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Open Access 01-12-2010 | Research article

Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2transcripts

Authors: Phillip J Whiley, Christopher A Pettigrew, Brooke L Brewster, Logan C Walker, Amanda B Spurdle, Melissa A Brown, kConFab Investigators

Published in: BMC Medical Genetics | Issue 1/2010

Abstract

Background

Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge.

Methods

This study used a combination of RT-PCR analysis and splicing reporter minigene assays to assess five unclassified variants in the BRCA2 gene that we had previously predicted to disrupt an ESE using bioinformatic approaches.

Results

Analysis of BRCA2 c.8308 G > A (p.Ala2770Thr) by mRNA analysis, and BRCA2 c.8962A > G (p.Ser2988Gly), BRCA2 c.8972G > A (p.Arg2991His), BRCA2 c.9172A > G (p.Ser3058Gly), and BRCA2 c.9213G > T (p.Glu3071Asp) by a minigene assay, revealed no evidence for aberrant splicing.

Conclusions

These results illustrate the need for improved methods for predicting functional ESEs and the potential consequences of sequence variants contained therein.

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Spurdle AB, Couch FJ, Hogervorst FB, Radice P, Sinilnikova OM: Prediction and assessment of splicing alterations: implications for clinical testing. Hum Mutat. 2008, 29: 1304-13. 10.1002/humu.20901.CrossRefPubMedPubMedCentral

Yang Y, Swaminathan S, Martin BK, Sharan SK: Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model. Hum Mol Genet. 2003, 12: 2121-2131. 10.1093/hmg/ddg222.CrossRefPubMed

Liu HX, Cartegni L, Zhang MQ, Krainer AR: A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes. Nat Genet. 2001, 27: 55-8.PubMed

Fackenthal JD, Cartegni L, Krainer AR, Olopade OI: BRCA2 T2722R is a deleterious allele that causes exon skipping. American Journal Of Human Genetics. 2002, 71: 625-631. 10.1086/342192.CrossRefPubMedPubMedCentral

Farrugia DJ, Agarwal MK, Pankratz VS, Deffenbaugh AM, Pruss D, Frye C, Wadum L, Johnson K, Mentlick J, Tavtigian SV, Goldgar DE, Couch FJ: Functional assays for classification of BRCA2 variants of uncertain significance. Cancer Res. 2008, 68: 3523-31. 10.1158/0008-5472.CAN-07-1587.CrossRefPubMedPubMedCentral

Millevoi S, Bernat S, Telly D, Fouque F, Gladieff L, Favre G, Vagner S, Toulas C: The c.5242C > A BRCA1 missense variant induces exon skipping by increasing splicing repressors binding. Breast Cancer Res Treat. 2009

Cartegni L, Chew SL, Krainer AR: Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nature Reviews Genetics. 2002, 3: 285-298. 10.1038/nrg775.CrossRefPubMed

Pettigrew CA, Wayte N, Lovelock PK, Tavtigian SV, Chenevix-Trench G, Spurdle AB, Brown MA: Evolutionary conservation analysis increases the colocalization of predicted exonic splicing enhancers in the BRCA1 gene with missense sequence changes and in-frame deletions, but not polymorphisms. Breast Cancer Research: BCR. 2005, 7: R929-39. 10.1186/bcr1324.CrossRefPubMedPubMedCentral

Gorlov IP, Gorlova OY, Frazier ML, Amos CI: Missense mutations in cancer suppressor gene TP53 are colocalized with exonic splicing enhancers (ESEs). Mutat Res. 2004, 554: 175-83.CrossRefPubMed

10.

Pettigrew CA, Wayte N, Wronski A, Lovelock PK, Spurdle AB, Brown MA: Colocalisation of predicted exonic splicing enhancers in BRCA2 with reported sequence variants. Breast Cancer Res Treat. 2008, 110: 227-34. 10.1007/s10549-007-9714-5.CrossRefPubMed

11.

den Dunnen JT, Antonarakis SE: Nomenclature for the description of human sequence variations. Hum Genet. 2001, 109: 121-4. 10.1007/s004390100505.CrossRefPubMed

12.

