BACKGROUND: Monoallelic germline pathogenic variants (GPVs) in five Fanconi anemia (FA) genes (BRCA1/FANCS, BRCA2/FANCD1, PALB2/FANCN, BRIP1/FANCJ, and RAD51C/FANCO) confer an increased risk of breast (BC) and/or ovarian (OC) cancer, but the role of GPVs in 17 other FA genes remains unclear. METHODS: Here, we investigated the association of germline variants in FANCG/XRCC9 with BC and OC risk. RESULTS: The frequency of truncating GPVs in FANCG did not differ between BC (20/10,204; 0.20%) and OC (8/2966; 0.27%) patients compared to controls (6/3250; 0.18%). In addition, only one out of five tumor samples showed loss-of-heterozygosity of the wild-type FANCG allele. Finally, none of the nine functionally tested rare recurrent missense FANCG variants impaired DNA repair activities (FANCD2 monoubiquitination and FANCD2 foci formation) upon DNA damage, in contrast to all tested FANCG truncations. CONCLUSION: Our study suggests that heterozygous germline FANCG variants are unlikely to contribute to the development of BC or OC.

• MeSH
• dospělí MeSH
• genetická predispozice k nemoci * MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory prsu * genetika   MeSH
• nádory vaječníků * genetika   MeSH
• oprava DNA genetika   MeSH
• protein FANCG * genetika   MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• zárodečné mutace * MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

• MeSH
• cyklin E genetika   metabolismus   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * genetika   metabolismus   MeSH
• nádory * MeSH
• nestabilita genomu MeSH
• proteinfosfatasa 2C genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cílené sekvenování nové generace umožňuje analýzu stovek nádorových predispozičních genů u onkologicky nemocných s podezřením na výskyt dědičné formy onemocnění. Identifikace příčinných mutací má zásadní prognostický, ale i prediktivní význam u pacientů a jejich příbuzných. S podporou předchozího projektu AZV 16-29959A (2016-19) jsme ustanovili konsorcium 9 laboratoří unifikující vyšetření nádorové predispozice pomocí CZECANCA panelu, vytvořili jsme společnou databázi genotypů analyzovaných jednotným bioinformatickým postupem u >6200 vysoce rizikových onkologických pacientů, provedli jsme analýzu >700 vzorků nenádorových kontrol identifikující genetické pozadí v ČR a připravili jsme in vitro modelové systémy pro hodnocení variant nejasného významu. Cílem navazujícího projektu je pokračování těchto aktivit, umožňující zevrubnou charakterizaci architektury nádorové predispozice u různých typů nádorů v ČR, určení klinické významnosti populačně specifických variant, identifikaci rizik u doposud nejasně charakterizovaných predispozičních genů s cílem zlepšení péče o vysoce rizikové osoby.; Targeted next gene sequencing enables to analyze hundreds of cancer predisposition genes in high-risk individuals. Identification of causal mutation has critical prognostic a predictive significance for the patient and their relatives. Supported by previous grant AZV 16-29959A (2016-19), we established a consortium of 9 Czech clinical laboratories unifying cancer predisposition analysis by CZECANCA (Czech cancer panel for clinical application) approach, developed joined database of over 6200 genotypes of analyzed patients, analyzed over 700 controls enabling to identify population-specific genetic background and prepared model systems for in vitro analyses of variants of uncertain significance in several genes. Proposed project aims to continue in these activities to comprehensively characterize genetic architecture of cancer predisposition in patients with various cancers in the Czech Republic, to identify clinically important population-specific mutations, and to elucidate cancer risks associated with mutations in poorly characterized predisposition genes.

• Klíčová slova
• NGS, NGS, bioinformatika, bioinformatics, nádorová predispozice, cancer predisposition, dědičné nádory, funkční analýzy, hereditary tumors, functional analyses,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Cell cycle checkpoints, oncogene-induced senescence and programmed cell death represent intrinsic barriers to tumorigenesis. Protein phosphatase magnesium-dependent 1 (PPM1D) is a negative regulator of the tumour suppressor p53 and has been implicated in termination of the DNA damage response. Here, we addressed the consequences of increased PPM1D activity resulting from the gain-of-function truncating mutations in exon 6 of the PPM1D. We show that while control cells permanently exit the cell cycle and reside in senescence in the presence of DNA damage caused by ionising radiation or replication stress induced by the active RAS oncogene, RPE1-hTERT and BJ-hTERT cells carrying the truncated PPM1D continue proliferation in the presence of DNA damage, form micronuclei and accumulate genomic rearrangements revealed by karyotyping. Further, we show that increased PPM1D activity promotes cell growth in the soft agar and formation of tumours in xenograft models. Finally, expression profiling of the transformed clones revealed dysregulation of several oncogenic and tumour suppressor pathways. Our data support the oncogenic potential of PPM1D in the context of exposure to ionising radiation and oncogene-induced replication stress.

