Alternatively spliced, truncated human BRCA2 isoforms contain a novel coding exon

Abstract

The protein truncation test (PTT) employs in vitro transcription and translation of amplified cDNA and exonic gDNA to reveal truncating germ-line mutations. In a series of PTT analyses, abnormal splicing in the region encompassing exons 20-23 of BRCA2 was discovered in leucocytes from high-risk breast cancer patients. Although sequencing of the genomic DNA in this region failed to reveal a detectable mutation in these patients, cDNA obtained from this region of BRCA2 uncovered numerous alternative splice isoforms. PTT analysis and nested RT-PCR using RNA from leucocytes from healthy individuals, normal tissue and breast and ovarian cancer tumours demonstrated the presence of these alternatively spliced transcripts in all cases. The splice forms appeared to be more prominent in RNA from aged blood, suggesting that isoform expression was conditional. It is therefore important to distinguish naturally occurring alternative splicing from true splice defects due to mutations when interpreting PTT results.

Characterisation of unclassified variants in the BRCA1/2 genes with a putative effect on splicing

“…So far, 18 BRCA1 ( [36] and references therein) and 8 BRCA2 [15,[37][38][39] transcript isoforms of unknown function have been identified both in normal and tumour tissues. However, little is known about their critical expression levels, expression variation among controls and their functions.…”

Prediction and assessment of splicing alterations: implications for clinical testing

“…Such a scenario would require additional in vitro quantitative expression studies. Since a number of naturally-occurring splice products are known to be produced for most genes, and since the type and amount of alternative/aberrant splice products can differ depending on cell type and assay conditions [Lastella et al, 2006;Speevak et al, 2003], it is essential to include a large number of controls assessed under similar assay conditions to compare the expression of ''aberrant'' splice products against the quantity of naturally-occurring splice products. Ideally, samples from more than one variant carrier should also be tested.…”

“…Another less tractable issue is whether in vitro results generated from assays on cultured or even uncultured lymphocyte cells can be generalized to the target tissue in question [Claes et al, 2002;Speevak et al, 2003]. This is particularly important for subtle variation in expression of an alternative splice product, which may be due in part to tissue-specific variation in splicing due to tissuespecific expression of splicing factors.…”

Caution should be taken in the methodology used to confirm c.156_157insAlu BRCA2 mutation

“…They were very well aware of previous data showing a physiological BRCA2 transcript lacking exon 3 [5][6][7][8]. Their questions allowed us, in a reply [9], to explain that indeed, we had tried several primers for our RT-PCR confirmation step and eventually chose primers 1FcDNA/ 10RcDNA, first described by Nordling et al [10], because these never amplify the ubiquitous splicing transcript.…”

“…Peixoto et al report RT-PCR data in their paper, that corroborates our own data concerning the segregation of c.156_157insAlu BRCA2 mutation and cancer in individuals from positive families, but this should not be taken as the most appropriate methodology for the confirmation of this rearrangement. The primers used for their RT-PCR reactions amplify not only the pathogenic Alu-mediated splicing product, lacking exon 3, but also a physiologic, ubiquitous splicing product, also exon 3-deficient, observed in individuals with sporadic cancers and healthy persons [4][5][6][7][8]. Although both splicing products lack exon 3, only the one associated with c.156_157insAlu mutation includes at least the first part of BRCA2 exon 10 and this distinguishes them [9].…”