The Features of BRCA1 and BRCA2 Germline Mutations in Hakka Ovarian Cancer Patients: BRCA1 C.536 A>T Maybe a Founder Mutation in This Population

Introduction

Ovarian cancer is one of the most common cancers in women and a leading cause of death in women. Increased risk factors for cancer have led to an upward trend in the incidence of the disease globally.^1,2 There are several risk factors for ovarian cancer, such as genetic predisposition, ovulation, endometriosis, dietary factors, and ethnicity/race. Ovarian cancer can occur sporadically in any woman, including those without any notable risk factors.³

Ovarian cancer is thought to be divided into two main subtypes: type I and type II. Ovarian carcinoma type I includes patients with endometrioid carcinoma, low-grade serous carcinoma, low grade adenocarcinoma carcinoma, mucinous carcinoma, clear cell carcinoma, and transitional cell carcinoma, which are mostly confined to one ovary and are associated with a good prognosis. Ovarian carcinoma type II includes patients with high-grade serous carcinoma, undifferentiated carcinoma, carcinosarcoma, and high grade adenocarcinoma. Unlike ovarian cancer type I, it is highly aggressive and progresses rapidly. Ovarian carcinoma type II is poorly differentiated, and is generally absent in early ovarian lesions.^4,5 The diagnosis of ovarian cancer involves pelvic examination, detection of serum tumor markers detection, blood cell analysis, ultrasonography, computed tomography (CT), magnetic resonance imaging (MRI), and other imaging techniques as well as the joint application of these detection methods. However, these tests used in the early diagnosis and condition monitoring of ovarian cancer have certain limitations; hence, the early diagnostic markers and disease characteristics of ovarian cancer need to be studied and clarified.

Studies have identified that family history and genetic factors are important risk factors for ovarian cancer. Ovarian cancer susceptibility genes include breast cancer susceptibility gene 1 (BRCA1), breast cancer susceptibility gene 2 (BRCA2), RAD51 recombinase (RAD51) gene family,⁶ BRCA1-associated RING Domain protein 1 (BARD1) gene,⁷ and murine double minute (MDM) gene family.^8,9 The two major ovarian cancer susceptibility genes, BRCA1 (MIM #113705) and BRCA2 (MIM #600185), have been explored the most.¹⁰ BRCA1 is located on chromosome 17q21 (containing 22 exons) and encodes and expresses a multi-domain protein containing 1863 amino acids.¹¹ BRCA2 is located on chromosome 13q12-q13 (containing 27 exons) and encodes and expresses a multidomain protein containing 3418 amino acids. BRCA1 is similar to BRCA2, but there is no significant homology in the exon region. BRCA1 plays a crucial role in important cell activities and in maintaining gene stability.^12–14 BRCA2 is thought to play a key role in the repair of double-strand breaks and in the partial regulation of RAD51 response via chromosome recombination mechanism.^15–17 Corresponding studies have also confirmed the correlation between the genetic marker on the chromosome 17q and the susceptibility to breast and ovarian cancer in some patients with a family history of these diseases.¹⁸ The results of a genome-wide association analysis involving 15 high-risk breast cancer families revealed that BRCA2, localized on the chromosome 13q12-13, is associated with breast cancer.¹⁹ Both BRCA1 and BRCA2 are tumor-suppressor genes, and mutations in these genes are seen in some patients with cancer. Mutations in the human BRCA gene may be race-specific in a given region and region-specific in a given ethnic group.^20,21

The Hakka is a Han ethnic group with a unique genetic background and originate from the Hakka ancestors of the Han nationality in Central China. They migrated southward for many times and fused with the ancient Yue residents in Guangdong, Fujian and Jiangxi.²² Meizhou City is located in the northeast of Guangdong Province and has a large Hakka population. The BRCA1/2 mutations and the characteristics of BRCA1/2-associated ovarian cancer in the Hakka population remain unclear. This study retrospectively analyzed the results of the BRCA gene in patients with ovarian cancer among the Hakka population using next-generation sequencing.

