RESUMO
The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.
RESUMO
Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.
Assuntos
Anemia de Fanconi , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas de Cabeça e Pescoço , Humanos , Proteínas de Ciclo Celular/genética , DNA , Emetina/uso terapêutico , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Estudo de Associação Genômica Ampla , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.
Assuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Proteínas Inibidoras de Apoptose/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alcinos/farmacologia , Proteína 3 com Repetições IAP de Baculovírus/antagonistas & inibidores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Anemia de Fanconi/complicações , Anemia de Fanconi/imunologia , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/complicações , Neoplasias de Cabeça e Pescoço/imunologia , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oligopeptídeos/farmacologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAP/genética , Proteínas de Sinalização YAP/metabolismoRESUMO
Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanism by which FLCN prevents kidney cancer remains unknown. Here, we show that disrupting FLCN in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.
Assuntos
Proteínas de Transporte/genética , Células Epiteliais/metabolismo , Rim/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Transporte/metabolismo , Mutação em Linhagem Germinativa , Humanos , Interferons/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.
Assuntos
Anormalidades Múltiplas/etiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Troca de Cromátide Irmã , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proliferação de Células , RNA Helicases DEAD-box/química , DNA Helicases/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Estabilidade Proteica , Pseudogenes , RNA Helicases/genética , RNA Helicases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Síndrome , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In a process linked to DNA replication, duplicated chromosomes are entrapped in large, circular cohesin complexes and functional sister chromatid cohesion (SCC) is established by acetylation of the SMC3 cohesin subunit. Roberts Syndrome (RBS) and Warsaw Breakage Syndrome (WABS) are rare human developmental syndromes that are characterized by defective SCC. RBS is caused by mutations in the SMC3 acetyltransferase ESCO2, whereas mutations in the DNA helicase DDX11 lead to WABS. We found that WABS-derived cells predominantly rely on ESCO2, not ESCO1, for residual SCC, growth and survival. Reciprocally, RBS-derived cells depend on DDX11 to maintain low levels of SCC. Synthetic lethality between DDX11 and ESCO2 correlated with a prolonged delay in mitosis, and was rescued by knockdown of the cohesin remover WAPL. Rescue experiments using human or mouse cDNAs revealed that DDX11, ESCO1 and ESCO2 act on different but related aspects of SCC establishment. Furthermore, a DNA binding DDX11 mutant failed to correct SCC in WABS cells and DDX11 deficiency reduced replication fork speed. We propose that DDX11, ESCO1 and ESCO2 control different fractions of cohesin that are spatially and mechanistically separated.
Assuntos
Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Células Epiteliais/enzimologia , Fibroblastos/enzimologia , Acetiltransferases/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/metabolismo , Quebra Cromossômica , Segregação de Cromossomos , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , RNA Helicases DEAD-box/metabolismo , DNA Helicases/metabolismo , Ectromelia/enzimologia , Ectromelia/genética , Ectromelia/patologia , Células Epiteliais/patologia , Fibroblastos/patologia , Expressão Gênica , Humanos , Hipertelorismo/enzimologia , Hipertelorismo/genética , Hipertelorismo/patologia , Camundongos , Mitose , Modelos Biológicos , Mutação , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , CoesinasRESUMO
Head and neck squamous cell carcinomas (HNSCCs) coincide with poor survival rates. The lack of driver oncogenes complicates the development of targeted treatments for HNSCC. Here, we follow-up on two previous genome-wide RNA and microRNA interference screens in HNSCC to cross-examine tumor-specific lethality by targeting ATM, ATR, CHEK1, or CHEK2. Our results uncover CHEK1 as the most promising target for HNSCC. CHEK1 expression is essential across a panel of HNSCC cell lines but redundant for growth and survival of untransformed oral keratinocytes and fibroblasts. LY2603618 (Rabusertib), which specifically targets Chk1 kinase, kills HNSCC cells effectively and specifically. Our findings show that HNSCC cells depend on Chk1-mediated signaling to progress through S-phase successfully. Chk1 inhibition coincides with stalled DNA replication, replication fork collapses, and accumulation of DNA damage. We further show that Chk1 inhibition leads to bimodal HNSCC cell killing. In the most sensitive cell lines, apoptosis is induced in S-phase, whereas more resistant cell lines manage to bypass replication-associated apoptosis, but accumulate chromosomal breaks that become lethal in subsequent mitosis. Interestingly, CDK1 expression correlates with treatment outcome. Moreover, sensitivity to Chk1 inhibition requires functional CDK1 and CDK4/6 to drive cell cycle progression, arguing against combining Chk1 inhibitors with CDK inhibitors. In contrast, Wee1 inhibitor Adavosertib progresses the cell cycle and thereby increases lethality to Chk1 inhibition in HNSCC cell lines. We conclude that Chk1 has become a key molecule in HNSCC cell cycle regulation and a very promising therapeutic target. Chk1 inhibition leads to S-phase apoptosis or death in mitosis. We provide a potential efficacy biomarker and combination therapy to follow-up in clinical setting.
