1,721,017 research outputs found
Effect of BRCA1 missense variants on gene reversion in DNA double-strand break repair mutants and cell cycle-arrested cells of Saccharomyces cerevisiae
No full textEvaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11a, rad50a and rad51a. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51a strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50a mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11a mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-Arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-Arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies
RAD52 influences the effect of BRCA1/2 missense variants on homologous recombination and gene reversion in Saccharomyces cerevisiae
No full textThe breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are key players in the homologous recombination (HR) repair pathway and act as tumor suppressors by maintaining genome stability. The yeast Saccharomyces cerevisiae has no BRCA1/2 homolog; however, a number of HR genes are evolutionary conserved between human and yeast. Among them, RAD52 is involved in DNA double strand break (DSB) repair by HR, and promotes genome stability. We previously reported that the heterologous expression of cancer-associated BRCA1/2 missense variants in growing yeast cultures affects both spontaneous HR and gene reversion (GR) suggesting that yeast could be a reliable system to assess the functional impact of variants. Because inhibition of Rad52p is lethal in BRCA1/2 mutated tumors, and Rad52p is conserved between humans and yeast, we asked if the effect of BRCA1/2 variants on HR and GR could be affected by loss of RAD52. We found that the rad52∆ mutation predominantly suppressed the stimulation of HR in yeast by pathogenic BRCA1 variants but also facilitated increased GR by pathogenic variants. Conversely, the rad52∆ mutation stimulated HR by a pathogenic BRCA2 variant in yeast but had no effect on GR. These results demonstrate a functional interplay between the pathogenic BRCA1/2 variants and Rad52p in budding yeast, supporting the use of budding yeast as a suitable system for evaluating potential chemotherapeutic strategies
IN VIVO AND IN VITRO ANALYSIS OF THE BRCA1 UNCLASSIFIED VARIANTS (VUSs)
No full textBRCA1 acts a tumor suppressor gene and germ-line mutations which disrupt its functions culminate, after the loss of the wild type allele, in breast and ovarian cancer development. Although the precise biochemical functions of BRCA1 relevant for tumor suppression still remains to be clarified, it has been demonstrated to play a role in several cellular processes including: DNA DSBs repair, transcriptional regulation, chromatin remodeling, cell cycle checkpoints, ubiquitination and centrosome replication.
An increasing number of mutations leading to an amino acidic substitution in the protein (missense mutations) have been identified in hereditary breast/ovarian cancer patients and the pathogenetic role of such missense mutations remains largely undefined. We studied some BRCA1 variants of uncertain significance (VUSs) both by in vivo analysis and by in vitro analysis. In particular we carried out an in vivo molecular study on the tumor tissue from patients carrier of the VUSs and we performed three in vitro functional assays based on two BRCA1 cellular function:
the repair of DNA double strand break by Homologous Recombination and Non Homologous End Joining;
the transcriptional control.
It is important to perform systematic studies of the VUSs to assess pathogenicity of these variants found during genetic testing for BRCA1 hereditary breast and ovarian cancers
Evaluation of BRCA1 related DNA repair genes in breast and ovarian carcinogenesis
No full textBreast cancer in one of the most common cancer worldwide. Among women this tumor accounts for over 25% of cancer diagnosis and 15% of cancer related deaths.
5-10% of breast cancer cases are hereditary, with a germline mutation in a known cancer susceptibility gene. Inherited susceptibility to developing breast and ovarian cancer most commonly results from mutation in BRCA1 and BRCA2 genes which are inherited in an autosomal dominant way.
BRCA1 is a tumor suppressor gene which encodes for a nuclear protein involved in a wide array of cellular functions, including cell cycle regulation, DNA damage response, maintenance of the genomic stability, transcription regulation, replication, recombination and higher chromatin hierarchical control.
BRCA1 is a component of a protein complex termed BASC (BRCA1-Associated Genome Surveillance Complex) and, therefore it has been associated with a variety of proteins implicated in DNA damage response and repair mechanism, such as MLH1, MSH2, MSH6, BLM and the MRN complex constituted by RAD50, MRE11A and NBS1. Many proteins of this complex can bind damaged DNA structures and act as sensors of DNA damage.
The aim of this study is to determine if some of the DNA repair partners of BRCA1 may contribute to breast and ovarian cancer development and progression.