Materials and Methods

Participants

Enrollment for the ovarian cancer BRCA gene mutation screening trial was conducted between January 2018 and May 2021 of subjects visiting the Meizhou People’s Hospital (Huangtang Hospital), Meizhou Academy of Medical Sciences. Signed informed consent forms were obtained from all participants. The baseline data, including the general information, related medical history, hematological parameters, and staging (according to the AJCC 8th edition staging) of the enrolled subjects were collected. The ovarian cancer patients were categorized into three groups according to the pathological type: (1) type 1, patients with endometrioid, mucinous, clear cell, low-grade serous, and low grade adenocarcinoma, benign serous cystadenoma or Mullerian cystadenoma; (2) type 2, high grade serous, undifferentiated, carcinosarcoma, and high grade adenocarcinoma; and (3) others, including granulosa cell tumors, borderline tumors, and tumors with unavailable grade or histology data. The study was conducted on the basis of the Declaration of Helsinki, and was supported by the Ethics Committee of the Meizhou People’s Hospital (Huangtang Hospital).

Detection of Serum Tumor Markers and Inflammatory Markers in the Sampled Blood

The subjects’ blood samples (3 mL) were collected at the time of admission and at 2–3 days before treatment and the serum was immediately separated. Serum tumor markers analysis was performed on the Luminex 200 system (Luminex Corporation, Austin, USA) to measure the concentration of serum carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), carbohydrate antigen 125 (CA125), and alpha-fetoprotein (AFP) using relevant quantitative detection kits (TELLGEN Life Science and Technology Co., Ltd., China). After adding the samples and the labeled antibodies into each well, the reaction was performed in the dark at 37°C for 5 min, after which the fluorescence-encoded microspheres cross-linked with antibodies were added. After the reaction, the plate was incubated for 60 min in a darkroom at 37°C, the termination solution was added to terminate the reaction, and the microsphere signals of each well were read on the Luminex 200 system.

The blood sample (2 mL) was collected via venipuncture of an antecubital vein from each subject and collected in a tube containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. The erythrocyte correlative indices were detected by using the Sysmex XE-2100 Blood Analyzer (Sysmex Corporation, Japan) according to the standard operating procedures (SOP). The blood routine results were collected before treatment and the inflammation index was calculated according to the following formulas: SII = platelet × neutrophil/lymphocyte,

SIRI = monocyte × neutrophil/lymphocyte,

NLR = neutrophil/lymphocyte,

PLR = platelet/lymphocyte,

LMR = lymphocyte/monocyte.

BRCA1 and BRCA2 Mutations Were Detected by Next-Generation Sequencing (NGS)

The peripheral blood sample (2 mL) was collected from each participant and collected in a tube containing EDTA as an anticoagulant. Genomic DNA was extracted by using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. DNA concentration and purity were quantified using the Nanodrop 2000^TM Spectrophotometer (ThermoFisher Scientific, Waltham, MA). The DNA samples were sequenced after library construction, template preparation and template enrichment according to standard operating procedures of the Life Technology Company. Then, 200–1000 ng DNA was sheared prior to library construction for 150 bp fragments. The NGS libraries were constructed using the IonPlus Fragment Library Kit (Life Technologies, Carlsbad, CA). Next, ligation was performed with barcode adapters, followed by ligated fragments amplification, library purification and concentration. Next-generation sequencing was performed on the Ion Proton instrument (Life Technologies) and tested by the CapitalBio Corporation (Beijing, China). Data were analyzed by the Torrent Suite 4.4.3 and 5.0.4 (Life Technologies) using optimized parameters: minimal depth 300×, detection threshold of 2% and 1% for hotspots. Variant call files from the variant caller were loaded on a galaxy platform and annotated using the Safir2report tool. The sequencing results were then compared with the standard human genome data to obtain the mutation sequence information of the samples to be tested. According to the Human Genome Variation Society (HGVS) guidelines, the genetic variations in this study, were named using the following reference sequences: NM_007294.4 (BRCA1) and NM_000059.4 (BRCA2).

