RESUMO
Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, pulmonary cysts, pneumothoraces and renal cell carcinomas. Here, we reveal a novel hereditary disorder in a family with skin and mucosal lesions, extensive lipomatosis and renal cell carcinomas. The proband was initially diagnosed with BHD based on the presence of fibrofolliculomas, but no pathogenic germline variant was detected in FLCN, the gene associated with BHD. By whole exome sequencing we identified a heterozygous missense variant (p.(Cys677Tyr)) in a zinc-finger encoding domain of the PRDM10 gene which co-segregated with the phenotype in the family. We show that PRDM10Cys677Tyr loses affinity for a regulatory binding motif in the FLCN promoter, abrogating cellular FLCN mRNA and protein levels. Overexpressing inducible PRDM10Cys677Tyr in renal epithelial cells altered the transcription of multiple genes, showing overlap but also differences with the effects of knocking out FLCN. We propose that PRDM10 controls an extensive gene program and acts as a critical regulator of FLCN gene transcription in human cells. The germline variant PRDM10Cys677Tyr curtails cellular folliculin expression and underlies a distinguishable syndrome characterized by extensive lipomatosis, fibrofolliculomas and renal cell carcinomas.
Assuntos
Síndrome de Birt-Hogg-Dubé , Carcinoma de Células Renais , Neoplasias Renais , Lipomatose , Neoplasias Cutâneas , Humanos , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patologia , Carcinoma de Células Renais/genética , Genes Supressores de Tumor , Neoplasias Cutâneas/genética , Lipomatose/genética , Neoplasias Renais/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
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.
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In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCNPOS and FLCNNEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCNNEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.
Assuntos
Síndrome de Birt-Hogg-Dubé , Neoplasias Renais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/metabolismo , Síndrome de Birt-Hogg-Dubé/patologia , Efrinas , Receptores ErbB , Humanos , Neoplasias Renais/genética , Fosfosserina , Proteínas Proto-Oncogênicas , Serina-Treonina Quinases TOR , Proteínas Supressoras de Tumor , TirosinaRESUMO
BACKGROUND: CRISPR screens provide large-scale assessment of cellular gene functions. Pooled libraries typically consist of several single guide RNAs (sgRNAs) per gene, for a large number of genes, which are transduced in such a way that every cell receives at most one sgRNA, resulting in the disruption of a single gene in that cell. This approach is often used to investigate effects on cellular fitness, by measuring sgRNA abundance at different time points. Comparing gene knockout effects between different cell populations is challenging due to variable cell-type specific parameters and between replicates variation. Failure to take those into account can lead to inflated or false discoveries. RESULTS: We propose a new, flexible approach called ShrinkCRISPR that can take into account multiple sources of variation. Impact on cellular fitness between conditions is inferred by using a mixed-effects model, which allows to test for gene-knockout effects while taking into account sgRNA-specific variation. Estimates are obtained using an empirical Bayesian approach. ShrinkCRISPR can be applied to a variety of experimental designs, including multiple factors. In simulation studies, we compared ShrinkCRISPR results with those of drugZ and MAGeCK, common methods used to detect differential effect on cell fitness. ShrinkCRISPR yielded as many true discoveries as drugZ using a paired screen design, and outperformed both drugZ and MAGeCK for an independent screen design. Although conservative, ShrinkCRISPR was the only approach that kept false discoveries under control at the desired level, for both designs. Using data from several publicly available screens, we showed that ShrinkCRISPR can take data for several time points into account simultaneously, helping to detect early and late differential effects. CONCLUSIONS: ShrinkCRISPR is a robust and flexible approach, able to incorporate different sources of variations and to test for differential effect on cell fitness at the gene level. These improve power to find effects on cell fitness, while keeping multiple testing under the correct control level and helping to improve reproducibility. ShrinkCrispr can be applied to different study designs and incorporate multiple time points, making it a complete and reliable tool to analyze CRISPR screen data.
Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Reprodutibilidade dos Testes , Teorema de Bayes , Técnicas de Inativação de GenesRESUMO
Previously, we reported a series of families presenting with trichodiscomas, inherited in an autosomal dominant pattern. The phenotype was named familial multiple discoid fibromas (FMDF). The genetic cause of FMDF remained unknown so far. Trichodiscomas are skin lesions previously reported to be part of the same spectrum as the fibrofolliculoma observed in Birt-Hogg-Dubé syndrome (BHD), an inherited disease caused by pathogenic variants in the FLCN gene. Given the clinical and histological differences with BHD and the exclusion of linkage with the FLCN locus, the phenotype was concluded to be distinct from BHD. We performed extensive clinical evaluations and genetic testing in ten families with FMDF. We identified a FNIP1 frameshift variant in nine families and genealogical studies showed common ancestry for eight families. Using whole exome sequencing, we identified six additional rare variants in the haplotype surrounding FNIP1, including a missense variant in the PDGFRB gene that was found to be present in all tested patients with FMDF. Genome-wide linkage analysis showed that the locus on chromosome 5 including FNIP1 was the only region reaching the maximal possible LOD score. We concluded that FMDF is linked to a haplotype on chromosome 5. Additional evaluations in families with FMDF are required to unravel the exact genetic cause underlying the phenotype. When evaluating patients with multiple trichodisomas without a pathogenic variant in the FLCN gene, further genetic testing is warranted and can include analysis of the haplotype on chromosome 5.
Assuntos
Síndrome de Birt-Hogg-Dubé , Fibroma , Neoplasias Renais , Humanos , Neoplasias Renais/genética , Cromossomos Humanos Par 5/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Proto-Oncogênicas/genética , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patologia , Fibroma/genética , Proteínas de Transporte/genéticaRESUMO
BACKGROUND: Previously, it has been suggested that colorectal polyps and carcinomas might be associated with Birt-Hogg-Dubé syndrome. We aimed to compare the occurrence of colorectal neoplasms between Dutch patients with Birt-Hogg-Dubé syndrome and their relatives without Birt-Hogg-Dubé syndrome. METHODS: In all, 399 patients with a pathogenic FLCN mutation and 382 relatives without the familial FLCN mutation were included. Anonymous data on colon and rectum pathology was provided by PALGA: the Dutch Pathology Registry. RESULTS: No significant difference in the percentage of individuals with a history of colorectal carcinoma was found between the two groups (3.6% vs 2.6%, p = 0.54). There was also no significant difference between the age at diagnosis, diameter, differentiation and location of the colorectal carcinomas. Significantly more individuals with Birt-Hogg-Dubé syndrome underwent removal of colorectal polyps (12.2% vs 6.3%, p = 0.005). However, there was no significant difference between the number of polyps per person, the histology, grade of dysplasia and location of the polyps. CONCLUSION: Our data do not provide evidence for an increased risk for colorectal carcinoma in Birt-Hogg-Dubé syndrome, arguing against the need for colorectal surveillance. The difference in polyps might be due to a bias caused by a higher number of colonoscopies in patients with Birt-Hogg-Dubé syndrome.
Assuntos
Síndrome de Birt-Hogg-Dubé/complicações , Neoplasias Colorretais/epidemiologia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , PrevalênciaRESUMO
More than half of the human genome consists of repetitive sequences, with the ribosomal DNA (rDNA) representing two of the largest repeats. Repetitive rDNA sequences may form a threat to genomic integrity and cellular homeostasis due to the challenging aspects of their transcription, replication, and repair. Predisposition to cancer, premature aging, and neurological impairment in ataxia-telangiectasia and Bloom syndrome, for instance, coincide with increased cellular rDNA repeat instability. However, the mechanisms by which rDNA instability contributes to these hereditary syndromes and tumorigenesis remain unknown. Here, we review how cells govern rDNA stability and how rDNA break repair influences expansion and contraction of repeat length, a process likely associated with human disease. Recent advancements in CRISPR-based genome engineering may help to explain how cells keep their rDNA intact in the near future.
