NPJ Genomic Medicine最新文献_第6页

Severe traumatic injury is associated with profound changes in DNA methylation. 严重创伤与 DNA 甲基化的深刻变化有关。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-11-01 DOI: 10.1038/s41525-024-00438-4

Trine O Eskesen, Kristian Almstrup, Laurits Elgaard, Tobias Arleth, Mathilde L Lassen, Andreas Creutzburg, Alice Herrlin Jensen, Niklas Breindahl, Felicia Dinesen, Malene Vang, Erik Sørensen, Anders Wallin Paulsen, Tatiana Nielsen, Lars S Rasmussen, Martin Sillesen, Jacob Steinmetz

{"title":"Severe traumatic injury is associated with profound changes in DNA methylation.","authors":"Trine O Eskesen, Kristian Almstrup, Laurits Elgaard, Tobias Arleth, Mathilde L Lassen, Andreas Creutzburg, Alice Herrlin Jensen, Niklas Breindahl, Felicia Dinesen, Malene Vang, Erik Sørensen, Anders Wallin Paulsen, Tatiana Nielsen, Lars S Rasmussen, Martin Sillesen, Jacob Steinmetz","doi":"10.1038/s41525-024-00438-4","DOIUrl":"10.1038/s41525-024-00438-4","url":null,"abstract":"Whether DNA methylation changes follow human physical trauma is uncertain. We aimed to investigate if severe trauma was associated with DNA methylation changes. In a prospective, observational, clinical study, we included severely injured adults and adults undergoing elective surgery (controls). Blood was obtained from trauma patients (n = 60) immediately- and 30-45 days post-trauma, and from surgical patients (n = 57) pre-, post-, and 30-45 days post-surgery. Epigenome-wide DNA methylation profiling was performed and analyzed for significant differentially methylated CpGs and -regions (DMRs) within and between groups. Within the trauma group we identified 10,126 significant differentially methylated CpGs and 1169 DMRs. No significant differential methylation was found in the surgical group. In the trauma group, differentially methylated sites were enriched in genes and pathways involved in blood coagulation and inflammatory response. Severe trauma was associated with profound alterations in the DNA methylome of circulating leucocytes, and differential methylation was located in trauma-relevant genes.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"53"},"PeriodicalIF":4.7,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11530621/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564822","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}

引用次数: 0

Gut microbial and human genetic signatures of inflammatory bowel disease increase risk of comorbid mental disorders. 炎症性肠病的肠道微生物和人类基因特征会增加合并精神障碍的风险。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-29 DOI: 10.1038/s41525-024-00440-w

Junho Lee, Shin Ju Oh, Eunji Ha, Ga Young Shin, Hyo Jong Kim, Kwangwoo Kim, Chang Kyun Lee

{"title":"Gut microbial and human genetic signatures of inflammatory bowel disease increase risk of comorbid mental disorders.","authors":"Junho Lee, Shin Ju Oh, Eunji Ha, Ga Young Shin, Hyo Jong Kim, Kwangwoo Kim, Chang Kyun Lee","doi":"10.1038/s41525-024-00440-w","DOIUrl":"10.1038/s41525-024-00440-w","url":null,"abstract":"The high prevalence of comorbid mental disorders (CMDs) in patients with inflammatory bowel disease (IBD) is well-documented. This study delves into the intricate CMD-IBD relationship through comprehensive analyses using human variants, gut microbiome, and anxiety/depression estimates from a cohort of 507 IBD patients and 75 controls. Notably, patients with IBD, especially those with CMD, exhibited lower diversity than controls. We identified 106 differentially abundant taxa (DATs) in IBD patients compared to controls and 21 DATs distinguishing CMD-affected from CMD-free IBD patients. Microbial IBD-risk scores, reflecting an individual's microbial burden for IBD, revealed a significant enrichment of IBD-risk signatures in CMD-affected patients compared to CMD-free patients. Additionally, there was an IBD-risk variant potentially regulating the abundance of an IBD/CMD-associated DAT, suggesting an interplay between IBD-risk variants and dysbiosis in CMD. Our investigation underscores the pivotal role of IBD-associated gut dysbiosis in predisposing IBD patients to CMD, partially through genetic variant-mediated mechanisms.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"52"},"PeriodicalIF":4.7,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522461/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546549","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}

引用次数: 0

Clinical genome sequencing in patients with suspected rare genetic disease in Peru. 秘鲁疑似罕见遗传病患者的临床基因组测序。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-28 DOI: 10.1038/s41525-024-00434-8

