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cDNA analysis demonstrates that the BRCA2 intronic variant IVS4-12del5 is a deleterious mutation

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Abstract

Mutation screening of the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice. Classification of rare non-truncating sequence variants in the BRCA1 and BRCA2 genes is problematic because it is not known whether these subtle changes alter function sufficiently to predispose cells to cancer development. Several studies have reported the biochemical analysis of BRCA2 variants, which disrupted the 5′ and 3′splicing consensus elements (the GU-AG rule). However, little has been done to look into the consequences of variants located outside the 5′ and 3′ consensus splice sites. cDNA analysis demonstrates that the BRCA2*IVS4-12del5 splice site variant results in the deletion of exon 5, and the gene putatively produces a truncated BRCA2 protein of 164 amino acids instead of 3418 with the incorporation of 22 out of frame amino acids. The pattern of breast, melanoma, and pancreatic cancers in the paternal kindred is consistent with autosomal dominant inheritance of a deleterious BRCA2 mutation. Analysis of a tumor specimen indicates loss of heterozygosity (LOH). Sequence alignment indicates the deleted region is well conserved across different species. These results support the conclusion that BRCA2 IVS4-12del5 is a deleterious mutation. This study will shed light on the reclassification of intronic variants that do not disrupt the 5′ and 3′ splice sites (the GU-AG rule).

Introduction

Mutation screening of the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 is becoming an increasingly important part of clinical practice [1]. Whereas many disease-causing mutations are associated with increased risk of cancer, the contribution of the sequence variants to cancer risk remains largely undefined. Interpretation of these “variants of unknown significance” (VUSs) is problematic because it is not known whether these changes alter the function of the proteins sufficiently to predispose to cancer. As a result, carriers of VUSs and their family members cannot take advantage of the risk assessment, prevention, and therapeutic measures that are available to carriers of known deleterious mutations [2], [3]. This ambiguity is now also potentially problematic for individuals with late stage cancers who might benefit from investigational chemotherapies targeted to mutation carriers.

Pre-mRNA splicing is an essential step in gene expression. The importance of accurate splicing is illustrated by the fact that about 15–50% of human genetic diseases arise from mutations that affect normal mRNA splicing. These mutations reside either in consensus splice site sequences or in the more variable auxiliary elements [4]. More than 100 intronic variants with unknown clinical significance are reported throughout BRCA2 in the BIC database (Breast Cancer Information Core, http://research.nhgri.nih.gov/bic/). These intronic variants may interfere with RNA processing and result in non-functional BRCA2 protein. Several studies have reported the biochemical analysis of BRCA1 and BRCA2 intronic variants, which disrupt the 5′ and 3′splicing consensus elements (the GU-AG rule) [5], [6], [7]. However, the consequences of variants located outside the 5′ and 3′ consensus splice sites have not been well examined.

The location of the deleted nucleotides (−12 to −8) in the BRCA2 IVS4-12del5 variant is upstream from the 3′ end of the intron 4, and therefore would not seem to disrupt the 3′ splice site. This variant has been reported once in the literature [8], as well as in the BIC Mutation Database and in several unrelated families by Myriad Genetics Laboratories. The IVS4-12del5 variant has not been tested for co-segregation with breast cancer. Therefore, the clinical significance of this intronic variant is unknown. Our cDNA analysis demonstrates that the BRCA2 IVS4-12del5 variant is a deleterious mutation as it results in the deletion of exon 5.

Section snippets

Subject

The patient described here is a 48-year-old female who was diagnosed with breast cancer at age 44. Her paternal history includes her father's diagnosis of melanoma at age 43, her grandfather's diagnosis of pancreatic cancer at age 72, her aunt's diagnosis of breast cancer in her 70s, and a first cousin's diagnosis of breast cancer at age 45 (Fig. 1). The genetic variant BRCA2 IVS4-12del5 was identified through clinical full-sequence analysis of BRCA1 and BRCA2 (BRACAnalysis, Myriad Genetic

Analysis of RNA transcripts

The effect of IVS4-12del5 on RNA splicing was evaluated by amplifying regions of BRCA2 from cDNA derived from the patient. PCR was designed to generate fragments that contained exons 3–7, which are most likely affected by this variant. We obtained one single PCR product from the 14 control cDNAs (lanes 1–14, Fig. 2C). However, in the patient sample, an additional band unique to the patient was detected (pt, Fig. 2C). This fragment represents an aberrant RNA splicing product attributable to

Discussion

The positions and sequences of the consensus cis-acting elements help to define the splice sites of a typical intron. The consensus elements include the 5′ splice site, the branch point, the polypyrimidine tract, the 3′ splice site, and exon splicing enhancers (ESEs) if present. All consensus elements are recognized; U1 snRNP interacts with the 5′ splice site, SF1 (splicing factor 1) binds the branch point, U2AF (U2 auxiliary factor, a dimer of 65 and 35 kDa subunits), binds the polypyrimidine

Conflict of interest

None of the authors have any financial interests or other relationships of a commercial nature that they believe could be construed as resulting in an actual, potential, or apparent conflict of interest with regard to the manuscript submitted for review.

Acknowledgments

We thank Justyna Sadowska and Lishi Chen for their technical assistance in this project.

Partially supported by the Niehaus foundation and the Breast Cancer Research Foundation.

References (11)

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