首诊为急性缺血性脑卒中患者的骨髓增殖性肿瘤相关基因突变研究

目的 分析首诊为急性缺血性脑卒中(AIS)患者的骨髓增殖性肿瘤(MPN)相关JAK2、MPL和CALR基因突变发生情况.方法 收集378例AIS患者的外周血标本和临床资料.用等位基因特异性PCR和Sanger基因测序法检测患者基因组DNA中JAK2 V617F、JAK2 Exon12、MPL Exon10和CALR Exon9基因突变,并分析突变阳性患者的病史、临床资料、外周血细胞分类计数以及其他辅助检查结果.结果 共检测到5例(1.3％)患者JAK2 V617F突变阳性,1例(0.3％)CALR基因突变阳性,总阳性率为1.6％.其中2例JAK2 V617F突变阳性患者的外周血血红蛋白水平达到MPN的诊断标准.结论 约1.6％的首诊为AIS的患者可检测到JAK2 V617F或CALR突变,这部分患者可能处于MPN的早期阶段.     

新疆维吾尔自治区高风险三阴性乳腺癌30例BRCA基因突变及临床病理特征对比

目的 了解新疆维吾尔自治区高风险三阴性乳腺癌（TNBC）BRCA基因突变情况及BRCA基因突变者与未突变者临床病理特征的差异.方法 以新疆维吾尔自治区30例符合高风险TNBC标准的患者为研究对象,抽取外周静脉血,提取基因组DNA,对BRCA1/2基因的全部编码序列进行扩增.变性高效液相色谱分析（DHPLC）预筛BRCA1/2基因突变,经DNA测序证实.对比分析BRCA突变者与未突变者的临床病理特征.结果 30例患者中,5例（16.7％）BRCA基因发生致病性突变,其中BRCA1突变4例（13.3％）,BRCA2突变1例（3.3％）;25例（83.3％）未发现BRCA基因突变.突变者与未突变者相比,临床病理分期早,差异有统计学意义（P=0.040）.结论 新疆维吾尔自治区高风险TNBC BRCA1基因突变率高,对于高风险TNBC患者建议进行BRCA基因检测;高风险TNBC BRCA基因突变者与未突变者相比,可能在临床病理特征方面存在差异,应考虑个体化治疗.     

嗜铬细胞瘤患者VHL基因突变的分析

目的 探讨散发性嗜铬细胞瘤患者VHL基因突变检测及其意义.方法 从41例散发性嗜铬细胞瘤患者的外周血及嗜铬细胞瘤组织中提取基因组DNA,进行VHL基因测序分析,存在基因突变者再进行家系成员筛查.以50例健康志愿者外周血提取的DNA样本作为对照.结果 3例散发性嗜铬细胞瘤患者的VHL基因第2外显子存在基因突变,其中1例为572位核苷酸由G突变为C,该突变导致120位编码氨基酸由精氨酸转变为苏氨酸;2例为623位核苷酸处插入一重复核苷酸T(TITGTtG),导致下游读码框移位.其余38例患者和50例健康志愿者未发现VHL基因突变.进一步对3例发生基因突变患者的家系成员进行筛查,分别筛查出3例G572C携带者、3例623(TTTGTtG)携带者.结论 散发性嗜铬细胞瘤患者中存在可能致病的VHL基阏突变,建议对散发性嗜铬细胞瘤患者进行VHL基因检测分析.VHL基因检测有望作为临床遗传性嗜铬细胞瘤基因诊断的指标之一.     

家族性副神经节瘤中SDHD SDHB 基因突变分析

目的:探讨SDHD 、SDHB 基因突变在我国副神经节瘤（PGL ）患者中的发生状况,为PGL 分子诊断和分子遗传学的深入研究提供基础。方法:收集天津医科大学附属肿瘤医院2006年4 月～2007年12月间收治的具有完整临床及病理资料的8 例散发性副神经节瘤患者、1 例家族性副神经节瘤患者及其4 位亲属的外周血标本。留取 18例正常人外周血标本作为对照。扩增 SDHD、SDHB 基因各外显子,纯化后直接进行DNA序列测定,将测序结果与美国生物技术信息中心公布的标准序列比对。结果:家族性PGL 病例检测到SDHD 基因杂合性错义突变,8 例散发性副神经节瘤病例未检测到SDHD 基因外显子突变。2 例散发性PGL 病例携带SDHB 基因第一外显子同义突变,发病时间早于不伴该同义突变者,且病理上均为恶性。家族性病例未检测到SDHB 基因突变。结论:家族性PGL 病例携带SDHD 基因突变,而散发性PGL 病例中未发现SDHD 基因突变,提示SDHD 基因突变可能是国人家族性副神经节瘤发病的分子基础之一。散发性副神经节瘤病例检测到SDHB 基因第一外显子同义突变A6A,有该突变的患者病理上均为恶性,提示A6A 可能影响副神经节瘤的表型。      

高通量基因测序技术检测外周血循环肿瘤DNA基因突变在非小细胞肺癌中的应用

目的:探究高通量基因测序技术检测非小细胞肺癌外周血循环肿瘤DNA基因突变的应用价值。方法:临床纳入2017年1月至2018年9月在我院就诊的40例晚期非小细胞肺癌患者作为研究对象,所有患者入院后均经肺组织活检或气管镜检查确诊为晚期非小细胞肺癌。对患者进行病理组织石蜡切片DNA（tDNA）检测,并采集患者肘静脉血使用高通量基因测序技术检测患者外周血循环肿瘤ctDNA基因情况。对比分析tDNA与ctDNA检测对患者DNA基因突变的准确性,探讨非小细胞肺癌患者进行高通量基因测序技术检测外周血循环肿瘤DNA基因突变的应用价值。结果:40例非小细胞肺癌的外周血循环肿瘤DNA基因突变检测与组织石蜡切片比较,两种方法检测率差异无统计学意义（P>0.05）。在高通量基因测序技术检查外周血循环肿瘤DNA中,21外显子测序结果:61号替代突变2573G→T,62/63/68号替代突变L858R（2573T→G）。19外显子测序结果:50号样品突变为del E746→A750+2235G→A,60号样品突变为del E746→A750,70号样品突变为del L747→T751,80号样品突变为del L747→S752＋2257C→T。结论:非小细胞肺癌外周血循环肿瘤DNA基因突变进行高通量基因测序技术对具体的基因突变或缺失具有较高准确性,可实时监测肿瘤DNA基因突变情况,且具有无创性、可重复应用等优点。     

肺癌外周血EGFR突变检测及其临床意义

目的:通过血浆循环DNA的基因突变检测,筛选表皮生长因子受体(E-GFR)突变型肺癌患者,探讨突变特征及其在肺癌靶向治疗中的意义.方法:选取2009年9月至2010年5月苏州大学附属第一医院及上海市三甲类医院收治的96例肺癌患者的血浆以及其中59例相对应的肿瘤组织中提取DNA,采用PCR扩增和基因测序的方法检测EGFR基因的突变.结果:96例肺癌血样中检测出EGFR突变17例,其突变率为17.7%.在这些突变的样本中,外显子19和21突变分别占88.2%(15/17)和11.8%(2/17),其中直接测序法检测出EGFR纯合突变3例[L858R 2例,del E746-A750(1)1例],杂合突变14例.对14例杂合突变样本进一步通过单克隆基因测序法确定其突变类型为del E746-A750(2)9例、del E746-A750(1)3例、del L747-S752 2例.EGFR基因突变多见于肺腺癌(包括腺鳞癌)患者,与患者的性别与吸烟史无明显相关性.59例肺癌患者肿瘤组织的进一步分析证明,血浆EGFR基因突变类型与患者自身肿瘤的突变类型相同,表明血浆DNA检测到的EGFR突变与原发肿瘤检测到的突变相一致.结论:肺癌患者的血浆循环DNA与相对应的肿瘤组织DNA的EGFR基因突变类型一致;此外,相对于传统的"金标准"直接测序法而言,单克隆基因测序的方法能够更精确地判断基因的突变类型.这种简便且微创地检测血浆循环DNA的方法可应用于肺癌EG.FR基因突变的诊断,从而预测靶向治疗的疗效.     

JAK2 V617F阴性骨髓增殖性肿瘤患者MPL exon10突变检测

 目的　探讨MPL exon10突变在JAK2 V617F阴性骨髓增殖性肿瘤（MPN）中的发生情况。方法　对235例MPN患者进行JAK2 V617F检测,对检出的103例阴性患者应用等位基因特异性聚合酶链反应（ASP-PCR）联合测序检测MPL exon10已知基因突变MPLW515K／L;应用DNA单链构象多态性聚合酶链反应（SSCP-PCR）联合测序检测MPL exon10未知突变。结果　103例JAK2 V617F阴性MPN 患者中MPLW515K／L检出1例MPLW515K（TGG→AAG）,为原发性骨髓纤维化（PMF）患者;MPL exon10未知突变检测发现1例原发性血小板增多症（ET）患者存在新的突变类型,即MPL核苷酸1491-1492位之间插入12个碱基（CTGGTGATCGCT）,且为纯合突变。结论　JAK2 V617F阴性MPN患者在MPL 基因exon10区域内除已知W515K/L突变外尚存在新的突变位点,但突变率较低。     

肺癌细胞学及血液标本表皮生长因子受体基因突变对比研究

目的:研究肺癌患者渗出液细胞学及血液标本中表皮生长因子受体(EGFR)第18、19、21外显子基因突变频率和类型以及与肿瘤组织学标本的关系。方法:收集肺癌患者渗出液细胞学标本53例及血液标本24例,提取DNA,聚合酶链反应扩增EGFR外显子18、19、21序列,用直接测序法检测基因序列,分析EGFR基因突变频率和类型以及与肿瘤组织学标本的关系。结果:53例细胞学标本检测到15例EGFR基因突变（28.3%,15/53）,18外显子突变1例,19外显子突变5例,21外显子突变9例,细胞学标本与肺腺癌组织学标本突变率（32.2%）相比,差异没有显著性（P=0.624）。24例血液标本中检测到1例突变（4.2%,1/24）,位于21外显子,血液标本与肿瘤组织学标本突变率相比,差异有显著性(P=0.006)。结论:肺癌患者渗出液细胞学标本适用于直接测序法检测EGFR突变,血液标本不适于用直接测序法检测肿瘤EGFR基因突变状态。     

信息动态

目的 提高对骨髓增殖性肿瘤诊断和治疗的认识.方法 回顾性分析1例原发性血小板增多症患者的临床资料并复习相关文献.结果 该患者血象、骨髓象符合MPN表现,除外继发性血小板增多症,但JAK2 V617F突变为阴性.结论 JAK2 V617F、JAK2 Exon12、MPL、CALR基因突变联合检测可提高MPN的诊断率.     

骨髓增殖性肿瘤120例JAK2 V617F基因突变的临床分析

目的 探讨JAK2 V617F基因突变在骨髓增殖性肿瘤(MPN)患者中的发生率及临床意义.方法 采用骨髓细胞学和活组织检查方法分析120例患者的骨髓病理状况,监测费城染色体(Ph染色体)和bcr-abl融合基因.从患者骨髓抽提DNA,采用荧光定量PCR技术检测JAK2 V617F基因突变.结果 所有患者均呈现出MPN各自类型的典型特征.Ph染色体和bcr-abl融合基因检测均为阴性.120例MPN患者中JAK2 V617F基因突变的阳性率为66.7％(80/120),其中真性红细胞增多症(PV)为72.7％(16/22),原发性血小板增多症(ET)为66.0％(62/94),4例原发性骨髓纤维化(PMF)患者中2例阳性.JAK2 V617F突变阳性PV患者的外周血白细胞计数(P=0.001)和血小板计数(P=0.010)均高于阴性患者;JAK2 V617F突变阳性ET患者的白细胞计数高于阴性患者(P=0.006);PMF中JAK2V617F突变阳性和阴性患者间各项指标差异均无统计学意义(均P＞0.05).结论 JAK2 V617F基因突变检测有助于bcr-abl阴性MPN的诊断和鉴别诊断,使患者能够在早期被发现和治疗.     

胃癌组织TIN2基因突变研究

目的 TIN2是端粒保护蛋白复合体中的一员,其在胃癌组织中表达异常.本研究检测胃癌组织中TIN2基因的突变位点,探索其在胃癌发生发展中所起的作用.方法 选取南华大学附属第二医院2012-05-03-2013-05-14收治的胃癌患者组织标本50例,均为胃腺癌.提取50例胃癌组织标本中的DNA,进行PCR扩增,对TIN2基因的6号外显子进行测序,通过序列分析,找到TIN2基因的突变位点,并对其突变位点进行生物信息学的分析.结果 在22%(11例)的胃癌标本中发现了TIN2基因6号外显子中两个基因突变位点,其中6%(3例)胃癌标本中TIN2基因6号外显子第90位碱基C/C突变为C/T,16%(8例)胃癌标本中TIN2基因6号外显子第241位碱基G/G突变为A/A.通过生物信息学分析,发现两者对TIN2蛋白质的功能皆有影响,但TIN2基因6号外显子中第90位碱基C/C突变为C/T比TIN2基因6号外显子中第241位碱基G/G突变为A/A对蛋白质功能的影响更大,即这个突变很有可能是有害的,发现TIN2基因的突变与胃癌患者的性别、年龄、分化程度和临床分期无关,P>0.05.结论 发现了TIN2基因6号外显子中的两个突变位点,其对蛋白质的功能皆有影响.TIN2基因6号外显子中第90位碱基C/C突变为C/T为新发现的突变类型,其对蛋白质的功能影响更大.TIN2基因的突变可能在胃癌的发生发展中起一定的作用.     

二氢嘧啶脱氢酶编码基因DPYD＊ 5及＊ 9A突变频率的研究

目的氟尿嘧啶（5-Fu）是各种肿瘤治疗中应用最为广泛的抗肿瘤药物之一。体内80%以上的5-Fu在肝脏内经由二氢嘧啶脱氢酶（DPD）分解代谢。DPYD基因是DPD酶的编码基因,本研究旨在考察DPYD基因的＊5及＊9A位点在中国汉族乳腺癌患者中的突变频率。方法本研究收集100例乳腺癌患者的外周静脉血,应用基因测序的方法对93例乳腺癌患者DNA标本进行了DPYD＊5及＊9A位点的测序。结果对于DPYD＊5的1627A〉G突变位点,有37例发生突变,其中7例为纯合子突变,30例为杂合子突变;对于DPYD＊9A的85T〉C突变位点,有19例发生突变,其中1例为纯合子突变,18例为杂合子突变。有9例患者呈现DPYD＊5/＊9A联合突变。结论在中国汉族乳腺癌患者中（93例）,DPYD＊5的突变频率为39.8%,DPYD＊9A的突变频率是20.4%,DPYD＊5/＊9A联合突变的发生频率为9.7%。     

<Emphasis Type="Italic">AXIN2</Emphasis> polymorphism and its association with prostate cancer in a Turkish population

Polymorphism of AXIN2, a component of Wnt signaling, has been shown to play a role in tumorigenesis and dysregulated in cancer cells. In order to find out if AXIN2 polymorphism is a risk factor for prostate cancer, we analyzed eight polymorphic regions of this gene in 84 patients with prostate cancer and compared the results with 100 healthy controls in a Turkish population using PCR–RFLP methods. The genotype frequencies and risk factors of prostate cancer and control groups were analyzed by Chi-square test. We found a statistically significant result between prostate cancer risk and AXIN2 Intron2-956 + 16A/G (rs35285779) SNP. The frequency of the homozygous G/G (0%) and heterozygous A/G (18%) genotypes was significantly less in patients with prostate cancer than in healthy controls (7 and 32%, respectively) (P < 0.05) for this SNP. When compared with the wild-type A/A genotype of the controls, prostate cancer patients with the A/G and G/G genotype showed reduced risk of cancer; the adjusted odds ratio (OR) for patients with the homozygous G/G genotype was 0.87 (95% CI: 0.81–0.95) and for heterozygous A/G genotype was 0.42 (95% CI: 0.20–0.85). We found no statistically significant association between controls and prostate cancer for other seven SNPs of AXIN2 including Exon1-148 C/T (rs2240308), Exon1-432 T/C (rs2240308), Exon5-1365 G/A (rs9915936), Exon5-1386 C/T (rs1133683), Intron5-1712 + 19 T/G, Exon7-2062 C/T, and Intron7-2141 + 73 G/A (rs4072245) (P > 0.05). These results suggest that the AXIN2 Intron2 rs35285779 SNP is associated with development of prostate cancer as a protective SNP, while an association between other seven SNPs of the AXIN2 and risk of prostate cancer was not observed.     

DNA修复基因XRCC1单核苷酸多态性与非霍奇金淋巴瘤遗传易感性的关系

 目的　探讨DNA修复基因XRCC1 R280H、XRCC1 TSS+29C／T单核苷酸多态性与非霍奇金淋巴瘤（NHL）易感性的关系。方法　运用MassARRAY技术对73例NHL病例和540名健康对照的DNA修复基因XRCC1多态性进行检测,比较其不同基因型与淋巴瘤患病风险的关系。结果　XRCC1 R280H中G、A基因频率在对照组和病例组中分布差异有统计学意义（P＝0.001）,而XRCC1 TSS+29C／T中T、C的基因频率在两组中的分布差异无统计学意义 （P＝0.383）。进一步的分析表明,在XRCC1 R280H中,与携带GG野生纯合子基因型者比较,携带至少一个A等位基因者（GA或AA）患淋巴瘤的风险显著降低（P＜0.001,OR＝0.309,95 % CI＝0.168～0.567）。而在XRCC1 TSS+29C／T中,CC和CT与基因TT比较,携带C基因者会增加淋巴瘤的发病风险（P＝0.472,OR＝1.262,95 % CI＝0.669～2.379）。结论　DNA修复基因XRCC1 R280H的基因多态性与NHL的发生密切相关。     

乳腺良恶性病变GSTP1 基因的突变和多态性分析*

目的:检测谷胱甘肽S-转移酶P1（glutathione S-transferase P 1，GSTP1）基因各外显子的突变和多态性，探讨其与乳腺癌发生的关系。方法:选取乳腺癌及其相应癌旁增生组织标本各50例，良性增生组织标本50例，正常乳腺组织50例和正常人（志愿者）血液淋巴细胞15例为研究对象，运用PCR-SSCP 技术和DNA测序的方法检测GSTP1 基因外显子突变和多态性。结果:1）在3/50例（6%）乳腺癌组织中检测到GSTP1 基因外显子3 的11个位点发生突变，其中8 个突变位点是错义突变（MS），均可导致氨基酸的改变；2 个突变位点是同义突变（SS），氨基酸未发生改变；1 个突变位点发生移码突变（FS），造成氨基酸读码框顺序的改变，并产生新的终止密码子，使蛋白发生截短，造成功能缺失。在5/50例（10%）乳腺癌组织中检测到外显子4 的12个位点发生突变，其中6 个为错义突变（MS），均可导致氨基酸的改变；6 个移码突变（FS），突变热点在221 位碱基，发生率最高（33%）。 外显子6、7 和剪接点未见突变。在正常人外周血淋巴细胞、正常乳腺组织、癌旁和非癌旁乳腺增生组织中均未检测到该基因突变。2）在12/50例（24%）乳腺癌，5/50例（10%）乳腺癌旁增生，3/50例（6%）乳腺良性增生，3/50例（6%）正常乳腺组织和1/15例（6.7%）正常人淋巴细胞检测到GSTP1 基因外显子5 第313 核苷酸多态性（A→G，导致Ile105Val）。 结论:研究结果提示:1）GSTP1 基因外显子突变可能与部分乳腺癌的发生有关；2）GSTP1 第5 外显子的多态性与乳腺癌易感性可能有关。      

Detection of p53 gene mutation in plasma of patients with gastric cancer

Objective:To investigated p53 gene mutation in plasma of gastric cancer patients. Methods: DNA extracted from plasma and matched tumor and tumor-adjacent non-cancerous tissues of 96 gastric cancer patients, and DNA from 20 healthy volunteers were studied. Exon 5, 6, 7, and 8 of p53 were amplified by Polymerase Chain Reaction (PCR). The mutation status was analyzed by denaturing high-performance liquid chromatography (DHPLC), followed by direct sequencing of cases with aberrant chromatographic patterns. Results: Heterozygous mutations of p53 gene were detected in 19.9% (19/96) of primary tumor tissues and 5.2% (5/96) of corresponding plasma. All p53 gene mutations detected in plasma DNA consisted with mutations in the matched primary tumor samples.Neither the tumor-adjacent gastric mucosa tissues nor control plasma from healthy volunteers showed p53 gene mutation. No correlation was found between p53 mutation status and clinicopathological features of gastric cancer patients. Conclusion: p53 gene mutation in plasma can be detected in tissues and plasma of gastric cancer patients, which could be applied in screening and surveillance of this disease.     

Increased level of chromosomal damage after irradiation of lymphocytes from BRCA1 mutation carriers

Deleterious mutations in the BRCA1 gene predispose women to an increased risk of breast and ovarian cancer. Many functional studies have suggested that BRCA1 has a role in DNA damage repair and failure in the DNA damage response pathway often leads to the accumulation of chromosomal aberrations. Here, we have compared normal lymphocytes with those heterozygous for a BRCA1 mutation. Short-term cultures were irradiated (8Gy) using a high dose rate and subsequently metaphases were analysed by 24-colour chromosome painting (M-FISH). We scored the chromosomal rearrangements in the metaphases from five BRCA1 mutation carriers and from five noncarrier control samples 6 days after irradiation. A significantly higher level of chromosomal damage was detected in the lymphocytes heterozygous for BRCA1 mutations compared with normal controls; the average number of aberrations per mitosis was 3.48 compared with 1.62 in controls (P=0.0001). This provides new evidence that heterozygous mutation carriers have a different response to DNA damage compared with noncarriers and that BRCA1 has a role in DNA damage surveillance. Our finding has implications for treatment and screening of BRCA1 mutation carriers using modalities that involve irradiation.     

Polymorphisms in Heat Shock Proteins A1B and A1L (HOM) as Risk Factors for Oesophageal Carcinoma in Northeast India

Background: To investigate polymorphisms in heat shock proteins A1B and A1L (HOM) and associated risk of oesophageal carcinoma in Northeast India. Materials and Methods: The study includes oesophageal cancer (ECA) patients attending general outpatient department (OPD) and endoscopic unit of Gauhati Medical College. Patients were diagnosed based on endoscopic and histopathological findings. Genomic DNA was typed for HSPA1B1267 and HSPA1L2437 SNPs using the polymerase chain reaction with restriction fragment length polymorphisms. Results: A total of 78 cases and 100 age-sex matched healthy controls were included in the study with a male: female ratio of 5:3 and a mean age of 61.4±8.5 years. Clinico pathological evaluation showed 84% had squamous cell carcinoma and 16% were adenocarcinoma. Dysphagia grades 4 (43.5%) and 5 (37.1%) were observed by endoscopic and hispathological evaluation. The frequency of genomic variation of A1B from wild type A/A to heterozygous A/G and mutant G/G showed a positive association [chi sq=19.9, p= <0.05] and the allelic frequency also showed a significant correlation [chi sq=10.3, with cases vs. controls, OR=0.32, p≤0.05]. The genomic variation of A1L from wild T/T to heterozygous T/C and mutant C/C were found positively associated [chi sq= 7.02, p<0.05] with development of ECA. While analyzing the allelic frequency, there was no significant association [chi sq= 3.19, OR=0.49, p=0.07]. Among all the risk factors, betel quid [OR =9.79, Chi square= 35.0, p<0.05], tobacco [OR = 2.95, chi square=10.6, p<0.05], smoking [OR=3.23, chi square=10.1, p<0.05] demonstrated significant differences between consumers vs. non consumers regarding EC development. Alcohol did not show any significant association [OR= 1.34, chi square=0.69, p=0.4] independently. Conclusions: It can be concluded that the present study provides marked evidence that polymorphisms of HSP70 A1B and HSP70 A1L genes are associated with the development of ECA in a population in Northeast India, A1B having a stronger influence. Betel quid consumption was found to be a highly significant risk factor, followed by smoking and tobacco chewing. Although alcohol was not a potent risk factor independently, alcohol consumption along with tobacco, smoking and betel nut was found to contribute to development of ECA.