Preliminary results obtained by our research group through functional assays in S. cerevisiae suggested that MSH2, MSH6, MRE11A, RAD50 and RAD51 genes might have a role in genomic instability induced by BRCA1 in yeast. Moreover a mutational screening revealed a surprisingly high frequency of MSH2 somatic mutations in breast and ovarian tumors from carriers of BRCA1 VUS (variants of unknown significance).
On the basis of these preliminary results the first task of this project is to perform a mutational analysis by next generation sequencing of MSH6, MRE11A, RAD50 and RAD51 genes in selected breast and/or ovarian tumors from BRCA1 VUS carriers, BRCA1 mutation carriers and BRCA1 wt individuals. This mutational screening identified 8 rare somatic variants pathogenic or predicted pathogenic distributed on 7 patients. 4/8 variants were located on MSH6 gene, 2/8 on RAD51, 1/8 on RAD50 and 1/8 on MRE11A.
In the second part of this study it was performed a GFP dependent homology directed repair assay whereby a recombination substrate is integrated into the genome. This functional assay was carried out in HeLa and MCF7 cell lines transfected with BRCA1 wild type and four BRCA1 VUS (p.Y179C, p.N550H, p.M1775R and p.A1789T). The results of this assay showed an increase in homologous recombination frequency associated to the pathogenic variants p.M1775R and p.A1789T in both cell lines.
Finally, the CRISPR-Cas9 system was used in order to obtain HeLa and MCF7 cell lines knockout for MSH2, but so far it was possible to isolate only HeLa MSH2 knockout clones. The MSH2 knockout clones will be tested with the homology directed repair assay to investigate the combined effect of MSH2 deficiency and BRCA1 VUS on homologous recombination.
On the basis of these results it is possible to conclude that the mismatch repair system seems to be frequently impaired in breast and ovarian tumors. Moreover the homologous recombination assay could be another powerful tool to help in the classification of BRCA1 VUS
Identification and analysis of pathogenic regulatory variants in promoters, introns and in 5’ and 3’ untranslated regions of BRCA1 and BRCA2 genes
No full textBackground - Germline mutations in breast cancer susceptibility genes BRCA1/2 confer a substantially increased lifetime risk of developing breast and/or ovarian cancer. The BRCA1/2 routinely clinical mutation screening is usually limited to the coding regions and intron–exon boundaries, which precludes the identification of mutations within the noncoding regulatory regions. These observations led us to assume that unknown genomic variations in the 5’, 3’ untranslated regions (UTRs) and introns of BRCA genes could be implicated in the inherited breast or ovarian cancer through their effect on BRCA gene expression. The activity of 5’ UTR and genomic flanking regions of BRCA1/2 is highly controlled by a self-regulating mechanism and by several transcription factors that act as activators or repressors. Aberrations in these regulatory regions may be associated with a deregulation in the transcriptional activity and in the mRNA levels of BRCA genes. Moreover the functional activity of BRCA genes is also regulated at the post transcriptional level: in particular the 3’UTR contains RNA-binding proteins and miRNA binding sites, and some germline variants in 3’UTR have been associated with breast cancer risk. In order to test this hypothesis, specific regulatory introns and 5’/3’ UTRs of BRCA genes were analyzed in patients highly selected for breast/ovarian cancer family history and tested negative for exonic mutations in these genes.
Materials and Methods - Genotyping analysis was performed by Next Generation Sequencing on genomic DNA of 80 index cases selected from breast and/or ovarian cancer patients with strong family history and wild type for BRCA1 and BRCA2. All variants emerged from the mutational screening of 5’/3’ UTRs and introns of BRCA1/2 were prioritized by bioinformatic tools. Following the bioinformatic prioritization, the effect of the interesting variants on gene regulation was evaluated by functional assays. A multifactorial analysis was performed to translate information from multiple sources to estimate the associated cancer risk of each regulatory variant.
Results - The mutation analysis of 80 patients showed 34 different variants: 19 in BRCA1 and 15 in BRCA2. 5 variants in BRCA1 and 8 variants in BRCA2 were novel and not present in dbSNP. 9 out of the 13 “no dbSNP” variants were resulted with significant change in bioinformatic prioritization.
3 variants in BRCA1 and 2 in BRCA2 were found in the promoter regions and were predicted to alter TF (transcription factors) binding site affinity. To examine the effect of these variants on promoter activity, luciferase reporter assays were performed.