Statistical Analyses

SPSS statistical software version 21.0 was used for data analyses. Continuous variable data are represented by mean ±SD, and analyzed using Student’s t-test or the Mann–Whitney U-test. The Chi-square test was applied for analyzing the categorical variables, which were then presented as percentages. P<0.05 was considered to indicate statistical significance.

Results

Population Characteristics

A total of 195 patients with ovarian cancer (all women) were included in the present study. There were 14 women (7.2%) <35 years of age, 54 cases (27.7%) between 35 and 50 years of age, and 127 women (65.1%) >50 years of age. None of the 195 patients had a family history of breast or ovarian cancer. The findings show that ovarian cancer mainly occurs in people >50 years of age. Some studies have observed that the risk of ovarian cancer decreases with the number of pregnancies.^23,24 We intended to analyze the relationship between the number of pregnancies and BRCA mutations in the patients with ovarian cancer. Accordingly, 8 women (4.1%) never had a pregnancy, 94 women (48.2%) had 1–3 pregnancies, 34 women (17.4%) had 4–5 pregnancies, 14 women (7.2%) had >5 pregnancies, and the remaining 45 (23.1%) were unknown. These results show that the risk of ovarian cancer decreases with the number of pregnancies. The CEA, CA199, AFP, CA125, NLR, LMR, and PLR levels of these patients were 11.56 ± 114.89 ng/mL, 144.34 ± 769.04 U/mL, 225.98 ± 1779.13 ng/mL, 733.15 ± 1553.06 U/mL, 4.22 ± 4.17, 3.61 ± 2.24, and 245.88 ± 169.87, respectively (Table 1).

Table 1 Clinical Characteristics of Ovarian Cancer Patients

Clinical Features of Patients with Different Types of Ovarian Cancer

There were 29 (14.9%) patients with type 1 ovarian cancer, 119 (61.0%) cases with type 2 ovarian cancer and 47 (24.1%) cases with other ovarian cancer types. The majority of the patients had type 2 ovarian cancer. It was found that 1 patient (3.4%) was <35 years of age, 6 patients (20.7%) were between 35 and 50 years of age, and 22 patients (75.9%) were >50 years of age in the type 1 group. On the other hand, in the type 2 group, 2 patients (1.7%) were <35 years of age, 28 patients (23.5%) were between 35 and 50 years of age, and 89 patients (74.8%) were >50 years of age. In the other types, there were 11 patients (23.4%) <35 years of age, 20 patients (42.6%) between 35 and 50 years of age, and 16 patients (34.0%) >50 years of age. There were significant differences in age distribution among the three groups of patients with ovarian cancer (P < 0.001). The results show that type 1 and type 2 ovarian cancers mainly occur in people >50 years of age, whereas the other types of cancer mainly occur in people in the age group of 35–50 years. Regarding the number of pregnancies, in the group of type 1 ovarian cancer, 17 women (58.6%) had never been pregnant or had 1–3 pregnancies, and 8 women (27.6%) had ≥4 pregnancies. In the type 2 ovarian cancer group, 55 women (46.2%) had never been pregnant or had 1–3 pregnancies, and 32 (26.9%) had ≥4 pregnancies. Although there was no significant difference in the number of pregnancies among the groups, these types of ovarian cancer were predominated by women with ≤3 pregnancies (Table 2). There were significant differences in CEA (P = 0.035) and AFP (P = 0.012) levels among the groups. The CEA level was the highest in patients with type 1 ovarian cancer (62.24 ± 296.74 ng/mL vs 2.87 ± 7.89 and 2.28 ± 2.26 ng/mL), whereas the AFP level was the lowest in patients with type 1 ovarian cancer (2.92 ± 1.50 ng/mL vs 16.83 ±120.70 and 893.17 ± 3565.47 ng/mL) (Table 2).