• MeSH
• buněčná smrt genetika   MeSH
• lidé MeSH
• myši MeSH
• nádorová transformace buněk * genetika   MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• poškození DNA * genetika   MeSH
• proliferace buněk genetika   MeSH
• proteinfosfatasa 2C * genetika   metabolismus   MeSH
• proteinfosfatasy genetika   metabolismus   MeSH
• stárnutí buněk * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

RAD18 is an E3 ubiquitin ligase that prevents replication fork collapse by promoting DNA translesion synthesis and template switching. Besides this classical role, RAD18 has been implicated in homologous recombination; however, this function is incompletely understood. Here, we show that RAD18 is recruited to DNA lesions by monoubiquitination of histone H2A at K15 and counteracts accumulation of 53BP1. Super-resolution microscopy revealed that RAD18 localizes to the proximity of DNA double strand breaks and limits the distribution of 53BP1 to the peripheral chromatin nanodomains. Whereas auto-ubiquitination of RAD18 mediated by RAD6 inhibits its recruitment to DNA breaks, interaction with SLF1 promotes RAD18 accumulation at DNA breaks in the post-replicative chromatin by recognition of histone H4K20me0. Surprisingly, suppression of 53BP1 function by RAD18 is not involved in homologous recombination and rather leads to reduction of non-homologous end joining. Instead, we provide evidence that RAD18 promotes HR repair by recruiting the SMC5/6 complex to DNA breaks. Finally, we identified several new loss-of-function mutations in RAD18 in cancer patients suggesting that RAD18 could be involved in cancer development.

• MeSH
• 53BP1 * metabolismus   genetika   MeSH
• chromatin * metabolismus   genetika   MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• dvouřetězcové zlomy DNA * MeSH
• histony * metabolismus   MeSH
• homologní rekombinace genetika   MeSH
• lidé MeSH
• oprava DNA spojením konců MeSH
• oprava DNA MeSH
• proteiny buněčného cyklu metabolismus   genetika   MeSH
• rekombinační oprava DNA MeSH
• replikace DNA MeSH
• ubikvitinace * MeSH
• ubikvitinligasy * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• buněčná diferenciace MeSH
• buněčný rodokmen MeSH
• hematopoetické kmenové buňky * cytologie   metabolismus   MeSH
• hematopoéza MeSH
• lidé MeSH
• proteinfosfatasy metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• komentáře MeSH
• úvodníky MeSH

• Publikační typ
• abstrakt z konference MeSH

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

• MeSH
• checkpoint kinasa 2 genetika   MeSH
• genetická predispozice k nemoci MeSH
• lidé MeSH
• missense mutace MeSH
• nádory prsu * epidemiologie   genetika   MeSH
• zárodečné buňky MeSH
• zárodečné mutace MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• metaanalýza MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Gen CHEK2 kóduje kinázu CHK2, fosforylující řadu intracelulárních proteinů v odpovědi na poškození DNA. Patogenní, zárodečné mutace v CHEK2 zvyšují riziko vzniku karcinomu prsu a některých dalších nádorů. Analýza CHEK2 je v současnosti součástí genetické analýzy nádorové predispozice v ČR. V předchozích analýzách jsme mutace v CHEK2 nalezli u více jak 6,5% nemocných s ca prsu a/nebo ovaria a ukázali jsme, že většinu alterací tvoří varianty nejasného významu (VUS). Pro jejich funkční klasifikaci jsme vyvinuli unikátní systém založený na kvantifikaci fosforylace KAP1 katalyzované CHK2 v buněčné linii. Ve studii uzpůsobíme modelový systém pro vysokokapacitní analýzu a provedeme klasifikaci všech VUS v CHEK2 detekovaných spolupracujícími pracovišti u pacientů v ČR a zahraničí. Výsledky studie zvýší diagnostickou výtěžnost analýz nádorové predispozice a umožní charakterizovat pacienty s patogenními mutacemi CHEK2, což je základním předpokladem pro genetické poradenství v rodinách nosičů, stanovení rizika nádorů asociovaných s mutacemi v genu CHEK2 a racionalizaci preventivních opatření.; The CHEK2 gene codes for a checkpoint kinase CHK2 phosphorylating various intracellular proteins in response to DNA damage. Pathogenic germ-line CHEK2 mutations confer an increased breast and other cancer risk. CHEK2 mutation analysis is currently an obligatory procedure required for diagnostics in Czech hereditary cancer patients. We have shown that the prevalence of germline CHEK2 variants exceeds 6.5% in high-risk breast/ovarian Czech cancer patients and that the majority of found alterations represent variants of unknown significance (VUS). We developed a unique cell-based approach quantifying CHK2-mediated phosphorylation of KAP1 protein in vivo that enables reliable functional classification of CHEK2 VUS. Here we aim to optimize the assay for the high throughput analysis and classify all CHEK2 VUS identified by Czech and international collaborators. This study will enable clinical characterization of individuals carrying deleterious CHEK2 mutations, increasing diagnostic utility of CHEK2 analysis, improving risk estimation, and enabling optimal follow-up and prevention set-up.

• Klíčová slova
• breast cancer, karcinom prsu, varianta nejasného významu, hereditary cancer syndromes, functional analysis, dědičné nádorové syndromy, nádorová predispozice, CHEK2, CHK2 kináza, funkční ananlýza, CHEK2, CHK2 kinase, cancer predisposition, variant of uncertain significance,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.

• MeSH
• akutní myeloidní leukemie * genetika   MeSH
• mutace MeSH
• myši MeSH
• nádorový supresorový protein p53 * genetika   MeSH
• poškození DNA MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH