5' Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints

Abstract

Background: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5' region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families.

Methods: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing.

Results: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin.

Conclusions: Taken together, our results firmly establish that the BRCA1 5' region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

Keywords: BRCA1 gene; large duplication; large genomic rearrangement; triplication; variant of unknown significance.

Figure 1

Schema representing the 5′ region of BRCA1 gene. Description of the 5′ region of the BRCA1 gene including NBR2 gene (purple) and BRCA1 pseudogene (ΨBRCA1) (dark red). (A) The positions of the two segmental deletions shown in the gray and in black boxes are the homologous regions with more than 80% identity. (B) Alu sequences are indicated as gray line.

Figure 2

Schema of the different LGR detected in French families. (A) This diagram gives the description of Table 2 large deletions detected in the 5′ region described in 14 families. Green lines represent deletions detected in the French families (F1 to 14) or previously reported in the literature (Lit1 to 6); all segments are represented proportionally. At top, the Alu sequences (AluSx, AluSg, AluY) were also positioned precisely to identify specific breakpoints. At bottom, the NBR2 gene is in purple, BRCA1 and pseudoBRCA1 in brown and segmental duplication in black. (B) Focus on the 5′ region of BRCA1 including exon 2, non-coding exons 1a and 1b, the AluSg position, bidirectional promoter, and first exon of the NBR2 gene in black, BRCA1 introns in grey and NBR2 introns in purple.

Figure 3

Molecular combing analysis of BRCA1 5′ region. Molecular combing was performed using high-resolution BRCA1 Genomic Morse Codes, as previously published [32]. The complete BRCA1 GMC covers a genomic region of 200 kb and is composed of 9 probes (S1–S7) of a distinct color (green, red, or blue). Triplication of exons 1 and 2 and part of the intron 2 of BRCA1 gene is visible as a tandem repeat triplication of the red and green signal of S7 and SYNT1 probes. Data are representative of the analyses performed on DNA from families F15, F16, F17, and F18. The sizes are estimated with this technology and are not defined precisely contrary to Sanger sequencing.

Figure 4

Sanger sequences analysis of three different 5–10 kb deletions with very close breakpoints harbored by individuals from five unrelated families. In red circles are nucleotides specific to the BRCA1 and NBR2 genes. The black vertical line is the breakpoint position and the horizontal line with arrows is a common region in which there is a breakpoint.

Figure 5

Structural organization of BRCA1 5′ region. (A) Proposed structural interaction to explain the non-allelic homologous recombination which leads to the 36,934 bp deletion reported here (in F1 and F5 families) and in the literature (Lit1, Lit2). BRCA1 and pseudoBRCA1 are represented in brown and NBR2 in purple. Under the genes, blue and grey boxes represent segmental duplications, with the blue box presenting more than 80% similarity. There is likely a chromatin loop which facilitates the recombination by bringing the two loci close together. (B) Analysis of the similarity of ΨBRCA1 and BRCA1 gene in the region with the reported breakpoints—chr17:43155400–43157973 and chr17:43118270–43121226. (1) Breakpoints identified in F2, F3, F4 and their distance to the segmental duplication with high homology (blue array); (2) breakpoints of F1, F5, Lit1 and Lit2.