Hereditary breast and ovarian cancer: assessment of point mutations and copy number variations in Brazilian patients

Abstract

Background: Germ line mutations in BRCA1 and BRCA2 (BRCA1/2) and other susceptibility genes have been identified as genetic causes of hereditary breast and ovarian cancer (HBOC). To identify the disease-causing mutations in a cohort of 120 Brazilian women fulfilling criteria for HBOC, we carried out a comprehensive screening of BRCA1/2, TP53 R337H, CHEK2 1100delC, followed by an analysis of copy number variations in 14 additional breast cancer susceptibility genes (PTEN, ATM, NBN, RAD50, RAD51, BRIP1, PALB2, MLH1, MSH2, MSH6, TP53, CDKN2A, CDH1 and CTNNB1).

Methods: Capillary sequencing and multiplex ligation-dependent probe amplification (MLPA) were used for detecting point mutations and copy number variations (CNVs), respectively, for the BRCA1 and BRCA2 genes; capillary sequencing was used for point mutation for both variants TP53 R337H and CHEK2 1100delC, and finally array comparative genomic hybridization (array-CGH) was used for identifying CNVs in the 14 additional genes.

Results: The positive detection rate in our series was 26%. BRCA1 pathogenic mutations were found in 20 cases, including two cases with CNVs, whereas BRCA2 mutations were found in 7 cases. We also found three patients with the TP53 R337H mutation and one patient with the CHEK2 1100delC mutation. Seven (25%) pathogenic mutations in BRCA1/2 were firstly described, including a splice-site BRCA1 mutation for which pathogenicity was confirmed by the presence of an aberrant transcript showing the loss of the last 62 bp of exon 7. Microdeletions of exon 4 in ATM and exon 2 in PTEN were identified in BRCA2-mutated and BRCA1/2-negative patients, respectively.

Conclusions: In summary, our results showed a high frequency of BRCA1/2 mutations and a higher prevalence of BRCA1 (64.5%) gene. Moreover, the detection of the TP53 R337H variant in our series and the fact that this variant has a founder effect in our population prompted us to suggest that all female breast cancer patients with clinical criteria for HBOC and negative for BRCA1/2 genes should be tested for the TP53 R337H variant. Furthermore, the presence of genomic structural rearrangement resulting in CNVs in other genes that predispose breast cancer in conjunction with BRCA2 point mutations demonstrated a highly complex genetic etiology in Brazilian breast cancer families.

Figure 1

Characterization of a novel BRCA1 splice site variant (c.560 + 2 T > A) in one HBOC patient. A: Sequencing pattern of BRCA1 exon 7 from blood cell genomic DNA showing the c.560 + 2 T > A mutation. B: Agarose gel showing RT-PCR products obtained from the cDNA of a tumor from patient MO-15 and one control sample (sporadic tumor negative for the c.560 + 2 T > A) using a forward primer in exon 6 and a reverse primer in exon 8 of BRCA1. An additional 186-bp cDNA fragment caused by the partial deletion of exon 7 was observed in the tumor sample of patient MO-15. C: Above, partial sequence of the expected fragment (258 bp) in the control tumor sample showing the exon 7–8 junction; below, partial sequence of the patient tumor cDNA showing the creation of a novel cryptic splice donor site causing the deletion of the last 62 bp of BRCA1 exon 7 in the aberrant transcript. D: Schematic representation of the premature stop codon (p.Ser127Thrfs*11) created in the BRCA1 mRNA after the frameshift deletion of the last 62 bp of exon 7 caused by the germ line splice site variant c.560 + 2 T > A. E: Amino acid sequence of the expected truncated protein (137 aa) showing the alteration of 10 amino acids (in red) and creation of a premature stop codon (*).

Figure 2

Schematic representations of BRCA1/2 proteins.BRCA1/2 proteins with their functional domains and the localization of all identified pathogenic mutations (red labels) and VUSs (black labels). Novel alterations are marked with an asterisk. The frequency of each alteration is represented by gray dots. The red and green bars represent the detected genomic rearrangements. The black bar represents the ATM, CDH1 and RAD50 binding domain.

Figure 3

BRCA1/2 mutation frequency. Mutation frequency according to age of cancer onset.

Figure 4

Array CGH and Duplex qPCR. Validation of three selected DNA copy number alterations detected using array CGH in HBOC patients. A: Chromosome 11 array CGH profile (lower panel) of a cancer patient (SM-46); the vertical blue bar indicates the affected genomic region, which is enlarged in the upper panel showing a deletion in ATM exon 4 (upper panel). B: Duplex qPCR for the ATM exon 4 and a reference gene; the ratio between the patient and control peaks of the melting curve was 0.63, which confirmed a one-copy deletion. C: Chromosome 10 array CGH profile (lower panel) of a cancer patient (SM-62) showing a deletion in PTEN exon 2 (upper panel); the blue vertical bar in the chromosome indicates the gene region, which is enlarged in the upper panel. D: Duplex qPCR for the PTEN exon 2 and reference gene; the ratio between the patient and control peaks of the melting curve was 0.64, confirming the one-copy deletion. Coordinates of the enlarged gene region are given according to the UCSC Feb. 2009 (GRCh37-Hg19) assembly.