人胶质母细胞瘤等位基因谱分析的研究

目的 探讨胶质母细胞瘤（glioblastoma,GBM)发病的分子遗传学机理，确定GBM的发生发展主要和哪些染色体或染色体区域有关，哪些染色体区域上可能存在与GBM相关的肿瘤抑制基因（tumor suppressor gene,TSG)。方法 应用聚合酶链反应技术，采用荧光标记的引物和377型DNA序列自动分析仪，对21例GBM的所有22对常染色体上共计382个微卫星位点进行了杂合性丢失（loss of heterozygosity,LOH)分析，相邻2个微卫星位点之间的平均遗传学距离为10cM。结果 在所有被检测的染色体臂上都观察到LOH，其中以染色体10q、10p、9p、17p和13q的LOH率最高（＞50％），这些染色体臂上已知的肿瘤抑制基因PTEN、DMBT1、p16、p53和Rb所在区域LOH率都较高；14q、3q、22q、11p、9q、19q上也存在较高的LOH率（＞40．5％）；首次发现多个共同微小丢失区域：9p22-23、10p12.2-14、10q21.3、13q12.1-14.1、13q14.3-31、17p11.2-12、17p13、3q24-27、11p12-13、14q31-32.3、14q21-24.1、22q13.2-13.3、4q35、4q31.1-31.2、6qtel、6q16.3。结论 GBM存在复杂的遗传学异常，涉及多条染色体臂。以10q、10p、9p、17p和13q的异常与GBM发生发展的关系最为密切。除了已知的肿瘤抑制基因PTEN、DMBT1、p16、p15、p53和Rb外，首次所发现的多个微小共同丢失区域上可能存在GBM相关的多个未知TSG。     

复发前后多形性胶质母细胞瘤的等位基因谱分析

目的：研究多形性胶质母细胞瘤（glioblastoma multiforme,GBM)复发前后的分子遗传学变化，了解基因组范围内哪些染色体区域可能与GBM复发有关。方法：应用聚合酶链反应技术为基础的杂合性丢失（loss of heterozygosity,LOH)分析法，采用荧光标记的引物和377型DNA序列自动分析仪，检测了1例复发前后GBM所有22对常染色体上多达382个微卫星位点。相邻2个微卫星位点之间的平均距离为10cM。结果：对原发肿瘤标本的等位基因谱分析显示，染色体9p21上D9S157位点和10q21.3-26.3上D10S537、D10S185、D10S192、D10S597、D10S587、D10S217位点存在LOH。对复发肿瘤标本的研究显示，不但9p21和10q21.3-26.3上LOH的范围扩大，而且在其它多条染色体上也出现了LOH（包括1q,7p,7q,21q,20p,20q,10p,19p,19q)。结论：染色体9p和10q可能在该例GBM的发生中起着重要作用。尽管该病例复发前后的病理诊断相同，复发后GBM存在着更广泛的分子遗传学异常改变，可能伴随着更多肿瘤抑制基因的失活。     

应用基因组扫描对胃癌19号染色体进行杂合性缺失分析

目的 检测胃癌患者19号染色体微卫星位点的杂合性缺失（less of heterozygosity,LOH),以初步确定19号染色体上与胃癌相关基因连锁最密切的微卫星多态位点。方法 应用多重PCR对44例原发性胃癌患者中覆盖19号染色体上的22个微卫星位点（遗传距离在1．1－10．9cM之间）进行扩增。聚丙烯酰胺凝胶电泳分离PCR产物,Gene Scan^TM,Genotype^TM软件进行分析。结果 19个位点（19／22）检测出LOH阳性,总LOH频率为59％（26／44）,其中D19S571位点的LOH频率最高（21．43％）。结论 高频的LOH位点附近,可能存在未知的胃癌相关基因。     

