NPJ Genomic Medicine最新文献

{"title":"Understanding the phenotypic variability in Niemann-Pick disease type C (NPC): a need for precision medicine.","authors":"Macarena Las Heras, Benjamín Szenfeld, Rami A Ballout, Emanuele Buratti, Silvana Zanlungo, Andrea Dardis, Andrés D Klein","doi":"10.1038/s41525-023-00365-w","DOIUrl":"10.1038/s41525-023-00365-w","url":null,"abstract":"<p><p>Niemann-Pick type C (NPC) disease is a lysosomal storage disease (LSD) characterized by the buildup of endo-lysosomal cholesterol and glycosphingolipids due to loss of function mutations in the NPC1 and NPC2 genes. NPC patients can present with a broad phenotypic spectrum, with differences at the age of onset, rate of progression, severity, organs involved, effects on the central nervous system, and even response to pharmacological treatments. This article reviews the phenotypic variation of NPC and discusses its possible causes, such as the remaining function of the defective protein, modifier genes, sex, environmental cues, and splicing factors, among others. We propose that these factors should be considered when designing or repurposing treatments for this disease. Despite its seeming complexity, this proposition is not far-fetched, considering the expanding interest in precision medicine and easier access to multi-omics technologies.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"21"},"PeriodicalIF":5.3,"publicationDate":"2023-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10421955/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9987873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Familial co-segregation and the emerging role of long-read sequencing to re-classify variants of uncertain significance in inherited retinal diseases.","authors":"Pankhuri Gupta, Kenji Nakamichi, Alyssa C Bonnell, Ryan Yanagihara, Nick Radulovich, Fuki M Hisama, Jennifer R Chao, Debarshi Mustafi","doi":"10.1038/s41525-023-00366-9","DOIUrl":"10.1038/s41525-023-00366-9","url":null,"abstract":"<p><p>Phasing genetic variants is essential in determining those that are potentially disease-causing. In autosomal recessive inherited retinal diseases (IRDs), reclassification of variants of uncertain significance (VUS) can provide a genetic diagnosis in indeterminate compound heterozygote cases. We report four cases in which familial co-segregation demonstrated a VUS resided in trans to a known pathogenic variant, which in concert with other supporting criteria, led to the reclassification of the VUS to likely pathogenic, thereby providing a genetic diagnosis in each case. We also demonstrate in a simplex patient without access to family members for co-segregation analysis that targeted long-read sequencing can provide haplotagged variant calling. This can elucidate if variants reside in trans and provide phase of genetic variants from the proband alone without parental testing. This emerging method can alleviate the bottleneck of haplotype analysis in cases where genetic testing of family members is unfeasible to provide a complete genetic diagnosis.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"20"},"PeriodicalIF":5.3,"publicationDate":"2023-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10412581/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9975638","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"T-cell priming transcriptomic markers: implications of immunome heterogeneity for precision immunotherapy.","authors":"Hirotaka Miyashita, Razelle Kurzrock, Nicholas J Bevins, Kartheeswaran Thangathurai, Suzanna Lee, Sarabjot Pabla, Mary Nesline, Sean T Glenn, Jeffrey M Conroy, Paul DePietro, Eitan Rubin, Jason K Sicklick, Shumei Kato","doi":"10.1038/s41525-023-00359-8","DOIUrl":"10.1038/s41525-023-00359-8","url":null,"abstract":"<p><p>Immune checkpoint blockade is effective for only a subset of cancers. Targeting T-cell priming markers (TPMs) may enhance activity, but proper application of these agents in the clinic is challenging due to immune complexity and heterogeneity. We interrogated transcriptomics of 15 TPMs (CD137, CD27, CD28, CD80, CD86, CD40, CD40LG, GITR, ICOS, ICOSLG, OX40, OX40LG, GZMB, IFNG, and TBX21) in a pan-cancer cohort (N = 514 patients, 30 types of cancer). TPM expression was analyzed for correlation with histological type, microsatellite instability high (MSI-H), tumor mutational burden (TMB), and programmed death-ligand 1 (PD-L1) expression. Among 514 patients, the most common histological types were colorectal (27%), pancreatic (11%), and breast cancer (10%). No statistically significant association between histological type and TPM expression was seen. In contrast, expression of GZMB (granzyme B, a serine protease stored in activated T and NK cells that induces cancer cell apoptosis) and IFNG (activates cytotoxic T cells) were significantly higher in tumors with MSI-H, TMB ≥ 10 mutations/mb and PD-L1 ≥ 1%. PD-L1 ≥ 1% was also associated with significantly higher CD137, GITR, and ICOS expression. Patients' tumors were classified into \"Hot\", \"Mixed\", or \"Cold\" clusters based on TPM expression using hierarchical clustering. The cold cluster showed a significantly lower proportion of tumors with PD-L1 ≥ 1%. Overall, 502 patients (98%) had individually distinct patterns of TPM expression. Diverse expression patterns of TPMs independent of histological type but correlating with other immunotherapy biomarkers (PD-L1 ≥ 1%, MSI-H and TMB ≥ 10 mutations/mb) were observed. Individualized selection of patients based on TPM immunomic profiles may potentially help with immunotherapy optimization.