32例三阴性乳腺癌患者易感基因胚系突变分析

目的:应用全外显子测序技术初步探讨三阴性乳腺癌(TNBC)患者易感基因突变情况。方法:收集本院就诊的32例TNBC患者,均经临床手术病理确诊。采集患者外周血提取基因组DNA进行全外显子组测序,通过生物信息学分析筛选与乳腺肿瘤相关的易感基因变异。结果:32例TNBC患者中14例检测到BRCA1/2罕见变异,明确致病性或可疑致病变异6例,突变携带频率为18.8%。其中BRCA1:c.5468-1_5474del和c.4749_4750del是较常见的突变;BRCA2:c.6027A>C为新的变异;BRCA2:c.3794G>T、c.7901T>A,BRCA1:c.4616T>C首次在中国人群中发现。除了BRCA1/2变异外,还检测到83个乳腺肿瘤易感基因变异,每个患者携带2.6个变异。2个以上患者携带的乳腺癌易感基因包括ALK、APC、CDH1、PTCH2、RB1CC1、RAD51D、RAD54L、TSC1等。结论:BRCA1/2是TNBC患者最重要的易感基因,其他与DNA损伤修复相关的基因突变可能与TNBC患者的表型有一定的相关性。     

Diagnosing,discarding, or de-VUSsing: A practical guide to (un)targeted metabolomics as variant-transcending functional tests

PurposeFor patients with inherited metabolic disorders (IMDs), any diagnostic delay should be avoided because early initiation of personalized treatment could prevent irreversible health damage. To improve diagnostic interpretation of genetic data, gene function tests can be valuable assets. For IMDs, variant-transcending functional tests are readily available through (un)targeted metabolomics assays. To support the application of metabolomics for this purpose, we developed a gene-based guide to select functional tests to either confirm or exclude an IMD diagnosis.MethodsUsing information from a diagnostic IMD exome panel, Kyoto Encyclopedia of Genes and Genomes, and Inborn Errors of Metabolism Knowledgebase, we compiled a guide for metabolomics-based gene function tests. From our practical experience with this guide, we retrospectively selected illustrative cases for whom combined metabolomic/genomic testing improved diagnostic success and evaluated the effect hereof on clinical management.ResultsThe guide contains 2047 metabolism-associated genes for which a validated or putative variant-transcending gene function test is available. We present 16 patients for whom metabolomic testing either confirmed or ruled out the presence of a second pathogenic variant, validated or ruled out pathogenicity of variants of uncertain significance, or identified a diagnosis initially missed by genetic analysis.ConclusionMetabolomics-based gene function tests provide additional value in the diagnostic trajectory of patients with suspected IMD by enhancing and accelerating diagnostic success.     

Detection and typing of human papillomavirus DNA in paired urine and cervical scrapes

The prevalence of human papillomavirus (HPV) in paired cervical scrape and urine specimens from 144 women attending a clinic for genitourinary medicine was determined by polymerase chain reaction (PCR) and nested PCR, using degenerate and general primer pairs localized within the L1 region. HPV typing was by restriction fragment length polymorphism (RFLP), type-specific PCR (HPV 6, 11, 16, 18, 33), and partial DNA sequencing of PCR products. HPV DNA was detected in 114 (84%) women. HPV DNA was detected in the specimens of 58 patients after amplification with MY09/MY11 primers and in a further 54 patients after nested PCR with the GP5 + /GP6 + primers. A total of 106/136 (78%) of women had HPV DNA positive cervical scrapes and 89 (65%) had HPV DNA positive urine specimens. Both the urine and cervical specimens of 81 women were positive. In 25 women HPV DNA was detected in the cervical specimen only, and in 8 women HPV DNA was detected in the urine specimens only. A total of 108 specimens from 75 patients were typed. For 33 patients HPV typing was achieved in both the cervical and the urine specimens and 19 women had identical types in paired specimens. Multiple HPV infections could be detected in 15 (20%) of 75 women where either the cervical and urine specimen or both of the specimens could be typed. More then one HPV type was found in 8 specimens and from multiple sites (cervix and urinary tract) in the same patients on 7 occasions. The results of this study indicate that the detection of HPVs in the urogenital tract can be maximised through the testing of both cervical scrapes and urine specimens in conjunction with the use of a nested PCR to increase the sensitivity of HPV DNA detection. Also, urine cannot be a direct substitute for a cervical scrape as different HPV types are often detected in the urine compared with those detected in the cervix.     