具有家族史鼻咽癌4p15.1-4q12区等位基因杂合性丢失分析

目的确定有家族史鼻咽癌患者4p15．1-4q12区域等位基因杂合性丢失（loss of heterozygosity，LOH）的分布和频率，为进一步缩小该区域内易感基因的范围提供新的线索和依据。方法收集具有家族史的鼻咽癌患者石蜡包埋的活检组织标本，采用显微切割的方法在肿瘤组织石蜡切片上分离肿瘤细胞和正常淋巴细胞，选定7个定位于4p15．1-4q12区域内的高密度微卫星位点，多重PCR扩增和丙烯酰胺凝胶电泳后，Genescan软件对各位点LOH进行分析。结果25例具家族史鼻咽癌患者中，23例在4p15．1-4q12区至少存在一个微卫星位点的LOH（92％）。其中D4S2382位点LOH的频率最高，达到56％；D4S350和D4S1547位点LOH频率均约为50％。最小共同缺失区位于位点D4S350和D4S1547之间。结论鼻咽癌4p15．1—4q12区域内的易感基因可能位于微卫星位点D4S350和D4S1547附近。     

非小细胞肺癌6号染色体长臂的基因扫描分析

目的 探讨6号染色体长臂上与非小细胞肺癌发生相关的微卫星位点。方法 应用多重PCR对41例非小细胞肺癌中6号染色体长臂上的18个微卫星位点进行扩增。PCR产物应用聚丙烯酰胺凝胶电泳分离，电泳结果用Gene Scam^TM,Genotyper^TM软件进行分析，结果 各个位点有明显的不同的杂合性缺失（loss of heterozygosity,LOH）频率，从3.85%到38.45%不等，总LOH频率为58.5%(24/41)。LOH频率超过20％的位点共有8个，主要分布的6q24及6q27。具体范围为6q24-6q25.3[D6S1699(35%)、D6S409(23.33%)、D6S441(33.33%)]及6q26-27[D6S1550(38.45%)、D6S264(20%)、D6S1585(25%)、D6S446(33.33%)、D6S281(30.77%]。结论 6q24及6q27附近可能存在与非小细胞肺癌发生相关的肿瘤抑制基因。     

髓母细胞瘤10号染色体的杂合性丢失分析

目的探讨髓母细胞瘤的遗传学异常及其发病机制。方法通过微卫星分析（microsatellite analysis）方法,应用7个分别位于10号染色体长臂上PTEN（10q23）和DMBT1（10q25）基因位点的特异性标记物,分析18例髓母细胞瘤的杂合性丢失（loss of heterozygosity,LOH）。结果18例髓母细胞瘤中,位于10q23上的LOH比率为24％（9／37可分析标记）;位于10q25上的LOH比率为47％（9／19可分析标记）。结论在髓母细胞瘤中,染色体10q25上高比率的杂合性丢失提示,位于该位点上DMBT1基因的遗传学改变可能在髓母细胞瘤的发病机制中起着重要的作用。     

髓母细胞瘤8号染色体短臂的杂合性丢失

目的研究髓母细胞瘤8号染色体的遗传学异常,寻找与该肿瘤发病机制有关的杂合性丢失位点。方法通过微卫星分析（microsatellite analysis）方法,应用19个位于8号染色体短臂（8p）上的多态性标记物,检测髓母细胞瘤的杂合性丢失（loss of heterozygosity,LOH）。结果在所检测的23例髓母细胞瘤中,21例为原发肿瘤,2例为复发肿瘤。染色体8p总的LOH比率为51%（124个LOH/243个可分析位点）。我们在8p22-23.2之间发现了一个高比率的共同丢失区,其长度为18.14 cM。结论染色体8p22-23.1上很可能存在重要的抑癌基因,该基因的丢失可能与髓母细胞瘤发病有关。     

前列腺癌及前列腺上皮内瘤6号染色体等位基因杂合子丢失分析

目的：检测原发性前列腺癌及高级别前列腺上皮内瘤（PIN）6号染色体等位基因杂合子丢失（LOH）及其意义。方法：经显微切割技术切获取前列腺癌及PIN各10例患者DNA,采用聚合酶链反应（PCR）及微卫星多态性技术,对6号染色体上的20个微卫星标志位点LOH进行检测。结果：10例原发性前列腺中有8例在6号染色上至少有1个位点检测到LOH,6p21-6q23及6q25-6q27为2个高频LOH区,10例高级别PIN检测6号染色体20个位点,有5例各有1个位点检测到LOH,结论：前列腺癌中存在6号染色体的高频LOH区,分别位于6q21-6q23,6q25-6q27区,编码细胞周期素C及胰岛素样生长因子Ⅱ受体的基因为此2区侯选的抑癌基因,它们可能与前列腺癌的发生发展有关。     

