胃癌及癌前病变组织染色体7q31.1杂合性缺失的意义

目的 检测胃癌及不典型增生细胞染色体7q31.1区域的杂合性缺失（LOH）,绘制胃癌及癌前病变7q31.1区域等位基因缺失图谱,确定其常见最小缺失区域,探索胃黏膜上皮癌变过程不同阶段的分子遗传学改变。方法 在胃癌及胃黏膜组织石蜡切片上行显微切割,获得胃癌及胃黏膜上皮不典型增生细胞。用高密度微卫星标志结合PCR技术检测胃癌及癌前病变细胞染色体7q31.1杂合性缺失,绘制胃癌及癌前病变染色体7q31.1等位基因的缺失图谱。结果 发现胃癌染色体7q31.1至少有一个位点存在杂合性缺失的21例,占70.0%（21/30）;D7S2459、D7S523、D7S2502、D7S486、D7S480、D7S650、D7S2486各位点杂合性缺失频率分别为10.0%、6.7%、23.3%、43.3%、26.7%、26.7%、20.0%;缺失图谱分析显示胃癌常见最小缺失区域位于D7S2502~D7S480之间。在胃黏膜不典型增生组织中,至少一个位点等位基因缺失的11例,占36.7%（11/30）,其中缺失频率最高的微卫星位点是D7S480为23.3%(7/30);不同程度胃黏膜不典型增生患者中染色体7q31.1 LOH阳性率比较差别有统计学意义（P<0.01）。结论 胃癌染色体7q31.1常见最小缺失区域在D7S2502~D7S480之间,在D7S486附近可能存在与胃癌相关的抑癌基因。在胃黏膜癌前病变阶段（不典型增生）可检测出染色体7q31.1区域的杂合性缺失,7q31.1 LOH可能是胃癌发生极早期的分子事件之一。     

原发性胃癌染色体7q31的最小共同缺失区域及其意义

背景与目的:先前的研究显示7号染色体长臂(7q)在原发性胃癌有高频缺失;位于7q31的D7S486是7q上最高频的杂合性缺失(lossofheterozygosity,LOH)位点,且该位点的LOH频率与肿瘤的淋巴结转移显著相关;推测D7S486位点附近可能存在胃癌相关的抑癌基因(tumorsuppressorgene,TSG)。为能在更小的区域内找寻胃癌相关的TSGs,本研究通过检测原发性胃癌在7q31区域内微卫星标记位点的LOH情况,确定胃癌的最小共同缺失区域,并分析它们在胃癌发病中的可能作用。方法:以D7S486位点为中心,在位于其上下的7q31区域内选取平均遗传距离约0.5厘摩(centimorgan,cM)的12个微卫星标记。显微切割78例原发性胃癌和相应的正常胃粘膜组织,分别提取DNA;进行多重PCR扩增,聚丙烯酰胺凝胶电泳分离PCR产物,以GeneScan、Genotyper软件分析各微卫星位点的LOH情况。根据LOH结果作图,确定胃癌在7q31内的最小共同缺失区域,并与临床病理指标联系,分析区域缺失在胃癌发病中的可能作用。结果:12个微卫星标记位点均可在原发性胃癌中出现LOH,总的LOH频率为41.7%(40/72)。LOH的最高频位点是D7S486(位于7q31.2),为30.4.%(17/56);次高频位点是D7S650(位于7q31.3),为21.1%(8/38)。原发性胃癌在7q31内有两个最小共同缺失区域,分别为D7S2543～D7S486和D7S480～D7S650(长度均约为90kb)。D7S2543～D7S486区域缺失的频率与胃癌患者的临床分期和淋巴结转移显著相关(P=0.01和P=0.03);D7S480～D7S650区域缺失的频率与胃癌患者的临床分期显著相关(P=0.03),且该区域缺失仅出现于临床Ⅲ/Ⅳ期、T3/T4期或淋巴结转移的患者。结论:原发性胃癌在染色体7q31上存在两个最小共同缺失区域,分别为D7S2543～D7S486和D7S480～D7S650;在这两个区域内可能存在胃癌发展密切相关的抑癌基因。     