Assuntos
DNA Ribossômico/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Dano ao DNA , Reparo do DNA , Replicação do DNA , Estudos de Associação Genética , Predisposição Genética para Doença , Instabilidade Genômica , Genômica/métodos , Humanos , Transcrição GênicaRESUMO
BACKGROUND: Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. RESULTS: We present "rscreenorm", a method that standardizes the functional data ranges between screens using assay controls, and subsequently performs a piecewise-linear normalization to make data distributions across all screens comparable. In simulation studies, rscreenorm reduces false positives. Using two multiple-cell lines siRNA screens, rscreenorm increased reproducibility between 27 and 62% for hits, and up to 5-fold for non-hits. Using publicly available CRISPR-Cas screen data, application of commonly used median centering yields merely 34% of overlapping hits, in contrast with rscreenorm yielding 84% of overlapping hits. Furthermore, rscreenorm yielded at most 8% discordant results, whilst median-centering yielded as much as 55%. CONCLUSIONS: Rscreenorm yields more consistent results and keeps false positive rates under control, improving reproducibility of genetic screens data analysis from multiple cell lines.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Testes Genéticos/métodos , Genômica/métodos , RNA Interferente Pequeno/genética , Humanos , Reprodutibilidade dos TestesRESUMO
E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Fatores de Transcrição E2F/metabolismo , Retroalimentação Fisiológica , Fase S , Animais , Proteínas Cdh1/genética , Proteínas Cdh1/metabolismo , Células Cultivadas , Ciclinas/metabolismo , Fatores de Transcrição E2F/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and TFIIH around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a new TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryo-EM and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.
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Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.
Assuntos
Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Mitógenos/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteína p107 Retinoblastoma-Like/fisiologia , Proteína p130 Retinoblastoma-Like/fisiologia , Animais , Apoptose/fisiologia , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Ciclina A/antagonistas & inibidores , Ciclina A/metabolismo , Ciclina B/efeitos dos fármacos , Ciclina B/metabolismo , Ciclina B1 , Inibidor de Quinase Dependente de Ciclina p21/farmacologia , Inibidor de Quinase Dependente de Ciclina p27/farmacologia , Dano ao DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Fase G2/efeitos dos fármacos , Fase G2/fisiologia , Camundongos , Camundongos Knockout , Mitógenos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Proteína do Retinoblastoma/genética , Proteína p107 Retinoblastoma-Like/genética , Proteína p130 Retinoblastoma-Like/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.
Assuntos
Proteínas Associadas a CRISPR , Neoplasias , Humanos , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Edição de Genes , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , DNA/genética , Mutação , Endonucleases/genética , Neoplasias/diagnóstico , Neoplasias/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Several hereditary disorders, with highly variable and sometimes difficult to recognize manifestations, can present with a spontaneous pneumothorax. Options to perform DNA-testing have changed rapidly, as a result of which physicians of diverse disciplines are coming into contact with hereditary disorders. CASE DESCRIPTION: Two patients with a history of multiple spontaneous pneumothoraxes were seen at the outpatient clinic of the department of Clinical Genetics. Based on family history and physical examination, a suspicion of an underlying hereditary disorder arose. Birt-Hogg-Dubé syndrome and vascular Ehlers-Danlos syndrome were diagnosed through DNA-testing. Based on this, additional screening advices were given and DNA-testing became possible in the family. CONCLUSION: A spontaneous pneumothorax may be a manifestation of an underlying hereditary disorder. With attention to clinical symptoms and family history, physicians can contribute to timely diagnosis. In many cases this results in significant health benefits for both the patient and affected family members, such as screening for kidney cancer in the case of Birt-Hogg-Dubé syndrome.