肝细胞癌8号染色体微卫星变异及其与临床病理特征的关系

目的　探讨肝细胞癌 (HCC)微卫星变异的特点及其与临床病理表现的相关性。方法采用毛细管电泳DNA分析系统 ,对 5 6例HCC中 8号染色体上 10个微卫星的杂合子丢失 (LOH)、微卫星不稳定性 (MSI)和等位基因失衡 (AI) 3种变异特征进行检测。结果　 5 6例HCC在 8号染色体上 10个基因位点发生LOH的总频率为 6 6 1% (37/ 5 6 ) ,LOH以D8S2 6 1最高为 5 3 5 % (2 3/ 4 3) ,其次为D8S172 1(5 2 5 % )和D8S1771(5 2 5 % )。D8S2 77基因位点 ,血清HBsAg阳性患者的LOH频率显著高于HBsAg阴性者 (P <0 0 1) ,D8S2 6 1、D8S2 98和D8S1733基因位点 ,血清HBsAg阴性患者的LOH频率显著高于HBsAg阳性者 (P <0 0 1) ;D8S2 98和D8S1771基因位点 ,直径 >3cm肿瘤的LOH率明显高于≤ 3cm组 (P <0 0 5和P <0 0 1) ;在D8S172 1基因位点 ,无包膜或包膜不完整的肿瘤的LOH显著高于包膜完整的肿瘤 (P <0 0 1) ;D8S2 98和D8S1771基因位点 ,肝内转移者的LOH明显高于无肝内转移者 (P <0 0 5 )。MSI的频率为 12 5 % (7/ 5 6 ) ,AI的频率为 19 6 % (11/ 5 6 )。结论HCC在 8号染色体上存在广泛的微卫星变异 ,其中LOH方式在HCC的发生和发展过程中起重要作用 ,MSI的作用次之。特定基因位点的LOH与临床和病理学参数有一定的相关性     

头颈部鳞癌的微卫星不稳定性和杂合性丢失的初步研究

目的:探讨头颈部鳞癌的微卫星不稳定性(MSI)及杂合性丢失(LOH)。方法:选择来自3、5、6、8、9、13、17和18号染色体的15个微卫星标志对36例头颈部鳞癌标本和相应的外周血进行微卫星分析。结果:36例头颈部鳞癌中,27.8%(10/36)分别有1-8个位点存在MSI,MSI发生率较高的位点为:D17S520(22.9%)、D6S105(16.7%)和D8S264(13.9%)。在9p21-p22和3p14等处存在一定的LOH。微卫星异常的检出率与肿瘤分期、分级无相关性。结论:提示MSI是头颈部鳞癌中较为常见的遗传学变化,染色体9p21-p22和3p14区域可能存在与头颈部鳞癌有关的抑癌基因。     

Frequent loss of heterozygosity on chromosome 5 in non-small cell lung carcinoma.

AIMS: Loss of heterozygosity (LOH) at specific chromosomal regions strongly suggests the existence of tumour suppressor genes at the relevant segment. Frequent LOH on chromosome 5q has been reported in a wide variety of human tumours, including those of the lung. The aim of this study was to screen for LOH and to clarify the location of putative tumour suppressor genes on chromosome 5 implicated in the genesis and/or development of non-small cell lung carcinoma. METHODS: Thirty three patients with advanced non-small cell lung carcinoma were screened for LOH with a panel of 21 microsatellite DNA markers spanning the entire chromosome 5, using semi-automated fluorochrome based methodology. RESULTS: Twenty of the non-small cell lung carcinoma samples displayed LOH for one or more informative locus. LOH involving only 5q was found in 10 of 14 of the informative samples. Deletions involving 5p only were not present in the samples under study. There was no evidence of microsatellite instability in any of the analysed loci. These results indicate the presence of five distinct segments displaying high frequencies of deletion on chromosome 5, namely: 5q11.2-q12.2, 5q15 (D5S644 locus), 5q22.3-q23.1, 5q31.1, and 5q35.3. Eight of 14 samples had simultaneous interstitial deletions in at least two different regions. Moreover, concomitant deletion of three and four distinct regions was displayed in three of 14 and two of 14, respectively, of the informative samples. CONCLUSION: Allelic deletion on chromosome 5 is a frequent event in patients with non-small cell lung carcinoma. These results suggest the involvement of these five regions, either independently or simultaneously, in both lung squamous cell carcinoma and lung adenocarcinoma.     