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"19"},"PeriodicalIF":5.3,"publicationDate":"2023-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10409760/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9969777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Scaling-up and future sustainability of a national reproductive genetic carrier screening program.","authors":"Zoe Fehlberg,&nbsp;Stephanie Best,&nbsp;Janet C Long,&nbsp;Tahlia Theodorou,&nbsp;Catherine Pope,&nbsp;Peter Hibbert,&nbsp;Sharon Williams,&nbsp;Lucinda Freeman,&nbsp;Sarah Righetti,&nbsp;Alison D Archibald,&nbsp;Jeffrey Braithwaite","doi":"10.1038/s41525-023-00357-w","DOIUrl":"https://doi.org/10.1038/s41525-023-00357-w","url":null,"abstract":"<p><p>An understanding of factors influencing implementation is essential to realise the benefits of population-based reproductive genetic carrier screening programs. The aim of this study was to synthesise data collected during the Australian Reproductive Genetic Carrier Screening Project (Mackenzie's Mission) to track how priorities shifted over time and identify important factors during scaling-up and for sustainment. We used a multi-method qualitative approach to integrate longitudinal project data collected from 10 project committees with 16 semi-structured interviews conducted with study team members. Both datasets were analysed using the Consolidated Framework for Implementation Research (CFIR) to identify constructs of interest within early, mid-point, and future implementation phases. Several CFIR constructs were present across implementation. The complexity of implementation presented challenges that were overcome through a quality-designed and packaged product, formal and informal networks and communication, and access to knowledge and information. Addressing the diverse consumer needs through resources and increasing community and non-genetic speciality engagement remained a priority throughout and for future sustainment. Going forward, further addressing program complexities and securing funding were emphasised. By applying an implementation framework, findings from this study may be useful for future effort towards building and/or sustaining reproductive genetic carrier screening programs.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"18"},"PeriodicalIF":5.3,"publicationDate":"2023-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10390466/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9927752","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chromatin regulators in the TBX1 network confer risk for conotruncal heart defects in 22q11.2DS.","authors":"Yingjie Zhao, Yujue Wang, Lijie Shi, Donna M McDonald-McGinn, T Blaine Crowley, Daniel E McGinn, Oanh T Tran, Daniella Miller, Jhih-Rong Lin, Elaine Zackai, H Richard Johnston, Eva W C Chow, Jacob A S Vorstman, Claudia Vingerhoets, Therese van Amelsvoort, Doron Gothelf, Ann Swillen, Jeroen Breckpot, Joris R Vermeesch, Stephan Eliez, Maude Schneider, Marianne B M van den Bree, Michael J Owen, Wendy R Kates, Gabriela M Repetto, Vandana Shashi, Kelly Schoch, Carrie E Bearden, M Cristina Digilio, Marta Unolt, Carolina Putotto, Bruno Marino, Maria Pontillo, Marco Armando, Stefano Vicari, Kathleen Angkustsiri, Linda Campbell, Tiffany Busa, Damian Heine-Suñer, Kieran C Murphy, Declan Murphy, Sixto García-Miñaúr, Luis Fernández, Zhengdong D Zhang, Elizabeth Goldmuntz, Raquel E Gur, Beverly S Emanuel, Deyou Zheng, Christian R Marshall, Anne S Bassett, Tao Wang, Bernice E Morrow","doi":"10.1038/s41525-023-00363-y","DOIUrl":"10.1038/s41525-023-00363-y","url":null,"abstract":"<p><p>Congenital heart disease (CHD) affecting the conotruncal region of the heart, occurs in 40-50% of patients with 22q11.2 deletion syndrome (22q11.2DS). This syndrome is a rare disorder with relative genetic homogeneity that can facilitate identification of genetic modifiers. Haploinsufficiency of TBX1, encoding a T-box transcription factor, is one of the main genes responsible for the etiology of the syndrome. We suggest that genetic modifiers of conotruncal defects in patients with 22q11.2DS may be in the TBX1 gene network. To identify genetic modifiers, we analyzed rare, predicted damaging variants in whole genome sequence