Analysis of BRCA1 large genomic rearrangements to identify individuals at risk of breast cancer


 Background. Breast cancer (BC) is a malignant neoplasm in which genetic and environmental aspects contribute to its development. In many cases, pathogenic mutations in BRCA1 are caused by large genomic rearrangements such as copy number variations, a mechanism of gene inactivation that increases the risk of BC. Therefore, identifying women at high risk of BC through genetic testing is of great importance. This study aimed to identify large BRCA1 rearrangements in patients with early-onset or family history of BC. Methods. Peripheral blood from patients with (n= 38) and without (n= 30) BC was analyzed using the multiplex ligation-dependent probe amplification assay to detect large genomic rearrangements in BRCA1. Results. One patient with sporadic BC showed an ambiguous deletion in exon 12 of the BRCA1 gene. To validate this result, full sequencing of this region was performed. The mutation detected by MLPA resulted in a false positive, showing that large genomic rearrangements in BRCA1 were absent in all subjects. Moreover, in this same patient, we detected the presence of c.4308T>C (rs1060915) polymorphism. Conclusions. In our patients with BC, large genomic rearrangements in BRCA1 are held unaccountable for the development of the disease. We identified the presence of single nucleotide polymorphism, c.4308T>C (rs1060915), in a patient with sporadic BC.

Conclusions. In our patients with BC, large genomic rearrangements in BRCA1 are held unaccountable for the development of the disease. We identi ed the presence of single nucleotide polymorphism, c.4308T>C (rs1060915), in a patient with sporadic BC.

Background
Breast cancer (BC) is the most common malignant neoplasia in women worldwide. Environment and genetic background play important roles in the development of this disease and as a result, for the vast majority of women is not possible to identify a speci c cause [1]. Approximately 5-10% of BC is identi ed of hereditary origin, with most mutations occurring in the tumor-suppressor gene BRCA1 (MIM#113705).
Mutations in this gene also confer an increased risk in other types of cancer such as ovarian, fallopian tube, colon, prostate, male breast and pancreatic [2]; however, these alterations are mainly identi ed in individuals with family history of breast cancer, ovarian cancer, bilateral BC and early-onset BC [3,4]; moreover, BC phenotypes such as medullary carcinoma and triple-negative (estrogen receptor, progesterone receptor and Her2/neu negative) have also been related with BRCA1 variations in 18% of patients under 50 years of age [3].
A selective group of these variations is known as large genomic rearrangements (LGRs).
LGRs are responsible for as much as 23% of all identi ed BRCA1 mutations [5]. A type of LGR is copy number variation (CNV), occurs when a segment of DNA, 1 kb or longer, repeat at a different number in comparison with the reference genome [6]. Individuals that present pathogenic LGRs in BRCA1 (0-27%) have an estimated lifetime risk of 60% for BC, 59% for ovarian cancer and 83% for contralateral BC [7,8].
It is well known that patients who receive treatment in an early stage of BC have higher survival rates. The identi cation of potentially pathogenic mutations in BRCA1 would allow taking clinical actions and preventive measurements such as prophylactic mastectomy, in the patient himself and his relatives [3]. Therefore, identifying women who are at high risk of BC through genetic testing has a very important implication. This study aimed to identify BRCA1 rearrangements in patients with early-onset or family history of breast cancer.

Study groups
A cross-sectional, observational and comparative study was carried out to evaluate the association The study population was divided into three groups, hereditary BC (HBC, n=18), sporadic BC (SBC, n=20) and healthy patients (HP, n=30). The groups included women from 18 to 50 years of age. In the case of BC patients (HBC and SBC), all patients were 50 years old or younger at the time of initial diagnosis, with histopathological con rmation, and for the speci c case of HBC, meeting at least one of the criteria listed in the NOM-041-SSA2-2011 [9].
Multiplex ligation-dependent probe ampli cation assay Genomic DNA was isolated from blood samples with the Gustincich method [10]. The DNA concentration was evaluated by the A260/A280 absorbance ratio with a Nanodrop-1000™ (NanoDrop Technologies) spectrophotometer. BRCA1 CNV was quanti ed by multiplex ligation-dependent probe ampli cation (MLPA) using the P002-D1 probe mix assay according to instructions provided by the manufacturer (MRC Holland). Ampli ed products were separated using an ABI PRISM 310 Genetic Analyzer™ (Applied Biosystems) and interpreted using the GeneMapper Software v4.0 (Applied-Biosystems) and the Coffalyser.net Software v14.0 (MRC Holland). Healthy patient data was used as a control.
PCR and gene sequencing Speci c primers for Exon 12 (forward 5´-AGGTGATTTCAATTCCTGTGCT-3´ and reverse 5´-GGCTCCATAATTACCCATGTGC-3´) were designed to con rm the predicted deletion detected from MLPA data. These primers can amplify the whole exon and MLPA probe hybridization site. To locate the deletion site as well as to con rm the data from MLPA analysis, resultant PCR products were subjected to direct sequencing.

Statistical analyses
To determine the statistical association between the variables and BC risk, we used analysis of variance. Chi-square, odds ratios (OR) and 95% con dence intervals (CI) were also estimated.

Results
A total of 38 unrelated women were diagnosed with BC, including women with HBC and SBC, this represented a 55.88% of the total study population ( We performed MLPA to analyze the presence of CNV in BRCA1. Results showed an ambiguous deletion (rate 0.49) in exon 12 that is distinguished when observing the different peak amplitudes of MLPA products ( Figure 1). To further con rm this deletion, exon 12 was fully sequenced and compared with the reference sequence of BRCA1 (NG_005905) searching for coincidences in the basic local alignment search tool (BLAST) published by the National Centre for Biotechnology Information. Results showed no rearrangement in exon 12 and indicated that the MLPA probe hybridization site was intact. Furthermore, the sequencing of the ampli ed product revealed a single nucleotide polymorphism, c.4308T>C (rs1060915) (Figure 2).

Discussion
In México, few studies have investigated the contribution of BRCA1 mutations to BC. To our knowledge, there are no published studies about the prevalence of CNV in BRCA1 in women from Sinaloa. With our research, we contributed to clarifying this situation.
In our study, only women diagnosed with BC presented a family history of cancer (breast, ovary, colon, pancreas and/or prostate cancer) related to mutations in BRCA1, providing further evidence about the importance of genetics as a risk factor for this pathology [11].
Large exon rearrangements have been investigated for more than a decade with unclear results; at the beginning, usage of conventional PCR yielded variable results due to the ampli cation of wild-type alleles in heterozygous cases [12]. Currently, new techniques such as MLPA are used to solve this problem, however, attention is required to recognize ambiguous data and exon deletions. Moreover, single nucleotide polymorphisms (SNPs) may also affect the correct ampli cation, leading to false apparent deletion in a single exon [13]. Thus, the results always need to be validated by another method.
Even though MLPA analysis did not show CNV in the BRCA1 gene, a larger study is necessary before its contribution in hereditary BC could be called null. Nowadays, more than 100 rearrangements have been described for BRCA1 [14]. In the case of the Hispanic population, the prevalence of CNV is over 10% and is mainly caused by a founder deletion of exons 9-12 [15]. When a pathogenic mutation is identi ed, preventive measures, such as prophylactic mastectomy, can be taken to reduce the risk of BC [4].
We detected an SNP (rs1060915), which is most prevalent in the South Asian population (alternative allele G=0.50), but also detected in lower frequency in American (G=0.36) [16]. This polymorphism acts as a benign mutation in women with BC, being a silent mutation that does not cause changes in the encoded amino acid (S1436S). The rs1060915 polymorphism is reported as a part of a common haplotype in BRCA1 [17,18,19], but its signi cance remains unclear.

Conclusions
In this study, CNV in BRCA1 was absent in all studied subjects, we recommend a large study sample to reveal novel or reported genomic rearrangements among Mexican mestizo population. We identi ed the presence of rs1060915 polymorphism by direct sequencing a sample corresponding to a patient with sporadic BC. All participants reviewed and signed the informed consent.

Consent for publication
Not applicable

Availability of data and materials
The datasets generated and/or analysed during the current study are not publicly available due to the privacy policies of the health institutions involved but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests All authors read and approved the nal manuscript.   Single nucleotide polymorphism (SNP) c.4308T>C in exon 12 of BRCA1. SNP (Y) detected in a patient with sporadic breast cancer.