A novel candidate tumour suppressor locus at 9q32-33 in bladder cancer: localization of the candidate region within a single 840 kb YAC

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, implicating the presence of a tumour suppressor gene or genes on 9q. To define the location of a tumour suppressor locus on 9q in TCC, we screened 156 TCCs of the bladder and upper urinary tract by detailed deletion mapping using 31 microsatellite markers on 9q. Partial deletions of 9q were found in 10 TCCs (6%), and LOH at all informative loci on 9q was found in 77 TCCs (49%). In five low grade superficial bladder tumours, the partial deletion was localized to D9S195 located at 9q32-33, with retention of heterozygosity at all other informative loci including D9S103, D9S258, D9S275 and GSN. We constructed a yeast artificial chromosome (YAC) contig covering the deleted region in these five tumours and placed another four unmapped microsatellite markers on this contig map. Using these markers, we further defined the common deleted region to the interval between D9S1848 and AFMA239XA9. The region is covered by a single YAC (852e11), whose size is estimated to be 840 kb. Our data should expedite further fine mapping and identification of the candidate tumour suppressor gene at 9q32-33.      

Mutation at chromosome 11q23 in human non-familial breast cancer: A microdissection microsatellite analysis

Allelotypic evaluation of loss of heterozygosity (LOH) has been instrumental in the identification of tumour suppressor genes. Here we report a high incidence of LOH at chromosome 11q23 in non-familial breast cancers with in situ, invasive, and metastatic tumour cells microdissected from archival haematoxylin and eosin (H & E) sections for polymerase chain reaction (PCR)-LOH analysis at polymorphic microsatellite loci. Ninety-four cases of non-familial breast cancer were examined at the D11S29 microsatellite locus on chromosome 11q23. Eighty-three cases (88 per cent) were informative and 35 cases overall (42 per cent) had LOH at this locus, comprising 23 per cent of in situ, 36 per cent of invasive, and 28 per cent of metastatic cancers. The DNA from those cancer cells with LOH was amplified at microsatellite loci D11S554 (11p12–p11.2) and D11S534 (11q13). In 19 of 67 cases overall (28 per cent), LOH occurred solely at 11q23. There was an association between LOH at 11q23 and tumour size ≥2 cm (P<0.01) in the overall results and the invasive cancers. The data revealed heterogeneity for LOH at D11S29 in in situ, invasive, and metastatic cells from the same case. In general, however, there was concordance between LOH (or its absence) in in situ and invasive disease. We conclude that the distal part of the long arm of chromosome 11 contains a region involved in breast carcinogenesis and that there is molecular heterogeneity at this chromosomal region in individual breast cancer cells.     

A high degree of chromosomal instability at 13q14 in cutaneous squamous cell carcinomas: indication for a role of a tumour suppressor gene other than Rb.

BACKGROUND/AIMS: Loss of function of the retinoblastoma (Rb) tumour suppressor gene, located on chromosome 13, is common in many inherited and sporadic forms of cancer. Inactivation of its gene product by oncogenic human papillomaviruses (HPV) plays a key role in the genesis of cervical cancer. It has been shown previously that non-melanoma skin cancers of renal transplant recipients and immunocompetent patients with skin cancer also frequently harbour potentially oncogenic HPV types. This study aimed to examine the integrity of the Rb gene in histologically confirmed squamous cell carcinomas (SCCs) from renal transplant recipients and immunocompetent patients with skin cancer. METHODS: Loss of heterozygosity (LOH) at the Rb locus was examined in 13 histologically confirmed SCCs using the D13S153 microsatellite marker, which is located in exon 2 of the Rb gene. Loss of a second marker, D13S118, distal telomerically to the Rb gene at 13q14.3 was also analysed. RESULTS: Of the 13 HPV associated SCCs examined 11 were informative (two SCCs were homozygous for both microsatellite markers). LOH at the D13S153 locus was found in four of the 10 informative SCCs and LOH at the D13S118 locus was found in five of the 11 informative cases. Overall, seven of the 11 informative cases showed LOH at one or other locus. This represents a high degree of chromosomal instability in these tumours. The expression of the Rb gene product in the 11 informative cases was analysed immunohistochemically. Expression of Rb was detected in 10 of the 11 SCCs examined. No correlation between the HPV status of the tumours and the expression of Rb was found. Although the only SCC not to express Rb also demonstrated LOH at the D13S153 locus, the remaining SCCs that had LOH at 13q14 were able to express RB: CONCLUSION: Another tumour suppressor gene located at 13q14 might be responsible for the genesis of these tumours.     

