强直性脊柱炎一家系全外显子组测序分析

目的： 对一强直性脊柱炎（ankylosing spondylitis,AS）家系进行全外显子测序,筛选该家系的易感基因,为其发病机制提供理论依据。方法： 收集1组AS家系成员的临床资料,其中男性患者2例,年龄分别为48岁和18岁,病程分别为23年和4年。提取相关家系成员外周血DNA进行全外显子测序,测序结果与人类数据库比对,过滤掉同义突变及高频突变,整合家系成员单核苷酸非同义突变,寻找致病基因。结果： 家系成员共得原始数据80 G,数据具有较高质量值,通过对家系患者与正常人测序结果比对分析,同时经过多个生物数据库数据过滤,发现JAK2基因12号外显子上存在的杂合突变c.1709A>G（p.Tyr570Cys）为该家系的可能致病基因突变。另外,该家系MUC3A基因c.1151T>C突变可能是该家系患病成员肠道症状的原因之一。结论： 运用全基因组外显子测序寻找AS易感基因是可行的,JAK2基因c.1709A>G突变可能是导致该家系AS的致病突变及位点。     

miR-181a-5p通过HOXB4抑制骨肉瘤细胞HOS增殖和迁移

目的: 研究miR-181a-5p对HOS骨肉瘤细胞增殖、周期和迁移的影响及其机制。方法: 采用实时定量PCR检测hFOB1.19成骨细胞和HOS、U2OS、MG63骨肉瘤细胞系中miR-181a-5p及HOXB4的表达情况。利用Lipofectamine 2000将miR-181a-5p mimics和miR-181a-5p inhibitor分别转染至人骨肉瘤HOS细胞中(分别为过表达组和抑制剂组),并设置miR 阴性对照组;CCK-8法检测各组细胞的增殖能力变化,流式细胞术检测各组细胞的细胞周期变化,划痕愈合实验以及Transwell迁移实验检测各组细胞的迁移能力变化。Targetscan网站预测miR-181a-5p的靶向基因,并通过双荧光素酶报告基因系统及Western blot验证靶向关系。结果: 与成骨细胞hFOB1.19相比,miR-181a-5p在骨肉瘤细胞HOS、U2OS和MG63中低表达(P<0.05),而HOXB4在骨肉瘤中高表达(P<0.05)。与阴性对照组相比,过表达miR-181a-5p抑制骨肉瘤HOS细胞的增殖和迁移能力,并且处于细胞周期S期的细胞减少(P<0.05)。敲低miR-181a-5p促进骨肉瘤HOS细胞的增殖和迁移能力,并且处于S期细胞增加(P<0.05)。生物信息学预测及双荧光素酶报告基因系统验证HOXB4为miR-181a-5p的下游靶基因(P<0.05)。Western blot显示,过表达miR-181a-5p的HOS细胞中,HOXB4蛋白表达低于阳性对照组(P<0.05),而敲低miR-181a-5p的HOS细胞中HOXB4蛋白表达高于对照组(P<0.05)。结论: miR-181a-5p在骨肉瘤细胞中低表达,过表达miR-181a-5p能够抑制骨肉瘤细胞HOS增殖、周期和迁移能力,该作用可能通过靶向HOXB4发挥作用。     

CT用于免疫检查点抑制剂相关性肺炎

目的 评价CT在免疫检查点抑制剂相关性肺炎(CIP)诊疗中的价值。方法 观察7例经程序性细胞死亡蛋白1(PD-1)/程序性死亡配体1(PD-L1)治疗后诊断CIP患者的临床资料及CT影像学表现,评价CT用于诊疗CIP的价值。结果 7例CIP的临床分级包括2级1例、3级4例及4级2例;均见多发病灶累及双肺多叶、多段;3例病灶呈对称分布,4例呈非对称分布。影像学分型包括磨玻璃型3例、实变型2例(多发斑片亚型、肺实变亚型各1例)及网格型2例;5例伴胸膜增厚,5例纵隔淋巴结受累,3例伴牵拉性支气管扩张,2例伴小叶间隔增厚,2例伴多发小叶中心结节,1例伴胸腔积液。结论 CT结合免疫检查点抑制剂治疗史可辅助诊断CIP,为确定治疗方案提供依据。     

胰腺癌肝转移核心基因的筛选与验证

背景与目的 胰腺癌是预后极差的恶性肿瘤,其5年生存率约为11.5%,有将近半数的患者在初诊时已出现远处转移,而肝转移则占到其中的37.0%~41.9%。探索新的胰腺癌肝转移的生物标志物可能有助于提高患者治疗的效果。因此,本研究通过生物信息学方法寻找在胰腺癌肝转移过程起关键作用的基因并验证。方法 下载GEO数据库中的胰腺导管腺癌（PDAC）高通量测序数据集GSE151580（该数据集中包含胰腺癌肝转移病灶组织样本和原发病灶组织样本）,使用R语言“limma”包筛选出肝转移病灶组织样本和原发病灶组织样本间的差异表达基因。对差异表达基因进行GO和KEGG功能富集。利用STRING数据库构建蛋白质间的相关作用关系,使用Cytoscape对蛋白质相互作用网络进行可视化展示并利用CytoHubba插件根据MCC拓扑分析方法,挑选MCC分数最高的前10位基因,确定为候选的核心基因。利用TCGA、GEPIA、UALCAN和HPA数据库的验证对候选的核心基因加以验证。结果 总共纳入分析基因数为46 512个,符合筛选条件的差异表达基因数为491个,其中上调162个,下调329个。挑选MCC分数最高的前10位基因后,通过候选基因经验证显示,APOB基因在肿瘤组织中高表达（P<0.05）,其表达产物主要定位于细胞质和细胞膜,免疫组化中等强度阳性。APOB基因的突变与患者的M分期有关,表现为该基因突变组中,M1患者构成比更大（P=0.022 1）;而该基因的表达与患者的总生存（OS）率和无病生存（DFS）率均无明显关系（均P>0.05）。此外,APOA4基因表达产物也主要定位于细胞质和细胞膜,免疫组化染色呈中等强度阳性。APOA4基因的突变与患者的TNM分期有关,表现为突变组中,TNM分期更早（P=0.018 3）。该基因低表达患者的DFS更高（HR=1.75,P=0.025）,但与患者的OS无关（P>0.05）。结论 APOB基因可能与胰腺癌的肝转移相关,有望作为胰腺癌肝转移早期筛查的分子标志物。APOA4基因与胰腺癌患者的DFS相关,有望成为新的分子标志物用于评价患者预后,监测肿瘤复发,或作为潜在的基因治疗靶点。     

原发性局灶节段性肾小球硬化患者ACTN4基因变异和多态性

目的 了解ACTN4基因变异和多态性在原发性局灶节段性肾小球硬化（FSGS）发病中的作用。 方法 选取FSGS患者82例，另设70例健康人作为对照组。盐析法提取外周血基因组DNA，PCR扩增后测序，与基因数据库进行匹配，寻找可能致病变异位点。氯酚法提取患者父母头发DNA，间接免疫荧光法检测患者肾组织辅肌动蛋白4(α-actinin-4)表达水平。单核苷酸多态（SNP）位点经Hardy-Weinberg平衡检验后行基因频率、基因型和临床表型关联分析。 结果 发现1例患者单核苷酸变异184T>A(Ser62Thr)，1例5’UTR变异1-34C>T。对照组和患者父母未发现相同变异。1个疾病易感SNP位点484+87C>G。变异者肾组织α-actinin 4表达水平分别较对照组和非变异FSGS组下降。变异基因型和野生基因型尿蛋白量（24 h）的差异有统计学意义[（7.90±1.60 ）比(4.50±0.46) g/24 h， P ＜ 0.01]。此外，还发现6个新的变异和另1个SNP位点，但未引起氨基酸改变。 结论 原发性FSGS患者中存在ACTN4基因变异位点和疾病易感SNP位点。ACTN4基因变异在原发性FSGS发病中可能起重要作用。     

