粪便DNA甲基化检测对结直肠癌诊断及预后评估 临床价值研究进展

<正>随着医学科学的发展,表观遗传学逐渐成为肿瘤诊断及预后评估领域的热点。基因甲基化是肿瘤发生发展机制的表观遗传学因素领域的重要内容之一~([1])。DNA甲基化主要因为在DNA甲基转移酶的作用下,将S-腺苷甲硫氨酸的甲基基团转移到胞嘧啶和鸟嘌呤二核苷酸胞嘧啶的5位碳原子上,与3位鸟嘌呤形成mCpG~([2]);CpG岛异常高甲基化是肿瘤抑制基因失活的重要途径,在目前已知的多种人类恶性肿瘤发生与发展的机制研究中均已被证实,CpG岛异常高甲基化的存在,导致     

DNA甲基化与大肠癌发生关系的研究

DNA的甲基化参与基因的表达调控它的异常与肿瘤等疾病的发生有关,抑癌基因启动子区的异常甲基化和癌基因的去甲基化均影响肿瘤发生发展。在大肠癌的发生发展过程中,也伴随着DNA的异常甲基化。本文对目前常见的基因甲基化及其对大肠癌的作用和影响以及在治疗和预后所起的作用作一综述。     

MSP扩增中3种DNA提取法的比较

目的比较标准酚-氯仿、TripureDNA提取法、强碱法3种DNA提取法,研究哪种方法更简单方便,更适合做DNA甲基化特异性PCR（MSP）。方法分别用标准酚-氯仿、TripureDNA提取法、强碱法提取Jurkat细胞株中的DNA,并对3种方法提取的DNA含量、纯度及所需时间进行比较。结果3种方法提取DNA的纯度和完整性都较好,标准酚-氯仿法提取的DNA含量最低,且最费时;强碱法提取的DNA含量最高,并最省时。结论这3种DNA提取法提取的DNA都能够达到做MSP的要求,比较而言,强碱法更具优越性。     

nMSP-DHPLC检测粪便SEPT9基因甲基化及其在结直肠癌诊断中的应用

目的探讨粪便SEPT9(septin 9)基因甲基化检测在结直肠癌早期诊断中的应用价值。方法常规提取87例结直肠癌手术切除癌组织、癌旁正常结肠组织及其术前粪便DNA,应用巢式甲基化特异性PCR(nested methyl-ation specific PCR,nMSP)结合变性高效液相色谱(denatured high performance liquid chromatography,DHPLC)技术检测结直肠癌组织、癌旁组织及粪便SEPT9基因甲基化水平;比较结直肠癌组织和粪便中SEPT9甲基化检测在结直肠癌诊断中的敏感性和特异性。结果在87例结直肠癌中,癌组织SEPT9检出率为80.5%(70/87),癌旁组织检出率为8.0%(7/87),二者比较差异有统计学意义(P<0.01),特异性为91.9%;结直肠癌粪便SEPT9基因甲基化检测结果与组织检测结果基本一致。SEPT9基因甲基化与大于65岁老年患者和右半结肠癌显著相关。结论结直肠癌组织SEPT9基因甲基化异常发生率高,尤其是老年和右半结肠癌患者;粪便SEPT9基因甲基化异常可作为结直肠癌早期诊断的肿瘤标志物。     

细胞线粒体DNA缺失鉴定的Southern杂交方法的建立

目的 建立一种简便、快捷、准确检测细胞线粒体DNA缺失的方法 .方法 将线粒体DNA所编码呼吸链中细胞色素氧化酶(COX)的亚单位COX Ⅰ和COX Ⅱ分别作为Southern杂交的分子探针,鉴别细胞株的线粒体DNA是否完全缺失.进行生长营养缺陷鉴定,并从RNA及蛋白水平对结果 进行验证.结果 Southern杂交显示,SK-Hep1细胞可见COX Ⅰ、COXⅡ杂交条带ρ°SK-Hep1细胞(线粒体缺失细胞)未见杂交条带形成,作为对比试验的生长营养缺陷法、PCR、Northern杂交和Western杂交,所得结果 与Southern杂交结果 一致.结论 成功建立了鉴定细胞线粒体DNA缺失的Southern杂交方法 ,该方法 能简便、快捷、准确地鉴定细胞线粒体DNA缺失.     

