RESUMEN
BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers.
Asunto(s)
Proteína BRCA2 , ADN de Cadena Simple , Unión Proteica , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animales , Ratones , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Inestabilidad Cromosómica , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Cisplatino/farmacología , Daño del ADN , Mutación Missense , Femenino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Células Madre Embrionarias de Ratones/metabolismo , Línea Celular Tumoral , Mitomicina/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Complejo de la Endopetidasa ProteasomalRESUMEN
The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.
Asunto(s)
Neoplasias de la Mama , Edición Génica , Animales , Humanos , Ratones , Femenino , Virulencia , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Exones/genética , Codón , Nucleótidos , Neoplasias de la Mama/genética , Predisposición Genética a la Enfermedad , Proteína BRCA1/genéticaRESUMEN
RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.
Asunto(s)
ADN de Neoplasias/biosíntesis , Resistencia a Antineoplásicos , Inestabilidad Genómica , Neoplasias/tratamiento farmacológico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/metabolismo , Origen de Réplica , Células A549 , Animales , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Sistemas CRISPR-Cas , Roturas del ADN de Doble Cadena , Reparación del ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Mutación , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Unión Proteica , Interferencia de ARN , Recombinasa Rad51/genética , TransfecciónRESUMEN
Advances in molecular diagnostics have led to improved diagnosis and molecular understanding of hereditary cancers in the clinic. Improving the management, treatment, and potential prevention of cancers in carriers of predisposing mutations requires preclinical experimental models that reflect the key pathogenic features of the specific syndrome associated with the mutations. Numerous genetically engineered mouse (GEM) models of hereditary cancer have been developed. In this review, we describe the models of Lynch syndrome and hereditary breast and ovarian cancer syndrome, the two most common hereditary cancer predisposition syndromes. We focus on Lynch syndrome models as illustrative of the potential for using mouse models to devise improved approaches to prevention of cancer in a high-risk population. GEM models are an invaluable tool for hereditary cancer models. Here, we review GEM models for some hereditary cancers and their potential use in cancer prevention studies.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis , Síndrome de Cáncer de Mama y Ovario Hereditario , Síndromes Neoplásicos Hereditarios , Humanos , Femenino , Animales , Ratones , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/diagnóstico , Predisposición Genética a la Enfermedad , Síndromes Neoplásicos Hereditarios/genética , MutaciónRESUMEN
Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.
Asunto(s)
Proteína BRCA2 , Neoplasias de la Mama , Cordoma , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Animales , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Cordoma/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Femenino , Genes BRCA2 , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Humanos , RatonesRESUMEN
Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.
Asunto(s)
Replicación del ADN/fisiología , Resistencia a Antineoplásicos/efectos de los fármacos , Eliminación de Gen , Genes BRCA1 , Genes BRCA2 , Neoplasias/patología , Proteínas Nucleares/deficiencia , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Cisplatino/farmacología , ADN/biosíntesis , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , ADN Helicasas/genética , Reparación del ADN/efectos de los fármacos , Reparación del ADN/genética , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/genética , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Femenino , Recombinación Homóloga , Proteína Homóloga de MRE11 , Ratones , Neoplasias/genética , Proteínas Nucleares/genética , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genéticaRESUMEN
The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.
Asunto(s)
Neoplasias de la Mama , Animales , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/epidemiología , Estudios de Cohortes , Femenino , Genes BRCA2 , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Humanos , Malasia , RatonesRESUMEN
Next-generation sequencing of Sri Lankan families with inherited cancer syndromes resulted in the identification of five BRCA2 variants of unknown clinical significance. Interpreting such variants poses significant challenges for both clinicians and patients. Using a mouse embryonic stem cell-based functional assay, we found I785V, N830D, and K2077N to be functionally indistinguishable from wild-type BRCA2. Specific but mild sensitivity to olaparib and reduction in homologous recombination (HR) efficiency suggest partial loss of function of the A262T variant. This variant is located in the N-terminal DNA binding domain of BRCA2 that can facilitate HR by binding to dsDNA/ssDNA junctions. P3039P is clearly pathogenic because of premature protein truncation caused by exon 23 skipping. These findings highlight the value of mouse embryonic stem cell-based assays for determining the functional significance of variants of unknown clinical significance and provide valuable information regarding risk estimation and genetic counseling of families carrying these BRCA2 variants.
Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células Madre Embrionarias de Ratones/metabolismo , Mutación , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/patología , Animales , Proteína BRCA2/metabolismo , Bioensayo/métodos , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/metabolismo , Supervivencia Celular , Estudios de Cohortes , Femenino , Recombinación Homóloga , Humanos , Ratones , Síndromes Neoplásicos Hereditarios/epidemiología , Síndromes Neoplásicos Hereditarios/metabolismo , Sri Lanka/epidemiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A pathogenic mutation in BRCA2 significantly increases the risk of breast and ovarian cancers making it imperative to examine the functional consequences of variants of uncertain clinical significance. Variants that are predicted to result in a truncated protein are unambiguously classified as pathogenic. We have previously shown how a pathogenic splice site variant known to generate a premature termination codon (PTC) in exon 9 and a nonsense mutation at exon 7, can generate functional BRCA2 by skipping exons 4-7 and restoring the reading frame. Using a well-established mouse embryonic stem cell-based assay, we functionally characterize here one splice site mutation and 11 pathogenic BRCA2 variants that are either nonsense mutation or generate PTC in different exons ranging from exons 4 to 7. Our study shows that five variants can restore the open reading frame by exon skipping and generate a functional protein. This suggests further need to exercise prudence when classifying clearly pathogenic variants.
Asunto(s)
Proteína BRCA2/genética , Codón sin Sentido , Células Madre Embrionarias/metabolismo , Neoplasias Ováricas/genética , Empalme Alternativo , Animales , Proteína BRCA2/metabolismo , Supervivencia Celular/genética , Codón sin Sentido/genética , Exones , Femenino , Técnicas de Inactivación de Genes , Ratones , Ratones Noqueados , Mutación , Sitios de Empalme de ARNRESUMEN
Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Inmunoprecipitación de Cromatina , ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Humanos , Análisis por Matrices de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , TranscriptomaRESUMEN
Human breast cancer susceptibility gene, BRCA2, encodes a 3418-amino acid protein that is essential for maintaining genomic integrity. Among the proteins that physically interact with BRCA2, Partner and Localizer of BRCA2 (PALB2), which binds to the N-terminal region of BRCA2, is vital for its function by facilitating its subnuclear localization. A functional redundancy has been reported between this N-terminal PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2, which undermines the relevance of the interaction between these two proteins. Here, we describe a genetic approach to examine the functional significance of the interaction between BRCA2 and PALB2 by generating a knock-in mouse model of Brca2 carrying a single amino acid change (Gly25Arg, Brca2G25R) that disrupts this interaction. In addition, we have combined Brca2G25R homozygosity as well as hemizygosity with Palb2 and Trp53 heterozygosity to generate an array of genotypically and phenotypically distinct mouse models. Our findings reveal defects in body size, fertility, meiotic progression, and genome stability, as well as increased tumor susceptibility in these mice. The severity of the phenotype increased with a decrease in the interaction between BRCA2 and PALB2, highlighting the significance of this interaction. In addition, our findings also demonstrate that hypomorphic mutations such as Brca2G25R have the potential to be more detrimental than the functionally null alleles by increasing genomic instability to a level that induces tumorigenesis, rather than apoptosis.
Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama/genética , Proteínas Nucleares/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Apoptosis/genética , Proteína BRCA1/genética , Proteína BRCA2/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Técnicas de Sustitución del Gen , Predisposición Genética a la Enfermedad , Inestabilidad Genómica/genética , Humanos , Ratones , Mutación , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers.
Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama/genética , Anemia de Fanconi/genética , Predisposición Genética a la Enfermedad , Empalme Alternativo/genética , Animales , Neoplasias de la Mama/patología , Exones/genética , Anemia de Fanconi/patología , Técnicas de Sustitución del Gen , Mutación de Línea Germinal , Humanos , Ratones , Mutación , Linaje , Sitios de Empalme de ARNAsunto(s)
Proteína BRCA1/genética , Proteína BRCA2/genética , Predisposición Genética a la Enfermedad , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Femenino , Síndrome de Cáncer de Mama y Ovario Hereditario/epidemiología , Síndrome de Cáncer de Mama y Ovario Hereditario/patología , HumanosRESUMEN
BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.
Asunto(s)
Proteína BRCA1/genética , Biología Computacional/métodos , Gráficos por Computador , Internet , Mutación Missense , Programas Informáticos , Neoplasias de la Mama/genética , Análisis Mutacional de ADN , Sistemas de Administración de Bases de Datos , Bases de Datos Genéticas , Conjuntos de Datos como Asunto , Femenino , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Humanos , Neoplasias Ováricas/genéticaRESUMEN
Fanconi anemia (FA) is a rare recessive disorder with chromosomal instability, congenital abnormalities, and a high cancer risk. The breast cancer susceptibility gene BRCA2 (FANCD1) is one of the 16 genes involved in this recessive disease. We have identified a novel mutation of the splice donor site of intron 1 in the noncoding region of BRCA2 in a Japanese FA family. This mutation may account for the FA phenotype in a patient originally reported to have biallelic mutations in BRCA2. Subsequent functional studies revealed that one of the mutations, K2729N, was a neutral change. As reported here, a more careful analysis resulted in the identification of a novel splice site mutation. Functional analysis using a mouse embryonic stem cell-based assay revealed that it causes aberrant splicing, reduced transcript levels and hypersensitivity to DNA damaging agents, suggesting that it is likely to be pathogenic. Although similar pathogenic variants in the noncoding region of BRCA1 and 2 were not identified in a cohort of 752 familial breast cancer cases, we still think this finding is relevant for mutation analysis in Hereditary Breast and Ovarian Cancer Syndrome families in a diagnostic setting.
Asunto(s)
Proteína BRCA2/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Síndrome de Cáncer de Mama y Ovario Hereditario/diagnóstico , Síndrome de Cáncer de Mama y Ovario Hereditario/genética , Animales , Proteína BRCA1/genética , Secuencia de Bases , Células Cultivadas , Análisis Mutacional de ADN , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Mutación Missense , Linaje , Sitios de Empalme de ARNRESUMEN
The implementation of next-generation sequence analysis of disease-related genes has resulted in an increasing number of genetic variants with an unknown clinical significance. The functional analysis of these so-called "variants of uncertain significance" (VUS) is hampered by the tedious and time-consuming procedures required to generate and test specific sequence variants in genomic DNA. Here, we describe an efficient pipeline for the generation of gene variants in a full-length human gene, BRCA2, using a bacterial artificial chromosome. This method permits the rapid generation of intronic and exonic variants in a complete gene through the use of an exon-replacement strategy based on simple site-directed mutagenesis and an effective positive-negative selection system in E. coli. The functionality of variants can then be assessed through the use of functional assays, such as complementation of gene-deficient mouse-embryonic stem (mES) cells in the case of human BRCA2. Our methodology builds upon an earlier protocol and, through the introduction of a series of major innovations, now represents a practical proposition for the rapid analysis of BRCA2 variants and a blueprint for the analysis of other genes using similar approaches. This method enables rapid generation and reliable classification of VUS in disease-related genes, allowing informed clinical decision-making.