YTH基因家族在肝癌中的表达和预后价值

背景与目的 YTH基因家族的所有成员都属于m6A阅读蛋白,负责参与肿瘤发生发展过程中的甲基化调控。然而,YTH基因家族在肝癌中的表达情况和具体作用仍有待进一步阐明。本文旨在通过生物信息学方法探究YTH家族成员在肝癌中的表达与预后价值,及其与免疫浸润及相关功能的关系。方法 用UALCAN数据库分析YTH基因家族在肝癌及其亚组中的表达差异。采用Kaplan-Meier plotter数据库分析YTH基因家族在肝癌中的预后价值。使用cBioPortal数据库进行YTH基因家族的共表达谱和遗传学变异分析。通过WebGestalt数据库进行YTH基因家族的功能和通路富集分析。YTH基因家族与肝癌中的免疫细胞浸润的相关性分析采用TIMER 2.0数据库完成。结果 肝癌中YTHDC1、YTHDC2、YTHDF1、YTHDF2、YTHDF3的mRNA表达均较正常肝组织明显升高,并与临床TNM分期及肿瘤分级密切相关,其在1、2、3期/级中表达逐级升高,而在4期/级中表达水平下降。预后分析显示,YTHDF1和YTHDF2的高表达均与患者更短的总生存期（OS）和无复发生存期（RFS）明显相关。TCGA数据库和cBioPortal工具分析显示,YTHDF3基因变异率为25%,是YTH家族中最高的。GO功能富集分析显示YTH基因家族的主要癌症相关功能指向代谢及生物合成分解方向,尤其是激素代谢、固醇类代谢、脂代谢、药物分解代谢等。通过TIMER 2.0数据库分析多数YTH家族成员的表达与CD4⁺ T细胞、CD8⁺ T细胞、B细胞、中性粒细胞以及树突细胞的浸润呈正相关,与巨噬细胞浸润呈负相关。结论 YTH基因家族在肝癌中存在分子和表达谱的失调。YTH基因家族成员（尤其是YTHDF1和YTHDF2）是肝癌潜在的预后标志物和新的治疗靶点,这些发现可为肝癌机制与靶向治疗的研究提供新的途径。     

浸润生长与无浸润生长胰腺实性假乳头状瘤MRI表现

目的 比较浸润与无浸润生长胰腺实性假乳头状瘤（SPTP）的MRI表现。方法 回顾性分析经手术病理证实的浸润生长SPTP患者20例（浸润组）和无浸润生长SPTP患者26例（无浸润组）的平扫及动态增强MRI特点。结果 浸润与无浸润组患者性别、年龄、病灶位置、病灶大小、类型、延迟期强化类型差异均无统计学意义（P均>0.05）,而病灶形态、周围侵犯情况差异有统计学意义（P均<0.05）。结论 SPTP形态不规则及侵犯周围组织时,提示恶性可能。即使病灶<30 mm且无周围侵犯征象,也不能排除肿瘤浸润。     

比较基因组杂交揭示骨肉瘤相关基因变异

比较基因组杂交是诊断肿瘤发生发展的方法,能够发现与骨肉瘤相关的基因变异,从而为基因治疗提供依据.通过比较基因组杂交发现骨肉瘤患者基因扩增多于缺失,经常发生变异的基因与骨肉瘤密切相关.4q变异是儿童骨肉瘤共同的畸变;8q扩增与骨肉瘤复发和侵袭性相关;17p扩增(除17p13.1外)与骨肉瘤恶性程度相关;凋亡相关因子与骨肉瘤发生发展、预后密切相关;环氧化酶-2在骨肉瘤的高表达为骨肉瘤新辅助化疗提供依据;高频的基因变异位点主要与癌基因、抑癌基因、细胞生长和细胞周期调控、细胞凋亡、端粒、细胞因子、肿瘤免疫、肿瘤血管生成、肿瘤转移相关因子等密切相关.     