RESUMO
BRCA2 encodes a protein with a fundamental role in homologous recombination that is essential for normal development. Carrier status of mutations in BRCA2 is associated with familial breast and ovarian cancer, while bi-allelic BRCA2 mutations can cause Fanconi anemia (FA), a cancer predisposition syndrome with cellular cross-linker hypersensitivity. Cancers associated with BRCA2 mutations can acquire chemo-resistance on relapse. We modeled acquired cross-linker resistance with an FA-derived BRCA2-mutated acute myeloid leukemia (AML) platform. Associated with acquired cross-linker resistance was the expression of a functional BRCA2 protein variant lacking exon 5 and exon 7 (BRCA2ΔE5+7), implying a role for BRCA2 splicing for acquired chemo-resistance. Integrated network analysis of transcriptomic and proteomic differences for phenotyping of BRCA2 disruption infers impact on transcription and chromatin remodeling in addition to the DNA damage response. The striking overlap with transcriptional profiles of FA patient hematopoiesis and BRCA mutation associated ovarian cancer helps define and explicate the 'BRCAness' profile.
Assuntos
Processamento Alternativo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Resistencia a Medicamentos Antineoplásicos , Genes BRCA2 , Mutação , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Éxons , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Íntrons , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Recidiva Local de Neoplasia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fenótipo , Splicing de RNA , Transcrição GênicaRESUMO
Fanconi anaemia (FA) is a hereditary disease featuring hypersensitivity to DNA cross-linker-induced chromosomal instability in association with developmental abnormalities, bone marrow failure and a strong predisposition to cancer. A total of 17 FA disease genes have been reported, all of which act in a recessive mode of inheritance. Here we report on a de novo g.41022153G>A; p.Ala293Thr (NM_002875) missense mutation in one allele of the homologous recombination DNA repair gene RAD51 in an FA-like patient. This heterozygous mutation causes a novel FA subtype, 'FA-R', which appears to be the first subtype of FA caused by a dominant-negative mutation. The patient, who features microcephaly and mental retardation, has reached adulthood without the typical bone marrow failure and paediatric cancers. Together with the recent reports on RAD51-associated congenital mirror movement disorders, our results point to an important role for RAD51-mediated homologous recombination in neurodevelopment, in addition to DNA repair and cancer susceptibility.
Assuntos
Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Anemia de Fanconi/enzimologia , Mutação de Sentido Incorreto , Hidrolases Anidrido Ácido , Sequência de Bases , Dano ao DNA , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Anemia de Fanconi/genética , Humanos , Masculino , Dados de Sequência Molecular , Recombinação Genética , Adulto JovemRESUMO
Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31(comet). A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/fisiologia , Troca de Cromátide Irmã/fisiologia , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Segregação de Cromossomos , Humanos , Mitose/fisiologia , Morfolinas/farmacologia , Nocodazol/farmacologia , Paclitaxel/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Troca de Cromátide Irmã/efeitos dos fármacos , Moduladores de Tubulina/farmacologiaRESUMO
Failure to repair DNA damage or defective sister chromatid cohesion, a process essential for correct chromosome segregation, can be causative of chromosomal instability (CIN), which is a hallmark of many types of cancers. We investigated how frequent this occurs in head and neck squamous cell carcinoma (HNSCC) and whether specific mechanisms or genes could be linked to these phenotypes. The genomic instability syndrome Fanconi anemia is caused by mutations in any of at least 16 genes regulating DNA interstrand crosslink (ICL) repair. Since patients with Fanconi anemia have a high risk to develop HNSCC, we investigated whether and to which extent Fanconi anemia pathway inactivation underlies CIN in HNSCC of non-Fanconi anemia individuals. We observed ICL-induced chromosomal breakage in 9 of 17 (53%) HNSCC cell lines derived from patients without Fanconi anemia. In addition, defective sister chromatid cohesion was observed in five HNSCC cell lines. Inactivation of FANCM was responsible for chromosomal breakage in one cell line, whereas in two other cell lines, somatic mutations in PDS5A or STAG2 resulted in inadequate sister chromatid cohesion. In addition, FANCF methylation was found in one cell line by screening an additional panel of 39 HNSCC cell lines. Our data demonstrate that CIN in terms of ICL-induced chromosomal breakage and defective chromatid cohesion is frequently observed in HNSCC. Inactivation of known Fanconi anemia and chromatid cohesion genes does explain CIN in the minority of cases. These findings point to phenotypes that may be highly relevant in treatment response of HNSCC.
