遗传病二代测序临床检测全流程规范化共识探讨(2)——样品采集处理及检测

二代测序技术(next generation sequencing,NGS)具有极高的检测通量,相对低的检测成本,高度的准确性(accuracy)和精准性(precision),是遗传病临床检测的有力工具之一。NGS实验室的检测流程是否规范,将直接影响NGS数据的稳定性、可靠性和有效性,将决定其是否能被用于遗传病的临床辅助诊断或筛查。因此.作为遗传病基因检测的重要环节之一,NGS实验室中流程的规范化与标准化非常重要。2019年5月,在第二届基因检测联盟会议上,针对如何规范NGS检测流程,从事遗传病临床诊治、实验室检测以及第三方基因检测机构的专家进行了全面充分的讨论,旨在规范基于NGS的基因检测流程,对检测流程中的前、中、后三个阶段(包括样本采集/接收/保存、NGS建库、上机测序及数据质控)的操作与实施提出了专业性的指导意见,以规范NGS技术在遗传病基因检测领域中的应用。本文根据此次研讨会上各行业专家的讨论,总结并发布NGS实验室检测流程的规范共识,以促进NGS实验室流程的规范化和标准化,推进我国NGS实验室在遗传病基因检测领域的快速和专业化发展。     

Clinical signification of high-mobility group box 1 protein (HMGB1) expression in infiltrating ductal carcinoma breast tissue

Objective:Exploring the clinical signification of high-mobility group box 1 protein (HMGB1) expression in infiltrat-ing ductal carcinoma (IDC) breast tissue. Methods:The expression of HMGB1 protein in IDC breast tissue was detected by immunohistochemistry, and the relations among size of tumour, lymph node metastasis, clinical staging, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) were also analyzed. Results:Forty-six cases out of 60 cases of IDC breast tissue showed positive or strong positive HMGB1 expression (76.67%), statistical significance was observed between HMGB1 expression with clinical staging (P<0.01), lymph node metastasis (P<0.01), breast cancer ER (P<0.05) and HER-2 (P<0.05), however same conclusion can not be drawn between HMGB1 with either size of tumour or PR expression (P>0.05) in IDC breast tissue. Spearman analysis showed negative correlation between HMGB1 expression and ER, and positive correlation between HMGB1 expression and clinical staging, lymph node metastasis together with HER-2. Conclusion:It’s promising that HMGB1 expression in IDC tissue can be one of biological indicators of poor prognosis.     

人全外显子组测序IDT捕获探针的性能验证

目的探讨已建立的二代测序技术进行人全外显子组测序项目对变异的检出性能。 方法对4例实验室能力比对验证样本和4例已知结果样本进行全外显子组检测,分析点变异和插入缺失变异,从检测准确度、检测灵敏度、检测特异性、精密度和可报告范围等方面进行性能评估。数据质量控制以目标区域覆盖率、捕获特异性和平均深度为基础。 结果数据输出量大于8G,实现了目标序列区域99.7%以上的覆盖率,捕获特异性86%以上,平均测序深度100×以上,Q30大于90%,对SNV的检出率达100%,50 bp以下Indel检出率达100%。 结论本标准操作在验证范围内对变异的检出能力达到应用的要求。     

Over expression of amphiregulin promoted malignant progression in gastric cancer

Human Amphiregulin (AREG) was found to be over expressed in many types of cancer patients including gastric cancer (GC). However, its role in GC development remained unknown. In this study, over expression of AREG in MGC803 and SGC7901 GC cells were conducted by gene transfection through utilizing gateway recombinant cloning technology. Meanwhile, AREG knocked down in BGC823 and MKN45 GC cells were achieved by lentvirus-mediated shRNA oligos infection. The results showed that over expression of AREG promoted cancer cells proliferation, invasion and migration, prevented apoptotic cell death and facilitated cell cycle progression. However, the above AREG-mediated oncogenic phenotypes were reversed while AREG was knocked down in cancer cells. Furthermore, the expression of AREG in GC cells was associated with higher level of activation of both ERK/JNK/p38 and PI3K/Akt signaling pathways, which were also diminished by AREG knocked down. Finally, the result from study in vivo showed that the silencing AREG expression by AREG knocked down inhibited tumor growth in nude mice. Collectively, the data provided a convincingly laboratory evidence that over expression of AREG promoted malignant procession via activation of ERK/JNK/p38 and PI3K/Akt signaling pathways. Thus AREG possessed an oncogenic activity and could be served as a target for gastric cancer therapy.     

