Short Communication
BRCA1 promoter deletions in young women with breast cancer and a strong family history: A population-based study

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Abstract

Women diagnosed with breast cancer before the age of 40 years who have a strong family history of breast and/or ovarian cancer were selected from an Australian population-based case-control-family study for large deletion screening within the BRCA1 promoter. Deletions within the BRCA1 promoter region are usually not detected by the methods applied in routine clinical mutation detection strategies. Fifty-one of the 66 women (77%) who met our inclusion criteria were tested for promoter deletions using linkage disequilibrium analysis of two BRCA1 polymorphic sites (C/G1802 and Pro871Leu) and multiplex ligation-dependent probe amplification. Two cases of BRCA1 promoter deletion involving exons 1A-2 and exons 1A-23 were detected. The morphology of the breast cancers arising in these women with BRCA1 promoter deletions was consistent with the morphology associated with other germline BRCA1 mutations. Large genomic deletions that involve the promoter regions of BRCA1 make up 20% (2/10) of all known BRCA1 mutations in this group of young women with a strong family history of breast and ovarian cancer. Our data support the inclusion of testing for large genomic alterations in the BRCA1 promoter region in routine clinical mutation detection within BRCA1.

Introduction

Large genomic alterations within BRCA1 are now known to represent a non-trivial proportion of deleterious, clinically relevant, BRCA1 mutations. Early studies of genomic alterations reported several large deletions, many with evidence of founder effects (for example, Ref. 1), but the molecular methods were laborious, technically challenging and usually required a large amount of genomic DNA. More recently, Hogervorst et al.2 described a multiplex ligation-dependent probe amplification (MLPA) test to identify large genomic alterations in BRCA1. This methodology is easily applied to genomic DNA and larger scale screening for genomic alterations in BRCA1.

As it is the site of transcription initiation, a genetic mutation within the promoter could potentially disrupt normal expression of the BRCA1 protein. The 5′ end of BRCA1 lies head to head with the NBR2 gene (Next to BRCA1 gene 2) and the two genes share a bi-directional promoter that is capable of transcription in both directions. Two promoter sites, α and β, exist for BRCA1 resulting in the transcription of alternate mRNA transcripts with varying first exons (1A or 1B).3, 4 The relevance of mutation screening within the BRCA1 promoter has also been demonstrated. In a family known to be BRCA1-linked and to lack the BRCA1 transcript from the allele associated with disease susceptibility, Swensen et al.5 identified a 14 kb deletion in BRCA1 that removed exons 1A, 1B and 2. Brown et al.6 using a method described by Catteau et al.7 that utilised the strong linkage disequilibrium identified between the two BRCA1 variants, C1802G and C2731T (Pro871Leu), reported a large genomic deletion in the 5′ region of BRCA1 that included NBR2, ψBRCA1 and NBR1 in an Australian multiple-case breast cancer family.6, 7

The proportion of women with breast cancer, especially young women, whose cancers are attributable to deletions within the promoter region of BRCA1 is currently unknown. Although methods vary between laboratories and services, deletions within the BRCA1 promoter region are usually not detected by the methods applied in the majority of routine clinical mutation detection strategies. To quantitate this proportion, we identified 66 women from our Australian population-based case-control-family study of breast cancer who were diagnosed with breast cancer before the age of 40 years and had a strong family history of breast and/or ovarian cancer (two or more first- or second-degree relatives affected with breast and/or ovarian cancer). Previous mutation screening performed on the germline DNA of these 66 cases had included screening within BRCA1, BRCA2 and ATM using a variety of mutation detection techniques.8, 9 This identified that 15 (23%) of these 66 cases have detectable BRCA1 (n = 8), BRCA2 (n = 6) or specific ATM (n = 1)9 mutations. The study was approved by the ethics committees of The University of Melbourne and The Cancer Council Victoria.

Section snippets

The Australian breast cancer family registry

The Australian breast cancer family registry (ABCFR) includes a population-based, case-control-family study of breast cancer (in which cases, control subjects and their relatives were administered the same questionnaire) with an emphasis on early-onset disease that was carried out in Melbourne and Sydney, Australia.10, 11, 12 Sixty-six women were identified within the ABCFR to be diagnosed with breast cancer before the age of 40 years and to have a strong family history of breast and/or ovarian

Promoter deletion screening

Genotyping for BRCA1 variants, C1802G (β-promoter, GenBank Accession No. U37574), and C2731T (Pro871Leu, exon 11, U14680), was performed for 51/66 women who met our criteria (Table 1) using the method described by Catteau et al.7

Multiplex ligation-dependent probe amplification (MLPA)

MLPA was carried out on 125 ng of DNA using the SALSA P002-BRCA1 Exon Copy Number Test Kit (MRC-Holland) and analysed on an ABI 3730 analyzer using Genotyper software. For data analysis, peak areas of each probe were normalised by dividing each peak area by the total

Results

Genotyping for BRCA1 variants, C1802G and C2731T (Pro871Leu, exon 11) was performed for 51/66 women who met our criteria (Table 1) using the method described by Catteau et al.7 Of these tested cases, 46 (90%) were found to be in accordance with the expected linkage disequilibrium, and 28 (55%) of genotypes were uninformative for assessment of BRCA1 promoter deletions as they were homozygous at both polymorphic sites (indicated by # in Table 2). Five cases (10%) were not in linkage

Discussion

We have identified two women diagnosed with breast cancer before the age of 40 years, with a strong family history of breast cancer, who carry large genomic deletions in BRCA1 that involved the promoter region. MLPA detected both of these deletions. The BRCA1 1A-23 deletion could not be detected using the linkage disequilibrium genotypying method as both variants, in the promoter and exon 11, were hemizygous due to the deletion. The other mutation detected by MLPA was a BRCA1 1A-2 deletion.

Conflict of interest statement

None declared.

Acknowledgements

The Australian Breast Cancer Family Study was supported by the National Health and Medical Research Council of Australia, the New South Wales Cancer Council, the Victorian Health Promotion Foundation (Australia), and the U.S. National Cancer Institute, National Institutes of Health, under Request for Application CA-95-003 as part of the Breast Cancer Family Registry, and through cooperative agreements with the Fox Chase Cancer Center; Huntsman Cancer Institute, Columbia University; Northern

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