For c.-232-176A>T and c.-232-55G>A BRCA1 variants, electrophoretic mobility shift assays (EMSA) also revealed different DNA-protein complex when the nuclear extracts were incubated with the biotinylated variant probe. The variants produced a significant change in the mobility of DNA-protein complexes compared to wild type.
Conclusion - The major challenge is to determine the significance and the contribution of non-coding variants in breast and ovarian cancer susceptibility. This could be relevant to identify inherited mutations in such patients with a strong family history without alterations in routinely analyzed BRCA1/2 regions, to propose an adaptive follow-up for them and their families and to design targeted therapeutic strategies.
Research is urgently needed to reduce the incidence and mortality of breast cancer. Genetic testing represents a powerful tool to increase the efficiency of prevention, through an effective risk prediction; moreover it may help in diagnosis and in the development of new and more specific therapeutic approaches. The final goal of this project is to develop clinically useful tools for the classification of sequence variants that map in regulatory regions of BRCA1/2 genes. Gaining deeper insights in breast cancer susceptibility will improve the ability to identify those families at increased risk and the efficiency of cancer prevention for BRCA carrier
Characterization of breast cancer susceptibility genes in men by Next-Generation Sequencing
No full textMale breast cancer (MBC) is a rare disease with an incidence of 1/105 year. MBC tends to occur between 60-70 years old and to express oestrogen and progesterone receptors with occasionally epidermal growth factor receptor 2 amplification. The rarity of MBC has precluded large-scale association studies, thus genetic predisposition remain not well understood.
In order to better define genetic risk factors in men, a germline investigation in 81 MBC cases was performed through the screening of 24 genes involved in breast cancer predisposition, genome stability maintenance and DNA repair mechanisms by Next Generation Sequencing.
Clinical pathological data and family history of 81 MBC cases were collection revealed an average age of onset was 61.3 years and breast cancer family history in 35 cases. Overall, our genetic screening let to attribute a genetic cause to breast cancer in 23% of cases. In total, 19 patients carried a pathogenic mutation in 4 genes: BRCA2, BRIP1, MUTYH and PMS2. As expected, a positive family history is a strong predictor of germline BRCA2 mutations. Moreover, 14 variants of unknown clinical significance (VUS) in 9 genes (BARD1, BRCA1, BRCA2, BRIP1, CHEK2, ERCC1, NBN, PALB2, PMS1) were predicted as potentially pathogenic by in-silico analysis leading to 40% the mutation detection rate. Understanding the potential pathogenicity of VUS represents an extremely urgent question for the management of breast cancer risk in MBC cases and their own families
Genetic profile of DNA repair genes in Triple Negative Breast Cancer
No full textAbstract
Triple Negative Breast Cancer (TNBC), negative for estrogen, progesterone and Her2 receptor, constitute 10–20% of all breast cancers and more frequently affect younger patients. TNBC tumors are generally large in size, higher grade cancers with lymph node involvement at diagnosis, and are biologically more aggressive than other subtypes. Treatment of TNBC patients has been challenging due to the heterogeneity of the disease and the absence of well-defined molecular targets. TNBCs share many phenotypic features with BRCA1-related and basal-like tumors, such as receptors negativity, expression of basal cytokeratins CK5/6, and a similar gene expression profile. This similarity is defined BRCAness and suggests that TNBC and BRCA-related cancer may also share molecular and genetic features and common deficiency in DNA repair genes. In addition to BRCA1 and BRCA2, mutations in other genes involved in DNA repair may predispose to breast and ovarian cancer and the therapy response can be determined by germline or somatic alterations in these genes. So, the aim of this thesis is to investigate which genes involved in DNA repair are mutated in TNBC and to correlate a genetic signature with the response to neoadjuvant or adjuvant therapy.
The entire coding sequences and intron-exon junctions of 24 genes involved in the main DNA repair pathways (BRCA1, BRCA2, CDH1, MRE11A, MSH2, PARP1, CHEK2, MSH6, RAD52, PTEN, STK11, ERCC1, TP53, PMS1, PMS2, NBN, RAD50, BRIP1, BARD1, MLH1, MUTYH, RAD51C, TP53BP1 and PALB2) were screened using PGM Ion Torrent platform. Some of these are considered breast and ovarian cancer susceptibility genes, while others are associated with breast tumorigenesis. Moreover a set of genes was analyzed for the presence of large rearrangements by MLPA. Two cohort of patients diagnosed of TNBC have been analyzed: 19 patients, with and without family history of breast cancer, treated with neoadjuvant chemotherapy including taxane and anthracycline for germline mutations and 37 unselected TNBC patients with a follow up >5 years for somatic and germline mutations.
