变性高效液相色谱技术检测非小细胞肺癌患者表皮生长因子受体基因突变的研究

 目的　探讨采用变性高效液相色谱（DHPLC）技术检测表皮生长因子受体（EGFR）基因突变的优势。方法　应用DHPLC技术检测49例非小细胞肺癌（NSCLC）患者EGFR基因第19与21外显子突变情况,并应用DNA直接测序法验证DHPLC检测基因突变的准确性。结果　49例NSCLC患者中,应用DHPLC检测出13例EGFR基因突变;其中第19外显子缺失突变10例（76.92 %）;第21外显子替代突变3例（23.08 %）。DNA直接测序法突变检测结果与DHPLC一致,DHPLC检测EGFR基因突变灵敏度为100 %。结论　DHPLC技术可以快速、准确、大规模筛选EGFR基因突变。     

肝硬化及肝癌p53基因突变的实验研究

目的 :研究肝硬化及肝癌p53基因的突变情况。方法 :选择 80例肝硬化、肝癌标本 ,分别以PCR SSCP法 ,双链DNA序列测定法研究其p53基因外显子的突变情况及突变位点。结果 :62例肝癌标本p53总突变率为 19 4 % ,其中 ,早、中、晚期突变率分别为 10 5%、15 0 %、35 0 % ;18例肝硬化标本p53总突变率为 5 6% ;第 7外显子的突变发生在 2 4 9位密码子第 3号碱基上 ,为G :C→T :A的颠换突变 ;第 8外显子的突变发生在 2 73位密码子第 1号碱基上 ,为C :G→T :A的转换突变。结论 :p53基因突变发生在肝细胞发生形态学改变之初 ,随着肝癌的进展逐渐积累 ,突变率呈上升趋势 ,故p53基因突变很可能是启动癌变过程的重要因素之一。     

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.     

抑癌基因PTEN突变在软组织肉瘤中的研究

目的本研究探讨软组织肉瘤中PTEN突变及与软组织肉瘤发生发展的相关性。方法应用聚合酶链反应．单链构象多态性分析（PCR．SSCP）方法以及DNA测序技术,扩增86例软组织肉瘤PTEN基因的第5～8外显子,根据构象改变检测其基因突变情况。结果86例软组织肉瘤中检测到2例突变：第8外显子第351位密码子由T→C的错义突变,突变使蛋白结构由苯丙氨酸突变为赖氨酸;第8外显子第334位密码子由A→T的错义突变,突变使蛋白结构由天门冬酰胺突变为赖氨酸。结论软组织肉瘤中存在PTEN抑癌基因的突变,但突变率很低,可能在STSs的发生发展中不起主要作用。     

多发性骨髓瘤骨髓RAS基因突变检测分析

目的:检测NRAS和KRAS基因在多发性骨髓瘤(MM)患者中的突变类型,探讨其在MM病理发生发展过程中的作用。方法:采用多聚酶链反应和DNA序列测定的方法,对50例MM患者骨髓细胞基因组中的NRAS基因第1和第2外显子以及KRAS基因的外显子进行检测,并与标准参考序列进行对比。结果:50例MM患者中有3例(6%)Ⅲ期男患者发现NRAS基因突变,分别为第1外显子编码区第34位核苷酸的杂合突变c.34G>A,导致第12密码子发生GCT>AGT错义突变,从而产生氨基酸Gly>Ser(G12S)的改变;c.38G>A杂合突变,导致第13密码子发生GGT>GAT,Gly>Asp(G13D)的错义突变;c.182A>T杂合突变,导致位于第2外显子中的第61密码子发生CAA>CTA,Gln>Leu(Q61L)的错义突变。未检测到KRAS基因外显子区的突变。结论:NRAS突变可能与MM的病理进程相关,NRAS G12S、G13D和Q61L突变在MM发生和发展中的功能和作用还有待进一步的深入研究。     

中国肺腺癌患者上皮生长因子受体基因突变的研究

目的:分析我国肺腺癌患者上皮生长因子受体(EGFR)基因突变的发生率和突变类型。方法:在上海、杭州和昆明等地收集61例肺腺癌及其正常肺组织,采用PCR扩增和基因测序方法对组织DNA中EGFR外显子19~21基因突变进行分析。结果:正常肺组织中EGFR基因均为野生型,肺腺癌组织中EGFR基因突变检测率为47.5%(29/61),其中外显子19和21突变分别占突变总数的55.2%(16/29)和44.8%(13/29),外显子20未检测到突变。外显子19突变发生在第746~752位密码子,均为碱基缺失突变,有6种不同类型。外显子21突变全部是第858位密码子碱基替换突变。EGFR基因突变与患者性别和年龄无显著相关性。但昆明和上海等地患者的基因突变存在明显差异。结论:EGFR基因突变是一种肿瘤特异性的体细胞遗传改变,突变发生率约占肺腺癌总数的一半,其中以外显子19和21突变为主。我国EGFR基因突变存在地域差异。     

