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Germline mutation in the RAD51B gene confers predisposition to breast cancer.

Golmard L, Caux-Moncoutier V, Davy G, Al Ageeli E, Poirot B, Tirapo C, Michaux D, Barbaroux C, d'Enghien CD, Nicolas A, Castéra L, Sastre-Garau X, Stern MH, Houdayer C, Stoppa-Lyonnet D - BMC Cancer (2013)

Bottom Line: Detected variants were characterized by Sanger sequencing analysis.Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys.No RAD51D and XRCC2 gene mutations were detected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Curie, Department of Tumour Biology, 26 rue d'Ulm, 75248, Paris, Cedex 05, France. lisa.golmard@curie.net.

ABSTRACT

Background: Most currently known breast cancer predisposition genes play a role in DNA repair by homologous recombination. Recent studies conducted on RAD51 paralogs, involved in the same DNA repair pathway, have identified rare germline mutations conferring breast and/or ovarian cancer predisposition in the RAD51C, RAD51D and XRCC2 genes. The present study analysed the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) to estimate their contribution to breast and ovarian cancer predisposition.

Methods: The study was conducted on 142 unrelated patients with breast and/or ovarian cancer either with early onset or with a breast/ovarian cancer family history. Patients were referred to a French family cancer clinic and had been previously tested negative for a BRCA1/2 mutation. Coding sequences of the five genes were analysed by EMMA (Enhanced Mismatch Mutation Analysis). Detected variants were characterized by Sanger sequencing analysis.

Results: Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys. No RAD51D and XRCC2 gene mutations were detected. These mutations and variants were detected in families with both breast and ovarian cancers, except for the RAD51B c.475C > T/p.Arg159Cys variant that occurred in a family with 3 breast cancer cases.

Conclusions: This study identified the first RAD51B mutation in a breast and ovarian cancer family and is the first report of XRCC3 mutation analysis in breast and ovarian cancer. It confirms that RAD51 paralog mutations confer breast and ovarian cancer predisposition and are rare events. In view of the low frequency of RAD51 paralog mutations, international collaboration of family cancer clinics will be required to more accurately estimate their penetrance and establish clinical guidelines in carrier individuals.

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mRNA analysis for RAD51C splicing mutations showing exon skipping. (A) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.706-2A > G mutation with two types of mRNA: wild type mRNA and mRNA with exon 5 skipping (right). (B) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.1026 + 5_1026 + 7del mutation with two types of mRNA: wild type mRNA and mRNA with exon 8 skipping (right).
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Figure 1: mRNA analysis for RAD51C splicing mutations showing exon skipping. (A) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.706-2A > G mutation with two types of mRNA: wild type mRNA and mRNA with exon 5 skipping (right). (B) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.1026 + 5_1026 + 7del mutation with two types of mRNA: wild type mRNA and mRNA with exon 8 skipping (right).

Mentions: All variants detected on DNA were tested by in silico splicing effect prediction according to a previously published pipeline [30]: a greater than 15% decrease of the MaxEntScan score for donor/acceptor splice sites and a greater than 5% decrease of the SpliceSiteFinder-like score for donor/acceptor splice sites were considered to be significant with 96% sensitivity and 83% specificity. Three variants were likely to alter splicing according to this pipeline: RAD51C c.706-2A > G and c.1026 + 5_1026 + 7del, and RAD51B c.452 + 3A > G. Exon skipping was confirmed by mRNA analysis for the two RAD51C mutations (Figure 1). No RNA was available to study the impact of the RAD51B c.452 + 3A > G mutation but, using immunohistochemistry with anti-RAD51B antibody, loss of expression of RAD51B protein was observed in breast carcinoma cells from the patient bearing this mutation, as compared with that detected in the nucleus of normal duct cells (Figure 2).


Germline mutation in the RAD51B gene confers predisposition to breast cancer.