宫颈癌3、6、11和18号染色体遗传不稳定性的研究

背景与目的：人乳头瘤病毒的感染以及细胞内的遗传性改变是宫颈癌的主要病因。本研究拟分析人宫颈癌组织部分染色体位点遗传不稳定性,以为宫颈癌相关基因的定位以及筛选宫颈癌诊断分子标志提供依据。方法：应用3、6、11和18号染色体上的8个微卫星多态性位点,对50例原发性宫颈癌活检标本进行杂合性丢失（loss of heterozygosity,LOH)和微卫星不稳定性（microsatellite instability,MI)分析。结果：一个或多个位点发生LOH的样本占66％（33／50）,D18s474(18q21)位点的LOH率最高,达40．5％,其它位点的LOH频率为：D3s1478(3p21.3-21.2,31.7%),D3s1766(18q21.32,15.0%),d6s260(5p23,23.3%),D11s925(11q22-23,17.9%),D18s35(18121.1-21.31,8.7%),D18s64(18q21.32,16.7%),D18s68(18q22.1,27.3%).M1发生频率很低,仅为8％（4／50）。结论：宫颈癌染色体特定区域存在不同频率的LOH,而MI是宫颈癌中的低频事件。染色体3p和18q上的LOH高频区可能存在潜在的宫颈癌相关抑癌基因。     

胃癌7号染色体长臂的杂合性缺失分析

目的:检测胃癌患者7号染色体长臂微卫星位点的杂合性缺失(loss of heterozygosity,LOH),以初步确定7号染色体长臂上与胃癌相关基因连锁最密切的微卫星多态位点及LOH的临床意义.方法:在70例原发性胃癌中应用多重PCR技术扩增覆盖整个7号染色体长臂的9个微卫星位点(平均遗传距离为10cm),聚丙烯酰胺凝胶电泳分离PCR产物,用GeneScan、Genotyper软件进行分析.结果:9个微卫星位点的LOH均可发生于原发性胃癌,总的LOH频率为34.3%(24/70),其中D7S486和D7S798位点的LOH频率较高,分别为24.0%(12/50)和19.2%(5/26);总的LOH频率随临床分期而显著增高(P=0.046),D7S486位点的LOH频率在淋巴结转移者显著高于无淋巴结转移者(P=0.015).结论:在7号染色体长臂D7S486和D7S798位点附近,可能存在与胃癌发展相关的抑癌基因.     

人卵巢癌3号染色体短臂杂合性丢失的研究

目的探讨3号染色体短臂(3p14)等位基因杂合性丢失(10ss 0f heterozygosity,LOH)与人卵巢癌发生及发展之间的相关性研究.方法采用聚合酶链反应并结合二核苷酸重复序列多态性方法,分别对31例卵巢癌及24例卵巢良性肿瘤患者的组织标本DNA中3p14上3个微卫星位点(D3S1234、D3S1300、D3S1312)杂合性丢失(LOH)进行检测,同时还随机地检测31例卵巢癌中21例患者的血清DNA中3p14的LOH.结果31例卵巢癌组织DNA中21例(67.7%)至少在1个微卫星位点中出现杂合性丢失,11例卵巢癌患者(35.5%)有2个以上微卫星位点出现LOH.癌组织DNA中基因杂合性丢失频率与癌细胞分化程度呈正相关,与肿瘤病理类型及FIGO分期无关.24例卵巢良性肿瘤组织及血清DNA中均未出现3p位点上的杂合性丢失.21例卵巢癌血清DNA与肿瘤组织DNA 3p14基因3个微卫星位点杂合性丢失率之间存在明显的相关性(P＜0 05).结论鉴于人卵巢癌3p14出现杂合性丢失率与其癌细胞分化恶性程度相关以及卵巢癌患者血清DNA与癌组织DNA中3p14出现LOH相一致,故本文实验结果提示3p14 LOH的检测可能具有对卵巢癌患者的临床诊断潜在性价值.     

T细胞淋巴瘤6号染色体微卫星DNA的杂合性缺失研究

目的：对T细胞淋巴瘤（T—cell lymphoma，TCL）6号染色体上6个微卫星多态标志物进行等位基因杂合性缺失（loss of heterozygosity，LOH）分析，以明确该区域是否存在与人类TCL发生发展相关的抑癌基因。方法：选取6号染色体上6个微卫星多态标志D6S251、D6S275、D6S287、D6S267、D6S262、D6S264，采用石蜡组织基因组DNA抽提、PCR扩增，变性聚丙烯酰胺凝胶垂直电泳、银染法分别检测了42例TCL中肿瘤组织与相应正常组织基因组DNA的LOH状况。结果：42例TCL中13例（13／42，30．95％）至少在1个位点出现LOH，以D6D262最高（10．3％），其次为D6S287（10．0％）和D6S267（7．3％）。而不同临床病理分型的TCL其LOH发生差异无统计学意义（P〉0．05）。结论：在6号染色体上的6个微卫星标志中D6S287、D6S262和D6S267周围的6q21-6q23压域发生杂合性缺失率较高，位于6q21区编码CyclinC的基因可能是此区与TCL发生发展相关的候选抑癌基因；尤其是6q21-6q22．1区域可能存在与TCL相关的抑癌基因。可能与TCL的发生发展有关。     