同时多层成像-弥散加权成像用于直肠癌的可行性

目的 探讨同时多层成像（SMS）-弥散加权成像（DWI）用于直肠癌的可行性。方法 回顾性分析45例经病理证实直肠癌患者的常规DWI及SMS-DWI资料，比较2种DWI所示病灶边缘锐利度、变形、伪影、病灶显著性和整体图像质量主观评分，以及图像质量客观评价及组织表观弥散系数（ADC）值的差异。结果 常规DWI与SMS-DWI之间，图像主观评分差异均无统计学意义（P均>0.05），而客观评价参数，包括图像对比信噪比及非瘤直肠组织ADC值差异均有统计学意义（P均<0.05），图像信噪比、信号强度比及病灶ADC值差异均无统计学意义（P均>0.05）。结论 SMS-DWI可用于评估直肠癌。     

汉族人群青少年特发性脊柱侧凸家系中存在复合型杂合DOCK9基因突变

目的探讨青少年特发性脊柱侧凸(AIS)家系中可能的致病基因突变。方法对一个汉族AIS家系的3名成员(先证者及其表型正常的父母)进行全基因组测序,筛选潜在的致病基因突变,并通过Sanger测序验证所有发现的突变。结果在AIS家系中发现DOCK9基因存在复合型杂合基因突变c.3259TC(p.F1087L)和c.2465AG(p.Y822C)。患者的父母是未出现AIS表型的突变基因携带者,父亲携带c2465AG突变,而母亲携带c.3259TC突变。结论复合型杂合DOCK9基因突变可能导致AIS的发生,其在AIS发生机制中的作用有待于进一步探索。     

Novel SOST gene mutation in a sclerosteosis patient and her parents

IntroductionSclerosteosis (OMIM 269500) is a rare autosomal recessive condition characterized by increased bone density associated with syndactyly. It is linked to a genetic defect in the SOST gene coding for sclerostin. So far, six different loss-of-function mutations in SOST have been reported in patients with sclerosteosis. Our objective was to sequence and identify mutation in the SOST and LRP5 genes which are known to be causal for craniotubular hyperostosis in a patient from India.Patient and methodsA 22 year old woman presented with typical features of sclerosteosis in form of progressive visual and hearing loss, syndactyly and radiographs revealing increased density of bone. Genomic sequencing of the SOST gene as well as exons 2, 3 and 4 of the LRP5 gene was performed.ResultsWe identified a novel homozygous mutation in the ^SOST gene, characterized as one nucleotide insertion resulting in a frame shift mutation and loss of functional sclerostin. Her parents were also found to have a similar but heterozygous mutation in the ^SOST gene.ConclusionA novel frame shift mutation in the ^SOST gene causing loss of functional sclerostin was identified in a patient with sclerosteosis and her parents.     

Double Heterozygous Mutations in the BRCA2 and ATM Genes: A Case Report and Review of the Literature

IntroductionGermline mutations of the BRCA1 and BRCA2 genes are responsible for about a quarter of hereditary breast cancers (BCs). In this study, we aimed to determine the importance of rare double heterozygous (DH) pathogenic variant carriership in BRCA2and ATM genes in a patient diagnosed with BC and pancreas cancer (PC).Case ReportA 54-year-old female patient was diagnosed with BC at the age of 34 years and with PC at the age of 48 years. The multigene panel and next-generation sequencing technique were used to evaluate the status of the patient''s cancer susceptibility genes. Pathogenic variants c.537dup (p.Ile180Tyrfs*3) in the BRCA2 gene and c.5065C>T (p.Gln1689Ter) in the ATM gene were detected as DH in the patient. Co-segregation analysis was performed on the relatives of the patient using Sanger sequencing.Discussion/ConclusionMultiple primary malignant neoplasms can be encountered more frequently in DH pathogenic variant carriers, and the diagnosis of malignancies can be made at an earlier age through surveillance guided by genetic testing. In this rare case, more patient studies are needed to determine the contribution of DH in BRCA2 and ATM genes to the phenotype.     