The mutational screening in the neoadjuvant setting, have identified 5 patients with clearly pathogenetic mutation in BRCA1, RAD51c and PALB2 accounting for a 20% of the total. Other 15 predicted pathogenetic variants in DNA repair genes were found. Data suggest a potential correlation between the presence of germline pathogenetic mutations, indicative of DNA repair defects, and a positive outcome after neoadjuvant therapy. Furthermore, this approach allowed the identification of 3 novel mutations predicted deleterious by in silico analysis and never reported in literature. From the second set of patients, BRCA1 and BRCA2 mutations were found in 7 patients and one patient, respectively. Also germline mutation in BRIP1 and CDH1 were found. Overall in 56 TNBC patients screened, this gene panel has evidenced that around 25% of them harbour a germline mutation in DNA repair genes. In addition to that, many interesting somatic variants were found in 8 genes (BRCA2, BRIP1, CDH1, MLH1, MSH2, NBN, PMS1, TP53) in this set of TNBC tissues. In particular TP53 was mutated in 68% of patients, and this condition was associated with a worse outcome. The large rearrangement analysis confirmed that PTEN and TP53 loss, along with EGFR gain, are very frequent events in TNBC.
This gene panel approach has proved useful to identify a large number of germline mutations in TNBC patients. In the next future, the genetic screening of TNBC patients, even without family history of breast cancer, may be a real option in order to design personalized therapy for individuals with a “BRCAness phenotype. Moreover, these results may provide new insights into TNBC pathogenesis and may help to further improve the accuracy of treatments
CARATTERIZZAZIONE DEL RUOLO PATOLOGICO DI MUTAZIONI MISSENSO DEL GENE BRCA1
No full textL’analisi mutazionale di 276 individui con storia familiare di tumore della mammella e/o dell’ovaio, attraverso il sequenziamento diretto delle intere regioni codificanti e delle giunzioni esone-introne dei geni BRCA1 e BRCA2, ha portato all’identificazione di numerose varianti missenso sia nuove che gia descritte. L’analisi in silico di queste varianti, ha permesso di evidenziare 8 potenziali varianti di splicing (2 in BRCA1 e 6 in BRCA2) e 7 varianti BRCA1 uniche non sinonime con alta probabilità di essere deleterie.
Lo scopo dello studio è di valutare l’effetto di queste 15 varianti missenso sulle funzioni di BRCA1 e BRCA2 attraverso l’analisi dell’espressione del mRNA, per le 8 varianti di splicing, ed un saggio funzionale in un modello in vivo in linee cellulari umane e di lievito per le 7 varianti missenso.
L’eventuale inattivazione funzionale, permette la classificazione di una variante come patogenetica e quindi di valutare più accuratamente il rischio di tumore nei soggetti carrier.
L’analisi del processamento del mRNA ha rivelato che BRCA1 c.IVS11+1G>A e BRCA2 c.IVS2+1G>A, c.IVS13-2A>G, c.IVS21+4A>G e c.9345G>A sono vere mutazioni deleterie. Lo studio dei saggi funzionali in lievito ed nell’uomo, suggeriscono che le varianti Y179C, I1766S e A1789T possono alterare le normali funzioni di BRCA1, mentre per la variante N132K ulteriori studi dovranno essere condotti
Definizione di una “signature” genetica correlata con la risposta alla terapia neoadiuvante in tumori della mammella triplo negativi con metodologia NGS.