肺癌6号染色体长臂的杂合性缺失研究

目的 检测原发性肺癌中6号染色体长臂的杂合性缺失。方法 应用PCR－SSLP－银染的方法,选用6号染色体长臂上的5对多态性微卫星标记,对36例原发性肺癌进行了杂合性缺失的研究。结果 36例肺癌中有19例至少在一个位点发生LOH,占52.8％,其中有1例同时在三个位点（D6S310、D6S314、D6S281）发生杂合性缺失。5个位点的LOH频率分别为：16.7%、13.9%、19.4％、5.6%和19.4％。结论 6号染色体长臂的杂合性缺失在原发性肺癌中是常见的染色体改变,并且在丢失的位点附近有一种或几种肿瘤抑制基因与人类原发性肺癌的发生与发展相关联。     

喉癌中13号染色体杂合性丢失研究

目的　为初步限定喉癌中 13号染色体抑癌基因的缺失区域和为发现及定位抑癌基因提供线索和依据。方法　应用聚合酶链反应 ( PCR) ,筛查了 58例喉癌 (包括 3例原位癌 )组织中染色体13q上 D13S765( 13q13) ,RB1.2 0 ( 13q14.2 ) ,D13S133( 13q14.3) ,D13S318( 13q2 1) 4个座位微卫星多态标记的杂合性丢失。结果　 3例原位癌中无一例发生 13q的杂合性丢失 ,55例浸润癌中在 13q一个以上座位出现杂合性丢失的频率为 4 5% ( 2 4 / 53) ,D13S765座位的杂合性丢失的频率最高 ,为 52 %( 2 2 / 4 2 )。结论　喉癌组织中染色体 13q缺失区域在 D13S765( 13q13)座位附近 ,RB1的近端 ,即在D13S765座位附近存在着与喉癌发生发展密切相关的抑癌基因 ,可能包括 RB1基因 ,它的失活与浸润期喉癌发生发展密切相关。     

鼻咽癌全基因组杂合性缺失分析

目的：分析鼻咽癌（nasopharyngeal carcinoma,NPS)全基因组染色体杂合性缺失（loss of heterozygosity,LOH),定位NPC发生高频率LOH的区域,为定位NPC相关基因提供分子遗传学依据。方法：用PCR微卫星多态性分析（335个位点）技术检测98例NPC肿瘤基因组DNA等位基因缺失。结果：在22对染色体中,19对染色体在所选取位点中有至少1个位点LOH频率≥30%,3对染色体（15号,20号和22号）无LOH频率大于30%的位点;在335个位点中,4个位点LOH频率≥60%,其中3个在3号染色体,1个在9号染色体;5个位点LOH频率介于50%-59%,其中3个在3号染色体,5号和11号染色体各1个位点;22个位点LOH频率介于40%-49%,52个位点LOH频率介于30%-39%,轼52个位点LOH频率低于30%,提示为背景缺失;LOH频率≥30%的位点主要集中于12个染色体臂,分布于：1p36-34,3p24-26,3p14-21,3q25-27,4q35-31,5q15-21和5q32-33,8p22-23,9p21-23和9q33-34,11p12-14,11q13-23,13q13-14和13q31-32,14q11-13,14q23-24,14q32。本研究新报道在染色体区带1p,5q和19q等发生高频率LOH,LOH精细图谱提示这些区域内可能存在与NPC相关的TSG。结论：NPC肿瘤细胞在多染色体区带发生高频率LOH是常见的分子事件,提示在这些缺失区,可能存在与NPC发生,发展过程中起重要作用的肿瘤抑制基因。     

原发性肝癌8号和16号染色体杂合子丢失的研究

目的:分析人肝细胞肝癌(HCC)组织中染色体8和16部分染色体片段的杂合子丢失及与临床病理关系,初步筛选HCC相关的抑癌基因,为HCC的早期诊断、预后预警提供可能的新分子标记物.方法:应用聚合酶链反应-变性聚丙烯酰胺凝胶-银染法分析45例HCC组织标本中分别位于染色体8和16上的具有高度多态性微卫星位点的杂合性丢失(LOH)状态.结果:发生LOH的总频率为68.89％ (31/45),其中D16S511位点的LOH发生率最高为53.33％ (24/45),其次是D8S261( 39.02％,16/41)和D8S499(34.88％,15/43).结论:染色体16q23、8p22-21.3及8p12区域的LOH发生频率高,可能存在与HCC发生发展相关的新的抑癌基因,特定位点的遗传变异可能与HBV感染、临床病理恶性程度等预后因素相关.     