Identification of tumor suppressor loci on the long arm of chromosome 15 in primary small cell lung cancer

Small cell lung cancer (SCLC) frequently shows a loss of heterozygosity (LOH) on chromosome 15q. In order to define the commonly affected region on chromosome 15q, we tested 23 primary SCLCs by microsatellite analysis. By analyzing 43 polymorphic microsatellite markers located on chromosome 15q, we found that 14 (60.8%) of 23 tumors exhibited a LOH in at least one of the tested microsatellite markers. Two (14.3%) of the 14 tumors were found to have more than a 50% LOH on chromosome 15q. LOH was observed in five commonly deleted regions on 15q. Of those regions, LOH from D15S1012 to D15S1016 was the most frequent (47.8%). LOH was also observed in more than 20-30% of tumors at four other regions, from D15S1031 to D15S1007, from D15S643 to D15S980, from D15S979 to D15S202, and from D15S652 to D15S642. Four of the 23 tumors exhibited shifted bands in at least one of the tested microsatellite markers. Shifted bands occurred in 3.2% (29 of 914) of the loci tested. Our data suggests the presence of at least five tumor suppressor loci on chromosome 15q in SCLC, and further that these may play an important role in SCLC tumorigenesis.     

Sublocalization of putative tumor suppressor gene loci on chromosome arm 14q in neuroblastoma.

RFLP and microsatellite analysis with 23 polymorphic markers spanning the entire long arm of chromosome 14 in 108 neuroblastomas showed allelic loss in 19 out of 107 (18%) informative tumors, placing 14q among the most frequently affected chromosomal regions in neuroblastoma. One minimal deletion region could be sublocalized in 17 of 19 cases between markers D14S1 and D14S16, and a second one between markers D14S17 and D14S23 in band 14q32. Furthermore, breakpoints in bands 14q23 and 14q12 were detected. These results suggest the presence of at least two putative tumor suppressor gene loci on chromosome 14. Survival analyses revealed no prognostic impact of allelic loss of 14q in neuroblastoma. Genes Chromosomes Cancer 26:40-46, 1999.     

Region-specific loss of heterozygosity on chromosome 19 is related to the morphologic type of human glioma

Allelic mutation on chromosome 19 has previously been reported as a frequent genetic event in human glial tumors. In an effort to localize specific regions of importance on this chromosome better, 13 highly polymorphic genetic markers distributed along the length of chromosome 19 were used for evaluation of loss of heterozygosity (LOH) and microsatellite instability in a total of 100 brain tumors, including 75 astrocytomas (55 grade 4; 7 grade 3; 5 grade 2; 6 grade 1; and 2 other), 17 oligodendrogliomas (1 grade 4; 5 grade 3; 10 grade 2; and 1 grade 1), and 8 mixed oligoastrocytomas (MOA) (3 grade 4; 2 grade 3; and 3 grade 2). No microsatellite expansion was observed in these glial tumors for any of the chromosome 19 loci examined. LOH for loci on chromosome 19 was detected in 23/74 informative astrocytomas (31%), 11/17 oligodendrogliomas (65%), and 3/8 MOA (38%). Partial deletion of chromosome 19 occurred more frequently (31/37 cases) than did loss of one whole copy of the chromosome, and a morphology-specific pattern of LOH was observed. In 12/14 (86%) instances of chromosome 19 deletion in oligodendrogliomas and MOA, the 19q arm showed LOH, whereas the 19p arm showed no loss for all informative loci. Conversely, in 17/23 (74%) instances of chromosome 19 deletion in astrocytomas, the 19p arm showed LOH, whereas the 19q arm showed no loss for one or more loci. Thus, loss of 19q and retention of 19p are strongly associated with oligodendroglioma and MOA, whereas loss of 19p and retention of distal 19q is associated with astrocytoma. These data indicate that two or more tumor suppressor genes may reside on chromosome 19, one on 19p important in the development of astrocytomas, and one on 19q important in oligodendrogliomas and MOA.