrual cycle [63], or fertility [64] between BRCA1/2 mutation carriers and non-carriers [65,66].
BRCA1/2 genes are important biomarkers for assessing the risk of breast cancer, ovarian cancer, and other related cancers, significantly influencing the choice of individualized treatment for patients.Identifying the hotspot mutation of BRCA in China's Hakka population is advantageous for the development of a targeted testing kit focused on specific sites, enabling faster and more cost-effective testing.In addition to identifying hotspot mutations in BRCA in the Chinese Hakka population, this study also found that family history, bilateral cancer, HER2-, and Ki67 ≥ 15% are significant independent predictors of BRCA pathogenic variants through logistic regression.Based on these clinical features, it is possible to efficiently identify patients who require BRCA gene testing.These results provide a basis and reference for clinical consultation and treatment strategies.However, there are some shortcomings in this study.First, this study used the method of inquiry to determine whether the subject was Hakka, and did not analyze the population genetic information of the subjects.Second, this study is based on a single-center retrospective study, and the inclusion of research objects inevitably has selection bias.Third, due to the small number of breast cancer patients carrying BRCA gene variants, this study was unable to analyze the differences in clinical characteristics between patients carrying BRCA1 gene variants and those carrying BRCA2 gene variants.In the future, a larger sample size of BRCA gene mutation research should be carried out in China to find more BRCA variants, improve the knowledge of the BRCA variation spectrum of Chinese Hakka, and provide a reference for the prevention and treatment of related cancers.

Conclusions
This article summarizes the BRCA1/2 mutation sites, clinical and pathological characteristics, menstruation, and fertility status among breast cancer patients in the Chinese Hakka population.The most prevalent pathogenic variant of the BRCA1 among breast cancer patients was c.2635G > T, while the most common pathogenic variant of the BRCA2 was c.5164_5165del.A statistical analysis of BRCA1/2 mutation carriers and non-carriers in Chinese Hakka breast cancer patients revealed that family history, bilateral cancer, HER2-, and Ki67 ≥ 15% were significant independent predictors of BRCA1/2 pathogenic variants.It is strongly recommended that breast cancer patients with a family history, of bilateral cancer, HER2-, and Ki67 ≥ 15% undergo testing for mutations of the BRCA1/2 genes.It complemented the BRCA gene mutation information in the Chinese population.The findings regarding the relationship between BRCA variation and clinicopathological features of breast cancer patients can provide a valuable reference for clinicians in diagnosis and treatment.

Fig. 1
Fig. 1 Locations and frequency of mutation sites in BRCA1 and BRCA2.(A) Twenty-five mutation sites in the BRCA1 gene (B) Twenty-six mutation sites in the BRCA2 gene.C The number and proportion of different variant types in the BRCA1 gene.D The number and proportion of different variant types in the BRCA2 gene

Fig. 2
Fig. 2 Age at onset of breast cancer and clinicopathologic characteristics of BRCA1/2 mutation carriers and non-carriers.A Age at onset of breast cancer by mutation status.B Clinicopathologic characteristics of BRCA1 mutation carriers, BRCA2 mutation carriers, and non-carriers

Fig. 3
Fig. 3 Logistic regression analysis of factors that influence BRCA1/2 carriers.A Univariate logistic regression.B Multivariate logistic regression

Table 1
Clinical characteristics in gBRCA1/2 carriers and noncarriers

Table 2
Menstrual and reproductive status of BRCA1/2 mutation carriers and noncarriers