巴斯德处理后坐骨神经再生的组织学研究

目的　观察大鼠坐骨神经经巴斯德温热处理后 ,在组织形态学方面的变化。　方法大鼠 12mm长左侧坐骨神经 ,进行 6 0℃、30min的巴斯德处理。分为处理后 1周群、6周群 ,各群分别为 15只。计算各群对照部 (处理部起的中枢 )、处理部以及末梢部 (处理部起的末梢 )中有髓轴突数、有髓轴突直径和有髓轴突面积比率 ,并用电生理和透射电镜进行组织形态学检查。　结果　(1)有髓轴突数 :1周群中 ,末梢部较对照部显著减少 (P <0 .0 1) ;末梢部中 ,6周群比 1周群显著增加 (P <0 .0 1)。 (2 )有髓轴突直径 :1周群中 ,末梢部较对照部显著减小 (P <0 .0 1) ;末梢部中 ,6周群比 1周群显著增大 (P <0 .0 1)。 (3)有髓轴突面积比率 :1周群中 ,末梢部较对照部显著减小 (P<0 .0 1) ;末梢部中 ,6周群比 1周群显著增大 (P <0 .0 1)。 (4 )肌电图 :处理后 1周与正常相比 ,神经传导速度明显延长 ,延长幅度为 13.2 % ;动作电位幅度明显降低 2 .95倍 (3.7mV)。处理后 6周与正常相比 ,神经传导速度仍然有明显延长 ,延长幅度为 8.3% ;6周后动作电位幅度已经恢复正常。 (5 )透射电镜 :1周群处理部中 ,髓鞘明显分层、增厚而凝固坏死 ,轴浆显著减少 ,雪旺细胞基底膜、神经外膜发生变性。 6周群末梢部中 ,显示多数再生有髓轴突。　结论     

三种全自动粪便分析仪对粪便隐血和幽门螺杆菌抗原检测的效果比较

 目的 探讨科域KU-F20、爱威AVE-562、沃文特FA280三台不同品牌型号粪便分析仪检测粪便隐血实验(occult blood test,OB)和粪便幽门螺杆菌(helicobacter pylori,HP)抗原,与人工法比较的一致性。方法 收集2020-06至2020-09住院患者粪便标本1851例,分别采用仪器法和人工法进行粪便OB和HP抗原检测,计算每台仪器的灵敏度、特异度、符合率,并进行差异比较及一致性分析。结果 KU-F20、AVE-562、FA280检测粪便OB与人工法比较,灵敏度分别为0.96、0.91、0.99,特异度分别是0.75、0.84、0.63,符合率分别是0.91、0.89、0.92,配对卡方检验分别是P=0.424、0.099、＜0.01,Kappa值分别是0.73、0.71、0.71。KU-F20、AVE-562、FA280检测粪便HP与人工法比较,灵敏度分别为0.88、0.85、0.90,特异度分别是0.60、0.95、0.66,符合率分别是0.80、0.88、0.83,配对χ²检验分别是P=0.689、＜0.01、0.56,Kappa值分别是0.48、0.73、0.56。结论 三台仪器检测粪便OB同人工法比较,一致性显著;AVE-562检测粪便HP抗原同人工法比较,一致性显著。     

宫颈癌前病变PAX1基因DNA甲基化检测与HPV-DNA分型检测的效果比较

目的 分析PAX1基因甲基化与HPV-DNA分型检测在宫颈癌前病变诊疗中的特征及潜在关联。方法 选择2021-06至2022-03在解放军总医院第三医学中心妇产科行液基薄层细胞检测(TCT)和人乳头瘤病毒-DNA(HPV-DNA)分型检测的患者。纳入研究TCT结果异常,意义不明的非典型鳞状细胞(ASCUS)和(或)HR-HPV阳性患者194例,宫颈组织病变筛查结果异常并转诊阴道镜下取病理活检的患者共130例。所有患者进行TCT、HPV-DNA分型检测、定量PAX1基因甲基化检测。结果 CIN3+(CIN3和宫颈癌)患者的PAX1基因甲基化数值显著高于正常、CIN1、CIN2患者,按照活检病理级别从高到低PAX1基因甲基化数值依次分别为18.21(17.56,18.66)和20.5(20.07,20.80),20.41(20.19,20.72),20.21(19.77,20.42);TCT结果提示ASCUS的患者共45例,CIN2+(CIN2、CIN3和宫颈癌)PAX1基因甲基化数值显著高于正常/CIN1,按照活检病理级别从高到低PAX1基因甲基化数值依次分别为18.87(17.80,2...     