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Síndrome de Birt-Hogg-Dubé , Neoplasias Renais , Pneumotórax , Humanos , Pneumotórax/etiologia , Pneumotórax/genética , Síndrome de Birt-Hogg-Dubé/complicações , Síndrome de Birt-Hogg-Dubé/diagnóstico , Síndrome de Birt-Hogg-Dubé/genética , AnamneseRESUMO
BACKGROUND: We present a family consisting of a father and his two children with an exceptional phenotype of childhood renal cell carcinoma and brain tumors. Extensive genetic testing revealed two inherited tumor predisposition syndromes in all three family members: Birt-Hogg-Dubé syndrome and Li-Fraumeni syndrome. The corresponding genes (FLCN and TP53) are both located on the short arm of chromosome 17. METHODS: We describe the phenotype and performed single nucleotide polymorphism (SNP)-based loss of heterozygosity (LOH) analysis of the tumors. RESULTS: All examined tumors showed somatic loss of the wild-type alleles of both FLCN and TP53. CONCLUSIONS: We hypothesize that a synergistic effect of both mutations caused the unusual phenotype of childhood renal cell carcinoma in this family. This family emphasizes the importance of further genetic testing if a tumor develops at an unexpected young age in an inherited cancer predisposition syndrome.
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Neoplasias Encefálicas , Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Proteínas Supressoras de Tumor/genética , Predisposição Genética para Doença , Células Germinativas/metabolismo , Células Germinativas/patologia , Proteína Supressora de Tumor p53/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
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
Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply 'switch' upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas dos Microfilamentos/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Antígenos CD , Caderinas/genética , Caderinas/metabolismo , Proteínas Cdc20 , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Cinetocoros/metabolismo , Camundongos , Microscopia , Prenilação , Interferência de RNA , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Budding uninhibited by benzimidazoles (BUB1) contributes to multiple mitotic processes. Here, we describe the first two patients with biallelic BUB1 germline mutations, who both display microcephaly, intellectual disability, and several patient-specific features. The identified mutations cause variable degrees of reduced total protein level and kinase activity, leading to distinct mitotic defects. Both patients' cells show prolonged mitosis duration, chromosome segregation errors, and an overall functional spindle assembly checkpoint. However, while BUB1 levels mostly affect BUBR1 kinetochore recruitment, impaired kinase activity prohibits centromeric recruitment of Aurora B, SGO1, and TOP2A, correlating with anaphase bridges, aneuploidy, and defective sister chromatid cohesion. We do not observe accelerated cohesion fatigue. We hypothesize that unresolved DNA catenanes increase cohesion strength, with concomitant increase in anaphase bridges. In conclusion, BUB1 mutations cause a neurodevelopmental disorder, with clinical and cellular phenotypes that partially resemble previously described syndromes, including autosomal recessive primary microcephaly, mosaic variegated aneuploidy, and cohesinopathies.
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Segregação de Cromossomos , Microcefalia , Aneuploidia , Segregação de Cromossomos/genética , Humanos , Microcefalia/genética , Mutação , Proteínas Serina-Treonina Quinases/genéticaRESUMO
In 2020, the Nobel Prize in Chemistry was awarded to American molecular biologist Jennifer Doudna and her French colleague Emmanuelle Charpentier for their fundamental research on CRISPR, an ingenious bacterial immune system. Studies into the working mechanism of CRISPR led to many Eureka moments. Through smart biotechnological engineering, CRISPR became suitable for applications in 'DNA surgery': the targeted editing of the genetic code. Here, we discuss emerging medical CRISPR applications for the treatment of human genetic disorders, including in vivo therapy. This Nobel Prize-winning discovery is powerful, adaptable and accurate, and clinical trials are being launched at an amazing pace. However, extensive research is needed on safe clinical use and possible side effects of CRISPR. In addition, the regulations on market authorization and reimbursement are not yet tailored to this very personal and potentially expensive therapy. Whereas challenges remain, CRISPR gene therapy will continue to rapidly mature as a clinical reality.
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas , DNA , Feminino , Terapia Genética , HumanosRESUMO
ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.