Allelic loss on chromosome 1 is associated with tumor progression of cervical carcinoma.

BACKGROUND: Alterations in chromosome 1 are common in human malignancies. The frequency of loss of heterozygosity (LOH) on chromosome 1 in cervical carcinoma and its clinical significance are not clearly understood. METHODS: LOH on chromosome 1 was studied in 100 cervical carcinomas by the polymerase chain reaction (PCR) using 29 highly polymorphic microsatellite markers spaced approximately 10 centimorgans apart. Loci with high frequencies of LOH were identified and the findings were correlated with clinicopathologic characteristics. RESULTS: LOH on chromosome 1 at 1 or more loci was detected in 93% of tumors. The frequencies of LOH at locus D1S2829 (1p31), D1S2663 (1p36.3), and D1S2725 (1q25) exceeded 30%, and 12 other loci exhibited frequencies of LOH of 20-30%. Advanced stage tumors had a significantly higher percentage of informative microsatellite markers with LOH than early stage tumors. Of the 29 microsatellite markers studied, 4 loci had a significantly higher frequency of LOH in Stage III and IV tumors than in earlier stage tumors. CONCLUSIONS: Frequent aberrations on chromosome 1 in cervical carcinoma suggest that inactivation of tumor suppressor genes is important in cervical tumorigenesis. Higher frequencies of LOH in Stage III and IV tumors suggest that chromosome 1 changes are late events in cervical carcinoma. The findings of this study are consistent with earlier reports that suggest that tumor suppressor genes are present at 1p36.3 and 1p31. To the authors' knowledge, the high frequency of LOH mapped to 1q25 has not been reported previously. Its significance awaits further clarification.     

宫颈癌患者基因组遗传不稳定性分析

目的 研究中国宫颈癌高发区宫颈癌患者基因组遗传不稳定性改变，为寻找宫颈癌相关内源因子提供依据。方法 从GenBank中选取8对微卫星DNA引物，采用PCR-变性PAGE-银染方法检测50例宫颈癌(来自高发区)活检组织及其对照(同病例血液)样品的杂合性丢失(LOH)和微卫星不稳定(MI)。结果 LOH总检出率为66％(33／50)，其中，D18S474(染色体18q21)LOH达40．5％；染色体3p21．2—3p21．3中微卫星位点D3S1478，LOH达31．7％；并且在原位癌与浸润癌中，LOH分布也有一定差异。MI在宫颈癌患者基因组中仅存在少数微卫星位点，总的发生率较低，为8％(4／50)。结论 除高危型人乳头瘤病毒(HPVhr)感染外，细胞内源基因的改变在宫颈癌发生发展过程中也起着重要作用。高频LOH位点18q21 D18S474、3p21．2-3p21．3 D3S1478可能存在潜在的宫颈癌抑癌基因。     

Detailed deletion mapping at chromosome 11q23 in colorectal carcinoma

Loss of heterozygosity (LOH) is frequent at the chromosomal region 11q22-q23 in several types of tumours of diverse cell origin. Previous investigations of LOH at this chromosomal region in colorectal carcinoma have been contradictory in their findings, and have only included between 1-4 loci. In order to define any regions of LOH on 11q23, we investigated 16 loci between D11S940 and D11S934 on the long arm of chromosome 11 using microsatellite analysis. Of 57 colorectal carcinomas specimens, 36 (63.2%) demonstrated LOH at one or more marker, with the highest frequencies of LOH at D11S1340 (41.0%), located between 105.13-111.97 Mb from the centromere, and D11S924 (37.1%) and D11S4107 (40.5%), both located approximately 113 Mb from the centromere. No statistically significant associations between LOH and age-of-presentation or Dukes' stage were found. LOH was observed in colorectal tumours of all Dukes' stages, including Dukes' stages A and B, suggesting that the inactivation of a tumour suppressor gene(s) on 11q23 occurs in the early stages of colorectal carcinoma. These results confirm the presence of putative tumour suppressor gene(s) at chromosome 11q23, involved in the carcinogenesis of colorectal carcinoma, and will facilitate future identification of candidate genes.     