Detection of a rare point mutation in Ki-ras of a human bladder cancer xenograft by polymerase chain reaction and direct sequencing

Summary This paper represents the first report of a codon 59 mutation in Ki-ras from a spontaneous human transitional cell carcinoma of the bladder. Point mutations have the potential to activate the ras genes if they occur in critical coding regions. These include the sequences of codons 12, 13, 59, 61 and 63. Mutations in codons 12, 13 and 61 have been reported in a wide variety of human cancers, including transitional cell carcinoma of the bladder. However mutations in codon 59 have been reported only in retroviral Ki-ras and as a result of in vitro mutagenesis experiments. We have used the polymerase chain reaction and direct sequencing to detect mutations of Ki-ras, and allele-specific restriction analysis to detect mutations of N-ras in xenografts and continuous cell lines established from bladder cancer biopsies of ten different patients as well as in direct biopsy specimens from five human bladder tumours. For studies of Ki-ras, a 139 bp fragment which spanned the critical codons 12 and 13 and a 128 bp fragment that spanned the sequences of codon 59, 61 and 63 were enzymatically amplified and then sequenced. No N-ras mutations were detected. A heterozygous mutation of Ki-ras at codon 59 GCA G/ACA was detected in one line. This mutation is being expressed and appears stable as it was detected over several xenograft passages and was present in paraffin-embedded tissue from the primary tumour of the patient. The biological significance of the mutation in bladder cancer is currently under study.     

DNA测序建立甘肃当归、大黄种子rRNA基因图谱的研究

目的：对甘肃地道性中药材当归、大黄种子中rRNA基因内转录间隔区进行碱基序列测定，以期建立从分子水平对中草药种子进行鉴定的一种新方法。方法：提取当归、大黄种子中核DNA，利用特异性引物对其rRNA基因内转录间隔区进行套式PCR扩增，并将扩增产物进行碱基序列测定。结果：经琼脂糖凝胶电泳证实：以上述方法可获得当归、大黄种子DNA中rRNA基因内转录间隔区PCR扩增产物；并经测序后得到了当归、大黄种子rRNA基因内转录间隔区的碱基序列，且具有明显的差异和可对比性。结论：不同植物性中药材种子rRNA基因内转录间隔区碱基序列可做为从分子水平进行鉴定的标记。     

改进的亚硫酸氢钠测序法检测HepG2 RASSF1A基因CpG岛甲基化状态

目的 改进亚硫酸氢钠测序法，并检验改进后方法在甲基化检测中的可行性。方法 提取人肝癌细胞株HepG2基因组DNA，亚硫酸氢钠化学修饰，针对修饰后Ras相关家族1A基因（RASSF1A）启动子序列设计特异引物并结合沉降PCR扩增，T-A载体克隆、测序。结果 HepG2细胞RASSF1A基因启动子CpG岛的16个CpG位点均被甲基化。结论 改进后亚硫酸氢钠测序法能明显减少非特异性扩增，提高PCR效率，更适于基因甲基化状态的检测。     

江苏省乙型肝炎病毒基因型分布与临床相关性

目的：了解江苏省乙型肝炎病毒（HBV）基因型分布及其临床相关性。方法：选择江苏省HBV-DNA阳性慢性HBV感染者215例，其中HBsAg携带者3例，急性乙型肝炎3例，慢性乙型肝炎166例（轻度80例、中度50例、重度36例）。肝硬化27例，原发型肝癌12例，重症肝炎4例，采用S区基因测序法检测HBV的基因型。结果：215例HBV-DNA阳性血清标本中，B基因型72例（33．5％），C基因型132例（61．4％），B＋C基因犁11例（5．1％）；未发现A、D、E、F、G、H型；C基因型肝癌＋肝硬化＋重症肝炎＋慢性乙肝（中度＋重度）显著高于B型与B＋C型（P〈0．05），C基因型的病程明显较B基因型和B＋C基因型长（F=17．615），γ-干扰素B基因型感染者的血清含量比C基因型和B＋C基因型感染者的血清含量显著高（F=13．652），S2＋S3＋S4方面C基因型比B基因型和B＋C基因型显著增多（P〈0．05），结论：江苏省存在HBV的B、C和B＋C三种基因型；以C基因型为江苏省优势毒株；并且C基因型病程长，肝纤维化程度明显，严重的肝病以及原发性肝癌的患者中所占比例显著高于其他基因型。     

重亚硫酸盐修饰直接测序技术检测p16基因甲基化的研究

目的建立稳定的重亚硫酸盐直接测序技术，检测p16基因甲基化状况。方法提取正常人全血基因组DNA．建立起稳定的重亚硫酸盐直接测序平台，利用该技术检测结直肠癌细胞株pl6基因甲基化状况。结果应用列联表的Fisher，Exact Test比较3种纯化方法的测序结果，乙醇／醋酸钠和过柱纯化与SAP—ExonI纯化法比较，测序结果差异有统计学意义（P〈0．05）；应用重亚硫酸盐直接测序技术，可检测出p16基因目的片段中CpG岛所有CpG位点的甲基化状况。结论应用SAP—ExonI纯化法，建立了稳定的重亚硫酸盐直接测序技术，并可检测出目的片段中CpG岛所有CpG位点的甲基化状况。     

基于化学成分与肠道菌群探讨消癌解毒方中黄连-乌梅药对的配伍机制

目的 探讨黄连-乌梅药对与消癌解毒方水煎液中部分生物碱类成分的含量差异,并比较药对与全方对小鼠肠道菌群的影响。方法 采用Extend-C₁₈(100 mm×2.1 mm,1.8 μm)色谱柱,以0.1%甲酸水（A）-乙腈（B）进行梯度洗脱,流速0.3 mL/min,柱温35 ℃,进样量2 μL;离子化模式为电喷雾离子化（ESI）,以正离子模式检测,开展方法学验证和含量测定研究。按照给药不同将小鼠分为正常组、全方高浓度组、全方低浓度组、药对高浓度组和药对低浓度组,连续灌胃给药7 d,每组分别收集粪便样本,进行16S rRNA基因测序。结果 建立的含量测定方法在一定浓度范围内线性关系良好,方法学验证结果均符合相关要求,木兰花碱与非洲防己碱在黄连、药对以及全方中的含量分别为:(4.433±0.133)(8.905±0.154) mg/g,（3.545±0.033）（9.170±0.051） mg/g,（5.287±0.038）（13.861±0.690） mg/g。全方组和药对组中拟杆菌门Bacteroides的丰度均高于正常组,而全方组和药对组中厚壁菌门Firmicutes的丰度均低于正常组。药对高浓度组中的疣微菌门Verrucomicrobia和变形菌门Proteobacteria的丰度高于其他组。结论 液质联用方法和相关参数简便、可靠,可用于有关成分的含量检测。与单味药中含量相比,经过药对配伍后,非洲防己碱含量增加,而木兰花碱含量则略有降低;经过全方配伍后,2种成分的含量均增加。此外,肠道菌群实验表明无论是药对还是全方在给药后,会不同程度地影响肠道菌群内部占比改变,其可能对调节肠道系统平衡,具有一定帮助作用,从而为进一步阐释黄连-乌梅在消癌解毒方中配伍机制提供了参考和依据。