Identification of a novel germline BRCA2 variant in a Chinese breast cancer family

Abstract Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer‐related deaths in women worldwide. In this study, a large Chinese pedigree with breast cancer including a proband and two female patients was recruited and a familial history of breast cancer was collected by questionnaire. Clinicopathological assessments and neoadjuvant therapy‐related information were obtained for the proband. Blood samples were taken, and gDNA was extracted. The BRCA1/2 and PALB2 genes were screened using next‐generation sequencing by a targeted gene panel. We have successfully identified a novel, germline heterozygous, missense mutation of the gene BRCA2: c.7007G>T, p.R2336L, which is likely to be pathogenic in the proband and her elder sister who both had breast cancer. Furthermore, the risk factors for developing breast cancer in this family are discussed. Thus, genetic counselling and long‐term follow‐up should be provided for this family of breast cancer patients as well as carriers carrying a germline variant of BRCA2: c.7007G>T (p.R2336L).

developing countries in over the last few decades. 3,4 In China, breast cancer is also the most common cancer in women; more than 1.6 million women have been diagnosed with breast cancer, and 1.2 million women are dying of breast cancer each year. 5,6 Patients in China currently account for 12.2% of all newly diagnosed breast cancers and 9.6% of all deaths from breast cancer worldwide. 5 Breast cancer is a multi-factorial disease that occurs because of various risk factors, such as sex, age, ethnicity, environmental factors, diet and lifestyle. 7,8 Although non-hereditary factors are the significant drivers of the observed international and interethnic differences in the disease incidence, genetics, including a personal or family history of breast or ovarian cancer and inherited mutations in BRCA1 (Breast Cancer 1 gene, OMIM 113705), 9 BRCA2 (Breast Cancer 2 gene, OMIM 600185) 10 and other breast cancer susceptibility genes such as PALB2 (Partner And Localizer Of BRCA2, OMIM 610355), 11 account for 6% to 10% of breast cancer cases. A woman's life-time risk of developing breast and/or ovarian cancer is greatly increased if she inherits a harmful mutation in the genes BRCA1/2 or PALB2. Breast cancer is also influenced by somatic gene mutations and chromosome instability. 12 In this study, we have successfully identified a germline mutation in the BRCA2 gene in a large Chinese family with breast cancer, expanding the knowledge and information about genetic mutations related to breast cancer.

| Ethical statement, patient information and DNA preparation
This study was approved by the Ethical Committees in the Southwest Medical University of China with written informed consent from the participants and was conducted in line with the tenets of the Declaration of Helsinki (2013 version). A Chinese pedigree of breast cancer including a proband (Figure 1, pedigree III: 11, arrow) was recruited, and the family history of breast cancer was collected by questionnaire. Clinicopathological assessments and neoadjuvant therapy information were obtained for the proband, such as haematoxylin and eosin (HE) staining, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Fresh peripheral blood samples (2 ml each) were taken from each individual, and gDNA was extracted using our previously described phenol/chloroform method. [13][14][15] Blood samples from one hundred healthy individuals were taken for DNA extraction as controls.

| Targeted NGS, Sanger sequencing and cosegregation analysis
All of the exons and exon-intron boundaries of the BRCA1/2 and PALB2 genes were screened with next-generation sequencing (NGS) by a targeted gene panel using an Illumina HiSeq × 10 NGS platform (Illumina, CA, US). Gene capture was up to 500 × depth and had greater than 99% coverage. For mutation identification, the sequenced results were aligned to reference sequences of the  Table 1). A PCR product with 553 bp for BRCA2 was amplified using gDNA as a template. The PCR products were directly subjected to sequencing with the Sanger method on an ABI-3500DX sequencer (Applied Biosystems Inc, Foster City, CA, USA) using the specific primer BRCA2-7007L (Table 1). 16,17 All unrelated, ethnically matched controls were also sequenced using the aforementioned primer, and then, co-segregation analysis was performed based on the sequencing results and clinical phenotypes in this pedigree.

| RE SULTS
We recruited a Chinese breast cancer family, including 3 female patients and 25 healthy individuals (Figure 1, II:3, III:3, III:11, Table 2 The clinical information of the two patients, including current age, the year diagnosed, unilateral status, treatment strategy, tumour grade and size, lymph node status, recurrence, IHC results for biomarkers and FISH results, is listed in Table 2. By IHC analysis based on biomarker testing of the cancer tissues, the proband's tumour was positive for Ki67, ER, PR and HER-2, whereas other markers were negative (Table 2). Both patients were initially diagnosed with stage II disease (Table 2). Treatments were used immediately when diagnosed, and the recurrences were not found over three years for the proband (III: 11) and five years for her sister (III:3) ( Table 2).    Table 3.
Sequencing of DNA materials from nine other female members in this family including the proband's sister with breast cancer was also performed. The representative results by Sanger sequencing are shown in Figure 2A-J. In addition to proband III:11 and her sister III:3 who were affected with breast cancer, another five women in this family, including the proband's mother, three sisters and one niece, who all appeared to be normal, also carried the same heterozygous, missense mutation of the gene BRCA2: c.7007G > T.
The other 3 women in this family with normal phenotypes carried two wild-type alleles ( Figure 2). DNA from III:13 was not available.
Sequencing of DNA materials from male members of this family was also performed and revealed that IV:3 had wild-type alleles of BRCA2, whereas IV:10 had the same heterozygous mutation of BRCA2: c.7007G>T ( Figure 2K,L, respectively). One hundred unrelated, healthy individual controls did not show this variant by Sanger sequencing (data not shown).

Searches of the Conserved Domain Database (CDD) in NCBI
were executed. Comparing the human BRCA2 protein to nine other species indicated that it is highly conserved in the chimpanzee, rhesus monkey, dog, cow, mouse, rat and chicken ( Figure 3A).
Comprehensively, this study shows that the heterozygous variant of BRCA2: c.7007G>T (p.R2336L) might cause breast cancer in this Chinese pedigree ( Figure 3B, arrow).

| D ISCUSS I ON
The

PA RTI C I PATE
The study was approved by Southwestern Medical University, and written informed consent was obtained from all patients, in compliance with the recommendations of the Helsinki Declaration.

CO N S E NT FO R PU B LI C ATI O N
Written informed consent was obtained from the participants for publication of their medical data and images.