A recombination-based method to characterize human BRCA1 missense variants

Guidugli, Lucia; Rugani, C.; Lombardi, Grazia; Aretini, Paolo; Galli, Alvaro; Caligo, Maria A.

doi:10.1007/s10549-010-1112-8

Cited by 6 publications

(7 citation statements)

References 32 publications

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“…In yeast cells both these mutations reverted the growth suppression (small colony) phenotype, but only M1775R induced homologous recombination [14]. In HeLa cells A1789T significantly altered the non-homologous end-joining activity as compared to BRCA1 wild-type [12]. …”

Section: Methodsmentioning

confidence: 99%

“…Five aliquots of the same clone of HeLa G1 cells were transiently transfected with the pcDNA3-BRCA1 wild-type (wt) vector, five with the pcDNA3-BRCA1-M1775R derivative vector and five with the pcDNA3-BRCA1-A1789T derivative vector as described by Guidugli et al [12]. …”

Section: Methodsmentioning

confidence: 99%

“…In a previous work we examined the expression profiles induced by these two mutations in yeast cells [11]. In a recent paper from Guidugli et al [12] these two variants were tested in a homologous recombination and a non-homologous end-joining assay in Hela cells. The A1789T variant significantly altered the non-homologous end-joining activity as compared to BRCA1 wild-type.…”

Section: Introductionmentioning

confidence: 99%

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Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

et al. 2012

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BackgroundBRCA1 (breast cancer 1, early onset) missense mutations have been detected in familial breast and ovarian cancers, but the role of these variants in cancer predisposition is often difficult to ascertain. In this work, the molecular mechanisms affected in human cells by two BRCA1 missense variants, M1775R and A1789T, both located in the second BRCT (BRCA1 C Terminus) domain, have been investigated. Both these variants were isolated from familial breast cancer patients and the study of their effect on yeast cell transcriptome has previously provided interesting clues to their possible role in the pathogenesis of breast cancer.MethodsWe compared by Human Whole Genome Microarrays the expression profiles of HeLa cells transfected with one or the other variant and HeLa cells transfected with BRCA1 wild-type. Microarray data analysis was performed by three comparisons: M1775R versus wild-type (M1775RvsWT-contrast), A1789T versus wild-type (A1789TvsWT-contrast) and the mutated BRCT domain versus wild-type (MutvsWT-contrast), considering the two variants as a single mutation of BRCT domain.Results201 differentially expressed genes were found in M1775RvsWT-contrast, 313 in A1789TvsWT-contrast and 173 in MutvsWT-contrast. Most of these genes mapped in pathways deregulated in cancer, such as cell cycle progression and DNA damage response and repair.ConclusionsOur results represent the first molecular evidence of the pathogenetic role of M1775R, already proposed by functional studies, and give support to a similar role for A1789T that we first hypothesized based on the yeast cell experiments. This is in line with the very recently suggested role of BRCT domain as the main effector of BRCA1 tumor suppressor activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

et al. 2012

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“…1 The recombination substrate contains the neo gene and two defective hyg gene sequences inactivated by 10-bp insertions, either at a unique PvuI site (hyg1) or at a unique SacII site (hyg2);. the two mutated hyg genes are in direct repeat orientation and are separated by a sequence containing the amino-glycoside phosphotransferase (Neo) gene conferring resistance to G418; an intrachromosomal recombination event occurring by gene conversion between the two hyg sequences results in restoration of one of the mutant hyg genes to wild type; the intrachromosomal deletion of the DNA sequence between the two mutated hyg genes leading to the formation of a Hyg R wild type (Hygwt) with loss of intervening sequence [18,20]. The figure is adapted from Guidugli et al [20] Results…”

Section: Discussionmentioning

confidence: 99%

“…In this article, we proposed a novel functional HR-based assay, previously used to classify BRCA1 UCVs [20], which determines the effect of the transient overexpression of the BRCA2 variant on the spontaneous HR. We assume that this analysis will give more information on how BRCA2 may induce genome instability and, therefore, on the basic mechanism of the BRCA2-induced tumourigenesis.…”