A novel VCP mutation as the cause of atypical IBMPFD in a Chinese family

IntroductionInclusion-body myopathy (IBM) with Paget's disease of bone (PDB) and frontotemporal dementia (FTD), designated as IBMPFD, is a rare, autosomal dominant disorder (MIM 605382). IBMPFD is caused by mutations in the gene that encode valosin-containing protein (VCP). We investigated a Chinese family in which multiple members were diagnosed with PDB and suffered from weakness of the limbs. However, no members of this family were diagnosed with FTD. We made a preliminary diagnosis of PDB, but failed to identify an SQSTM1 mutation in any of the patients. We used whole-exome sequencing to identify the pathogenic gene mutation affecting the Chinese male proband.Materials and methodsAltogether, 254 subjects, including one 56-year-old male proband, four affected, related individuals and additional nine family members from a non-consanguineous Chinese family, and 240 healthy donors were recruited and genomic DNA was extracted. All eight exons and the exon–intron boundaries of the SQSTM1 gene were amplified by polymerase chain reaction (PCR) and directly sequenced in five patients (II13, II4, II5, II8, II9). Using whole-exome sequencing, we identified a novel mutation in VCP as the disease-causing mutation. We confirmed the result by sequencing a 500-bp region of the promoter and the coding region of VCP in all 254 of the participants using Sanger sequencing.ResultsNo mutation in the SQSTM1 gene was identified in the five patients examined using direct Sanger sequencing. However, through whole-exome sequencing we were able to identify a novel missense mutation in exon 3 of the VCP gene (p.Gly97Glu) in the Chinese male proband. This mutation was confirmed using Sanger sequencing. The proband, four affected individuals and three unaffected individuals carried this mutation. We were able to correctly diagnose the patients with atypical IBMPFD. Structural analysis of the p.Gly97Glu mutation in the VCP protein showed that the affected amino‐acid is located in the interface of the protein. This abnormality may therefore interfere with protein function.ConclusionsThis is the first report of a family from China with IBMPFD. A novel VCP mutation was found as the cause of atypical IBMPFD in a Chinese family. Our findings confirm that VCP gene mutations can be a pathogenic cause of IBMPFD.     

Comprehensive data analysis of genomics,epigenomics, and transcriptomics to identify specific biomolecular markers for prostate adenocarcinoma

BackgroundMultiomics data analysis based on high-throughput sequencing technology has become a hotspot in tumor investigation. The present study aimed to explore prognostic biomarkers via investigating DNA copy number variation (CNV) and methylation variation (MET) data in prostate cancer.MethodsWe obtained the messenger RNA (mRNA) expression, CNV, and methylated data of prostate adenocarcinoma (PRAD) samples via The Cancer Genome Atlas (TCGA)-PRAD cohort. We calculated and assessed the associations between CNV and RNA sequencing (RNA-seq), and between MET and RNA-seq via Pearson correlation coefficients. We then used the “iCluster” package to perform multigroup cluster analysis with CNVcor gene CNV data, METcor gene methylation data, and CNVcor and METcor gene mRNA data. The univariate Cox analysis was used to screen significant hub genes, and multivariate Cox analysis was used to construct risk a model. The nomogram was constructed based on “rms” package, and the immune infiltrating patterns were compared between high- and low-risk groups.ResultsA total of 477 PRAD samples with complete CNV, methylation, mRNA, and matched clinical information were included in our study. A list of 10,073 CNVcor genes and 9841 METcor genes were confirmed with a significance level of P<0.01. We found that CNVcor is more likely to appear on chromosome (chr)8, chr17, and chr10, while METcor is more likely to appear on chr1, chr19, and chr17. Based on the core genes, we finally classified the samples into 4 subtypes, incorporating iC1 (iCluster) (92 samples), iC2 (79 samples), iC3 (165 samples), and iC4 (141 samples). Furthermore, we constructed the prognostic model for PRAD based on the 5 genes (IER3, AOX1, PRKCDBP, UBD, and FBLN5). Nomograms incorporating risk score and other clinical variables were further constructed, and these nomograms exhibited superior predictive ability. We further compared the differential immune infiltrating patterns in 2 risk groups and found significantly low levels of infiltrating cluster of differentiation (CD)8⁺ T cells in high-risk samples.ConclusionsOur study integrated the multi-omics data to elucidate the molecular features of PRAD and pivotal genes for predicting prognosis.     