多重PCR-SSCP法检测妇科肿瘤患者hMSH2基因突变

目的：建立经济快捷地筛查基因突变的技术，研究妇科肿瘤患者hMSH2基因突变情况，寻找与妇科肿瘤发生相关的分子标记物。方法：通过PCR优化策略，建立多重PCR—SSCP法；收集42名妇科肿瘤病人的基础资料及血液标本，抽提血液DNA，用多重PCR法扩增hMSH2基因第六、第七外显子，并进行单链构象多态性分析(SSCP)及DNA序列分析。结果：检测出2位妇科肿瘤病人在hMSH2基因第七外显子有杂合性突变，且为Leu→Phe的错义突变。未发现hMSH2基因第六外显子上存在突变。结论：多重PCR—SSCP是筛查基因突变的一种快速有效的方法，hMSH2基因第七外显子上的突变可能是与妇科肿瘤发生相关的一种分子标记物。     

肝硬化及肝癌p53基因突变的实验研究

目的：研究肝硬化及肝癌p53基因的突变情况。方法：选择80例肝硬化、肝癌标本,分别以PCR－SSCP法,双链DNA序列测定法研究其p53基因外显子的突变情况及突变位点。结果：62例肝癌标本p53总突变率为19．4％,其中,早、中、晚期突变率分别为10．5％、15．0％、35．0％;18例肝硬化标本p53总突变率为5．6％;第7外显子的突变发生在249位密码子第3号碱基上,为G：C→T：A的转换突变。结论：p53基因突变发生在肝细胞发生形态学改变之初,随着肝癌的进展逐渐积累,突变率呈上升趋势,故p53基因突变很可能是启动癌变过程的重要因素之一。     

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。     

肝癌非高发区安徽省肝细胞癌p53基因249密码子点突变的研究

目的：研究ｐ５３基因２４９密码子在肝癌非高发区安徽省的点突变的发生率。方法：应用ＰＣＲ－ＲＦＬＰ技术肝细胞癌ｐ５３基因２４９密码子突变的频率。结果：所检测的３８例肝细胞癌均无出现ｐ５３基因第七外显子纯合性缺失,４例有ｐ５３基因２４９密码子的点突变,且均为杂合型突变,突变率为１０．５％（４／３８）。结论：本资料说明,在肝细胞癌非高发区中,肝细胞癌中ｐ５３基因第七外显子发生的点突变并非为高发事件。提示     

Germline mutations of the STK11 gene in Korean Peutz-Jeghers syndrome patients

Peutz-Jeghers syndrome (PJS) is an autosomal dominantly inherited disease characterized by hamartomatous gastrointestinal polyps and mucocutaneous pigmentation, with an increased risk for various neoplasms, including gastrointestinal cancer. Recently, the PJS gene encoding the serine/threonine kinase STK11 (also named LKB1) was mapped to chromosome 19p13.3, and germline mutations were identified in PJS patients. We screened a total of ten Korean PJS patients (nine sporadic cases and one familial case including two patients) to investigate the germline mutations of the STK11 gene. By polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing analysis, three kinds of mis-sense mutation and a frame-shift mutation were identified: codon 232 (TCC to CCC) in exon 5, codon 256 (GAA to GCA) in exon 6, codon 324 (CCG to CTG) in exon 8, and a guanine insertion at codon 342 resulting in a premature stop codon in exon 8. These mis-sense variants were not detected in 100 control DNA samples. Furthermore, we found an intronic mutation at the dinucleotide sequence of a splice-acceptor site: a one base substitution from AG to CG in intron 1, which may cause aberrant splicing. Most reported germline mutations of the STK11 gene in PJS patients were frame-shift or non-sense mutations resulting in truncated proteins. Together, these findings indicate that germline mis-sense mutations of the STK11 gene are found in PJS patients in addition to truncating mutations. The effects of these mutations on protein function require further examination. In summary, we found germline mutations of the STK11 gene in five out of ten Korean PJS patients.     