Section: Discussionmentioning

confidence: 99%

Effect of the overexpression of BRCA2 unclassified missense variants on spontaneous homologous recombination in human cells

Balia

Galli

Caligo

2011

Breast Cancer Res Treat

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Breast Cancer 2 gene (BRCA2) mutation carriers have a 45% chance of developing breast cancer and a 11% risk of developing ovarian cancer by the age of 70. While hundreds of BRCA2-truncating mutations have been associated with an increased cancer risk in carriers, the contribution of unclassified variants (UCVs) to cancer risk remains largely undefined. BRCA2-defective cells show a high degree of chromosome instability. Although a functional assay based on the BRCA2 capability to stimulate DSB-induced homologous recombination (HR) as a way to classify UCVs has been proposed, so far no data are available concerning the effect of BRCA2 UCVs on spontaneous HR. In this study, we proposed a novel functional HR-based assay that determines the effect of the transient overexpression of the BRCA2 variant on spontaneous HR. This assay will help one in the difficult task of classifying UCVs, and it will give more information on how BRCA2 may induce genome instability and on the basic mechanism of BRCA2-induced tumourigenesis. We chose 11 BRCA2 UCVs not previously described or classified in other articles, and distributed along the entire BRCA2-coding region. They are as follows: G173V, D191V, S286P, M927V, T1011R, L1019V, N1878K, S2006R, R2108C, G2353R and V3091I. Basically, because the expression of BRCA2wt and the neutral variants did not increase spontaneous HR, we classified the variants G173V, S286P, M927V, T1011R and L1019V as HR-negative and presumed that they were not pathogenic. The HR-positive variants, D191V, N1878K, S2006R, R2108C, G2353R, and V3091I, which increased HR as much as the cancer-associated variant G2748D, could probably be classified as pathogenic. We observed that all our variants in the C-terminus of the protein behaved differently from the wt, suggesting a role for this protein region in spontaneous HR.

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Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway

Guglielmi

Cerri

Evangelista

et al. 2013

Breast Cancer Res Treat

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M1775R and A1789T are two missense variants located within the BRCT domains of BRCA1 gene. The M1775R is a known deleterious variant, while the A1789T is an unclassified variant that has been analyzed and classified as probably deleterious for the first time by our group. In a previous study, we described the expression profile of HeLa G1 cells transfected with the two variants and we found that they altered molecular mechanisms critical for cell proliferation and genome integrity. Considering that the mutations in the BRCA1 C terminus (BRCT) domains are associated to a phenotype with an altered ability in the DNA double-strand break repair, we chose three of the genes previously identified, EEF1E1, MRE11A, and OBFC2B, to be tested for an homologous recombination (HR) in vitro assay. For our purpose, we performed a gene expression knockdown by siRNA transfection in HeLa cells, containing an integrated recombination substrate (hprtDRGFP), for each of the target genes included BRCA1. The knockdown of BRCA1, OBFC2B, MRE11A, and EEF1E1 reduces the HR rate, respectively, of 97.6, 28.6, 41.8, and 42.3 % compared to cells transfected with a scrambled negative control duplex and all these differences are statistically significant (P < 0.05; Kruskal-Wallis test). Finally, we analyzed the effect of target gene depletion both on HR that on cell survival after DNA-damage induction by ionizing radiation. The clonogenic assay showed that the down-regulation of the target genes reduced the cell survival, but the effect on the HR, is not evident. Only the BRCA1-siRNA confirmed the inhibition effect on HR. Overall these results confirmed the involvement of MRE11A in the HR pathway and identified two new genes, OBFC2B and EEF1E1, which according to these data and the knowledge obtained from literature, might be involved in BRCA1-pathway.

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A recombination-based method to characterize human BRCA1 missense variants

Cited by 6 publications

References 32 publications

Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

Effects on human transcriptome of mutated BRCA1 BRCT domain: A microarray study

Effect of the overexpression of BRCA2 unclassified missense variants on spontaneous homologous recombination in human cells

Identification of two novel BRCA1-partner genes in the DNA double-strand break repair pathway

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