Assuntos
Carcinoma de Células Escamosas/genética , Instabilidade Cromossômica/genética , Anemia de Fanconi/genética , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cromátides/genética , Dano ao DNA/genética , Reparo do DNA/genética , Anemia de Fanconi/patologia , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Masculino , Mutação , Estadiamento de Neoplasias , Troca de Cromátide Irmã , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
The encouraging response rates of BRCA1- and BRCA2-mutated cancers toward PARP inhibitors make it worthwhile to identify other potential determinants of PARP inhibitor responsiveness. Since the Fanconi anemia (FA) pathway coordinates several DNA repair pathways, including homologous recombination in which BRCA1 and BRCA2 play important roles, we investigated whether this pathway harbors other predictors of PARP inhibitor sensitivity. Lymphoblastoid cell lines derived from individuals with FA or clinically related syndromes, such as Warsaw breakage syndrome, were tested for PARP inhibitor sensitivity. Remarkably, we found a strong variability in PARP inhibitor sensitivity among different FANCD1/BRCA2-deficient lymphoblasts, suggesting that PARP inhibitor response depends on the type of FANCD1/BRCA2 mutation. We identified the DNA helicases FANCM and DDX11 as determinants of PARP inhibitor response. These results may extend the utility of PARP inhibition as effective anticancer treatment.
Assuntos
RNA Helicases DEAD-box/genética , DNA Helicases/genética , Inibidores Enzimáticos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína BRCA2/genética , Linhagem Celular Transformada , Anemia de Fanconi/genética , Feminino , Fluorbenzenos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Masculino , Ftalazinas/farmacologiaRESUMO
Fanconi anemia (FA) is a genetically heterogeneous syndrome associated with increased cancer predisposition. The underlying genes govern the FA pathway which functions to protect the genome during the S-phase of the cell cycle. While upregulation of FA genes has been linked to chemotherapy resistance, little is known about their regulation in response to proliferative stimuli. The purpose of this study was to examine how FA genes are regulated, especially in relation to the cell cycle, in order to reveal their possible participation in biochemical networks. Expression of 14 FA genes was monitored in two human cell-cycle models and in two RB1/E2F pathway-associated primary cancers, retinoblastoma and basal breast cancer. In silico studies were performed to further evaluate coregulation and identify connected networks and diseases. Only FANCA was consistently induced over 2-fold; FANCF failed to exhibit any regulatory fluctuations. Two tools exploiting public data sets indicated coregulation of FANCA with BRCA1. Upregulation of FANCA and BRCA1 correlated with upregulation of E2F3. Genes coregulated with both FANCA and BRCA1 were enriched for MeSH-Term id(s) genomic instability, microcephaly, and Bloom syndrome, and enriched for the cellular component centrosome. The regulation of FA genes appears highly divergent. In RB1-linked tumors, upregulation of FA network genes was associated with reduced expression of FANCF. FANCA and BRCA1 may jointly act in a subnetwork - supporting vital function(s) at the subcellular level (centrosome) as well as at the level of embryonic development (mechanisms controlling head circumference).
RESUMO
Fanconi anemia (FA) is a rare genomic instability syndrome. Disease-causing are biallelic mutations in any one of at least 15 genes encoding members of the FA/BRCA pathway of DNA-interstrand crosslink repair. Patients are diagnosed based upon phenotypical manifestations and the diagnosis of FA is confirmed by the hypersensitivity of cells to DNA interstrand crosslinking agents. Customary molecular diagnostics has become increasingly cumbersome, time-consuming and expensive the more FA genes have been identified. We performed Whole Exome Sequencing (WES) in four FA patients in order to investigate the potential of this method for FA genotyping. In search of an optimal WES methodology we explored different enrichment and sequencing techniques. In each case we were able to identify the pathogenic mutations so that WES provided both, complementation group assignment and mutation detection in a single approach. The mutations included homozygous and heterozygous single base pair substitutions and a two-base-pair duplication in FANCJ, -D1, or -D2. Different WES strategies had no critical influence on the individual outcome. However, database errors and in particular pseudogenes impose obstacles that may prevent correct data perception and interpretation, and thus cause pitfalls. With these difficulties in mind, our results show that WES is a valuable tool for the molecular diagnosis of FA and a sufficiently safe technique, capable of engaging increasingly in competition with classical genetic approaches.