of 456 cases with conotruncal defects and 537 controls, with 22q11.2DS. We then performed gene set approaches and identified chromatin regulatory genes as modifiers. Chromatin genes with recurrent damaging variants include EP400, KAT6A, KMT2C, KMT2D, NSD1, CHD7 and PHF21A. In total, we identified 37 chromatin regulatory genes, that may increase risk for conotruncal heart defects in 8.5% of 22q11.2DS cases. Many of these genes were identified as risk factors for sporadic CHD in the general population. These genes are co-expressed in cardiac progenitor cells with TBX1, suggesting that they may be in the same genetic network. The genes KAT6A, KMT2C, CHD7 and EZH2, have been previously shown to genetically interact with TBX1 in mouse models. Our findings indicate that disturbance of chromatin regulatory genes impact the TBX1 gene network serving as genetic modifiers of 22q11.2DS and sporadic CHD, suggesting that there are some shared mechanisms involving the TBX1 gene network in the etiology of CHD.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"17"},"PeriodicalIF":4.7,"publicationDate":"2023-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10354062/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10202134","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Atypical splicing variants in PKD1 explain most undiagnosed typical familial ADPKD.","authors":"Yvonne Hort,&nbsp;Patricia Sullivan,&nbsp;Laura Wedd,&nbsp;Lindsay Fowles,&nbsp;Igor Stevanovski,&nbsp;Ira Deveson,&nbsp;Cas Simons,&nbsp;Andrew Mallett,&nbsp;Chirag Patel,&nbsp;Timothy Furlong,&nbsp;Mark J Cowley,&nbsp;John Shine,&nbsp;Amali Mallawaarachchi","doi":"10.1038/s41525-023-00362-z","DOIUrl":"https://doi.org/10.1038/s41525-023-00362-z","url":null,"abstract":"<p><p>Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of kidney failure and is primarily associated with PKD1 or PKD2. Approximately 10% of patients remain undiagnosed after standard genetic testing. We aimed to utilise short and long-read genome sequencing and RNA studies to investigate undiagnosed families. Patients with typical ADPKD phenotype and undiagnosed after genetic diagnostics were recruited. Probands underwent short-read genome sequencing, PKD1 and PKD2 coding and non-coding analyses and then genome-wide analysis. Targeted RNA studies investigated variants suspected to impact splicing. Those undiagnosed then underwent Oxford Nanopore Technologies long-read genome sequencing. From over 172 probands, 9 met inclusion criteria and consented. A genetic diagnosis was made in 8 of 9 (89%) families undiagnosed on prior genetic testing. Six had variants impacting splicing, five in non-coding regions of PKD1. Short-read genome sequencing identified novel branchpoint, AG-exclusion zone and missense variants generating cryptic splice sites and a deletion causing critical intron shortening. Long-read sequencing confirmed the diagnosis in one family. Most undiagnosed families with typical ADPKD have splice-impacting variants in PKD1. We describe a pragmatic method for diagnostic laboratories to assess PKD1 and PKD2 non-coding regions and validate suspected splicing variants through targeted RNA studies.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"16"},"PeriodicalIF":5.3,"publicationDate":"2023-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10328916/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9804103","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tumor collagens predict genetic features and patient outcomes.","authors":"Kevin S Guo,&nbsp;Alexander S Brodsky","doi":"10.1038/s41525-023-00358-9","DOIUrl":"https://doi.org/10.1038/s41525-023-00358-9","url":null,"abstract":"<p><p>The extracellular matrix (ECM) is a critical determinant of tumor fate that reflects the output from myriad cell types in the tumor. Collagens constitute the principal components of the tumor ECM. The changing collagen composition in tumors along with their impact on patient outcomes and possible biomarkers remains largely unknown. The RNA expression of the 43 collagen genes from solid tumors in The Cancer Genome Atlas (TCGA) was clustered to classify tumors. PanCancer analysis revealed how collagens by themselves can identify the tissue of origin. Clustering by collagens in each cancer type demonstrated strong associations with survival, specific immunoenvironments, somatic gene mutations, copy number variations, and aneuploidy. We developed a machine learning classifier that predicts aneuploidy, and chromosome arm copy number alteration (CNA) status based on collagen expression alone with high accuracy in many cancer types with somatic mutations, suggesting a strong relationship between the collagen ECM context and specific molecular alterations. These findings have broad implications in defining the relationship between cancer-related genetic defects and the tumor microenvironment to improve prognosis and therapeutic targeting for patient care, opening new avenues of investigation to define tumor ecosystems.