J Breast Cancer.  2017 Sep;20(3):310-313. 10.4048/jbc.2017.20.3.310.

A Novel Germline Mutation in BRCA1 Causes Exon 20 Skipping in a Korean Family with a History of Breast Cancer

• Yoon KA
• Kong SY
• Lee EJ
• Cho JN
• Chang S
• Lee ES

• KMID: 2438999
• DOI: http://doi.org/10.4048/jbc.2017.20.3.310

Abstract

Germline mutations in the BRCA1 and BRCA2 genes are strong genetic factors for predispositions to breast, ovarian, and other related cancers. This report describes a family with a history of breast and ovarian cancers that harbored a novel BRCA1 germline mutation. A single nucleotide deletion in intron 20, namely c.5332+4delA, was detected in a 43-year-old patient with breast cancer. This mutation led to the skipping of exon 20, which in turn resulted in the production of a truncated BRCA1 protein that was 1773 amino acids in length. The mother of the proband had died due to ovarian cancer and had harbored the same germline mutation. Ectopically expressed mutant BRCA1 protein interacted with the BARD1 protein, but showed a reduced transcriptional function, as demonstrated by the expression of cyclin B1. This novel germline mutation in the BRCA1 gene caused familial breast and ovarian cancers.

Keyword

BRCA1 protein; Breast neoplasms; Germ-line mutation

MeSH Terms

Adult
Amino Acids
BRCA1 Protein
Breast Neoplasms*
Breast*
Cyclin B1
Exons*
Genes, BRCA1
Genes, BRCA2
Germ-Line Mutation*
Humans
Introns
Mothers
Ovarian Neoplasms
Amino Acids
BRCA1 Protein
Cyclin B1

Figure

• 

  Figure 1 Pedigree of the patient's family, showing multiple individuals with breast and ovarian cancer. The proband (arrow) and her family members harboring the mutation (c.5332+4delA) in BRCA1 gene are marked in red. Male is represented by a square and female by a circle. Numbers in parentheses indicate age at cancer diagnosis.

• 

  Figure 2 Mutation analysis of exon 20 in BRCA1 gene. (A) Sequence analysis of the mutant BRCA1 gene shows a deletion in c.5332+4delA (genomic DNA) and exon 20 skipping (RNA). Exon 20 sequences are colored in yellow. (B) The schematic view shows the variant localization and the mutated mRNA skipping 55 bp of exon 20.

• 

  Figure 3 Functional analyses of mutant BRCA1 protein. (A) Mutant BRCA1 protein (exon 20 deletion) showed reduced protein level. BRCA1-deficient MDA-MB-436 cells were transfected with a vector expressing HA-tagged BRCA1 wild-type (WT) or exon20 skipping mutant or untagged mutant BRCA1 (exon 20 deletion) mRNA. A point mutation of BRCA1 that causes an amino acid substitution of leucine to phenylalanine (L52F) was used as a missense mutant control. After 24 hours, the cells were treated with DMSO (control) or MG132 (1 µM). The arrows indicate BRCA1 protein and the asterisk indicates a nonspecific band. (B) WT or mutant BRCA1 is immunoprecipitated with anti-BARD1 antibody. Immunoprecipitated WT and mutant BRCA1 were detected by Western blotting with an anti-BRCA1 antibody (boxed lane). (C) Expression level of cyclin B1 was compared among cells transfected with WT or mutant BRCA1. Relative expression of cyclin B1 was measured by real-time reverse transcription polymerase chain reaction compared to glyceraldehyde 3-phosphate dehydrogenase after exogenous expression of WT or mutant BRCA1. DMSO=dimethyl sulfoxide; Ex20del=exon 20 deletion; BARD1=BRCA1 associated RING domain1; GFP=green fluoresence protein (control DNA); IP=immunoprecipitation; IB=immunoblotting. *Nonspecific band.

Reference

1. Garber JE, Offit K. Hereditary cancer predisposition syndromes. J Clin Oncol. 2005; 23:276–292.

2. Lux MP, Fasching PA, Beckmann MW. Hereditary breast and ovarian cancer: review and future perspectives. J Mol Med (Berl). 2006; 84:16–28.

3. Hall MJ, Reid JE, Burbidge LA, Pruss D, Deffenbaugh AM, Frye C, et al. BRCA1 and BRCA2 mutations in women of different ethnicities undergoing testing for hereditary breast-ovarian cancer. Cancer. 2009; 115:2222–2233.

4. Narod SA. Modifiers of risk of hereditary breast cancer. Oncogene. 2006; 25:5832–5836.

5. Couch FJ, DeShano ML, Blackwood MA, Calzone K, Stopfer J, Campeau L, et al. BRCA1 mutations in women attending clinics that evaluate the risk of breast cancer. N Engl J Med. 1997; 336:1409–1415.

6. Ford D, Easton DF, Bishop DT, Narod SA, Goldgar DE. Risks of cancer in BRCA1-mutation carriers. Breast Cancer Linkage Consortium. Lancet. 1994; 343:692–695.

7. Chen S, Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. J Clin Oncol. 2007; 25:1329–1333.

8. Langston AA, Malone KE, Thompson JD, Daling JR, Ostrander EA. BRCA1 mutations in a population-based sample of young women with breast cancer. N Engl J Med. 1996; 334:137–142.

9. Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet. 2002; 3:285–298.

10. Houdayer C, Caux-Moncoutier V, Krieger S, Barrois M, Bonnet F, Bourdon V, et al. Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants. Hum Mutat. 2012; 33:1228–1238.

11. 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; 27:55–58.

12. Chang S, Biswas K, Martin BK, Stauffer S, Sharan SK. Expression of human BRCA1 variants in mouse ES cells allows functional analysis of BRCA1 mutations. J Clin Invest. 2009; 119:3160–3171.

13. Coquelle N, Green R, Glover JN. Impact of BRCA1 BRCT domain missense substitutions on phosphopeptide recognition. Biochemistry. 2011; 50:4579–4589.

14. Del Valle J, Campos O, Velasco A, Darder E, Menéndez M, Feliubadaló L, et al. Identification of a new complex rearrangement affecting exon 20 of BRCA1. Breast Cancer Res Treat. 2011; 130:341–344.

15. MacLachlan TK, Somasundaram K, Sgagias M, Shifman Y, Muschel RJ, Cowan KH, et al. BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression. J Biol Chem. 2000; 275:2777–2785.