Received on June 16, 2009
Accepted on October 8, 2009

Molecular Diagnostics and Genetics

Alternative Splicing and Molecular Characterization of Splice Site Variants: BRCA1 c.591C>T as a Case Study

¹ Laboratorio de Oncología Molecular, Hospital Clínico San Carlos, Madrid, Spain
² Laboratorio de Oncología Molecular, and Servicio de Oncología Médica, Hospital Clínico San Carlos, Madrid, Spain

^* To whom correspondence should be addressed. E-mail: mdhoya{at}hotmail.com.

BACKGROUND: Deleterious mutations in BRCA1 (breast cancer 1, early onset; MIM 113705) increase human breast and ovarian cancer [HB(O)C] risk; however, many variants cannot be readily classified as deleterious or neutral. Unclassified variants (UVs) pose serious problems in genetic counseling. RNA-splicing analysis is essential for the assessment of many UVs.

METHODS: Denaturing gradient gel electrophoresis was used to genotype the BRCA1 c.591C>T variant in 685 index cases of HB(O)C families, 326 sporadic breast cancer cases, and 450 healthy controls from Spain. In silico tools were used to predict the effect of the c.591C>T variant on splicing. In vitro splicing analysis was performed in 7 c.591C>T carriers and 10 noncarriers. cDNAs were PCR-amplified with primers designed to detect BRCA1 alternative splicing isoforms. The products were analyzed by capillary electrophoresis. Peak areas were used to quantify the relative abundance of each isoform. Sequencing through exonic single-nucleotide polymorphisms (SNPs) enabled us to discriminate wild-type and variant transcripts.

RESULTS: c.591C>T was detected in HB(O)C cases (1.5%), breast cancer cases (0.3%), and controls (0.9%). c.591C>T induced BRCA1 exon 9 skipping and modified the relative expression of (9,10), (9,10,11B), 11B, and full-length isoforms. The mean ratio of (9,10) to the full-length isoform increased from 0.25 in noncarriers to 1.5 in carriers. The mean (9,10,11B)/11B ratio increased from 0.2 to 4. Overall expression levels of c.591C>T and wild-type alleles were similar.

CONCLUSIONS: Our data support a nonpathogenic role for the BRCA1 c.591C>T variant. Naturally occurring alternative splicing isoforms need to be considered when assessing the role of BRCA1 UVs on splicing.