Jeny Bazalar-Montoya, Mario Cornejo-Olivas, Milagros M Duenas-Roque, Nelson Purizaca-Rosillo, Richard S Rodriguez, Karina Milla-Neyra, Carlos A De La Torre-Hernandez, Elison Sarapura-Castro, Carolina I Galarreta Aima, Gioconda Manassero-Morales, Giulliana Chávez-Pasco, Luis Celis-García, Jorge E La Serna-Infantes, Evgenii Chekalin, Erin Thorpe, Ryan J Taft

{"title":"Clinical genome sequencing in patients with suspected rare genetic disease in Peru.","authors":"Jeny Bazalar-Montoya, Mario Cornejo-Olivas, Milagros M Duenas-Roque, Nelson Purizaca-Rosillo, Richard S Rodriguez, Karina Milla-Neyra, Carlos A De La Torre-Hernandez, Elison Sarapura-Castro, Carolina I Galarreta Aima, Gioconda Manassero-Morales, Giulliana Chávez-Pasco, Luis Celis-García, Jorge E La Serna-Infantes, Evgenii Chekalin, Erin Thorpe, Ryan J Taft","doi":"10.1038/s41525-024-00434-8","DOIUrl":"10.1038/s41525-024-00434-8","url":null,"abstract":"There is limited access to molecular genetic testing in most low- and middle-income countries. The iHope program provides clinical genome sequencing (cGS) to underserved individuals with signs or symptoms of rare genetic diseases and limited or no access to molecular genetic testing. Here we describe the performance and impact of cGS in 247 patients from three clinics in Peru. Although most patients had at least one genetic test prior to cGS (70.9%), the most frequent was karyotyping (53.4%). The diagnostic yield of cGS was 54.3%, with candidate variants reported in an additional 22.3% of patients. Clinical GS results impacted clinician diagnostic evaluation in 85.0% and genetic counseling in 72.1% of cases. Changes in management were reported in 71.3%, inclusive of referrals (64.7%), therapeutics (26.3%), laboratory or physiological testing (25.5%), imaging (19%), and palliative care (17.4%), suggesting that increased availability of genomic testing in Peru would enable improved patient management.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"51"},"PeriodicalIF":4.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11519459/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142522530","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}

引用次数: 0

SLC16A8 is a causal contributor to age-related macular degeneration risk. SLC16A8 是老年性黄斑变性风险的成因之一。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-28 DOI: 10.1038/s41525-024-00442-8

Navid Nouri, Bailey Hannon Gussler, Amy Stockwell, Tom Truong, Gyeong Jin Kang, Kristen C Browder, Yann Malato, Abdoulaye Sene, Sherri Van Everen, Charles C Wykoff, David Brown, Arthur Fu, James D Palmer, Jose Ronaldo Lima de Carvalho, Ehsan Ullah, Ranya Al Rawi, Emily Y Chew, Wadih M Zein, Bin Guan, Mark I McCarthy, Jeffrey W Hofmann, Shawnta Y Chaney, Heinrich Jasper, Brian L Yaspan

{"title":"SLC16A8 is a causal contributor to age-related macular degeneration risk.","authors":"Navid Nouri, Bailey Hannon Gussler, Amy Stockwell, Tom Truong, Gyeong Jin Kang, Kristen C Browder, Yann Malato, Abdoulaye Sene, Sherri Van Everen, Charles C Wykoff, David Brown, Arthur Fu, James D Palmer, Jose Ronaldo Lima de Carvalho, Ehsan Ullah, Ranya Al Rawi, Emily Y Chew, Wadih M Zein, Bin Guan, Mark I McCarthy, Jeffrey W Hofmann, Shawnta Y Chaney, Heinrich Jasper, Brian L Yaspan","doi":"10.1038/s41525-024-00442-8","DOIUrl":"10.1038/s41525-024-00442-8","url":null,"abstract":"Age-related macular degeneration (AMD), a complex neurodegenerative disease, is a leading cause of visual impairment worldwide with a strong genetic component. Genetic studies have identified several loci, but few causal genes with functional characterization. Here we highlight multiple lines of evidence which show a causal role in AMD for SLC16A8, which encodes MCT3, a retinal pigment epithelium (RPE) specific lactate transporter. First, in an unbiased, genome-wide analysis of rare coding variants we show multiple SLC16A8 rare variants are associated with AMD risk, corroborating previous borderline significant reports from AMD rare variant studies. Second, we report a novel SLC16A8 mutation in a three-generation family with early onset macular degeneration. Finally, mis-expression in multiple model organisms shows functional and anatomic retinal consequences. This study highlights the important role for SLC16A8 and lactate regulation towards outer retina/RPE health and highlights a potential new therapeutic opportunity for the treatment of AMD.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"50"},"PeriodicalIF":4.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11519927/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142522531","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}