Golmard L, Caux-Moncoutier V, Davy G, Al Ageeli E, Poirot B, Tirapo C, Michaux D, Barbaroux C, d'Enghien CD, Nicolas A, Castéra L, Sastre-Garau X, Stern MH, Houdayer C, Stoppa-Lyonnet D - BMC Cancer (2013)

mRNA analysis for RAD51C splicing mutations showing exon skipping. (A) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.706-2A > G mutation with two types of mRNA: wild type mRNA and mRNA with exon 5 skipping (right). (B) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.1026 + 5_1026 + 7del mutation with two types of mRNA: wild type mRNA and mRNA with exon 8 skipping (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016303&req=5

Figure 1: mRNA analysis for RAD51C splicing mutations showing exon skipping. (A) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.706-2A > G mutation with two types of mRNA: wild type mRNA and mRNA with exon 5 skipping (right). (B) Electropherograms of Sanger sequencing analysis for a control sample with wild type RAD51C mRNA only (left) and for RAD51C c.1026 + 5_1026 + 7del mutation with two types of mRNA: wild type mRNA and mRNA with exon 8 skipping (right).
Mentions: All variants detected on DNA were tested by in silico splicing effect prediction according to a previously published pipeline [30]: a greater than 15% decrease of the MaxEntScan score for donor/acceptor splice sites and a greater than 5% decrease of the SpliceSiteFinder-like score for donor/acceptor splice sites were considered to be significant with 96% sensitivity and 83% specificity. Three variants were likely to alter splicing according to this pipeline: RAD51C c.706-2A > G and c.1026 + 5_1026 + 7del, and RAD51B c.452 + 3A > G. Exon skipping was confirmed by mRNA analysis for the two RAD51C mutations (Figure 1). No RNA was available to study the impact of the RAD51B c.452 + 3A > G mutation but, using immunohistochemistry with anti-RAD51B antibody, loss of expression of RAD51B protein was observed in breast carcinoma cells from the patient bearing this mutation, as compared with that detected in the nucleus of normal duct cells (Figure 2).

Bottom Line: Detected variants were characterized by Sanger sequencing analysis.Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys.No RAD51D and XRCC2 gene mutations were detected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Curie, Department of Tumour Biology, 26 rue d'Ulm, 75248, Paris, Cedex 05, France. lisa.golmard@curie.net.

ABSTRACT

Background: Most currently known breast cancer predisposition genes play a role in DNA repair by homologous recombination. Recent studies conducted on RAD51 paralogs, involved in the same DNA repair pathway, have identified rare germline mutations conferring breast and/or ovarian cancer predisposition in the RAD51C, RAD51D and XRCC2 genes. The present study analysed the five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3) to estimate their contribution to breast and ovarian cancer predisposition.

Methods: The study was conducted on 142 unrelated patients with breast and/or ovarian cancer either with early onset or with a breast/ovarian cancer family history. Patients were referred to a French family cancer clinic and had been previously tested negative for a BRCA1/2 mutation. Coding sequences of the five genes were analysed by EMMA (Enhanced Mismatch Mutation Analysis). Detected variants were characterized by Sanger sequencing analysis.

Results: Three splicing mutations and two likely deleterious missense variants were identified: RAD51B c.452 + 3A > G, RAD51C c.706-2A > G, RAD51C c.1026 + 5_1026 + 7del, RAD51B c.475C > T/p.Arg159Cys and XRCC3 c.448C > T/p.Arg150Cys. No RAD51D and XRCC2 gene mutations were detected. These mutations and variants were detected in families with both breast and ovarian cancers, except for the RAD51B c.475C > T/p.Arg159Cys variant that occurred in a family with 3 breast cancer cases.

Conclusions: This study identified the first RAD51B mutation in a breast and ovarian cancer family and is the first report of XRCC3 mutation analysis in breast and ovarian cancer. It confirms that RAD51 paralog mutations confer breast and ovarian cancer predisposition and are rare events. In view of the low frequency of RAD51 paralog mutations, international collaboration of family cancer clinics will be required to more accurately estimate their penetrance and establish clinical guidelines in carrier individuals.

Show MeSH
Related in: MedlinePlus