Distinct BRCA1 rearrangements involving the BRCA1 pseudogene suggest the existence of a recombination hot spot

Abstract

The 5' end of the breast and ovarian cancer-susceptibility gene BRCA1 has previously been shown to lie within a duplicated region of chromosome band 17q21. The duplicated region contains BRCA1 exons 1A, 1B, and 2 and their surrounding introns; as a result, a BRCA1 pseudogene (PsiBRCA1) lies upstream of BRCA1. However, the sequence of this segment remained essentially unknown. We needed this information to investigate at the nucleotide level the germline deletions comprising BRCA1 exons 1A, 1B, and 2, which we had previously identified in two families with breast and ovarian cancer. We have analyzed the recently deposited nucleotide sequence of the 1.0-Mb region upstream of BRCA1. We found that 14 blocks of homology between the tandemly repeated copies (cumulative length = 11.5 kb) show similarity of 77%-92%. Gaps between blocks result from insertion or deletion, usually of repetitive elements. BRCA1 exon 1A and PsiBRCA1 exon 1A are 44.5 kb apart. In the two families with breast and ovarian cancer mentioned above, distinct homologous recombination events occurred between intron 2 of BRCA1 and intron 2 of PsiBRCA1, leading to 37-kb deletions. Breakpoint junctions were found to be located at close but distinct sites within segments that are 98% identical. The mutant alleles lack the BRCA1 promoter and harbor a chimeric gene consisting of PsiBRCA1 exons 1A, 1B, and 2, which lacks the initiation codon, fused to BRCA1 exons 3-24. Thus, we report a new mutational mechanism for the BRCA1 gene. The presence of a large region homologous to BRCA1 on the same chromosome appears to constitute a hot spot for recombination.

Figure 1

Schematic representation of the duplicated regions showing the homology between NBR1 and NBR2, and between ΨBRCA1 and BRCA1. Blackened boxes represent exons, and shaded boxes represent blocks of homology, which were arbitrarily numbered. Repetitive elements, i.e., Alu, L1/L2, LTR, SVA, and MIR sequences are also shown. Block 11, which contains the breakpoints of both deletions in families F32 and F3514, is emphasized with an asterisk (*). The schema is drawn to scale.

Figure 2

BRCA1 color bar code of DNA samples from families F32 and F3514. Two allele populations could be visualized under a microscope when combed DNA samples were hybridized with fluorescent probes generated from PAC 103O14 (green) and cosmid D06121 (red) and from long-range PCR products NBR2 LR2-4 (blue), BRCA1 LR9-12 (blue), BRCA1 LR16-19 (deep pink), and BRCA1 LR24-3′ (blue). The sizes of the probes are indicated. The expected normal bar code for the wild-type allele was ΨBRCA1-NBR2-BRCA1 exons 1–2, 9–12, 16–19, and 24, as shown in A and C, for F32 and F3514, respectively. This pattern was found in 18 full signals (50%) for F32 and 26 full signals (58%) for F3514. An abnormal, shorter signal, without the 5′ part of the normal pattern, as shown in B and D, for F32 and F3514, respectively, was also found in 18 full signals (50%) for F32 and 19 full signals (42%) for F3514, corresponding to the mutant alleles with the deletion.

Figure 3

Representation of the 37-kb deletion in F32 and F3514. Drawn-to-scale schema of the normal and mutant alleles at the NBR1 and BRCA1 loci, showing the location of the 36,934-bp deletion. The size of the NBR1, ΨBRCA1, NBR2, and BRCA1 genes are given in parentheses in the normal allele schema.