引用次数: 0

Comprehensive reanalysis for CNVs in ES data from unsolved rare disease cases results in new diagnoses. 对来自未解决罕见病病例的 ES 数据中的 CNV 进行全面的重新分析，从而得出新的诊断结果。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-26 DOI: 10.1038/s41525-024-00436-6

German Demidov, Burcu Yaldiz, José Garcia-Pelaez, Elke de Boer, Nika Schuermans, Liedewei Van de Vondel, Ida Paramonov, Lennart F Johansson, Francesco Musacchia, Elisa Benetti, Gemma Bullich, Karolis Sablauskas, Sergi Beltran, Christian Gilissen, Alexander Hoischen, Stephan Ossowski, Richarda de Voer, Katja Lohmann, Carla Oliveira, Ana Topf, Lisenka E L M Vissers, Steven Laurie

{"title":"Comprehensive reanalysis for CNVs in ES data from unsolved rare disease cases results in new diagnoses.","authors":"German Demidov, Burcu Yaldiz, José Garcia-Pelaez, Elke de Boer, Nika Schuermans, Liedewei Van de Vondel, Ida Paramonov, Lennart F Johansson, Francesco Musacchia, Elisa Benetti, Gemma Bullich, Karolis Sablauskas, Sergi Beltran, Christian Gilissen, Alexander Hoischen, Stephan Ossowski, Richarda de Voer, Katja Lohmann, Carla Oliveira, Ana Topf, Lisenka E L M Vissers, Steven Laurie","doi":"10.1038/s41525-024-00436-6","DOIUrl":"10.1038/s41525-024-00436-6","url":null,"abstract":"We report the results of a comprehensive copy number variant (CNV) reanalysis of 9171 exome sequencing datasets from 5757 families affected by a rare disease (RD). The data reanalysed was extremely heterogeneous, having been generated using 28 different enrichment kits by 42 different research groups across Europe partnering in the Solve-RD project. Each research group had previously undertaken their own analysis of the data but failed to identify disease-causing variants. We applied three CNV calling algorithms to maximise sensitivity, and rare CNVs overlapping genes of interest, provided by four partner European Reference Networks, were taken forward for interpretation by clinical experts. This reanalysis has resulted in a molecular diagnosis being provided to 51 families in this sample, with ClinCNV performing the best of the three algorithms. We also identified partially explanatory pathogenic CNVs in a further 34 individuals. This work illustrates the value of reanalysing ES cold cases for CNVs.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"49"},"PeriodicalIF":4.7,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513043/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504916","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}

引用次数: 0

Biallelic GGGCC repeat expansion leading to NAXE-related mitochondrial encephalopathy. 双倍性 GGGCC 重复扩增导致 NAXE 相关线粒体脑病。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-25 DOI: 10.1038/s41525-024-00429-5

Kokoro Ozaki, Yukiko Yatsuka, Yoshinobu Oyazato, Atsushi Nishiyama, Kazuhiro R Nitta, Yoshihito Kishita, Takuya Fushimi, Masaru Shimura, Shohei Noma, Yohei Sugiyama, Michihira Tagami, Moe Fukunaga, Hiroko Kinoshita, Tomoko Hirata, Wataru Suda, Yasuhiro Murakawa, Piero Carninci, Akira Ohtake, Kei Murayama, Yasushi Okazaki

{"title":"Biallelic GGGCC repeat expansion leading to NAXE-related mitochondrial encephalopathy.","authors":"Kokoro Ozaki, Yukiko Yatsuka, Yoshinobu Oyazato, Atsushi Nishiyama, Kazuhiro R Nitta, Yoshihito Kishita, Takuya Fushimi, Masaru Shimura, Shohei Noma, Yohei Sugiyama, Michihira Tagami, Moe Fukunaga, Hiroko Kinoshita, Tomoko Hirata, Wataru Suda, Yasuhiro Murakawa, Piero Carninci, Akira Ohtake, Kei Murayama, Yasushi Okazaki","doi":"10.1038/s41525-024-00429-5","DOIUrl":"https://doi.org/10.1038/s41525-024-00429-5","url":null,"abstract":"Repeat expansions cause at least 50 hereditary disorders, including Friedreich ataxia and other diseases known to cause mitochondrial dysfunction. We identified a patient with NAXE-related mitochondrial encephalopathy and novel biallelic GGGCC repeat expansion as long as ~200 repeats in the NAXE promoter region using long-read sequencing. In addition to a marked reduction in the RNA and protein, we found a marked reduction in nascent RNA in the promoter using native elongating transcript-cap analysis of gene expression (NET-CAGE), suggesting transcriptional suppression. Accordingly, CpG hypermethylation was observed in the repeat region. Genetic analyses determined that homozygosity in the patient was due to maternal chromosome 1 uniparental disomy (UPD). We assessed short variants within NAXE including the repeat region in the undiagnosed mitochondrial encephalopathy cohort of 242 patients. This study identified the GGGCC repeat expansion causing a mitochondrial disease and suggests that UPD could significantly contribute to homozygosity for rare repeat-expanded alleles.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"48"},"PeriodicalIF":4.7,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11512015/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504915","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}