抑癌基因FHIT第8和第5外显子在原发性肝癌中纯合性缺失与点突变的研究

目的　对 2 0例原发性肝细胞肝癌 (HCC)的抑癌基因 FHIT外显子 5、外显子 8的纯合性缺失和点突变进行检测。方法　收集 HCC手术标本 ,提取癌细胞 DNA,采用 PCR方法研究 FHIT外显子 5、外显子 8的纯合性缺失 ;使用 PCR- SSCP方法研究 FHIT外显子 5和外显子 8的点突变。结果　发现在 2 0例 HCC中外显子 5的纯合性缺失率为 10 .0 (2 / 2 0 ) ,外显子 8的纯合性缺失率为 30 .0 % (6 / 2 0 ) ;有 1例外显子 5和外显子 8均缺失 ;FHIT的异常率为 35 .0 % (7/ 2 0 )。在被检的肿瘤组织细胞中 ,未发现 FHIT外显子 5和外显子 8存在点突变。在被检的正常细胞中未发现 FHIT缺失和点突变的情况。结论　 FHIT的缺失只发生在 HCC的肿瘤细胞中。在 HCC中 ,外显子 (尤其是外显子 8)的纯合性缺失是 FHIT基因失活的重要方式之一。点突变可能不是 HCC中 FHIT失活的主要方式     

P16 gene deletions and point mutations in patients with agnogenic myeloid metaplasia (AMM).

Studies of p16 alterations with homozygous deletions and mutation analysis were done in 32 patients with agnogenic myeloid metaplasia (AMM) including six patients in leukemic phase. No homozygous deletions were found and, one patient was found to have a shift band in exon 2C fragment by PCR-SSCP analysis. Further sequence analysis demonstrated that the mutated band was a point mutation of G to A in exon 2 codon 140 (GCG-->ACG) causing an amino acid substitution of alanine to threonine demonstrating this patient either carried an mutated gene in one allele as a polymorphism (heterozygous carrier of a mutant p16 gene) or carried a mutant p16 gene clone. This study demonstrates that p16 alterations with homozygous deletions and mutations were very rare in patients with AMM. A single patient found to have a shifted band by PCR-SSCP may be represented as a coincidence or as a polymorphism with a heterozygous carrier of mutated p16 gene, predisposable to AMM or as a mutant p16 gene which can be infrequently observed in this disease.     

Detection of Homozygous Deletions and Mutations in the CDKN2A Gene in Hydatidiform Moles

OBJECTIVE To investigate homozygous deletions and mutations in the CDKN2A gene(p16 INK4a and p14 ARF gene)in hydatidiform moles. METHODS A total of 38 hydatidiform mole samples and 30 villi samples were examined for homozygous deletions in the CDKN2A gene by PCR and for mutations by DHPLC. RESULTS i)Among 38 hydatidiform mole samples, homozygous deletions in the p16 INK4a exon 1 were identified in 5 cases(13.2%),while no homozygous deletions were found in the p16I NK4aexon 1 of 30 early-pregnancy samples.The rates of those deletions in hydatidiform compared to early-pregnancy villi samples was statistically significant(P=0.036).ii)No homozygous deletions in the p14 ARF exon 1 or p16 INK4a exon 2 were found in any of the hydatidiform moles or early-preganancy samples.iii) In all hydatidiform moles and early-pregnancy villi samples,no mutations were detected by DHPLC. CONCLUSION We suggest there may be a close correlation between homozygous deletions in the CDKN2A gene and occurrence of hydatidiform moles variation in the CDKN2A gene is mainly caused by homozygous deletions,while mutations may be not a major cause.     

Alterations of CDKN2(p16/MTS1) exon 2 in human hepatocellular carcinoma

To determine the state of CDKN2(p16/MTS1) gene in human hepatocellular carcinoma (HCC), the polymerase chain reaction (PCR), dot blot hybridization, PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing techniques were applied to investigate the homozygous deletions and intragenic mutations of CDKN2 exon 2 in tumor and non-tumor liver tissues from 24 patients with HCCs. Alterations of CDKN2 exon 2 were found in 10 (2 homozygous deletions and 8 intragenic mutations) of the 24 tumor samples (41.7%), no alteration was found in any of the non-tumor liver tissues. All of the intragenic mutations were located at codon 81 of CDKN2 with GC bases deletions confirmed by DNA sequencing. A different sequence at codon 53-54 (C-G rather than G-C) was also found in all of the liver and tumor tissues. Our study indicates that alterations of CDKN2 gene is not a rare phenomenon in HCC, however, it might play an important role in the development of HCC and codon 81 might be a mutational hotspot of CDKN2 gene in HCC.     

原发性胃癌p16INK4a基因缺失和突变的研究

目的探讨p16INK4a基因缺失和突变在胃癌发病机制中所起的作用。方法采用多重PCR、PCR-SSCP和DNA测序对62例胃癌、癌旁组织及10例正常胃黏膜标本中p16INK4a基因纯合性缺失和突变进行检测。结果62例胃癌中发现p16INK4a基因第一外显子和第三外显子各有2例纯合性缺失,缺失率6.5%(4/62),PCR-SS-CP和DNA测序发现1例p16INK4a基因第一内含子区碱基插入,突变率1.6%(1/62),癌旁和正常胃黏膜均未发现缺失和突变。结论在原发性胃癌中,p16INK4a基因纯合性缺失率很低、突变罕见。     