Assuntos
Exoma , Anemia de Fanconi/genética , Genótipo , Sequência de Bases , Ciclo Celular , Anemia de Fanconi/diagnóstico , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Linhagem , Reprodutibilidade dos TestesRESUMO
Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.
Assuntos
Epistasia Genética/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação G da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Mutação/genética , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quebra Cromossômica/efeitos dos fármacos , Embrião de Mamíferos , Feminino , Fertilidade/genética , Fibroblastos/citologia , Fluorbenzenos/farmacologia , Testes Hematológicos , Humanos , Masculino , Camundongos , Ovário/metabolismo , Ftalazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Testículo/metabolismoRESUMO
DNA interstrand crosslink repair requires several classes of proteins, including structure-specific endonucleases and Fanconi anemia proteins. SLX4, which coordinates three separate endonucleases, was recently recognized as an important regulator of DNA repair. Here we report the first human individuals found to have biallelic mutations in SLX4. These individuals, who were previously diagnosed as having Fanconi anemia, add SLX4 as an essential component to the FA-BRCA genome maintenance pathway.
Assuntos
Anemia de Fanconi/genética , Recombinases/genética , Alelos , Camptotecina/farmacologia , Criança , Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico/química , Humanos , Imunoprecipitação , Masculino , Mitomicina/farmacologia , Mutação , FenótipoRESUMO
The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.
Assuntos
Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Quebra Cromossômica , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Mutação/genética , Troca de Cromátide Irmã/genética , Xeroderma Pigmentoso/genética , Adolescente , Sequência de Bases , Pré-Escolar , RNA Helicases DEAD-box/deficiência , DNA Helicases/deficiência , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Neoplasias/genética , Linhagem , Fenótipo , Polônia , Gravidez , SíndromeRESUMO
Fanconi anemia (FA) is a recessively inherited disease characterized by multiple symptoms including growth retardation, skeletal abnormalities, and bone marrow failure. The FA diagnosis is complicated due to the fact that the clinical manifestations are both diverse and variable. A chromosomal breakage test using a DNA cross-linking agent, in which cells from an FA patient typically exhibit an extraordinarily sensitive response, has been considered the gold standard for the ultimate diagnosis of FA. In the majority of FA patients the test results are unambiguous, although in some cases the presence of hematopoietic mosaicism may complicate interpretation of the data. However, some diagnostic overlap with other syndromes has previously been noted in cases with Nijmegen breakage syndrome. Here we present results showing that misdiagnosis may also occur with patients suffering from two of the three currently known cohesinopathies, that is, Roberts syndrome (RBS) and Warsaw breakage syndrome (WABS). This complication may be avoided by scoring metaphase chromosomes-in addition to chromosomal breakage-for spontaneously occurring premature centromere division, which is characteristic for RBS and WABS, but not for FA.
RESUMO
The Fanconi anemia (FA) core complex member FANCM remodels synthetic replication forks and recombination intermediates. Thus far, only one FA patient with FANCM mutations has been described, but the relevance of these mutations for the FA phenotype is uncertain. To provide further experimental access to the FA-M complementation group we have generated Fancm-deficient mice by deleting exon 2. FANCM deficiency caused hypogonadism in mice and hypersensitivity to cross-linking agents in mouse embryonic fibroblasts (MEFs), thus phenocopying other FA mouse models. However, Fancm(Delta2/Delta2) mice also showed unique features atypical for FA mice, including underrepresentation of female Fancm(Delta2/Delta2) mice and decreased overall and tumor-free survival. This increased cancer incidence may be correlated to the role of FANCM in the suppression of spontaneous sister chromatid exchanges as observed in MEFs. In addition, FANCM appeared to have a stimulatory rather than essential role in FANCD2 monoubiquitination. The FA-M mouse model presented here suggests that FANCM functions both inside and outside the FA core complex to maintain genome stability and to prevent tumorigenesis.