引用次数: 0

Systematic decision frameworks for the socially responsible use of precision medicine. 以对社会负责的方式使用精准医疗的系统决策框架。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-05 DOI: 10.1038/s41525-024-00433-9

Ian S Peebles, David B Kinney, Emily Foster-Hanson

引用次数: 0

A genotype imputation reference panel specific for native Southeast Asian populations. 专门针对东南亚本地人群的基因型推算参考面板。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-10-05 DOI: 10.1038/s41525-024-00435-7

Alvin Cengnata, Lian Deng, Wai-Sum Yap, Lay-Hong Renee Lim, Chee-Onn Leong, Shuhua Xu, Boon-Peng Hoh

{"title":"A genotype imputation reference panel specific for native Southeast Asian populations.","authors":"Alvin Cengnata, Lian Deng, Wai-Sum Yap, Lay-Hong Renee Lim, Chee-Onn Leong, Shuhua Xu, Boon-Peng Hoh","doi":"10.1038/s41525-024-00435-7","DOIUrl":"10.1038/s41525-024-00435-7","url":null,"abstract":"We report the development of a \"Southeast Asian Specific (SEA-specific) Reference Panel\" through a \"Cross-panel Imputation\" approach, consisting of 2550 samples derived from the GA100K, SG10K, and the Peninsular Malaysia Orang Asli (OA) datasets, covering 113,851,450 variants. The SEA-specific panel produced more high confidence variants than 1000 Genomes Project (1KGP) when imputing the OA (8.9 million SEA-specific vs 8.1 million 1KGP) and the Singapore Genome Variation Project (SGVP) (12.5 million SEA-specific vs 11.8 million 1KGP) genotyping datasets. Further, the SEA-specific panel imputed SNPs with better estimated quality scores (INFO, DR2 and R2) on the OA genotyping dataset when comparing with TOPMED and the Human Genome Diversity Project, but performed similarly on SGVP dataset. This panel also exhibited higher recall and non-reference disconcordance rates, indicating the influence of ancestry closeness of the reference panel. However, we note that the imputation accuracy may be compromised by the size of the reference panel.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"47"},"PeriodicalIF":4.7,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11455956/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142378134","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}

引用次数: 0

Gene-environmental influence of space and microgravity on red blood cells with sickle cell disease. 太空和微重力对镰状细胞病红细胞的基因环境影响

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-09-30 DOI: 10.1038/s41525-024-00427-7

Norris E Igbineweka, Jack J W A van Loon

{"title":"Gene-environmental influence of space and microgravity on red blood cells with sickle cell disease.","authors":"Norris E Igbineweka, Jack J W A van Loon","doi":"10.1038/s41525-024-00427-7","DOIUrl":"10.1038/s41525-024-00427-7","url":null,"abstract":"A fundamental question in human biology and for hematological disease is how do complex gene-environment interactions lead to individual disease outcome? This is no less the case for sickle cell disease (SCD), a monogenic disorder of Mendelian inheritance, both clinical course, severity, and treatment response, is variable amongst affected individuals. New insight and discovery often lie between the intersection of seemingly disparate disciplines. Recently, opportunities for space medicine have flourished and have offered a new paradigm for study. Two recent Nature papers have shown that hemolysis and oxidative stress play key mechanistic roles in erythrocyte pathogenesis during spaceflight. This paper reviews existing genetic and environmental modifiers of the sickle cell disease phenotype. It reviews evidence for erythrocyte pathology in microgravity environments and demonstrates why this may be relevant for the unique gene-environment interaction of the SCD phenotype. It also introduces the hematology and scientific community to methodological tools for evaluation in space and microgravity research. The increasing understanding of space biology may yield insight into gene-environment influences and new treatment paradigms in SCD and other hematological disease phenotypes.","PeriodicalId":19273,"journal":{"name":"NPJ Genomic Medicine","volume":"9 1","pages":"44"},"PeriodicalIF":4.7,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11442622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142350888","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}

引用次数: 0

Native Hawaiian and Pacific Islander populations in genomic research. 基因组研究中的夏威夷原住民和太平洋岛民。

IF 4.7 2区医学

NPJ Genomic Medicine Pub Date : 2024-09-30 DOI: 10.1038/s41525-024-00428-6

Edra K Ha, Daniel Shriner, Shawneequa L Callier, Lorinda Riley, Adebowale A Adeyemo, Charles N Rotimi, Amy R Bentley

引用次数: 0