Frequent genetic alterations and reduced expression of the Axin1 gene in oral squamous cell carcinoma: involvement in tumor progression and metastasis

The aims of this study were to analyze the genetic alterations and expression levels of the Axin1 gene in oral squamous cell carcinoma (OSCC), to evaluate its clinical importance and to clarify whether the Axin1 gene is involved in the pathogenesis of OSCC. Mutation analysis of the Axin1 gene was performed by denaturing high performance liquid chromatography (DHPLC) and DNA sequencing in 44 OSCC samples. Meanwhile, Axin1 protein expression was investigated by immunohistochemistry in these samples. Aberrant profiles were detected by DHPLC screening in 26 different OSCC cases. After sequencing analysis, four mutations and five polymorphisms were identified. One case of poorly differentiated OSCC with metastasis contained two mutations: one revealed a T>G substitution at nucleotide 324 in exon 1, resulting in a glycine to stop codon substitution at amino acid residue 108; the other revealed an A>G heterozygous mutation in intron 7, located very near to exon 8. In another two patients with moderately differentiated OSCC and metastasis, a G>T heterozygous mutation at codon 488 in exon 5 and a C>G substitution at the intron 5+26 position was detected, respectively. Five polymorphisms were all frequent and localized at positions of codon 254 (GAT--> GAC), codon 429 (GTC-->ATC), codon 525 (GAC-->GAT), codon 609 (GCT--> GCC), and intron 4+17 (G>A), with a frequency of 39%, 8%, 6%, 13% and 9%, respectively. Immunoreactivity for Axin1 was strongly positive in normal stratified squamous epithelium but significantly reduced expression of Axin1 was shown in most of the 44 tumor specimens (35/44), especially in poorly differentiated tumors with metastasis. These results suggest that mutational inactivation and reduced expression of the Axin1 gene may play a pivotal role in OSCC carcinogenesis and metastasis.     

脆性组氨酸三联体基因与食管癌遗传易感性的关系初探

目的检测７３对食管癌标本中脆性组氨酸三联体（ｆｒａｇｉｌｅｈｉｓｔｉｄｉｎｅｔｒｉａｄｇｅｎｅ,ＦＨＩＴ）基因相关的多态性位点的杂合性丢失。方法以ＲＴＰＣＲ和ＤＮＡ序列分析,检测１４例食管癌标本及２个食管癌细胞系中ＦＨＩＴ基因５′ＵＴＲ及编码区的缺失情况。结果３ｐ１４．２的Ｄ３１４８０、Ｄ３Ｓ１４８１、Ｄ３Ｓ１２３４位点有较高频率的杂合性丢失。在１４例标本中,有９例发现ＦＨＩＴｃＤＮＡ的全部或部分丢失,其中７例存在外显子１～４的丢失,５例有外显子５～９（编码区）的丢失。在食管癌细胞系ＥＣ８７３３中发现了ＦＨＩＴ全基因的丢失。结论ＦＨＩＴ基因的改变在食管癌的发生中是一种常见的遗传现象,但其是否就是食管癌的易感基因还需进一步探讨。     

p16 mutations/deletions are not frequent events in prostate cancer.

Cyclin-dependent kinase-4 inhibitor gene (p16INK4) has recently been mapped to chromosome 9p21. Homozygous deletions of this gene have been found at high frequency in cell lines derived from different types of tumours. These findings suggested therefore, that p16INK4 is a tumour-suppressor gene involved in a wide variety of human cancers. To investigate the frequency of p16INK mutations/deletions in prostate cancer, we screened 20 primary prostate tumours and four established cell lines by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis for exon 1 and exon 2. In contrast to most previous reports, no homozygous deletions were found in prostate cancer cell lines, but one cell line (DU145) has revealed to a mutation at codon 76. Only two SSCP shifts were detected in primary tumours: one of them corresponds to a mutation at codon 55 and the other one probably corresponds to a polymorphism. These data suggest that mutation of the p16INK4 gene is not a frequent genetic alteration implicated in prostate cancer development.     

Abnormal transcripts of FHIT gene in Chinese brain tumors

FHIT located at chromosome 3p14.2 was discovered and proposed as a candidate tumor suppressor gene in several cancers. To determine whether the FHIT gene at 3p14.2 is altered in Chinese brain tumors, we examined 13 brain tumors for deletions within FHIT locus. Evaluation of the FHIT gene in the panel of brain tumors led to a comprehensive mutation analysis. The complete sequence of the FHIT gene was determined and deletions between exon 5-8 were found in all 13 cases. In addition, single point mutation of amino acid from two glioblastoma and one atypical meningioma cases and multiple amino acid mutations from one pituitary tumor were observed. Our results support the hypothesis that FHIT gene alteration is involved in tumorigenic development of human neoplasms.