DNA分子水平的放射效应研究

点介绍癌基因与全基因DNA辐射损伤的比较,有氧和乏氧下、剂量率、LET、加热等因素对DNA修复作用的机制及p53基因研究等的最新研究进展。许多研究结果进一步论证了DNA辐射损伤类型的多样性,其损伤类型受环境因素影响。随后的修复则视其损伤在DNA中的位置,可能是决定细胞命运的关键。     

食管鳞状细胞癌预后甲基化基因生物标志物筛选

 目的 鉴定食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)甲基化生物标志物,预测ESCC患者预后。方法 从癌症基因组图谱数据库(the cancer genome atlas, TCGA)下载ESCC和正常样本基因组甲基化数据及临床信息。所有ESCC样本随机分为验证组和训练组。采用Cox比例风险回归模型和随机生存森林算法在训练组中鉴定甲基化生物标志物。并使用时间依赖性ROC曲线来评估该模型的性能。在验证组中对该模型进行验证。通过GO功能注释,探讨DNA甲基化标志物的生物学功能。结果 鉴定出差异甲基化基因283个,并从中筛选出与生存相关的4个甲基化基因(RRAGB、SYP、ERCC6L和RNASEH2CP1)作为预后的生物标志物。训练组和验证组ROC曲线下面积(AUC)分别为0.984和0.83。该生物标志物能够将训练组患者分为高风险组、低风险组,并在验证组中得到相似的结果。多因素Cox回归分析表明,甲基化基因生物标志物是ESCC患者预后的独立预测因素。功能分析表明,这些标志物基因参与转录调控与DNA结合。结论 筛选出预测ESCC预后的甲基化基因标志物,是独立的预后预测因子。     

An improved rapid method for DNA recovery from cotton swabs

We present a novel rapid method for the recovery of cellular and free DNA from cotton swabs based on a simple elution buffer containing a high molecular weight polymer and detergent combined with a short proteinase K digestion to release cellular DNA. This method shows increased yields approaching 80% recovery of the input DNA compared to the QIAamp DNA Mini kit standard extraction protocol for swabs which has a recovery of 20–30%. The buffer components in the described method are compatible with direct PCR analysis of the isolated DNA without further purification. Recovery efficiencies were estimated by qPCR.     

Development of a forensically useful age prediction method based on DNA methylation analysis

Forensic DNA phenotyping needs to be supplemented with age prediction to become a relevant source of information on human appearance. Recent progress in analysis of the human methylome has enabled selection of multiple candidate loci showing linear correlation with chronological age. Practical application in forensic science depends on successful validation of these potential age predictors. In this study, eight DNA methylation candidate loci were analysed using convenient and reliable pyrosequencing technology. A total number of 41 CpG sites was investigated in 420 samples collected from men and women aged from 2 to 75 years. The study confirmed correlation of all the investigated markers with human age. The five most significantly correlated CpG sites in ELOVL2 on 6p24.2, C1orf132 on 1q32.2, TRIM59 on 3q25.33, KLF14 on 7q32.3 and FHL2 on 2q12.2 were chosen to build a prediction model. This restriction allowed the technical analysis to be simplified without lowering the prediction accuracy significantly. Model parameters for a discovery set of 300 samples were R² = 0.94 and the standard error of the estimate = 4.5 years. An independent set of 120 samples was used to test the model performance. Mean absolute deviation for this testing set was 3.9 years. The number of correct predictions ±5 years achieved a very high level of 86.7% in the age category 2–19 and gradually decreased to 50% in the age category 60–75. The prediction model was deterministic for individuals belonging to these two extreme age categories. The developed method was implemented in a freely available online age prediction calculator.     

Application of droplet digital PCR method for DNA methylation-based age prediction from saliva

The recent studies reported that DNA methylation markers show changes with age, and expected that the DNA methylation markers can be effectively used for estimation of age in forensic genetics. In this study, we applied droplet digital PCR (ddPCR) method to investigate the DNA methylation pattern in the CpG sites, and we constructed an age prediction model based on the ddPCR method. The ddPCR is capable of highly sensitive quantitation of nucleic acid and detection of sequence variations in gene by separating the sample into large number of partitions and clonally amplifying nucleic acids in each partition. We extracted DNA from saliva samples collected from several age groups. The DNA was bisulfite converted and subjected to ddPCR using specifically designed primers and probes. The methylation ratio of each sample was calculated and correlation between the methylation ratio and the chronological age was analyzed. In the results, methylated DNA ratio at the 4 CpG sites (cg14361627, cg14361627, cg08928145 and cg07547549) showed strong correlation with chronological age. Percent-methylation values at 4 CpG markers and chronological ages of the 76 individuals were analyzed by multiple regression analysis, and we constructed an age prediction model. We observed a strong correlation (Spearman’s rho = 0.922) between predicted and chronological ages of 76 individuals with a MAD from chronological age of 3.3 years. Collectively, the result in this study showed the potential applicability of ddPCR to predict age from saliva.     