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"15"},"PeriodicalIF":5.3,"publicationDate":"2023-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10326062/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9796186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrating somatic CNV and gene expression in breast cancers from women with PTEN hamartoma tumor syndrome.","authors":"Takae Brewer,&nbsp;Lamis Yehia,&nbsp;Peter Bazeley,&nbsp;Charis Eng","doi":"10.1038/s41525-023-00361-0","DOIUrl":"https://doi.org/10.1038/s41525-023-00361-0","url":null,"abstract":"<p><p>Women with germline PTEN variants (PTEN hamartoma tumor syndrome, PHTS) have up to 85% lifetime risk of female breast cancer (BC). We previously showed that PHTS-derived BCs are distinct from sporadic BCs both at the clinical and genomic levels. In this study, we examined somatic copy number variations (CNV) and transcriptome data to further characterize the somatic landscape of PHTS-derived BCs. We analyzed exome sequencing data from 44 BCs from women with PHTS for CNV. The control group comprised of 558 women with sporadic BCs from The Cancer Genome Atlas (TCGA) dataset. Here, we found that PHTS-derived BCs have several distinct CNV peaks compared to TCGA. Furthermore, RNA sequencing data revealed that PHTS-derived BCs have a distinct immunologic cell type signature, which points toward cancer immune evasion. Transcriptomic data also revealed PHTS-derived BCs with pathogenic germline PTEN variants appear to have vitamin E degradation as a key pathway associated with tumorigenesis. In conclusion, our study revealed distinct CNV x transcript features in PHTS-derived BCs, which further facilitate understanding of BC biology arising in the setting of germline PTEN mutations.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"14"},"PeriodicalIF":5.3,"publicationDate":"2023-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10322985/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9858861","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigating genomic medicine practice and perceptions amongst Australian non-genetics physicians to inform education and implementation.","authors":"Amy Nisselle,&nbsp;Emily King,&nbsp;Bronwyn Terrill,&nbsp;Belinda Davey,&nbsp;Belinda McClaren,&nbsp;Kate Dunlop,&nbsp;Debra Graves,&nbsp;Sylvia Metcalfe,&nbsp;Clara Gaff","doi":"10.1038/s41525-023-00360-1","DOIUrl":"https://doi.org/10.1038/s41525-023-00360-1","url":null,"abstract":"<p><p>Genomic medicine is being implemented on a global scale, requiring a genomic-competent health workforce. To inform education as part of implementation strategies to optimize adoption of genomics by non-genetics physicians, we investigated current practices, perceptions and preferences relating to genomic testing and education. Australian non-genetics physicians completed an online survey; we conducted univariate and multivariate analyses of determinants of confidence and engagement with genomic medicine. Confident or engaged respondents were more likely to be pediatricians, have completed continuing genomics education (CGE) and/or have genomics research experience. Confident or engaged respondents were also more likely to prefer to request genomic testing with support from genetics services than other models. Respondents who had completed CGE and were engaged reported higher confidence than those who were not engaged. We propose a progression of genomic competence aligned with service delivery models, where education is one enabler of mastery or independence to facilitate genomic tests (from referral to requesting with or without clinical genetics support). Workplace learning could provide additional impetus for adoption.</p>","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"13"},"PeriodicalIF":5.3,"publicationDate":"2023-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10290687/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10072057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Author Correction: Accurate detection of circulating tumor DNA using nanopore consensus sequencing.","authors":"Alessio Marcozzi,&nbsp;Myrthe Jager,&nbsp;Martin Elferink,&nbsp;Roy Straver,&nbsp;Joost H van Ginkel,&nbsp;Boris Peltenburg,&nbsp;Li-Ting Chen,&nbsp;Ivo Renkens,&nbsp;Joyce van Kuik,&nbsp;Chris Terhaard,&nbsp;Remco de Bree,&nbsp;Lot A Devriese,&nbsp;Stefan M Willems,&nbsp;Wigard P Kloosterman,&nbsp;Jeroen de Ridder","doi":"10.1038/s41525-023-00356-x","DOIUrl":"https://doi.org/10.1038/s41525-023-00356-x","url":null,"abstract":"","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"8 1","pages":"12"},"PeriodicalIF":5.3,"publicationDate":"2023-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10247772/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9602617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}