Table 2 Clinical Characteristics of Participants with Type 1, Type 2, and Other Ovarian Cancer

The Frequencies and Distributions of the BRCA Gene Mutations

The protein coding region and exon-intron boundary region of the BRCA1 and BRCA2 genes of the patients were sequenced using next-generation sequencing. There were 64 patients (64/195, 32.8%) with BRCA gene mutations, among whom 32 patients (32/64, 50.0%) had BRCA1 gene mutation/mutations, 27 patients (27/64, 42.2%) had BRCA2 gene mutation/mutations, and 5 patients (5/64, 7.8%) had both mutations. The numbers of patients with type 1, type 2, and other types of ovarian cancer who had BRCA mutations were 7, 42, and 15, respectively. The numbers of patients with type 1 ovarian cancer who had BRCA1, BRCA2, and both mutations were 3 (42.9%), 4 (57.1%), and 0(0), respectively. The corresponding numbers of patients with type 2 ovarian cancer and other types of cancer were 23 (54.8%), 15 (35.7%), 4 (9.5%) and 6 (40.0%), 8 (53.3%), 1 (6.7%), respectively. These results show that type 1 ovarian cancer with BRCA mutation mainly involves the BRCA2 gene, while type 2 ovarian cancer with BRCA mutation mainly involves the BRCA1 gene. The frequencies and distributions of BRCA1 and BRCA2 gene mutations are presented in Table 3.

Table 3 The Frequencies and Distributions of BRCA1 and BRCA2 Gene Mutations

In this study, a total of 52 variants of the BRCA gene were detected. The sequence analysis revealed 22 distinct pathogenic mutations detected in 26 patients, 2 likely pathogenic variants in 2 patients, 12 variants of uncertain significance (VOUS) in 20 patients, and 16 likely benign variants in 24 patients. The mutations were predominantly seen in exons 8, 14, and 17 of the BRCA1 gene and exons 10, 11, 14 and 15 of the BRCA2 gene (Figure 1A and B). Among the BRCA1 gene variants, 15 (62.5%) were pathogenic variants, 2 (8.3%) were likely pathogenic variants, 5 (20.8%) were likely benign variants, and 2 (8.3%) were VOUSs. On the other hand, among the BRCA2 mutations, 7 (25.0%) were pathogenic variants, 11 (39.3%) were likely benign variants, and 10 (35.7%) were VOUSs (Figure 1C).

Figure 1 All the BRCA1 (A) and BRCA2 (B) variants and their locations on the protein coding region and exon intron boundary region identified in ovarian cancer patients, and the overall frequencies of the ClinVar grades of variants in ovarian cancer patients (C). Gray boxes represent untranslated regions and green boxes represent coding exons.

Table 4 provides detailed information on each patient with BRCA mutation/mutations, including ClinVar information (pathogenic, likely pathogenic, likely benign, and VOUS) pertaining to the BRCA1 and BRCA2 genes. Data on mutations, mutation types, family history of cancer, and number of pregnancies are presented. In this study, 10 patients (15.63%, 10/64) carried the BRCA1 gene c.536 A>T variant, 4 patients (6.25%, 4/64) carried the BRCA1 gene c.2635 G>T variant, 4 patients (6.25%, 4/64) carried the BRCA1 gene c.2566 T>C variant, and 3 patients (4.69%, 3/64) carried the BRCA2 gene c.5785 A>G variant, 3 patients (4.69%, 3/64) carried the BRCA2 gene c.8187 G>T variant. Based on these observations, BRCA1 c.536 A>T might be a founder mutation in this population.

Table 4 All Mutations and the Characteristics of Ovarian Cancer Patients

Discussion

Ovarian cancer is one of the most common malignant tumors that threaten women’s health and quality of life and has the highest mortality rate among gynecological tumors.²⁵ BRCA is an important gene that determines the genetic susceptibility to cancer by participating in the regulation of DNA damage and repair, cell growth and apoptosis and by playing an indispensable role in maintaining the genetic stability of cells.^26,27 Mutations in the BRCA gene can lead to ovarian cancer. Screening for BRCA gene mutations can effectively assess and predict the risk for ovarian cancer, intervene to reduce the incidence of the disease, and guide precise treatment.

Worldwide, the incidence of BRCA mutations in patients with ovarian cancer is approximately 10%-15%. BRCA mutations are chiefly distributed in Europe and North America, among which the incidence of BRCA1 mutation is significantly higher than that of BRCA2.²⁸ The incidence of BRCA1/2 mutations among Mexican patients with ovarian cancer is approximately 28%, of which BRCA1 constitutes 88% and BRCA2 constitutes 12%.²⁹ Another study on patients with ovarian cancer in Mexico found that the incidence of BRCA1/2 mutations was 33%, of which 66.1% was BRCA1 and 33.9% was BRCA2.³⁰ These differences may be related to the geographical distribution of the selected population. The prevalence of BRCA mutations among Spanish patients with ovarian cancer was 16%, of which the prevalence of BRCA2 mutations was 63%, which is contrary to most studies.³¹ The prevalence of BRCA mutations in Israeli Arab patients with ovarian cancer was 32%.³² The prevalence of BRCA mutations in Korean patients with ovarian cancer was 24.6%.³³ At present, there are several studies based on large samples in China. Shi et al³⁴ performed BRCA1/2 gene detection in 916 patients with epithelial ovarian cancer. The results showed that the incidence of BRCA1/2 gene mutation was 16.7%, of which BRCA1 mutation accounted for 13.1%, BRCA2 mutation accounted for 3.9%, and the simultaneous presence of both mutations accounted for 0.3%. Wu et al³⁵ conducted the BRCA test on 826 patients with ovarian cancer in a multiCenter nationwide study, and the incidence of BRCA1/2 gene mutations was found to be 28.5%, of which the BRCA1 mutation accounted for 20.8% and the BRCA2 mutation accounted for 7.6%. Bu et al³⁶ attempted to detect BRCA mutation in 547 patients with ovarian cancer and found the incidence of BRCA1/2 gene mutations to be 23.6%, of which BRCA1 mutation accounted for 15.4% and BRCA2 mutation accounted for 8.2%. The proportion of BRCA mutations was 5.41% in breast and ovarian cancer in a Hakka population.³⁷ The proportion of patients with BRCA mutation in Hong Kong was 15.3%.³⁸ In this study, the incidence of BRCA1/2 gene mutations was 32.8% in patients with ovarian cancer, of which BRCA1 mutation accounted for 50.0%, BRCA2 mutation accounted for 42.2%, and both mutations accounted for 7.8%. In the future, multicenter BRCA gene mutation studies should be conducted in China with a larger sample size by adopting unified standards so as to create a BRCA gene mutation database consistent with the characteristics of the Chinese population.

To the best of our knowledge, this study is the largest on BRCA1 and BRCA2 gene sequencing in patients with ovarian cancer for mutation screening analysis in the Chinese Hakka population. According to researches, mutations in the BRCA1 gene are concentrated in exons 8, 11, 22, and 24 and mutations in the BRCA2 gene are concentrated in exons 10, 11, 14, and 21 in Mainland China populations.^39–43 Our results suggested that mutations in the BRCA1 gene mainly occur in exons 8, 14, and 17, whereas mutations in the BRCA2 gene chiefly occur in exons 10, 11, 14, and 15. The findings indicate that the BRCA genes have different mutation hotspots in different ethnic groups and regions.

In addition, some variants were present at higher frequencies when compared with the other variants. It was observed that 10 patients (15.63%, 10/64) carried the BRCA1 gene c.536 A>T variant, 4 patients (6.25%, 4/64) carried the BRCA1 gene c.2635 G>T variant, 4 patients (6.25%, 4/64) carried the BRCA1 gene c.2566 T>C variant, and 3 patients (4.69%, 3/64) carried the BRCA2 gene c.5785 A>G variant, 3 patients (4.69%, 3/64) carried the BRCA2 gene c.8187 G>T variant. Whether these mutations are founder and hotspot mutations in the patients with ovarian cancer in the Chinese Hakka population remains to be confirmed. Founder mutations in the BRCA genes have been reported in many nations and ethnic groups worldwide. It has been documented that BRCA1 c. 5470_5477del8 is a BRCA1 founder mutation of ovarian cancer in the Chinese population;^34,44 this mutation may be the BRCA1 founder mutation unique to Asians.⁴⁵ The mutation BRCA2 c.3109 C>T is a founder mutation in the Southern Chinese population.⁴⁶ Another study showed that the most common mutation was BRCA1 ex9-12del, a Mexican founder mutation.³⁰ Mutational data show that the most frequently recorded BRCA1 c.5266dupC mutation is the founder mutation in Italian,⁴⁷ Northeastern Romanian,⁴⁸ and Turkish populations.⁴⁹ Slavic BRCA1 and BRCA2 founder mutations include BRCA1 c.5266dupC, BRCA1 c.4034delA, and BRCA1 c.68_69delAG.⁵⁰ BRCA1 c.4136_4137delCT and c.1140dupG represent the founder mutations in the Middle Eastern population.⁵¹ BRCA2 c.3922G>T is a founder mutation in the Puerto Rican population.⁵² BRCA1 c.5266dupC and c.181T>G are founder mutations in the Polish population.⁵³ BRCA1 c.798_799delTT might be a founder mutation in the North African population.⁵⁴ BRCA1 c.3319G>T might be a founder mutation in the Western Danish population.⁵⁵ In the present study, the BRCA1 c.536 A>T mutation was observed which might be considered a founder mutation in this ovarian cancer population. The recurrent BRCA1 mutation reported herein has rarely been observed in other ethnic groups.

In general, the mutation frequency of the BRCA1/2 gene in the Hakka patients with ovarian cancer in Southern China is different from that in other ethnic groups. Moreover, differences exist in the exon regions where the mutations occur. This study provides a basis and serves as a reference for clinical counseling and for devising individualized prevention and treatment strategies to combat ovarian cancer. Identifying founder and recurrent mutations is an important way to improve genetic counseling because molecular testing can target the founder and recurrent mutations, thereby enabling faster and less expensive testing. As the frequency of founder mutations increases, molecular testing can analyze a large number of cases and provide accurate information on the relationship between the patient’s mutation status and disease risk, thereby improving disease management. Owing to the small sample size of this study, the distributions of the BRCA gene mutations among the patients with ovarian cancer in the Hakka population of Southern China have not been entirely revealed. The distributions of the BRCA gene mutations in different populations, and the relationship between mutation status and disease risk and pathological features need to be investigated further.

In this population, the significance of identifying the founder mutations mainly lies in reducing the cost of population screening. Genetic screening can be performed by first focusing on the founder mutation. In this manner, genetic screening can be easily implemented in the Hakka population. Although this study has shed light on the founder BRCA1 mutation in the Hakka population, we cannot rule out the possibility that other founder BRCA mutations may exist in the larger patient population. However, given the economic advantages of genetic screening, we believe that this study would pave the way for future studies in the Hakka population.

Conclusions

In this study, the BRCA gene mutations were found to account for a certain proportion of the patients with ovarian cancer in the Hakka population of Southern China. The BRCA1 c.536 A>T mutation was detected among in 10/64 (15.63%) of the individuals with BRCA mutation/mutations in the cohort and can, therefore, be considered a founder mutation in this ovarian cancer population. This recurrent BRCA1 mutation has rarely been observed in other ethnic groups. Understanding the frequency of BRCA1 and BRCA2 gene mutations in the Hakka patients with ovarian cancer will provide valuable data for clinical consultation and for devising individualized therapeutic strategies for patients with ovarian cancer.