Lastella P, Surdo NC, Resta N, Guanti G, Stella A: In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects. BMC Genomics. 2006, 7: 243-10.1186/1471-2164-7-243.CrossRefPubMedPubMedCentral

13.

Anczukow O, Buisson M, Salles MJ, Triboulet S, Longy M, Lidereau R, Sinilnikova OM, Mazoyer S: Unclassified variants identified in BRCA1 exon 11: Consequences on splicing. Genes Chromosomes Cancer. 2008, 47: 418-26. 10.1002/gcc.20546.CrossRefPubMed

14.

Bonnet C, Krieger S, Vezain M, Rousselin A, Tournier I, Martins A, Berthet P, Chevrier A, Dugast C, Layet V, Rossi A, Lidereau R, Frebourg T, Hardouin A, Tosi M: Screening BRCA1 and BRCA2 unclassified variants for splicing mutations using reverse transcription PCR on patient RNA and an ex vivo assay based on a splicing reporter minigene. J Med Genet. 2008, 45: 438-46. 10.1136/jmg.2007.056895.CrossRefPubMed

15.

Brown MA, Jones KA, Nicolai H, Bonjardim M, Black D, McFarlane R, de Jong P, Quirk JP, Lehrach H, Solomon E: Physical mapping, cloning, and identification of genes within a 500-kb region containing BRCA1. Proc Natl Acad Sci USA. 1995, 92: 4362-6. 10.1073/pnas.92.10.4362.CrossRefPubMedPubMedCentral

16.

Bonatti F, Pepe C, Tancredi M, Lombardi G, Aretini P, Sensi E, Falaschi E, Cipollini G, Bevilacqua G, Caligo MA: RNA-based analysis of BRCA1 and BRCA2 gene alterations. Cancer Genetics And Cytogenetics. 2006, 170: 93-101. 10.1016/j.cancergencyto.2006.05.005.CrossRefPubMed

17.

Tesoriero AA, Wong EM, Jenkins MA, Hopper JL, Brown MA, Chenevix-Trench G, Spurdle AB, Southey MC: Molecular characterization and cancer risk associated with BRCA1 and BRCA2 splice site variants identified in multiple-case breast cancer families. Hum Mutat. 2005, 26: 495-495. 10.1002/humu.9379.CrossRefPubMed

18.

Burn TC, Connors TD, Klinger KW, Landes GM: Increased exon-trapping efficiency through modifications to the pSPL3 splicing vector. Gene. 1995, 161: 183-7. 10.1016/0378-1119(95)00223-S.CrossRefPubMed

19.

Caux-Moncoutier V, Pages-Berhouet S, Michaux D, Asselain B, Castera L, De Pauw A, Buecher B, Gauthier-Villars M, Stoppa-Lyonnet D, Houdayer C: Impact of BRCA1 and BRCA2 variants on splicing: clues from an allelic imbalance study. Eur J Hum Genet. 2009

20.

Fairbrother WG, Yeh RF, Sharp PA, Burge CB: Predictive identification of exonic splicing enhancers in human genes. Science. 2002, 297: 1007-13. 10.1126/science.1073774.CrossRefPubMed

21.

Zatkova A, Messiaen L, Vandenbroucke I, Wieser R, Fonatsch C, Krainer AR, Wimmer K: Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1. Hum Mutat. 2004, 24: 491-501. 10.1002/humu.20103.CrossRefPubMed

The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/80/prepub

Title: Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2transcripts
Authors: Phillip J Whiley
Christopher A Pettigrew
Brooke L Brewster
Logan C Walker
Amanda B Spurdle
Melissa A Brown
kConFab Investigators
Publication date: 01-12-2010
Publisher: BioMed Central
Published in: BMC Medical Genetics / Issue 1/2010
Electronic ISSN: 1471-2350
DOI: https://doi.org/10.1186/1471-2350-11-80

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Effect of BRCA2 sequence variants predicted to disrupt exonic splice enhancers on BRCA2transcripts

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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