Assessment of DNA methylation markers for forensic applications

ABSTRACT

DNA methylation displays promise in a variety of forensic applications, including chronological age estimation and identical twin differentiation. Knowledge of the age of a body fluid donor would offer intelligence information, as well as provide context to physical characteristic markers. Differentiation of identical twins would also be of considerable benefit as the DNA profile of identical twins cannot be distinguished when a correspondence between DNA profiles recovered from crime scene samples and a person is identified. Our research investigates and develops a methodology for DNA methylation analysis that could be implemented into forensic casework. Our results show that the differentiation of identical twins and chronological age prediction are both possible, with similar accuracies to other international research. Targeted multiplexing of bisulphite converted DNA in combination with massively parallel sequencing is demonstrated as a viable alternative to traditional methylation analysis techniques.     

Combining current knowledge on DNA methylation-based age estimation towards the development of a superior forensic DNA intelligence tool

The estimation of chronological age from biological fluids has been an important quest for forensic scientists worldwide, with recent approaches exploiting the variability of DNA methylation patterns with age in order to develop the next generation of forensic ‘DNA intelligence’ tools for this application. Drawing from the conclusions of previous work utilising massively parallel sequencing (MPS) for this analysis, this work introduces a DNA methylation-based age estimation method for blood that exhibits the best combination of prediction accuracy and sensitivity reported to date. Statistical evaluation of markers from 51 studies using microarray data from over 4000 individuals, followed by validation using in-house generated MPS data, revealed a final set of 11 markers with the greatest potential for accurate age estimation from minimal DNA material. Utilising an algorithm based on support vector machines, the proposed model achieved an average error (MAE) of 3.3 years, with this level of accuracy retained down to 5 ng of starting DNA input (~ 1 ng PCR input). The accuracy of the model was retained (MAE = 3.8 years) in a separate test set of 88 samples of Spanish origin, while predictions for donors of greater forensic interest (< 55 years of age) displayed even higher accuracy (MAE = 2.6 years). Finally, no sex-related bias was observed for this model, while there were also no signs of variation observed between control and disease-associated populations for schizophrenia, rheumatoid arthritis, frontal temporal dementia and progressive supranuclear palsy in microarray data relating to the 11 markers.     

Age determination through DNA methylation patterns in fingernails and toenails

Over the past decade, age prediction based on DNA methylation has become a vastly investigated topic; many age prediction models have been developed based on different DNAm markers and using various tissues. However, the potential of using nails to this end has not yet been explored. Their inherent resistance to decay and ease of sampling would offer an advantage in cases where post-mortem degradation poses challenges concerning sample collection and DNA-extraction. In the current study, clippings from both fingernails and toenails were collected from 108 living test subjects (age range: 0–96 years). The methylation status of 15 CpGs located in 4 previously established age-related markers (ASPA, EDARADD, PDE4C, ELOVL2) was investigated through pyrosequencing of bisulphite converted DNA. Significant dissimilarities in methylation levels were observed between all four limbs, hence both limb-specific age prediction models and prediction models combining multiple sampling locations were developed. When applied to their respective test sets, these models yielded a mean absolute deviation between predicted and chronological age ranging from 5.48 to 9.36 years when using ordinary least squares regression. In addition, the assay was tested on methylation data derived from 5 nail samples collected from deceased individuals, demonstrating its feasibility for application in post-mortem cases. In conclusion, this study provides the first proof that chronological age can be assessed through DNA methylation patterns in nails.     

鼠疫耶尔森菌DNA标识序列的鉴定及其应用研究

目的确定鼠疫耶尔森菌DNA标识序列,以用于鼠疫耶尔森菌的快速检测和鉴定。方法通过芯片比较基因组杂交和PCR验证,鉴定鼠疫耶尔森菌DNA标识序列,针对标识序列设计特定的PCR引物。结果鼠疫耶尔森菌基因组中存在3个区段,共28条基因,均是鼠疫耶尔森菌DNA标识序列。针对其中3条基因设计PCR引物,能特异性扩增出鼠疫耶尔森菌,与来自其他非鼠疫耶尔森菌的DNA无交叉反应。结论鼠疫耶尔森菌存在DNA标识序列,并能用于鼠疫耶尔森菌的快速检测与鉴定。