Hybrid minigene splicing assay verifies the pathogenicity of a novel splice site variant in the COL1A1 gene of a chinese patient with osteogenesis imperfecta type I

BackgroundOsteogenesis imperfecta (OI) is a rare genetic bone disease associated with brittle bones and fractures. Among all known types, OI type I is the most common type and characterized by increased bone fragility, low bone mass, distinctly blue-gray sclera, and susceptibility to conductive hearing loss beginning in adolescence. Mutations in genes encoding type I collagen (COL1A1 and COL1A2) contribute to the main pathogenic mechanism of OI.MethodsSubtle mutation of the COL1A1 gene in the proband was detected by targeted next-generation sequencing (NGS) and confirmed by Sanger sequencing. We then assessed the effect of the mutation on the splicing of the COL1A1 gene by bioinformatics prediction and hybrid minigene splicing assay (HMSA).ResultsA novel splice site mutation c.1821+1 G>C was discovered in the proband by NGS and further confirmed by Sanger sequencing, which was also simultaneously identified from the proband's mother and elder sister. Bioinformatics predicted that this mutation would result in a disappearance of the 5′ donor splice site in intron 26, thereby leading to abnormal splicing and generation of premature stop codon. The follow-up experimental data generated by HMSA was consistent with this prediction.ConclusionOur study identified a novel splice site mutation that caused OI type I in the proband by abnormal splicing and demonstrated that combined applications of NGS, bioinformatics and HMSA are comprehensive and effective methods for diagnosis and aberrant splicing study of OI.     

FAT1 and MSH2 Are Predictive Prognostic Markers for Chinese Osteosarcoma Patients Following Chemotherapeutic Treatment

Osteosarcoma is characterized by diverse genetic mutations, including single-nucleotide variants (SNVs), which can complicate clinical outcomes of the treatment. This study identified key mutations or polymorphisms in genes that correlate with osteosarcoma prognoses. A total of 110 patients with osteosarcoma were assigned to “good” or “poor” cohorts depending on their 5-year disease-free survival (DFS) after surgery and chemotherapeutic treatment. We performed next-generation sequencing analysis of tumor tissues for prognosis-associated SNVs in 315 tumorigenesis-related genes, followed by modeling of clinical outcomes for these patients using random forest classification via a support vector machine (SVM). Data from the Chinese Millionome Database were used to compare SNV frequency in osteosarcoma patients and healthy people. SVM screening identified 17 nonsynonymous SNVs located in 15 genes, of which rs17224367 and rs3733406 (located in MSH2 and FAT1, respectively) were strongly correlated with osteosarcoma prognosis. These results were verified in a 26-patient validation cohort, confirming that these SNVs could be used to predict prognosis. These results demonstrated that two SNVs located in MSH2 and FAT1 are associated with prognosis of osteosarcoma patients. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Fluorescence In Situ Hybridization for the Origin of De Novo Renal Cell Carcinoma in a Kidney Allograft: A Case Report

BackgroundWe present a rare case of de novo renal cell carcinoma that developed in an allograft kidney 14 years after transplantation.Case reportA 39-year-old man underwent living donor kidney transplantation from his mother. After 14 years, routine screening ultrasonography revealed a solid mass of 30-mm diameter in the kidney allograft. Partial nephrectomy was performed by clamping the renal artery under in situ cooling. Tissue histology revealed clear cell carcinoma with negative surgical margins. We explored the tumor's genetic origin using fluorescence in situ hybridization to analyze the X and Y chromosomes of the tumor cells. Postoperative hemodialysis was avoided, and the patient's serum creatinine level remained stable.ConclusionsFluorescence in situ hybridization clearly indicated that the tumor originated from the donor and that the tumor vasculature originated from the recipient. The patient recovered well and remains without any tumor recurrence.