No full textRecenti studi evidenziano come mutazioni in geni coinvolti nel riparo del DNA, oltre ai principali geni di predisposizione BRCA1 e BRCA2, possono aumentare il rischio di sviluppare cancro alla mammella e alle ovaie. La risposta a determinate terapie dipende fortemente da eventuali alterazioni genetiche germinali o tumore-specifico all'interno di questi geni. Alcuni tumori sporadici, o in pazienti non selezionati per storia familiare, mostrano proprietà cliniche, patologiche e molecolari simili ai tumori ereditari BRCA-mutati, una condizione definita BRCAness. Tra questi ci sono alcuni tra i tumori che non esprimono i recettori ormonali (ER e PR) e Her2, definiti tripli negativi (Triple Negative Breast Cancer, TNBC): essi rispondono alla chemioterapia con agenti alchilanti, in modo simile ai BRCA mutati, ma hanno un alto rischio di recidiva e di progressione della malattia. Al fine di sviluppare terapie più mirate per questo specifico sottotipo di tumore, potrebbe essere rilevante individuare una correlazione tra la risposta alla terapia neoadiuvante con antracicline e taxani ed eventuali mutazioni germinali in geni principalmente coinvolti nel riparo del DNA. Con il fine di caratterizzare i TNBC è stata testata la presenza di mutazioni germinali nei geni BRCA1, BRCA2, CHEK2, BRIP1, PALB2, TP53, PTEN, STK11, CDH1, BARD1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50, RAD51C, RAD52, 53BP1, PARP1, ERCC1, coinvolti principalmente nei meccanismi di riparazione del DNA, tramite la tecnologia Next Generation Sequencing (NGS) “Ion Torrent” in pazienti trattati con terapia neoadiuvante con taxani e antracicline. Sono state selezionate 16 pazienti con TNBC in terapia neoadiuvante con almeno 3 cicli di FEC (Fluorouracile, Epirubicina, Ciclofosfamide) e taxani presso il Polo Oncologico del Santa Chiara di Pisa. Per ciascuna paziente è stato collezionato un campione di sangue periferico, da cui è stato estratto il DNA genomico. La strategia adottata per il sequenziamento è quella del target-resequencing. La costruzione delle librerie è stata effettuata con PCR multiplex. Con il software “Ion AmpliSeq™ Designer” è stato costruito un pannello di primers per analizzare le sequenze codificanti e le regioni fiancheggianti gli esoni dei geni selezionati. Le librerie di ampliconi sono state costruite in modo tale di poter analizzare più pazienti contemporaneamente, utilizzando dei barcode nucleotidici. Ogni libreria è stata purificata e amplificata clonalmente sulla superficie di microsfere, successivamente caricate su un chip semiconduttore. Il sequenziamento è stato effettuato con il sequenziatore “Personal Genome Machine” (PGM). La raccolta e l’elaborazione dei dati è stata eseguita dalla workstation Torrent Suite (TS). I file VCF sono stati generati dal sofware “Ion Reporter 1.6” ed i file BAM e BAI sono stati visualizzati con Interactive Genome Viewer (IGV), per identificare eventuali artefatti di sequenza. L’annotazione delle varianti è stata eseguita con wANNOVAR e con Variant Effect Predictor (VEP). In seguito al sequenziamento NGS sono state ottenute 232 varianti. Di queste, 110 varianti sono di tipo esonico, 132 varianti cadono in regioni non codificanti. Per selezionare le varianti probabilmente deleterie, le varianti esoniche sono state filtrate con i software di predizione di deleteriosità e conservazione SIFT, PolyPhen-2, LRT, Mutation Taster e PhyloP per rimuovere le varianti neutrali. Sono state considerate anche le varianti di splicing e le In/Del eterozigosi non localizzate in regioni omopolimeriche. Sono state selezionate 34 varianti uniche e 5 presenti in più pazienti. In seguito a sequenziamento diretto sono state confermate 28 mutazioni. Per approfondire il significato funzionale di queste varianti è stato fatto un confronto con la letteratura scientifica e con i principali database di varianti (LOVD, BIC, dbSNP), concludendo che 12 varianti predette deleterie hanno un effetto neutrale, mentre le restanti 16 sono probabilmente deleterie. In cinque pazienti non è stata identificata alcuna mutazione deleteria. È possibile osservare la tendenza ad una migliore risposta alla terapia nei pazienti con almeno una mutazione patogenetica. Questo risultato andrebbe confermato aumentando la coorte dei pazienti analizzati per poter osservare differenze statisticamente significative.
Questo studio ha evidenziato le potenzialità dell’approccio NGS, per l’analisi di molti geni in tempi ristretti. In questo lavoro sono state identificate 6 mutazioni mai osservate prima che necessitano di un’ulteriore indagine per chiarine il significato funzionale, e l’eventuale loro coinvolgimento nella tumorigenesi. Inoltre, ha permesso di identificare delle mutazioni con chiaro significato patogenetico, anche su geni che normalmente non sono analizzati nella consulenza genetica per il tumore alla mammella, in pazienti senza sospetta familiarità. Questo lavoro può porre le basi per uno studio più ampio che potrebbe avere ripercussioni utili dal punto di vista clinico e che potrebbe indirizzare verso nuove scelte terapeutiche
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