多中心血糖检测电子化质量监控系统的建立与实施

目的 建立多中心血糖检测电子化质量监控系统, 为大规模流行病学调查中多地区血糖和其他指标实验室检测质控系统的建立提供借鉴。方法 在中国慢性病及其危险因素监测项目中, 利用电子信息化技术建立基于网络的血糖检测质量控制系统, 包括血糖检测实验室性能验证数据、血糖检测实时质控数据输入、传输和质量评估反馈, 对302个监测点血糖检测质量和有效性进行评估。结果 参与该项目的所有监测点, 满足了质量要求并成功地完成了血糖重复性精密度监测初步评估。仅有少数监测点需要强化训练和重新进行中间精密度检测性能评估。从各监测点每日质控的数据分析来看, 绝大部分监测点实验室所采用的血糖监测系统具有良好的室内重复性。结论 为确保多中心的血糖数据的完整性和可靠性, 电子化血糖质控系统的建立和适当质量保证程序可以帮助识别监测点是否可以满足室内精密度质量标准, 确保其能够胜任血糖实验室检测工作, 及时评估各地区血糖检测的质量;同时发现, 实验室人员的培训和有效沟通非常重要。后续可以根据研究项目的需要增加并完善监控系统的功能, 降低因使用不同血糖检测方法导致的室间分析偏倚。     

Telemedicine across borders: A systematic review of factors that hinder or support implementation

PurposeInnovative technologies to deliver health care across borders have attracted both evangelists and sceptics. Our aim was to systematically identify factors that hinder or support implementation of cross-border telemedicine services worldwide in the last two decades.MethodsTwo reviewers independently searched ten databases including MEDLINE and EMBASE, in June 2011 including citations from 1990 onwards when at least an abstract was available in English. We also searched ELDIS and INTUTE databases and Internet search engines to identify grey literature. We included studies which (a) described the use of telemedicine to deliver cross-border healthcare and, or (b) described the factors that hinder or support implementation of cross-border telemedicine services. All study designs were included. Two reviewers independently assessed titles and abstracts of articles identified. Papers were allocated to one of four reviewers who extracted relevant data and validated it. We took a qualitative approach to the analysis, conducting a narrative synthesis of the evidence.Results6026 records were identified of which 5806 were excluded following screening of titles and abstracts. We assessed 227 full text articles, excluding 133 because they were fatally flawed or did not meet the inclusion criteria, producing a final sample of 94. They involved 76 countries worldwide, most involving collaborations between high and low or middle income countries. Most described services delivering a combination of types of telemedicine but specialties most represented were telepathology, telesurgery, Emergency and trauma telemedicine and teleradiology. Most link health professionals, with only a few linking professionals directly to patients. A main driver for the development of cross-border telemedicine is the need to improve access to specialist services in low and middle income countries and in underserved rural areas in high income countries. Factors that hinder or support implementation clustered into four main themes: (1) legal factors; (2) sustainability factors; (3) cultural factors; and (4) contextual factors.ConclusionsNational telemedicine programmes may build infrastructure and change mindsets, laying the foundations for successful engagement in cross-border services. Regional networks can also help with sharing of expertise and innovative ways of overcoming barriers to the implementation of services. Strong team leadership, training, flexible and locally responsive services delivered at low cost, using simple technologies, and within a clear legal and regulatory framework, are all important factors for the successful implementation of cross-border telemedicine services.     

PDCA提升分子诊断自动审核通过率的效果研究

目的探讨PDCA提升分子诊断自动审核通过率的效果。方法基于PDCA思路,使用系统导出的自动审核效能数据作为现状调查数据,以此确定目标、分析原因并确定主因,然后在此基础上制订措施并实施,再次使用自动审核效能数据进行效果分析,不达标者再次进行原因分析,进入PDCA循环,目标达到者固化措施,总结并制订下一步计划。结果分子诊断自动审核通过率由31.2%提升至66.2%,报告单审核效率提升了60.0%,检测项目标本周转时间(TAT)缩短2.2 h。结论使用PDCA循环结合实验室预警效能分析能够有效推进自动审核工作,该方法亦可推广用于实验室其他方面的改进。     

人类白细胞抗原-B等位基因多态性与江苏地区汉族人群2型糖尿病的相关性研究

目的  探讨人类白细胞抗原（HLA-B）等位基因多态性与江苏地区汉族人群2型糖尿病的相关性。方法  采用聚合酶链反应直接测序分型法（PCR-SBT）对118例江苏地区汉族2型糖尿病（T2DM）个体进行HLA-B等位基因分型，计算各等位基因频率和血清型频率，并与中华骨髓库江苏分库汉族志愿者人群（644例和20 248例）进行统计学比较。结果  HLA-B?鄢1501的等位基因频率在江苏地区汉族T2DM人群中明显升高（8.90% vs 3.03%，Pc = 0.000，■=3.13，95%CI=1.80～5.42，Power=98.23%），而HLA-B?鄢5801的等位基因频率则明显下降（1.69% vs 8.62%，Pc =0.000，■=0.18，95%CI=0.07～0.50，Power=91.07%）；相对应地，HLA-B?鄢15的血清型频率在江苏地区汉族T2DM人群中明显升高（21.18% vs 13.03%，Pc =0.022，■=1.79，95%CI=1.26～2.55，Power=90.15%），而HLA-B?鄢58的血清型频率则明显下降（1.69% vs 8.62%，Pc =0.000，■=0.18，95%CI= 0.07～0.50，Power=91.09%）。结论  HLA-B?鄢1501等位基因可能与江苏地区汉族人群2型糖尿病的易感性有关，而HLA-B?鄢5801则可能有保护作用。这些结果首次提示HLA-I类基因多态性可能与中国人群2型糖尿病有关。