Loss of heterozygosity occurs at the D11S29 locus on chromosome 11q23 in invasive cervical carcinoma.

Allelotypic detection of loss of heterozygosity (LOH) has been used to identify putative tumour-suppressor genes. Loci on human chromosome 11q23 are frequently altered in malignant disease, and LOH has been reported at an anonymous D11S29 locus at 11q23 in a proportion of breast and ovarian cancers and malignant melanomas. Previous studies have reported a high frequency of LOH in cervical carcinoma mapping to 11q23. Using polymerase chain reaction techniques employing probes for a recently described polymorphic dinucleotide microsatellite within this locus, we have searched for LOH in 69 cases of invasive cervical carcinoma. Genomic material was microdissected from sections cut from archival paraffin-embedded material, using the patients'' constitutional genotype as a control Sixty-two (90%) of the cases were informative, and LOH occurred in 25/62 (40%) of tumours. Loss of an arm or single chromosome 11 is a well-recognised event in cervical carcinoma, and by employing other microsatellite polymorphisms mapping to 11q13 and 11p11-p12 we excluded those cases with widespread allelic loss. By doing so, LOH at D11S29 was found in 16/53 (30%) of tumours. The findings suggest a putative tumour-suppressor gene on 11q involved in cervical carcinogenesis.     

Frequent loss of the long arm of chromosome 18 in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common and deadly cancers in Japan. In this study we performed fluorescent in situ hybridization (FISH) and loss of heterozygosity (LOH) analysis for chromosome 18q in ESCC cells to investigate allelic imbalance of chromosome 18q in ESCC. In the FISH analysis, only one signal for chromosome 18q was detected in TE-1 esophageal cancer cells, whereas two signals were detected in TE-2 cells. Two of five resected ESCC samples from patients showed loss of one copy of chromosome 18q. To construct a precise deletion map of chromosome 18q, LOH analysis was performed using 30 microsatellite markers localized to chromosome 18q. LOH was observed in 31 of 46 ESCC samples (67.4%) for at least one locus on chromosome 18q. LOH frequency for individual markers varied from 18.5% (D18S460) to 48.4% (D18S866). Thirteen of 46 ESCC samples (28.3%) showed the loss of most of the long arm of chromosome 18. Lymph node metastasis and vein invasion were significantly associated with the deletion of chromosome 18q. Loss of chromosome 18q may play an important role in the progression of ESCC.     

p16 expression in Barrett's esophagus and esophageal adenocarcinoma: association with genetic and epigenetic alterations

Our previous work has shown that a number of genes locating on 1q21 were down-regulated in esophageal squamous cell carcinomas (ESCC) and they may involve in carcinogenesis. To determine whether chromosome 1q LOH occurs in ESCC, we analyzed LOH in 61 ESCCs using 18 microsatellite markers on chromosome 1q. Forty-six of 61 (75.4%) tumors presented LOH at one or more loci. A significant association was found between chromosome 1q LOH and histopathological grade. LOH on D1S3466 had a negative correlation with family history, whereas LOH on D1S2777 positively correlated with smoking. These results suggest this region harbor putative tumor suppressor gene(s) contributing to tumorigenesis and differentiation in ESCC.     

Loss of heterozygosity on chromosome 10q23 and mutation of the phosphatase and tensin homolog deleted from chromosome 10 tumor suppressor gene in Korean hepatocellular carcinoma patients

Loss of heterozygosity (LOH) in the 10q23 chromosomal region was analyzed in 18 tissue samples from Korean hepatocellular carcinoma (HCC) patients. LOH at the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) region (D10S215, AFMa086wg9 and D10S541) was found in 8 of the 18 (44.4%) HCCs. LOH (20%) and microsatellite instability (26.7%) were also frequently found at the D10S2177 locus, which is located on the telomere side of the PTEN region. LOH was found in other loci, such as AFM280we1 and D10S2281. The presence of LOH in regions other than the PTEN region on chromosome 10q23 suggested the presence of additional tumor suppressor gene(s). PTEN mutation was found in only a subset of HCCs: A single base insertion at the end of the 5'-end splice signal (AG-GUAAGUU) in intron 5 and a silent mutation in exon 6 (codon 188, CTG-Val to CTA). Our data collectively suggest that the genetic alterations of chromosome 10q23, including the PTEN gene, could be important in hepatocarcinogenesis in the Korean population.