Asunto(s)
Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Estudios de Asociación Genética/métodos , Pruebas Genéticas/métodos , Variación Genética , Animales , Línea Celular , Células Madre Embrionarias/metabolismo , Femenino , Expresión Génica , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Empalme del ARN , Selección GenéticaRESUMEN
Single-nucleotide substitutions and small in-frame insertions or deletions identified in human breast cancer susceptibility genes BRCA1 and BRCA2 are frequently classified as variants of unknown clinical significance (VUS) due to the availability of very limited information about their functional consequences. Such variants can most reliably be classified as pathogenic or non-pathogenic based on the data of their co-segregation with breast cancer in affected families and/or their co-occurrence with a pathogenic mutation. Biological assays that examine the effect of variants on protein function can provide important information that can be used in conjunction with available familial data to determine the pathogenicity of VUS. In this report, we have used a previously described mouse embryonic stem (mES) cell-based functional assay to characterize eight BRCA2 VUS that affect highly conserved amino acid residues and map to the N-terminal PALB2-binding or the C-terminal DNA-binding domains. For several of these variants, very limited co-segregation information is available, making it difficult to determine their pathogenicity. Based on their ability to rescue the lethality of Brca2-deficient mES cells and their effect on sensitivity to DNA-damaging agents, homologous recombination and genomic integrity, we have classified these variants as pathogenic or non-pathogenic. In addition, we have used homology-based modeling as a predictive tool to assess the effect of some of these variants on the structural integrity of the C-terminal DNA-binding domain and also generated a knock-in mouse model to analyze the physiological significance of a residue reported to be essential for the interaction of BRCA2 with meiosis-specific recombinase, DMC1.
Asunto(s)
Proteína BRCA2/genética , Neoplasias de la Mama/genética , Células Madre Embrionarias/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Proteína BRCA2/química , Proteínas de Ciclo Celular , Supervivencia Celular , Células Cultivadas , Mapeo Cromosómico , Secuencia Conservada , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Femenino , Estudios de Asociación Genética , Humanos , Funciones de Verosimilitud , Masculino , Ratones , Ratones Transgénicos , Mitomicina/farmacología , Modelos Moleculares , Mutágenos/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Cuaternaria de Proteína , Homología Estructural de ProteínaRESUMEN
Ectodermal appendages, such as the mammary gland (MG), are thought to have evolved from hair-associated apocrine glands to serve the function of milk secretion. Through the directed differentiation of mouse embryonic stem cells (mESCs), here, we report the generation of multilineage ESC-derived mammary organoids (MEMOs). We adapted the skin organoid model, inducing the dermal mesenchyme to transform into mammary-specific mesenchyme via the sequential activation of Bone Morphogenetic Protein 4 (BMP4) and Parathyroid Hormone-related Protein (PTHrP) and inhibition of hedgehog (HH) signaling. Using single-cell RNA sequencing, we identified gene expression profiles that demonstrate the presence of mammary-specific epithelial cells, fibroblasts, and adipocytes. MEMOs undergo ductal morphogenesis in Matrigel and can reconstitute the MG in vivo. Further, we demonstrate that the loss of function in placode regulators LEF1 and TBX3 in mESCs results in impaired skin and MEMO generation. In summary, our MEMO model is a robust tool for studying the development of ectodermal appendages, and it provides a foundation for regenerative medicine and disease modeling.
Asunto(s)
Proteínas Hedgehog , Células Madre Embrionarias de Ratones , Ratones , Animales , Proteínas Hedgehog/metabolismo , Glándulas Mamarias Animales , Células Epiteliales , Diferenciación Celular , OrganoidesRESUMEN
Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in BRCA2 mutation carriers, cells undergoing loss of heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor of BRCA2 whose overexpression supports the viability of Brca2-null cells. Mechanistically, we showed that MLH1 interacts with Flap endonuclease 1 (FEN1) and competes to process the RNA flaps of Okazaki fragments. Together, they restrained the DNA2 nuclease activity on the reversed forks of lagging strands, leading to replication fork (RF) stability in BRCA2-deficient cells. In these cells, MLH1 also attenuated R-loops, allowing the progression of stable RFs, which suppressed genomic instability and supported cell viability. We demonstrated the significance of their genetic interaction by the lethality of Brca2-mutant mice and inhibition of Brca2-deficient tumor growth in mice by Mlh1 loss. Furthermore, we described estrogen as inducing MLH1 expression through estrogen receptor α (ERα), which might explain why the majority of BRCA2 mutation carriers develop ER-positive breast cancer. Taken together, our findings reveal a role of MLH1 in relieving replicative stress and show how it may contribute to the establishment of BRCA2-deficient breast tumors.