     

Electromyography and Fos immunostaining study establish a possible functional link between trigeminal proprioception and the oculomotor system in rats

The objective of this study was to explore whether there was a functional link between trigeminal proprioception and the oculomotor system mediated through jaw muscle afferents. Electromyography (EMG) was undertaken of the levator palpebrae (LP) and superior rectus (SR), and Fos expression was detected in the brainstem following consecutive down-stretching of the lower jaw at 2-4 Hz in rats. Retrograde tracing was undertaken of the interstitial nucleus of Cajal and Darkschewitsch nucleus (INC/DN) pre-oculomotor neurons. EMG-like responses were recorded from the LP/SR during down-stretching of the lower jaw at 2-4 Hz in 3 out of 11 rats. Fos expression was induced by consecutive down-stretching of the lower jaw at 2-4 Hz for 20-30 seconds. Interestingly, Fos expression was distributed mainly in the bilateral INC/DN area. We also examined Fos-like immunoreactivity in central mesencephalic and paramedian pontine reticular formation that harbors premotor neurons controlling horizontal eye movement, but no Fos-like staining was observed therein. By injection of retrograde tracers into the oculomotor nucleus combined with Fos immunostaining, double labeled pre-oculomotor neurons were visualized to distribute in the INC/DN. In conclusions, there may exist a trigeminal proprioceptive – oculomotor system neural circuit through jaw muscle afferents in rats. Judging from Fos distribution pattern, this pathway might be related to vertical and torsional eye movements.     

高三酰甘油血症-腰围表型临床特征及其与糖尿病前期和糖尿病的关系研究

背景　近年,我国糖尿病前期和糖尿病的患病率逐年上升,若能早期发现其危险因素,予以干预可减少相关疾病的发生。高三酰甘油血症-腰围表型（HTGW）与血糖之间的关系尚未形成定论。目的　探讨HTGW的临床特征及其与糖尿病前期及糖尿病的关系。方法　于2012年6—10月在珠海市香洲区湾仔镇采用多阶段分层整群随机抽样法选取2 142例年龄18~75岁的社区居民为研究对象。根据腰围和三酰甘油水平将参与者分为3组：组1、组2及组3。组1：男性腰围≤90 cm或女性腰围≤85 cm且血清三酰甘油<2.0 mmol/L;组2：男性腰围≤90 cm或女性腰围≤85 cm且血清三酰甘油≥2.0 mmol/L或男性腰围>90 cm或女性腰围>85 cm且血清三酰甘油<2.0 mmol/L;组3：男性腰围>90 cm或女性腰围>85 cm且血清三酰甘油≥2.0 mmol/L。比较不同组别居民的临床特征,采用Logistic回归分析HTGW与糖尿病前期和糖尿病的关系。结果　2 142例参与者中169例因为缺失三酰甘油或腰围数据被剔除。最后纳入1 973例参与者,其中糖尿病172例、糖尿病前期143例,组1参与者1 141例,组2参与者624例,组3参与者208例。组2年龄、男性占比、高血压者占比、糖尿病前期者占比、糖尿病者占比、饮酒者占比、收缩压、舒张压、体质指数（BMI）、腰围、空腹血糖、C反应蛋白、总胆固醇、三酰甘油、低密度脂蛋白胆固醇、血肌酐、血尿酸高于组1,教育状况为高中以上者占比、高密度脂蛋白胆固醇低于组1,差异有统计学意义（P<0.05）。组3年龄、高血压者占比、糖尿病前期者占比、糖尿病者占比、吸烟者占比、饮酒者占比、收缩压、BMI、腰围、空腹血糖、总胆固醇、三酰甘油、血肌酐、血尿酸高于组1和组2,高密度脂蛋白胆固醇低于组1和组2,男性占比、受教育程度为高中以上者占比、舒张压、C反应蛋白高于组1,差异有统计学意义（P<0.05）。Logistic回归分析结果显示,在未调整的模型中,调整年龄、性别、高血压病史、吸烟、饮酒、体育锻炼模型,调整年龄、性别、高血压病史、吸烟、饮酒、体育锻炼、BMI模型中,HTGW分组均为糖尿病前期及糖尿病发生的影响因素（P<0.05）。结论　HTGW与糖尿病前期和糖尿病密切相关,应注意加强对该类人群的血脂控制及腹围控制,以降低糖尿病前期和糖尿病的患病率,减少心血管事件的发生。